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Serotonergic projections and serotonin receptor expression
in the reticular nucleus of the thalamus in the rat
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Journal:
Manuscript ID:
Wiley - Manuscript type:
Complete List of Authors:
Draft
Research Article
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Date Submitted by the
Author:
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Rodríguez, José; IKERBASQUE, Basque Foundation for Science;
University of the Basque Country UPV/EHU,, Department of
Neurosciences; Institute of Experimental Medicine, Neuroscience
Noristani, Harun; The University of Manchester, Faculty of Life
Science
Hoover, Walter; Florida Atlantic University, Center for Complex
Systems and Brain Sciences
Linley, Stephanie; Florida Atlantic University, Department of
Psychology
Vertes, Robert; Florida Atlantic University, Center for Complex
Systems and Brain Sciences
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Reticular nucleus, Thalamus, Serotonin, Serotonin receptors, Raphe
Abstract:
The reticular nucleus (RT) of the thalamus, a thin sheet of
GABAergic neurons located between the external medullary lamina
and the internal capsule of the thalamus, has functionally distinct
afferent and efferent connections with thalamic nuclei, the
neocortex, the basal forebrain and the brainstem. RT is critically
positioned to rhythmically pace thalamocortical networks leading to
the generation of spindle activity during the early phases of sleep
and during absence (spike-wave) seizures. Serotonin, acting on 5HT1A receptors on parvalbumin-containing cells of RT, has been
implicated in this rhythmicity. However, the precise source(s) of 5HT afferents to the RT remains to be determined. In the present
study, we injected the retrograde tracer, Fluorogold, into dorsal and
ventral regions of RT to determine the origins of raphe input to RT.
We further characterized the distribution of 5-HT fibers to RT by
using immunohistochemistry for 5-HT and for the 5HT transporter
(SERT) detection. Finally, we described the presence of the two
major post-synaptic 5-HT receptors in RT, 5-HT1A and 5-HT2A
receptors. Our results show that the dorsal raphe nucleus and the
supralemniscal nucleus (B9) of the midbrain are the principal
sources of raphe projections to RT. In addition, serotonergic fibers
(5-HT and SERT positive) were richly distributed throughout RT,
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Keywords:
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and 5-HT1A and 5-HT2A receptors were highly expressed on RT
neurons and dendrites. These findings suggest a significant 5-HT
modulatory influence on GABAergic neurons of RT in the control of
rhythmical (or spindle) activity in thalamocortical systems directly
associated with sleep and possibly with absence seizures.
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Rodríguez et al.
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Serotonergic projections and serotonin receptor expression in the reticular
nucleus of the thalamus in the rat
J.J. Rodríguez1,2,3* Harun. N. Noristani4, Walter B. Hoover5, Stephanie B. Linley6,
Robert P. Vertes5
IKERBASQUE, Basque Foundation for Science, 48011, Bilbao, Spain. 2Department
1
of Neurosciences, University of the Basque Country UPV/EHU, 48940, Leioa, Spain.
3
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Institute of Experimental Medicine, ASCR, Videnska 1083, 142 20, Prague, Czech
Republic. 4Faculty of Life Sciences, The University of Manchester, Manchester,
5
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United Kingdom.
Center for Complex Systems and Brain Sciences, Florida Atlantic University, Boca
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Raton, Florida 33431
*
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Number of text pages: 17
Number of Tables: 1
Number of Figures: 4
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Department of Psychology, Florida Atlantic University, Boca Raton, Florida 33431
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Correspondence to: José J. Rodríguez, IKERBASQUE, Department of
Neuroscience, University of the Basque Country UPV/EHU. Technological Park,
Bldg. 205, Floor -1. Laida Bidea. 48170-Zamudio. Bizkaia, Spain. Tel: +34946018305,
Fax:
+34-946018289.
E-mail
address:
j.rodriguezarellano@ikerbasque.org
Running title: 5-HT innervation of reticular nucleus of thalamus
Keywords: Reticular nucleus, thalamus, serotonin, serotonin receptors, serotonin
transporter, raphe, supralemniscal nucleus, plasticity.
