Am J Physiol Regul Integr Comp Physiol 293: R1495–R1503, 2007.
First published August 1, 2007; doi:10.1152/ajpregu.00393.2007.
Experimental dissociation of neural circuits underlying conditioned avoidance
and hypophagic responses to lithium chloride
Linda Rinaman and Victoria Dzmura
Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania
Submitted 6 June 2007; accepted in final form 29 July 2007
(LICL) IS commonly used in experiments to
study central neural pathways and mechanisms involved in
“sickness behavior.” Systemic administration of LiCl in rats
dose-dependently inhibits food intake, activates the hypothalamic-pituitary-adrenal (HPA) stress axis, and promotes conditioned flavor avoidance (CFA) (2, 3, 6, 9, 10, 34, 41, 50, 51,
54). CFA represents a learned association between a novel
flavor and an aversive postingestive event (48). Because toxic,
rancid, and other potentially dangerous foods coexist with most
animals, CFA is believed to have evolved to protect animals
(especially nonemetic species such as rats) from poisoning
themselves out of the gene pool (47). The ability of LiCl to
inhibit food intake and to support robust conditioned avoidance
of associated flavors is evidence that LiCl has aversive quali-
ties and that it recruits central neural circuits that underlie both
hypophagia and adaptive associative learning.
It is widely held that LiCl-induced hypophagia is a direct
consequence of LiCl-induced malaise. However, relatively low
doses of LiCl that do not inhibit ad libitum or deprivationinduced food intake can nevertheless support conditioned taste
aversion and avoidance behavior in rats (2, 41), and an experimental dissociation between the ability of LiCl to produce
CFA and to inhibit food intake has been reported (6). In the
latter study, LiCl-induced CFA was abolished in rats with
aspiration lesions of the chemosensitive area postrema (AP),
although LiCl still promoted hypophagia and increased plasma
levels of oxytocin (6). Thus, LiCl inhibits food intake and
recruits hypothalamic endocrine responses in AP-lesioned rats
that are either incapable of experiencing LiCl-induced malaise
or are incapable of associating malaise with a novel flavor.
First-order central nervous system (CNS) regions that are
activated to express the immediate-early gene product c-Fos
after LiCl treatment include the AP, which lacks a blood-brain
barrier, and the subjacent nucleus of the solitary tract (NST).
Activated AP and NST neurons include noradrenergic (NA)
neurons that are immunoreactive for dopamine beta hydroxylase (DbH), and non-NA neurons (30, 36). NA and non-NA
neurons in the AP and NST relay LiCl-initiated signals to other
brain regions involved in sickness behavior, CFA learning, and
stress responses, including the lateral parabrachial nucleus
(laPBN), the central nucleus of the amygdala (CeA), and the
paraventricular nucleus of the hypothalamus (PVN). Conditioned taste aversion and CFA responses to LiCl in rats require
an intact AP, PBN, and amygdala (3, 6, 13, 17, 31, 32, 40 – 42,
58). However, the requisite neural substrates for LiCl-induced
hypophagia are less well defined.
A novel immunotoxin comprising an antibody to the NA
synthetic enzyme DbH conjugated to saporin toxin (DSAP)
destroys NA neurons selectively and site-specifically (1, 11,
12, 25, 35, 39, 56). A study from our laboratory used DSAP
lesions to reveal that NA neurons in the caudal NST are
necessary for exogenous CCK to inhibit food intake in rats,
likely because of disruption of caudal brain stem circuits that
mediate CCK-induced hypophagia (35). Caudal medullary NA
neurons also are necessary for CCK to activate c-Fos expression in the PVN but are unnecessary to activate the laPBN and
CeA, in which CCK-induced c-Fos expression appeared to be
increased normally in rats with complete bilateral DSAP lesions of the caudal NST (35). The present study used a similar
DSAP-lesioning approach to test the hypothesis that the neural
substrates of LiCl-induced hypophagia require NA neurons
Address for reprint requests and other correspondence: L. Rinaman, Univ. of
Pittsburgh, A210 Langley Hall, Pittsburgh, PA 15260 (e-mail: Rinaman
@pitt.edu).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
vagus; nucleus of the solitary tract; hypothalamus; amygdala; parabrachial nucleus; saporin toxin; viscerosensory; sickness behavior;
stress; hypothalamic-pituitary-adrenal axis
LITHIUM CHLORIDE
http://www.ajpregu.org
0363-6119/07 $8.00 Copyright © 2007 the American Physiological Society
R1495
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on September 30, 2016
Rinaman L, Dzmura V. Experimental dissociation of neural
circuits underlying conditioned avoidance and hypophagic responses to lithium chloride. Am J Physiol Regul Integr Comp
Physiol 293: R1495–R1503, 2007. First published August 1, 2007;
doi:10.1152/ajpregu.00393.2007.—We previously reported that noradrenergic (NA) neurons in the nucleus of the solitary tract (NST) are
necessary for exogenous CCK octapeptide to inhibit food intake in
rats. To determine whether NST NA neurons also are necessary for
lithium chloride (LiCl) to inhibit food intake and/or to support
conditioned avoidance behavior, saporin toxin conjugated to an antibody against dopamine beta hydroxylase (DSAP) was microinjected
bilaterally into the NST to ablate resident NA neurons. DSAP and
sham control rats subsequently were tested for the ability of LiCl
(0.15M, 2% body wt) to inhibit food intake and to support conditioned
flavor avoidance (CFA). LiCl-induced hypophagia was significantly
blunted in DSAP rats, and those with the most extensive loss of NST
NA neurons demonstrated the most attenuated LiCl-induced hypophagia. Conversely, LiCl supported a robust CFA that was of similar
magnitude in sham control and DSAP rats, including rats with the
most extensive NA lesions. A terminal c-Fos study revealed intact
LiCl-induced c-Fos expression in the lateral parabrachial nucleus and
central amygdala in DSAP rats, despite significant loss of NST NA
neurons and attenuated c-Fos activation of corticotropin-releasing
hormone-positive neurons in the paraventricular nucleus of the hypothalamus (PVN). Thus, NST NA neurons contribute significantly to
LiCl-induced hypophagia and recruitment of stress-responsive PVN
neurons but appear to be unnecessary for CFA learning and expression. These findings support the view that distinct central nervous
system circuits underlie LiCl-induced inhibition of food intake and
conditioned avoidance behavior in rats.
R1496
NA NEURONS, HYPOPHAGIA, AND CONDITIONED AVOIDANCE
within the caudal dorsomedial medulla, whereas the same NA
neurons are unnecessary for the ability of LiCl to recruit AP
and laPBN neural circuits that underlie CFA learning.
MATERIALS AND METHODS
Animals
Experimental Groups and Body Weights
Rats received bilateral injections of toxin (DSAP; Advanced Targeting Systems, San Diego, CA; n ⫽ 27) or vehicle (0.15 M NaCl;
sham control; n ⫽ 16) into the caudal NST, as described below. Each
rat was subsequently used in a CFA experiment, in a food intake
experiment, and in a terminal c-Fos study. Thus, each rat received
three LiCl treatments over the course of the study.
Rats weighed 187–212 g at the time of DSAP or sham surgery,
240 –276 g at the time of CFA and food intake experiments, and
265–297 g at the time of the terminal c-Fos study. Body weights did
not differ significantly at any experimental time point in DSAP vs.
sham control rats. A previous report from our laboratory described a
transient anorexia and BW loss that emerged in some rats ⬃1 wk after
bilateral DSAP injections into the caudal NST (35). The initial
anorexia observed in that study resolved, and all rats gradually
recovered normal body weight growth curves (35). With that result in
mind, all rats in the present study were offered a palatable liquid diet
(described in CFA experiment) in addition to their regular chow for 2
days before brain stem injection surgery and for 5–7 days after
surgery. The lack of lesion-related body weight loss in the present
study (see RESULTS) could be due to the dietary intervention and/or to
the smaller number of DSAP injection sites (i.e., four per rat as
described below, vs. six per rat in our previous study). A smaller
number of DSAP injection sites were used in the present study in an
effort to reduce toxin spread to NA neurons within the ventrolateral
medulla.
