Received: 26 June 2018

| Revised: 11 January 2019

| Accepted: 30 January 2019

DOI: 10.1111/ejn.14368

RESEARCH REPORT

Hippocampal activation of 5‐HT1B receptors and BDNF

production by vagus nerve stimulation in rats under chronic
restraint stress

Hwa Chul Shin1* | Byung Gon Jo1* | Chan‐Yong Lee2 | Kang‐Woo Lee1 |
1
Uk Namgung

1
Department of Oriental Medicine, Daejeon
University, Daejeon, Korea
Abstract
2
Department of Microbiology and A growing body of evidence shows that the electrical stimulation of the vagus nerve
Biotechnology, Daejeon University, can improve mental illness including depression. Here, we investigated whether the
Daejeon, Korea
vagus nerve stimulation (VNS) is involved in regulating the responsiveness of hip-
Correspondence pocampal neurons in rats under chronic restraint stress (CRS). c‐Fos protein signals
Uk Namgung, Department of Oriental were detected 2 hr after VNS in 5‐HT1A receptor‐positive neurons in the dorsal raphe
Medicine, Daejeon University, Daejeon,
nucleus (DRN) as well as in the nucleus tractus solitarius (NTS). Chronic VNS was
Korea.
Email: [email protected] performed on a daily basis for 2 weeks using an implanted microelectrode in rats that
had undergone CRS for 2 weeks. We found that the levels of both 5‐HT1B receptors
Funding information
Daejeon University, Grant/Award Number:
and phospho‐Erk1/2 were decreased in parallel in the hippocampal neurons of CRS
20173711 animals and then increased to the baseline levels by chronic VNS. Hippocampal in-
duction of 5‐HT1B receptors and phospho‐Erk1/2 by VNS was diminished after the
injection of 5,7‐dihydroxytryptamine (5,7‐DHT), a neurotoxin of serotonergic neu-
rons, into the DRN. Hippocampal production of brain‐derived neurotrophic factor
(BDNF) was also upregulated by VNS, but the treatment of 5,7‐DHT abrogated the
effects of VNS on BDNF induction. VNS in CRS animals improved the behavioral
scores in forced swimming test (FST) compared to sham‐stimulated control. Our re-
sults suggest that VNS‐mediated serotonergic input via 5‐HT1B receptors into the
hippocampal neurons may activate BDNF pathway and improve depressive‐like be-
haviors in CRS animals.

KEYWORDS
animal model, BDNF, depression, dorsal raphe nucleus, Phospho‐Erk1/2

Abbreviations: 5,7‐DHT, 5,7‐dihydroxytryptamine; BDNF, brain‐derived neurotrophic factor; CRS, chronic restraint stress; DRN, dorsal raphe nucleus;
NTS, nucleus tractus solitarius; VNS, vagus nerve stimulation.

*These authors contributed equally.

Edited by Marco Capogna. Reviewed by Kinga Gavel, Pawel Matulewicz, Anna Serefko and Mikhail Paveliev.
All peer review communications can be found with the online version of the article.

1820 | © 2019 Federation of European Neuroscience Societies wileyonlinelibrary.com/journal/ejn Eur J Neurosci. 2019;50:1820–1830.
and John Wiley & Sons Ltd
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SHIN et al.    1821

