AN ABSTRACT OF THE THESIS OF
Christopher A. Lowry for the degree of Doctor of Philosophy in Zoology presented
on June 2, 1995. Title: Neurobiology of Stress: Central Actions of CorticotropinReleasing Factor in an Amphibian.
Abstract Approved:
:rg_747 :.._
Redacted for Privacy
Frank L. Moore
Corticotropin-releasing factor (CRF) is a 41-amino acid peptide that elicits a
broad array of neuroendocrine, autonomic and behavioral responses that are also
observed in the adaptive response to stress. The studies in this thesis characterize the
effects of CRF on locomotor activity in a urodele amphibian, Taricha granulosa, as
well as the neurochemical and electrophysiological correlates of this behavior. These
studies establish that the effect of CRF on locomotor activity in T. granulosa is doseand time-dependent and is completely blocked by administration of the specific CRF
receptor antagonist, a-helical CRF9_41. In addition, a-helical CRF9_41 suppresses
stress-induced locomotor activity suggesting that an endogenous CRF-like
neuropeptide may contribute to the initiation and expression of stress-induced
locomotor activity. This conclusion is supported by the observation that CRF infused
directly into the lateral ventricle of T. granulosa rapidly (within 2-4 minutes) alters
medullary neuronal activity associated with increased locomotion.
Interactions between CRF, opioidergic, and monoaminergic systems in the
control of locomotor activity were investigated using behavioral, pharmacological, and
biochemical approaches. These studies demonstrated that endogenous opioid peptides
can suppress CRF-induced locomotor activity through activation of the mu-type opioid
receptor. Microdissection and high performance liquid chromatography with
electrochemical detection techniques were used to characterize the distribution of
catecholamines and indoleamines in the central nervous system of T. granulosa; other
studies using these techniques determined that CRF-induced locomotor activity is
associated with site-specific changes in the tissue concentrations of serotonin,
dopamine, and 5-hydroxyindoleacetic acid
monoaminergic neurotransmitters and a
serotonin metabolite. These changes in monoamines and 5-hydroxyindoleacetic acid
were restricted to the striatum and dorsomedial hypothalamus. The dorsomedial
hypothalamus in vertebrates is an important structure for the coordination of the
autonomic and behavioral responses to stress and therefore the CRF-induced
neurochemical changes in this region may have important consequences for the
physiological and behavioral state of the organism. Together, these studies support a
role for CRF as a neuromodulator or neurotransmitter in the regulation of locomotor
responses to stressful stimuli in T. granulosa.
Neurobiology of Stress: Central Actions of Corticotropin-Releasing Factor in an
Amphibian
by
Christopher A. Lowry
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed June 2, 1995
Commencement June 1996
Doctor of Philosophy thesis of Christopher A. Lowry presented on June 2, 1995
APPROVED:
Redacted for Privacy
Major Professor, representing Zoology
Redacted for Privacy
Chair of Depa
ent of
Redacted for Privacy
Dean of Graduate Sc
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Redacted for Privacy
uthor
ACKNOWLEDGEMENTS
I gratefully acknowledge my major professor, Dr. Frank Moore. Frank has
provided an ideal environment for my scientific and personal development. I am
especially grateful for his reviews of abstracts and revisions of manuscripts as well as
fellowship and grant applications; I have benefited greatly from this ongoing process.
I would also like to thank my committee members, Bob Mason, Tom Murray,
and Larry Ryan for interactions that have maintained my enthusiasm for this
sometimes overwhelming task of pursuing an advanced degree. I owe a great deal to
many excellent scientists for training throughout my tenure as a graduate student. I
thank Jim Rose who has incredible patience and a commitment to sharing his
expertise through teaching. I thank Ken Renner for his commitment to excellence in
our collaborations. I thank Pierre Deviche for developing in me an appreciation for
the value of collaboration (but may we never have to perform instantaneous sampling
again for the rest of our lives). I thank Tom Zoeller for sharing his expertise in the
application of in situ hybridization, as well as his patience in answering all of my
questions. I thank Linda Muske for sharing her expertise in immunohistochemical
techniques, as well as her enthusiasm for science. I thank Mike Moore for his
support for a project on Mount Graham in Eastern Arizona; I also thank Rosemary
Knapp, Eva Lacey, and Sarah Wood ley for tolerating long days climbing steep
ravines as well as braving wasps, rattlesnakes, and the Arizona State Police. I thank
former graduate students in our laboratory including Sunny Boyd, Glen Davis, Larry
Miller, Miles Orchinik, Cathy Propper, and Mitch Smith for sharing portions of my
life as a graduate student, and for continued friendships. I thank the faculty and staff
of the Zoology Department at Oregon State University as well as fellow graduate
students for a sense of community and mutual support. I thank many undergraduates
and high school students who have shared in the labors of research while I have been
here, including Cyrelle Bostrom, Luke Burke, Charlie Dean, Chris Henderson, Mark
Higby, Mark McKim, Lori McKinney, Marc Miller, Thao T. Pham, Richard Ralph,
Bev Schmidt, and Josh Tacchini. I thank Carla Richardson not only for the precision,
thoroughness, and high quality of her work but for here patience with me when I was
under pressure. I thank Jeb, Barrett, and Anthony Baugher, and Amber and Jason
Babcock for showing me the best way to go newting. I thank countless friends in the
community of Corvallis who have made my experience here an immensely enjoyable
one.
I thank Mary Curvey for her assistance and the National Institutes of Health
for financial support to conduct the studies included in this thesis as well as additional
studies which will be published elsewhere. I also thank Sigma-Xi for a Grant-in Aid
of Research.
Finally, I thank my family. I thank my parents, Charles E. and Sandra J.
Lowry for the freedom and encouragement to pursue my own goals. I thank my
older brother Anthony and my younger brother Jay for their friendship.
CONTRIBUTION OF AUTHORS
Chapter II is reproduced with the written permission of Elsevier Science
Publishers B.V. and the journal Brain Research. The experimental design, execution
of experiments, as well as analysis and interpretation of data for these studies were a
product of a collaboration with Dr. Pierre Deviche. Dr. Deviche and Dr. Frank
Moore provided editorial comments which were useful in the preparation of the
manuscript for publication.
Chapter III is reproduced with the written permission of Academic Press, Inc.
and the journal Hormones and Behavior. I conducted the experiments and analysed
the data in the laboratory of Dr. Frank Moore. Dr. Moore provided guidance
throughout these studies and provided editorial comments which were useful in the
preparation of the manuscript for publication.
The studies discussed in Chapter IV were conducted by me in the laboratory of
Dr. James D. Rose at the University of Wyoming. Dr. James Rose provided me with
the technical training necessary for the performance of these chronic recording
studies. All animal surgeries were performed by Dr. Rose. Drs. Moore and Rose
provided editorial comments which were useful in the preparation of the manuscript.
Chapter V is reproduced with the written permission of S. Karger AG and the
journal Brain, Behavior and Evolution. I conducted the experiments, including the
microdissections, in the laboratory of Dr. Moore. Dr. Ken Renner at the University
of Southwest Missouri provided quantitative measurements of specific monoamines in
prepared samples using high performance liquid chromatography with electrochemical
detection. Drs. Moore and Renner provided editorial comments which were useful in
the preparation of the manuscript for publication.
The studies discussed in Chapter VI were conducted in the laboratory of Dr.
Frank Moore. Dr. Renner, with the assistance of Kathy Kedzie, provided quantitative
measurements of specific monoamines in prepared samples using high performance
liquid chromatography with electrochemical detection. Dr. Miles Orchinik assisted in
the experimental design and execution of studies involving corticosterone. Dr. Moore
has provided editorial comments which were useful in the preparation of the
manuscript.
TABLE OF CONTENTS
Page
I. GENERAL INTRODUCTION
1
II. EFFECTS OF CORTICOTROPIN-RELEASING FACTOR (CRF)
AND OPIATES ON AMPHIBIAN LOCOMOTION
5
6
7
9
Abstract
Introduction
General Materials and Methods
Specific Methods and Results
Discussion
Acknowledgements
11
19
22
III. CORTICOTROPIN-RELEASING FACTOR (CRF) ANTAGONIST
SUPPRESSES STRESS-INDUCED LOCOMOTOR ACTIVITY IN AN
AMPHIBIAN
23
24
25
27
29
37
Abstract
Introduction
General Materials and Methods
Specific Methods and Results
Discussion
Acknowledgements
IV. CORTICOTROPIN-RELEASING FACTOR ENHANCES LOCOMOTION
AND MEDULLARY NEURONAL FIRING IN AN AMPHIBIAN . . .
Abstract
Introduction
Methods
Results
Discussion
Acknowledgements
V. CATECHOLAMINES AND INDOLEAMINES IN THE CENTRAL
NERVOUS SYSTEM OF A URODELE AMPHIBIAN: A
MICRODISSECTION STUDY WITH EMPHASIS ON THE
DISTRIBUTION OF EPINEPHRINE
Abstract
Introduction
Materials and Methods
Results
40
.
41
42
43
45
48
60
67
68
69
70
75
83
TABLE OF CONTENTS (Continued)
Discussion
Acknowledgements
Abbreviations
Abbreviations Used in Figures
VI. ELEVATION OF SEROTONIN, DOPAMINE, AND SELECTED
METABOLITE CONCENTRATIONS IN THE DORSOMEDIAL
HYPOTHALAMUS FOLLOWING CORTICOTROPIN-RELEASING
FACTOR OR CORTICOSTERONE ADMINISTRATION
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
89
120
120
120
123
124
125
127
131
140
154
VII. DISCUSSION
155
BIBLIOGRAPHY
159
LIST OF FIGURES
Page
Figure
II.1
11.2
11.3
11.4
Locomotor activity of male rough-skinned newts, defined as the number
of crossings in an open field arena during 3 consecutive minutes of
observation per animal
12
Locomotor activity in response to an i.c.v. injection of CRF (25 ng)
preceded by an i.p. injection of a control solution (Sal) or naloxone
(Nal; 200 ,c(g in B)
15
Locomotor activity in response to icy injections of a control solution or
CRF (25 ng) preceded by an ip injection of a control solution (Sal) or
bremazocine (Bre; 10 pg) in A; or preceded by a control solution (Sal)
or morphine (Mor; 10 AO in B
17
Locomotor activity in response to icy injections of CRF preceded by ip
injections of control solution (Sal), morphine (Mor; 10 ptg) or a
solution of morphine (Mor; 10 fig) and naloxone (Mor-Nal; 200 itg) .
. .
18
Corticotropin-releasing factor (CRF; 25ng) stimulated locomotor
activity when injected icy by itself (P < 0.001; Dunn's Test) but had
no effect when injected icy concurrently with ahCRF (CRF-A;
250 or 500 ng)
30
111.2
Locomotor activity of male rough-skinned newts
32
111.3
Locomotor activity of male rough-skinned newts tested 35 min after
drug treatment with control solution, clonidine (10 or 100 ng), CRF (25
ng), or CRF and clonidine together (n = 26)
36
Diagrammatic dorsal view of the brainstem of an adult male T.
granulosa, with black dots illustrating the location of recording sites in
the raphe region and adjacent reticular formation
49
IV.2
CRF-enhanced firing in a neuron with movement-related firing
50
IV.3
Diverse patterns of unit activity change in five simultaneously-recorded
rostromedial medullary neurons during CRF-induced locomotor
activation
53
111.1
IV.1
LIST OF FIGURES (Continued)
Behavioral and single-unit activity before and after infusion of saline
into the lateral ventricle, showing an absence of effect on neuronal
firing or behavior
56
IV.5
CRF-induced increases in firing rate in a medullary neuron that were
independent of behavioral state
58
V.1
Camera lucida drawings of brain tissues of an adult male T. granulosa
illustrating diagrammatically the relationships between the intact brain
and individual 300 ktm sections which were prepared for
microdissection
78
The ratios of metabolites to monoamines in the brain of adult male T.
granulosa were higher in regions of the brain with low concentrations
of the parent amines
90
IV.4
V.2
V.3
VIA
VI.2
VI.3
VI.4
Schematic representation of multiple biogenic amine-metabolizing
systems in the mammalian (above) and urodele amphibian (below)
infundibular hypothalami based on histochemical studies
112
Effects of CRF (25 ng) on monoamine and monoamine metabolite
levels in microdissected brain regions measured approximately 35 min
following treatment
133
Effects of CRF (50 ng) on monoamine and monoamine metabolite
levels in microdissected brain regions measured approximately 35 min
following treatment
135
Effects of corticosterone on monoamine and monoamine metabolite
levels in microdissected brain regions measured 20 min following
treatment
136
Effects of CRF or corticosterone on norepinephrine and epinephrine
concentrations in the dorsomedial hypothalamus
138
LIST OF TABLES
Table
Page
II.1
Locomotor activity in response to an i.p. injection of 0 or 200 itg
naloxone
13
111.1
Locomotor activity 2 hr after drug treatment (0, 25, 125, or 250 ng
ahCRF) either before (15°C) or during (28°C), Experiment IV; 30°C,
Experiment V) warm stress (n = 15)
34
111.2
Locomotor activity in response to icy injections
of clonidine
35
V.1
Monoamine levels and monoamine metabolite levels in microdissected
brain regions of T. granulosa
85
V.2
Percent epinephrine relative to norepinephrine in major
brain regions
88
VI.1
Locomotor activity in response to an i.c.v. injection
of saline or CRF
132
PREFACE
This thesis is composed of manuscripts which have been published, are in
press, are submitted for publication, or are prepared for publication. Chapter II was
published as Lowry, C.A., Deviche, P., and Moore, F.L. (1990) Effects of
corticotropin-releasing factor (CRF) and opiates on amphibian locomotion. Brain
Res., 513: 94-100. Chapter III has been submitted for publication and is currently
under review, Lowry, C.A., Rose, J.D., and Moore, F.L. (1995) Corticotropinreleasing factor enhances locomotion and medullary neuronal firing in an amphibian.
Chapter IV was published as Lowry, C.A., and Moore, F.L. (1991) Corticotropinreleasing factor (CRF) antagonist suppresses stress-induced locomotor activity in an
amphibian. Horm. Behay., 25: 84-96. Chapter V is in press, Lowry, C.A., Renner,
K.J., and Moore, F.L. (1995) Catecholamines and indoleamines in the central
nervous system of a urodele amphibian: A microdissection study with emphasis on the
distribution of epinephrine. Brain Behay. Evol. Chapter VI is prepared for
submission, Lowry, C.A., Orchinik, M., Burke, K.A., Renner, K.J., and Moore,
F.L. (1995) Elevation of serotonin, dopamine, and selected metabolites in the
dorsomedial hypothalamus following corticotropin-releasing factor of corticosterone
administration.
Portions of this work have appeared in preliminary form: 1) Lowry, C.A.,
Deviche, P., and Moore, F.L. (1988) Interaction between the opioid system and
corticotropin-releasing factor in the control of locomotion in an amphibian. Amer.
Zool. 28: 186A. 2) Lowry, C.A., Deviche, P., and Moore, F.L. (1990)
Neuroendocrine modulation of locomotor activity in an amphibian: Corticotropin-
releasing factor and opioids. Soc. Neurosci. Abstr. 16: 907. 3) Lowry, C.A.,
Renner, K.J., Laughlin, L.M., and Moore, F.L. (1991) CRF alters monoamine and
monoamine metabolite levels in discrete motor areas in amphibian brain. Soc.
Neurosci. Abstr. 17: 1418. 4) Lowry, C.A., Rose, J.D., and Moore, F.L. (1994)
Corticotropin-releasing factor alters medullary neuronal activity associated with
swimming and walking behaviors. Soc. Neurosci. Abstr. 20: 373. 5) Moore, F.L.,
Orchinik, M., and Lowry, C. (1995) Functional studies of corticosterone receptors in
neuronal membranes. Receptor, 5: 21-28.
Neurobiology of Stress: Central Actions of Corticotropin-Releasing Factor in an
Amphibian
CHAPTER I: GENERAL INTRODUCTION
In mammals CRF elicits neuroendocrine, autonomic, and behavioral events
that are also observed in the adaptive response to stress (reviewed by Taylor and
Fishman, 1988). Immunocytochemical studies suggest a homologous distribution of
CRF perikarya and fibers among vertebrates including a wide distribution of CRF-like
immunoreactivity in hypothalamic and extrahypothalamic areas (reviewed by Peter,
1986). These studies, in addition to the widespread distribution of CRF receptors in
the rat brain (De Souza, 1987), are consistent with possible neuromodulator functions
for CRF in addition to hypophysiotrophic functions.
To our knowledge only one nonmammalian species, the rough-skinned newt
(Taricha granulosa), has been used to study the behavioral effects of CRF. In this
species CRF elicits behavioral responses that are similar to responses observed in
rats. In rats and T. granulosa CRF suppresses male sex behavior (Sirinathsinghji,
1987; Moore and Miller, 1984) and stimulates locomotor activity (Sutton et al., 1982;
Moore et al., 1984). The stimulatory effects of CRF on locomotor activity persist in
hypophysectomized animals indicating a mechanism independent of the hypothalamic-
pituitary axis (Eaves et al., 1985; Moore et al., 1984).
Several studies have examined interactions between CRF and other
neurochemical systems. For example, the opioid system can modulate CRF-induced
locomotor activity, however, CRF-induced locomotor activity is not dependent on the
activation of opioid receptors (Koob et al., 1984; Saunders and Thornhill, 1986;
2
Lowry et al., 1990). Monoaminergic systems also can modulate CRF-induced
locomotor activity (Koob et al., 1984; Imaki et al., 1985, 1987), however, the
conclusion from these studies and others is that CRF-induced locomotor activity is not
dependent on the activation of dopaminergic or noradrenergic receptors (Swerdlow
and Koob, 1985; Butler et al., 1990; Cole and Koob, 1988; reviewed by Swerdlow et
al., 1986). Although administration of serotonergic 5-HTIA agonists does not alter
CRF-induced locomotor activity in rats (Lazosky and Britton, 1991), the possiblity
that CRF-induced locomotor activity is dependent on the activation of other
serotonergic receptor systems has not been thoroughly addressed.
Recent studies suggest that CRF stimulates 5-HT release and tryptophan
hydroxylase activity in the rat (Corley et al., 1990; Singh et al., 1992; Lavicky and
Dunn, 1993). These findings are consistent with recent work in our laboratory in
which the 5-HT uptake inhibitor fluoxetine had no effect on locomotor activity of
saline-injected animals but greatly enhanced locomotor activity in CRF-injected
animals (Lowry et al., 1993). The most parsimonious interpretation of the behavioral
data is that CRF stimulated 5-HT release in newt brain and fluoxetine enhanced
serotonergic neurotransmission by preventing reuptake from the synaptic cleft.
Neurochemical studies have described associations between CRF-induced
locomotor activity and site-specific changes in monoaminergic systems (Dunn and
Berridge, 1987; Kalivas et al., 1987; Barrett et al., 1989; Matsuzaki et al., 1989;
Shimizu and Bray, 1989; Emoto et al., 1993; Lavicky and Dunn, 1993; Lee et al.,
1994; reviewed by Dunn and Berridge, 1990). These studies demonstrated that CRF
3
alters catecholamine utilization in several brain areas. A few of these studies also
have described CRF-induced effects on serotonin metabolism (Barrett et al., 1989;
Shimizu and Bray, 1989; Lavicky and Dunn, 1993). In summary, although changes
in monoamine metabolism have been associated with CRF-induced locomotor activity,
the neurochemical mechanisms by which CRF stimulates locomotor activity are
unclear.
In addition to studies of the potential neurochemical mechanisms of CRFinduced effects on behavior, several studies have investigated the potential
neuroanatomical substrates for initiation of CRF-induced effects. In studies using
rodents CRF effectively stimulates locomotor activity when injected into the
hippocamupus (Lee and Tsai, 1989), amygdala (Lee and Tsai, 1989), ventral
tegmental area (Kalivas et al., 1987), paraventricular hypothalamic nucleus (Krahn et
al., 1988), and cerebral ventricle (Tazi et al., 1987b). The multiplicity of sites in
which CRF administration effectively stimulates locomotor activity in rodents suggests
an anatomical redundancy in the substrate for CRF action. Anatomical redundancy in
the substrate for CRF action was also suggested based on data from a study of CRF
activation of the autonomic nervous system in the rat (Brown, 1986). In this study,
Brown made site-specific injections of CRF throughout the rostrocaudal extent of the
rat brain. Although the injections were most effective when given in hypothalamic
areas and the third ventricle, injections in many other areas were also effective. In
the study by Brown, no site was more effective than the third ventricle for increasing
plasma norepinephrine levels. Other studies also suggest that the third ventricle and
4
the ventral forebrain are important neuroanatomical substrates for CRF-induced
effects (Tazi et al., 1987b; Spadaro et al., 1990).
In summary, the neuropeptide corticotropin-releasing factor (CRF) initiates
physiological and behavioral responses to stress. The mechanisms by which CRF
affects brain function and the neuroanatomical sites involved, however, are not clear.
The studies presented in this thesis use behavioral, pharmacological,
electrophysiological, and biochemical approaches to characterize the effect of CRF on
locomotor activity, with the ultimate aim of providing new information on possible
neurochemical mechanisms and neuroanatomical sites involved in this behavior. This
research involves the use of an amphibian model with the advantages of a structurally
simple brain and a behavioral response that is robust and easily quantified. The
neuroendocrine and behavioral effects of CRF are similar in amphibians and
mammals, suggesting that an understanding of CRF's effects in amphibians will
contribute to a general understanding of CRF actions in other animals, including
humans.
5
CHAPTER II: EFFECTS OF CORTICOTROPIN-RELEASING FACTOR (CRF)
AND OPIATES ON AMPHIBIAN LOCOMOTION
Christopher A. Lowry, Pierre Deviche, and Frank L. Moore
Department of Zoology, Oregon State University, Corvallis, OR 97331-2914
Copyright 1990 by Elsevier Science Publishers B.V.
All rights of reproduction in any form reserved.
6
ABSTRACT
Male rough-skinned newts (Taricha granulosa) were used as a model for the
study of the neuroendocrine regulation of locomotion. Intracerebroventricular (icy)
injections of nanogram quantities of corticotropin-releasing factor (CRF)
dose-dependently increased locomotion as measured in a circular open-field test arena.
In other studies animals received intraperitoneal (ip) injections of saline or naloxone,
a synthetic opioid antagonist, followed by icy injections of saline or CRF. With
1-minute intervals between injections, neither ip saline nor naloxone injections
modified the stimulatory effects of CRF injections on locomotor activity. In contrast,
with 20-minute intervals between injections, the naloxone-plus-CRF injected newts
displayed more locomotor activity than the saline-plus-CRF injected newts, suggesting
that the opioid system modulated the behavioral effects of CRF. An ip injection of
bremazocine, an opiate k-receptor agonist, suppressed spontaneous locomotion but not
CRF-induced locomotion. In contrast, an ip injection of morphine, an opiate
g-receptor agonist, did not affect spontaneous locomotion but reduced CRF-induced
locomotion, indicating further that the opioid system may modulate the behavioral
effects of CRF in this amphibian. The present study provides the first evidence that
both CRF and opioids may be involved in the regulation of amphibian locomotor
activity.
7
INTRODUCTION
Corticotropin-releasing factor (CRF), a 41-amino acid peptide originally
isolated from the ovine hypothalamus (Spiess et al., 1981; Vale et al., 1981),
stimulates the release of adrenocorticotropic hormone (ACTH) and 13- endorphin from
the adenohypophysis (Fryer et al., 1983; Rivier et al., 1982; Tonon et al., 1986; Vale
et al., 1981). In addition to these neuroendocrine effects, CRF elicits autonomic
(Brown et al., 1982) and behavioral (Berridge and Dunn, 1986; Britton et al., 1982)
events associated with the 'stress response'. Behavioral effects of CRF include the
inhibition of sexual behaviors and the stimulation of locomotor activity in amphibians
and mammals (Koob and Bloom, 1985; Moore and Miller, 1984; Moore et al., 1984;
Rivier and Vale, 1984; Sutton et al., 1982).
Studies of the effects of intracerebroventricular (icy) injections of CRF on
locomotor activity of nonmammalian vertebrates are limited to one species, the
rough-skinned newt (Taricha granulosa; Moore et al., 1984). In this species,
injections of synthetic ovine CRF stimulate locomotor activity in both intact and
hypophysectomized animals.
The effects of icy injections of CRF on mammalian locomotor activity depend
upon the dose administered and the testing environment. For example, an icy
injection of CRF dose-dependently increases locomotion in rats tested in photocell
cages (Koob and Bloom, 1985; Sutton et al., 1982), while rats tested in novel open
field environments exhibit a biphasic response to CRF injection: low doses of the
8
peptide stimulate the behavior (Sutton et al., 1982; Veldhuis and De Wied, 1984)
whereas high doses inhibit it (Sutton et al., 1982).
Opioid substances also influence mammalian locomotion. In rodents,
administration of the opioid receptor antagonist, naloxone, either has no effect
(Abercrombie et al., 1988), or decreases (Amir et al., 1979; Arnsten and Segal,
1979; Ukai and Kameyama 1985) spontaneous locomotion, suggesting that
endogenous opioids sometimes act to stimulate spontaneous locomotor activity.
Although many opioid substances can stimulate spontaneous locomotor activity, as
reviewed in Olson, et al., 1987, the synthetic opioid k-receptor agonists bremazocine
(Romer et al., 1977), tifluadom (Romer et al., 1982), U-50,488 (Clark and Pasternak,
1988; Vonvoigtlander et al., 1983) consistently suppress this behavior in rodents
(Castellano and Pavone, 1985; Castellano et al., 1984; Jackson and Cooper, 1986;
Vonvoigtlander et al., 1983). Likewise, dynorphin, an endogenous opioid peptide
which binds the k-receptor (Chavkin et al., 1982), suppresses locomotion in rodents
(Ukai and Kameyama, 1985; Zwiers et al., 1981). These observations are consistent
with the proposal that activation of the opioid k-receptor decreases spontaneous
locomotor activity (Mansour et al., 1988).
The effects of A-receptor agonists on spontaneous locomotor activity in
mammals are more complex. The opioid A-receptor agonists morphine (Martin et al.,
1976), DAGO (Handa et al., 1981) and FK 33,825 (Romer et al., 1977) given at
lower doses stimulate spontaneous locomotor activity in the rat (Babbini and Davis,
1972; Locke and Holtzman, 1986). These same substances, however, given at higher
9
doses to rats (Babbini and Davis, 1972; Locke and Holtzman, 1986) or morphine at
all doses given to DBA/2J mice (Gwynn and Domino, 1984) cause an initial
suppression of spontaneous locomotion followed by a period of stimulation.
In mammals, CRF and the opioid system may interact to regulate locomotion.
Naloxone treatment in rats either has no effect (Koob et al., 1984) or attenuates
(Saunders and Thornhill, 1986) the stimulation of locomotion caused by CRF
administration.
It is not known whether the apparent functional interaction between CRF and
opioids in the control of locomotor activity in rodents is a widespread phenomenon
among vertebrates. This study examines the possibility that such an interaction exists
in an amphibian. Agonists for the k- and 14- receptors were chosen for study based on
their effects on spontaneous locomotion in vertebrates and on binding studies which
indicate that opioid receptors in amphibians are predominantly of theµ and
benzomorphan preferring or kl a types (Ruegg et al., 1981; Simon et al., 1982). In
this study an amphibian (T. granulosa) was tested using an open-field testing
procedure to investigate the effects on locomotion of CRF and of opioids specific for
the p,- and k-receptor types administered either separately or in combination.
GENERAL MATERIALS AND METHODS
Adult male T. granulosa, averaging 15 g in body weight, were collected
locally (Benton County, Oregon) from freshwater ponds and were maintained in the
laboratory for approximately 48 h in two circular holding tanks (85 cm in diameter),
each containing 100 1 of dechlorinated tap water. These tanks were in a room with
10
controlled photoperiod (14 h light/10 h dark; lights on at 06.30 h) and an ambient
temperature of 19°C. Newts were not handled or disturbed until immediately prior to
treatment. Experiments were initiated between 11.00 and 15.30 h.
Synthetic ovine CRF was supplied by Drs. J. Rivier and W. Vale (Salk
Institute for Biological Studies, San Diego, CA); naloxone hydrochloride was
provided by Endo Pharmaceuticals (Glenolden, NY); (±) bremazocine hydrochloride
was provided by Dr. D. ROmer (Sandoz; Basel, Switzerland); and morphine sulphate
was purchased from Mallinckrodt Chemical Works (St. Louis, MO). Drugs were
dissolved in amphibian Ringer's solution (saline). Control injections consisted of the
appropriate volume of saline alone: 2µl for intracerebroventricular (icy) or 100 i.41
for intraperitoneal (ip) injections. Intracerebroventricular injections were given
through a 0.5 mm hole at the junction of the parietal and frontal bones in the cranial
midline using a microsyringe with a tip diameter of 50 inn as described in Moore and
Miller, 1983. Solutions (2 Al) were infused over a period of 5 seconds.
