NEUROSCIENCE

RESEARCH ARTICLE
X. Li et al. / Neuroscience 441 (2020) 217–225

GABAergic Neurons in the Dorsal Raphe Nucleus that Express

5-HT3A Receptors Participate in Responses to Stress Hormones
Xiaotao Li, a,b,c*y Shanping Chen, a,dy Haiyang Yang, a,d Xiang Li, a Kwok-Fai So b,e and Liping Wang a*
a
Shenzhen Key Lab of Neuropsychiatric Modulation, Guangdong Provincial Key Laboratory of Brain Connectome and Behavior, CAS
Key Laboratory of Brain Connectome and Manipulation, CAS Center for Excellence in Brain Science and Intelligence Technology, the
Brain Cognition and Brain Disease Institute, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences; Shenzhen-Hong
Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, 518055, China
b
School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
c
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA
d
University of Chinese Academy of Sciences, Beijing, 100049, China
e
Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, GD, China

Abstract—The dorsal raphe nucleus (DRN) participates in stress responses and in mood regulation via its ascend-
ing release of serotonin (5-HT) onto neural circuits within the forebrain. Although the 5-HT DRN region is easily
defined via 5-HT-expressing DRN neurons, the neuroarchitecture and microcircuitry that confer its multifunction-
ality have remained incompletely understood and have required further investigation. In this present study, neu-
rochemical interactions within different subregions of the rat DRN were precisely analyzed. We found that 97.5%
of GABAergic neurons in the DRN expressed ionotropic 5-HT3A receptors (5-HT3ARs), whereas there were only
rare parvalbumin (PV)-positive or somatostatin (SOM)-positive GABAergic neurons. Furthermore, corticosterone
administration into male rats as a rodent model of depression induced significantly higher c-Fos expression in 5-
HT3AR-positive GABAergic neurons compared to that in 5-HT neurons within the DRN. Taken together, our find-
ings suggest that 5-HT3AR-positive GABAergic neurons in the DRN participate in responses to stress hormones
in a rat model of depression. Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: dorsal raphe nucleus, serotonin, GABA, 5HT3AR, stress, corticosterone.

INTRODUCTION gamma-aminobutyric acid (GABA), glutamate, and/or

The dorsal raphe nucleus (DRN) is a key source of
serotonin (5-HT) and plays a vital role in both stress
responses and mood regulation through controlling its
ascending release of 5-HT into the forebrain (Michelsen
et al., 2007). Currently available antidepressants, such
as selective serotonin reuptake inhibitors (SSRIs), are lar-
gely based on the modulation of the 5-HT DRN system to
reduce 5-HT turnover (Araragi and Lesch, 2013). Aside
from 5-HT neurons, many other types of neurons are also
located within the DRN including those expressing
*Corresponding authors address: Brain Cognition and Brain Disease
Institute, Shenzhen Institutes of Advanced Technology, Chinese
Academy of Sciences, Shenzhen-Hong Kong Institute of Brain
Science-Shenzhen Fundamental Research Institutions, Shenzhen,
GD, China (X. Li and L. Wang).
E-mail addresses: xtli@mit.edu (X. Li), lp.wang@siat.ac.cn (L. Wang).
y
Equally contributed to this study.
Abbreviations: DRI, interfascicular part of dorsal raphe nucleus; DRN,
dorsal raphe nucleus; DRV, ventral part of dorsal raphe nucleus;
DRVL, ventrolateral part of dorsal raphe nucleus; GABA, gamma-
aminobutyric acid; PV, parvalbumin; SOM, somatostatin; SSRIs,
selective serotonin reuptake inhibitors.
dopamine and participate in 5-HT-mediated multifunction-
alities (Michelsen et al., 2007). The 5-HT DRN is easily
defined by a distribution of 5-HT-positive neurons; how-
ever, the neuroarchitecture and microcircuitry underlying
5-HT functionality have not been fully elucidated.
As regulators of 5-HT production and release,
GABAergic interneurons and afferent terminals
innervating the DRN both play inhibitory roles via robust
synaptic contacts with 5-HT DRN neurons (Harandi
et al., 1987) via signaling through GABAergic receptors
(e.g. GABA-A receptors) (Gao et al., 1993). Previous
research has shown that some negative stressors induce
activation of GABAergic DRN neurons, which in turn inhi-
bit the activities of their neighboring 5-HT neurons (Roche
et al., 2003). There are several subtypes of GABAergic
neurons located in both cortical and subcortical regions
of mammalian brains (Rudy et al., 2011), including those
that express the Ca2+-binding protein, parvalbumin (PV),
as well as those expressing the neuropeptide, somato-
statin (SOM), or ionotropic 5-HT3A receptors (5-
HT3ARs). PV/GABA-positive neurons were located

https://doi.org/10.1016/j.neuroscience.2020.05.055
0306-4522/Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved.