depression or depression‐like symptoms in both human and

1 | IN T RO D U C T ION
experimental animals.
Chronic restraint stress (CRS) is widely used to examine
In the vagus nerve, afferent nerve fibers account about 80%
stress‐induced depression‐like behavior (Chiba et al., 2012)
and send sensory inputs from major internal organs to the
and related neurobiological factors including stress‐related
neurons in the nucleus tractus solitarius (NTS). While some
hormones and neurotransmitters in a brain (Toth & Neumann,
of these signals are transmitted into the vagal motor neurons
2013). We reasoned that electrical stimulation of the vagus
in the brainstem for feedback regulation of internal organs,
nerve may activate neurobiological responses in the brain in
others are connected to the nuclei in the upper brain areas.
CRS animals and contribute to behavioral improvement, as
Accumulating evidence indicates that the vagus nerve acts
has been implicated in human patients of depression. So far,
as a bridge connecting between the brain and gut including
the effects of VNS have not been explored by using animal
enteric nervous system (Han et al., 2018), which is termed as
models of depression while there are several reports show-
brain‐gut axis, and the vagus nerve activity may be involved
ing that the application of VNS in normal animals induced
in neuroimmune modulation (Breit, Kupferberg, Rogler, &
antidepressant‐like and anxiolytic activities (Furmaga et al.,
Hasler, 2018). Ever since the vagus nerve stimulation (VNS)
2011; Grimonprez et al., 2015; Krahl, Senanayake, Pekary,
was investigated initially for the control of epileptic seizure in
& Sattin, 2014). Here, we investigated VNS in CRS animals
dogs (Zabara, 1985a,1985b), it has been applied for the treat-
and tried to delineate the relationship between VNS‐induced
ments of mental illness such as treatment‐resistant depres-
neural pathway and behavioral consequences.
sion, memory improvement, and epileptic seizures (Carreno
& Frazer, 2017; Clark, Naritoku, Smith, Browning, & Jensen,
1999; George et al., 2000; Penry & Dean, 1990). 2 | M ATERIAL S AND M ETHO D S
While there has been growing concern on clinical applica-
tion of VNS for the last two decades, its use is largely empirical 2.1 | Experimental animals
and is weakly supported by scientific evidence. Application Sprague‐Dawley rats (male, 200–250 g) were purchased
of acute VNS in normal rats induced c‐Fos signals in NTS, from Samtako (Seoul, Korea), and maintained in animal
parabrachial nucleus, paraventricular nucleus of hypothala- room with regulated temperature (22°C), 60% humidity, and
mus ,and locus coeruleus (Cunningham, Mifflin, Gould, & a 12‐h light and 12‐h dark cycle. All protocols involving live
Frazer, 2008), suggesting the activation of ascending central and postoperative animal care were approved by the Daejeon
pathways by VNS. Chronic VNS in rats activated adrener- University Institutional Animal Use and Care Committee,
gic neurons in the locus coeruleus and serotonergic neurons and were in accordance with the Animal‐Use Statement
in the dorsal raphe nucleus (DRN), and serotonergic nerve and Ethics Committee Approval Statement for Animal
was required for VNS‐mediated antidepressant‐like effects Experiments provided by Daejeon University (Protocol num-
(Furmaga, Shah, & Frazer, 2011; Manta, Dong, Debonnel, & ber: DJUARB2014‐036; Daejeon, Korea). A total of 102
Blier, 2009). Other studies provide evidence that VNS affects rats were used for the present investigation. The number of
levels of depression‐related biological parameters in human rats assigned to individual experimental groups are listed in
patients. For instance, the levels of adrenocorticotropic hor- Table 1.
mone (ACTH) in the serum, which were significantly in-
creased by corticotropin‐releasing hormone (CRH) challenge
in chronically depressed patients compared with healthy con-
2.2 | CRS and VNS
trol group, were reduced by VNS (O'Keane, Dinan, Scott, We conducted acute and chronic VNS surgeries in rats. For
& Corcoran, 2005). It was also reported in human subjects VNS surgery, animals were anesthetized with a mixture of
that serum levels of some proinflammatory cytokines were ketamine (80 mg/kg) and xylazine (5 mg/kg) and the left
associated with severity of depressive symptoms, and VNS vagus nerve was exposed around ascending carotid artery.
was effective in regulating pro‐ and anti‐inflammatory cy- Acute VNS surgery was performed by placing the exposed
tokines (Corcoran, Connor, O'Keane, & Garland, 2005; nerve on a bipolar hook electrode of nichrome wire (Olofsson
Suarez, Krishnan, & Lewis, 2003). However, in animal stud- et al., 2015). The electrical current (10 mA, 5 Hz with 5 ms of
ies, acute or chronic VNS induced the expression of IL‐1β in pulse duration, 5 min; A‐M Systems Inc., Sequim, USA) was
the hypothalamus and hippocampus and also increased the applied once and animal was killed 2 hr later for immuno-
expression of CRH and ACTH leading to increased serum fluorescence analysis. Chronic VNS was applied to animals
corticosterone levels (De Herdt et al., 2009; Hosoi, Okuma, that had undergone CRS. CRS was given to rats as described
& Nomura, 2000). Taken together, VNS may influence hor- previously (Shin et al., 2017). Briefly, rats were placed in a
monal regulation through the hypothalamic‐pituitary‐adrenal well‐ventilated cylindrical shape of Plexiglas container for
axis and production of inflammatory cytokines in relation to 6 hr (9:00 a.m.–15:00 p.m.) each day for 14 days. Following
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1822    SHIN et al.

TABLE 1 The number of animals used in the present study

Histology Behavioral tests

Animal groups Experiments Western blotting IFS H&E FST OFT

Acute VNS CTL + acute VNS 2
Chronic VNS CTL 4 2 10 8
CRS + VNS 4 2 10 8
CRS + Sham 4 2 10 8
CRS + VNS + DMT 4 2
CRS + VNS + saline 4 2
BDNF injection CRS + BDNF 8
CRS + Vehicle 8
FST, forced swimming test; H&E, hematoxylin and eosin staining; IFS, immunofluorescence staining; OFT, open field test.