Depending on the experiment, newts received one or two injections and, unless
otherwise stated, were tested starting 30 minutes following the final injection. During
the periods between the two injections in Experiment V and from final injection to
testing in all experiments, newts were isolated in temporary circular holding tanks (25
cm in diameter) containing 5 1 dechlorinated tap water.
To measure locomotor activity newts were placed individually in a water-filled
circular runway with an inside diameter of 70 cm and an outside diameter of 85 cm.
Each test arena contained 30 1 dechlorinated tap water and was marked with radial
11
lines to define 16 equal sectors. Starting 4 minutes after placement, locomotor
activity was quantified by counting the total number of lines crossed during 3
consecutive minutes. Locomotion consisted of a combination of walking and
swimming.
Kruskal-Wallis one-way analysis of variance was used to test for statistical
differences among treatment groups within each experiment (n = 15 unless otherwise
stated). When appropriate, comparisons between two experimental groups were made
using the Mann-Whitney U-test (Siegel, 1956). Two-tailed probabilities P < 0.05
were considered statistically significant.
SPECIFIC METHODS AND RESULTS
Experiment I. Dose-response study: icy injections of CRF
To determine if CRF would elicit a dose-dependent behavioral response,
locomotor activity was measured 30 minutes after an icy injection. The experiment
was conducted in two parts. In Part I newts received 0, 25, 50 or 150 ng CRF;
newts in Part II received 0, 6.25, 12.5, or 25 ng CRF. Data obtained from identical
treatments (0 and 25 ng) in Parts I and II were combined (n = 30) for statistical
analyses since corresponding groups were not different.
Centrally administered CRF stimulated locomotor activity in a dose-dependent
pattern (Fig.II.1A). Significant differences were observed between the treatment
groups (T = 41.27; P < 0.0001). Comparison of CRF-injected newts with control
newts revealed that doses ranging from 12.5 to 150 ng increased locomotor activity.
12
The dose of 25 ng, equivalent to about 1.7 ,u,glkg body weight, resulted in a
maximum response and was used in subsequent experiments.
A
0
E.25
12.5
25
50
150
CRF (ng)
B
ED Soling
40
7.
12:J CRF(25rvg)
>
30
O
0
w
al
20
E
o0
o
_J
180
30
Time (min)
360
FIGURE ILL Locornotor activity of male rough-sldnned newts, defined as the
number of crossings in an open field arena during 3 consecutive minutes of
observation per animal. Results are presented as median values (±) interquartile
ranges. (A) Locomotor activity 30 minutes after icy injection of various doses of
CRF (n = 15 except groups which received 0 or 25 ng CRF for which n = 30). (B)
Time course of the effect of CRF on locomotor activity. Animals received an icy
injection of a control solution (Saline) or of 25 ng CRF (n =15). *: P < 0.05,
2-tailed probability for comparison of control and corresponding CRF-treated groups
(Mann-Whitney U-test).
13
Experiment II. Time-course study: icy injection of CRF
To determine the temporal relationship between an icy injection and the
stimulation of locomotor activity, newts were injected with saline or 25 ng CRF and
then were tested 5, 30, 180, and 360 minutes after the injection. Between tests,
newts were returned to the temporary holding tanks.
An icy injection of CRF resulted in significant increases in locomotor activity
compared to a saline injection. This effect was apparent starting 30 minutes and
lasting for at least 360 minutes after injection (Fig.II.1B). Subsequent experiments
measured locomotor activity starting 30 minutes after injection of CRF.
TABLE ILL Locomotor activity in response to an ip injection of 0 or 200 fig
naloxone
Newts were tested either 20 minutes or 50 minutes following treatment. Values
represent median locomotor activity and interquartile range for each group. No
statistically significant differences between the treatment groups were observed.
Treatment
No injection
Control injection
200,ug naloxone
Median locomotor activity
(crossings13 min)
20 min
50 min
2 (0-10)
2 (0-8)
2 (0-6)
2 (0-7)
2 (0-10)
7 (2-8)
Experiment III. Injection of naloxone
To investigate a possible involvement of opioids in amphibian spontaneous
locomotor activity, newts were injected ip with 200 itg naloxone. Two control groups
were included: one group (handled controls) consisted of newts that were handled but
14
not injected; the other group (saline-injected controls) consisted of newts that were
handled and injected ip with amphibian Ringer's solution. Half of the newts in each
treatment group were tested for locomotor activity at 20 minutes after the injection;
the remaining newts were tested for locomotor activity at 50 minutes after the
injection.
Results are presented in Table I. No statistical differences were observed
between any of the 3 groups tested 20 minutes (T = .51; P > 0.75) or 50 minutes (T
= 4.20; P > 0.10) after treatment.
Experiment IV. Sequential injections of naloxone and CRF: short interinjection
interval
To determine if the stimulatory effects of CRF on locomotion are modulated
by the opioid system, sequential injections of naloxone and CRF were administered to
newts. The first injection was ip and consisted of 0, 40, or 200 fig naloxone. The
second injection was icy and consisted of saline or 25 ng CRF. The interval between
the injections was approximately 1 minute; testing was conducted 30 minutes after the
second injection.
Results of the experiment are presented in Fig.II.2A. There were statistical
differences among the four experimental groups (T = 22.17; P < 0.0001). The
three groups of newts which received an injection of CRF each moved more than the
control group (Sal/Sal). Neither injection of saline nor injection of naloxone affected
the CRF-induced stimulation of locomotor activity.
15
A
(1 min Intoned)
40
20 10 0
B
Sal
Sal
Sal
CRF
Nal
Nal
(4Oug)
(200u0
CRF
CRF
(20 min Intorval)
40 30 -
2010-
a
0
Sal
Sal
Sal
CRF
Nal
CRF
FIGURE 11.2. Locomotor activity in response to an icy injection of CRF (25 ng)
preceded by an ip injection of a control solution (Sal) or naloxone (Nal; 200 jig in B).
The approximate time interval between injections was either 1 minute (A;n = 15) or
20 minutes (B;n = 15). See Fig.II.1 for additional comments. Different superscripts
represent significant differences between treatment groups, 2-tailed probability <
0.05 (Maim-Whitney U-test).
Experiment V. Sequential injections of naloxone and CRF: 20 minute interinjection
interval
To insure that naloxone would reach its binding sites before CRF was
administered, the interval between the injections was increased. As in Experiment
IV, newts were given sequential injections of naloxone and CRF. In this experiment,
however, the interval between injections was 20 minutes rather than 1 minute.
Results are illustrated in Fig.II.2B. There were significant differences between
the three experimental groups (T = 17.05; P < 0.0002). Newts injected with control
16
solution, then CRF (Sal/CRF), moved more than newts that did not receive CRF
(Sal/Sal; P < 0.05). Newts injected with 200 kg naloxone, then CRF, moved more
than both control newts (Sal/Sal; P < 0.0001) and newts treated with control solution
and CRF (Sal/CRF; P < 0.05). Therefore, the stimulatory effects of CRF were
enhanced by prior treatment with naloxone.
Experiment VI. Sequential injections of bremazocine and CRF
To test if an exogenously administered opiate k-receptor agonist could alter the
behavioral response to CRF, newts received 2 injections: the first injection was ip
and consisted of saline or 10 ttg bremazocine; the second injection was icy and
consisted of saline or 25 ng CRF. A short interinjection interval (approximately 1
minute) was used to avoid possible complications resulting from the release of
endogenous opioids associated with initial injection and/or handling of the animals.
In addition, because in the previous experiment the effects of naloxone were observed
50 minutes after its injection, newts in this experiment were tested 50 minutes after
treatment with bremazocine.
Results of the experiment are presented in Fig.II.3A. There was a significant
treatment effect (T = 34.00; P < 0.0001). Treatment with bremazocine followed by
an icy injection of control solution decreased locomotor activity. An icy injection of
CRF increased locomotor activity whether the injection was preceded by a control
injection or by an injection of 10 Ag bremazocine.
17
A
40
5S
E
0 0,
30
O
20
o
o
10
E.
b
I al
0
Sal
Sal
Bre
Sal
Sal
CRF
CRF
Sal
Sal
Mor
Sal
Sal
CRF
CRF
Bre
B
40 -
30 20 10 0
Mor
FIGURE 11.3. Locomotor activity in response to icy injections of a control solution
or CRF (25 ng) preceded by an ip injection of a control solution (Sal) or bremazocine
(Bre; 10 Ag) in A; or preceded by a control solution (Sal) or morphine (Mor; 10 Ag)
in B. The time interval between injections was approximately 1 minute. See Fig.II.1
for additional comments. Different superscripts represent significant differences
between treatment groups, 2-tailed probability < 0.05 (Mann-Whitney U-test).
Experiment VII. Sequential injections of morphine and CRF
To determine whether an exogenously administered opiate A-receptor agonist
could alter the behavioral response to CRF, Experiment VI was replicated using
morphine (10 Ag) instead of bremazocine. The experiment was conducted in two
parts. Part I (n=15) and Part II (n=15) were identical but performed on separate
days.
Results of the experiment are presented in Fig.II.3B. There was a significant
treatment effect (T = 41.49; P < 0.0001). Morphine had no effect on spontaneous
18
locomotor activity but reduced the behavioral stimulation induced by CRF (P <
0.05).
Experiment VIII. Sequential injections of morphine/naloxone and CRF
To determine if the reduction of CRF-induced locomotion caused by morphine
was naloxone-reversible and therefore due to activation of an opiate receptor, three
treatment groups were formed based on the nature of the initial injection. The initial
injection consisted of either (1) a control solution, (2) morphine (10 Ag) alone, or (3)
a solution of morphine (10 pg) and naloxone (200 pg). In each case the initial
injection was followed by an icy injection of 25 ng CRF. The experiment was
conducted in two parts. Part I (n=15) and Part II (n=15) were identical but
performed on subsequent days.
>.
5.
,--.
t< rEo
L
Or
8
E
40
c
,,
.c
O0
30_
la
a
b
20
r'g
O c'..3
o s_.
10
__I
0
Sal
CRF
Mor
CRF
NalMor
CRF
FIGURE H.4. Locomotor activity in response to icy injections of CRF preceded by
ip injections of control solution (Sal), morphine (Mor; 10 p,g) or a solution of
morphine (Mor; 10 pg) and naloxone (Mor-Nal; 200 AO. The time interval between
injections was approximately 1 minute. See Fig.II.1 for additional comments.
Different superscripts represent differences between treatment groups, 2-tailed
probability < 0.05 (Mann-Whitney U-test).
19
Results are presented in Fig.II.4. There was a significant treatment effect (T
= 6.92; P < 0.05). As in the previous experiment, prior treatment with morphine
reduced the behavioral stimulation induced by CRF (P < 0.05). This effect of
morphine was not evident in the presence of the specific opiate receptor antagonist
naloxone, suggesting that the suppressive effect of morphine was mediated by an
opiate receptor.
DISCUSSION
Results of this study indicate that in rough-skinned newts, an icy injection of
CRF stimulates locomotor activity in a dose and time-related pattern. Injection of
doses ranging from 12.5 to 150 ng CRF increased locomotion in newts tested 30
minutes after treatment and administration of a dose of 25 ng of the peptide stimulated
locomotor activity for up to 6 h. These results are consistent with previous results
(Moore et al., 1984) and further characterize the stimulatory effects of CRF on
locomotion in T. granulosa. They are also consistent with the effects of the peptide
in rats tested in photocell cages (Sutton et al., 1982).
Our investigation of the interaction between CRF and the opioid system in the
regulation of locomotion is the first to be performed on any nonmammalian
vertebrate. The opioid receptor antagonist, naloxone, when given immediately before
CRF, did not affect CRF-induced stimulation of locomotor activity in newts. This
result would suggest that the stimulatory effects of CRF in the newt are not modulated
by the opioid system. However, when naloxone was given 20 minutes before CRF,
20
naloxone enhanced the stimulatory effects of CRF on locomotor activity. This
observation suggests that handling and/or injecting the newts with a control solution
20 minutes before injecting CRF stimulated the release of one or more endogenous
opioids which then reduced CRF's potential to stimulate locomotion.
Indeed, opioids are released in response to a multitude of physical stressors in
mammals, as reviewed in Olson, et al. (1986) and in Amir et al. (1980), and at least
one study has demonstrated opioid involvement in stress-induced behavioral alterations
in an amphibian (Pezalla and Dicig, 1984). Under the experimental conditions of this
study, naloxone by itself did not affect spontaneous locomotor activity. The
endogenous opioid substances which were apparently released in response to handling
or injection may not have been present in sufficient amounts to affect spontaneous
locomotion, yet they may have been present in sufficient amounts to affect
CRF-induced stimulation of locomotion. Treatment with naloxone before treatment
with CRF would under these circumstances negate the inhibitory effects of the
endogenous opioids, allowing the full expression of CRF-induced stimulation of
locomotor activity.
An interaction between CRF and naloxone in the regulation of locomotion has
been demonstrated in rats (Saunders and Thornhill, 1986). In this species, naloxone
given 10 minutes before an icy injection of CRF attenuates the stimulatory effect of
the peptide (Saunders and Thornhill, 1986). This attenuation is consistent with other
studies indicating that in rodents, naloxone administration can decrease locomotion
(Amir et al., 1979; Arnsten and Segal, 1979; Ukai and Kameyama, 1985). In
21
contrast, naloxone treatment under some conditions increases locomotion in newts
(Deviche et al., 1989); this observation is consistent with the fact that in newts,
naloxone injection enhances the stimulatory influence of CRF on locomotion. Thus,
while in mammals opioids appear to enhance the effects of CRF (Saunders and
Thornhill, 1986), the opposite may be true in amphibians.
To study the mechanism involved in the interaction between CRF and the
opioid system in the newt, we examined the effects produced by the administration of
the opioid k-receptor agonist, bremazocine (Romer et al., 1980). Bremazocine
injection, when followed immediately by an icy injection of saline, decreased
spontaneous locomotor activity. Bremazocine did not, however, affect the stimulation
of locomotor activity induced by CRF. These results suggest that although opioid
k-receptors play a role in the regulation of amphibian locomotion, they do not
modulate the influence of CRF on this behavior.
A counterpart to the mammalianµ opioid receptor has been characterized in
urodele amphibians (Audigier et al., 1980). Based on experiments performed in the
present study, using morphine, activation of A-type receptors may reduce the potential
for CRF to stimulate locomotor activity in the newt. This finding is interesting in
light of evidence which suggests that in mammals CRF-induced behavioral activation
may involve stimulation of norepinephrine-containing cells of the locus coeruleus
(Valentino et al., 1983; Velley et al., 1988), a brain nucleus in which both the opioid
k- and A-receptor subtypes have been located (reviewed in Mansour et al., 1988).
22
Activation of the pc receptor hyperpolarizes locus coeruleus cells (Pepper and
Henderson, 1980) and decreases single cell activity (Abercrombie et al., 1988; Korf
et al., 1974; North et al., 1987), while application of either k-type (McFadzean et al.,
1987) or 6-type (North et al., 1987) receptor agonists has no effect on single cell
activity. Only opioid agonists selective for the pc-receptor appear to be able to affect
locus coeruleus cells directly and this effect is inhibitory.
The nature of the neurohormonal regulation of these norepinephrine-containing
cells in the mammal corresponds to the neurohormonal regulation of locomotor
behavior in this amphibian.
ACKNOWLEDGEMENTS
We thank Drs. J. Rivier, W. Vale, D. Romer, and Endo Pharmaceuticals for
their generous gifts of materials used in these studies. We also thank Miles Orchinik
and Cathy Propper for valuable criticism of the manuscript and Dr. Gary De Lander
for assistance in experimental design. This research was supported in part by an
NICHD grant, RO1 HD13508.
23
CHAPTER III: CORTICOTROPIN-RELEASING FACTOR (CRF)
ANTAGONIST SUPPRESSES STRESS-INDUCED LOCOMOTOR ACTIVITY
IN AN AMPHIBIAN
Christopher A. Lowry and Frank L. Moore
Department of Zoology, Oregon State University, Corvallis, OR 97331-2914
Copyright 1991 by Academic Press, Inc.
All rights of reproduction in any form reserved.
24
ABSTRACT
Intracerebroventricular (icy) injections of corticotropin-releasing factor (CRF;
25 ng) given to male rough-skinned newts (Taricha granulosa) stimulated locomotor
activity tested in a circular arena starting 35 min after the injection. The CRF
receptor antagonist, a-helical CRF9_41 (ahCRF; 250 or 500 ng), injected icy
concurrently with CRF, blocked CRF-induced locomotor activity. In contrast, icy
injection of ahCRF had no effect on spontaneous locomotor activity. Other studies
examined the effect of ahCRF on the elevated locomotor activity that was observed
when the animals were stressed (handled or placed in warm water). The CRF
antagonist dose-dependently attenuated the response to either handling or warm stress
tested 2 h after drug treatment. We also examined the effect of the a2-adrenergic
agonist, clonidine, on spontaneous and CRF-induced locomotor activity. Clonidine
injected icy dose-dependently suppressed spontaneous locomotor activity but not CRF-
induced locomotor activity. These studies support the hypothesis that endogenous
CRF is involved in mediating stress-induced locomotor activity and indicate that the
effects of CRF on locomotor activity are independent of activation of the a2adrenergic system.
25
INTRODUCTION
Since the identification and characterization of corticotropin-releasing factor
(CRF; Vale et al., 1981), CRF-like immunoreactivity has been identified in
representatives of each major vertebrate group (reviewed by Peter, 1986). Further,
the neuroanatomical distribution of CRF-immunoreactive cell bodies and fibers
appears to be very similar among vertebrates, including the distribution of
CRF-containing neurons in extrahypothalamic areas (Peter, 1986). This widespread
distribution of CRF in the central nervous system suggests that CRF, or CRF-like
substances, may have additional functions besides that of controlling the
hypothalamic-pituitary-adrenal axis.
There is evidence that in mammals CRF can stimulate the release of
adrenocorticotropic hormone (ACTH) and 0-endorphin from the anterior pituitary
(Vale et al., 1981). In addition, the administration of CRF elicits electrophysiological
(reviewed by Siggins et al., 1985), neurochemical (Dunn and Berridge, 1987; Kalivas
et al., 1987), behavioral (Britton et al., 1982; Morley and Levine, 1982;
Sirinathsinghji, 1987; Sirinathsinghji et al., 1983; Sutton et al., 1982; Veldhuis and
De Wied, 1984), and autonomic (Brown et al., 1982) responses. These diverse
effects of CRF have led to the hypothesis that CRF may initiate and coordinate a
generalized response to stressful stimuli (Gold and Chrousos, 1985; Koob and Bloom,
1985).
In contrast to the many studies with mammals, few studies have investigated
non-hypophysiotropic functions for CRF in nonmammalian vertebrates. However, in
26
the urodele amphibian, T. granulosa, the administration of CRF suppresses male
sexual behavior and stimulates male locomotor activity (Moore and Miller, 1984;
Moore et al., 1984; Lowry et al., 1990). The effects of CRF on locomotor activity
in this species are dose-related and time dependent (Lowry et al., 1990), and appear
to be centrally mediated since icy injections of CRF stimulate locomotor activity in
hypophysectomized newts (Moore et al., 1984).
In mammals the CRF-receptor antagonist, a-helical CRF9_41 (ahCRF; Rivier et
al., 1984) suppresses stress-induced neuroendocrine (Rivier et al., 1986), autonomic
(Brown et al., 1985) and behavioral (Berridge and Dunn 1987; Ka lin et al., 1988;
Krahn et al., 1986; Tazi et al., 1987a; Winslow et al., 1989) effects. These studies
suggest a role for an endogenous CRF in regulating neuroendocrine, autonomic and
behavioral responses to stressful stimuli in mammals. To examine whether an
endogenous CRF-like substance has a role in regulating the behavioral response to
stressful stimuli in a nonmammalian vertebrate, we investigated the effect of ahCRF
on stress-induced locomotor activity in T. granulosa.
Several studies, electrophysiological (Valentino and Foote, 1988),
neurochemical (Dunn and Berridge, 1987), and behavioral (Imaki et al., 1987; Cole
and Koob, 1988; Ehlers et al., 1987; Butler et al., 1990), suggest that behavioral
effects of CRF in mammals are mediated at least in part by an activation of
norepinephrine-containing cells. The neurochemical mechanism by which CRF elicits
behavioral effects in nonmammalian vertebrates is unknown. However, since the
neuromodulatory control of CRF-induced locomotor activity in male newts is similar
27
to the neuromodulatory control of norepinephrine-containing cells in mammals; that
is, both are suppressed by A-opioid receptor agonists (Abercrombie et al., 1988;
Lowry et al., 1990; North and Williams, 1984) and unaffected by k-opioid receptor
agonists (Lowry et al., 1990; McFadzean et al., 1987), we hypothesized that CRF
induces locomotor activity in male newts by an activation of
norepinephrine-containing cells. Since the single cell activity of
norepinephrine-containing cells in mammals is suppressed by a2-adrenergic agonists as
well as p,-opioid receptor agonists (Aghajanian and Wang, 1986), we investigated the
effect of an a2-adrenergic agonist on CRF-induced locomotor activity in male newts.
GENERAL MATERIALS AND METHODS
Adult male newts (T. granulosa; approximately 15 g in body weight) were
collected from local freshwater ponds and transported to two large water-filled
holding tanks. These tanks (85 cm in diameter with approximately 100 1
dechlorinated tap water and an ambient temperature of 15 °C) and tanks used for
testing were in a room with a controlled photoperiod (12 h light/12 h dark; lights on
at 07:00 h).
Testing for locomotor activity was done in one of two types of circular testing
tanks. These tanks contained either a small circular arena (inside diameter 10 cm;
outside diameter 25 cm) or a large circular arena (inside diameter 70 cm; outside
diameter 85 cm). Both arenas contained dechlorinated tap water (15 °C) to a depth of
approximately 15 cm. Small arenas were marked with 8 equally-spaced radial lines,
large arenas were marked with 16 radial lines. Small arenas were used in
28
experiments requiring testing of unhandled animals. Locomotor activity consisting of
integrated swimming and walking movements was recorded as the number of lines
crossed during a 3 min test period.
Experimental conditions were varied based on objectives of individual
experiments. In experiments I and VII, experimental conditions were selected to
optimize the behavioral response to exogenous CRF. These conditions, including the
interval between treatment and placement in a testing arena (30 min), and the interval
between placement and testing (5 min), were established in previous work (Lowry et
al., 1990). In experiments II-V, experimental conditions were selected to optimize
the behavioral effects of endogenous CRF. Two treatments were given in these
experiments, an initial drug treatment followed by a stress treatment designed to cause
the release of endogenous CRF. Since previous results (Lowry et al., 1990) suggest
that as a result of endogenous opioid release, endogenous CRF would have no
behavioral effect if released within 30 minutes of drug treatment, the interval between
drug treatment and testing was increased to 2 hours. In addition since we were
interested in the effects of both drug treatment and stress treatment in these
experiments, animals were tested immediately following stress treatment.
Synthetic ovine CRF was supplied by Drs. J. Rivier and W. Vale (Salk
Institute for Biological Studies, San Diego, CA); ahCRF and clonidine hydrochloride
were purchased from Sigma Chemical Co. (St. Louis, MO). In experiments using
ahCRF, drugs were dissolved in amphibian Ringer's with 0.001 M HC1. In other
experiments drugs were dissolved in amphibian Ringer's alone. In each case solutions
29
were prepared the day of the experiment. For treatment, newts were selected
randomly from large holding tanks and given intracerebroventricular (icy) injections
through a 0.5 mm hole in the cranial midline at the junction of the parietal and frontal
bones as described by Moore and Miller (1983). Solutions (2 id) were injected over a
5 s period using a microsyringe with a tip diameter of approximately 50 Am.
Two-factor experiments with completely randomized designs (Experiments I,
II, VII) were analyzed with a nonparametric two-factor analysis of variance (Scheirer,
Ray, and Hare, 1984). Two-factor experiments with repeated measures on one factor
(Experiments III, IV, V), were analyzed using the Friedman test for two or more
observations per experimental unit (Marascuilo and McSweeney, 1977). Differences
within levels of the blocking factors were determined using Kruskal-Wallis one-way
analysis of variance (Siegel, 1956; Experiment III) or nonparametric monotonic trend
analysis (Ferguson, 1965; Experiments IV, V). Experiment VI was analyzed with a
Kruskal-Wallis one-way analysis of variance.
When appropriate, nonparametric
multiple comparisons were made using Dunn's Test (Dunn, 1964). Two-tailed
probabilities < 0.05 were considered statistically significant.
SPECIFIC METHODS AND RESULTS
Experiment I. CRF-antagonist: Effects on spontaneous and CRF-induced locomotor
activity
To test whether ahCRF would affect spontaneous locomotor activity, newts
received a single icy injection of saline or one of two doses of the CRF antagonist
(250 or 500 ng; n = 15). Treated newts were isolated for 30 min in temporary
30
holding tanks (25 cm in diameter) containing 5 I dechlorinated tap water, then placed
in large testing arenas and tested 5 min after placement.
O Solin
50
CRF(25 ng)
40
30
++
20
10
0
Control
ohCRF
(250 n9)
ohCRF
(500 nq)
TREATMENT
FIGURE III.1. Corticotropin-releasing factor (CRF; 25ng) stimulated locomotor
activity when injected icy by itself (*P < 0.001; Dunn's Test) but had no effect when
injected icy concurrently with ahCRF (CRF-A; 250 or 500 ng). Newts were tested in
water-filled arenas 35 min after treatment (n = 15). Locomotor activity was
quantified as the number of crossings during a 3 min test period and is expressed as
the median value (±) interquartile range. +P < 0.01, + +P < 0.001, two-tailed
probabilities for comparison of CRF-injected control and corresponding
ahCRF-treated groups (Dunn's Test).
To test whether ahCRF would affect CRF-induced locomotor activity, newts
received either a single icy injection of CRF (25 ng) or a single icy injection of a
combination of CRF (25 ng) and one of two doses of ahCRF (250 or 500 ng; n =
15). Newts were then handled and tested as described in the above paragraph. There
was a significant interaction between CRF and the CRF antagonist (df = 2, H =
12.56, P < 0.001). As shown in Fig.III.1, ahCRF decreased locomotor activity but
only in CRF-injected animals.
31
Experiments II-III. CRF-antagonist: Effects on spontaneous and handling-induced
locomotor activity
Experiment II. To test whether handling would cause an increase in locomotor
activity and, pending an increase, to determine whether ahCRF would suppress
handling-induced locomotor activity, icy injections of either saline or 250 ng ahCRF
(n = 26) were given to newts. Following treatment, newts were placed individually
in small testing arenas.
Two h after drug treatment newts from half of each treatment group were
subjected to mild handling-stress, the remaining newts were left undisturbed.
Handling consisted of grasping the newt lightly behind the forelimbs with the index
and middle fingers and placing it in the palm of the opposite hand; light pressure was
applied by the thumb on the neck of the newt during a 30 s period. This procedure
was intended to restrain rather than to inflict pain by squeezing the newt.
Time-matched pairs of handled and unhandled newts were tested simultaneously,
immediately upon replacement of the handled newt in the small testing arena. Results
are presented in Fig.III.2A. There was a significant effect of handling (df = 1, H =
13.73, P < 0.001). Subsequent comparison of unhandled and handled newts
indicated that handling significantly increased locomotor activity in saline-injected
newts (z = 3.49, P < 0.01) but not ahCRF-injected newts (z = 1.82, P > 0.10).
Experiment III. To determine whether the effect of ahCRF on handling-induced
locomotor activity was dose-related, newts received an icy injection of 0, 250 or 500
ng ahCRF (n = 15). These groups were tested before and after handling, 2 h after
32
A
I
Saline
I
ahCRF
(250 ng)
Saline
ahCRF
(250 ng)
HANDLED
NOT HANDLED
8
5
c
E
<
CI Before Handling
IZ3 After Handling
20
0
O
.