217
218 X. Li et al. / Neuroscience 441 (2020) 217–225
around the lateral wings of the rat DRN (Tan et al., 2011),
and SOM/GABA-positive neurons have been found in the
mouse DRN (Fu et al., 2010b). In contrast, although high
densities of 5-HT3AR/GABA-positive neurons have been
found in both the neocortex and amygdala (Mascagni and
McDonald, 2007; Lee et al., 2010), this subpopulation has
not yet been identified in the rat DRN.
A primary function of the 5-HT raphe system is in
arousal-mediated modulation of neural circuits (Rueter
et al., 1997). The 5-HT system often acts through com-
plex signaling to modulate state-dependent moods and
behaviors, such as during the release of stress hormones.
An overload of stress hormones leads to irregular 5-HT
synthesis in the raphe system (Donner et al., 2012). Cor-
tisol is a glucocorticoid stress hormone in humans, and
corticosterone represents its analog in rodents (Pariante
and Miller, 2001). Rodents subjected to high-dose corti-
costerone administration exhibit depression-like behavior
that is similar to that of patients with Cushing’s disease,
who have abnormally high levels of cortisol in the blood
(Orth, 1995). Additionally, stress-related corticotrophin
releasing factor (CRF) in the DRN has been shown to
modulate 5-HT neural activity via CRF signaling
(Hammack et al., 2002), which might act together with
GABAergic signaling within the DRN (Maier and
Watkins, 2005).
In the present study, we found that the majority of
GABAergic neurons within the rat DRN expressed 5-
HT3ARs, whereas there was only a small number of
PV/GABA-positive neurons adjacent to the ventral
DRN and almost no SOM-positive cells were found.
Furthermore, we found that corticosterone administration
into male rats as a rodent model of depression induced
significantly higher c-Fos expression in 5-HT3AR-positive
GABAergic neurons compared to that in 5-HT neurons
within the DRN.
EXPERIMENTAL PROCEDURES
Animals
Adult male Sprague-Dawley (SD) rats (250–350 g,
n = 32 in total) were used in the present study. Rats
were kept in a 12:12 light/dark cycle with food and water
provided ad libitum. All experiments were performed in
accordance with policies on the utilization of animals
and humans in neuroscience research and were
approved by the Faculty Committee on the Use of Live
Animals in Teaching and Research (CULATR) at The
University of Hong Kong, as well as by the faculty
counterpart at the Shenzhen Institute of Advanced
Technology (Chinese Academy of Sciences, China).
Corticosterone administration and c-Fos detection
For corticosterone administration into SD rats,
corticosterone (CORT, C2505; Sigma-Aldrich, USA) at a
concentration of 40 mg/kg dissolved in sesame oil
(Sigma-Aldrich, USA) was injected daily into SD rats for
14 days, according to the methods reported in our
previous work (Yau et al., 2014). Briefly, a stock emulsion
of CORT was prepared daily by vortexing corticosterone
in sesame oil for 10 min, followed by 60 min of sonication.
Prior to every injection, the emulsion was vortexed briefly
and injections were made subcutaneously into the neck
region of each rat at 4:30 pm–6:30 pm in every day. Con-
trol treatment consisted of sesame oil without CORT.
Each injection in rats were less than 1 ml of liquid and
the needle with 21G size was kept in the rat body no more
than 1 minute. For c-Fos detection, at the last day of 15-
day experiments, rats were perfused to sacrifice with
deep anesthesia in the morning after the light condition
for 1.5 h.
Tissue collection and preparation
Following deep anesthesia with over-dose pentobarbital
sodium, rats were perfused with saline (pH 7.4) followed
by 4% paraformaldehyde (PFA) in 0.01 M of phosphate-
buffer saline (PBS, pH 7.4). Rat brains were harvested
and stored in 4% PFA at 4 °C overnight. The next day,
brains were rinsed in 0.01 M of PBS and were then
stored in 30% sucrose in 0.01 M of PBS until they sank.
The hindbrain including the midbrain raphe complex was
then serially sectioned at 30-mm intervals using a
microtome. Brain sections were collected as six
alternate sets of slices (with each set containing one
section at 180-mm intervals throughout the hindbrain)
and were stored at 20 °C in a cryoprotectant storage
buffer (30% ethylene glycol, 30% sucrose dissolve in
0.01 M of PBS until they were used for immunostaining
(Bouwknecht et al., 2007). For the experiment of c-Fos
quantification, one sixth of DRN serial sections each rat
was taken to conduct co-labelling stain with TPH or GABA
antibody. The animal number (n) was no less than 5 each
group.
Immunofluorescence staining
Tissues were rinsed three times in 0.01 M of PBS (10 min
per wash) and were then incubated for 1 h in blocking
solution with 5% donkey serum in PBS containing 0.1%
Triton-X-100, followed by incubation of primary
antibodies for 48 h at 4 °C. The primary antibodies used
in this study are listed in Table 1. The tissue sections
were then washed three times in PBS (10 min per
wash) and were incubated in corresponding secondary
antibodies (1:500, Molecular Probes) for 2 h at room
temperature. All sections were then washed three times
in PBS (10 min per wash) and were finally cover-slipped
with aqueous mounting medium (Dako Corp.,
Carpinteria, California, USA). Moreover, anti-GAD67
and anti-SOM antibody staining in the DRN was
performed with a tyramide signal amplification kit (TSA,
T20934, Molecular probes) to amplify signals, according
to the manufacturer’s protocol (Fu et al., 2010a).
Immunoperoxidase staining
Serial sections that included the midbrain raphe complex
were used for immunoperoxidase staining. The primary
antibodies that were used are listed in Table 1. The
procedure that we used for double immunostaining of c-
Fos and tryptophan hydroxylase (TPH) was as follows.
 X. Li et al. / Neuroscience 441 (2020) 217–225 219