CRS, animals were randomly assigned into chronic VNS‐ Hyde, & MacLeod, 1979; Kiianmaa & Fuxe, 1977; Vergnes
and sham‐treated groups (CRS + VNS and CRS + Sham, re- & Kempf, 1982).
spectively), together with untreated control group (CTL). For For the injection of BDNF into the hippocampal CA1
chronic VNS, a custom‐built, coiled platinum nerve cuff elec- area, rats were anesthetized with ketamine (80 mg/kg) and
trode (Inner diameter; 0.75 mm; Microprobes, Gaithersburg, xylazine (5 mg/kg) and placed onto the stereotaxic ap-
MD, USA) was implanted by referring to an experimental paratus. BDNF (500 ng/μl in 0.1% BSA and 0.9% saline,
procedure described by Zhang et al. (2013). The nerve cuff Sigma‐Aldrich, MO, USA) or an equivalent volume of saline
was wrapped around the exposed nerve, and the platinum vehicle was bilaterally injected into the hippocampal CA1
wire, which was placed between the skin and muscle around area (coordinate, AP: −4.2 mm, ML: 2.0 mm, DV: 1.8 mm)
the neck, was extended all the way back to the neck where (Alonso et al., 2005; Paxinos & Watson, 1998) with a flow
the end of the wire was connected to the stimulation unit rate of 0.25 μl/min for 2 min by using micropump (Harvard
(A‐M Systems Inc., Carlsborg, WA, USA). After suture, ani- Instrument).
mals were recovered from anesthesia and returned to animal
room. Electrical stimulation of the vagus nerve was initiated
3 days after the implantation of the simulation electrode with
2.4 | Western blot analysis
the same stimulation condition as acute stimulation and was Hippocampal tissues were rapidly dissected from rats and
given for 14 days. For sham control, the left vagus nerves of sonicated in triton lysis buffer (20 mM Tris; pH 7.4, 137 mM
CRS animals were exposed and then closed by suturing the NaCl, 25 mM β‐glycerophosphate; pH 7.14, 2 mM sodium
skin. pyrophosphate, 2 mM ethylenediaminetetraacetic acid, 1 mM
Na3VO4, 1% Triton X‐100, 10% glycerol, 5 μg/ml leupeptin,
5 μg/ml aprotinin, 3 μM benzamidine, 0.5 mM dithiothrei-
2.3 | Focal administration of drugs
tol, 1 mM phenylmethanesulfonyl fluoride). The supernatant
To remove serotonergic neurons in the DRN, we anesthetized was taken after centrifugation at 8,050 g for 10 min at 4°C.
rats with ketamine (80 mg/kg) and xylazine (5 mg/kg) and Protein (15 μg) was resolved by SDS polyacrylamide gel
placed in the stereotaxic apparatus. The dorsal skin on the electrophoresis and subjected to immunoblotting. Antibodies
area of hindbrain was exposed and the skull was perforated used were anti‐5‐HT1B receptor (rabbit‐polyclonal, 1:2000;
by using a drill. Glass microcapillary tube filled with 5,7‐di- Abcam, Cambridge, UK), p‐Erk1/2 (mouse‐monoclonal; rab-
hydroxytryptamine (5,7‐DHT) solution (5 mM in 0.9% saline bit‐polyclonal, 1:2000, Cell Signaling Technology, Danvers,
containing 1% ascorbic acid, Chemodex, Gallen, Switzerland) MA, USA), anti‐BDNF (rabbit‐monoclonal, 1:2000; Abcam),
was stereotaxically guided into the DRN (from the bregma and anti‐actin (monoclonal, 1:10,000; MP Bio, Aurora, USA)
to posterior: −8.0 mm; L: 0 mm; DV: −5.8 mm) (Paxinos & primary antibodies, and horseradish peroxidase (HRP)‐con-
Watson, 1998) and drug solution containing 5,7‐DMT was jugated secondary antibodies (goat anti‐rabbit; 1:1000; Santa
injected with a flow rate of 1 μl/min for 5 min by using a Cruz Biotech, or anti‐mouse; 1:000, Amersham Biosciences,
micropump (Pump 11 Elite; Harvard Apparatus, Holliston, Buckinghamshire, UK). Protein bands were quantified by
MA, USA). The amount of injection of 5,7‐DMT (4.8 μg) densitometric analysis of scanned images of X‐ray film
was similar to doses which were injected (4–5 μg) into raphe by using the i‐Solution software (Image & Microscope
nucleus and hypothalamus of rats in previous studies (File, Technology, Daejeon, Korea).
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SHIN et al.    1823

and placed in a transparent cylindrical chamber (height 50 cm,

2.5 | Immunofluorescence staining
diameter 25 cm) filled with water at 27°C (depth 40 cm).
Animals were killed with an overdose of ketamine and xyla- Scores of immobility and climbing duration were measured for
zine and transcardially perfused with 4% paraformaldehyde 5 min following an initial 2 min of adjustment in water.
and 4% sucrose in phosphate‐buffered saline (PBS). The brain For open field test (OFT), rats in three experimental
was dissected and immersed in 15% sucrose solution over- groups (CTL, CRS + Sham, CRS + VNS) were prepared as
night. Sagittal or coronal brain sections (10 or 20 μm thickness) described above and were subjected to the test as described
were prepared using a Cryostat (Leica, Wetzlar, Germany) previously (Cheng et al., 2017; Shin et al., 2017). Briefly,
and thaw‐mounted on gelatin‐coated slides. Pretreatment, animals were adjusted by placing in a test room for 1 hr and
blocking, and antibody reactions were performed as described placed into a square chamber (70 × 70 × 40 cm3). Behavioral
previously (Chang, Kim, & Namgung,2012). Primary anti- scores were measured for 10 min following an initial 5 min
bodies used were anti‐c‐Fos (monoclonal, 1:400; Abcam), of adjustment. Spontaneous locomotor activity was mea-
anti‐5‐HT1A receptor (rabbit‐polyclonal, 1:400; Abcam), sured for 10 min by monitoring the total distance of move-
anti‐5‐HT1B receptor (rabbit‐polyclonal, 1:400; Abcam), ment by using video tracking software (SMART 3.0; Panlab
anti‐phospho‐Erk1/2 (mouse‐monoclonal; rabbit‐polyclonal, S.L.U.), and the frequency of rearing, a behavior that the rat
1:400, Cell Signaling Technology), and anti‐NF‐200 (mouse‐ stands upright on its hind legs, was counted for 10 min.
monoclonal, 1:400, Sigma‐Aldrich) antibodies, and second-
ary antibodies were fluorescein‐goat anti‐mouse (1:400;
Molecular Probes, Eugene, USA) and rhodamine‐goat anti‐
2.7 | Statistical analysis
rabbit (1:400; Invitrogen, Carlsbad, USA) antibodies. Nuclear Data were presented as mean ± standard error of mean
staining was performed using Hoechst dye 33258 (bisbenzi- (SEM). The number data among animal groups were com-
mide; 2.5 μg/ml; Sigma) for 10 min before the final washing pared by Student's t test or one‐way ANOVA followed by
step. Fluorescence images were visualized and captured by Tukey's multiple comparison test (GraphPad Prism 7.00),
digital camera attached to fluorescence microscope (Nikon, and statistically significant differences were set at p < 0.05.
Japan), and transferred to a computer for analysis by using
i‐Solution software (Image and Microscope Technology,
Daejeon, Korea). 3 | RESULTS