NST microinjection. DSAP or vehicle microinjections were made
bilaterally into the NST at four sites in each rat, with two injection
sites in the left NST and two in the right. DSAP was freshly prepared
from a frozen stock solution within 2 h of injection. Injection sites
encompassed the caudal medial NST region that contains the highest
incidence of c-Fos expression by DbH-positive NA neurons after LiCl
treatment, corresponding to the location of the A2 NA cell group (30,
37). Rats were anesthetized (2–5% halothane in 100% oxygen) and
secured in a stereotaxic frame using blunt ear bars, with the nose
ventroflexed. The skin over the dorsal neck surface was shaved,
sterilized, and incised, and the neck muscles were retracted and
bluntly dissected to expose the meninges overlying the dorsal surface
of the caudal medulla. With the aid of a surgical microscope, the
meninges were cut with a sterile needle to reveal the AP. A glass
micropipette tip (outer diameter 50 –75 ␮m) filled with DSAP or
vehicle and affixed to a 1.0-␮l Hamilton syringe was positioned on the
midline at the caudal limit of the AP, then moved 0.25 mm lateral and
0.5 mm below the medullary surface for the first injection site. A
second more rostral injection site was located 0.25 mm below the
lateral border of the AP at its widest rostrocaudal extent (35). The two
NST injection sites were then duplicated contralaterally. At each
injection site, 50 nl of sterile 0.15 M NaCl vehicle containing 0 or 5
ng DSAP was delivered by manual pressure injection over ⬃30 s. The
AJP-Regul Integr Comp Physiol • VOL
293 • OCTOBER 2007 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on September 30, 2016
All procedures conformed to the National Institutes of Health
guidelines and were approved by the University of Pittsburgh Animal
Care and Use Committee. Data from 43 adult male Sprague-Dawley
rats (Harlan Laboratories, Indianapolis, IN) are included in this report.
Singly housed rats had ad libitum access to water and pelleted rat
chow (Purina, St. Louis, MO), except during food intake and CFA
experiments, as detailed below. Colony rooms were maintained at
22–23°C and kept on a 12:12-h light-dark cycle, with lights on from
0700 to 1900.
pipette tip was left in place for 2 min after each microinjection, then
withdrawn. The skin incision was closed with sutures after the final
injection. Rats were returned to their home cages after recovery from
anesthesia. In addition to their regular chow diet, a palatable liquid
diet (Vanilla flavored Ensure, 25 ml/day; Abbott Laboratories, Abbott
Park, IL) was offered for 2 days before and for 5–7 days after DSAP
or sham injection surgery. All rats consumed both liquid and solid
diets, and DSAP and sham control rats gained equivalent amounts of
body weight during the postsurgical period.
CFA experiment. Two to three weeks after NST microinjection
surgery, DSAP and sham control rats were tested for the ability of
LiCl to support CFA. A two-bottle choice paradigm (8) was used to
determine whether rats avoid consuming water that contains flavors
previously paired with LiCl treatment. Flavor exposure during CFA
training and testing was conducted near the end of the light cycle of
the photoperiod, between 1600 and 1800. Rats were acclimated for
3– 4 days to gentle handling before the start of the CFA experiment.
Rats were water deprived for 22 h. Approximately half of the rats
within each NST injection group (i.e., DSAP or sham) were then
presented with a single bottle of almond-flavored tap water to drink
from a graduated tube, and the others were presented with a single
bottle of vanilla-flavored water (0.5% McCormick brand almond or
vanilla extract). The left-right position of the drinking tube on each
cage was switched after 15 min, with cumulative intake recorded at
the 30-min time point. Thirty minutes after the end of this initial
single-flavor exposure session, all rats were injected intraperitoneally
with 0.15 M NaCl (2% body weight). Plain tap water was returned 30
min later, and rats had ad libitum water access for the next 24 h. Rats
were then water deprived for 22 h, followed by presentation of a
single bottle containing the alternate flavor to drink for 30 min, with
the bottle position on each cage switched after 15 min. Thirty minutes
after the end of this second single-bottle flavor exposure session, rats
were injected intraperitoneally with 0.15 M LiCl (2% body weight).
Plain water was returned 30 min later, and rats had ad libitum water
access for 24 – 48 h.
For the final two-bottle choice test, rats were water deprived for
22 h, and then given 30-min simultaneous access to two bottles of
water, one containing the saline-paired flavor (either almond or
vanilla) and the other containing the alternate LiCl-paired flavor. The
volume consumed by each rat from each bottle was recorded after 30
min of access, with bottle positions switched at the 15-min time point.
Rats then were returned to ad libitum water access.
Flavor preference ratios displayed by each rat during the two-bottle
choice test were averaged within each treatment group to obtain group
preference ratios (means ⫾ SE) for intake of saline-paired flavors
relative to LiCl-paired flavors. Outcomes indicating significantly
shifted preference ratios (e.g., 75%:25%) were interpreted as evidence
for conditioned avoidance of the flavor represented by the lower value
in the ratio. Paired t-tests were used to determine whether differences
in preference for saline-paired vs. LiCl-paired flavors were statistically significant, with significance set at P ⬍ 0.05.
Food intake experiment. One week after completing the CFA
experiment, DSAP and sham control rats were tested for the ability of
LiCl treatment to inhibit food intake. For this purpose, rats were
moved from their original colony room to a new environment with the
same 12:12-h light-dark cycle. Rats were housed individually in clear
Plexiglas boxes (25 cm ⫻ 30 cm floor, 22 cm height) with stainlesssteel rod floors, each equipped with a computer-driven pellet delivery
and monitoring system (Med Associates, St. Albans, NY). Drinking
water was available ad libitum from sipper tubes within each box.
Food access was restricted to a daily 3-h period beginning at lights out
(1900), when a single 45-mg chow pellet (Precision Dustless Pellets;
Bio-Serv, Frenchtown, NJ) was delivered automatically to a shallow
feeding trough in each cage, thereby breaking a laser photobeam
crossing the base of the trough. A new pellet was delivered automatically each time the preceding pellet was removed. The cumulative
number of pellet deliveries (i.e., photobeam breaks) was recorded
NA NEURONS, HYPOPHAGIA, AND CONDITIONED AVOIDANCE
AJP-Regul Integr Comp Physiol • VOL
Laboratories, West Grove, PA), Elite Vectastain avidin-biotin reagents (Vector Laboratories, Burlingame, CA) and a nickel-enhanced
diaminobenzidene (DAB) reaction. After c-Fos immunostaining, sections were incubated for 48 h at 4°C in a monoclonal mouse anti-DbH
antibody (1:50,000; Chemicon, Temecula, CA) and processed for
brown immunoperoxidase localization of cytoplasmic DbH using
biotinylated donkey anti-mouse IgG (1:500), Elite Vectastain reagents, and a nonenhanced DAB reaction. Additional adjacent sets of
tissue sections from each rat were processed similarly for dual
immunoperoxidase localization of c-Fos and CGRP (1:50,000; Peninsula Laboratories, San Carlos, CA), or for c-Fos and corticotropinreleasing hormone (CRH; 1:20,000; Peninsula Laboratories). CGRP
provides a distinct neurochemical marker for the viscerosensory
laPBN-to-CeA projection pathway (22, 59), and in our previous study,
the CGRP-positive pathway was activated normally in DSAPlesioned rats after CCK treatment (35). CRH immunostaining identifies parvocellular PVN neurons that comprise the apex of the HPA
stress axis, and in our previous study, CCK-induced activation of
CRH neurons was markedly attenuated in DSAP-lesioned rats after
CCK treatment (35). Immunoreacted sections were mounted out of
buffer onto charged glass microscope slides (SuperFrost Plus; Fisher
Scientific, San Jose, CA), dehydrated in a graded ethanol series,
defatted in xylene, and coverslipped with Histomount (VWR, Pittsburgh, PA).