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Abbreviations
3rd ventricle
5-Hydroxytryptamine, serotonin
anterodorsal nucleus of thalamus
anteromedial nucleus of thalamus
aqueduct
anteroventral nucleus of thalamus
central gray of the medulla oblongata
choroid plexus
caudal linear nucleus
central medial nucleus of thalamus
caudal ventral medulla
dorsal 3rd ventricle
dorsal raphe nucleus
dorsal raphe nucleus, dorsal part
dorsal raphe nucleus, lateral part
dorsal raphe nucleus, ventral part
fornix
interanteromedial nucleus of thalamus
internal capsule
lamboid septal zone
laterodorsal nucleus of thalamus, ventrolateral part
lateral paragigantocellular nucleus
lateral ventricle
mediodorsal nucleus of thalamus
median raphe nucleus
pararubral nucleus
paraventricular nucleus of thalamus
reuniens nucleus of thalamus
rhomboid nucleus of thalamus
raphe magnus
raphe obscurus
raphe pallidus
reticular nucleus of thalamus, dorsal part
reticular nucleus of thalamus, ventral part
rostral ventrolateral medulla
5-HT transporter
supralemniscal nucleus (B9)
ventral anterior lateral nucleus of thalamus
ventrolateral nucleus
ventral posterolateral nucleus of thalamus
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rR
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3V
5-HT
AD
AM
Aq
AV
CGM
chp
Cli
CM
CVl
D3V
DR
DRd
DRL
DRv
fx
IAM
ic
LD
LDVL
LPGi
LV
MD
MR
PaR
PV
RE
RH
RMg
Rob
Rpa
RTd
RTv
RVL
SERT
SLN
VAL
VL
VPL
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Abstract
The reticular nucleus (RT) of the thalamus, a thin sheet of GABAergic neurons
located between the external medullary lamina and the internal capsule of the
thalamus, has functionally distinct afferent and efferent connections with thalamic
nuclei, the neocortex, the basal forebrain and the brainstem. RT is critically
positioned to rhythmically pace thalamocortical networks leading to the generation of
spindle activity during the early phases of sleep and during absence (spike-wave)
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seizures. Serotonin, acting on 5-HT1A receptors on parvalbumin-containing cells of
RT, has been implicated in this rhythmicity. However, the precise source(s) of 5-HT
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afferents to the RT remains to be determined. In the present study, we injected the
retrograde tracer, Fluorogold, into dorsal and ventral regions of RT to determine the
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origins of raphe input to RT. We further characterized the distribution of 5-HT fibers
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to RT by using immunohistochemistry for 5-HT and for the 5HT transporter (SERT)
detection. Finally, we described the presence of the two major post-synaptic 5-HT
receptors in RT, 5-HT1A and 5-HT2A receptors. Our results show that the dorsal
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raphe nucleus and the supralemniscal nucleus (B9) of the midbrain are the principal
sources of raphe projections to RT. In addition, serotonergic fibers (5-HT and SERT
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positive) were richly distributed throughout RT, and 5-HT1A and 5-HT2A receptors
were highly expressed on RT neurons and dendrites. These findings suggest a
significant 5-HT modulatory influence on GABAergic neurons of RT in the control of
rhythmical (or spindle) activity in thalamocortical systems directly associated with
sleep and possibly with absence seizures.
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Introduction
The thalamus is the gateway to the forebrain/cortex and is involved in an array of
functions including the gating of sensory information, arousal, attention, learning and
memory (Exner et al., 2001; Guillery et al., 1998; Hampstead and Koffler, 2009;
Jones, 2007; McAlonan et al., 2000; Van der Werf et al., 2003; van Groen et al.,
2002). Serotonin-containing (5-HT) neurons are primarily located in raphe nuclei of
the brainstem with processes that distribute widely throughout the brain, subcortically
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and cortically (Morin and Meyer-Bernstein, 1999; Vertes, 1991; Vertes et al., 1999;
Vertes and Linley, 2007; Vertes and Linley, 2008). Thalamic 5-HT projections mainly
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originate from the small to medium sized 5-HT neurons located in the dorsal (DR)
and median raphe (MR) nuclei of the rostral pons and midbrain (Dahlstroem and
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Fuxe, 1964; Gonzalo-Ruiz et al., 1995; Morin and Meyer-Bernstein, 1999;
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Peschanski and Besson, 1984; Vertes, 1991; Vertes et al., 1999; Vertes and Martin,
1988). Early studies described a relatively restricted pattern of distribution of DR/MR
fibers to the thalamus; that is, to the anterior nuclei, mediodorsal nucleus, midline
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and intralaminar nuclei, habenula, laterodorsal nucleus and lateral geniculate
complex (LGN) (Azmitia and Segal, 1978; Vertes, 1991; Vertes et al., 1999; Vertes
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and Martin, 1988).