To determine whether VNS affected the activation in as-

2.6 | Forced swimming test and open
cending neuronal pathways in a brain, we prepared sagittal
field test
brain sections 2 hr after VNS and analyzed c‐Fos protein
Forced swimming test was performed as previously described signals by immunofluorescence staining. c‐Fos signals
(Bogdanova, Kanekar, D'Anci, & Renshaw, 2013; Shin et al., were clearly induced in neurons of the NTS and the DRN
2017). Each rat was placed in a transparent cylindrical cham- (Figure 1). 5‐HT1A receptors are known to be expressed in
ber (height 50 cm, diameter 25 cm) filled with water at 27°C neurons of the raphe nucleus and involved in regulating
(depth 40 cm). Twenty four hours before the test, animals were serotonergic neuronal function as an autoreceptor (Artigas,
adjusted in water for 15 min. Swimming test was conducted 2013). Our data show that c‐Fos signals in the DRN were
for 5 min following an initial 2 min of adjustment in water, and largely colocalized with neurons expressing 5‐HT1A recep-
the immobility time and climbing time were analyzed by using tors, suggesting the activation of serotonergic neurons in
a Smart version 2.5 video tracking system (Panlab, Barcelona, the DRN by VNS.
Spain). Immobility time was determined by measuring the pe- Serotonergic neurons in the raphe nucleus project axons
riod during which animals kept maintaining the head above the into several forebrain areas including the hippocampus. To
water without any additional actions. Duration of climbing be- determine whether VNS affected serotonergic neuronal ac-
havior was determined by measuring the time during which an- tivity in the hippocampus in association with depression‐like
imals displayed upward‐directed movements of the forepaws behavior, we compared production levels of hippocampal 5‐
along the side of the swim chamber. FST was also performed HT1B receptors among animal groups of CTL, CRS + Sham,
in CRS + BDNF and CRS + vehicle groups of rats. CRS was and CRS + VNS. Levels of 5‐HT1B receptors were signifi-
given to rats for 14 days (9:00 a.m.–15:00 p.m.) by placing in a cantly decreased in CRS + Sham animals compared with un-
well‐ventilated cylindrical shape of Plexiglas container for 6 hr treated CTL (p = 0.049), and then elevated to a similar level
for 14 consecutive days. Then 24 hr later, BDNF or saline was as that of CTL (p = 0.022, CRS + Sham vs. CRS + VNS)
injected into the hippocampus as described above, and 24 hr (Figure 2a). Examination of the production of 5‐HT1B recep-
later, CRS + BDNF and CRS + Vehicle groups of rats were tors in the hippocampal subfields revealed that clear signals
subjected to FST. Animals were adjusted for 1 hr in a test room were detected in CA1 area of control group and CRS + VNS
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1824    SHIN et al.

F I G U R E 1 Immunofluorescence
images of c‐Fos and 5‐HT1A receptor signals
in brain tissues. Sagittal brain sections
were prepared from rats 2 hr after VNS and
used for immunofluorescence staining by
antibodies as indicated in the figures. DRN,
dorsal raphe nucleus; NTS, nucleus tractus
solitaries. [Colour figure can be viewed at
wileyonlinelibrary.com]