E
o
10 -
Ci
Saline
ahCRF
(250 ng)
ahCRF
(500 ng)
TREATMENT
FIGURE 111.2. Locomotor activity of male rough-skinned newts. Data shown are
median values and interquartile ranges. See Fig.III.1 for description of locomotor
activity. A: locomotor activity of unhandled and handled (30 s) newts 2 h after
treatment with a control solution or ahCRF (250 ng; n = 13). *P < 0.05, two-tailed
probability for comparison of unhandled and corresponding handled groups (Dunn's
Test). B: locomotor activity (2 h after drug treatment with 0, 250 or 500 ng ahCRF;
n =15) of newts before and after a 30 s period of handling. Handling caused a
significant increase in locomotor activity (df =1, X2 = 20.70, P < 0.001, Friedman
Test for multiple observations per experimental unit). *P < 0.05, two-tailed
probability for comparison of saline-injected controls and corresponding
ahCRF-treated groups (Dunn's Test).
33
drug treatment. Results are presented in Fig.III.2B. As in Experiment II, the
handling procedure caused a significant increase in locomotor activity (df = 1, X2 =
20.70, P < 0.001). Analysis of the effects of drug treatment indicated that ahCRF
affected locomotor activity in handled animals (df = 2, H = 8.68, P < 0.05) but not
in unhandled animals (df = 2, H = 2.49, P > 0.25). Among the handled animals,
only a dose of 250 ng ahCRF significantly decreased locomotor activity when
compared with control animals (z = 2.31, P < 0.05).
Experiments IV and V. CRF-antagonist: Effects on warm stress-induced locomotor
activity
Experiment IV. To determine whether ahCRF would elicit a dose-dependent
change in locomotor activity induced by an alternative form of stress, placement in a
higher ambient temperature, newts received an icy injection of 0, 25, 125, or 250 ng
ahCRF (n = 15). Newts were then placed individually in small testing arenas (15 °C)
and tested 2 h after drug treatment. Each animal was tested twice, once at 15 °C and
again after being transferred to a second small testing arena maintained at 28 °C.
Animals were transferred by grasping the tip of the tail, a handling technique which
by itself does not affect locomotor activity (data not shown). Results are presented in
Table 1. Exposure to warm water significantly increased locomotor activity (df = 1,
72 = 36.30, P < 0.001). Although ahCRF had no effect on locomotor activity of
unstressed animals, there was a significant nonparametric monotonic trend for higher
doses of ahCRF to reduce warm stress-induced locomotor activity (z = 1.96; P <
0.05).
34
TABLE MA
Locomotor activity in response to ahCRF treatment in warm-stressed
newts. Locomotor activity 2 h after drug treatment (0, 25, 125 or 250 ng ahCRF)
either before (15 °C) or during (28 °C, Experiment IV; 30 °C, Experiment V) warm
stress (n =15). See Fig.III.1 for a description of locomotor activity. Data shown are
median values and interquartile ranges (line crossings/3 min). Handling increased
locomotor activity in both experiments, (Expt. IV, df = 1, X2 =35.3, P < 0.001;
Expt. V, df = 1, X2 = 85.2, P < 0.001). In addition, in both experiments there was
a significant monotonic trend for higher doses of ahCRF to suppress stress-induced
increases in locomotor activity (Expt. IV, z = 1.96, P < 0.05; Expt. V, z = 2.14, P
.
< 0.05).
Locomotor activity
CRF antagonist (ng)
Experiment IV
0
25
125
250
Experiment V
0
25
125
250
15°C
28°C
Increase
0 (0-8)
6 (0-15)
0 (0-4)
0 (0-9)
24 (9-34)
23 (13-44)
14 (8-40)
11 (1-31)
19 (9-26)
15 (8-34)
14 (2-31)
6 (1-19)
15°C
30°C
Increase
67 (51-84)
62 (50-89)
57 (38-82)
49 (32-58)
62 (51-76)
62 (45-89)
57 (35-70)
48 (32-58)
0 (0-6)
0 (0-3)
0 (0-5)
0 (0-1)
Experiment V. To determine whether the ability of ahCRF to reduce warm
stress-induced locomotor activity is dependent on the intensity of the warm stress,
Experiment IV was replicated using a warm stress of 30 °C rather than 28 °C (n =
15). Results are presented in Table 1. Although increases in locomotor activity in
response to warm stress were greater in this experiment, (df = 1, X2 = 85.2, P <
0.001), there was again a significant nonparametric monotonic trend for ahCRF to
reduce warm stress-induced locomotor activity (z = 2.14; P < 0.05). In other
similar experiments (data not shown), higher doses of ahCRF (0.5, 1 and 5 Ag) did
35
not significantly reduce the locomotor activity of unstressed animals or warm
stress-induced locomotor activity.
Experiment VI. Dose response study: icy injection of clonidine
To determine whether the a2-adrenergic agonist, clonidine, would dosedependently affect spontaneous locomotor activity, newts were given either an icy
injection of saline (n=28) or 0.01, 0.1, 1, or 10 fig clonidine (n=15). Newts were
isolated in temporary holding tanks for 30 min then placed in large testing arenas and
tested 5 min after placement. Results are presented in Table 111.2. There was a
significant treatment effect (df = 4, H = 20.44, P < 0.001). A dose of 1 Ag
clonidine significantly suppressed spontaneous locomotor activity (z = 3.37; P <
0.05).
TABLE 111.2. Locomotor activity in response to icy injections of clonidine.
Locomotor activity tested 35 min after icy injection of saline (n = 28) or 0.01, 0.1, 1
or 10 ng clonidine (n = 15). See Fig.III.1 for a description of locomotor activity.
Data shown are mean responses + SEM (line crossings/3 min). *P < 0.05, twotailed probability for the comparisons between the control group and treatment groups
(Dunn's Test).
Clonidine (p.,g)
Control
0.01
0.1
1
10
Locomotor
activity
4.25
4.86
0.80
0.20
0.33
(1.26)
(1.67)
(0.31)
(0.20)*
(0.13)
36
Experiment VII. Injection of clonidine and CRF
To determine whether clonidine would affect CRF-induced locomotor activity,
newts were given icy injections of either saline, clonidine alone (10 or 100 ng), CRF
alone (25 ng), or clonidine and CRF in combination (n =26). Newts were handled
and tested as in experiment VI. Results are presented in Fig.III.3. There was no
interaction between clonidine and CRF (df =2, X2 =1.90, P > 0.30), however, they
had independent effects on locomotor activity (clonidine, df = 2, X2 = 6.06, P <
0.05; CRF, df = 1, X2 = 49.20, P < 0.001). CRF stimulated locomotor activity (z
= 3.15, P < 0.01) while the higher dose of clonidine suppressed it (z = 2.61; P <
0.05).
50
0 Saline
C2D CRF(25 ng)
40
30--
20
10
0
Control
Clonidine
(10 ng)
Clonidine
(100 ng)
TREATMENT
FIGURE 111.3. Locomotor activity of male rough-skinned newts tested 35 min after
drug treatment with control solution, clonidine (10 or 100 ng), CRF (25 ng) or CRF
and clonidine together (n=26). Data shown are median values and interquartile
ranges. See Fig.III.1 for a description of locomotor activity. *P < 0.001, two-tailed
probability for comparison of CRF injected and saline-injected control groups, +P <
0.05, two-tailed probability for comparisons of clonidine-injected groups with
corresponding control groups (Dunn's Test).
37
DISCUSSION
This study provides the first evidence that stress-induced increases in
nonmammalian locomotor activity are mediated in part by CRF. Results from this
study also indicate that the stimulatory effects of CRF and the sedative effects of
clonidine are independent.
As described previously in mammals (Sutton et al., 1982; Veldhuis and De
Wied, 1984) and the urodele amphibian, T. granulosa (Moore et al., 1984; Lowry et
al., 1990), icy injections of CRF stimulate locomotor activity. In this study using
male newts the CRF receptor antagonist (ahCRF), injected icy at doses that did not
affect spontaneous locomotor activity, suppressed CRF-induced locomotor activity.
Similar effects of ahCRF have been demonstrated in male rats (Britton et al., 1986b).
Because studies with ahCRF have shown that it competes with CRF for binding at
CRF receptors (Rivier et al., 1984), the effectiveness of this antagonist in these
behavioral studies suggests that CRF receptors in mammals and amphibians have
similar specificity.
Although CRF has profound effects on locomotor activity in mammals, only
one study has demonstrated a suppression of stress-induced locomotor activity by
ahCRF (Winslow et al, 1989). In the present study we examined the effects of
ahCRF on locomotor activity induced by two different environmental stimuli,
handling and exposure to warm ambient temperature. In each case, ahCRF
significantly reduced stress-induced locomotor activity. This suggests that a CRF-like
substance was released in response to environmental stress and contributed to the
38
observed increase in locomotor activity. This hypothesis is consistent with studies of
mammals that indicate that following exposure to stress, CRF is released in specific
brain areas associated with the central nervous system response to stress (Chappell et
al., 1986).
The effects of ahCRF on stress-induced locomotor activity were dose related.
It is interesting, however, that doses higher than 250 ng did not significantly reduce
either handling stress-induced locomotor activity (0.5 ilg, Experiment III) or warm
stress-induced locomotor activity (0.5, 1 and 5 itg, data not shown). These
observations are consistent with studies in mammals in which high doses of ahCRF
are less effective (Berridge and Dunn, 1987) or ineffective (Ka lin et al., 1988) in
reversing stress-induced behaviors. One possible explanation for these observations is
that ahCRF may have partial agonist activity (Winslow et al., 1989; Kahn et al.,
1988).
Exposure to warm temperatures stimulates ectotherms to search for cooler
locations (Reynolds and Caster lin, 1979). The involvement of CRF in this response
had not been studied. Although ahCRF significantly reduced warm stress-induced
locomotor activity in the newt, it is clear that ahCRF did not completely suppress this
response. This may be due to an increased thermoregulatory response or to an
increased nociceptive response during exposure to warm temperatures. Both
thermoregulatory and nociceptive responses have been shown to be highly dependent
on the activation of endogenous opioid systems in vertebrate and invertebrate species
(Kavaliers and Hirst, 1986; Kavaliers, 1989), and many opioid substances can
39
stimulate locomotor activity (see Olson et al., 1987). Regardless, at least one
component of the warm stress-induced increase in locomotor activity appears to be
mediated by an endogenous CRF-like substance. The handling and warm stress
experiments together suggest that a CRF-like substance may be a component of the
behavioral response to stressful environmental stimuli in general.
One hypothesis for the mechanism of CRF-induced behavioral arousal in
mammals involves activation of norepinephrine-containing cells (see introduction).
These cells can be suppressed by activation of presynaptic a2-adrenergic receptors
(Cedarbaum and Aghajanian, 1977) which can result in an inhibition of the release of
norepinephrine from nerve terminals (reviewed by Langer, 1981). Although the
a2-adrenergic system has been implicated in CRF-induced locomotor activity in rats
by studies using adrenergic receptor antagonists (Imaki et al., 1987), no studies to
date have examined the effect of a2-adrenergic agonists on CRF-induced locomotor
activity. In our study we found that the selective a2-adrenergic agonist, clonidine,
suppressed spontaneous but not CRF-induced locomotor activity. This suggests that
CRF may stimulate locomotor activity independent of a2-adrenergic systems in T.
granulosa, consistent with studies in rodents in which treatment with a selective
noradrenergic neurotoxin, N-(2-cloroethyl)-N-ethyl-2-bromobenzyl amine
hydrochloride (DSP-4), failed to alter CRF-induced increases in emotionality in an
open field test (Bakke, Bogsnes and Murison, 1990) or CRF-induced decreases in
exploratory behavior (Berridge and Dunn, 1989).
40
Results from experiments in this study suggest that the function of CRF as a
regulator of stress-induced behavior has been conserved during vertebrate evolution.
The neuroendocrine mechanism by which CRF induces these changes in T. granulosa
remains unresolved.
ACKNOWLEDGMENTS
We thank Drs. J. Rivier and W. Vale for kindly providing CRF used in this
study. We also thank Christie A. Lowry for reviewing early drafts of the manuscript.
This research was supported in part by an NSF Grant No. BNS 8909173 awarded to
F.L. Moore.
41
CHAPTER IV: CORTICOTROPIN-RELEASING FACTOR ENHANCES
LOCOMOTION AND MEDULLARY NEURONAL FIRING IN AN
AMPHIBIAN
Christopher A. Lowry', James D. Rose2, and Frank L. Moore'
'Department of Zoology, Oregon State University, Corvallis, OR 97331-2914
2Departments of Psychology and Zoology-Physiology, University of Wyoming,
Laramie, WY 82071
42
ABSTRACT
Corticotropin-releasing factor (CRF) administration has been shown to act
centrally to enhance locomotion in rats and amphibians. The present study used an
amphibian, the roughskin newt (Taricha granulosa) to characterize changes in
medullary neuronal activity associated with CRF-induced walking and swimming in
animals chronically implanted with fine-wire microelectrodes. Neuronal activity was
recorded from the raphe and adjacent reticular region of the rostral medulla. Under
baseline conditions most of the recorded neurons showed low to moderate amounts of
neuronal activity during periods of immobility and pronounced increases in firing that
were time-locked with episodes of walking. These neurons sometimes showed further
increases in discharge during swimming. Injections of CRF but not saline into the
lateral ventricle produced a rapidly-appearing increase in walking and pronounced
changes (mostly increases) in firing rates of the medullary neurons. CRF produced
diverse changes in patterns of firing in different neurons, but for these neurons as a
group, the effects of CRF showed a close temporal association with the onset and
expression of the peptide's effect on locomotion. In neurons that were active
exclusively during movement prior to CRF treatment, the post-CRF increase in firing
was evident during episodes of walking; in other neurons that also were spontaneously
active during immobility prior to CRF infusion, post-CRF activity changes were
evident during immobility as well as during episodes of locomotion. Thus, a
principal effect of CRF was to potentiate the level of locomotion-related medullary
neuronal firing. Due to the route of administration CRF may have acted on multiple
43
central nervous system sites to enhance locomotion, but the results are consistent with
neurophysiological effects involving medullary locomotion-regulating neurons.
INTRODUCTION
Corticotropin-releasing factor (CRF) has a wide range of functions associated
with the initiation and coordination of neuroendocrine, autonomic, and behavioral
responses to stressful stimuli (reviewed by Dunn and Berridge, 1990). A CRFinduced increase in locomotor activity has been studied in rats (Sutton et al., 1982;
Veldhuis and De Wied, 1984; Diamant and De Wied, 1991; Saunders and Thornhill,
1986; Sherman and Ka lin, 1987) and the urodele amphibian, Taricha granulosa
(Moore et al., 1984; Lowry et al., 1990, 1991, 1993; Lowry and Moore, 1991). In
rats and T. granulosa, CRF-induced increases in locomotor activity are dose-related,
time-dependent (Sutton et al., 1982; Lowry et al., 1990), and persist in
hypophysectomized animals (Eaves et al., 1985; Moore et al., 1984). In addition,
CRF- and stress-induced locomotor activity are suppressed by the
intracerebroventricular administration of a-helical CRF, a competitive CRF antagonist
(Rivier et al., 1984; Britton et al., 1986b; Winslow et al., 1989; Lowry and Moore,
1991). These studies and others (Britton et al., 1985, 1986a) provide evidence that
CRF can act at sites within the central nervous system to stimulate locomotion in
diverse vertebrate species.
Studies using extracellular recording, lesion, as well as chemical and electrical
excitation approaches have described multiple neuroanatomical sites involved in the
initiation and expression of locomotion in vertebrates. These sites include the
44
preoptic basal forebrain, lateral hypothalamus, dorsomedial hypothalamus, the
perifornical area, the zona incerta, the ventral tegmental area, the central gray, the
dorsal and median raphe, and the area around the pedunculopontine nucleus (for
references, see Sinnamon, 1990). Several of these sites share a common feature in
that they have excitatory projections to the medial reticulospinal system (Orlovskii,
1970). The convergence of excitatory inputs regulating the initiation and expression
of locomotion to the medial reticulospinal system, the involvement of raphe neuronal
systems in the regulation of locomotion (Sinnamon, 1984; Jacobs and Fornal, 1993;
Viana Di Prisco et al., 1994), and the identification of raphe structures as potential
sites of action for CRF (Sharkey et al., 1989; Corley et al., 1990; Singh et al., 1992)
led us to select the medial reticular and raphe regions as recording sites for an initial
investigation of the neurophysiological correlates of CRF-induced locomotion.
Based on these previous studies our working hypothesis was that CRF
stimulates locomotion through direct or indirect actions on rostral medullary
locomotor control systems; this led to the prediction that CRF action would be
reflected in locomotion-related firing of neurons in or near the raphe region. Thus, in
the present study we investigated the effects of CRF on locomotion and behaviorally-
correlated single unit activity of rostromedial medullary neurons. These results have
been published in part in abstract form (Lowry et al., 1994).
45
METHODS
Subjects and surgical preparation
Adult male roughskin newts (T. granulosa) were collected in Benton County,
Oregon, and held in the laboratory for several months prior to use under conditions
described previously (Rose et al., 1993). Implant surgery was conducted with the
newts anesthetized in 0.1% MS-222. A cannula guide consisting of a short length of
polyethylene tubing plugged with a stainless steel insert was positioned in contact with
the surface of the forebrain over a lateral ventricle. Recording electrodes consisted of
twisted pairs of 50 Am Diamel-insulated nichrome wires with impedances of
approximately 500 kOhms at 1000 Hz. Either one or two pairs of microwires were
implanted on the rostral medullary surface at the midline or slightly lateral to the
midline. The implant assembly was embedded in dental cement that was anchored by
a single 0-90 stainless-steel screw placed in the skull. The microwire electrodes were
glued together and extended from the implant for approximately 6 cm before
terminating in an Amphenol micro-miniature connector strip.
Recording procedure
Following surgery animals were placed in the recording apparatus while still
anesthetized. Brain activity was monitored to determine when animals had fully
recovered from the anesthetic. Data collection was begun approximately 30 min to 1
h later. Recording was conducted with the newts in a well-water-filled rectangular
plastic pan (6x34x42 cm). Activity from the microwires was led to a preamplifier
46
through Microdot Mininoise coaxial cables and amplified with a Grass P511
preamplifier with filters set at 100-1 kHZ due to the relatively low frequency
characteristics of single neuron spikes in this species. During recording a manuallyactivated pressure transducer was used to signal the onset and offset of walking or
swimming and a continuous voice narrative was recorded to describe procedures and
the animals' behavior. Data storage, off-line analysis and activity quantification
procedures were similar to those previously explained (Rose, 1992), but a brief
description follows. Each recording site typically yielded activity from several
individual neurons, each of which was identified from tape recordings by means of a
Bak dual-window, time-amplitude window discriminator. The properties of single
neuron activity and effects of CRF infusions were analyzed from chart records of
standard pulses that were electronically integrated (330 msec time constant) and were
displayed on the polygraph together with the transducer signal indicating the presence
or absence of walking or swimming.
In each experiment, a sample of activity was recorded from several minutes to
one hour prior to infusion of CRF (50 ng in 1µl amphibian Ringer's solution) or
vehicle into the lateral ventricle. In some experiments, the 30 ga. infusion needle was
inserted and the solution immediately injected. In other experiments, the infusion
needle was inserted, left in place for varying lengths of time and the infusion was
accomplished remotely with a microsyringe without further handling of the newt.
Leakage of the solutions from the injection needle was prevented by bracketing the
volume of solution between small air bubbles. In separate experiments a cannula was
47
inserted without an infusion or a 1 Al volume of vehicle was injected 20 min prior to
CRF as control procedures.
At the end of each recording experiment, newts were
deeply anesthetized in MS-222, and the brains removed. Cannula placements and
recording electrode sites were histologically located in frozen sections stained with
neutral red.
Statistics
Statistical analysis of behavior was performed from samples of behavior
classified as immobility, walking, or swimming at 10 sec intervals beginning 5 min
prior to a treatment and extending to 20 min afterward, yielding 150 time samples per
animal (except for one newt where only 144 samples were available). The ChangePoint Test for a Sequence of Binomial Variables (Siegel and Caste llan, 1988) was
used to determine if the amount of locomotion (walking or swimming) changed after
CRF infusion or control procedures.
Changes in unit activity were evaluated by comparing the firing rate of each
neuron (quantified from polygraph records of a raster-stepper driven by the spike
discriminator) during comparable behavioral states before and after CRF or control
treatments. In order for a unit's activity to have been considered altered, second-bysecond firing rates for a post-treatment sample of firing from the integrator records
had to be uniformly higher or lower than an activity sample of comparable duration
recorded just prior to saline or CRF infusion or just prior to cannula placement in the
case where there was no infusion. For corroborative analysis, several units were
analyzed using the Change-Point Test for Continuous Variables (Siegel and Caste llan,
48
1988). For this test, the behavior-correlated firing rates were calculated for each 10
sec interval of the entire sampling period. The criterion for a sample of unit activity
to be used in an analysis was that the corresponding episode of behavior had to be at
least 2 sec in duration. The baseline for comparison with post-treatment firing rates
was the mean firing rate for a given behavioral state during the 5 min period prior to
a treatment. Conclusions about the behavioral and neurophysiological effects of
treatment are based on trials where at least 20 min of post-treatment data were
available for analysis.
RESULTS
Locations of recorded neurons
The results are based on recordings from 33 single units from a total of 9 sites
in the rostral medulla (n = 9 male T. granulosa). Locations of recording sites are
illustrated in Fig.IV.1. Of these units, 28 were recorded in experiments where a
single infusion was given (CRF, n=19; saline, n=9). Within these groups six units
were recorded following remote infusion of CRF; five units were recorded following
remote infusion of saline. The remaining units in these groups were recorded
following a brief period during which the infusion needle was placed in the cannula
guide and CRF or saline was infused while the animal was restrained. Two units
were recorded in an experiment where remote infusion of saline was followed, 20 min
later, by a remote infusion of CRF. Three units were recorded in an experiment
where a cannula was placed with no infusion as an additional control procedure.
49
FIGURE IV.1. Diagrammatic dorsal view of the brainstem of an adult male T.
granulosa, with black dots illustrating the location of recording sites in the raphe
region and adjacent reticular formation. The cerebellum has been omitted from the
figure for clarity. Scale bar, 1 mm.
Unit firing patterns dependent on behavioral state
The principal distinguishing feature of these neurons under baseline conditions
was the degree to which their activity was movement-dependent.
The majority of the
33 units (73%) were tonically active to some degree in the absence of movement.
The remaining units only fired occasionally, or did not fire at all, during immobility.
Sixty-seven percent of the neurons in this study showed changes in firing that were
associated with the transition to walking in the aquatic testing environment. These
neurons showed an onset of firing (or for the tonically-active cells increased firing)
50
FIGURE IV.2. CRF-enhanced firing in a neuron with movement-related firing. A.
Each tracing from top to bottom shows a 58 sec chart record of activity recorded
from this neuron. The vertical deflections on the chart are integrated spike
discriminator pulses showing the rate of single neuron firing (vertical calibration =
10 spikes/sec). The horizontal lines beneath the integrator records signify episodes of
movement, as indicated. Before CRF infusion, this medullary neuron fired small
bursts of spikes exclusively during movement. Records at 11 and 22 min after CRF,
when locomotion was enhanced, show elevated firing during walking. B. Histograms
illustrating changes in behavior and firing rate of this neuron over a 72 min period.
Placement of the cannula was performed during the 2 min, 15 sec period immediately
prior to CRF infusion (onset of CRF infusion indicated by arrowhead, 30 sec infusion
period). The effect of CRF infusion on behavior and unit activity lasted
approximately 40 min. C. Digitized spike waveform from the neuron (45 IN
amplitude; 2.5 msec spike duration).
51
FIGURE IV.2
A
Pre CRF
Walking
CRF + 2 min
._k
Head
Movement
Backward
Movement
Walking
Walking
CRF + 11 min
Walking
Walking
CRF + 22 min
Walking
CRF + 42 min
Walking
C
B
ci)
0
I Do
75
(1)
50
25 Cr)
1'1
0
0
tr-itiOr
10
I0
o
A 10
E
CRF
20
30
40
Time (min)
50
60
70
52
during walking. This walking-related firing consisted of either a relatively steady or a
randomly fluctuating rate of overall activity that was not temporally correlated with
the stepping pattern. Twenty-seven percent of the neurons that showed increases in
firing that were associated with the onset of walking displayed a further increase in
activity during swimming (see units CRF8-2, CRF8-4, Fig.IV.4). There was no
apparent pattern of movement-related unit activity based on the location of the
recording electrode; the patterns recorded from electrodes in more lateral positions
within the medulla were similar to those recorded from electrodes in midline
positions.
Although in most cases the intensity of unit activity was dependent on the
behavioral state, this firing showed no specificity for the types of movement patterns
that were executed. For example, units that increased firing rates during periods of
walking also increased firing rates during orientation movements of the head, shifts in
body position, single limb movements and backward locomotion (see Fig.IV.2). In
addition, we did not observe any cases of phasic firing (firing that was phase-locked
with rhythmic locomotor activities). The chief distinction between firing associated
with one type of movement or another was that the more pronounced and sustained
movements (walking, swimming) were associated with the highest level of firing.
Sensory responsiveness of rostral medullary neurons
Although sensory responses were not systematically assessed, it was clear that the
medullary neurons tended to fire in response to tactile stimuli applied to the face, feet
or cloaca, as seen in immobilized newts (Rose et al., 1993). More generalized
53
FIGURE W.3. Diverse patterns of unit activity change in five simultaneouslyrecorded rostromedial medullary neurons during CRF-induced locomotor activation.
There was a significant change in movement-related firing by each of these neurons as
well as a significant increase in walking by this newt following CRF infusion. The
top five histograms show a 35 min sample of firing rate for each neuron. The bottom
two histograms show sequential 10 sec samples of walking and swimming,
respectively. The histograms of neuronal activity also illustrate transiently increased
firing by units 1, 2 and 5 during a 60 sec period of handling (onset indicated by
arrow) associated with placement of the infusion needle in the guide cannula 6.5 min
after the beginning of each record. The onset of a 30 sec remote CRF infusion
(without handling of the newt) is shown by the vertical line through all histograms.
CRF infusion caused an increased locomotion-related firing rate (Unit 1) that mirrored
the time course of the CRF effect on walking. Units 2 and 5 showed increased
locomotion-related firing rates after the CRF infusion that were evident quickly and
reached a maximum within 5 min as the behavioral effect of CRF became more
evident, and then gradually showed decreased locomotion-related firing rates
throughout the remainder of the recording period. Units 3 and 4 displayed no notable
changes in firing rates during handling or immediately after CRF infusion, but their
firing rates slowly decreased as the CRF-induced locomotion became more
pronounced; these neurons eventually ceased firing altogether. Note the difference in
ranges of the vertical axes indicating different dynamic ranges of firing in these units.
Unit 1 was moderately active while Units 2 and 4 fired at low rates during periods of
walking and immobility. Unit 3 displayed bursts of activity at the onset of movement
and was only active during movement or sensory stimulation. Unit 5 fired primarily
during walking but also fired at a low rate during periods of immobility. Scale: 1
msec, 20 IN (Units 2-5), 10 AV (Unit 1).
54
FIGURE IV.3
300
Unit
1
250
200
150
100
50
0
100
80
60
40
20
0
100
Unit 5
80
60
4o
20
30
Time (min)
55
stimulation associated with manual restraint of the newt during cannula insertion was
monitored for 26 units. The majority of these neurons (69%) increased their firing
rates substantially during the period of handling, even if the newt was not moving.
Typically, unit activity returned to baseline levels immediately following the release
of the animal, although the activity of some units appeared to be suppressed for
several minutes following handling or other forms of sensory stimulation.
Effects of CRF administration on behavioral and unit activity
Corticotropin-releasing factor infusion increased the incidence of walking to a highly
significant degree in five out of six CRF-injected newts [CRF2, P <0.001; CRF3,
P<0.001; CRF4, P=0.05, CRF5, Fig.IV.5, P<0.001; CRF6, Fig.IV.3, P<0.005;
CRF7 did not respond, P >0.05). Visual inspection of behavioral records in the
statistically significant cases indicated that an increase in walking behavior was clearly
evident within 2-3 min of CRF infusion. Only one animal (CRF2) showed increased
swimming, a behavior that was much less frequent than walking either before or after
CRF administration; the effect on swimming behavior in this animal was delayed
approximately 20 min and was much less pronounced than the effect on walking
behavior. In the five newts showing increased wal activity of the 14 units studied
following application of these control procedures (see Fig.IV.2).
Changes in unit activity during periods of immobility following CRF infusion
Examination of integrator records of firing rates for units with firing during
periods of immobility (i.e. "spontaneous discharge") revealed that three neurons
56
FIGURE IV.4. Behavioral and single-unit activity before and after infusion of saline
into the lateral ventricle, showing an absence of effect on neuronal firing or behavior.