Quantitative analysis of immunolabeling. Cell counting was performed to document DSAP lesion extent and LiCl-induced c-Fos
expression in each animal. Counts of DbH-positive neurons in the
NST and VLM were performed bilaterally in every sixth section
(210-␮m frequency) through the rostrocaudal extent of the AP (3 or 4
tissue sections per rat). Cells were counted using a 20⫻ microscope
objective on a Zeiss Axioplan 2 microscope. All DbH-immunopositive profiles that were clearly perikaryal (rather than dendritic or
axonal) were counted, regardless of whether their nucleus was visible
in the section. This cell-counting strategy was adopted to avoid
undercounting of DbH-positive neurons that were c-Fos-negative,
because c-Fos-positive nuclei are more readily visible than are unstained nuclei. Perikaryal DbH-positive profiles were ovoid shaped
and at least as large in diameter as representative c-Fos-labeled nuclei
within the section. LiCl-induced neural activation within the AP and
NST was quantified by counting c-Fos-immunopositive profiles
within each region bilaterally in the same tissue sections. The proportion of DbH-positive NST and VLM cells that were colabeled for
nuclear c-Fos was determined. Cells were considered c-Fos-positive if
their nucleus contained detectable blue/black immunolabeling, regardless of labeling intensity, and c-Fos-negative if they displayed no
visible nucleus or a nucleus lacking c-Fos labeling. Thus, the reported
proportion of DbH-positive neurons that were double-labeled for
c-Fos is likely an underestimation of the actual proportion. However,
the magnitude of this potential underestimation should not differ
between experimental treatment groups.
c-Fos labeling in the visceral sensory region of the pontine PBN
was quantified by determining the number of CGRP-positive laPBN
neurons that were activated to express c-Fos, using counting criteria
similar to those described above. Tissue sections were examined
bilaterally through the rostrocaudal extent of cellular CGRP labeling
in the laPBN (3 or 4 sections spaced by 210 ␮m per rat). c-Fos
activation in the hypothalamus was quantified by determining the
number of CRH-immunopositive neurons within the medial parvocellular PVN (mpPVN) that contained nuclear c-Fos labeling. For each
rat, two tissue sections (spaced by 210 ␮m) through the mpPVN that
contained the highest density of CRH immunolabeling were selected
for analysis. c-Fos activation in the amygdala was determined using
CGRP fiber immunolabeling to distinguish the cytoarchitectural
boundaries of the CeA. All c-Fos-positive profiles within those CeA
boundaries were counted in tissue sections through the rostrocaudal
extent of fibrous CGRP labeling (5–7 sections spaced by 210 ␮m in
each rat).
293 • OCTOBER 2007 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on September 30, 2016
automatically at 30-min intervals during the 3-h feeding period. Cage
trays beneath the open-rod floors were routinely inspected at the end
of the feeding period to confirm that delivered pellets were actually
consumed. Data were collected and stored using Med PC software
(Med Associates). Rats were acclimated to the new environment and
feeding schedule for 1 wk before testing, by which time stable daily
3-h food intakes of ⬃20 –22 g per rat (i.e., ⬃7.5– 8.5% body weight)
were achieved.
On testing day 1, rats were injected intraperitoneally with 0.15 M
NaCl (2% body wt) at 1830. Food access was initiated 30 min later,
at lights out (1900). Cumulative pellet delivery data were collected for
3 h. On testing day 2 (24 h later), rats were injected intraperitoneally
with 0.15 M LiCl (2% body wt) at 1830. Food access was initiated 30
min later, at lights out (1900). Cumulative pellet delivery data were
collected for 3 h. Thus, each DSAP or sham control rat served as its
own control for the effects of intraperitoneal saline vs. intraperitoneal
LiCl on subsequent food intake. Rats were returned to their original
home cages with ad libitum chow access after testing day 2.
Food intake data (i.e., the number of 45-mg pellets consumed over
3 h) were combined a priori according to NST injection group (DSAP
vs. sham control) and expressed as group means ⫾ SE at each 30-min
time point. Group- and treatment-related differences in food intake
were tested for statistical significance by using ANOVA, with NST
injection (DSAP vs. sham), intraperitoneal treatment (NaCl or LiCl),
and time (30-min intervals) as independent variables. When F values
indicated significant main effects and interactions among these variables on food intake, ANOVAs were followed up with post hoc t-tests
using Dunn’s (Bonferroni) correction for multiple comparisons. Differences were considered significant when P ⬍ 0.05. Feeding data
also were analyzed by correlating the magnitude of LiCl-induced
feeding suppression at the final 3-h time point with DSAP lesion
extent, defined by the number of DbH-positive NST neurons (see
RESULTS).
LiCl-induced c-Fos experiment. A terminal c-Fos study was performed 7–10 days after the end of the food intake experiment.
Previous work demonstrated that little or no c-Fos labeling is present
within the AP, NST, PBN, CeA, or PVN in either DSAP or sham
control rats perfused after control intraperitoneal injections of 0.15 M
NaCl (35). Thus, all rats in the present study (n ⫽ 27 DSAP, n ⫽ 16
sham control) were injected intraperitoneally with 0.15 M LiCl (2%
body wt) before perfusion. LiCl injections were made between 0900
and 1000. Rats were returned to their home cages immediately after
intraperitoneal injection and left undisturbed for 90 –120 min. Rats
then were anesthetized with pentobarbital sodium (Nembutal; 50
mg/kg ip) and perfused through the heart with 50 –100 ml of 0.15 M
NaCl followed by 500 ml of fixative solution (26) containing 4%
paraformaldehyde, 1.4% lysine, and 0.2% sodium metaperiodate in
0.1 M sodium phosphate buffer (hereafter, buffer). Fixed brains were
removed from the skull, postfixed overnight, then cryoprotected for
24 – 48 h in 20% sucrose. A freezing-stage microtome was used to cut
coronal sections with a thickness of 35 ␮m from the caudal extent of
the NST through the rostral extent of the corpus callosum. Sections
were collected in six serially adjacent sets and stored at 20°C in
cryopreservant solution (55).
Immunocytochemistry. Primary and secondary antisera were diluted in buffer containing 0.3% Triton-X100 and 1% normal donkey
serum. Dual immunoperoxidase localization of cytoplasmic DbH and
nuclear c-Fos labeling was performed to simultaneously assess the
extent of NST lesions (i.e., the loss of DbH labeling) and to determine
LiCl-induced neuronal activation (i.e., c-Fos labeling). For this purpose, one set of tissue sections (1:6 frequency) from each DSAP and
sham control rat was incubated for 48 h at 4°C in rabbit anti-c-Fos
antiserum (provided by P. J. Larsen and J. D. Mikkelsen, Denmark;
1:50,000). The specificity of this antibody for c-Fos protein has been
reported (38). Sections were rinsed and processed for blue/black
immunoperoxidase localization of nuclear c-Fos labeling using biotinylated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch
R1497
R1498
NA NEURONS, HYPOPHAGIA, AND CONDITIONED AVOIDANCE
In each rat, regional cell count data were expressed as the number
of cells counted within each specified brain region summed bilaterally, divided by the number of analyzed tissue sections through the
specified region, to obtain “average per section” values. Group data
were combined a priori by NST injection group (i.e., DSAP vs. sham
control) and expressed as mean counts ⫾ SE for each brain region.