Table 1. Primary antibodies used in this present study. Abbreviations are as follows: IFC, immunofluorescence; IP, immunoperoxidase

Name Cal Num & Host Dosage Description

company

serotonin (5HT) S5545, Sigma rabbit 1:1000 for IFC Immunostain for 5-HT cells (Zohar et al., 2015)
tryptophan hydroxylase T8575, Sigma sheep 1:1000 for IFC A key enzyme for 5-HT synthesis (Paul et al., 2011)
(TPH) 1:10 000 for IP
c-Fos PC38 (Ab-5), rabbit 1:1000 for IFC Immunostain for activated neurons after stimuli (Paul et al., 2011)
Calbiochem 1:3000 for IP
corticotropin releasing ab8901, rabbit 1:500 for IFC CRF involves stress response (Zhang et al., 2016)
factor (CRF) Abcam
glutamic acid MAB 5406, mouse 1:1000 for IFC A key enzyme for GABA synthesis (Xu et al., 2006)
decarboxylase-67 Chemicon 1:2000 for IP
(GAD-67)
gamma-aminobutyric A0310, Sigma mouse 1:1000 for IFC Immunostain for GABA cells (Gonchar et al., 2007)
acid (GABA)
5-HT3A, an ionotropic ab13897, rabbit 1:200 for IFC A sole ionotropic serotonin receptor among at least 14 types of 5HT
serotonin receptor abcam receptors (Wu et al., 2017; Farahani et al., 2019)
parvalbumin (PV) P3088, Sigma mouse 1:1000 for IFC a Ca2+-binding protein, parvalbumin (Rowniak et al., 2015)
somatostatin (SOM) AB5494, rabbit 1:100 for IFC Immunostain for SOM cells (Puighermanal et al., 2017)
Millipore