groups, but the signal intensity was much reduced in durations of immobility and climbing behaviors. Immobility
CRS + Sham group (Figure 2b). Intense signals were seen in time in CRS animals was significantly increased com-
the soma of CA1 neurons and also in the stratum radiatum (ar- pared to control animals (p = 0.0001), and then decreased
rows in Figure 2b). We also found that the levels of phospho‐ by VNS (p = 0.0061) (Figure 4a). Climbing duration was
Erk1/2 protein in the hippocampus were significantly reduced significantly decreased in CRS group compared with un-
in CRS + Sham group compared to control (p = 0.0023), treated control group and elevated by VNS ((p = 0.0007 and
and then upregulated by VNS (p = 0.0013, CRS + Sham vs. p = 0.005, respectively) (Figure 4b). To determine whether
CRS + VNS) (Figure 2c). It was observed that, in both CTL or not the behavioral scores in FST were affected by animals’
and CRS + VNS groups, signals of phospho‐Erk1/2 and 5‐ spontaneous locomotor activity, we analyzed the distance
HT1B receptors were highly colocalized in hippocampal CA1 of movement and rearing behavior in OFT. Comparison of
neurons, but the signal intensities of both proteins were much movement among CTL, CRS + Sham, and CRS + VNS
weaker in CRS + Sham group (Figure 2d). groups did not show any significant differences (p = 0.98
To further investigate whether serotonergic raphe neu- for CTL vs. CRS + Sham; p = 0.81 for CRS + Sham vs.
rons were required for the responses of hippocampal neurons CRS + VNS; Figure 4c). Also, the measurement of rearing
in CRS + VNS animals, we injected a neurotoxin 5,7‐DHT behavior showed no significant differences between CTL and
into the DRN area to destroy serotonergic neurons and in- CRS + Sham (p = 0.36) and CRS + Sham and CRS + VNS
vestigated hippocampal proteins. Injection of 5,7‐DHT into (p = 0.75) (Figure 4d), suggesting that the results obtained
the DRN area of CRS + VNS animals resulted in a loss of from the FST were not affected by their spontaneous loco-
many neurons in the DRN (Figure 3a). Levels of 5‐HT1B motor activity.
receptors and phospho‐Erk1/2 in CRS+VNS animals were To further examine whether hippocampal expression of
significantly decreased by 5,7‐DHT injection compared BDNF mimics the antidepressant effect of VNS, we injected
with saline control (p = 0.019 and p = 0.035, respectively, BDNF into the hippocampus of CRS animals and analyzed
Figure 3b,c). Previous studies reported that hippocampal FST scores. There were some changes in immobility and
production of BDNF is closely related to anti‐depression‐ climbing durations in BDNF‐injected group compared to
like effects in animal model of depression (Shirayama, vehicle‐treated group (14.3% decrease and 24.3% increase,
Chen, Nakagawa, Russell, & Duman, 2002). BDNF level respectively) but the differences were not statistically signif-
was significantly decreased in CRS animals compared to icant (p = 0.11 and p = 0.23, respectively, t test unpaired)
control animals and then elevated by VNS (p = 0.035 and (Figure 4e,f).
p = 0.012, respectively, Figure 3d). Then, the treatment of
5,7‐DHT suppressed BDNF induction by VNS (p = 0.04).
Taken together, these data indicate that the activation of 4 | DISCUSSION
serotonergic neurons in the DRN may be involved in VNS‐
mediated activation of hippocampal neurons in terms of The primary objective of current investigation was to identify
increases in 5‐HT1B receptor, phospho‐Erk1/2, and BDNF. molecular targets affected by VNS in the hippocampal neu-
To further investigate the effects of VNS on behav- rons of CRS animals. As CRS is widely used to investigate
ioral consequences, we conducted FST and measured the depressive‐like behaviors associated with persistent stress,
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SHIN et al.    1825

F I G U R E 2 Regulation of 5‐HT1B receptors and phospho‐Erk1/2 in hippocampal tissues by VNS in CRS animals. (a) Western blot analysis
of 5‐HT1B receptors. Protein extracts were prepared from hippocampi in animal groups of control (CTL), CRS + Sham, and CRS + VNS. (b)
Immunofluorescence images of 5‐HT1B receptor signals in hippocampal CA1 area of CTL, CRS + Sham, and CRS + VNS groups of animals.
(c) Western blot analysis of phospho‐Erk1/2. Protein extracts were prepared from hippocampi in animal groups of CTL, CRS + Sham, and
CRS + VNS. (d) Immunofluorescence images of 5‐HT1B receptor and Erk1/2 signals in the hippocampal CA1 subfield of CTL, CRS + Sham,
and CRS + VNS groups of animals. In (a) and (c), images in the upper panel are the representatives from four independent experiments, and
quantification plots of relative protein band intensity to actin are shown in the lower panel. *p < 0.05, **p < 0.01 (One‐way ANOVA). [Colour
figure can be viewed at wileyonlinelibrary.com]
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1826    SHIN et al.

FIGURE 3 Effects of 5,7‐DMT injection on hippocampal production of 5‐HT1B receptors, phospho‐Erk1/2, and BDNF in CRS + VNS
animals. (a) Hemotoxylin and eosin (H & E) staining of sagittal brain sections in DRN area. (b–d) Western blot analyses of hippocampal proteins
from animal groups as indicated in the figure. In (b) and (d), images in the upper panel are the representatives from four independent experiments,
and quantification plots of relative protein band intensity to actin are shown in the lower panel. *p < 0.05 (One‐way ANOVA). [Colour figure can
be viewed at wileyonlinelibrary.com]