Between-group differences in cell count values within each region
were tested for statistical significance by using separate t-tests, with
NST injection condition (i.e., DSAP or vehicle) as the independent
variable. Differences were considered significant when P ⬍ 0.05.
RESULTS
DSAP Lesions Do not Alter the Ability of LiCl to
Support CFA
Rats in both surgical groups consumed similar volumes of
novel almond- or vanilla-flavored water during initial presentation on single-bottle CFA training days (i.e., 13.5 ml ⫾ 1.2
consumed on a saline-paired day, 13.3 ml ⫾ 1.4 consumed on
LiCl-paired day; range 11–18 ml; paired t-test P ⬎ 0.05).
There were no significant effects of lesion group (DSAP vs.
sham) or flavor pairing order (vanilla ⫹ intraperitoneal saline
followed by almond ⫹ intraperitoneal LiCl, or vice versa) on
cumulative 30-min fluid intake during training. Thus, vanillaand almond-flavored waters were isopreferred by DSAP and
sham control rats, and surgical group had no effect on the
volumes consumed of either flavor after water deprivation.
In two-bottle choice tests, DSAP and sham control rats
drank significantly lower volumes of flavors that previously
were paired with intraperitoneal LiCl (DSAP 2.7 ml ⫾ 0.3;
sham 3.2 ml ⫾ 0.4) vs. their intake of intraperitoneal salinepaired flavors (DSAP 15.3 ml ⫾ 0.8; sham 14.1 ml ⫾ 0.9)
(P ⬍ 0.01 for each group when comparing intakes of LiCl- vs.
saline-paired flavors in paired t-tests) (Fig. 1). The resulting
flavor preference ratios for saline-paired vs. LiCl-paired flavors
were ⬃80%:20% in sham control rats and ⬃85%:15% in
DSAP rats (Fig. 1). Thus, DSAP and sham control rats displayed similarly robust CFA responses to flavors previously
paired with LiCl treatment.
DSAP Lesions Attenuate LiCl-Induced Hypophagia
DSAP and sham control rats consumed similar numbers
of food pellets over the 3-h monitoring period after intraTable 1. Average number of DbH-positive NA neurons per
tissue section
NST Injection Group
NST (A2/C2 cell group)
VLM (A1/C1 cell group)
Sham Control (n ⫽ 16)
DSAP (n ⫽ 27)
52.97⫾2.40
25.42⫾1.55
27.02⫾3.44*
22.86⫾1.12
Values are group means ⫾ SE. *Significantly (48.9%) fewer than in sham
controls (t-test; P ⬍ 0.05). DbH, dopamine beta hydroxylase; NA, noradrenergic; NST, nucleus of the solitary tract; VLM, ventrolateral medulla.
AJP-Regul Integr Comp Physiol • VOL
Fig. 1. Average group preference ratios (means ⫾ SE) in two-bottle choice
tests between flavors that were previously paired with intraperitoneal injection
of saline (0.15M NaCl, 2% body wt) or with intraperitoneal injection of LiCl
(0.15M, 2% body wt). The dashed line indicates the expected ratio of
50%:50% if there were no effect of flavor pairing condition on intake
preferences during the choice test. Sham control (n ⫽ 16) and DSAP rats (n ⫽
27) show equivalent and significantly lower preferences for LiCl-paired flavors
(solid bars) vs. saline-paired flavors (open bars). Thus, DSAP and sham control
rats display a similarly robust conditioned avoidance of LiCl-paired flavors.
See Fig. 3 for a breakdown of preference ratio results as a function of DSAP
lesion extent, which also reveals no differences among groups.
peritoneal injection of 0.15 M NaCl (Fig. 2, upper dashed
lines) [F(1,41) ⫽ 0.147, P ⬎ 0.05]. As expected, intraperitoneal injection of LiCl in sham control rats markedly suppressed
their food intake vs. intake by the same rats after intraperitoneal NaCl (Fig. 2) [F(1,15) ⫽ 71.46, P ⬍ 0.05]. The hypophagic effect of LiCl treatment in sham control rats was
maintained throughout the 3-h postinjection monitoring period,
ranging from ⬃70% inhibition of feeding at the 30-min time
point to ⬃45% inhibition at the final 3-h time point (Fig. 2).
The rate of eating (as evidenced by the slope of the lines
connecting time points; Fig. 2) was suppressed to the greatest
extent during the first 30 min of feeding in both sham and
DSAP rats after LiCl compared with feeding after saline
injections. Between the 30- and 60-min time points, DSAP rats
ate at a somewhat higher rate than sham rats, which produced
the statistically significant group difference in food intake
evident at the 60 min and later time points (Fig. 2). The ability
of LiCl to inhibit food intake in DSAP rats was significantly
attenuated at most time points (Fig. 2, asterisks), although
DSAP rats as a group still exhibited LiCl-induced hypophagia
across the 3-h monitoring period [F(1,26) ⫽ 53.39, P ⬍ 0.05].
However, within the 3-h monitoring period, neither group
compensated for the reduced food intake that was most evident
during the initial 30 – 60 min of the 3-h monitoring period.
The suppressive effect of LiCl on cumulative food intake at
the final 3-h time point was more markedly attenuated in rats
with more complete DSAP lesions (Fig. 3; lesion data described further, below). A significant correlation existed between lesion extent and the magnitude of LiCl-induced hypophagia at this time point in DSAP rats (Pearson r ⫽ ⫺0.531;
293 • OCTOBER 2007 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on September 30, 2016
DSAP toxin injections eliminated a significant proportion of
DbH-positive NST neurons. Quantitative analysis revealed an
average loss of ⬃49% of the NST NA neuronal population in
DSAP rats relative to sham controls (Table 1). However,
DSAP lesion extent was variable across animals. Lesion data
are detailed in DSAP-Induced Loss of DbH-Immunopositive
NA Neurons.
NA NEURONS, HYPOPHAGIA, AND CONDITIONED AVOIDANCE
R1499
LiCl-Induced c-Fos Expression in DSAP and Sham
Control Rats
P ⬍ 0.05), such that larger lesions were associated with less
feeding suppression after LiCl compared with intake after
saline injections. Conversely, grouping animals by DSAP lesion extent did not reveal any differences among groups in
results from the two-bottle choice test that demonstrated similarly robust CFA responses to LiCl (see Fig. 3).
DSAP-Induced Loss of DbH-Immunopositive NA Neurons
Bilateral toxin injections were associated with an average
49% reduction of DbH-positive NST neurons in DSAP rats
compared with sham controls (Table 1, Fig. 4, A and B). The
loss of DbH-positive NST neurons in DSAP rats was statistically significant (t-test, P ⬍ 0.01). Marked loss of DbHpositive neurons within the AP also was observed in DSAP rats
(Fig. 4, A and B), although this was not quantified. Loss of
DbH-positive NST neurons in individual DSAP rats ranged
from 3% to 84% relative to average counts in sham controls.
Thus, the overall effect of bilateral DSAP toxin injections to
eliminate DbH-positive NST neurons was significant but was
variable across animals within the DSAP group.
Compared with results from our previous study (35), in
which six toxin injection sites were made in each animal,
DSAP lesion extent in the present study was more variable
(i.e., 3– 84% loss of DbH-positive NST neurons), and we
obtained a larger proportion of DSAP rats with NA lesions that
were less than 50% complete. However, DSAP lesions in the
present study were more anatomically restricted than in our
previous report, with no significant loss of A1/C1 neurons in
the caudal VLM (Table 1).