Tissue serial sections were washed three times (15 min
per wash) with 0.01 M of PBS in 12-well plates during
light shaking. Then, sections were rinsed in 1% H2O2 in
PBS for 15 min, followed by washing in PBS for 15 min
and pre-incubating in 0.1% PBST for 1 h. Sections were
then incubated overnight at room temperature with a
rabbit anti-c-Fos antibody (1: 3000) in 0.1% PBST. After
a 16-h incubation, the tissue sections were then washed
three times in PBS (10 min per wash), followed by
incubation with a biotinylated goat anti-rabbit secondary
antibody (1:200, E043201, Dako) in PBS for 90 min.
Tissue sections were then washed three times in PBS
(15 min per wash) and were then incubated in an
avidin–biotin-peroxidase complex (Elite ABC reagent,
1:200; PK-6100, Vector Laboratories) in PBS for 90 min.
Tissue sections were then washed three times in PBS
(15 min per wash) and incubated in a peroxidase
chromogen substrate (Vector SG, SK4700, Vector
Laboratories; diluted per the manufacturer’s instructions)
in PBS for 20 min. After the chromogen reaction, tissue
sections were immediately washed three times in PBS
(15 min each wash). After another treatment of 1%
H2O2 followed by the pre-incubation, DRN sections were
incubated with a sheep anti-TPH antibody (1: 10,000) in
0.1% PBST overnight at room temperature. All
subsequent steps were identical to those described
above for anti-c-Fos staining, except for the specifics of
the secondary antibody and chromogen reaction steps,
in which a biotinylated rabbit anti-sheep secondary
antibody (BA-6000, 1:200; Vector Laboratories) in PBS
for 90 min and a peroxidase chromogen substrate (DAB
substrate kit, SK-4100, Vector Laboratories) in distilled
water were used for 5 min. Finally, sections were
washed three times in PBS (15 min per wash) to stop
the reaction and were then mounted on microscope
slides and dehydrated. The color reaction of the c-Fos
immunostaining was blue-black and localized to the
nucleus, whereas TPH immunostaining was orange-
brown and localized to the cytoplasm. The
immunostaining process of GAD-67 (1:2000) co-
labelling with c-Fos was similar to that described
previously (Bouwknecht et al., 2007).
DRN location and analysis
One-sixth of the DRN sections were immunostained with
a rabbit anti-5HT antibody (S5545, Sigma) or a sheep
anti-TPH antibody (T8575, Sigma), which were used to
indicate the DRN region filled with serotoninergic
neurons. According to the different distances from the
bregma, the different subregions of the DRN can be
divided into the rostral, middle, and caudal DRN.
Additionally, these three subregions can be further
divided into the dorsal part of dorsal raphe nucleus
(DRD), ventral part of dorsal raphe nucleus (DRV),
ventrolateral part of dorsal raphe nucleus (DRVL),
interfascicular part of dorsal raphe nucleus (DRI), and
caudal part of dorsal raphe nucleus (DRC). The DRN
anatomy described in the present study was mainly
according to a classic rat stereotaxic atlas (Paxinos
et al., 1980).
Data acquisition and analysis
The images with immunofluorescence were captured
under a two-photo confocal microscope (LSM 710, Carl
Zeiss). Whereas the data with immunoperoxidase
staining were collected under the Neurolucida software
(MBF Bioscience). Statistical analysis was performed by
GraphPad Prism 7 (GraphPad Software Inc., SD, CA)
and all data were reported as the mean ± standard
error of the mean (SEM). For the normality of data
distributions, Shapiro-Wilk normality test was used to
evaluate. More details of the normality test were shown
at table S1. Thus Student’s t-test with Welch’s
correction was used for two groups where those
variances were not equal. Analyses of variance
(ANOVA) were used and followed by Sidak’s multiple
220 X. Li et al. / Neuroscience 441 (2020) 217–225

Fig. 1. Immunostaining delineating the DRN and its subsets of neurons. Co-labelled staining with 5-HT and TPH antibodies in DRN sections (A1–
A3). 5-HT cells mostly co-expressed glutamate (B). Whereas 5-HT cells were embedded with many GAD-67 positive puncta (C), as well as some
CRF fibers (D). (E1–E3) Immunoperoxidase staining with a TPH antibody was used to define the subregions of the DRN. Scale bars are as follows:
A–D, 20 mm; E1–E3, 50 mm.

comparisons test where appropriate. A p-value of <0.05
was considered at the statistically significant level.
RESULTS
The majority of GABAergic neurons in the DRN
express 5-HT3ARs
One-sixth of rat DRN sections were immunostained for
identifying 5-HT neurons within the DRN. Each TPH-
positive cell co-labeled with 5-HT staining (Fig. 1A1–
A3). Thus the TPH antibody with sheep host could be
used for 5-HT stain with more co-labelling antibodies
from other hosts. In the rat DRN, most 5-HT cells co-
expressed glutamate (Fig. 1B), and the 5-HT cells were
embedded with many GAD-67+ puncta (Fig. 1C) and
CRF+ fibers as well (Fig. 1D), which is in agreement
with findings from some previous study (Liu et al.,
2014). Moreover, according to a classic rat stereotaxic
atlas (Paxinos et al., 1980), different subregions of the
DRN were defined (Fig. 1E1–E3). All quantified analysis
 X. Li et al. / Neuroscience 441 (2020) 217–225 221

5-HT3ARs (Fig. 2E–G, H1–H3).