we asked whether chronic VNS applied to CRS animals was Genetic polymorphism of human 5‐HT1B receptor gene was
involved in regulating neuronal responses and further linked shown to be associated with major depression (Huang et al.,
to behavioral consequences. Specifically, we examined sero- 2003), and activation of 5‐HT1B receptors by administration
tonergic receptors in the hippocampal neurons and found that of selective agonist induced antidepressant‐like effect in mice
production of 5‐HT1B receptors was reduced after chronic (Chenu et al., 2008). Yeast two‐hybrid screening identified
stress but then elevated by VNS. Production of phospho‐ the interaction of 5‐HT1B and 5‐HT4 receptors with p11 in
Erk1/2 and BDNF was similarly regulated by VNS in CRS several brain areas, and further interaction of p11 with down-
animals. These changes appeared to relate with antidepres- stream effector molecules was implicated by anti‐depres-
sion‐like responses, thus implying that VNS may be linked to sive activity in experimental animals (Svenningsson, Kim,
both hippocampal neuronal activation and behavioral effects. Warner‐Schmidt, Oh, & Greengard, 2013; Svenningsson
Among seven serotonin receptor families (5‐HT1—5‐HT7 et al., 2006).
receptors), 5‐HT1A and 5‐HT1B receptors in 5‐HT1 family In the present study, we found that hippocampal pro-
have been well characterized as potential mediators regulat- duction of 5‐HT1B receptors is regulated by chronic VNS
ing depression. Mice deficient in 5‐HT1A receptors showed in CRS animals. Electrical stimulation of the vagus nerve
elevated anxiety and antidepressant‐like response (Heisler around the ascending carotid artery activated neurons in the
et al., 1998; Ramboz et al., 1998) and developmental ex- DRN as demonstrated by c‐Fos induction. These neurons
pression of 5‐HT1A receptors induced to rescue anxiolytic were positive to 5‐HT1A receptors, suggesting that they are
behavior in adult animal (Gross et al., 2002). Like 5‐HT1A autoreceptors that may act to regulate the release of neu-
receptors, 5‐HT1B receptors act as both presynaptic autore- rotransmitters including serotonin in the axon terminals
ceptors and non‐serotonergic postsynaptic heteroreceptors, (Artigas, 2013; Haj‐Dahmane, Hamon, & Lanfumey, 1991).
thus function to regulate the release of neurotransmitters in Following VNS, serotonergic neurons in the DRN may fa-
several brain areas (Ruf & Bhagwagar, 2009; Sari, 2004). cilitate signal transmission to several brain areas including
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SHIN et al.    1827

F I G U R E 4 Effects of VNS on
immobility and climbing scores and
spontaneous movement in CRS animals.
Rats were subjected to the FST, and
durations of immobility (a) and climbing
(b) were measured separately for 5 min test
period. Scores of total distance of movement
(c) and rearing (d) were recorded for 5 min
and compared among CTL, CRS + Sham,
and CRS + VNS groups of rats. In (e) and
(f), CRS rats were injected with BDNF
or saline vehicle and subjected to FST to
measure the duration of immobility (e)
and climbing (f) behaviors. Number of
animals per each group in (a) and (b), (c)
and (d), and (e) and (f) were 10, 8, and
8, respectively. *p < 0.05, **p < 0.01,
***p < 0.001 One‐way ANOVA in (a‐d) and
unpaired t test in (e‐f). ns, not significant

hippocampus and upregulate the production of heterosynap- Activation of 5‐HT1 receptors induces the phosphorylation
tic 5‐HT1B receptors. Previous histological studies showed of Erk2 through the involvement of βγ subunit of G protein
that moderate levels of 5‐HT1B receptor are expressed in (Mendez, Kadia, Somayazula, El‐Badawi, & Cowen, 1999;
the hippocampal CA1 neurons (Voigt, Laurie, Seeburg, & Wirth, Holst, & Ponimaskin, 2017), raising the possibil-
Bach, 1991) and low levels of expression in the dentate ity that increased phosphorylation of Erk1/2 may act as a
granule cells (Egeland, Warner‐Schmidt, Greengard, & downstream effector of 5‐HT1B receptors in hippocampal
Svenningsson, 2011). Here, our data showed that hippocam- neurons of CRS + VNS animals. In addition, previous stud-
pal levels of 5‐HT1B receptors were upregulated by VNS in ies reported that VNS increased ΔFosB immunoreactivity
CRS animals, showing intense signals in the striatum py- in several brain areas including noraderenergic locus coeru-
ramidale and radiatum of CA1 neurons. It was previously leus and parabrachial nucleus (Furmaga, Sadhu, & Frazer,
reported that the interaction of 5‐HT1B receptors with p11 2012; Furmaga et al., 2011). Thus, the possibility that
activates annexin A2 and SMARCA3 pathway to induce neuronal inputs such as noradrenergic activity from locus
transcriptional activation of target genes, which is further coeruleus increase Erk1/2 phosphorylation in hippocampal
linked to antidepression‐like effects (Svenningsson et al., neurons cannot be ruled out (Kong, Ruan, Qian, Liu, & Le,
2013). Whether VNS‐induced activation of 5‐HT1B recep- 2010).
tors is related to p11‐associated antidepressant pathway re- Increases in BDNF production in several brain areas in-
mains to be explored. cluding hippocampus are known to be related to antidepres-
Our data further showed that hippocampal neurons in- sant activity in experimental animals (Nestler et al., 2002;
creasing to the production of 5‐HT1B receptors by VNS also Tsankova et al., 2006). In one study, hippocampal BDNF
upregulated phospho‐Erk1/2 signals, as has been demon- expression was attenuated after CRS, and then elevated by
strated in rat hippocampus (Carreno & Frazer, 2014). combined administration of quetiapine and venlafaxine for
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1828    SHIN et al.