Episodes of walking and swimming are shown in the two bottom histograms.
Insertion of the infusion needle into the cannula preceded the illustrated period of
recording by 30 sec. Activity of four different neurons that were recorded
simultaneously from a single site (Units 1-4) are illustrated in the top four histograms.
The spike waveform for each of these units is shown at the right of these histograms.
These neurons showed no significant changes in firing rates after saline infusion
(indicated by the vertical line through all histograms). All these neurons were
moderately active during periods of immobility and to varying degrees, more active
during periods of locomotion. Units 2 and 4 displayed highest levels of firing during
episodes of swimming. Calibration: 1 msec, 10 AV.
57
FIGURE IV.4
]
58
FIGURE IV.5. CRF-induced increases in firing rate in a medullary neuron that were
independent of behavioral state. The top tracing in each 58 sec record shows
integrated neuronal activity (vertical calibration = 10 spikes/sec), with horizontal
lines beneath each integrator record signifying walking or swimming. Firing rates are
illustrated for a brief period prior to CRF infusion (A) and at five successive times
afterward (B-E). As was typical for newts in this study, bouts of locomotion were
often characterized by an initial period of walking followed immediately by a
transition to a period of swimming (A,B,D,E). Bouts of swimming were usually
followed by periods of immobility (A,B,E). Prior to CRF infusion, this neuron was
active during periods of immobility as well as locomotion, showing no reliable
association between firing rate and movement. CRF infusion resulted in a significant
increase in walking and concurrently elevated neuronal firing rate during immobility
as well as locomotion, with a maximum effect at approximately 25-30 min.
59
FIGURE IV.5
A
Pre CRF
Walking
Swimming
B
CRF +9 min
Walking
Swimming
C
CRF +23 min
Walking
D
CRF +31 min
Walking
Swimming
E
Walking
Swimming
CRF +51 min
60
showed increased tonic firing and three showed decreased tonic firing after CRF
infusion. Latencies for changes in firing rates during immobility were determined for
five neurons with the Change-Point Test for Continuous Variables. Three of these
units showed increased discharge and two showed decreased discharge following CRF
infusion with latencies ranging from 3 min, 10 sec to 13 min, 10 sec. The majority
of these neurons showed changes in less than 4.5 min. For all 14 neurons recorded
during control procedures (all of which showed some degree of spontaneous activity),
quantified values of firing rates during periods of immobility were essentially identical
before and after control procedures.
DISCUSSION
Behavior-related properties of medullary neurons
A conspicuous feature of these medullary neurons was the close association
between their firing and episodes of movement. Although most cells exhibited some
degree of firing during immobility, most showed a pronounced increase in activity
that was associated with changes in movement intensity and duration but not
specifically to movement type, a pattern similar to the movement-related firing of
ventromedial midbrain neurons in hamsters (Rose, 1985). Thus, in this urodele
amphibian as in cyclostomes (Grillner et al., 1988), teleost fish (Grillner et al., 1988;
Mos and Roberts, 1994), and mammals that have been studied (Ross and Sinnamon,
1984; Millhorn, 1986), these medullary neurons show properties consistent with the
modulatory control of central pattern generators, including the central pattern
61
generator for locomotion. Since this study is the first to record neuronal activity
during behavior in a species showing both quadrupedal locomotion as well as
swimming, which involves waves of trunk and tail flexion as in fish, it is noteworthy
that the same neurons were active during both forms of movement.
CRF effects on neuronal activity and behavior
This study provides the first description of single neuron activity correlates of
CRF-induced behavior in a nonmammalian vertebrate. In newts that displayed a
pronounced increase in walking in response to CRF administration, the great majority
of the recorded rostromedial medullary neurons displayed pronounced changes,
usually increases, in movement-related firing. In contrast, in animals that failed to
show a behavioral response to CRF or to control treatments, none of the recorded
single units displayed altered neuronal activity. This close association between neural
and behavioral effects of CRF was also seen in the timing of the peptide's effects;
behavioral and neurophysiological effects were comparably rapid, beginning in some
cases within 3-4 min of CRF infusion.
The time course of CRF effects on neuronal activity was more complex than
that on behavior. Neurons exhibited increases
or in a few cases decreases
in
activity that spanned the time course of the CRF effect on behavior, but also showed
changes involving initial increases in activity followed by decreases.
Thus, while the
overall neuronal effect of CRF corresponded with the behavioral effect, the peptide's
effect on individual neurons differed in form, probably due to sampling of
heterogeneous neuron types with this recording technique. Of particular importance,
62
the diversity in the neurophysiological effects of CRF shows that these changes were
not simply a reflection of the intensity of sensory feedback from more pronounced
locomotion.
Our results showing predominantly increased neuronal activity following
CRF infusion are similar to results from previous neurophysiological studies that
investigated the effects of CRF on diverse brain regions of the rat central nervous
system, including the preoptic region, hypothalamus, locus coeruleus, solitary tract
complex, hippocampus, and cortex (Eberly et al., 1983; Siggins et al., 1985; Siggins,
1990; Valentino, 1990). The latency of the fastest electrophysiological effects
observed in the present study (as early as 3-4 min) was similar to latencies of
electrophysiological effects of direct administration of CRF to neural tissue in other
vertebrates (Valentino, 1990). The presence of behavior-related neuronal activity
properties allowed for detection of more diverse patterns of CRF action than would be
possible with preparations that use brain slices or anesthetized animals. For example,
some units which under baseline conditions displayed enhanced activity during
episodes of walking displayed progressively less activity during CRF-induced
locomotion and ultimately ceased activity altogether, illustrating that not all
locomotion-related neuronal activity is enhanced by CRF administration (CRF6-3,
CRF6-4, Fig.IV.3). Since CRF may alter
and in some cases suppress -- a variety
of sensory, integrative, and motor processes involved in the regulation of complex
physiological and behavioral responses (reviewed by Dunn and Berridge, 1990), it is
not surprising that we observed diverse neuronal responses in the present study.
63
In the present study one of six animals did not respond behaviorally or
neurophysiologically to CRF administration (CRF7). This failure to respond could
reflect a mechanical error in the administration of the peptide, or alternatively, the
physiological condition of this animal may have altered the behavioral and
neurophysiological responses to CRF. For example, the nonresponsive animal was
handled for cannula placement approximately 30 min prior to CRF administration; in
all other cases in the present study CRF was administered within four min of cannula
placement. We have shown previously that exposure to a stressful stimulus 20 min
but not 1 min prior to administration of CRF inhibits CRF-induced locomotion in a
naloxone- reversible manner (Lowry et al., 1990). Thus, it is possible that during the
initial handling associated with placement of the cannula in the nonresponsive animal
the release of endogenous opiates may have suppressed the subsequent behavioral and
neurophysiological responses to CRF administration.
CRF actions in the central nervous system
The present study focused on the raphe and paramedian region of the rostral
medulla due to evidence for locomotion-controlling neurons in these regions. One
characteristic of the raphe region that appears to be evolutionarily conserved across all
vertebrate classes is the presence of a complex of serotonergic neuronal cell bodies.
This complex of neuronal cell bodies provides a widespread serotonergic innervation
of the central nervous system through ascending projections within the medial
forebrain bundle (Steinbusch, 1981, 1984; Steinbusch and Nieuwenhuys, 1983; Parent
1981). Although most of the recording sites in this study were optimally positioned
64
to record from serotonergic raphe neurons (Yoshida et al., 1983; Ueda et al., 1984;
Faso lo et al., 1986; Corio et al., 1992), we did not detect neurons firing with the
highly regular, relatively slow patterns characteristic of mammalian serotonergic
raphe neurons (Jacobs and Fornal, 1991).
Although it remains unresolved whether or not CRF administration altered
neuronal activity of raphe serotonergic neurons, there is independent evidence that
CRF-induced locomotion in T. granulosa is associated with a widespread activation of
serotonergic neuronal systems. Intracerebroventricular injections of CRF dosedependently enhance locomotion and alter serotonin or 5-hydroxyindoleacetic acid (5-
HIAA; a serotonin metabolite) concentrations in the ventral forebrain and dorsomedial
hypothalamus, as measured by microdissection and high performance liquid
chromatography with electrochemical detection techniques (Lowry et al., 1991). In
related studies, the specific serotonin reuptake inhibitor, fluoxetine (Fuller and Wong,
1977), enhanced locomotor activity in T. granulosa treated with a behaviorally
subthreshold dose of CRF (but not in saline-treated animals); this effect was
associated with a widespread depletion of serotonin throughout the central nervous
system (Lowry et al., 1993). In rats, CRF infused into the lateral ventricles dosedependently increases the activity of tryptophan hydroxylase, the rate-limiting enzyme
for serotonin synthesis (Corley et al., 1990; Singh et al., 1992). These studies
suggest that the central actions of CRF involve the activation of serotonergic neuronal
systems.
65
There is evidence that serotonin facilitates locomotor activity in vertebrates by
actions at caudal brainstem as well as spinal levels (Harris-Warrick and Cohen, 1985;
Grillner et al., 1987, Cazalets et al., 1992). Of particular importance to this study is
the evidence that serotonin potentiates firing of reticulospinal neurons in response to
excitatory input (Viana Di Prisco et al., 1992). Activation of reticulospinal neurons is
one means of initiating locomotion (Ross and Sinnamon, 1984; Grillner et al., 1988)
and in the thoroughly-studied lamprey model, serotonin-containing neurons may
synapse directly on locomotion-triggering reticulospinal neurons (Viana Di Prisco et
al., 1994). In the present study with T. granulosa the increases in the maximal
discharge rate of locomotion-related medullary reticular neurons are consistent with
prior studies of serotonin effects on motor output (Grillner et al., 1987; Cazalets et
al., 1992; Viana Di Prisco et al., 1992, 1994; Schotland and Grillner, 1993; HarrisWarrick and Cohen, 1985; Jacobs and Fornal, 1993). Further neurophysiological
studies that directly manipulate serotonin action coupled with CRF administration will
be required to establish the neurochemical processes underlying the neural effects of
CRF.
Multiple neuroanatomical sites may be involved in the expression of CRF-
induced locomotion. In T. granulosa CRF-induced locomotor activity is associated
with site-specific changes in concentrations of neurotransmitters (or their metabolites)
in the basal forebrain and the dorsomedial infundibular hypothalamus (Lowry et al.,
1991; 1993). These sites, as well as the dorsal and median raphe nuclei and the
medial reticulospinal system are important locations for the initiation and expression
66
of locomotion in vertebrates (Sandner et al., 1979; Di Scala et al., 1984; Ross and
Sinnamon, 1984; Sinnamon, 1984; Brudzynski and Mogenson, 1985; Sinnamon,
1987; Swerdlow and Koob, 1987; Lammers et al., 1988; Marcie llo and Sinnamon,
1990). Multiple neuroanatomical levels of CRF regulation of locomotor activity
would allow for greater refinement in the neurobehavioral control effected by this
peptide.
Summary and conclusions
Infusions of CRF (but not saline) into the lateral ventricle stimulated a rapidlyappearing increase in walking and pronounced changes (mostly increases) in firing of
rostromedial medullary neurons. In general, the neuronal effects of CRF showed a
close temporal association with the onset and expression of the peptide's effect on
locomotion. In neurons that were active exclusively during movement prior to CRF
treatment, the post-CRF increase in firing was evident during episodes of walking. In
other neurons that also were spontaneously active during immobility prior to CRF
infusion, post-CRF activity changes were evident during immobility as well as during
episodes of locomotion. The results of the present study together with previous
investigations of T. granulosa suggest that CRF acts centrally at multiple
neuroanatomical sites to enhance the action of brainstem reticular and spinal
locomotor-controlling regions, thereby producing more frequent and sustained
episodes of locomotion.
67
ACKNOWLEDGEMENTS
This research was supported in part by NIMH predoctoral fellowship to CAL
(BNR-1F31MH10288) and research grants to JDR (NIH NS13748) and FLM (NSF
IBN 93-19633). We thank Drs. J. Rivier and W. Vale for their generous gift of CRF
used in this study. We also kindly thank Lorie Smucker for her excellent technical
assistance.
68
CHAPTER V: CATECHOLAMINES AND INDOLEAMINES IN THE
CENTRAL NERVOUS SYSTEM OF A URODELE AMPHIBIAN: A
MICRODISSECTION STUDY WITH EMPHASIS ON THE DISTRIBUTION OF
EPINEPHRINE
Christopher A. Lowry', Kenneth J. Renner2, and F.L. Moore'
'Department of Zoology, Oregon State University, Corvallis, OR 97331-2914
2Biomedical Sciences Department, Southwest Missouri State University, Springfield,
MO 65804-0094
2Current address, Department of Biology, University of South Dakota, Vermillion,
SD 57069-2390
Copyright 1995 by S. Karger A.G.
All rights of reproduction in any form reserved.
69
ABSTRACT
Individual brain nuclei and regions of the central nervous system of adult male
roughskin newts (Taricha granulosa) were microdissected and the concentrations of
norepinephrine, epinephrine, 3,4-dihydroxyphenylacetic acid, dopamine, 5hydroxyindoleacetic acid, and serotonin were determined using high performance
liquid chromatography (HPLC) with electrochemical detection. The pattern of
distribution of these catecholamines and indoleamines revealed many similarities
between this urodele and other vertebrates. The highest concentrations of biogenic
amines were observed in brainstem, hypothalamic, and basal forebrain structures; the
lowest concentrations were observed in the internal granule layer of the olfactory bulb
and pallial structures of the telencephalon. High concentrations of catecholamines and
indoleamines were found in hypothalamic periventricular regions that are known to
include cerebrospinal fluid-contacting, monoamine-containing neuronal cell bodies.
The rostral diencephalon, which included the preoptic recess organ, had high
concentrations of the primary catecholamines, norepinephrine and dopamine, and
extremely high concentrations of the secondary catecholamine epinephrine. The
dorsomedial infundibular hypothalamic region, which included the paraventricular
organ, had high concentrations of dopamine and serotonin. The lateral infundibular
hypothalamic region, which included the nucleus infundibularis dorsalis, had high
concentrations of each of the biogenic amines. The results revealed unique patterns
of distribution for each of the catecholamines and indoleamines studied, and provided
evidence that regions of the hypothalamus that include cerebrospinal fluid-contacting,
70
monoamine-containing neuronal cell bodies are focal regions for the metabolism of
multiple biogenic amines.
INTRODUCTION
Catecholamine and indoleamine systems have been visualized using the
original Falck-Hillarp formaldehyde histofluorescence technique (Falck et al., 1962;
Falck and Owman, 1965), or variations of this technique (De la Torre and Surgeon,
1976; Loren et al., 1980; Scholer and Armstrong, 1982) in the central nervous system
of several amphibians including Xenopus laevis (Goos and van Halewijn, 1968;
Terlou and Ploemacher, 1973), Rana esculenta (Braak, 1970), Rana temporaria
(Bartels, 1971; Parent, 1973; Prasada Rao and Hartwig, 1974), Bufo poweri (Chacko
et al., 1974), Rana pipiens (Parent, 1975), Ambystoma mexicanum (Sims, 1977),
Necturus maculosus (Dube and Parent, 1982), and Triturus alpestris (Corio and
Doerr-Schott, 1988; see also Lamas et al., 1988). Immunohistochemical techniques
have been used to describe the distribution of catecholamine-synthesizing enzymes
(Wilde lt et al., 1973a) in Rana catesbeiana (Yoshida et al., 1983; Can et al., 1991),
Triturus cristatus carnifex (Franzoni et al, 1986), Rana ridibunda and Pleurodeles
waltlii (Smeets and Gonzalez, 1990; Gonzalez and Smeets, 1991, 1994; Gonzalez et
al., 1994a), T. alpestris (Corio et al., 1992), X. laevis (Gonzalez and Smeets, 1993,
1994; Gonzalez et al., 1993a, 1994a), Siren lacertina (Gonzalez and Smeets, 1994),
and Typhlonectes compressicauda (Gonzalez et al., 1994b). Immunohistochemistry
has also been used to describe the distribution of specific amines, dopamine,
norepinephrine, and serotonin (5-hydroxytryptamine, 5-HT; Steinbusch et al., 1978;
71
Steinbusch and Tilders, 1987), in R. catesbeiana (Yoshida et al., 1983; Ueda et al.,
1984a), T. cristatus carnifex (Faso lo et al., 1986), R. ridibunda and P. waltlii (Smeets
and Gonzalez, 1990; Gonzalez and Smeets, 1991, 1994; Gonzalez et al., 1994a), T.
alpestris (Corio et al., 1992), X. laevis (Gonzalez and Smeets, 1993, 1994; Gonzalez
et al., 1993a, 1994a), and S. lacertina (Gonzalez and Smeets, 1994).
The distribution of biogenic amines in these amphibians reveals many
similarities to a generalized vertebrate pattern (reviewed by Parent, 1979; Parent,
1981; Parent, 1984; Parent et al., 1984; Parent, 1986; Gonzalez and Smeets, 1994).
Principle components of this pattern are ascending fiber systems that extend from
monoaminergic cell bodies in brainstem nuclei. These components are evident in
anuran and urodele amphibians with an ascending serotonergic system arising from
columns of neurons situated along the raphe and mesencephalic reticular formation
(Yoshida et al., 1983; Ueda et al., 1984a; Faso lo et al., 1986; Corio et al., 1992), an
ascending dopaminergic system (presumed homologue of the mesostriatal and
mesolimbocortical systems of mammals) arising from the ventromedial tegmentum of
the rostral midbrain, rostral or dorsomedial to the root of the oculomotor nerve
(Yoshida et al., 1983; Franzoni et al., 1986; Smeets and Gonzalez, 1990; Carr et al.,
1991; Gonzalez and Smeets, 1991, 1994; Gonzalez et al., 1993a,b), as well as
ascending noradrenergic and adrenergic systems arising from lateral regions of the
medulla oblongata (Franzoni et al., 1986; Yoshida et al., 1983; Gonzalez et al.,
1993b; Gonzalez and Smeets, 1994). In addition, recent evidence suggests that
anuran and urodele amphibians also may have an ascending noradrenergic system
72
homologous to the isthmocortical (locus coeruleus) system of other vertebrates (Dube
et al., 1990, Gonzalez and Smeets, 1993, 1994). In general, these ascending
monoaminergic systems appear to be more extensive and complex in birds and
mammals than in fishes, amphibians, and reptiles (Parent et al., 1984).
Another aspect of the vertebrate pattern of monoamine distribution is the
presence of monoaminergic systems, characterized immunohistochemically by the
presence of monoamine-synthesizing enzymes and the monoamines themselves, that
arise from neurons in the hypothalamus (for amphibians see Yoshida et al., 1983;
Franzoni et al., 1986; Gonzalez and Smeets, 1991, 1994; and Gonzalez et al., 1993a;
for a review of monoaminergic neuronal systems in the hypothalamus of mammals,
see Everitt et al., 1992).
Studies of monoamine-containing systems in the hypothalamus reveal some
apparent differences between nonmammalian and mammalian vertebrates. Associated
with the hypothalamic monoaminergic systems in nonmammalian vertebrates, but not
mammalian vertebrates, are bipolar monoamine-containing neurons with apical
processes that contact the cerebrospinal fluid (CSF) (reviewed by Parent et al., 1984).
In amphibians, immunohistochemical studies found immunoreactivities to dopamine
(Smeets and Gonzalez, 1990; Gonzalez and Smeets, 1991, 1994; Corio et al., 1992;
Gonzalez et al., 1993a), norepinephrine (Gonzalez and Smeets, 1993, 1994), and 5-
HT (Sano et al., 1983; Shimizu et al., 1983; Yoshida et al., 1983; Ueda et al.,
1984a; Faso lo et al., 1986; Corio et al., 1992) in these hypothalamic CSF-contacting
neurons.
73
Conspicuously absent from the patterns of distribution of aminergic systems
described above is a description of the distribution of epinephrine in the vertebrate
central nervous system. Although immunohistochemistry has been used to detect
epinephrine in the adrenal medulla (Verhofstad et al., 1980, 1983), this technique has
not been used successfully to visualize epinephrine in the central nervous system
(Wilde lt et al., 1980; Steinbusch and Tilders, 1987). The lack of success in
immunohistochemical studies, as well as the relatively low sensitivity of the FalckHillarp formaldehyde histofluorescence technique for the detection of epinephrine
(Falck et al., 1962), has limited our understanding of the regional distribution of
epinephrine in amphibians. Information available on the regional distribution of
epinephrine in urodele amphibians is limited to high performance liquid
chromatography with electrochemical detection (HPLC-ED) studies which measured
concentrations of epinephrine in major subdivisions of the brain (olfactory bulb,
telencephalon, hypothalamus, and midbrain or brainstem) in Chtyptobranchus
alleganiensis, Ambystoma tigrinum, and N. maculosus (Cooney et al., 1985; Hamilton
et al., 1987; Norris et al., 1992).
As indirect evidence for epinephrine-containing neuronal structures,
immunohistochemical studies have used antibodies raised against the epinephrineforming enzyme, phenylethanolamine N-methyltransferase (PNMT) to visualize
PNMT-immunoreactive neuronal structures. Unfortunately, although
immunohistochemical studies have proven successful for visualizing the distribution of
PNMT- immunoreactive neuronal systems in mammals (Hokfelt et al., 1974, 1984a;
74
Howe et al., 1980; Ross et al., 1984; Foster et al., 1985; Ruggiero et al., 1985,
1986, 1992; Kitahama et al., 1985, 1986, 1988; Tillet, 1988; Iwamoto et al., 1989;
Komori et al., 1990; Dormer et al., 1993), this approach has had limited success in
nonmammalian vertebrates. Of the studies that have described the distribution of
PNMT-immunoreactive structures in nonmammalian vertebrates (Yoshida et al., 1983;
Steeves et al., 1987; Sas et al., 1990; Smeets and Jonker, 1990; Smeets and
Steinbusch, 1990; Gonzalez and Smeets, 1994), only the studies by Yoshida and
colleagues of R. catesbeiana (1983) and by Gonzalez and Smeets of P. waltlii (1994)
describe the distribution of PNMT-immunoreactive structures in amphibians. These
studies, however, focus on the distribution of medullary PNMT-immunoreactive
neuronal cell bodies; the distribution of PNMT-immunoreactive fibers throughout the
amphibian central nervous system has not been described. Furthermore, PNMT
immunoreactivity and PNMT activity are not reliable indicators of the regional
distribution of epinephrine in vertebrate brain (Fuller and Hemrick-Luecke, 1983;
Sved, 1989).
Microdissection techniques combined with HPLC-ED lack the neuroanatomical
resolution of histofluorescence and immunohistochemical techniques but complement
them by providing valuable information in the form of independent, quantitative
estimates of neurotransmitters or metabolites in specific brain areas (Brownstein and
Palkovits, 1984).
The present study used microdissection and HPLC-ED procedures to determine
the neuroanatomical distribution of specific catecholamines, indoleamines, and
75
selected metabolites in the brain of the male roughskin newt (T. granulosa), providing
for the first time in any amphibian detailed quantitative information on the relative
concentrations of monoamines in specific brain areas. This information complements
previous histochemical and biochemical studies of the central nervous system in
amphibians and is valuable for comparisons with studies which have used similar
techniques to examine other vertebrate species. Our main objective was to determine,
using quantitative measures, which brain areas are characterized by relatively high
concentrations (and, conversely, which brain areas are characterized by relatively low
concentrations) of specific biogenic amines. We focus attention on the regional
distribution of epinephrine and the aminergic content of regions of the hypothalamus
that include cerebrospinal fluid-contacting, monoamine-containing (CSF-MA) neuronal
structures. These data have been presented in part in preliminary form (Lowry et al.,
1991).
MATERIALS AND METHODS
The original research reported herein was performed in accordance with the
U.S. Public Health Service's Guide to the Care and Use of Laboratory Animals.
Preparation of tissue for microdissection
Adult male roughskin newts (Taricha granulosa) were collected in February,
1990 (Benton County, Oregon). For preparation of tissues animals were killed by
rapid decapitation and each animal was rapidly dissected (1-2 min) to remove the
intact brain and rostral spinal cord. These were then embedded in Tissue-Tee
76
O.C.T. Compound, frozen on dry ice, and stored in airtight microcentrifuge tubes at
80 °C until sectioning. Serial 300 µm sections of frozen brain were cut at -12 °C in a
cryostat. Sections were placed on gelatin-coated glass slides and collectively thaw-
mounted by briefly warming the slide. Sections were refrozen within approximately
30 sec. Slides were sealed in slide boxes and stored at -80 °C.
Preparation of microdissection atlas
The microdissection atlas (Fig.V.1), was prepared using adult male T.
granulosa (n=4) that were treated as above, with the following modifications. Intact
heads were fixed for 24 h in modified Zamboni fixative (4% paraformaldehyde, 3%
sucrose and 7.5% picric acid in 0.1 M sodium phosphate buffer (SPB), pH 7.4).
After 24 h the intact brain and spinal cord was removed, washed 2x12 h in SPB, and
stored in SPB containing 30% sucrose and 0.1% sodium azide. Tissue was sectioned
(20 pan) and sections were thaw-mounted on gelatin-coated glass slides, immersed in
SPB containing 4% paraformaldehyde and 1.5% sucrose for 5 min, and then stained
using the Lillie modification of the Weigert method (Lillie, 1965). The atlas
illustrated in Fig. V. lb is a series of camera lucida drawings prepared from sections
from a single animal. Brain regions were identified using descriptions of the urodele
amphibian telencephalon (Herrick, 1927; Northcutt and Kicliter, 1980), diencephalon
and midbrain (Herrick, 1917), and hindbrain (Herrick, 1930), as well as the
comprehensive study by Herrick (1948). The glyoxylic acid-induced
histofluorescence technique (De la Torre and Surgeon, 1976; De la Torre, 1980) was
used to verify the precise locations in T. granulosa of the preoptic recess organ (PRO;
77
Vigh-Teichmann et al., 1969), paraventricular organ (PVO; Vigh-Teichmann and
Vigh, 1983; nucleus organi paraventricularis, Vigh et al., 1967; periventricular organ,
Smeets and Gonzalez, 1990), and nucleus infundibularis dorsalis (NID; Terlou and
Ploemacher, 1973). The dissection of the PVO included the rostral region of this
complex, the organon vasculosum hypothalami (Hanke, 1976).
Microdissection of discrete brain regions
Microdissection of individual brain regions (April, 1990; n=8 animals) was
accomplished using the methods described for the microdissection of mammalian
brain tissues (Palkovits, 1973; Palkovits and Brownstein, 1982) as previously applied
in T. granulosa (Zoeller and Moore, 1986). Slides representing an entire brain were
placed on a cold stage (Thermoelectric Cold Plate, TCP-2, Thermoelectrics
Unlimited, Inc.) and maintained at -10 °C. Using a stereomicroscope (25X
magnification), brain regions were identified using the ventricular systems and white
matter and gray matter as landmarks. Tissue punches (300 Am i.d.) were expelled
into 60 Al of acetate buffer (pH 5) containing 0.5 x 10-7 M internal standard, amethyldopamine, and then stored at -80 °C until analyzed for monoamine content. If
a brain region was damaged during preparation of the tissue for microdissection and
landmarks could not be recognized, the region was not sampled.
Monoamine assay
Norepinephrine (NE), epinephrine (E), 3,4-dihydroxyphenylacetic acid
(DOPAC), dopamine (3,4-dihydroxyphenylethylamine; DA), 5-hydroxyindoleacetic
78
FIGURE V.1. Camera lucida drawings of brain tissues of an adult male T.
granulosa illustrating diagrammatically the relationships between the intact brain and
individual 300 itm sections which were prepared for microdissection. Figure V.1A.
Camera lucida drawings illustrating dorsal (above) and lateral (below) views. The
brain was illustrated while the meninges were intact and therefore melanocytes are
visible as large irregularly-shaped dots on the diencephalic and brainstem surfaces.
The reticular structure in the center of the caudal telencephalic lobes is the choroid
plexus. The numbered scale indicates the section numbers that correspond to 300 itm
transverse sections. Figure V.1B. Camera lucida drawings of transverse sections
from the serially sectioned (20 itm) brain illustrated in Figure V.1A following removal
of the meninges and the choroid plexus. The sections illustrated are approximately
300 itm apart. The dotted lines represent (except in the lower regions of sections 21
and 22 where they represent connective tissue associated with the pituitary) borders
between gray matter and white matter which provide excellent landmarks for
microdissection of tissues. Numerical labels indicate the section number
corresponding to Figure V.1A followed by the section number in the series of 20 /Am
sections used for illustration. The location of microdissected punches (300 tan i.d.) is
indicated on the right hand side of each section. Microdissection of all areas was
bilateral except for the anterior preoptic area, rostral ventral hypothalamus, and the
interpeduncular nucleus. Stippling within the microdissected regions represents
relative concentrations of epinephrine in these regions (light stippling, <10 pg/i2g
protein; medium stippling, 10-20 pg/itg protein; and solid, >20 pg/ktg protein). Scale
bar = 1 mm.