Another major 5-HT cell group of the midbrain is the B9 group (or
supralemniscal nucleus, SLN). Serotonergic SLN cells are primarily located within
and dorsal to the medial lemniscus (Vertes and Crane, 1997). Like DR/MR, there is
evidence for SLN (B9) projections to the thalamus, specifically to the laterodorsal,
central lateral, ventrolateral and posterior nuclei as well as LGN (Nagata, 1986;
Sawchenko et al., 1983; Vertes and Crane, 1997; Vertes et al., 2010; Willoughby
and Blessing, 1987).
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The reticular nucleus of thalamus (RT) is a thin sheet of gamma-amino butyric
acid containing (GABAergic) neurons located between the external medullary lamina
and the internal capsule of the thalamus (Barone et al., 1994; de Biasi et al., 1986;
Guillery et al., 1998; Houser et al., 1980; McAlonan and Brown, 2002; Mitrofanis,
1992; Spreafico et al., 1991). RT has been shown to regulate the flow of information
between the thalamus and the cortex, and accordingly, reportedly serves a direct
role in processes of attention, arousal and sleep state control (Horner et al., 1997;
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Huguenard and McCormick, 2007; McAlonan and Brown, 2002; McAlonan et al.,
2000; Rodrigo-Angulo et al., 2008). RT neurons receive collateral projections from
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thalamocortical and corticothalamic fibers, as well as, afferents from cholinergic and
5-HT neurons of the basal forebrain and brainstem (Aznar et al., 2003; Hallanger
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and Wainer, 1988). RT GABAergic neurons exhibit two distinct types of activity
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depending on the behavioural state of the animal: (1) rhythmic high frequency bursts
of action potentials, associated with slow-wave sleep, inattentiveness or drowsiness;
and (2) tonic single-spike activity, associated with arousal and attentiveness
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(Huguenard and McCormick, 2007; McCormick and Wang, 1991; Rodrigo-Angulo et
al., 2008). The local application of 5-HT to RT suppresses the burst firing of RT
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neurons and thereby promotes the occurrence of single-spike activity, mediated
through a decrease in K+ currents (McCormick and Pape, 1990; McCormick and
Wang, 1991). This effect is blocked by the administration of the 5-HT2A receptor
antagonist, ketanserin, suggesting a direct involvement of 5-HT2A receptors in the
response (McCormick and Wang, 1991).
Despite evidence for physiological actions of 5-HT at RT, reports have
conflicted with respect to the 5-HT innervation of RT (Cropper et al., 1984; Lavoie
and Parent, 1991; Steinbusch, 1981). For instance, studies in the rat have shown a
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low to moderate density of 5-HT fibers in RT (Cropper et al., 1984; Steinbusch,
1981), whereas a report in primates demonstrated a high density 5-HT axons in RT
(Lavoie and Parent, 1991). These discrepancies may involve species differences or
possibly differences in immunohistochemical procedures used to detect serotonergic
fibers (see, Vertes et al., 2010). Early studies used antiserum against 5-HT (Cropper
et al., 1984; Steinbusch, 1981) for the detection of 5-HT processes, and this is
reportedly a less sensitive marker for 5-HT fibers than is antiserum directed against
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the serotonin transporter (SERT) (Nielsen et al., 2006; Noristani et al., 2010; Vertes
et al., 2010). In this regard, we recently identified (Vertes et al., 2010) moderate
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numbers of 5-HT fibers in RT (or the rostral RT) using SERT immunohistochemical
procedures. This is consistent with the findings of 5-HT receptors, including 5-HT1A
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and 5-HT2A receptors on RT cells (Bonnin et al., 2006; Li et al., 2004), mainly on
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GABAergic parvalbumin-positive neurons of RT (Aznar et al., 2003). Despite the
foregoing, the precise cellular origin of 5-HT neurons giving rise to 5-HT innervation
of RT remains to be determined -- as does the specific complement of 5-HT
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receptors on cells of RT.
Accordingly, the present study examined the origin of raphe neurons
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projecting to RT, and further characterized the patterns of distribution of 5-HT+ and
SERT+ fibers in RT as well as the distribution of 5-HT1A and 5-HT2A receptors in RT.
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Materials and Methods
Animals
Nineteen male Sprague-Dawley rats (SD; Charles River, Wilmington, MA) weighing
275–350 g were used for the retrograde tracing experiments and ten male SD rats
were used for the identification of 5-HT fibers and receptors in RT. Experiments were
approved by the Florida Atlantic University and Manchester University Institutional
Animal Care and Use Committee and conform to all Federal regulations and the
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National Institute of Health guidelines for the care and use of laboratory animals as
well as to the United Kingdom Animals (Scientific Procedures) Act of 1986 under the
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License from the Home Office.