AJP-Regul Integr Comp Physiol • VOL
Fig. 3. Hypophagia at the 3-h time point in sham control and DSAP rats after
LiCl treatment is expressed on the y-axis as a percentage of food intake by the
same rats at the same time point after intraperitoneal saline injection. DSAP
rats were divided into three groups based on noradrenergic (NA) lesion extent
[i.e., percent reduction in the number of dopamine beta hydroxylase (DbH)positive nucleus of the solitary tract (NST) neurons compared with the average
number in sham control rats]. One group comprises DSAP rats within the
lower quartile of lesion extent (i.e., up to 25% NA cell loss; n ⫽ 6). The second
group comprises DSAP rats within the middle two quartiles of lesion extent
(i.e., 26 –75% NA cell loss; n ⫽ 15). The third group comprises DSAP rats
within the top quartile of lesion extent (i.e., more than 75% cell loss; n ⫽ 6).
There was a significant effect of lesion extent on LiCl-induced hypophagia
[F(2,24) ⫽ 5.83; P ⫽ 0.009]. Conversely, grouping animals by DSAP lesion
extent did not reveal any differences among groups in results from the
two-bottle choice test, which demonstrated similarly robust conditioned flavor
avoidance (CFA) responses to LiCl (see also Fig. 1). *Significantly less
hypophagia than in sham controls or in DSAP rats with lesions in the lower
25% (t-tests; P ⬍ 0.05); ^Significantly less hypophagia than in all other groups
(t-tests; P ⬍ 0.05).
293 • OCTOBER 2007 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on September 30, 2016
Fig. 2. Cumulative food intake assayed at six time points in sham control (n ⫽
16) and DSAP rats (n ⫽ 27) after intraperitoneal injection of saline (0.15M
NaCl, 2% body wt; dashed lines) or LiCl (0.15 M, 2% body wt; solid lines).
Compared with intake after saline injections, LiCl significantly reduced food
intake in both sham (open symbols) and DSAP rats (solid symbols) at every
time point. However, the magnitude of LiCl-induced hypophagia was significantly attenuated in DSAP rats vs. sham controls at most time points
examined. *Significantly more food intake by DSAP rats after LiCl treatment
vs. intake in sham control rats at the same time points after LiCl (P ⬍ 0.05).
Hindbrain. The average numbers of c-Fos-positive profiles
counted per tissue section within the AP and NST were similar
in DSAP and sham control rats after LiCl treatment (Table 2,
Fig. 4, C and D), evidence that DSAP lesions eliminated a
relatively small subset of AP and NST neurons activated by
LiCl. By extension, DbH-positive NA neurons represent a
relatively small subset of the AP and NST neurons activated by
LiCl. Despite the average 49% loss of DbH-positive NST
neurons in DSAP rats (Table 1), the proportion of remaining
DbH-positive NST neurons activated in DSAP rats after LiCl
treatment (i.e., 70.5%) was statistically similar to the proportion activated in sham controls (i.e., 64.4%; Table 2). DbHpositive NA neurons within the caudal VLM also were similarly activated after LiCl treatment in DSAP and sham control
rats (i.e., 34.8% and 38.0%, respectively; Table 2).
Hypothalamus. DSAP lesions were associated with reduced
DbH immunolabeling throughout magnocellular and parvocellular subdivisions of the PVN (Fig. 5, A and B). DSAP lesions
also were associated with reduced PVN c-Fos expression after
LiCl treatment, including a significant attenuation of c-Fos expression by CRH-immunopositive neurons within the mpPVN
(Table 2, Fig. 5, C and D). LiCl activated 44.1% fewer
R1500
NA NEURONS, HYPOPHAGIA, AND CONDITIONED AVOIDANCE
CRH-positive neurons in DSAP rats compared with CRH
activation in sham controls (Table 2; P ⬍ 0.05). Conversely,
LiCl-induced c-Fos expression within the lateral magnocellular
PVN appeared similarly robust in DSAP and sham control rats
(Fig. 5, C and D). A significant correlation existed between NA
lesion extent in DSAP rats and LiCl-induced activation of
CRH-positive neurons (Pearson r ⫽ ⫺0.404; P ⬍ 0.01), such
that rats with larger lesions exhibited less CRH neural activation. LiCl-induced CRH activation in DSAP rats also was
positively correlated with the degree of LiCl-induced hypophagia assessed at the 3-h time point (Pearson r ⫽ 0.407;
P ⬍ 0.05).
Fig. 5. Color photomicrographs depicting DbH fiber and terminal immunolabeling (A and B) and LiCl-induced c-Fos expression (C and D) within the
hypothalamic paraventricular nucleus (PVN) in a representative sham control
rat (A and C) and in a representative DSAP rat (B and D). Tissue sections in
C and D are double-labeled for c-Fos (blue-black nuclear label) and corticotropin-releasing hormone (CRH; brown). Note the reduced DbH fiber immunolabeling (A and B) and reduced CRH c-Fos expression (C and D) in the
DSAP rat. Sections shown are taken from the same cases used for Figs. 4 and
6. dc, dorsal cap; lm, lateral magnocellular; mp, medial parvocellular; vp,
ventral parvocellular; 3, third ventricle.
Pons and amygdala. In contrast to the reduced activation of
c-Fos labeling in CRH-positive mpPVN neurons in DSAP rats,
c-Fos expression within the laPBN (Fig. 6, A and B) and CeA
(Fig. 6, C and D) was not attenuated in DSAP rats. Quantitative
analysis confirmed that similar numbers of CGRP-positive
laPBN neurons and similar numbers of cells within the CGRPrich region of the CeA were activated to express c-Fos in
DSAP and sham control rats after LiCl treatment (Table 2,
Fig. 6).
DISCUSSION
Table 2. Neural population activated to express Fos
NST Injection Group
AP neurons, avg. #/section
NST neurons, avg. #/section
DbH-positive NST neurons, %
DbH-positive VLM neurons, %
CRH-positive mpPVN neurons, avg.
#/section
CGRP-positive laPBN neurons, avg.
#/section
Neurons within the CGRP-rich zone of CeA,
avg. #/section
Sham Control
(n ⫽ 16)
DSAP
(n ⫽ 27)
208.6⫾13.8
362.8⫾11.1
64.4⫾4.2
38.0⫾5.1
227.9⫾16.2
323.3⫾9.6
70.5⫾7.5
34.8⫾4.7
56.7⫾5.7
31.7⫾4.9*
82.7⫾10.2
76.9⫾8.3
264.6⫾18.5
233.3⫾13.2
Values are group means ⫾ SE. *Significantly (44.1%) less than in sham
controls (t-test, P ⬍ 0.05). mpPVN, medial parvocellular paraventricular
nucleus; laPBN, lateral parabrachial nucleus; CeA, central nucleus of the
amygdala.
AJP-Regul Integr Comp Physiol • VOL
Results from this study support the view that NA neurons in
the caudal dorsomedial medulla contribute to LiCl-induced
hypophagia and also are important for conveying LiCl-related
interoceptive signals to CRH-positive neurons in the mpPVN.
Conversely, DSAP lesions of this NA cell population did not
affect LiCl-induced activation of neurons within the laPBN or
CeA and did not reduce the ability of LiCl to support CFA.
These results are striking, as they illustrate a unique experimental situation in which this “nauseogenic,” malaise-inducing
agent maintains its ability to support robust conditioned avoidance in animals in which hypophagic responses to the same
agent are significantly attenuated.