I
1
2
1
3
1
4
1
5
1
6
1
1
'
8
1
9 110 111 112 113 114 115116 111 118 119
1
20
1 211221 23124125126 121 128129 130131 '32'33 1341351 36/31138139 140 1 411 42 143144 1451
80
FIGURE V.1B
81
FIGURE V.1B (Continued)
82
acid (5-HIAA), and serotonin (5-hydroxytryptamine; 5-HT) were analyzed using
HPLC-ED. The method of analysis was based on a previously described procedure
(Renner and Luine, 1984; Renner and Luine, 1986) with several modifications. The
tissue samples were thawed and centrifuged at 15,000 x g for 2 min. The supernatant
was treated with 2µl of 0.02% ascorbate oxidase (Boehringer Mannheim) to
minimize ascorbic acid contributions to the solvent front (McKay et al., 1984) and
directly injected into a Waters chromatographic system (Waters Associates, Inc.).
Chromatographic separation was accomplished using a C-18, 4µm radial compression
cartridge and a mobile phase consisting of (wt /vol), 0.84% sodium acetate, 1.2%
citric acid, 0.015 % octanesulfonic acid sodium salt (Eastman Kodak), 0.02%
disodium EDTA, in (vol /vol) 15% methanol in water. Electrochemical detection was
provided by a laboratory built potentiostat and a glassy carbon electrode (Bioanalytical
Systems) set at +0.7 V with respect to an Ag/AgC1 reference electrode. The tissue
pellet was dissolved in 0.3 N NaOH and analyzed for protein content according to the
method of Bradford (1976).
Calculations
The pg/cm peak heights of known concentrations of the standards were
determined from the mean peak heights of 3 chromatograms for each respective
standard. The a-methyldopamine internal standard was injected 3 times to determine
the peak height for 100 % sample recovery. Amine concentrations were calculated
from the standard values and corrected for % recovery and injection volume using a
Waters 730 Data Module. The amine concentrations were divided by Ag protein to
83
yield pg amine /µg protein. Outlier values identified using the Grubbs method were
not included in reported values (two-tailed probability, P < 0.05; Grubbs and Beck,
1972).
RESULTS
Table I summarizes the concentrations of monoamines and monoamine
metabolites in microdissected regions of the central nervous system of male T.
granulosa. Concentrations of norepinephrine were highest in microdissected areas in
the preoptic-hypothalamic region, including the microdissected regions that contained
the PRO and the NID, and in the brainstem. Norepinephrine concentrations were
moderately high in the ventral striatum, amygdala pars medialis and amygdala pars
lateralis of the basal forebrain, thalamus and habenula of the diencephalon, and the
microdissected region that included the PVO. The lowest concentrations of
norepinephrine were found in the internal granule layer of the olfactory bulb and
pallial structures of the telencephalon.
Concentrations of epinephrine were highest in the microdissected regions that
contained the PRO and the NID. Relatively high epinephrine concentrations were
observed in the remaining areas of the diencephalon, amygdala complex, and
brainstem. Epinephrine concentrations were lower in the remaining telencephalon,
including the dorsal striatum, ventral striatum, and septal structures.
Table II shows the percentage of epinephrine (relative to the total amount of
epinephrine and norepinephrine) in the major subdivisions of the brain. In general,
norepinephrine concentrations exceeded epinephrine concentrations in the
84
telencephalon, but norepinephrine concentrations were exceeded by epinephrine
concentrations in most diencephalic regions.
Concentrations of dopamine were highest in areas in the basal forebrain
(striatum, septal structures, and amygdala complex), and in the preoptic-hypothalamic
region (ventral hypothalamus and the microdissected regions that contained the PRO,
the PVO, and the NID). All other regions measured had relatively low concentrations
of dopamine.
The highest concentrations of 5-HT were observed in the raphe region of the
brainstem (interpeduncular nucleus), and the microdissected regions that contained the
PVO and the NID. High 5-HT concentrations also were observed in the striatum, the
microdissected region that included the PRO, the tectum, and the ventral tegmentum.
Moderately high 5-HT concentrations were observed in microdissected areas
throughout the remaining basal forebrain, diencephalon, and brainstem. The lowest
levels of 5-HT, as with the catecholamines, were found in the internal granule layer
of the olfactory bulb and pallial structures of the telencephalon. In general, 5-HT
concentrations were higher than catecholamine concentrations.
Concentrations of the dopamine metabolite 3,4-dihydroxyphenylacetic acid
(DOPAC) were highest in microdissected tissues of the basal forebrain, diencephalon
and hindbrain structures. The metabolite to monoamine ratio, [DOPAC] /[DA], which
has often been interpreted as an index of utilization or turnover of dopamine, was
lowest in areas with the highest concentrations of dopamine (Fig.V.2a). The lowest
85
TABLE V.1. Monoamine Levels and Monoamine Metabolite Levels* in
Microdissected Brain Regions of T. granulosa. *Mean values ± S.E.M. expressed as
pg/tig protein; bold-faced and italicized text are used to facilitate identification of the
regions with the highest and lowest concentrations, respectively, for each of the amine
neurotransmitters. Abbreviations: DA, dopamine, 3,4-dihydroxyphenylethylamine;
DOPAC, 3,4-dihydroxyphenylacetic acid; E, epinephrine; 5-HIAA, 5hydroxyindoleacetic acid; 5-HT, serotonin, 5-hydroxytryptamine; NE, norepinephrine.
NE
BRAIN REGION
DOPAC
E
DOPAC/DA
5-HIAA
3.36 ± 0.46
0.563 ± 0.056
4.12 ± 0.43
22.75 ±
1.98
0.191 ± 0.011
± 0.13
0.627 ± 0.069
2.25 ± 0.26
9.10 ±
0.78
0.250 ± 0.019
DA
5-HT
5-HIAA/5-HT
Telencephalon
± 0.10
Internal Granule Layer, Medial
5.26 ± 0.91
1.47 ±
0.15
1.73
Internal Granule Layer, Lateral
3.18 ± 0.40
0.99 ±
0.17
1.20 ± 0.08
Medial Pa Ilium
4.19 ± 0.41
2.04 ±
0.10
2.11
± 0.20
4.09 ± 0.55
0.450 ± 0.055
4.86 ± 0.38
22.64 ±
2.50
0.199 ± 0.016
Dorsal Pa Ilium
3.52
± 0.27
1.53 ±
0.12
1.81
± 0.23
3.12 ± 0.26
0.576 ± 0.045
2.67 ± 0.29
12.05 ±
1.83
0.240 ± 0.031
Lateral Pa Ilium
4.49 ± 0.42
2.18
±
0.35
2.86 ± 0.33
5.44 ± 0,40
0.553 ± 0.093
3.60 ± 0.26
17.30 ±
3.17
0.256 i 0.040
Dorsal Striatum
9.88 ± 1.70
3.62 ±
0.83
5.46 ± 1.84
14.55 ± 1.48
0.274 ± 0.074
5.44 ± 0.32
72.81 ±
8.38
0.083 ± 0.011
Ventral Striatum
13.08 ± 2.67
6.80
±
1.02
3.41
± 0.39
20.20 ± 1.72
0.177 ± 0.020
4.99 ± 0.37
68.36 ±
3.96
0.078 ± 0.008
7.79 ± 0.97
5.09
±
1.07
4.22 ± 0.60
13.30 ± 1.42
0,332 ± 0.052
8.74 ± 1.09
53.84 ±
3.73
0.159 ± 0.012
Septum
2.01
Amygdala Pars Medians
19.14 ± 4.14
22.83 ±
4.31
5.73 ± 1.44
17.04 ± 3.66
0.452 ± 0.083
10.28 ± 1.69
45.17 ±
4.89
0.208 ± 0.024
Amygdala Pars Lateralis
21.32 ± 3.52
14.62 ±
3.74
4.82 ± 0.76
12.14 ± 1.96
0.412 ± 0.060
8.10 ± 0.61
52.04 ±
6.41
0.145 ± 0.012
Preoptic Recess Organ
37.44 ± 5.81
73.28 ±
10.93
9.03 ± 1.78
25.03 ± 3.15
0.339 ± 0.052
8.04 ± 0.95
59.82 ±
11.59
0.178 ± 0.019
Thalamus
16.93 ± 2.34
22.66 ±
3.07
4.53 ± 1.06
8.54 ± 1.59
0.525 ± 0.099
6.76 ± 0.76
52.17 ±
3.72
0.128 ± 0.016
Habenula
20.28 ±
5.75
12.94 ±
2.56
7.57 ± 2.06
5.72 ± 0.71
1.241
± 0.309
9.96 ± 2.29
48.59 ±
12.88
0.184 ± 0,059
8.33 ± 1.35
13.30 ±
2.10
3.82 ± 0.93
11.92 ± 3.06
0.212 ± 0.025
5.56 ± 0.97
53.64 ±
6.90
0.114 ± 0.022
Nucleus Infundibularis Dorsalis
24.94 ± 2.91
39.77 ±
5.32
6.31 ± 0.78
29.26 ± 3.66
0.317 ± 0.090
12.62 ± 2.21
103.51 ±
16.52
0.130 ± 0.021
Paraventricular Organ
14.64 ± 1.78
20.20
2.30
3.49 ± 0.63
28.30 ± 8.53
0.184 ± 0.043
5.05 ± 0.57
80.63 ±
8.30
0.065 ± 0.011
Diencephalon
Ventral Hypothalamus
±
Midbrain
'rectum
6.69 ± 0.70
9.68 ±
1.27
1.67
± 0.17
5.92 ± 0.26
0.330 ± 0.041
Dorsal Tegmentum Mesencephali
5.89 ± 0.35
60.80 ±
6.08
0.107 ± 0.008
11.43 ± 2.20
49.97 ±
3.40
0.261 ± 0.049
9,06 ± 1.15
7.85 ±
0.75
3,59 ± 0.74
8.75 ± 0.33
0.418 ± 0.105
4.38 ± 0.44
48.87 ±
6.72
0.103 ± 0.020
Interpeduncular Nucleus (Raphe)
13.73 ± 2.26
11.96 ±
0.63
3.46 ± 0.64
6.33 ± 0.99
0,501 ± 0.101
5.50 ± 0.96
121.73 ±
21.01
0.041 ± 0.012
Ventral Tegmentum
24.77 ± 4.12
15.88 ±
0.95
4.16 ± 0.51
7.51 ± 0.74
0.553 ± 0.061
7.45 ± 1.07
69.82 ±
3.01
0.112 ± 0.015
Dorsal Tegmentum
15.41 ± 3.45
16.03 ±
2.88
5.39 ± 1.24
8.43 ± 0.88
0.528 ± 0.109
5.81
± 0.98
46.01 ±
4.46
0.110 1 0.010
Isthmic Tegmentum
Hindbrain
88
TABLE V.2. Percent epinephrine relative to norepinephrine in major brain
regions
Region
Epinephrine
(% of Total)*
# Microdissected
Regions (n)
Olfactory bulb
(Internal granule layer)
22.79 + 0.95
2
Pallium
31.91 + 0.80
3
Subpallium
39.13 ± 0.45
5
Diencephalon
57.22 ± 3.88
6
Midbrain and
Hindbrain
48.43 + 3.29
5
*Values for percent methylated amine (concentration of epinephrine x 100/total
concentration of epinephrine and norepinephrine) represent mean values + S.E.M.
using values from Table V.1.
[DOPAC] /[DA] ratios occurred in the ventral striatum and the microdissected region
that included the PVO.
Concentrations of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA)
were high in microdissected tissues of the septal structures, amygdala complex,
diencephalon, dorsal tegmentum mesencephali, and ventral tegmentum. As with
dopamine the metabolite to monoamine ratio, [5-HIAA]/[5-HT], was lowest in
microdissected areas with the highest concentrations of 5-HT (Fig.V.2b). The lowest
(5- HIAA] /(5 -HT] ratios occurred in the raphe region and the microdissected region
that included the PVO.
89
DISCUSSION
The present microdissection with HPLC-ED study reveals the neuroanatomical
distribution of catecholamines and indoleamines in the central nervous system of an
amphibian, T. granulosa. These data complement previous histofluorescence and
immunohistochemical studies (reviewed by Parent et al., 1984; Parent, 1986;
Gonzalez and Smeets, 1994) by providing quantitative measures of biochemically
identified monoamines and their metabolites in specific brain areas. The overall
pattern in this amphibian resembles that of other vertebrates, including the most
fundamental characteristic of monoaminergic systems, that is, a widespread
distribution throughout the central nervous system (Parent, 1981, 1984; Parent et al.,
1984). These monoaminergic systems are not necessarily diffuse and nonspecific but,
instead, can be highly organized topographically (see Lindvall and Bjorklund, 1983;
Cunningham and Sawchenko, 1988).
The technique used in the present study has a high level of sensitivity. We
observed measurable levels of monoamines in all regions of the central nervous
system studied.
Regional distribution of norepinephrine
Early biochemical studies using quantitative fluorometric techniques were
unable to measure norepinephrine in amphibian brain tissues (R. pipiens, Hy la
cinerea, Bufo americanus, B. marinus, A. tigrinum, and N. maculosus; Bogdanski et
al., 1963). In contrast, in recent studies which used HPLC-ED techniques in urodele
90
A
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
5
10
15
20
25
30
100
125
150
[DOPAMINE]
B
0.4
0.3
0.2
0.1
0.0
0
25
50
75
[5-HT]
FIGURE V.2. The ratios of metabolites to monoamines in the brain of adult male T.
granulosa were higher in regions of the brain with low concentrations of the parent
amines. Mean values for each brain region were included. Coefficients of
correlation: (DOPAC]/(DA] vs (DA], -0.7309, P < 0.0005; (5-HIAA1/(5-HT] vs (5HT], -0.7729, P < 0.0001.
91
amphibians (A. tigrinum, Hamilton et al., 1987; Norris et al., 1992; Ambystoma
maculata, Amphiuma means, C. alleganiensis, N. maculosus, Plethodeon glutinosus,
and S. lacertina Cooney et al., 1985) norepinephrine was detected in whole brain
homogenates or in each major region of the central nervous system analyzed
(olfactory bulb, telencephalon, hypothalamus, and midbrain or brainstem). Our
microdissection and HPLC-ED study expands these findings by quantifying
norepinephrine in specific brain areas. These observations, together with
immunohistochemical studies of P. waltlii and X. laevis (Gonzalez and Smeets, 1993,
1994), suggest that noradrenergic systems are more extensive in some amphibians
than previously recognized.
Early histofluorescence studies failed to detect catecholamine (norepinephrine)
neuronal somata within the isthmic tegmentum of amphibians (Braak, 1970; Bartels,
1971; Parent, 1973; Terlou and Ploemacher, 1973; Chacko et al., 1974; Parent,
1975; Sims, 1977), or the presence of an isthmotelencephalic catecholaminergic
pathway (Parent, 1975; Dube and Parent, 1982). However, immunohistochemistry
provided evidence for noradrenergic neuronal somata within the isthmic tegmentum of
X. laevis, R. ridibunda, and P. waltlii (Gonzalez and Smeets, 1991, 1993, 1994).
These observations, together with previous descriptions of ascending projections from
the isthmic tegmentum to the midbrain tectum and telencephalon (Wilczynski and
Northcutt, 1977; Finkenstadt et al., 1983; Dube et al., 1990; Lazar and Kozicz, 1990)
led Gonzalez and Smeets to label the noradrenergic cell mass in the isthmic
tegmentum as the locus coeruleus. In the present study, the moderately high
92
norepinephrine concentrations in the thalamus, amygdala pars medialis and amygdala
pars lateralis are consistent with biochemical and immunohistochemical observations
in mammals in which the thalamic and amygdaloid structures receive moderately high
norepinephrine innervation originating in the locus coeruleus (Kobayashi et al., 1974;
Fallon et al., 1978; Lindvall and Bjorklund, 1978; Moore and Bloom, 1979); these
observations are consistent with the hypothesis that amphibians have a norepinephrine
cell body region with homology to the locus coeruleus of other vertebrates (Gonzalez
and Smeets, 1993; 1994).
Regional distribution of epinephrine
Our understanding of the regional distribution of epinephrine in the amphibian
central nervous system is incomplete (see Introduction). However, in all studies that
used either quantitative fluorometric techniques (Bufo arenarum, Segura and Biscardi,
1967; R. temporaria, Juorio, 1973) or HPLC-ED techniques (R. catesbeiana, Fuller
and Hemrick-Luecke, 1983; Scaphiopus holbrookii, Bufo terrestris, N. maculosus, C.
alleganiensis, Cooney et al., 1985; T. granulosa, present study) the hypothalamus
contains higher epinephrine concentrations than the cerebral hemispheres, brainstem,
or spinal cord. A similar pattern has been observed in several species of reptiles,
birds, and mammals (Fuller and Hemrick-Luecke, 1983).
The distribution of epinephrine described in the present study is similar to the
pattern of distribution described in microdissection studies of other vertebrates (Juorio
and Vogt, 1967; Koslow and Schlumpf, 1974; Mefford et al., 1978; 1982;
Brownstein and Palkovits, 1984), with the highest concentrations in microdissected
93
areas in the hypothalamus (preoptic-hypothalamic region in amphibians, see definition
by Herrick, 1948; discussion by Peter, 1986), thalamus, habenula, and brainstem.
The neuroanatomical distribution of adrenergic neuronal systems can be
inferred from immunohistochemical studies of the epinephrine-forming enzyme,
PNMT. The PNMT-immunoreactive neurons in the mammalian systems are typically
identified as two separate populations within the rostral medulla, the C 1 neurons in
the reticular formation of the ventrolateral medulla, and the C2 neurons in the
dorsomedial medulla, including the periventricular region, medial aspects of the
dorsal vagal motor nucleus, and portions of the medial and basal aspects of the
nucleus of the solitary tract (Hokfelt et al., 1974, 1984a; Ruggiero et al., 1985;
Iwamoto et al., 1989; Ruggiero et al., 1992; Dormer et al., 1993). A third group of
PNMT-immunoreactive neurons have been described in the rostral midline raphe of
some mammals, Rattus rattus, Sus scrofa, and canines (C3 Group; Howe et al., 1980;
Ruggiero et al., 1992; Dormer et al., 1993), but not other mammals (Kitahama et al.,
1985, 1986, 1988; Tillet, 1988). The distribution of PNMT- immunoreactive neuronal
cell bodies in these mammals is comparable to the distribution of medullary PNMTimmunoreactive neurons in the lizard Gekko gecko, the turtle Pseudemys scripta
elegans (Smeets and Jonker, 1990; Smeets and Steinbusch, 1990), and the white pekin
duck Anas platyrhynchos (Steeves et al., 1987). There is considerable interspecies
variation, however, in the precise location and number of groups of medullary
PNMT- immunoreactive neurons (see discussion by Smeets and Jonker, 1990).
94
Among the Amphibia PNMT-immunoreactive neurons have been described in
R. catesbeiana and P. waltlii (Yoshida et al., 1983; Gonzalez and Smeets, 1994).
The PNMT-immunoreactive neurons in the medulla oblongata of R. catesbeiana and
P. waltlii are located in the nucleus of the solitary tract and the neighboring reticular
formation (Yoshida et al., 1983; Gonzalez and Smeets, 1994). It is likely but not
certain that these brainstem neurons are a significant source of epinephrine throughout
the amphibian central nervous system. The only reference to ascending PNMTimmunoreactive fibers in amphibians is the presence of a few weakly stained fibers
which course through the ventral tegmentum to the basal forebrain in P. waltlii
(Gonzalez and Smeets, 1994). In addition, there is some debate regarding whether or
not PNMT activity or PNMT-immunoreactive structures are well correlated with
adrenergic systems (Fuller and Hemrick-Luecke, 1983; Sved, 1989). Although lesion
studies indicate that the majority of hypothalamic epinephrine originates from within
the central nervous system (Rattus rattus, Palkovits et al., 1980; Mefford et al.,
1981), the mechanisms through which hypothalamic tissues in T. granulosa and other
vertebrates accumulate high concentrations of epinephrine are uncertain.
Relative concentrations of norepinephrine and epinephrine
In the present study all diencephalic regions, except for the habenula, had
higher epinephrine concentrations than norepinephrine concentrations. Conversely,
within the rostral telencephalon including the internal granule layer of the olfactory
bulb, pallial structures and the striatum, norepinephrine concentrations were higher
than epinephrine concentrations. This pattern is similar to that observed in N.
95
maculosus, where norepinephrine concentrations exceeded epinephrine concentrations
in the cerebral hemispheres but not the hypothalamus (Cooney et al., 1985). Based
on the regions sampled in this study, the epinephrine concentration was less than 50%
of the total (relative to the sum of epinephrine and norepinephrine concentrations) in
T. granulosa (Table II).
Studies using quantitative fluorometric techniques on whole brain homogenates
(R. pipiens, H. cinerea, B. americanus, Bufo marinus, A. tigrinum, and N.
maculosus, Bogdanski et al., 1963) or homogenates of major regions of the central
nervous system (B. arenarum, Segura and Biscardi, 1967; R. temporaria, Juorio,
1973) demonstrated that the ratio of epinephrine to norepinephrine is unusually high
in amphibians (norepinephrine was not detectable, Bogdanski et al., 1963; or
concentrations of epinephrine were approximately 80% of the total; Juorio, 1973; cf
other nonmammalian vertebrates, Juorio and Vogt, 1967; Juorio, 1973). In contrast,
in mammals epinephrine concentrations are low compared to norepinephrine
concentrations (approximately 5-15% of the total; Vogt, 1954; Gunne, 1962).
Based on the more recent study by Cooney and colleagues (1985) and the
present study, there appears to be considerable variation among Amphibia with
respect to the relative concentrations of epinephrine and norepinephrine in the central
nervous system. In fact, in some urodele amphibians which belong to the suborders
Cryptobranchoidea and Salamandroidea (Duellman and Trueb, 1986), norepinephrine
can be considered to be the predominant amine relative to epinephrine (ie., T.
granulosa, present study; C. alleganiensis, where epinephrine is 38.4% of total; A.
96
maculata, where epinephrine is 41.7% of total; A. means, where epinephrine is
46.6% of total, Cooney et al., 1985). Thus, although it remains a cogent
generalization that in anuran amphibians epinephrine is the predominant amine relative
to norepinephrine, this is not a cogent generalization for Amphibia.
The predominance of norepinephrine over epinephrine in the mammalian
central nervous system is most extreme in the cerebral cortices (e.g. Mefford et al.,
1982). Since cortical structures are a major target of the locus coeruleus
(isthmocortical) noradrenergic pathways in vertebrates (i.e. the locus coeruleus gives
rise to the norepinephrine innervation of the entire neocortex in the rat; for references
see Lindvall and Bjorklund, 1978), it seems likely that, as suggested previously for
the midbrain dopaminergic system (Parent et al., 1984), hypertrophy of the neocortex
may account for the extensive development of the locus coeruleus complex in
mammals. Conversely, lack of extensive cortical targets in amphibians might account
in part for the difficulties encountered (reviewed by Parent, 1986; see discussions by
Gonzalez and Smeets, 1993, 1994) in the identification of noradrenergic
isthmotelencephalic pathways in amphibians.
Regional distribution of dopamine
In T. granulosa dopamine concentrations were highest in microdissected areas
of the striatum, septum and the amygdala complex in the basal forebrain, as well as in
diencephalic regions which contained CSF-MA neuronal cell bodies. Dopamine
concentrations were low in microdissected areas of the internal granule layer of the
olfactory bulb and pallial structures.
97
In mammals, the neurons that comprise the majority of the ascending
dopaminergic system are located ventrally at the mesodiencephalic junction and give
rise to well defined mesostriatal, mesolimbocortical, and mesodiencephalic systems
(reviewed by Parent, 1986; see Lindvall and Bjorklund, 1983). These nuclei
A8
(the retrorubral nucleus), A9 (the substantia nigra), and A10 (the ventral tegmental
area, a medial extension of the substantia nigra)
extend rostrally toward the
diencephalon from the level of the oculomotor nucleus (reviewed by Lindvall and
Bjorklund, 1983). A similar but smaller complex of dopaminergic neurons in the
ventromedial portion of the rostral midbrain tegmentum has been described in
amphibians using histofluorescence (Parent, 1973; Sims, 1977; Dube and Parent,
1982; reviewed by Parent, 1986) and immunohistochemical techniques (Yoshida et
al., 1983; Franzoni et al., 1986; Smeets and Gonzalez, 1990; Carr et al., 1991;
Gonzalez and Smeets, 1991, 1994; Gonzalez et al., 1993a, 1994a). Although not
described as such in all studies (i.e. Corio et al., 1992), and certainly not definitive
(see discussion by Gonzalez and Smeets, 1994; Gonzalez et al., 1994a), dopaminergic
neurons in the rostral ventral midbrain tegmentum of amphibians may represent a
complex which is homologous to the A8, A9, A10 complex described in mammals.
In rats microdissection and HPLC-ED studies found that the highest
concentrations of dopamine are observed in elements of the basal ganglia (olfactory
tubercle, nucleus accumbens, caudate-putamen, ventral striatum, anterior amygdala
area, and globus pallidus; Versteeg et al., 1976; Brownstein and Palkovits, 1984). In
T. granulosa we observed high concentrations of dopamine within microdissected
98
regions of the basal forebrain (the dorsal striatum, ventral striatum, septal structures,
and amygdala complex; Northcutt and Kicliter, 1980; nucleus accumbens, unpublished
observations). These observations are consistent with the hypothesis that T. granulosa
has functional mesostriatal and mesolimbic dopaminergic systems, however, more
detailed topographical, hodological (as in, for example, Gonzalez et al., 1993b), and
neurochemical studies (see Faso lo et al., 1990) are necessary before direct
comparisons can be made with a high degree of confidence between elements of the
basal forebrain of this amphibian and elements of the basal ganglia of mammals or
other vertebrates; (in particular, it should be noted that the use of the term ventral
striatum in the present study does not imply homology with similarly labeled
structures in amniotes). Interestingly, however, there are clear differences between
our observations in T. granulosa and those in R. rattus described by Brownstein and
Palkovits (1984). Most notably, although the concentrations of dopamine in the basal
forebrain of T. granulosa were high relative to most regions of the central nervous
system, the highest dopamine concentrations occurred in regions of the hypothalamus
that contain CSF-MA neuronal cell bodies.
Regional distribution of serotonin
In T. granulosa 5-HT concentrations in the telencephalon were greater than
catecholamine concentrations. This observation is consistent with studies of
amphibians using quantitative fluorometric (Bogdanski et al., 1963), histofluorescence
(Parent, 1975) and HPLC-ED methods (Hamilton et al., 1987; Norris et al., 1992).
In fact, in T. granulosa all regions contained appreciable levels of 5-HT, with the
99
highest concentrations in the areas which, based on previous studies, include 5-HT-
containing neurons (raphe region, PVO, NID). The pattern of 5-HT distribution is
similar to that observed in other vertebrates (Saavedra, 1977; Gaudin-Chazal et al.,
1979; Mefford et al., 1982; Brownstein and Palkovits, 1984) with high concentrations
in the raphe region, as well as in regions of the hypothalamus, ventral forebrain, and
brainstem. This pattern most likely represents a widespread innervation of the brain
by 5-HT neurons in the raphe region of the brainstem, a neuronal complex which has
been described as a major source of 5-HT innervation, via ascending projections
within the medial forebrain bundle, in virtually all vertebrate species studied
(reviewed by Parent et al., 1984).
Preoptic-Hypothalamic CSF-MA Neuronal Systems
The highest concentrations of monoamines in the brain of T. granulosa
(excluding the serotonergic raphe system) were observed in microdissected samples
from the preoptic-hypothalamic regions that contain CSF-MA perikarya in
amphibians.
Preoptic Recess Organ
The microdissected region of the PRO contained the highest concentrations of
norepinephrine and epinephrine compared to all other regions in the present study.
The concentrations of dopamine in this region were also relatively high, exceeded
only by the concentrations measured in the microdissected regions of the PVO and the
100
MD. In addition, the concentrations of 5-HT were moderately high in the region of
the PRO compared to other regions of the central nervous system.