We found that the population of PV/
GABA-positive cells was sparse in
the DRN (Fig. 2A, B), even though
dense intra-DRN PV+ terminals
were found (Fig. 2B). Most PV+
somata were located within two
areas of the medial longitudinal
fasciculus (mlf) adjacent to the
DRV subregion (Fig. 2A–C). In
contrast, SOM expression was
extremely weak within the DRN,
even after using a TSA amplification
kit to amplify any signal (Fig. 2D3
with TSA kit). However, 5-HT3ARs
robustly co-labeled with GABAergic
cells throughout the entire DRN
(Fig. 2E–G, H1–H3). Approximately
97.5% of GABAergic cells within
the DRN co-expressed 5-HT3ARs
(Fig. 2I, n = 6), which is similar to
findings of 5-HT3AR + GABAergic
cells in the neocortex and
amygdala reported by other
groups (Mascagni and McDonald,
2007; Rudy et al., 2011). Further-
more, we found that PV+ cells
accounted for approximately 2.0%
of all GABAergic cells in the DRN
(Fig. 2I, n = 6), and they were
mainly located at the boundary of
the DRV subregion. In contrast,
SOM/GABA-positive cells only
accounted for approximately 0.5%
of all GABAergic cells in the DRN
(Fig. 2I, n = 6), if considering some
extreme expression of SOM was
still available along the boundary
of DRV subregion.

Significant c-Fos expression in

5-HT3AR-positive GABAergic
neurons in the DRN following
corticosterone administration
A high dose of corticosterone
administration (40 mg/kg/day) for
14 days was used as a rodent
model of depression since this
Fig. 2. The majority of GABAergic cells in the DRN express 5-HT3ARs. (A) 5-HT cells (green) and PV protocol has been shown to
cells (red) are both shown at the DRN. (B) and (C) are enlarged images from (A). Note that most PV induce significant depression-like
cells (red) were not located in the middle DRN (B) but were found along the boundary of the DRV behavior in rats (Yau et al., 2014).
subregion (C). (D1–D3) SOM antibody was used to stain at the DRN using a TSA kit (green), but not
obvious expression. (E–G) and (H1–H3) 5-HT3AR antibody co-staining with GABAergic cells in the
We found that there was a signifi-
DRN. 5-HT3AR + GABAergic cells predominated in the DRN, with 97.5% of all GABAergic cells, as cant increase in c-Fos expression
shown at (I, n = 6). Scale bars are as follows: A-H, 20 mm. (For interpretation of the references to in the DRNs of rats following corti-
colour in this figure legend, the reader is referred to the web version of this article.) costerone administration (Fig. 3A,
Oil-ctrl group vs. CORT group,
of the rat DRN subregions in the present study were n = 5 each group, unpaired t-test, t (7.112) = 3.193,
based on these definitions (Abrams et al., 2004). p < 0.05), mainly due to an increase of c-Fos expression
Three key biomarkers of GABAergic neurons were used at the middle portion of each DRN (Fig. 3C, t (7.517)
to stain the DRN, including antibodies for parvalbumin = 3.072, p < 0.05), including at the DRVL and DRV parts
(PV, Fig. 2A–C), somatostatin (SOM, Fig. 2D1–D3), and of middle DRN (Fig. 3F, G, for DRVL part, t (7.890)
222 X. Li et al. / Neuroscience 441 (2020) 217–225