An earlier study reported that aspiration lesions of the AP
block the ability of LiCl to support CFA without significantly
affecting LiCl-induced hypophagia (6). That result is perhaps
easier to reconcile with the knowledge that many natural
conditions and experimental treatments inhibit food intake
293 • OCTOBER 2007 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on September 30, 2016
Fig. 4. Color photomicrographs depicting DbH immunolabeling (A and B) and
LiCl-induced c-Fos expression (C and D) within the dorsomedial medulla in a
representative sham control rat (A and C) and in a representative DSAP rat (B
and D). This DSAP rat had an 80% loss of DbH-positive neurons within the
nucleus of the solitary tract (NST). The overall distribution and density of
LiCl-induced c-Fos expression (blue-black nuclear label) within the area
postrema (AP) and medial NST are similar in the sham control and DSAP rat
(C and D), despite the marked loss of DbH-positive neurons in the DSAP rat
(A and B). Tissue sections in C and D are double-labeled for CGRP (brown
fibers). Sections shown are taken from the same cases depicted in Figs. 5 and
6. DMV, dorsal motor nucleus of the vagus; tr, solitary tract.
NA NEURONS, HYPOPHAGIA, AND CONDITIONED AVOIDANCE
R1501
NA Cell Loss, Hypophagia, and CFA
without also producing conditioned avoidance behavior, presumably because those situations and treatments are not aversive. On the other hand, conditioned avoidance also can occur
in the absence of demonstrable hyophagia. Certain doses of
LiCl that are subthreshold for inhibiting food intake in rats
have been reported to support comparatively mild but still
significant and dose-related conditioned avoidance (2, 9). Conversely, we are unaware of another experimental condition in
which a robust CFA induced by a relatively high dose of LiCl
is maintained without a similarly robust unconditioned hypophagic response, as occurred in rats with the most complete
DSAP lesions.
DSAP Lesion Efficacy and Specificity
Bilateral NST microinjections of DSAP were designed to
destroy NA neurons in the caudal medial NST, corresponding
to the location of the A2 cell group and the caudal portion of
the C2 cell group. Results from previous studies attest to the
efficacy and specificity of the conjugated DSAP toxin (1, 11,
12, 25, 35, 39, 56). In the present study, toxin-induced loss of
DbH cell and terminal immunostaining within the caudal
dorsomedial medulla (both AP and NST) and loss of DbH
terminal labeling in the PVN offer support for lesion efficacy.
Despite the loss of DbH-positive neurons, similar numbers of
c-Fos-positive NST and AP neurons were counted in DSAP
and sham rats after LiCl treatment, supporting the view that
NA neurons comprise a relatively small (albeit important)
subset of the total population of NST and AP neurons activated
by LiCl.
AJP-Regul Integr Comp Physiol • VOL
DSAP Lesions Reduce PVN Activation After LiCl
In addition to inhibiting food intake in rats, systemic LiCl
activates the HPA stress axis and also increases plasma levels
of oxytocin, and, to a lesser degree, vasopressin (6, 10, 50, 51,
54). Plasma hormone levels were not assayed in the present
study, but the significantly blunted c-Fos expression by CRHpositive mpPVN neurons in DSAP rats predicts a similarly
blunted HPA response to LiCl treatment. Indeed, a close
association between treatment-induced c-Fos activation in
CRH neurons and plasma corticosterone levels has been reported (1). Our finding of reduced c-Fos expression in mpPVN
CRH-positive neurons in DSAP rats after LiCl treatment is
consistent with a previous report of reduced mpPVN activation
in DSAP rats after CCK treatment (35). Conversely, despite
significant loss of DbH-immunoreactive fibers throughout the
PVN in DSAP rats, LiCl-induced c-Fos activation in the lateral
magnocellular PVN appeared to be intact, presumably due to
remaining inputs from neurons other than dorsomedial medullary NA neurons. Likely candidates include NA neurons in the
caudal VLM, which were not lesioned in DSAP rats and which
project directly to the vasopressin-rich core of the lateral
magnocellular PVN (4, 5, 44, 52).
DSAP Lesions Do not Attenuate laPBN or CeA Activation
After LiCl
In contrast to the decremented mpPVN activation in DSAP
rats after LiCl, activation of the CRGP-containing pathway
from laPBN to CeA (59) was quantitatively unaffected. We
interpret this dissociation as evidence that NA neurons in the
caudal dorsomedial medulla are unnecessary for conveying
LiCl-related viscerosensory signals to the CeA, probably because these signals are relayed through CGRP neurons in the
293 • OCTOBER 2007 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on September 30, 2016
Fig. 6. Color photomicrographs depicting LiCl-induced c-Fos expression
(blue-black nuclear label) within the laPBN (A and B) and CeA (C and D) in
a representative sham control rat (A and C) and in a representative DSAP rat
(B and D). Tissue sections are double-labeled for c-Fos and CGRP. CGRPpositive laPBN neurons (A and B) are known to project to the CeA, where
CGRP fiber and terminal labeling are coextensive with LiCl-induced c-Fos
expression (C and D). Note the similar c-Fos activation of CGRP-positive
laPBN neurons and within the CGRP-rich region of the CeA in DSAP and
sham control rats. Sections shown are taken from the same cases used for Figs.
4 and 5. BLA, basolateral amygdala; CeA, central nucleus of the amygdala;
laPBN, lateral parabrachial nucleus; scp, superior cerebellar peduncle.
As in our previous DSAP study examining the effect of NA
lesions on CCK-induced hypophagia (35), a positive correlation was found in the present study between the number of
surviving DbH-positive NA neurons in the caudal NST and the
extent of LiCl-induced hypophagia. As shown in Fig. 3, the
more effective the DSAP lesion, the more blunted the ability of
LiCl to inhibit food intake. Rats with the most complete DSAP
lesions (i.e., more than 75% loss of NA neurons) displayed
significantly less LiCl-induced hypophagia than sham control
rats or DSAP rats with less complete lesions. Thus, not only are
NA neurons in the caudal dorsomedial medulla activated by
LiCl and CCK, they underlie at least a significant portion of the
ability of these agents to inhibit food intake. It should be noted
that DSAP lesions in the present study were never 100%
complete; thus, it remains unclear whether NA neurons are
absolutely necessary for treatment-induced hypophagia. We
cannot rule out the possibility that more extensive NA lesions
would disrupt LiCl-induced CFA and/or c-Fos responses in the
laPBN and CeA. Considered together, however, these findings
support the view that NA neurons in the NST contribute
importantly to brain stem circuits that suppress food intake.
These circuits likely include known NA inputs to regions of the
medullary and pontine reticular formation (7) that contain the
premotor and motor neurons that ultimately control ingestive
behavior (49).
R1502
NA NEURONS, HYPOPHAGIA, AND CONDITIONED AVOIDANCE
AJP-Regul Integr Comp Physiol • VOL
mpPVN neurons but are unnecessary for the ability of LiCl to
support robust CFA learning and expression. Our findings
support the view that the behavioral effects of LiCl are mediated by CNS circuits that are at least partially dissociable.
GRANTS
This work was supported by National Institutes of Health Grant MH-59911.
REFERENCES
1. Banihashemi L, Rinaman L. Noradrenergic inputs to the bed nucleus of
the stria teminalis and paraventricular nucleus of the hypothalamus underlie hypothalamic-pituitary-adrenal axis but not hypophagic or conditioned avoidance responses to systemic yohimbine. J Neurosci 26: 11442–
11453, 2006.
2. Benoit SC, Air EL, Wilmer K, Messerschmidt P, Hodge KMB, Jones
MB, Eckstein DMM, McOsker CC, Seeley RJ, Woods SC, Sheldon
RJ. Two novel paradigms for the simultaneous assessment of conditioned
taste aversion and food intake effects of anorexic agents. Physiol Behav
79: 761–766, 2003.
3. Bernstein IL, Chavez M, Allen D, Taylor EM. Area postrema mediation
of physiological and behavioral effects of lithium chloride in the rat. Brain
Res 575: 132–137, 1992.
4. Cunningham ET Jr, Bohn MC, Sawchenko PE. Organization of adrenergic inputs to the paraventricular and supraoptic nuclei of the hypothalamus in the rat. J Comp Neurol 292: 651– 667, 1990.