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Fluorogold procedures
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Rats were anesthetised using a 75 mg/kg dose of sodium pentobarbital. Each rat
received an injection of Fluorogold (FG) in the reticular nucleus (RT) of the thalamus.
Fluorogold was dissolved in 0.1M sodium acetate buffer (pH 4.0–5.0) to yield a 4.0–
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5.0% concentration. Single injections of Fluorogold were made iontophoretically
using glass micropipettes with an outside tip diameter of 25-50µm. Positive direct
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current (5-10 µA) was applied through a Grass stimulator (model 88) coupled with a
high voltage stimulator (FHC, Bowdoinham, ME) at 2s ‘on’/ 2 s ‘off’ intervals for 2-10
min as described previously (Vertes et al., 2006). Ten rats received injections in the
dorsal RT (RTd) and 9 rats received injections in the ventral RT (RTv). Following a
survival time of 7 days, rats were deeply anesthetized with sodium pentobarbital.
The brain of the rats were fixed using aortic arch perfusion with 50 ml of 3.8% of
acrolein in a solution of 2% paraformaldehyde and 0.1M phosphate buffer (PB, Ph
7.4), followed by 250 ml of paraformaldehyde and 0.1M PB. The brains were then
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removed from the cranium and postfixed in 2% paraformaldehyde for 30 minutes.
Coronal sections of the brain were cut into 40–50 µm thickness using a vibrating
microtome (VT1000S, Leica, Milton Keynes, UK). Free floating brain sections in 0.1
M PB, pH 7.4 were collected and stored in cryoprotectant solution containing 25%
sucrose and 3.5% glycerol in 0.05M PB at pH 7.4. Coronal sections at levels -1.08
mm/-4.08 mm (reticular nucleus) and -6.84 mm/-8.40 mm (raphe nuclei) posterior to
bregma, were selected for immunohistochemistry according to the rat brain atlas of
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Paxinos and Watson (Paxinos and Watson, 2005). Brightfield photomicrographs of
injection sites and labeled fibers were taken with a Nikon DXM1200 camera mounted
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on a Nikon Eclipse E80 microscope. Patterns of labeling according to the number of
cells labeled were classified as negative, light (+), moderate (++), and dense (+++),
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with ‘light’ referring to a few labeled cells widely dispersed throughout the nuclei,
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‘dense’ as a heavy concentration of labeled cells generally occupying an important
portion of the nuclei, and ‘moderate’ between these two patterns.
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Antibodies
A primary antiserum directed against FG (rabbit anti-FluoroGold, Chemicon,
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Temecula, CA) was used for detection of retrogradely labeled projection neurons. 5HT positive fibers within the RT were studied using a polyclonal rabbit antibody
against a peptide sequence corresponding to amino acids 579-599 of rat 5-HT
transporter (Immunostar, Hudson, WI, USA) and a polyclonal rabbit antibody
antiserum generated against 5-HT (Immunostar, USA). The specificity of these
antibodies has been reported previously using immunohistochemistry (Mamounas et
al., 2000; Noristani et al., 2010; O'Rourke and Fudge, 2006) and western blots
(Albright et al., 2007). A polyclonal guinea pig antibody against a synthetic peptide
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conjugated to bovine thyroglobulin corresponding to amino acid 248-262 of mouse 5HT1A (BD Pharmingen, San Diego, CA, USA) and a monoclonal mouse antiserum
generated against a recombinant hybridoma fusion protein between glutathione Stransferase and peptide containing amino acid 1-72 of the human 5-HT2A (BD
Pharmingen, San Diego, CA, USA) were used for detection of 5-HT receptors within
RT. Previous reports have confirmed the specificity of these antibodies using
western-blot and immunocytochemistry (Madhavan et al., 2003; Peddie et al., 2008;
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Wu et al., 1998). To determine the specificity of the antibodies adsorption controls
were done by omission of primary and/or secondary antibodies also showed no
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immunoreactivity (data not shown).
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Immunohistochemistry
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The sections were incubated for 30 min in 30% methanol in 0.1M PB and 3%
hydrogen peroxide (H2O2) (Sigma Chemicals, St. Louis, MO). Sections were then
rinsed with 0.1M PB for 5 minutes and placed in 1% sodium borohydride (Aldrich,
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USA) for 30 minutes. The sections were then washed with PB profusely before
rinsing in 0.1M Trizma base saline (TS) for 10 minutes. Brain sections were then
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incubated in 0.5% albumin bovine serum BSA (Sigma Chemicals, St. Louis, MO) in
0.1M TS and 0.25% Triton (Sigma Chemicals, St. Louis, MO, x 100) for 30 minutes.