c-Fos cells increased from 32.3%

± 6.8% to 85.1% ± 2.1% follow-
ing corticosterone administration
(Fig. 4F, n = 6 in Oil-ctrl group,
n = 10 in CORT group, unpaired
t-test, t (5.993) = 8.157, p <
0.001); and the increase of the
percentage of these co-labeled
cells in total GABA cells from
1.9% ± 0.6% to 27.71% ± 3.0%
in the DRN following corticos-
terone administration (Fig. 4G,
n = 6 in Oil-ctrl group, n = 10 in
CORT group, F (1, 22) = 31.94,
p < 0.0001, two-way ANOVA
followed by Sidak’s multiple
comparisons test). In contrast, the
co-localization of c-Fos with TPH
+ serotonergic neurons were
not significantly changed follow-
ing corticosterone administra-
tion (Fig. 4A, B, E, and G). There
were kept approximately 30.0%
TPH/c-Fos+ cells and 4.0% c-
Fos/TPH + cells (Fig. 4E, G,
n = 5 each group). These findings
are similar to those from a
previous study (Torterolo et al.,
2000). Moreover, c-Fos + GABAer-
gic cells were evenly distributed
throughout the entire DRN (Fig. 4C),
including the DRD subregion
(Fig. 4D, D0 ), which corresponded
spatially with the distribution of
5HT3AR + GABAergic cells
(Fig. 2H1–H3).

DISCUSSION
Fig. 3. Increased c-Fos expression in the DRN following corticosterone administration. (A–G) Bar Quantification of subtypes of 5-HT
graphs represent changes in the numbers of c-Fos expression in different subregions of the DRN
following corticosterone administration (40 mg/kg/day for 14 days). Values represent means ± SEM
DRN neurons can be performed
(n = 5 in each group). Unpaired t-test with Welch’s correction was used: for DRN (A), *p = 0.0149, t precisely since the subregions
(7.112) = 3.193; for Middle portion of DRN (C), *p = 0.0165, t (7.517) = 3.072; for DRVL part (F), of the DRN have previously been
*p = 0.0320, t (7.890) = 2.599; for DRV part (G), *p = 0.0186, t (6.840) = 3.068. An image (a) defined with high spatial specifi-
showing the co-staining of c-Fos and TPH. The arrows indicate c-Fos+ staining in the nucleus cities (Abrams et al., 2004). We
(black), while the arrowhead represents a c-Fos-positive 5-HT cell (black + brown). Abbreviations are
as follows: Oil-ctrl: oil-injected control group; CORT: corticosterone-injected group. (For interpretation found that most GABAergic cells
in the DRN expressed 5-HT3ARs,
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
while PV-positive or SOM-positive
GABA cells were sparse (Fig. 2).
Furthermore, our results indicated
= 2.599, p < 0.05; for DRV part, t (6.840) = 3.068, that these 5HT3AR + GABAergic neurons were sensitive
p < 0.05). This finding implicates the middle DRN as to high levels of stress, since high-dose corticosterone
the main locus of increased c-Fos expression in the administration into rats (40 mg/kg) induced a significantly
DRN following corticosterone administration. In particular, increased expression of c-Fos in 5HT3AR + GABAergic
the number of c-Fos-positive cells in the DRVL increased neurons compared to that of 5-HT neurons within the
from 19.80 ± 3.62 to 32.40 ± 3.22 (Fig. 3F). DRN.
Furthermore, the increased c-Fos expression in the According to a previous study (Rudy et al., 2011),
DRN following corticosterone administration was there are three main subtypes of GABAergic neurons
predominantly localized to GAD67 + GABAergic located in both cortical and subcortical regions, which
neurons (Fig. 4C, D, F, and G), as the percentage of consist of PV+ cells, SOM+ cells, and 5-HT3AR+ cells.
these co-labeled cells in total Interestingly, we found that the population of PV cells in
 X. Li et al. / Neuroscience 441 (2020) 217–225 223