5. Cunningham ET Jr, Sawchenko PE. Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat
hypothalamus. J Comp Neurol 274: 60 –76, 1988.
6. Curtis KS, Sved AF, Verbalis JG, Stricker EM. Lithium chlorideinduced anorexia, but not conditioned taste aversions, in rats with area
postrema lesions. Brain Res 663: 30 –37, 1994.
7. Day HE, Campeau S, Watson SJ, Akil H. Distribution of alpha 1a-,
alpha 1b- and alpha 1d-adrenergic receptor mRNA in the rat brain and
spinal cord. J Chem Neuroanat 13: 115–139, 1997.
8. Deutsch JA, Hardy WT. Cholecystokinin produces bait shyness in rats.
Nature 266: 196, 1977.
9. Ervin GN, Birkemo LS, Johnson MF, Conger LK, Mosher JT, Menius
Jr JA. The effects of anorectic and aversive agents on deprivation-induced
feeding and taste aversion conditioning in rats. J Pharmacol Exp Ther 273:
1203–1210, 1995.
10. Flanagan LM, Verbalis JG, Stricker EM. Naloxone potentiation of
effects of cholecystokinin and lithium chloride on oxytocin secretion,
gastric motility, and feeding. Neuroendocrinology 48: 668 – 673, 1988.
11. Fraley GS, Dinh TT, Ritter S. Immunotoxic catecholamine lesions
attenuate 2DG-induced increase of AGRP mRNA. Peptides 23: 1093–
1099, 2002.
12. Fraley GS, Ritter S. Immunolesion of norepinephrine and epinephrine
afferents to medial hypothalamus alters basal and 2-deoxy-D-glucoseinduced neuropeptide Y and agouti gene-related protein messenger ribonucleic acid expression in the arcuate nucleus. Endocrinology 144: 75– 83,
2003.
13. Grigson PS, Reilly S, Scalera G, Norgren R. The parabrachial nucleus
is essential for acquisition of a conditioned odor aversion in rats. Behav
Neurosci 112: 1104 –1113, 1998.
14. Grill HJ, Carmody JS, Sadacca LA, Williams DL, Kaplan JM.
Attenuation of lipopolysaccharide anorexia by antagonism of caudal brain
stem but not forebrain GLP-1-R. Am J Physiol Regul Integr Comp Physiol
287: R1190 –R1193, 2004.
15. Herbert H, Moga MM, Saper CB. Connections of the parabrachial
nucleus with the nucleus of the solitary tract and the medullary reticular
formation in the rat. J Comp Neurol 293: 540 –580, 1990.
16. Herbert H, Saper CB. Cholecystokinin-, galanin-, and corticotropinreleasing factor-like immunoreactive projections from the nucleus of the
solitary tract to the parabrachial nucleus in the rat. J Comp Neurol 293:
581–598, 1990.
17. Ivanova SF, Bures J. Acquisition of conditioned taste aversion in rats is
prevented by tetrodotoxin blockade of a small midbrain regions centered
around the parabrachial nucleis. Physiol Behav 48: 543–549, 1990.
18. Jia HG, Rao ZR, Shi JW. An indirect projection from the nucleus of the
solitary tract to the central nucleus of the amygdala via the parabrachial
nucleus in the rat: a light and electron microscopic study. Brain Res 663:
181–190, 1994.
293 • OCTOBER 2007 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on September 30, 2016
laPBN that innervate the CeA. Direct NST projections to the
amygdala include a prominent NA component that is activated
by LiCl and other experimental treatments that inhibit food
intake and promote conditioned avoidance behavior (28 –30).
However, the laPBN is a major relay for ascending viscerosensory inputs from the caudal medulla to the amygdala (15, 18,
33, 43, 53). NST and AP inputs to the laPBN are predominantly excitatory, and arise from both NA and non-NA neurons
(15, 16, 19). Subpopulations of neurons within the NST and
laPBN project differentially to brain stem and forebrain target
regions, suggesting that different types of viscerosensory signals may be carried through different projection pathways. For
example, systemic administration of endotoxin in rats activates
10 times as many CeA-projecting neurons in the laPBN than
CeA-projecting neurons in the NST (53). Neurons within the
laPBN, including CGRP-positive neurons, project rather
weakly to the PVN and lateral hypothalamus but strongly to the
CeA and bed nucleus of the stria terminalis (BNST) in rats and
in humans (21, 22, 45, 59). As another example, NA inputs to
the mpPVN arise from caudal medullary NST and VLM
neurons that also provide collateralized axonal inputs to the
BNST, whereas NA inputs to the lateral magnocellular PVN
arise from a separate population of hindbrain NA neurons (1).
Although DbH-positive AP and NST neurons that project to
the laPBN and amygdala undoubtedly were lesioned (to varying degrees) in DSAP rats, non-NA neurons that project to
these regions presumably were spared. The latter include
projections arising from serotoninergic AP neurons that target
the laPBN (23) and from glucagon-like peptide-1 (GLP-1)positive NST neurons that target the laPBN and CeA (24, 27)
and are activated in intact rats after LiCl treatment (36). We did
not examine LiCl-induced activation of these neurons in the
present study. However, in our previous DSAP study, GLP-1
neurons remained intact and were activated to express c-Fos
after CCK treatment in rats with more extensive dorsal medullary NA lesions (35). Thus, GLP-1 signaling pathways may
contribute to the observed maintenance of LiCl-induced CFA
in DSAP rats. Indeed, infusion of GLP-1 into the amygdala
supports conditioned avoidance behavior in rats, and blockade
of amygdalar GLP-1 receptors attenuates the ability of systemic LiCl to produce conditioned avoidance (20). Conversely,
infusion of GLP-1 directly into the amygdala does not inhibit
food intake (20). If GLP-1 pathways are sufficient to maintain
CFA in rats with DSAP lesions, it is still unclear why they are
not sufficient to maintain LiCl-induced hypophagia. Two laboratories have reported that global blockade of central GLP-1
receptors attenuates LiCl-induced hypophagia (34, 46), but in
neither study was the hypophagia eliminated. It should be
noted that the hypophagic effect of systemic endotoxin is
significantly attenuated by pharmacological antagonism of
hindbrain but not forebrain GLP-1 receptors (14), that hindbrain NA neurons express GLP-1 receptors, and that central
infusion of GLP-1 activates these NA neurons (57). Thus,
central GLP-1 signaling pathways may participate in hypophagic responses to LiCl because they recruit hindbrain NA
neurons. It would be interesting to determine whether the
ability of central GLP-1 to inhibit food intake is attenuated in
rats with DSAP lesions of these neurons.
We conclude that NA neurons within the dorsomedial medulla (AP and/or NST) contribute importantly to LiCl-induced
hypophagia and also to recruitment of stress-responsive
NA NEURONS, HYPOPHAGIA, AND CONDITIONED AVOIDANCE
AJP-Regul Integr Comp Physiol • VOL
39. Ritter S, Bugarith K, Dinh TT. Immunotoxic destruction of distinct
catecholamine subgroups produces selective impairment of glucoregulatory responses and neuronal activation. J Comp Neurol 432: 197–216,
2001.
40. Ritter S, McGlone JL, Kelly KW. Absence of lithium-induced taste
aversion after area postrema lesion. Brain Res 201: 501–506, 1980.
41. Sakai N, Yamamoto T. Possible routes of visceral information in the rat
brain in formation of conditioned taste aversion. Neurosci Res 35: 53– 61,
1999.
42. Sakai N, Yamamoto T. Role of the medial and lateral parabrachial
nucleus in acquisition and retention of conditioned taste aversion in rat.
Behav Brain Res 93: 63–70, 1998.
43. Saper CB, Loewy AD. Efferent connections of the parabrachial nucleus
in the rat. Brain Res 197: 291–317, 1980.