Sections were incubated for 48 hours at room temperature in primary antisera
directed against either FG, SERT, 5-HT, 5-HT1A and 5-HT2A (rabbit anti-FluoroGold,
1:5000, Chemicon, Temecula, CA; rabbit anti-SERT, 1:2500, rabbit anti-5-HT,
1:5000, Immunostar, Hudson, WI, USA; guinea pig anti 5-HT1A, 1:500 and mouse
anti 5-HT2A, 1:500, BD Pharmingen, San Diego, CA, USA). Following incubation in
the primary antiserum, sections were washed (4 x 6 min) in 0.1M PB and then
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incubated in an appropriate secondary antiserum (Vector Labs, Burlingame, CA) at a
concentration of 1:400 in diluent for 1h. Sections were then washed again (4 x 6 min)
and incubated in avidin-biotin complex (Vector Labs) at a 1:100 concentration in
diluent for 30 min. After a final set of 4 x 6 min rinses, the peroxidase reaction
product was visualised by incubation in a solution containing 0.022% of 3,3’
diaminobenzidine (DAB, Aldrich, Milwaukee, WI), and 0.003% H2O2 in TBS for 6 min
as described previously (Rodriguez et al., 2008; Rodriguez et al., 2009). The
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reaction was stopped by rinsing the sections in 0.1M TS for 6 minutes followed by
0.1M PB for 15 minutes. Brain sections were permanently mounted onto chrome-
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alum gelatin coated slides and allowed to dehydrate overnight. Sections were then
dehydrated in ascending concentration of ethanol (50, 70, 80, 90, 95 and 100%), and
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finally xylene. Coverslips were applied using Entellan (Merck KGaA, Germany) and
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slides were left to dry overnight. Adjacent series of representative sections from each
rat was stained with toludine blue for anatomical reference. Sections were examined
using light microscopy. The injection sites and labeled cells were imaged on a Nikon
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Eclipse E80 microscope. The micrographs were prepared using Adobe Photoshop
7.0 (Mountain View, CA).
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Results
The patterns of labeled cells following injections of the retrograde tracer
Fluorogold in RT of the thalamus are described. Figure 1 shows representative
injection sites in dorsal and ventral regions of RT (RTd and RTv) (Fig. 1A,B). Figure
2 depicts retrogradely labeled cells in the supralemniscal nucleus (SLN) (Fig.
2A,C,E) and dorsal raphe nucleus (DR) (Fig. 2B,D,F) following retrograde injections
in RTd or RTv. Retrogradely labeled neurons in SLN were medium sized (15–25
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µm), varied in shape but were mainly fusiform-like or round (Fig. 2C,E). A few
neurons exhibited a single primary dendrite that extended directly out from, and
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parallel to, the polar regions of the cells and measured 75–100 µm in length (Fig.
2E). FG positive neurons in the DR were similar in size and shape to those of SLN,
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but most contained two to four primary dendrites extending from the neuronal soma
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(Fig. 2D, F).
Considerably more labeled cells were present in SLN and DR with RTv than
with RTd injections (Table 1). Very few labeled cells were detected in other raphe
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nuclei including the median raphe nucleus with either RTd or RTv injections (Table
1). Eight of ten cases with RTd injections produced labeling in SLN and DR, with on
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average more labeled cells in SLN than DR (Table 1A). Most of the RTd injections
resulted in approximately 1-5 labeled cells/section in DR. One case (RTd10),
however, produced more than 10 labeled cells/section in DR – and equivalent
numbers in SLN (Table 1A). Labeled neurons of DR were mainly restricted to the
dorsal subnucleus of DR (DRd) (Fig. 2B); whereas those of SLN were predominantly
localized to anteromedial regions of SLN (Fig. 2A), relatively few were seen at
caudal levels of SLN.