These observations are consistent with the description of CSF-MA neurons in
the PRO of several amphibians including A. mexicanum, Bombinator igneus, Bufo
bufo, P. waltlii, R. esculenta, and R. ridibunda (Vigh- Teichmann et al., 1969; see
also Braak, 1970; Bartels, 1971; Parent, 1973; Terlou and Ploemacher, 1973; Chacko
et al., 1974; Prasada Rao and Hartwig, 1974; Sims, 1977; Kikuyama et al., 1979;
Dube and Parent, 1982; Yoshida et al., 1983; Miyakawa et al., 1984; Ueda et al.,
1984a; Franzoni et al., 1986; Corio and Doerr-Schott, 1988; Lamas et al., 1988; Can
et al., 1991; Corio et al., 1992; Gonzalez and Smeets, 1994). These neurons are
distributed around the entire surface of the preoptic recess, from the most rostral
portion of the recess caudally to the margin of the neurosecretory preoptic nucleus
(Vigh-Teichmann et al., 1969). Several studies have described a rostral extension of
this nucleus with monoamine-containing neurons at the posterior and upper surface of
the anterior commissure ("PRO-additional cells" of Chacko et al., 1974; see also
Vigh-Teichmann et al., 1969; Dube and Parent, 1982; Lamas et al., 1988).
A monoamine neuronal component of rostral preoptic area CSF-contacting
neurons was not demonstrated in initial studies of fishes, reptiles, birds, or mammals
(Vigh-Teichmann et al., 1969) using the formaldehyde-induced histofluorescence
technique (Falck and Owman, 1965). More recent studies have, however, identified a
potential monoaminergic preoptic recess organ in elasmobranch (Scyliorhinus canicula
L., Wilson and Dodd, 1973), and teleost fishes (Anguilla vulgaris, Lefranc et al.,
101
1969), a few weakly fluorescent CSF-MA neurons in the preoptic recess area of the
turtle, Chtysemys picta (Parent and Poirier, 1971; Parent and Poitras, 1974), and
numerous tyrosine hydroxylase-immunoreactive, CSF-contacting neurons in the
preoptic recess area of the reptile, Caiman crocodilus (Brauth, 1988).
Based on the results of the present study, the possibility exists that regions
with high concentrations of epinephrine, especially the regions of the anterior preoptic
area and the caudal infundibular hypothalamus, may be important sites for the
synthesis, storage, transport or utilization of epinephrine in T. granulosa brain. In
comparison, based on measurements using gas chromatography-mass spectrometry
(Koslow and Schlumpf, 1974), radiometric methods (Van der Gugten et al., 1976), or
HPLC-ED techniques (Gereau et al., 1993), periventricular regions at the anterior
hypothalamic level and the infundibular hypothalamus of the rat contain several-fold
higher concentrations of epinephrine than other regions measured, suggesting these
regions may be important loci for the synthesis, storage, transport or utilization of
epinephrine in a mammalian species as well.
In addition to high concentrations of epinephrine in the microdissected region
that included the PRO, the concentrations of the primary catecholamines were also
relatively high. These observations are consistent with previous studies which have
identified CSF-MA neurons in the preoptic region using anti-tyrosine hydroxylase
antisera in R. catesbeiana (Nagatsu et al., 1982; Yoshida et al., 1983; Carr et al.,
1991), R. ridibunda and P. waltlii (Gonzalez and Smeets, 1991, 1994), and T.
alpestris (Corio et al., 1992). Although these observations and the reserpine-sensitive
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properties of CSF-contacting neurons in the preoptic recess organ are consistent with
the presence of catecholamines (Vigh-Teichmann et al., 1969), it was until recently
unclear which catecholamines were represented. Spectrofluorometric analysis in R.
temporaria (Prasada Rao and Hartwig, 1974) suggested that the catecholamine
localized in the cells of the PRO was dopamine, while the ability of repeated
treatments with DL-m-tyrosine to deplete the catecholamine content of these neurons
favored the presence of norepinephrine (Vigh- Teichmann et al., 1969). These initial
characterizations of the PRO are supported by the presence of dopamineimmunoreactive, cerebrospinal fluid-contacting neurons within the PRO of T. alpestris
(Corio et al., 1992), R. ridibunda, P. waltlii, and X. laevis (Gonzalez and Smeets,
1991, 1994), as well as norepinephrine-immunoreactive, dopamine-B-hydroxylase-
immunonegative cerebrospinal fluid-contacting neurons within the PRO of P. waltlii
(Gonzalez and Smeets, 1994). In addition, immunohistochemical studies have
identified 5-HT in CSF-contacting cells in the PRO of R. catesbeiana (Yoshida et al.,
1983; Ueda et al., 1984a). These observations and the quantitative measurements in
the present study suggest that the periventricular anterior preoptic area of amphibians
is an important locus for the metabolism of biogenic amines.
Paraventricular Organ
The region containing the PVO had high concentrations of dopamine and 5-
HT in T. granulosa. Cerebrospinal fluid-contacting neurons in the hypothalamic PVO
contain dopamine, identified using immunohistochemical techniques, but lack
immunoreactivity for tyrosine hydroxylase in representatives of teleost fishes
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including Gasterosteus aculeatus (Ekstrom et al., 1990), the electric mormyrid
teleost, Gnathonemus petersii (Meek et al., 1989), and the electric gymnotiform
teleost, Apteronotus leptorhynchus (Sas et al., 1990), in urodele and anuran
amphibians (Smeets and Gonzalez, 1990; Gonzalez and Smeets, 1991, 1994; Gonzalez
et al., 1993a), the lizard G. gecko and the turtle, P. scripta elegans (Smeets and
Steinbusch, 1990), and the bird, Gallus domesticus (Smeets and Gonzalez, 1990).
Studies in the anuran amphibian, X. laevis (Gonzalez and Smeets, 1993) and the
reptiles, G. gecko, and P. scripta elegans (Smeets and Steinbusch, 1989, 1990) have
determined that these neurons also contain norepinephrine and are immunonegative
for dopamine 13-hydroxylase (DBH), the enzyme which converts dopamine to
norepinephrine in noradrenergic neurons. It is uncertain if dopamine and
norepinephrine immunoreactivities are located within the same cell bodies (Gonzalez
and Smeets, 1994).
In addition, histofluorescence studies (Terlou and Ploemacher, 1973; Chacko
et al., 1974; Prasada Rao and Hartwig, 1974; Dube and Parent, 1982; Corio and
Doerr-Schott, 1988) and immunohistochemical studies (Sano et al., 1983; Shimizu et
al., 1983; Yoshida et al., 1983; Ueda et al., 1984a; Faso lo et al., 1986) indicate the
presence of 5-HT within CSF-MA PVO neurons. There does not, however, appear to
be a good correspondence between immunoreactivities for 5-HT and tryptophan
hydroxylase, the rate-limiting enzyme for biosynthesis of 5-HT, in this region of the
hypothalamus. For example, in the chick embryo, numerous CSF-contacting 5-HTcontaining neurons are located in the PVO, while the majority of tryptophan
104
hydroxylase-immunoreactive neurons are located lateral to the PVO. A much smaller
number of tryptophan hydroxylase- immunoreactive neurons are located in the PVO,
compared to 5-HT-immunoreactive neurons, suggesting that the presence of 5-HT-
containing cells in the PVO is probably not due to local intraneuronal synthesis of 5HT (Gabaldon et al., 1992; J.W. Wallace, personal communication).
Collectively, these studies indicate that CSF-contacting cells of the PVO in
amphibians contain dopamine, noradrenaline, and 5-HT, yet do not contain
immunoreactivity for enzymes normally required for biosynthesis of monoamines.
These observations and the ability of these neurons to accumulate radiolabeled
dopamine following intracerebroventricular injection in Rana nigromaculata and R.
catesbeiana (Nakai et al., 1977), has led to the proposal that the CSF-contacting cells
of the PVO accumulate catecholamines from the cerebrospinal fluid (i.e. dopamine,
Nakai et al., 1977; Smeets and Gonzalez, 1990; and norepinephrine, Gonzalez and
Smeets, 1993). This proposal is consistent with the observation that in reptiles the
dopamine synthesis inhibitor a-methyl-p-tyrosine, which significantly reduces
dopamine immunoreactivity in the dopaminergic cell bodies of the ventral midbrain
tegmentum, has little effect on dopamine immunoreactivity in CSF-MA neurons of the
PVO (Smeets et al., 1991).
Although discrete clusters of monoamine-accumulating, CSF-contacting
neurons have not been described in mammals, it is clear that specific populations of
non-monoaminergic neurons in the preoptic area and hypothalamus of mammals can
also accumulate monoamines (Fuxe and Ungerstedt, 1968; Lidbrink et al., 1974;
105
Chan-Palay, 1977; Kent and Sladek, 1978; Beaudet and Descarries, 1979; Karasawa
et al., 1994). It is also clear that monoamine-containing neurons project into the
cerebral ventricles of normal animals (Richards et al., 1973; Aghajanian and
Gal lager, 1975; Chan-Palay, 1976). It is not clear, however, whether the
monoamine-accumulating neurons that are located in the preoptic area and
hypothalamus of mammals project to the third ventricle. In fact, evidence suggests
that 5-HT-containing cerebrospinal fluid-contacting projections in mammals originate
in the midbrain median and dorsal raphe nuclei (Aghajanian and Gal lager, 1975).
Nonetheless, the conservation of these functional and structural features of
monoamine systems, that is, monoamine-accumulating capabilities of nonmonoaminergic preoptic area and hypothalamic neurons and CSF-contacting structural
elements in all classes of vertebrate organisms suggests that these features may be
important components of monoaminergic systems in the central nervous system (see
Steinbusch and Niewenhuys, 1983). As suggested by Parent and colleagues (Parent,
1984; Parent et al., 1984) monoamine-containing neurons may provide an important
link between the cerebrospinal fluid and neural tissues in all vertebrates.
Nucleus Infundibularis Dorsalis
Relatively high concentrations of norepinephrine, epinephrine, dopamine, and
5-HT were observed in the region of the NID (terminology of Terlou and
Ploemacher, 1973) of T. granulosa. Microspectrofluorometric characteristics of this
nucleus in X. laevis suggest that it has a similar monoaminergic composition as the
PVO, with both dopamine and 5-HT present (Terlou and van Kooten, 1974). In
106
support of these observations, immunohistochemical studies in amphibians have
identified dopamine (Corio et al., 1985; Corio et al., 1992) and 5-HT (Shimizu et al.,
1983; Yoshida et al., 1983; Ueda et al., 1984a; Faso lo et al., 1986; Corio et al.,
1992) in NID CSF-contacting neurons.
As with other hypothalamic CSF-MA neuronal systems in amphibians, it is
uncertain whether or not NID CSF-contacting neurons contain epinephrine. However,
the topographical position of NID neurons, in the caudal infundibular hypothalamus of
amphibians, is strikingly similar to the distribution of a population of cerebrospinal
fluid-contacting tyrosine hydroxylase-immunoreactive, PNMT-immunoreactive
neuronal cell bodies adjacent to the caudal infundibular recess of a gymnotiform
teleost fish, A. leptorhynchus (nucleus tuberis lateralis pars anterior; Sas et al., 1990),
and a population of PNMT-immunoreactive neurons in the rat (Foster et al., 1985).
The presumed hypothalamic adrenergic system of mammals is characterized by
PNMT-immunoreactive neurons within regions of the dorsal and medial
hypothalamus, as well as lateral, perifornical, zona incerta (Ross et al., 1984;
Ruggiero et al., 1985), and caudal arcuate nuclei (Foster et al., 1985). It was
suggested by Sas and colleagues (1990) that cerebrospinal fluid-contacting PNMTimmunoreactive neurons in the caudal infundibular hypothalamus of A. leptorhynchus
may correspond to the PNMT- immunoreactive neurons in the caudal arcuate nucleus
of the rat. These neurons in the rat, however, do not appear to contain other
catecholamine-synthesizing enzymes, including tyrosine hydroxylase, aromatic L-
amino acid decarboxylase (AADC), and DBH. Since the PNMT-immunoreactive
107
neurons described in the rat hypothalamus do not contain immunoreactivity for
tyrosine hydroxylase, it was suggested that these neurons may accumulate
norepinephrine from extracellular sources as a precursor for epinephrine synthesis
(Ruggiero et al., 1985), a proposal which is consistent with a hypothalamic source of
PNMT activity, independent of the medullary adrenergic cells (Brownstein et al.,
1976; Mefford et al., 1981; Saavedra et al., 1983; Masana and Mefford, 1989).
Furthermore, the possibility that these PNMT- immunoreactive neurons in the caudal
infundibular hypothalamus of the rat may synthesize, store, transport, or utilize
epinephrine is consistent with the observation that the arcuate nucleus is among the
regions of the rat central nervous system with the highest tissue concentrations of
epinephrine (Brownstein and Palkovits, 1984). In this context, further studies of
parvocellular monoamine-accumulating neuronal systems in the caudal arcuate nucleus
and ventral premammillary nucleus of the rat (see Fig.V.3), as well as potentially
homologous neuronal systems associated with the mammillary recess in the caudal
infundibular hypothalamus of nonmammalian vertebrates, promise to be rewarding in
our effort to understand hypothalamic monoamine-metabolizing systems.
The observations outlined above and others (see discussion below) suggest that
metabolism of multiple biogenic amines in the infundibular hypothalamus may be an
evolutionarily conserved feature of vertebrate brain.
108
Do hypothalamic monoamine-containing neurons contribute to the pattern of
monoamine distribution in extrahypothalamic regions?
Evidence suggests that hypothalamic monoamine-containing neuronal systems
in anuran and urodele amphibians contribute to the innervation of the median
eminence, intermediate pituitary (Terlou and Ploemacher, 1973; Prasada Rao and
Hartwig, 1974; Aronsson and Enemar, 1981; Dube and Parent, 1982; Tuinhof et al.,
1993; see also Artero et al., 1994; Tuinhof et al., 1994) and the larval pars distalis
(Aronsson and Enemar, 1981). Although in the urodele amphibian, T. cristatus,
retrograde labeling studies suggest that the region containing the PVO neurons
provides a major projection to the striatum (Dube et al., 1990), the full extent to
which these neurons contribute to the pattern of monoamine distribution in
hypothalamic and extrahypothalamic areas of the amphibian central nervous system is
uncertain. Likewise, with the exception of the well-characterized diencephalospinal
projection of mammalian (R. rattus) All dopaminergic neurons (Skagerberg and
Lindvall, 1985), the extent to which the hypothalamic dopaminergic (tyrosine
hydroxylase-immunoreactive, DBH-inununonegative) neuronal systems innervate
extrahypothalamic regions is uncertain in amphibians (Yoshida et al., 1983; Gonzalez
and Smeets, 1994) and in mammals (reviewed by Everitt et al., 1992).
The infundibular hypothalamus as a focal region for metabolism of biogenic amines
Our observation that the highest concentrations of norepinephrine, epinephrine,
dopamine, and 5-HT (excluding the serotonergic raphe system) occur in
periventricular regions of the diencephalon with CSF-MA neurons indicates that these
109
regions are important loci for metabolism of multiple biogenic amines. Based on
previous studies in mammalian and nonmammalian vertebrates, such a complex of
amine-metabolizing structures within the infundibular hypothalamus may be an
evolutionarily conserved trait of the vertebrate brain (see Fig.V.3).
Of particular interest are the striking similarities between amine-metabolizing
structures in the nonmammalian PVO and the mammalian dorsomedial hypothalamic
nucleus (DMN). These structures share a similar topographical position in the
periventricular region of the dorsal infundibular hypothalamus, well-removed caudally
from the hypothalamic neurosecretory nuclei (Vigh et al., 1967). In addition, these
regions are each characterized by discrete populations of monoamine-containing or
monoamine-accumulating neuronal cell bodies (for references see previous discussion
of PVO).
The presence of 5-HT-accumulating neurons in the DMN of the rat is
supported by studies in which 5-HT is visualized immunohistochemically following
systemic pretreatment with a monoamine oxidase inhibitor, either pargyline or
nialamide, and the precursors of 5-HT synthesis, either L-tryptophan (Frankfurt et al.,
1981; Steinbusch et al., 1982; Frankfurt and Azmitia, 1983; Steinbusch and
Nieuwenhuys, 1983; Sakumoto et al., 1984; Steinbusch, 1984; Arezki et al., 1985;
Ugrumov et al., 1988, 1989) or 5-hydroxytryptophan (5-HTP; Sakumoto et al., 1984;
Staines et al., 1986; Ugrumov et al., 1989; also in the cat, Denoyer et al., 1989;
Sakumoto et al., 1984), or by high dosages of monoamine oxidase inhibitor alone
(Ueda et al., 1984b; Ugrumov et al., 1989). Alternatively, following treatment of
110
rats with the 5-HT neurotoxin, 5,7-dihydroxytryptamine, which is resistant to
metabolism by monoamine oxidase, these neuronal cell bodies were visualized using a
monoclonal antibody to 5-HT which cross-reacts with the 5,7-dihydroxytryptamine
antigen (Howe et al., 1982). The presence of 5-HT-accumulating neurons in the rat
DMN is further supported by studies in which serotonin binding protein
a protein
that is believed to be an intrinsic and a specific component of serotonergic neurons
is visualized immunohistochemically within DMN neuronal cell bodies in the absence
of the pharmacological treatments necessary for the visualization of the same neurons
using anti-5-HT antisera (Kirchgessner et al., 1988).
Details about the mechanisms through which DMN neurons accumulate 5-HT
remain unclear, however, the ability of pretreatment with the selective 5-HT uptake
inhibitor, fluoxetine, to prevent the visualization of 5-HT immunoreactivity following
treatment with pargyline and L-tryptophan suggests that the 5-HT immunostaining is a
result of specific uptake of 5-HTP or 5-HT from extracellular fluids rather
thanintraneuronal synthesis from the precursor L-tryptophan (Ugrumov et al., 1988,
1989).
Congruent with the ability of neuronal cell bodies in the mammalian DMN to
accumulate 5-HT following administration of the 5-HT precursors, L-tryptophan or 5HTP, as well as to exhibit catecholamine histofluorescence or dopamine
immunoreactivity following administration of the precursor of dopamine synthesis, L-
3,4-dihydroxyphenylalanine (L-DOPA; Lidbrink et al., 1974; Karasawa et al., 1994),
neuronal cell bodies of the adult X. laevis PVO accumulate both radiolabeled 5-HTP
111
and L-DOPA (Peute and van Oordt, 1974), indicating that neuronal cell populations
within the dorsomedial hypothalamus of these representatives of Mammalia and
Amphibia may have similar functional characteristics.
There are similarities in the distribution of 5-HT-synthesizing enzymes
associated with the mammalian DMN and nonmammalian PVO. There are laterally
displaced tryptophan hydroxylase-immunoreactive neurons within the rostral DMN
(Weissmann et al., 1987), an observation which is consistent with the ability of
hypothalamic cell cultures from 16-day-old rat fetuses to synthesize 5-HT from
tryptophan (Becquet et al., 1990), and the observation that surgical isolation of the
medial basal hypothalamus in adult rats does not eliminate either tryptophan
hydroxylase or 5-HT content of the DMN (Brownstein et al., 1976). This
arrangement in the rat with presumed 5-HT-accumulating neurons located medially
within the DMN and tryptophan hydroxylase-immunoreactive neurons located laterally
within the DMN is very similar to the arrangement in the PVO of the chick embryo
(see previous discussion of PVO).
There are similarities in the distribution of catecholamine-synthesizing enzymes
associated with the mammalian DMN and nonmammalian PVO. The CSF-MA
neurons of the nonmammalian PVO are immunonegative for catecholamine-
synthesizing enzymes including tyrosine hydroxylase and DBH, but are clearly
associated with laterally displaced tyrosine hydroxylase-immunoreactive neurons
(PVO-accompanying cells in amphibians; Smeets and Gonzalez, 1990; Gonzalez and
Smeets, 1991, 1993, 1994; Gonzalez et al., 1993a; presumed homologue of the
112
FIGURE V.3. Schematic representation of multiple biogenic amine-metabolizing
systems in the mammalian (above) and urodele amphibian (below) infundibular
hypothalami based on histochemical studies.
Mammalian hypothalamus. Filled circles, dopaminergic cell group of the
arcuate nucleus (Al2) (Dahlstrom and Fuxe, 1964) and the dorsal hypothalamic
dopaminergic cell group (A13) (Fuxe et al., 1969); open circles, serotonin binding
protein-immunoreactive (Kirchgessner et al., 1988), 5-HT-accumulating cells within
the DMN (Fuxe and Ungerstedt, 1968; Chan-Palay, 1977; Beaudet and Descarries,
1979; Frankfurt et al., 1981; Howe et al., 1982; Steinbusch et al., 1982; Frankfurt
and Azmitia, 1983; Steinbusch and Nieuwenhuys, 1983; Sakumoto et al., 1984;
Steinbusch, 1984; Ueda et al., 1984b; Staines et al., 1986; Ugrumov et al., 1986,
1988, 1989; Denoyer et al., 1989). These cells are associated with catecholamineaccumulating cells (Lidbrink et al., 1974; Karasawa et al., 1994). These 5-HTand/or catecholamine-accumulating neurons in the DMN are associated with and may
correspond to tyrosine hydroxylase-immunonegative, aromatic L-amino acid
decarboxylase- (AADC)-immunoreactive neurons in the periventricular part of the
DMN (Group D12) described by Jaeger and colleagues (1984); filled circles with
ventricular contact, bipolar subependymal catecholamine-containing cells (Sladek,
1978; Knigge et al., 1980); asterisks, phenylethanolamine N-methyltransferase
(PNMT)-immunoreactive perikarya within the lateral and perifornical nuclei, zona
incerta, and dorsal and medial hypothalamus (Ruggiero et al., 1985; PNMTimmunoreactive perikarya in the caudal arcuate nucleus are not illustrated, Foster et
al., 1985). Within the infundibular hypothalamus, caudal to the level illustrated, are
parvocellular 5-HT-accumulating neurons which do not follow classical
neuroanatomical boundaries but are, instead, scattered around the mammillary recess
in the dorsocaudal extreme of the arcuate nucleus, extending into the region around
the premammillary nuclei (Chan-Palay, 1977; Maeda et al., 1984; Sakumoto et al.,
1984; tuberal and ventral subdivisions of the tuberomammillary nucleus according to
Staines et al., 1986), which may correspond to parvocellular AADC-immunoreactive
neurons (Group D9) described in this region by Jaeger and colleagues (1984). These
5-HT-accumulating neurons are adjacent to a group of medium-sized catecholamineaccumulating neurons within the ventral premammillary nucleus, extending dorsally
into the dorsal premammillary nucleus (Odake, 1967; Bjorklund et al., 1968; Fuxe
and Ungerstedt, 1968, Loizou, 1971; Hate lt and Fuxe, 1972; Lidbrink et al., 1974),
which may correspond to medium-sized AADC-immunoreactive neurons (Group D8)
described in this region by Jaeger and colleagues (1984; see also Karasawa et al.,
1994). Also at a level caudal to that illustrated, norepinephrine-containing cell bodies
are intermingled with dopamine-containing cell bodies in the caudal dopaminergic cell
group (All; Dahlstrom and Fuxe, 1964; Bjorklund and Nobin, 1973). Topography
of neuroanatomical structures is based on the atlas by Paxinos and Watson (1986).
Urodele amphibian hypothalamus. Filled circles, tyrosine hydroxylaseimmunoreactive cells commonly referred to as the PVO-accompanying cells which do
not appear to contact the CSF, and tyrosine hydroxylase- immunoreactive cells
frequently of the CSF-contacting type (Franzoni et al., 1986; Gonzalez and Smeets,
113
1991; Corio et al., 1992; Gonzalez and Smeets, 1994), associated with the NID (a
structure which is in turn closely associated with the primordial mammillary region,
Herrick, 1948; or more specifically with the primordial tuberomammillary nucleus,
Airaksinen and Panula, 1990); open circles with ventricular contacts, bipolar 5-HTcontaining CSF-contacting cells associated with the PVO and NID (Dube and Parent,
1982; Faso lo et al., 1986; Corio and Doerr-Schott, 1988; Corio et al., 1992); filled
circles with ventricular contacts, bipolar catecholamine-containing CSF-contacting
cells within the PVO and NID (Sims, 1977; Dube and Parent, 1982; Corio and
Doerr-Schott, 1988; Lamas et al., 1988; Gonzalez and Smeets, 1991, 1994; Corio et
al., 1992).
**
*
FIGURE V.3
\
k
\
*
\
*
*
** * *
/
1
114
115
mammalian A13 group in the caudal medial hypothalamus of C. crocodilus, Brauth,
1988). Likewise, the monoamine-accumulating perikarya within the DMN of the rat
are associated with laterally displaced tyrosine hydroxylase-immunoreactive neurons
(A13, A131; A141; flokfelt et al., 1984a,b).
Also described in the DMN of the rat, within the periventricular aspect of the
nucleus, are a population of AADC-immunoreactive neurons which are not
immunoreactive for tyrosine hydroxylase (D12 Group; Jaeger et al., 1984). These
cells may correspond to the catecholamine- or 5-HT-accumulating cells in the same
region (see discussion by Hokfelt et al., 1984a). This is an attractive hypothesis since
a single or multiple AADC enzyme(s) catalyze the conversion of L-DOPA to
dopamine and 5-HTP to 5-HT in mammalian tissues (HOkfelt et al., 1973b; Sourkes,
1987). In support of this hypothesis studies of cultured embryonic mouse
hypothalamus indicate that a population of cells which is tyrosine hydroxylase- and
tryptophan hydroxylase-immunonegative has capabilities for 5-HTP-uptake and
decarboxylation as well as 3H-5-HT accumulation (De Vitry et al., 1986). The
presence of AADC-immunoreactive neuronal cell bodies in the mammalian DMN is
consistent with the arrangement in the nonmammalian PVO. The CSF-MA neurons
of the PVO of the bullfrog, R. catesbeiana, and the posterior PVO of a teleost fish,
C. auratus, are immunonegative for tyrosine hydroxylase (Carr et al., 1991; Beltramo
et al., 1994), but contain immunoreactivities for AADC (Karii et al., 1987; Beltramo
et al., 1994).
116
In accordance with a role for these hypothalamic structures in the metabolism
of biogenic amines, both are characterized by the presence of monoamine oxidase
(MAO), an enzyme with a wide substrate selectivity which converts catecholamines to
their corresponding aldehydes, or 5-HT to the metabolite 5-HIAA (reviewed by Berry
et al., 1994). In MAO enzyme histochemical preparations, the intraventricular
projections of PVO neurons in X. laevis show a strong MAO activity, whereas the
neuronal cell bodies show a more moderate MAO activity (Terlou and Stroband,
1973). This is paralleled by the description of small MAO-B-positive neuronal cell
bodies within the DMN in studies of the cat using a sensitive enzyme histochemical
method (Kitahama et al., 1989).
Elegant studies have described the projections of the PVO in the dipnoan fish
(Protopterus dolloi; von Bartheld and Meyer, 1990), and the DMN of the rat (ter
Horst and Luiten, 1986). These studies of diverse species of vertebrates indicate that
there are impressive similarities in intrahypothalamic, ascending and descending
projections of the PVO and the DMN. In these studies intrahypothalamic projections
were described for the PVO and DMN to the median eminence, lateral regions of the
infundibular hypothalamus, as well as ventral and lateral aspects of the anterior
hypothalamic-preoptic area. Ascending projections were evidenced by terminal
labeling in the habenular nuclei and adjacent thalamic structures, as well as in
elements of the basal forebrain, including septal structures. Descending pathways
were observed extending caudally along the ventral brainstem, innervating multiple
mesencephalic, isthmic, and rhombencephalic regions. These included the ventral
117
mesencephalic tegmentum, the isthmic region (locus coeruleus and subcoeruleus
regions in the rat), the cerebellum, multiple raphe structures at mesencephalic and
lower brainstem levels, and portions of the reticular formation throughout the
rhombencephalon. Thus, the generalized projections of the PVO in P. dolloi, a
dipnoan fish, and the DMN in the rat are remarkably similar. Equally thorough
descriptions of the projections of the PVO in representatives of intermediate classes of
vertebrates are necessary to adequately evaluate the extent to which the
nonmammalian PVO and the mammalian DMN innervate homologous regions of the
central nervous system.
Based on connectivity as well as topographical, immunohistochemical,
biochemical, and functional characteristics of the nonmammalian PVO and the
mammalian DMN, we propose that these structures may be homologous, and that
they represent important components of an evolutionarily conserved complex of
amine-metabolizing structures in the infundibular hypothalamus.