44. Sawchenko PE, Swanson LW. The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the
rat. Brain Res Rev 4: 275–325, 1982.
45. Schwaber JS, Sternini C, Brecha NC, Rogers WT, Card JP. Neurons
containing calcitonin gene-related peptide in the parabrachial nucleus
project to the central nucleus of the amygdala. J Comp Neurol 270:
416 – 426, 1988.
46. Seeley RJ, Blake K, Rushing PA, Benoit W, Eng J, Woods SC,
D’Alessio D. The role of CNS glucagon-like peptide-1 (7–36) amide
receptors in mediating the visceral illness effects of lithium chloride.
J Neurosci 20: 1616 –1621, 2000.
47. Selcher JC, Weeber EJ, Varga AW, Sweatt JD, Swank M. Protein
kinase signal transduction cascades in mammalian associative conditioning. Neuroscientist 8: 122–131, 2002.
48. Smith GP. Feeding: control of eating. In: Encyclopedia of Neuroscience
(3rd ed.), Amsterdam: Elsevier, 2003.
49. Smith GP. The controls of eating: a shift from nutritional homeostasis to
behavioral neuroscience. Nutrition 16: 814 – 820, 2000.
50. Smotherman WP. Glucocorticoid and other hormonal substrates of conditioned taste aversion. Ann NY Acad Sci 443: 126 –144, 1985.
51. Sugawara M, Hashimoto K, Hattori T, Takao T, Suemaru S, Ota Z.
Effects of lithium on the hypothalamo-pituitary-adrenal axis. Endocrinologia Japonica 35: 655– 663, 1988.
52. Swanson LW, Sawchenko PW. Paraventricular nucleus: a site for the
integraton of neuroendocrine and autonomic mechanisms. Neuroendocrinology 31: 410 – 417, 1980.
53. Tkacs NC, Li J. Immune stimulation induces Fos expression in brainstem
amygdala afferents. Brain Res Bull 48: 223–231, 1999.
54. Verbalis JG, McHale CM, Gardiner TW, Stricker EM. Oxytocin and
vasopressin secretion in response to stimuli producing learned taste aversions in rats. Behav Neurosci 100: 466 – 475, 1986.
55. Watson RE, Wiegand ST, Clough RW, Hoffman GE. Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides 7: 155–159, 1986.
56. Wrenn CC, Picklo MJ, Lappi DA, Robertson D, Wiley RG. Central
noradrenergic lesioning using anti-DBH-saporin: anatomical findings.
Brain Res 740: 175–184, 1996.
57. Yamamoto H, Lee CE, Marcus JN, Williams TD, Overton JM, Lopez
ME, Hollenberg AN, Baggio L, Saper CB, Drucker DJ, Elmquist JK.
Glucagon-like peptide-1 receptor stimulation increases blood pressure and
heart rate and activates autonomic regulatory neurons. J Clin Invest 110:
43–52, 2002.
58. Yamamoto T, Shimura T, Sako N, Yasoshima Y, Sakai N. Neural
substrates for conditioned taste aversion in the rat. Behav Brain Res 65:
123–137, 1994.
59. Yasui Y, Saper CB, Cechetto DF. Calcitonin gene-related peptide
immunoreactivity in the visceral sensory cortex, thalamus, and related
pathways in the rat. J Comp Neurol 22: 487–501, 1989.
293 • OCTOBER 2007 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.3 on September 30, 2016
19. Kawai Y, Takagi H, Yanai K, Tohyama M. Adrenergic projection from
the caudal part of the nucleus of the tractus solitarius to the parabrachial
nucleus in the rat: immunocytochemical study combined with a retrograde
tracing method. Brain Res 459: 369 –372, 1988.
20. Kinzig KP, D’Alessio DA, Seeley RJ. The diverse roles of specific
GLP-1 receptors in the control of food intake and the response to visceral
illness. J Neurosci 22: 10470 –10475, 2002.
21. Krukoff TL, Harris KH, Jhamandas JH. Efferent projections from the
parabrachial nucleus demonstrated with the anterograde tracer Phaseolus
vulgaris leucoagglutinin. Brain Res Bull 30: 163–172, 1993.
22. LaCalle SD, Saper CB. Calcitonin gene-related peptide-like immunoreactivity marks putative visceral sensory pathways in human brain. Neuroscience 100: 115–130, 2000.
23. Lanca AJ, Van der Kooy D. A serotonin-containing pathway from the
area postrema to the parabrachial nucleus in the rat. Neuroscience 14:
1117–1126, 1985.
24. Larsen PJ, Tang-Christensen M, Holst JJ, Orskov C. Distribution of
glucagon-like peptide-1 and other preproglucagon-derived peptides in rat
hypothalamus and brainstem. Neuroscience 77: 257–270, 1997.
25. Madden CJ, Ito S, Rinaman L, Wiley RG, Sved AF. Lesions of the C1
catecholaminergic neurons of the ventrolateral medulla in rats using
anti-DbH saporin. Am J Physiol Regul Integr Comp Physiol 277: R1063–
R1075, 1999.
26. McLean IW, Nakane PK. Periodate-lysine-paraformldehyde fixative. A
new fixative for immunoelectron microscopy. J Histochem Cytochem 22:
1077–1083, 1974.
27. Merchenthaler I, Lane M, Shughrue P. Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat
central nervous system. J Comp Neurol 403: 261–280, 1999.
28. Myers EA, Banihashemi L, Rinaman L. The anxiogenic drug yohimbine
activates central viscerosensory circuits in rats. J Comp Neurol 492:
426 – 441, 2005.
29. Myers EA, Rinaman L. Trimethylthiazoline (TMT) supports conditioned
flavor avoidance and activates viscerosensory, hypothalamic, and limbic
circuits in rats. Am J Physiol Regul Integr Comp Physiol 288: R1716 –
R1726, 2005.
30. Myers EA, Rinaman L. Viscerosensory activation of noradrenergic
inputs to the amygdala in rats. Physiol Behav 77: 723–729, 2002.
31. Reilly S. The parabrachial nucleus and conditioned taste aversion. Brain
Res Bull 48: 239 –254, 1999.
32. Reilly S, Grigson PS, Norgren R. Parabrachial nucleus lesions and
conditioned taste aversion: evidence supporting an associative deficit.
Behav Neurosci 107: 1005–1017, 1993.
33. Richard S, Engblom D, Paues J, Mackerlova L, Blomqvist A. Activation of the parabrachio-amygdaloid pathway by immune challenge or
spinal nociceptive input: a quantitative study in the rat using Fos immunohistochemistry and retrograde tract tracing. J Comp Neurol 481: 210 –
219, 2005.
34. Rinaman L. A functional role for central glucagon-like peptide-1 receptors in lithium chloride-induced anorexia. Am J Physiol Regul Integr
Comp Physiol 277: R1537–R1540, 1999.
35. Rinaman L. Hindbrain noradrenergic lesions attenuate anorexia and alter
central c-Fos expression in rats after gastric viscerosensory stimulation.
J Neurosci 23: 10084 –10092, 2003.
36. Rinaman L. Interoceptive stress activates glucagon-like peptide-1 neurons that project to the hypothalamus. Am J Physiol Regul Integr Comp
Physiol 277: R582–R590, 1999.
37. Rinaman L, Hoffman GE, Dohanics J, Le WW, Stricker EM, Verbalis
JG. Cholecystokinin activates catecholaminergic neurons in the caudal
medulla that innervate the paraventricular nucleus of the hypothalamus in
rats. J Comp Neurol 360: 246 –256, 1995.
38. Rinaman L, Stricker EM, Hoffman GE, Verbalis JG. Central c-fos
expression in neonatal and adult rats after subcutaneous injection of
hypertonic saline. Neuroscience 79: 1165–1175, 1997.
R1503