antidepression effects (Xu et al., 2006), and in other stud- CONFLICT OF INTEREST
ies, administration of VNS to normal rats enhanced BDNF
The authors declare no conflicts of interest.
expression in the hippocampus (Biggio et al., 2009; Follesa
et al., 2007). Here, we demonstrate that the hippocampal
production of BDNF is decreased in CRS animals, which DATA ACCESSIBILIT Y
is then upregulated by VNS. How would the VNS be in-
The corresponding author is happy to provide data upon rea-
volved in regulating the hippocampal production of BDNF
sonable request.
in CRS animals? Previous studies showed that, after admin-
istration of tricyclic antidepressant imipramine into the mice
with chronic social defeat stress, BDNF gene expression AUTHOR CONTRIBUTIONS
was induced by the downregulation of histone deacetylase
5 (HDAC5) and subsequent increases of splice variants of HCS, BGJ, and KWL designed the study, conducted the ex-
BDNF mRNA through the chromatin remodeling of BDNF periments, and analyzed data. CYL designed the study and
gene promoter (Tsankova et al., 2006). In addition to acetyla- analyzed data. UN designed the study, analyzed data, and
tion and methylation, phosphorylation by Erk1/2 can modify wrote the manuscript. All the authors approved the final
histone proteins and epigenetic regulation of target gene ex- manuscript.
pression (Cheung, Allis, & Sassone‐Corsi, 2000). Thus, one
possible speculation is that VNS may be directly involved in ORCID
epigenetic regulation of BDNF gene expression in animal
models of depression. Alternatively, Erk1/2 activity, which Uk Namgung https://orcid.org/0000-0001-5253-1143
was induced through the activation of 5‐HT1B receptors after
chronic VNS in CRS animals, may phosphorylate histone 3
R E F E R E NC E S
and regulate target gene expression including BDNF (Cheung
et al., 2000). Alonso, M., Bekinschtein, P., Cammarota, M., Vianna, M. R., Izquierdo,
Our data show that application of VNS in CRS animals I., & Medina, J. H. (2005). Endogenous BDNF is required for long‐
term memory formation in the rat parietal cortex. Learning and
upregulates the production of 5‐HT1B receptors in the hippo-
Memory, 12, 504–510. https://doi.org/10.1101/lm.27305
campal neurons and further activates Erk1/2 and BDNF path- Artigas, F. (2013). Serotonin receptors involved in antidepressant ef-
way, as a possible downstream signaling event. Changes in fects. Pharmacology & Therapeutics, 137, 119–131. https://doi.
responsiveness of Erk1/2 and BDNF in CRS + VNS animals org/10.1016/j.pharmthera.2012.09.006
are well correlated with the improved immobility and climb- Biggio, F., Gorini, G., Utzeri, C., Olla, P., Marrosu, F., Mocchetti,
ing scores in the FST. Hippocampal administration of BDNF I., & Follesa, P. (2009). Chronic vagus nerve stimulation in-
in CRS animals resulted in decreasing immobility and in- duces neuronal plasticity in the rat hippocampus. International
Journal of Neuropsychopharmacology, 12, 1209–1221. https://doi.
creasing climbing scores in FST but failed to show statistical
org/10.1017/S1461145709000200
significance. It is thus speculated that VNS‐induced BDNF
Bogdanova, O. V., Kanekar, S., D'Anci, K. E., & Renshaw, P. F.
activity may be involved in generating antidepression‐like (2013). Factors influencing behavior in the forced swim test.
behavioral effect in CRS animals, but hippocampal BNDF it- Physiology & Behavior, 118, 227–239. https://doi.org 10.1016/j.
self would not be sufficient enough to lead to behavioral con- physbeh.2013.05.012
sequences. This notion is consistent with a previous report Breit, S., Kupferberg, A., Rogler, G., & Hasler, G. (2018). Vagus nerve
showing that hippocampal injection of BDNF potentiated an- as modulator of the brain‐gut axis in psychiatric and inflammatory
tidepression‐like activity of paroxetine as measured by swim- disorders. Frontiers in Psychiatry, 13(9), 44. https://doi.org/10.3389/
fpsyt.2018.00044
ming behavior in FST (Deltheil et al., 2009). VNS‐induced
Carreno, F. R., & Frazer, A. (2014). Activation of signaling pathways
serotonergic activity can affect other brain areas besides hip-
downstream of the brain‐derived neurotrophic factor receptor, TrkB,
pocampus, thus the exploration of the responsiveness of mo- in the rat brain by vagal nerve stimulation and antidepressant drugs.
lecular factors including BDNF in other brain areas is equally International Journal of Neuropsychopharmacology, 17, 247–258.
important as well. In conclusion, our study demonstrates that https://doi.org/10.1017/S1461145713000977
VNS can be applied successfully to animal models of depres- Carreno, F. R., & Frazer, A. (2017). Vagal nerve stimulation for treat-
sion and is useful to investigate molecular factors that are re- ment‐resistant depression. Neurotherapeutics: the Journal of the
lated to behavioral antidepressive activity. American Society for Experimental NeuroTherapeutics, 14, 716–
727. https://doi.org/10.1007/s13311-017-0537-8
Chang, I. A., Oh, M. J., Kim, M. H., Park, S. K., Kim, B. G., &
ACKNOWLEDGMENT Namgung, U. (2012). Vimentin phosphorylation by Cdc2 in
Schwann cell controls axon growth via β1-integrin activation.
This research was supported by the Daejeon University fund The FASEB Journal., 26, 2401–2413. https://doi.org/10.1096/
(Grant number 20173711) (Daejeon, Korea). fj.11-199018
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SHIN et al.    1829