Metabolites
The regions of the brain with the highest concentrations of monoamines, (DA)
and (5-HT), had the lowest ratios of metabolites, [DOPAC] and [5-HIAA], to
monoamines (Fig.V.2). This pattern has been described previously in mammals
(Gaudin-Chazal et al., 1979; Mefford et al., 1982). The lowest [DOPAC] /[DA]
ratios and the lowest [5-HIAA]/[5-HT] ratios were in regions of the brain with
monoamine-containing neuronal cell bodies, for example in the raphe region and the
region of the PVO for the serotonergic system. It is likely that these regions had low
118
ratios of metabolites to monoamines as a result of the relatively high synthesis,
storage and transport properties of these neurons, compared to the levels of synaptic
interactions.
The ratios of metabolites to monoamines in this species were approximately an
order of magnitude lower than those described in studies of mammals (cf. Gaudin-
Chazal et al., 1979; Mefford et al., 1982). This suggests that some aspects of
synthesis, storage, transport, and utilization of monoamine neurotransmitters in this
amphibian are different than in mammalian systems. Although few studies have made
direct comparisons of these processes in mammalian and amphibian systems, several
studies suggest that there are fundamental differences. For example, injections of
either reserpine, a catecholamine depleter, or a-methyl-p-tyrosine, an effective
catecholamine synthesis inhibitor in mammals, have little or no effect on total brain
concentrations of monoamines in the anuran amphibian R. pipiens using biochemical
(Baumgarten, 1972) or histofluorescence techniques (Parent, 1975). These
observations are supported by the inability of a-methyl-p-tyrosine (1.2, 9, or 18 mg;
intraperitoneally), in animals killed two and four h after treatment, to alter
concentrations of catecholamines in several microdissected brain regions of T.
granulosa (unpublished observations). As previously suggested (Baumgarten, 1972;
Parent, 1975) it may be that due to the slower metabolic rates of ectotherms, turnover
rates of monoaminergic systems are much slower and turnover cannot be quantified
when using sampling protocols developed for mammalian systems. Consequently,
119
chronic injections of pharmacological agents may be more efficient for manipulations
of monoamine neuronal systems in some amphibians.
Conclusions
Quantitative measurements of norepinephrine, epinephrine, dopamine, and 5-
HT in the central nervous system of T. granulosa revealed many similarities between
this urodele and other vertebrates, although the sources of these neurotransmitters
(single or multiple sources in hypothalamic or brainstem regions) have not been
determined. In addition, the highest concentrations of monoamines, with the
exception of 5-HT in the raphe region, were observed in the regions of the
hypothalamus that contain the CSF-contacting cells of the PRO, PVO, and NID. Of
particular interest is the observation that epinephrine concentrations were high in these
regions relative to other regions of the central nervous system, raising the possibility
that these regions may be important sites for the synthesis, storage, transport, or
utilization of epinephrine. In addition, regions which include CSF-MA neuronal
systems may be focal regions for metabolism of multiple biogenic amines. As such,
they may have important neuroendocrine, physiological or behavioral functions. An
understanding of the characteristics of these conserved systems in diverse vertebrate
classes may help distinguish functional aspects of hypothalamic aminergic systems
from technical artifacts, and lead to a more thorough understanding of catecholamine
and indoleamine systems in the vertebrate central nervous system.
120
ACKNOWLEDGMENTS
This work was supported in part by NIMH predoctoral fellowship to CAL
(BNR-1F31MH10288) and research grants to KJR (USPHS MH-44893) and FLM
(IBN 93-19633). We graciously thank L.M. Laughlin for excellent technical
assistance. We also thank William L. Henckler, Merck Sharp and Dohme Research
Laboratories, for the generous gift of a-methyldopamine.
ABBREVIATIONS
AADC, aromatic L-amino acid decarboxylase; CSF-MA, cerebrospinal fluidcontacting, monoamine-containing; DBH, dopamine B-hydroxylase; DOPAC, 3,4-
dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; HPLC-ED, high
performance liquid chromatography with electrochemical detection; 5-HT, serotonin,
5-hydroxytryptamine; 5-HTP, 5-hydroxytryptophan; L-DOPA, L-3,4dihydroxyphenylalanine; PNMT, phenylethanolamine N-methyltransferase
ABBREVIATIONS USED IN FIGURES
AM
amygdala
APL
amygdala pars lateralis
APM
amygdala pars medialis
APOA
anterior preoptic area (microdissected region that included the PRO)
Arc
arcuate hypothalamic nucleus
DA
dorsal hypothalamic area
DMN
dorsomedial hypothalamic nucleus
121
DP
dorsal pallium
DT
dorsal thalamus
EPL
external plexiform layer
f
fornix
GL
glomerular layer
H
habenula
is
internal capsule
IGL
internal glomerular layer
IPN
interpeduncular nucleus
LH
lateral hypothalamic area
LP
lateral pallium
ME
median eminence
mfb
medial forebrain bundle
ML
mitral layer
MP
medial pallium
mt
mammillothalamic tract
NA
nucleus accumbens
NID
nucleus infundibularis dorsalis
opt
optic tract
Pe
periventricular hypothalamic nucleus
PMV
premammillary nucleus, ventral
PVO
paraventricular organ
122
PRO
preoptic recess organ
S
septum
ST
striatum
T
tectum
TC
tuber cinereum area
TEG
tegmentum (dorsal and ventral regions)
TI
isthmic tegmentum
TM
dorsal tegmentum mesencephali
V
3rd ventricle
VH
ventral hypothalamus
VMH
ventromedial hypothalamic nucleus
VT
ventral thalamus
ZI
zona incerta
123
CHAPTER VI: ELEVATION OF SEROTONIN, DOPAMINE, AND SELECTED
METABOLITE CONCENTRATIONS IN THE DORSOMEDIAL
HYPOTHALAMUS FOLLOWING CORTICOTROPIN-RELEASING FACTOR
OR CORTICOSTERONE ADMINISTRATION
Christopher A. Lowry', Miles Orchinik2, Kathy A. Burke, Kenneth J. Renner3'4, and
Frank L. Moore'
'Department of Zoology, Oregon State University, Corvallis, OR 97331-2914
2Rockefeller University, New York, NY 10021-6399
3Biomedical Sciences Department, Southwest Missouri State University, Springfield,
MO 65804-0094
'Current address, Department of Biology, University of South Dakota, Vermillion,
SD 57069-2390
124
ABSTRACT
The male roughskin newt (Taricha granulosa) was used to investigate -- using
microdissection and high performance liquid chromatography with electrochemical
detection techniques
the effects of corticotropin-releasing factor (CRF) or
corticosterone administration on tissue concentrations of norepinephrine, epinephrine,
dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin, and 5-
hydroxyindoleacetic acid (5-HIAA). In experiments I and II we injected control
solutions or CRF (25 or 50 ng), measured locomotor activity 34 min after treatment,
then measured monoamine and monoamine metabolite levels in microdissected brain
areas. Compared to control animals CRF-injected animals were hyperactive and had
altered levels of monoamines or monoamine metabolites in two of 12 brain areas, the
ventral striatum and the dorsomedial hypothalamus. In the ventral striatum
concentrations of the serotonin metabolite 5-HIAA were lower in CRF- treated animals
than in control animals. In the dorsomedial hypothalamus, a region that contains
dopamine- and serotonin-accumulating neuronal cell bodies, dopamine and serotonin
concentrations were higher in CRF-treated animals than in control animals. In
experiment III male newts were treated as follows: 1) no injection, no handling, 2)
saline injection, or 3) 10 iLg corticosterone (20 min). Monoamine and monoamine
metabolite concentrations were similar in the unhandled and saline-injected controls.
In contrast, corticosterone-injected newts had elevated concentrations of serotonin, 5HIAA, and the dopamine metabolite DOPAC in the dorsomedial hypothalamus (but
not in seven other brain areas studied). Together these observations suggest that CRF
125
and corticosterone act at the level of the dorsomedial hypothalamus to regulate
serotonergic and dopaminergic systems.
INTRODUCTION
In addition to the well-established hypophysiotropic role for corticotropin-
releasing factor (CRF; Vale et al., 1981; Rivier et al., 1990), there is evidence that
this 41-amino acid neuropeptide acts within the central nervous system as a
neurotransmitter or neuromodulator. For example, there is evidence that (1) CRFimmunoreactive neuronal cell bodies and fibers, receptor proteins, and mRNA are
widely distributed throughout the central nervous system (Swanson et al., 1983; De
Souza et al., 1984, 1985; Merchenthaler, 1984; Skofitsch et al., 1985; Wong et al.,
1994), (2) CRF is localized within nerve terminals (Cain et al., 1991), (3)
depolarization of neuronal cell bodies containing CRF results in calcium-dependent
CRF release (Smith et al., 1986; Clarke et al., 1987; Widmaier et al., 1989;
Hillhouse and Reich lin, 1990; Hu et al., 1992; Cratty and Birk le, 1994; Gabr et al.,
1994), and (4) iontophoretic or intracerebroventricular (i.c.v.) application of CRF
alters the spontaneous firing rates of extrahypothalamic neurons in rats (Valentino et
al., 1983; Siggins et al., 1985; Siggins, 1990; see also Valentino and Wehby, 1988;
Valentino et al., 1991) and the urodele amphibian Taricha granulosa (Lowry et al.,
1994).
In addition to the evidence that CRF may act as a neurotransmitter or
neuromodulator, recent studies suggest that the glucocorticoid steroid hormone
corticosterone can rapidly modulate neuronal excitability within the central nervous
126
system. For example, iontophoretically applied corticosterone or cortisol rapidly
(within 1-10 sec) alters spontaneous and glutamate-induced increases in firing of
electrophysiologically identified units in the hypothalamic paraventricular nucleus
(Saphier and Feldman, 1988) as well as of single units within the medial tuberal
hypothalamus of the rat (Mandelbrod et al., 1974). Rapid electrophysiological effects
of corticosterone also have been described in studies of T. granulosa (Rose et al.,
1993). These rapid electrophysiological effects of corticosterone in the rat and in T.
granulosa may be mediated by specific binding sites for glucocorticoids in neuronal
membranes (Towle and Sze, 1983; Orchinik et al., 1991; 1992; Sze and Towle, 1993;
Orchinik and Mc Ewen, 1995).
Together these studies indicate that CRF and corticosterone can rapidly alter
neuronal activity in diverse vertebrate species. Among the many central actions of
CRF and corticosterone is the modulation of monoaminergic metabolism.
Microdialysis as well as studies using microdissection with high performance liquid
chromatography and electrochemical detection (HPLC-ED) techniques indicate that
both CRF and corticosterone can alter elements of the synthesis, storage, transport, or
utilization of catecholamines and indoleamines in vertebrates (Dunn and Berridge,
1987; Kalivas et al., 1987; Losada, 1988; Barrett et al., 1989; Matsuzaki et al.,
1989; Shimizu and Bray, 1989; Emoto et al., 1993; Lavicky and Dunn, 1993; Lowry
et al., 1993; Lee et al., 1994). Consistent with these observations CRF can stimulate
the activities of tyrosine hydroxylase (Olianas and Onali, 1989) and tryptophan
127
hydroxylase (Corley et al., 1990; Singh et al., 1992), the rate-limiting enzymes for
synthesis of dopamine and serotonin (5-hydroxytryptamine, 5-HT), respectively.
Although in T. granulosa CRF and corticosterone can act within minutes to
alter behavior (Moore and Miller, 1984; Moore et al., 1984, 1995; Boyd and Moore,
1990; Lowry et al., 1990, 1991, 1993, 1994; Orchinik et al., 1991), it is currently
unknown where in the central nervous system these behaviorally active
neurochemicals act. It is also unclear which neurotransmitter systems may be
involved in mediating the effects of CRF and corticosterone on behavior. As an
initial step in addressing these questions we have investigated, using T. granulosa, the
effects of i.c.v. CRF and intraperitoneal corticosterone administration on tissue
concentrations of norepinephrine, epinephrine, DOPAC, dopamine, 5-HIAA, and 5-
HT in microdissected brain regions. Portions of these data have been presented in
preliminary form (Lowry et al., 1991; Moore et al., 1995).
MATERIALS AND METHODS
Adult male roughskin newts (Taricha granulosa) were collected locally from
freshwater ponds on separate occasions for each of three experiments. Animals were
maintained in the laboratory for approximately 48 h in holding tanks containing
dechlorinated tap water. These tanks were in a room with controlled photoperiod (14
h light:10 h dark; lights on at 0630 h) and an ambient temperature of 19°C
(Experiments I and II), or in a room with natural photoperiod and an ambient
temperature of 23°C (Experiment III). Newts were not handled or disturbed until
immediately prior to treatment.
128
In Experiments I and II animals received i.c.v. injections of either amphibian
Ringer's solution or vehicle containing synthetic ovine corticotropin-releasing factor
(CRF; generous gift from Drs. Wylie Vale and Jean Rivier, Salk Institute for
Biological Studies, San Diego, CA; Experiment I, 25 ng; Experiment II, 50 ng;
n =10). Intracerebroventricular injections were given through a 0.5 mm hole at the
junction of the parietal and frontal bones in the cranial midline using a microsyringe
with a tip diameter of 50 ,um as described in Moore and Miller (1983). Solutions (2
Al) were infused over a period of 5 seconds. Following i.c.v. injection newts were
isolated in temporary circular holding tanks, (25 cm in diameter) containing 5 L of
dechlorinated tap water, until behavioral testing. For behavioral testing newts were
placed individually in a dechlorinated water-filled (10 cm depth) circular runway with
an inside diameter of 70 cm and an outside diameter of 85 cm; each testing arena was
marked with radial lines to define 16 equal sectors. Newts were placed in the testing
arena 30 minutes after treatment. Starting 4 minutes after placement, locomotor
activity was quantified by counting the total number of lines crossed during 3
consecutive minutes. Locomotion consisted of a combination of walking and
swimming movements. The Mann-Whitney U-test was used to test for statistical
differences in locomotor activity between treatment groups (Siegel, 1956). Two-tailed
probabilities P < 0.05 were considered statistically significant.
In Experiment III male newts were treated as follows: 1) no injection, no
handling, 2) saline injection, or 3) 10 Ag corticosterone (Sigma Chemical Company;
intraperitoneal injection, 0.1 ml volume; n=12). Following treatment, newts were
129
isolated in temporary circular holding tanks as described above, but were not tested
for locomotor activity; previous studies in T. granulosa indicate that intraperitoneal
injection of corticosterone does not alter locomotor activity (Moore, Roberts, and
Bevers, 1984).
Immediately after behavioral testing (Experiments I and II), or 20 min after
treatment (Experiment III), newts were killed by rapid decapitation. Each animal was
rapidly dissected (1-2 min) to remove the intact brain and rostral spinal cord. These
were then embedded in Tissue-TekR O.C.T. Compound, frozen on dry ice, and stored
in airtight microcentrifuge tubes at -80 °C until sectioning. Serial 300 Am sections of
frozen brain were cut at -12 °C in a cryostat. Sections were placed on gelatin-coated
glass slides and collectively thaw-mounted by briefly warming the slide. Sections
were refrozen within approximately 30 sec. Slides were sealed in slide boxes and
stored at -80 °C.
Microdissection of individual brain regions was accomplished using the
methods described for the microdissection of mammalian brain tissues (Palkovits,
1973; Palkovits and Brownstein, 1982) as previously applied in T. granulosa (Zoeller
and Moore, 1986; Lowry et al., 1995). Slides representing an entire brain were
placed on a cold stage (Thermoelectric Cold Plate, TCP-2, Thermoelectrics
Unlimited, Inc.) and maintained at -10 °C. Tissue punches (300 Am i.d.) were
expelled into 60 Al of acetate buffer (pH 5) containing internal standard, 0.5 x 10' M
a-methyldopamine (Experiment I and II), or 3.2 x 10' M 3,4-dihydroxybenzylamine
hydrobromide (Experiment III), and then stored at -80 °C until analyzed for
130
monoamine content. A microdissection atlas described previously for T. granulosa
(Lowry et al., 1995) was used. Brain regions were identified based on descriptions of
the urodele amphibian telencephalon (Herrick, 1927; Northcutt and Kicliter, 1980),
diencephalon and midbrain (Herrick, 1917), and hindbrain (Herrick, 1930), as well as
the comprehensive study by Herrick (1948).
Tissue concentrations of norepinephrine, epinephrine, DOPAC, dopamine, 5-
HIAA, and 5-HT were analyzed using HPLC-ED. The method of analysis was based
on a previously described procedure (Renner and Luine, 1984; Renner and Luine,
1986) with several modifications (Lowry et al., 1995). The tissue samples were
thawed and centrifuged at 15,000 x g for 2 min. The supernatant was treated with 2
Al of 0.02% ascorbate oxidase (Boehringer Mannheim) to minimize ascorbic acid
contributions to the solvent front (McKay et al., 1984) and directly injected into a
Waters chromatographic system (Waters Associates, Inc.).
Chromatographic
separation was accomplished using a C-18, 4µm radial compression cartridge and a
mobile phase consisting of (wt /vol), 0.84% sodium acetate, 1.2% citric acid, 0.015%
octanesulfonic acid sodium salt (Eastman Kodak), 0.02% disodium EDTA, in
(vol /vol) 15% methanol in water. Electrochemical detection was provided by a
laboratory built potentiostat and a glassy carbon electrode (Bioanalytical Systems) set
at +0.7 V with respect to an Ag/AgC1 reference electrode. The tissue pellet was
dissolved in 0.3 N NaOH and analyzed for protein content according to the method of
Bradford (1976).
131
The pg/cm peak heights of known concentrations of the standards were
determined from the mean peak heights of 3 chromatograms for each respective
standard. The internal standard was injected 3 times to determine the peak height for
100 % sample recovery. Amine concentrations were calculated from the standard
values and corrected for % recovery and injection volume using a Waters 730 Data
Module. The amine concentrations were divided by jig protein to yield pg amine /µg
protein. Outlier values identified using the Grubbs method were not included in
reported values (two-tailed probability, P < 0.05; Grubbs and Beck, 1972). In
Experiments I and II, Student's t-test was used to test for differences between
treatment groups in tissue concentrations of monoamines, monoamine metabolites, or
ratios of monoamines to their respective metabolites. In Experiment III, differences
among treatment groups were analysed using one-way analysis of variance; multiple
comparisons to the same control (saline versus unhandled, uninjected; saline versus
corticosterone) were made when appropriate using Dunnett's Test. Two-tailed
probabilities P < 0.05 were considered statistically significant.
RESULTS
Corticotropin-releasing factor (Figs.VI.1, VI.2) and corticosterone (Fig.VI.3)
administration resulted in site-specific changes in tissue concentrations of DOPAC,
dopamine, 5-HIAA, and 5-HT. With a single exception the CRF- and corticosteroneinduced changes in monoamine and monoamine metabolite concentrations were
restricted to the dorsomedial hypothalamus (DMH). In contrast to the differences
132
observed in the tissue concentrations of DOPAC, dopamine, 5-HIAA, and 5-HT there
were no differences in tissue concentrations of either norepinephrine or epinephrine.
TABLE VIA. Locomotor activity in response to an i.c.v. injection of saline or CRF
Experiment
Mean locomotor activity
(crossings/3 min)
Control injection
CRF
Experiment I
(25 ng CRF)
9.0 + 3.3
23.5
Experiment II
(50 ng CRF)
10.7
± 3.2
37.5
±6.0*
± 4.6*
Values represent mean locomotor activity ± S.E.M (n=10). *Significantly different
from saline-injected control, two-tailed probability < 0.05 (Mann-Whitney U-test).
following treatment with either CRF or corticosterone in any brain region (Fig.VI.4).
In Experiments I and II i.c.v. injection of CRF stimulated locomotor activity
compared to saline-injected controls (Table VI.1). Concurrently CRF had sitespecific effects on tissue concentrations of monoamines or monoamine metabolites that
were dependent on the dosage of CRF given. Injection of the lower dose of CRF (25
ng) did not alter 5-HT, 5-HIAA, or DOPAC concentrations in any of the brain
regions studied, however this dose resulted in elevated levels of dopamine in the
dorsomedial hypothalamus when compared to saline-injected controls (Fig.VI.1).
There was a corresponding decrease in the ratio of DOPAC/DA in this region (saline,
133
200
300
275
I
250
I
175
Saline
CRF (25 ng)
225
150
200
125
175
100
150
a.
125
75
100
I
75
50
50
25
25
0
0
MP VS
S
TH DMH LH DTM 1ST
R
TT
MP VS
S
R
TT
BRAIN REGION
BRAIN REGION
20
20
I
15
TH DMH LH DTM 1ST
I
Saline
CRF (25 ng )
I
15
I
Saline
111. CRF (25 ng)
10
10
5
0
MP VS
S
TH DMH LH DIM 1ST
BRAIN REGION
R
TT
MP
VS
S
TH DMH LH DTM !ST
R
TT
BRAIN REGION
FIGURE VI.1. Effects of CRF (25 ng) on monoamine and monoamine metabolite
levels in microdissected brain regions measured approximately 35 min following
treatment. Effects of i.c.v. injection of saline or CRF (25 ng) on 5-HT, 5hydroxyindoleacetic acid (5-HIAA), dopamine (DA), and 3-4-dihydroxyphenylacetic
acid (DOPAC) concentrations in microdissected brain regions of adult male T.
granulosa measured approximately 35 min following treatment. Injection of CRF
resulted in elevated concentrations of dopamine in the dorsomedial hypothalamus
compared to saline-injected controls. Abbreviations: DMH, dorsomedial
hypothalamus; DTM, dorsal tegmentum mesencephali; IST, isthmic tegmentum; LH,
dorsolateral hypothalamus (region containing the nucleus infundibularis dorsalis); MP,
medial pallium; R, raphe region; S, septum; TH, thalamus; TT, trigeminal
tegmentum; VS, ventral striatum.
134
0.29 ± 0.06; CRF, 0.13 ± 0.01; t=3.47, 11 df, P < 0.01). The higher dose of CRF
(50 ng) did not alter dopamine or DOPAC concentrations in any of the brain regions
studied, however this dose resulted in elevated levels of 5-HT in the dorsomedial
hypothalamus and lower levels of 5-HIAA in the ventral striatum (Fig.VI.2). There
was a corresponding decrease in the ratio of 5-HIAA/5-HT in the ventral striatum in
CRF-injected newts compared to saline-injected controls (saline, 0.13 ± 0.01; CRF,
0.08 ± 0.01; t=3.15, 16 df, P<0.01).
In Experiment III, there were differences in tissue concentrations of
monoamines and monoamine metabolites among treatment groups, but only in the
dorsomedial hypothalamus. Specifically, there were differences among the treatment
groups in tissue concentrations of 5-HT (F2,31=4.85, PG0.05), 5-HIAA (F2,30=7.017,
P<0.01), dopamine (F2,31=3.47, P < 0.05), and DOPAC (F2,31=3.34, P < 0.05) in
this region. There was no effect of handling and saline injection on tissue
concentrations of any of the monoamines or monoamine metabolites in any of the
eight brain regions studied compared to unhandled controls. There were, however,
significant increases in the tissue concentrations of 5-HT, 5-HIAA, and DOPAC in
the dorsomedial hypothalamus 20 min following corticosterone administration
compared to saline-injected controls (Fig.VI.3).
135
200
300
275
Saline
I
250
175
1111 CRF (50 ng)
225
200
125
175
100
150
125
75
1,10.05
100
P.0.10
50
75
50
MP VS
S
25
ri 1 I
25
0
Saline
NM CRF (50 ng)
150
0
TH DMH LH DTM 1ST
R
TT
MP VS
S
R
TT
BRAIN REGION
BRAIN REGION
20
20
I
I
Saline
I
CRF (50 ng)
15
15
10
10
5
5
0
TH DMH LH DIM 1ST
1
MP VS
S
TH DMH
BRAIN REGION
1ST
R
7
0
I
So line
CRF (50 ng)
ill
e i
111
TH DMH LH DTM 1ST
R
I
TT
BRAIN REGION
FIGURE VI.2. Effects of CRF (50 ng) on monoamine and monoamine metabolite
levels in microdissected brain regions measured approximately 35 min following
treatment. Effects of i.c.v. injection of saline or CRF (50 ng) on 5-HT, 5-HIAA,
dopamine, and DOPAC concentrations in microdissected brain regions of adult male
T. granulosa measured approximately 35 min following treatment. Injection of CRF
resulted in elevated concentrations of 5-HT in the dorsomedial hypothalamus and
lower levels of 5-HIAA in the ventral striatum compared to saline-injected controls.
See Fig.VI.1 for abbreviations.
136
FIGURE VI.3. Effects of corticosterone on monoamine and monoamine metabolite
levels in microdissected brain regions measured 20 min following treatment. A.
Effects of intraperitoneal injection of saline or corticosterone (10 AO on 5-HT and 5hydroxyindoleacetic acid (5-HIAA) concentrations in microdissected brain regions of
adult male T. granulosa measured 20 min following treatment. Although the handling
and injection of saline did not alter 5-HT or 5-HIAA concentrations in any of the
brain regions compared to unhandled controls, injection of corticosterone resulted in
elevated concentrations of both 5-HT and 5-HIAA in the dorsomedial hypothalamus
compared to saline-injected controls.
B. Effects of intraperitoneal injection of saline or corticosterone (10 p.g) on dopamine
and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations in microdissected brain
regions of adult male T. granulosa measured 20 min following treatment. As
observed for 5-HT and 5-HIAA, the handling and injection of saline did not alter
dopamine or DOPAC concentrations in any of the brain regions compared to
unhandled controls. Injection of corticosterone resulted in elevated concentrations of
DOPAC in the dorsomedial hypothalamus compared to saline-injected controls.
Although a one-way analysis of variance indicated significant differences among
treatment groups for the concentration of dopamine in the dorsomedial hypothalamus,
the comparison between saline injected controls and corticosterone-injected animals
was not significant (P = 0.07). Abbreviations: APM, amygdala pars medialis;
DMH, dorsomedial hypothalamus; LH, dorsolateral hypothalamus (region containing
the nucleus infundibularis dorsalis); NA, nucleus accumbens; NTS, nucleus of the
solitary tract; POA, preoptic area; R, raphe region; VS, ventral striatum.
137
FIGURE VI.3
300
300
275
250
I
1
LLI
225
275
Uninjected
Saline
Corticosterone
Uninjected
Saline
Corticosterone
225
200
0.05
200
175
175
150
150
125
125
13
100
100
75
75
50
50
A
25
0
j
250
VS
NA
APM POA DMH
25
0
LH
R
VS
NTS
NA
20
Uninjected
= Saline
( /1
I
Corticosterone
APM POA DMH
BRAIN REGION
LH
R
NTS
LH
R
NTS
BRAIN REGION
BRAIN REGION
NA
ARM BOA DMH
15
LH
R
NTS
I
VS
Uninjected
Saline
Corticosterone
I ii 111
NA
APM POA DMH
R RAIN REGION
138
raises the possibility that corticosterone may have similar effects on serotonin
metabolism in the mediobasal hypothalamus of nonmammalian and mammalian
vertebrates.
Neurochemical changes within the dorsomedial hypothalamic nucleus following
exposure to stressful stimuli
The effects of CRF (elevation of dopamine and 5-HT) on monoamine
concentrations in the dorsomedial hypothalamus of T. granulosa are strikingly similar
to the effects of exposure of rats to certain types of stressful stimuli. For example,
exposure of rats to electric foot shock results in a three- to four-fold increase in the
tissue concentration of 5-HT within the DMN (but not the adjacent lateral
hypothalamus) 24 h following exposure to electric foot shock (Shekhar et al., 1994).
Since this study used microdissection and HPLC-ED techniques as in the present
study, it is uncertain where within the DMN the accumulation of 5-HT is taking
place. For example, the elevation of 5-HT may be localized within neuronal
perikarya which are intrinsic to the DMN or, alternatively, within 5-HT fibers and
terminals arising from serotonergic neurons within the brainstem raphe.
In addition, exposure to a psychological stressor, fear-potentiated startle (but
not exposure to elevated plus-maze and social interaction tests) results in increases in
dopamine concentrations within the DMN after 24 h (Katner et al., 1994). This
change in dopamine concentration was evident even though there was no change in
tyrosine hydroxylase activity within the DMN compared to controls. These studies
indicate that accumulation of dopamine and 5-HT within the DMN may be important
139
FIGURE VI.4
150
150
I
125
Saline
CRF (25 ng)
100
100
75
75
50
50
25
25
r-11
0
MP
VS
S
Saline
I
ORE (25 ng)
125
TH DMH LH DTM 1ST
0
TT
R
MP VS
11
S
150
150
125
125
R
Tr
100
100
75
75
50
50
25
25
Saline
MP VS
S
TH DMH LH DTM 1ST
R
0
T1
CRF (50 ng)
r-sm
MP VS
BRAIN REGION
S
:11
LTA
TH DMH LH DTM 1ST
R
TT
BRAIN REGION
200
200
I
171
150
Uninjected
Saline
Corticosterone
175
150
125
125
100
100
75
75
a_
Cr,
1ST
BRAIN REGION
BRAIN REGION
175
1 1 ill 111
TH DMH LH DIM
50
50
25
25
ITI
VS
rri
NA
TI
APM
POA DMH
BRAIN REGION
Am.