In contrast, we did not detect any

noteworthy SOM expression in
the DRN at this study (Fig. 2D1–
D3). Although some previous stud-
ies have revealed SOM expression
in the DRN (Vincent et al., 1985),
those results only revealed a small
number of 5-HT cells with SOM
expression within the DRN
(Araneda et al., 1999), whereas
some SOM+ cells seem to be
located just outside the lateral
wings of the DRN (i.e. not within
the DRN directly) (Fu et al.,
2010b).
Ionotropic 5-HT3ARs have
been shown to be expressed in
most (if not all) GABAergic cells in
the mouse cortex that do not
express PV or SOM (Lee et al.,
2010). In our present study, most
GABAergic cells in the rat DRN
were also found to express 5-
HT3ARs (97.5%, Fig. 2I), although
the mouse DRN has not been
found to exhibit 5-HT3AR expres-
sion in a transgenic mouse
expressing 5-HT3A-GFP (Koyama
et al., 2017). That was most likely
to be species difference between
the rat and the mouse. The
5HT3AR + GABAergic cells in the
rat DRN that we found in our pre-
sent study may be similar to the
GABAergic cells in the rat amygdala
that have been previously reported
(Mascagni and McDonald, 2007).
Based on previous study, those
GABAergic cells expressing 5-
HT3ARs do not usually overlap
with PV-positive or SOM-positive
cells (Rudy et al., 2011), but do
tend to co-express vasoactive
Fig. 4. Increased c-Fos expression in the DRN following corticosterone administration is mainly
localized to GABAergic neurons. (A) and (B) show co-staining of c-Fos and TPH, while C and D show
intestinal peptide or neuropeptide
0
co-staining of c-Fos and GAD-67. Black arrows in (B) and (D ) show some c-Fos+ nuclei (black), Y (Vucurovic et al., 2010).
while the black arrowhead indicates c-Fos co-labelling with a TPH+ cell (B, black + brown); white In our present study, we further
arrows at (D) and (D0 ) indicate c-Fos co-labelling with GAD-67+ cells (black + brown), whereas white detected a significantly increased
arrowheads show GAD-67 cells without c-Fos expression (brown). Note that c-Fos+ GABAergic cells activation of 5HT3AR + GABAergic
are distributed throughout the entire DRN. (E–G) Bar graphs represent percentage changes in c-Fos
expression by means ± SEM, in the TPH-positive cells (n = 5 each group) and GAD-67-positive cells cells, but not TPH + 5-HT cells in
(n = 6 in Oil-ctrl group, n = 10 in CORT group). Compared to that in 5-HT cells, an increased DRN, following corticosterone
expression of c-Fos significantly occurred in GAD-67 + GABAergic cells (at (F) ***p = 0.0002, t administration (Figs. 3 and 4).
(5.993) = 8.157, unpaired t-test with Welch’s correction; at (G) ****p < 0.0001, F (1, 22) = 31.94, This finding is similar to the
two-way ANOVA followed by Sidak’s multiple comparisons test). Scale bars are as follows: A and C,
0
50 mm; B, D and D , 10 mm. Abbreviations are as follows: Oil-ctrl: oil-injected control group; CORT:
activation pattern of such cells
corticosterone-injected group. during rapid-eye-movement sleep
(Monti, 2010). Additionally,
GABAergic cells in the DRN that
the DRN was sparse (Fig. 2), although many PV+ termi- have been shown to exhibit high c-Fos expression have
nals were found (Fig. 2B). Most of these PV+ GABAergic been demonstrated to inhibit 5-HT activity in the DRN
cells were located along the boundary of the DRV subre- (Torterolo et al., 2000). That phenomenon has been
gion in close apposition to the mlf (Fig. 2A–C), which is demonstrated in stressed rats with high stress hormones,
similar to finding of a previous study (Tan et al., 2011). which may account for their depression-like responses
224 X. Li et al. / Neuroscience 441 (2020) 217–225
(Yau et al., 2014), similar to some other reports in rodents
(Ishida et al., 2002; Roche et al., 2003). Rodents sub-
jected to high-dose corticosterone administration
(40 mg/kg/day for 2–3 weeks) were usually used for the
study of depression-like behavior (Johnson et al., 2006).
Increased activation of 5HT3AR + GABAergic neurons
as we found using c-Fos indication may further inhibit 5-
HT synthesis, which may also be associated with
depression-like responses in rats (Detke et al., 1995).
Although the precise function of 5-HT3AR signaling in
DRN GABAergic neurons remains unclear, previous stud-
ies have shown that 5-HT3AR activation and deactivation
as indicated by differential c-Fos levels are associated
with depression-like and antidepressant-like responses,
respectively (Kos et al., 2006; Artigas, 2013), which sug-
gests that 5-HT3ARs may represent therapeutic targets in
depressed individuals.
ACKNOWLEDGMENTS
This work was supported by the Key-Area Research and
Development Program of Guangdong Province
(2018B030331001), the Commission on Innovation and
Technology in Shenzhen Municipality of China
(JCYJ20150630114942262), International Postdoctoral
Exchange Fellowship Program by the China
Postdoctoral Council (20160021), and the International
Partnership Program of Chinese Academy of Sciences
(172644KYS820170004). This was also supported by
grants from Project of International, as well as Hong
Kong, Macao & Taiwan Science and Technology
Cooperation Innovation Platform in Universities in
Guangdong Province (2013gjhz0002).
DECLARATION OF COMPETING INTEREST
The authors declare that they have no competing
interests.
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