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Similar to RTd, injections in RTv gave rise to labeled cells essentially confined
to DR and SLN of the raphe nuclei (Fig. 2C,D), and were more abundant in both
nuclei than shown for RTd injections. For DR, 5 of 9 injections resulted in greater
than 10 labeled cells/section, while the remaining 4 injections gave rise to 1-10
labeled neurons/section (Table 1B, Fig. 2D). As demonstrated for RTd, labeled cells
with RTv injections were primarily found in DRd. Some were also present in the
ventral DR (DRv), but exceedingly few were detected in lateral regions of DR – or
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the lateral wings of DR (Fig. 2D). Similar to DR, 3 of 9 SLN cases showed greater
than 10 FG-labeled neurons/section, while two cases exhibited 1-10 labeled
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cells/section (Table 1B, Fig. 2C). Labeled cells in SLN were predominantly localized
to anteromedial regions of SLN. As indicated, RTd or RTv injections produced only
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minor labeling in other serotonergic nuclei including MR and CLi (Table 1). A few
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labeled neurons were observed in raphe pallidus and raphe magnus with one RTv
case, but essentially none in the caudal raphe with other RTv cases (data not
shown).
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Presence of 5-HT and SERT immunolabeled fibers in RT
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5-HT and SERT immunoreactive fibres spread relatively homogeneously
throughout the extent of RT showing little difference in density in rostrocaudal or
dorsoventral planes (Fig. 3A,C). 5-HT labeling in RT, however, was less pronounced
than seen in some other nuclei of the thalamus including the anterior and midline
groups, but denser than present in lateral (relay) nuclei of the thalamus: the
posterior, ventral-anterolateral (VAL) and ventrobasal thalamus. The quality, and to
some extent the density, of labeling in RT (as well as in other regions of thalamus)
was superior following immunostaining for SERT compared to 5-HT, but patterns of
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labeling were generally equivalent for the two immune-techniques. 5-HT fibers in RT
showed morphological characteristics of fine fibers typical of dorsal raphe axons,
with small circular and regularly spaced varicosities (Fig. 3B,D) (Noristani et al.,
2010).
Distribution and expression of 5-HT1A and 5-HT2A receptors in RT
5-HT1A and 5-HT2A receptors were found throughout RT with no clear
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difference (or gradient) in the rostrocaudal or dorsoventral planes (Fig. 4A,C).
Labeled 5-HT1A and 5-HT2A receptors were mainly present on the somata of RT
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neurons, but were also observed on proximal and mid-dendritic segments of RT
neurons (Fig. 4C,D). There was, however, a relatively marked difference in the
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pattern of expression of the two receptors (Fig. 4A,C) in RT such that 5-HT1A
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receptors were more strongly expressed than 5-HT2A receptors and were mainly
present on the soma and proximal dendrites, whereas 5-HT2A receptors were
moderately expressed on cell bodies and more abundant on fine and medium-sized
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dendrites (Fig. 4B,D). 5-HT1A and 5-HT2A receptors were more densely expressed in
RT than in several other nuclei of the thalamus including VAL, the anterior and
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midline nuclei (Fig. 4A,C).
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Discussion
We describe the origin of raphe projections to the reticular nucleus of the
thalamus (RT) and the pattern of distribution of 5-HT fibers and 5-HT1A and 5-HT2A
receptors in RT. We showed that FG injections in RT (dorsal and ventral parts) gave
rise to relatively significant numbers of labeled cells in the dorsal raphe (DR) nucleus
and in the supralemniscal nucleus (B9) of the brainstem. There were, however,
differences in DR and SLN projections to the dorsal (RTd) and ventral (RTv).
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Specifically, RTv injections gave rise to significantly more labeled neurons in DR
than did RTd injections. The SLN labeling with RTd and RTv injections was mixed;
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that is, 4/10 cases with RTd injections resulted in > 5 labeled cells/section in SLN,
whereas 3/9 cases with RTv injections produced > 10 cells/section in SLN.
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Although moderate, the presently described DR projection to RT is
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inconsistent with previous reports using the anterograde tracers showing a relative
absence of DR projections to RT (Morin and Meyer-Bernstein, 1999; Peschanski and
Besson, 1984; Vertes, 1991). While it is generally recognized that different
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anatomical tracers can yield dissimilar results, it is also possible that the PHA-L
injections of the previous studies were not optimally placed in regions of DR giving
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rise to RT projections. For instance, we showed that labeled cells with RT injections
were mainly located in the dorsal subnucleus of DR and this could have been largely
missed in previous anterograde tracing studies.
On the other hand, our demonstration of moderate numbers of labeled cells in
SLN with RT injections is to, our knowledge, the first description of SLN-RT
projections. Further, the present findings that RT is moderately supplied by 5-HT
fibers and contains a fairly dense population of 5-HT1A and 5-HT2A receptors indicate
that the DR and SLN input to RT is mainly serotonergic.