LH
R
NTS
VS
NA APM POA DMH
BRAIN REGION
LH
R
NTS
140
DISCUSSION
We have identified site-specific effects of behaviorally active doses of CRF
and corticosterone administration on the tissue concentrations of specific monoamines
and monoamine metabolites in the central nervous system of male T. granulosa.
These effects were restricted to changes in tissue concentrations of DOPAC,
dopamine, 5-HIAA, and 5-HT within the dorsomedial hypothalamus and changes in
tissue concentrations of 5-HIAA in the ventral striatum.
The most remarkable observation in the present study is the site-specificity of
the effects of i.c.v. CRF and intraperitoneal corticosterone administration on tissue
concentrations of monoamines and monoamine metabolites. Depending on the
dosage, CRF administration resulted in either an increase in the concentration of
dopamine or 5-HT within the dorsomedial hypothalamus. Corticosterone
administration resulted in an increase in the concentrations of DOPAC, 5-HIAA, and
5-HT within the dorsomedial hypothalamus compared to vehicle-injected controls.
This is intriguing because the dorsomedial hypothalamus in vertebrates contains
populations of dopamine- and 5-HT-accumulating neuronal cell bodies
.
In
nonmammalian vertebrates these dopamine- and 5-HT-accumulating neuronal cell
bodies are components of a vascularized structure referred to as the paraventricular
organ; in mammalian vertebrates these cell bodies are localized within the
dorsomedial hypothalamic nucleus (DMN; for references, see Lowry et al., 1995).
Based on connectivity as well as topographical, immunohistochemical, biochemical,
and functional characteristics of the nonmammalian PVO and the mammalian DMN, it
141
appears that the monoamine-accumulating neurons in DMN and the neuronal
component of the PVO are homologous structures (Lowry et al., 1995).
The increases in dopamine and 5-HT in the dorsomedial hypothalamus
following i.c.v. CRF administration may reflect an accumulation of these
neurotransmitters by the dopamine- and serotonin-accumulating neuronal cell bodies in
this region of the hypothalamus or, alternatively, by serotonergic fibers and terminals
which arise from neuronal cell bodies within the brainstem raphe (Frankfurt and
Azmitia, 1983). The possibility that stress hormone-induced elevations of 5-HT and
dopamine concentrations in the dorsomedial hypothalamus may be localized within
neuronal cell bodies intrinsic to the dorsomedial hypothalamus is based on
descriptions of populations of dopamine- and 5-HT-accumulating neuronal cell bodies
in this region in mammals (Fuxe and Ungerstedt, 1968; Lidbrink et al., 1974; ChanPa lay, 1977; Beaudet and Descarries, 1979; Frankfurt et al., 1981; Howe et al.,
1982; Steinbusch et al., 1982; Frankfurt and Azmitia, 1983; Steinbusch and
Nieuwenhuys, 1983; Sakumoto et al., 1984; Steinbusch, 1984; Ueda et al., 1984;
Arezki, 1985; Staines et al., 1986; Ugrumov et al., 1986, 1988, 1989; Denoyer et
al., 1989; Karasawa et al., 1994) and amphibians (Peute and van Oordt, 1974; Nakai
et al., 1977). Although dopamine-accumulating neuronal cell bodies have been
described in the dorsomedial hypothalamus of the rat (Lidbrink et al., 1974), the
laboratory shrew, Suncus murinus (Karasawa et al., 1994), and the anuran
amphibians, Rana catesbeiana, Rana nigromaculata, and Xenopus laevis (Peute and
van Oordt, 1974; Nakai et al., 1977), the characteristics of the 5-HT-accumulating
142
neuronal cell bodies in this region have been described in more detail. The term "5HT- accumulating neuronal cell bodies" is used because, although these cells do not
normally contain tryptophan hydroxylase (the rate limiting enzyme for de novo
synthesis of 5 -HT) or 5-HT immunoreactivity, 5-HT immunoreactivity is observed
following systemic pretreatment with a monoamine oxidase inhibitor and 5-HT
precursors, either L-tryptophan or 5-hydroxytryptophan. Alternatively, 5-HT
immunoreactivity can be visualized within these cells following high dosages of
monoamine oxidase inhibitor alone (Ueda et al., 1984). These same neurons contain
immunoreactivity for serotonin binding protein
a protein that is believed to be an
intrinsic and a specific component of serotonergic neurons -- in normal animals
(Kirchgessner et al., 1988; Tamir et al., 1994)
.
Furthermore, systemic treatment of
rats with dopaminergic or noradrenergic agonists can result in an accumulation of 5HT immunoreactivity within DMN neuronal cell bodies (Arezki et al., 1985),
suggesting that 5-HT accumulation within these neurons may be regulated in vivo;
however, there is currently no physiological or behavioral function ascribed to this
unique population of neurons.
Although studies in mammals have not assessed the effects of i.c.v. CRF on
tissue concentrations of monoamines within the DMN, intraperitoneal injection of
corticosterone results in dramatic increases in tissue concentrations of 5-HT and 5HIAA in the mediobasal hypothalamus of the rat, a structure which includes the
DMN, measured 30 min after treatment (Losada, 1988). This observation, together
with the observation in the present study that corticosterone administration results in
143
an elevation of 5-HT and 5-HIAA in the dorsomedial hypothalamus of T. granulosa,
raises the possibility that corticosterone may have similar effects on serotonin
metabolism in the mediobasal hypothalamus of nonmammalian and mammalian
vertebrates.
Neurochemical changes within the dorsomedial hypothalamic nucleus following
exposure to stressful stimuli
The effects of CRF (elevation of dopamine and 5-HT) on monoamine
concentrations in the dorsomedial hypothalamus of T. granulosa are strikingly similar
to the effects of exposure of rats to certain types of stressful stimuli. For example,
exposure of rats to electric foot shock results in a three- to four-fold increase in the
tissue concentration of 5-HT within the DMN (but not the adjacent lateral
hypothalamus) 24 h following exposure to electric foot shock (Shekhar et al., 1994).
Since this study used microdissection and HPLC-ED techniques as in the present
study, it is uncertain where within the DMN the accumulation of 5-HT is taking
place. For example, the elevation of 5-HT may be localized within neuronal
perikarya which are intrinsic to the DMN or, alternatively, within 5-HT fibers and
terminals arising from serotonergic neurons within the the brainstem raphe.
In addition, exposure to a psychological stressor, fear-potentiated startle (but
not exposure to elevated plus-maze and social interaction tests) results in increases in
dopamine concentrations within the DMN after 24 h (Katner et al., 1994). This
change in dopamine concentration was evident even though there was no change in
tyrosine hydroxylase activity within the DMN compared to controls. These studies
144
indicate that accumulation of dopamine and 5-HT within the DMN may be important
components of the response to certain types of stressful stimuli. Although the
neurochemical responses to electric foot shock and fear potentiated startle in rats
appear similar to the neurochemical responses to CRF and corticosterone in the
present study, it is uncertain at this time whether the effects of electric foot shock or
fear potentiated startle on 5-HT and dopamine levels in the DMN involve the actions
of CRF, corticosterone, or other neuroactive chemicals.
Mechanisms for accumulation of dopamine and serotonin in the dorsomedial
hypothalamus
We are unable to conclude if CRF is acting independently from corticosterone
to elevate tissue concentrations of 5-HT and dopamine in the dorsomedial
hypothalamus. Corticotropin-releasing factor may stimulate the accumulation of
dopamine or serotonin in the dorsomedial hypothalamus through its actions on the
hypothalamic-pituitary-adrenal (HPA) axis and the consequent elevation of plasma
corticosterone levels or, alternatively, through direct or indirect actions within the
central nervous system independent from activation of the HPA axis. For example,
the DMN has reciprocal connections with the parvocellular CRF cell body region of
the paraventricular nucleus (Luiten et al., 1985; ter Horst and Luiten, 1986), contains
CRF receptors (Aguilera et al., 1990), and, together with the ventral premammillary
nucleus, is a region of the hypothalamus which is noted to express CRF-binding
protein (Potter et al., 1992); these observations suggest that the DMN may be an
important site for direct actions of CRF. In addition, studies in rats indicate that
145
activation of dopaminergic or B-adrenergic receptors can stimulate the accumulation of
5-HT in identified 5-HT-accumulating neuronal cell bodies within the DMN (Arezki
et al., 1985). Since CRF can activate dopaminergic and noradrenergic systems
(reviewed by Dunn and Berridge, 1990), it is possible that CRF may stimulate the
accumulation of 5-HT within intrinsic neuronal cell bodies within the DMN indirectly
through activation of dopaminergic or noradrenergic systems.
We are also unable to conclude if corticosterone is acting independently from
CRF to elevate tissue concentrations of DOPAC, 5-HIAA, and 5-HT in the
dorsomedial hypothalamus. Corticosterone may stimulate the accumulation of
DOPAC, 5-HIAA, and 5-HT, in the dorsomedial hypothalamus through the
stimulation of select CRF neuronal populations within the central nervous system or,
alternatively, through actions within the central nervous system independent of CRF
activation. For example, since corticosterone can activate some CRF neuronal
populations within the central nervous system (Lee et al., 1994; Makino et al., 1994;
Schulkin et al., 1994), corticosterone may stimulate the accumulation of DOPAC, 5HIAA, or 5-HT in the dorsomedial hypothalamus through its actions on CRF neurons.
Alternatively, corticosterone may act directly within the dorsomedial hypothalamus to
rapidly stimulate the uptake of 5-HT precursors. Based on studies using mice
glucocorticoids may regulate the uptake of tryptophan, the amino acid precursor of 5-
HT (Neckers and Sze, 1975; Towle and Sze, 1983). A single injection of
hydrocortisone acetate elevated the brain levels of tryptophan by 50% within 1 hour
and enhanced the accumulation of 5-HT in whole brain in the presence of a
146
monoamine oxidase inhibitor (Neckers and Sze, 1975). Treatment did not appear to
alter tryptophan hydroxylase or aromatic L-amino acid decarboxylase activity.
Injection of hydrocortisone acetate 60 minutes before sacrifice elevated tryptophan but
not tyrosine (the amino acid precursor for catecholamines) concentrations in
subcellular synaptosomal fractions of brain tissue, indicating that the effect was
selective for accumulation of the precursor of 5-HT. Consistent with these studies, in
vitro synaptosomal preparations, hydrocortisone acetate or corticosterone stimulated
uptake of L4311]-tryptophan by the synaptosomes (Neckers and Sze, 1975; Towle and
Sze, 1983). In contrast, androgen-, estrogen-, and progesterone-like steroids were
without effect. The highest concentration of corticosterone binding sites in the
synaptosomal plasma membrane preparation was found in the hypothalamus,
compared to binding capacities in the cortex and hippocampus (Towle and Sze, 1983),
suggesting that the hypothalamus may be an important site of action for the
corticosterone-induced uptake of 14311]-tryptophan. These results indicate that
glucocorticoids can regulate 5-HT accumulation through direct actions on
synaptosomal elements to stimulate uptake of L-tryptophan. A rapid effect of
corticosterone on the accumulation of L-tryptophan by neuronal structures within the
dorsomedial hypothalamus may account for the corticosterone induced elevation of 5-
HT and 5-HIAA observed in T. granulosa in the present study.
The dorsomedial hypothalamus as a substrate for CRF and stress-mediated effects
The observations that both CRF and corticosterone administration result in
elevation of monoamines or monoamine metabolites in the dorsomedial hypothalamus
147
are consistent with studies in mammals which have identified the dorsomedial
hypothalamus as an important substrate for the central nervous system response to
stress. Studies in rats have demonstrated that the DMN is an important
neuroanatomical site for the integration of physiological and behavioral responses to
stressful stimuli (Soltis and DiMicco, 1991; Inglefield et al., 1994). These studies in
rats are supported by the observations that exposure to a variety of stressors, or to
i.c.v. administration of CRF, results in altered c-fos expression (Pezzone et al., 1992;
Rivest et al., 1992; Imaki et al., 1993), and that exposure to a variety of stressors
also alters neurotransmitter and neuropeptide concentrations within this nucleus
(Siegel et al., 1987; McCarthy et al., 1993; Katner et al., 1994; Shekhar et al.,
1994). Intracerebroventricular administration of corticotropin-releasing factor,
immobilization stress for 60 min, or electric foot-shock stress induces c-fos mRNA
expression within the dorsomedial hypothalamus, as well as in regions of the limbic
system which receive projections from the DMH, including the lateral septal nucleus,
the hippocampus, the anterior corticomedial and medial amygdaloid nuclei, and the
paraventricular and supraoptic nuclei of the hypothalamus, within 30-60 min (Pezzone
et al., 1992; Imaki et al., 1993). C-fos expression was also enhanced in select
diencephalic and brainstem regions innervated by projections from the DMH,
including some thalamic nuclei, the habenula, the pontine nucleus and the locus
coeruleus.
The dorsomedial hypothalamus has particularly strong projections to the locus
coeruleus, as well as projections to the dorsal raphe nucleus, the pontine reticular
148
formation lateral to the medial raphe nucleus, the median raphe nucleus, the pontine
raphe nucleus, the nucleus raphe magnus, raphe obscuris, and raphe pallidus nucleus
(ter Horst and Luiten, 1986; Behzadi et al., 1990). This raises the possibility that
alterations in neuronal activity within the DMH, mediated by CRF or corticosterone,
may affect locus coeruleus noradrenergic and raphe serotonergic function.
The dorsomedial hypothalamus as a neuroanatomical substrate for maintenance of the
homeostatic condition in vertebrates
The present study provides no information about the functional consequences
of CRF or corticosterone-induced elevation of DOPAC, dopamine, 5-HIAA, or 5-HT
within the DMN; however, one of the most well characterized functional roles for the
DMH is the regulation of cardiovascular and behavioral responses to stress. These
studies indicate that endogenous gamma-aminobutyric acid (GABA) suppresses the
activity of a sympatho-excitatory mechanism in the DMH in rats which results in
tachycardic and pressor responses (Anderson and DiMicco, 1990; Soltis and DiMicco,
1991a,b; Stoltz-Potter et al., 1994). Thus, microinjection of drugs that impair
GABA-mediated synaptic inhibition in the DMH of rats generates cardiovascular and
behavioral responses that mimic the response to stress. The cardiovasucular
responses, marked tachycardia and modest elevation of arterial pressure, following
inhibition of GABA function are dependent on activation of local ionotropic and
metabotropic excitatory amino-acid receptors (Soltis and DiMicco, 1991b, 1992;
DiMicco and Monroe, 1994). Associated with the stress-induced tachycardia
responses attributed to the DMH are stress-induced behavioral responses which are
149
dependent on the DMH (Shekhar et al., 1987, 1993; Inglefield et al., 1994; Shekhar
amd Katner, 1995). The elevations of dopamine and 5-HT observed following
exposure to stressful stimuli as well as CRF or corticosterone administration may
have pronounced effects on these autonomic and behavioral processes. Since both
CRF and corticosterone alter the monoaminergic content of the dorsomedial
hypothalamus in T. granulosa, it is unlikely that the effects on monoaminergic
systems are responsible for CRF-induced locomotor activity. However, our
observation that both CRF and corticosterone result in an increase in the tissue
concentration of 5-HT within the DMN does not allow us to determine if CRF and
corticosterone have the same effect on synaptic release of 5-HT in this region; thus
synaptic release of 5-HT and consequent behavioral and physiological responses
mediated within the dorsomedial hypothalamus may be affected differently by CRF
and corticosterone. Regardless, since blockade of GABAA receptor function in the
region of the dorsomedial hypothalamus elicits escape-oriented locomotor activity
(Shekhar and DiMicco, 1987), it remains possible that CRF-induced accumulation of
dopamine or 5-HT may modulate escape-oriented locomotor activity or other
autonomic or behavioral processes.
For example, in a study using 5,7-dihydroxytryptamine (5,7-DHT; a selective
neurotoxin for serotonergic systems), hypothalamic lesions but not lesions of the
raphe region resulted in facilitation of female sexual behavior (Luine et al., 1983).
This suggests that serotonergic neuronal systems within the hypothalamus are critical
for regulation of female sexual behavior. This is consistent with lesion studies in the
150
urodele amphibian which indicate that the monoamine-accumulating neurons of the
dorsomedial hypothalamus are critical for the performance of male sexual behavior
(Dube et al., 1990). The observation in the present study that CRF and
corticosterone
which both have suppressive effects on the performance of sexual
behavior in male T. granulosa (Moore and Miller, 1984)
alter the tissue
concentrations of 5-HT in the dorsomedial hypothalamus is consistent with the
hypothesis that intrahypothalamic serotonergic systems regulate sexual behavior, and
raises the possibility that stress-related neurochemicals may regulate sexual behavior
in part through actions within the dorsomedial hypothalamus.
Associations between CRF-induced locomotor activity and monoaminergic systems in
vertebrates
In the present study i.c.v. CRF administration resulted in increased locomotor
activity compared to saline-injected controls. This demonstrates that the doses of
CRF used were behaviorally active. This behavioral effect of CRF is consistent with
previous studies in rats (Sutton et al., 1982; Veldhuis and De Wied, 1984; Saunders
and Thornhill, 1986; Sherman and KalM, 1987; Diamant and De Wied, 1991; Lee et
al., 1994) and the urodele amphibian, Taricha granulosa (Moore et al., 1984; Lowry
et al., 1990, 1993, 1994; Lowry and Moore, 1991). In rats and T. granulosa, CRFinduced increases in locomotor activity are dose-related, time-dependent (Sutton et al.,
1982; Lowry et al., 1990), and persist in hypophysectomized animals (Moore et al.,
1984; Eaves et al., 1985). In addition, CRF- and stress-induced locomotor activity
are suppressed by the intracerebroventricular administration of a-helical CRF, a
151
competitive CRF antagonist (Rivier et al., 1984; Britton et al., 1986b; Winslow et
al., 1989; Lowry and Moore, 1991). These studies and others (Britton et al., 1985,
1986a) provide evidence that CRF can act at sites within the central nervous system to
stimulate locomotion in diverse vertebrate species. The sites of action for CRFinduced locomotor activity remain unclear.
Although the neurochemical mechanisms through which CRF induces
locomotion have not been determined, the present study and previous studies (Dunn
and Berridge, 1987; Kalivas et al., 1987; Lee et al., 1994; Lowry et al., 1993;
Matsuzaki et al., 1989) clearly indicate that CRF-induced locomotor activity is
associated with changes in metabolism of multiple monoaminergic systems, including
noradrenergic, dopaminergic and serotonergic systems. For example, studies using
microdissection with HPLC-ED techniques indicate that i.c.v. CRF-induced locomotor
activity is associated with increased DOPAC or DOPAC:DA ratios in the prefrontal
cortex, septum, striatum, nucleus accumbens, hypothalamus and brainstem (Dunn and
Berridge, 1987; Kalivas et al., 1987; Matsuzaki et al., 1989), as well as increased 3methoxy-4-hydroxy-phenylglycol (MHPG) or MHPG:norepinephrine ratios in the
prefrontal cortex, amygdala, hypothalamus midbrain, locus coeruleus region, and
brainstem (Dunn and Berridge, 1987; Emoto et al., 1993). Studies using in vivo
microdialysis in freely moving rats indicates that i.c.v. CRF-induced locomotor
activity is associated with elevated dialysate concentrations of norepinephrine in the
hypothalamus (Lee et al., 1994). Consistent with this observation, i.c.v.
administration of CRF elevates dialysate concentrations of norepinephrine, dopamine,
152
and several of their measurable metabolites in the medial hypothalamus and medial
prefrontal cortex of rats (Lavicky and Dunn, 1993). Although dialysate
concentrations of serotonin were not determined, concentrations of the metabolite 5HIAA also were elevated in the medial hypothalamus and medial prefrontal cortex.
These studies, together with studies in T. granulosa (Lowry et al., 1993), provide
evidence that i.c.v. administration of CRF results in a concommittant activation of
dopaminergic, noradrenergic, and serotonergic systems.
Several studies have addressed the question of whether or not CRF-induced
locomotor activity is dependent on activation of monoaminergic receptor systems.
Studies have demonstrated that i.c.v. CRF-induced locomotor activity can be
suppressed by a-flupenthixol and haloperidol, dopamine receptor antagonists, but only
at doses that induced catalepsy (Koob et al., 1984; Imaki et al., 1985). In addition,
destruction of dopamine nerve terminals using 6-hydroxydopamine within the nucleus
accumbens did not block CRF-induced locomotor activity (Swerdlow and Koob,
1985). Microinjection of CRF directly into the dopamine cell body-containing region
of the ventral tegmental area (VTA) of rats also induces increases in locomotor
activity, but this effect also is insensitive to the dopaminergic receptor antagonist
haloperidol (Kalivas et al., 1987), providing further evidence that CRF- induced
locomotor activity is not dependent on activation of dopaminergic receptor systems.
The involvement of noradrenergic receptor systems in CRF-induced locomotor
activity is more equivocal. For example, behavioral studies in rats indicate that al
and «2 adrenergic receptor antagonists (phentolamine, prazosin, and yohimbine) can
153
suppress CRF-induced locomotor activity measured in a novel environment (Imaki et
al., 1987); however the observation that administration of the a2 adrenergic receptor
agonist, clonidine
which can result in an inhibition of norepinephrine release from
nerve terminals by acting on a2 receptors within the locus coeruleus (Langer, 1981) -,
suppresses spontaneous but not CRF-induced locomotor activity in T. granulosa
(Lowry et al., 1991) and the observation that microinfusion of CRF directly into the
locus coeruleus did not alter locomotor activity (Butler et al., 1990), suggest that
CRF-induced locomotor activity is not dependent on activation of the noradrenergic
system at the level of the locus coeruleus.
Studies in T. granulosa suggest that CRF-induced locomotion is associated
with a widespread activation of serotonergic neuronal systems. The selective
serotonin reuptake inhibitor fluoxetine (Fuller et al., 1991) enhances locomotor
activity in T. granulosa treated with a behaviorally subthreshold dose of CRF (but not
in saline-treated animals); this effect is associated with a widespread depletion of
serotonin throughout the central nervous system measured using HPLD-ED techniques
(Lowry et al., 1993). In rats, CRF infused into the lateral ventricles dosedependently increases the activity of tryptophan hydroxylase, the rate-limiting enzyme
for serotonin synthesis (Corley et al., 1990; Singh et al., 1992). Collectively, these
studies suggest that the central actions of CRF involve the activation of serotonergic
neuronal systems. It remains uncertain if CRF-induced locomotor activity is
dependent on the activation of serotonergic receptor systems.
154
Summary
In summary, the dorsomedial hypothalamus in vertebrates integrates behavioral
and autonomic responses to physiological or environmental challenges to the
organism. The accumulation of catecholamines and indoleamines within the
dorsomedial hypothalamus following exposure to stressful stimuli, i.c.v.
administration of CRF, or intraperitoneal administration of corticosterone in
vertebrate species may alter homeostatic processes regulated by this structure. The
mechanisms by which stress or stress-related neurochemicals alter the accumulation of
monoamines in the dorsomedial hypothalamus, as well as the precise neuroendocrine,
autonomic and behavioral consequences, are important questions for future
investigations.
ACKNOWLEDGEMENTS
This work was supported by NIMH predoctoral fellowship to CAL (BNR1F31MH10288) and research grants to KJR (USPHS MH-44893) and FLM (IBN 93-
19633). We kindly thank Drs. Wylie Vale and Jean Rivier for the generous gift of
CRF used in these studies.
155
CHAPTER VII: DISCUSSION
These studies establish that, in male T. granulosa, administration of the
neuropeptide CRF into the cerebral ventricle stimulates locomotor activity in a dose-
and time-dependent manner. In addition, these studies provide the first evidence that
an endogenous CRF may be involved in the adaptive locomotor responses to stressful
stimuli; administration of the CRF receptor antagonist a-helical CRF9_41 suppressed
the adaptive locomotor responses to handling and to exposure to a dangerously warm
ambient temperature. These studies as well as the rapid nature of the behavioral
responses to CRF administered directly into the lateral ventricle demonstrate that CRF
can act to alter locomotor activity in a time-frame consistent with a role for this
peptide in the "fight or flight" response. This rapid change in behavior elicited by
intraventricular administration of CRF is associated with rapid changes in medullary
neuronal activity, suggesting that there is a rapid functional reconfiguration of
neuronal activity in response to CRF administration in this amphibian.
Microdissection and high performance liquid chromatography with
electrochemical detection techniques were used to first describe the distribution of
catecholamines and indoleamines throughout the central nervous system of male T.
granulosa, then to identify site-specific effects of CRF and corticosterone
administration on tissue concentrations of specific monoamines. In the survey of
monoaminergic systems in male T. granulosa it was determined that the highest
concentrations of monoamines, with the single exception of 5-HT concentrations in
the serotonergic raphe system, are in regions of the hypothalamus that contain
156
cerebrospinal fluid-contacting, monoamine-containing neuronal cell bodies. A
thorough review of recent literature suggests that these hypothalamic monoaminecontaining neuronal systems may be more conserved evolutionarily than originally
believed. For example, since the original description of the paraventricular organ
(PVO) in nonmammalian vertebrates, it has been believed that there is no homologous
structure present in the central nervous system of mammalian vertebrates. Based on
connectivity as well as topographical, immunohistochemical, biochemical, and
functional characteristics of this region, however, the PVO of nonmammalian
vertebrates appears to be homologous to the dorsomedial hypothalamic nucleus
(DMN) of mammals.
This is an important observation because in studies of the effects of CRF- and
corticosterone-administration on the tissue concentrations of catecholamines and
indoleamines, the region containing the PVO consistently responded with elevations in
dopamine, DOPAC, 5-HT, and 5-HIAA. These responses are remarkably similar to
the neurochemical changes observed within the DMN of rats following exposure to
certain types of stressful stimuli. Together, these studies suggest that the dorsomedial
hypothalamus of vertebrates may be an important neuroanatomical site for the actions
of stress and stress-related neurochemicals on central nervous system function.
The changes in tissue concentrations of monoamines and monoamine
metabolites within the dorsomedial hypothalamus following administration of CRF or
corticosterone
or following exposure to stressful stimuli
may have important
neuroendocrine, physiological, or behavioral consequences for the organism.
157
Although it is impossible at this time to determine what those consequences may be,
studies of the DMH of rats demonstrate that excitation of this structure elicits 1)
increases in heart rate, respiration, and blood pressure (DiMicco and Abshire, 1987;
Anderson and DiMicco, 1990; Soltis and DiMicco, 1991a,b; Soltis and DiMicco,
1992; Shekhar, 1993; DiMicco and Monroe, 1994; Stoltz-Potter et al., 1994), 2)
hyperglycemic reactions accompanied by increases in plasma catecholamine levels
(Frohman and Bernardis, 1971; Hasegawa et al., 1993), 3) increases in brown fat
temperature (Kelly and Bielajew, 1991), 4) hyperthermia (Sellami and Beaurepaire,
1993), and 5) behavioral effects including a suppression of feeding (Takaki et al.,
1992), an increase in locomotor activity (Shekhar and DiMicco, 1987), as well as
increases in indices of fear and anxiety (Shekhar et al., 1987, 1990; Shekhar, 1993;
Inglefield et al., 1994). Since all of these physiological and behavioral effects also
have been identified as central actions of CRF (reviewed by Dunn and Berridge,
1990), it is reasonable to propose the hypothesis that CRF may stimulate elements
within the dorsomedial hypothalamus and thereby initiate and coordinate this suite of
physiological and behavioral responses. Testing the validity of this hypothesis
as
well as determining the cellular mechanisms through which CRF and corticosterone
alter tissue concentrations of dopamine, DOPAC, 5-HT, and 5-HIAA within the
dorsomedial hypothalamus
are important areas for future research.
In summary, these studies establish that administration of CRF into the
cerebral ventricle of male T. granulosa rapidly elicits behavioral and neurochemical
effects that are also observed following exposure of vertebrate organisms to stressful
158
stimuli. These observations and others described in this thesis provide evidence that
CRF actions are important in the adaptive behavioral response to stressful stimuli in
this amphibian and, consequently, that this functional role for CRF is an
evolutionarily ancient feature of this neuropeptide.
159
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