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Very few labeled neurons were identified in other raphe nuclei including the
median raphe (MR) nucleus. With respect to MR, these findings are consistent with
those of several previous reports, using various tracers, showing a virtual lack of MR
projections to RT (Morin and Meyer-Bernstein, 1999; Peschanski and Besson, 1984;
Vertes et al., 1999; Vertes and Martin, 1988).
Using antiserum against 5-HT or SERT, we observed a moderate density of
serotonergic fibers within RT. Morphologically; the 5-HT fibers innervating RT were
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fine with small and regularly spaced varicosities, characteristic of 5-HT axons from
the dorsal raphe nucleus (Bjarkam et al., 2005; Noristani et al., 2010). Previous
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reports in the rat, using 5-HT or SERT antisera, have similarly described a moderate
5-HT innervation of RT (Cropper et al., 1984; Steinbusch, 1981; Vertes et al., 2010).
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These findings in rodents, however, partially conflict with the demonstration of a high
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density of 5-HT fibers in the RT of the squirrel monkey (Lavoie and Parent, 1991).
This may largely involve species differences (Vertes et al., 2010).
The present findings of a relatively high density of 5-HT1A and 5-HT2A
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receptors in RT (i.e. higher than in other regions of the thalamus) are consistent with
the previous identification of 5-HT1A and 5-HT2A receptors in the rodent and human
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RT (Bonnin et al., 2006; Li et al., 2004; Wai et al., 2010). Aznar et al. (2003) showed
that 5-HT1A receptors are highly localized to GABAergic (parvalbumin-positive) RT
neurons, suggesting a serotonergic modulation of RT output (Aznar et al., 2003).
Supporting this, the local application of 5-HT in RT inhibits the burst firing of RT
neurons (associated with slow-wave sleep, inattentiveness, drowsiness) resulting in
the occurrence of single-spike activity seen during aroused and attentive states
(McCormick and Pape, 1990; McCormick and Wang, 1991). The administration of
the 5-HT2A receptor antagonist, ketanserin, blocks the switch from bursting to tonic
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RT activity. Together these findings suggest a serotonergic involvement in sleepwaking behavior via modulation of pacemaking GABAergic neurons of RT
(McCormick and Pape, 1990; McCormick and Wang, 1991).
In conclusion, we demonstrate moderate DR and SLN projections to RT,
mainly targeting the ventral RT. By actions on 5-HT1A and 5-HT2A receptors on
GABAergic cells of RT, serotonergic fibers to RT may serve an important role in the
modulation of sleep and waking states.
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ACKNOWLEDGEMENTS
The present study was supported by Government of the Basque Country grant (AE2010-1-28; AEGV10/16), Grant Agency of the Czech Republic (GACR 309/09/1696)
to JJR and by NSF grant IOS 0820639 to RPV. The authors would also like to thank
BBSRC for PhD studentship to H.N. Noristani.
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Figure legends
Figure 1: Brightfield micrographs showing Fluorogold injection sites in the dorsal (A)
and ventral (B) reticular nucleus of thalamus (RT). Schematic drawings of a section
through the rat brain at the level of the RT injection sites corresponding to -2.16 mm
posterior to Bregma (C) demonstrating the locations of the injections in the dorsal
and ventral RT. Brightfield micrograph of a representative section through the
diencephalon at the level of RT stained with Toludine Blue. Scale bars; A, B and D =
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1 mm. RTd: dorsal part of reticular nucleus of thalamus, RTv: ventral part of reticular
nucleus of thalamus; chp: choroid plexus; 3V: 3rd ventricle; D3V: dorsal 3rd ventricle;
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LV: lateral ventricle; PV: paraventricular nucleus of thalamus; CM: central medial
nucleus of thalamus; RH: rhomboid nucleus of thalamus; RE: reuniens nucleus of
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thalamus; VL: ventrolateral nucleus of thalamus; VPL: ventral posterolateral nucleus
internal capsule.
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of thalamus; LDVL: laterodorsal nucleus of thalamus, ventrolateral part; fx: fornix; ic:
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Figure 2: Brightfield micrographs showing Fluorogold (FG) retrogradely labeled cells
(arrows) in the supralemniscal nucleus (SLN, B9) (A,C), and in the dorsal raphe
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nucleus (mDR; B, D) following FG injections in the dorsal (A, B) and ventral (C, D)
RT. Modal average of 1-5 labeled cells/section for dorsal (B) and > 10 labeled
cells/section for the ventral RT (D). PaR: pararubral nucleus; Aq: aqueduct (Sylvius);
DRL: dorsal raphe nucleus; lateral part, DRd: dorsal raphe nucleus, dorsal part;
DRv: dorsal raphe nucleus; ventral part; SLN supralemniscal nucleus.
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Figure 3: Brightfield micrographs illustrating the distribution of serotonin-containing
fibers in RT following immunostaining against the serotonin transporter (SERT; A,B)
or 5-HT (C,D). VAL: ventral anterior lateral nucleus of thalamus; ic: internal capsule.
Figure 4: Brightfield micrographs the distribution of 5-HT1A (A,B) and 5-HT2A (C,D)
receptors in RT. 5-HT1A labelling is mainly restricted to the soma (asterisk) and
occasionally observed within proximal dendrites (arrows, B). 5-HT2A labelling,
Fo
however, is more commonly observed within proximal, medial and distal dendritic
processes (arrows) as well as in the soma (asterisk, D).
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Table 1. Summary Table showing raphe nuclei with Fluorogold positive neurons following the
injection of the tracer into dorsal (A) and ventral (B) parts of the reticular nucleus of thalamus
(RT), RTd and RTv, respectively. The average of positive cells per section is described as
follows: (-) = no cells, (+) = 1-5 positive cells, (++) = 5-10 positive cells, (+++) = 10 or more
positive cells.
A
DORSAL
RTd1
RTd2
RTd3
RTd4
RTd5
RTd6
RTd7
RTd8
RTd9
RTd10
Nuclei
MR
DR
CLi
SLN
+
+
+++
+
+
+
+
+
++
+
-
+
+
+
++
+
-
+++
+++
VENTRAL
RTv1
RTv3
Fo
RTv4
RTv5
RTv6
RTv7
RTv8
RTv9
Nuclei
MR
DR
CLi
SLN
+++
+
++
-
+++
-
+++
+++
+
+
+++
-
+
+++
+
+++
B
RTv2
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+++
-
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Figure 1: Brightfield micrographs showing Fluorogold injection sites in the dorsal (A) and ventral (B)
reticular nucleus of thalamus (RT). Schematic drawings of a section through the rat brain at the
level of the RT injection sites corresponding to -2.16 mm posterior to Bregma (C) demonstrating the
locations of the injections in the dorsal and ventral RT. Brightfield micrograph of a representative
section through the diencephalon at the level of RT stained with Toludine Blue. Scale bars; A, B and
D = 1 mm. RTd: dorsal part of reticular nucleus of thalamus, RTv: ventral part of reticular nucleus
of thalamus; chp: choroid plexus; 3V: 3rd ventricle; D3V: dorsal 3rd ventricle; LV: lateral ventricle;
PV: paraventricular nucleus of thalamus; CM: central medial nucleus of thalamus; RH: rhomboid
nucleus of thalamus; RE: reuniens nucleus of thalamus; VL: ventrolateral nucleus of thalamus; VPL:
ventral posterolateral nucleus of thalamus; LDVL: laterodorsal nucleus of thalamus, ventrolateral
part; fx: fornix; ic: internal capsule.
209x297mm (300 x 300 DPI)
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Figure 2: Brightfield micrographs showing Fluorogold (FG) retrogradely labeled cells (arrows) in the
supralemniscal nucleus (SLN, B9) (A,C), and in the dorsal raphe nucleus (mDR; B, D) following FG
injections in the dorsal (A, B) and ventral (C, D) RT. Modal average of 1-5 labeled cells/section for
dorsal (B) and > 10 labeled cells/section for the ventral RT (D). PaR: pararubral nucleus; Aq:
aqueduct (Sylvius); DRL: dorsal raphe nucleus; lateral part, DRd: dorsal raphe nucleus, dorsal part;
DRv: dorsal raphe nucleus; ventral part; SLN supralemniscal nucleus.
209x297mm (300 x 300 DPI)
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Figure 3: Brightfield micrographs illustrating the distribution of serotonin-containing fibers in RT
following immunostaining against the serotonin transporter (SERT; A,B) or 5-HT (C,D). VAL: ventral
anterior lateral nucleus of thalamus; ic: internal capsule.
209x297mm (300 x 300 DPI)
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Figure 4: Brightfield micrographs the distribution of 5-HT1A (A,B) and 5-HT2A (C,D) receptors in
RT. 5-HT1A labelling is mainly restricted to the soma (asterisk) and occasionally observed within
proximal dendrites (arrows, B). 5-HT2A labelling, however, is more commonly observed within
proximal, medial and distal dendritic processes (arrows) as well as in the soma (asterisk, D).
209x297mm (300 x 300 DPI)