Cheng, F., Ma, C., Wang, X., Zhai, C., Wang, G., Xu, X., … Wang, Q. caused by pharmacologically diverse antidepressants. Journal of
(2017). Effect of traditional Chinese medicine formula Sinisan on Pharmacology and Experimental Therapeutics, 341, 317–325.
chronic restraint stress‐induced nonalcoholic fatty liver disease: a https://doi.org/10.1124/jpet.111.188953
rat study. BMC Complementary and Alternative Medicine, 17, 203. Furmaga, H., Shah, A., & Frazer, A. (2011). Serotonergic and noradren-
https://doi.org/10.1186/s12906-017-1707-2 ergic pathways are required for the anxiolytic‐like and antidepres-
Chenu, F., David, D. J., Leroux‐Nicollet, I., Le Maître, E., Gardier, A. sant‐like behavioral effects of repeated vagal nerve stimulation in
M., & Bourin, M. (2008). Serotonin1B heteroreceptor activation in- rats. Biological Psychiatry, 70, 937–945. https://doi.org/10.1016/j.
duces an antidepressant‐like effect in mice with an alteration of the biopsych.2011.07.020
serotonergic system. Journal of Psychiatry and Neuroscience, 33, George, M. S., Sackeim, H. A., Rush, A. J., Marangell, L. B., Nahas, Z.,
541–550. Husain, M. M., … Ballenger, J. C. (2000). Vagus nerve stimulation:
Cheung, P., Allis, C. D., & Sassone‐Corsi, P. (2000). Signaling to chro- a new tool for brain research and therapy. Biological Psychiatry, 47,
matin through histone modifications. Cell, 103, 263–271. 287–295.
Chiba, S., Numakawa, T., Ninomiya, M., Richards, M. C., Grimonprez, A., Raedt, R., Portelli, J., Dauwe, I., Larsen, L. E.,
Wakabayashi, C., & Kunugi, H. (2012). Chronic restraint stress Bouckaert, C., … Vonck, K. (2015). The antidepressant‐like effect
causes anxiety‐ and depression‐like behaviors, downregulates of vagus nerve stimulation is mediated through the locus coeruleus.
glucocorticoid receptor expression, and attenuates glutamate Journal of Psychiatric Research, 68, 1–7. https://doi.org/10.1016/j.
release induced by brain‐derived neurotrophic factor in the pre- jpsychires.2015.05.002
frontal cortex. Progress in Neuro‐Psychopharmacology and Gross, C., Zhuang, X., Stark, K., Ramboz, S., Oosting, R., Kirby, L., …
Biological Psychiatry, 39, 112–119. https://doi.org/ 10.1016/j. Hen, R. (2002). Serotonin1A receptor acts during development to
pnpbp.2012.05.018 establish normal anxiety‐like behaviour in the adult. Nature, 416,
Clark, K. B., Naritoku, D. K., Smith, D. C., Browning, R. A., & Jensen, 396–400. https://doi.org/10.1038/416396a
R. A. (1999). Enhanced recognition memory following vagus nerve Haj‐Dahmane, S., Hamon, M., & Lanfumey, L. (1991). K+ channel and
stimulation in human subjects. Nature Neuroscience, 2, 94–98. 5‐hydroxytryptamine1A autoreceptor interactions in the rat dorsal
https://doi.org/10.1038/4600 raphe nucleus: an in vitro electrophysiological study. Neuroscience,
Corcoran, C., Connor, T. J., O'Keane, V., & Garland, M. R. (2005). The 41, 495–505.
effects of vagus nerve stimulation on pro‐ and anti‐inflammatory cy- Han, W., Tellez, L. A., Perkins, M. H., Perez, I. O., Qu, T., Ferreira, J.,
tokines in humans: a preliminary report. NeuroImmunoModulation, … de Araujo, I. E. (2018). A neural circuit for gut‐induced reward.
12, 307–309. https://doi.org/10.1159/000087109 Cell, 175, 665–678. https://doi.org/10.1016/j.cell.2018.08.049
Cunningham, J. T., Mifflin, S. W., Gould, G. G., & Frazer, A. (2008). Heisler, L. K., Chu, H. M., Brennan, T. J., Danao, J. A., Bajwa, P.,
Induction of c‐Fos and DeltaFosB immunoreactivity in rat brain Parsons, L. H., & Tecott, L. H. (1998). Elevated anxiety and antide-
by vagal nerve stimulation. Neuropsychopharmacology, 33, 1884– pressant‐like responses in serotonin 5‐HT1A receptor mutant mice.
1895. https://doi.org/10.1038/sj.npp.1301570 Proceedings of the National Academy of Sciences of the United
De Herdt, V., Puimege, L., De Waele, J., Raedt, R., Wyckhuys, T., States of America, 95, 15049–15054.
El Tahry, R., … Vonck, K. (2009). Increased rat serum corticos- Hosoi, T., Okuma, Y., & Nomura, Y. (2000). Electrical stimulation of
terone suggests immunomodulation by stimulation of the vagal afferent vagus nerve induces IL‐1beta expression in the brain and
nerve. Journal of Neuroimmunology, 212, 102–105. https://doi. activates HPA axis. American Journal of Physiology: Regulatory,
org/10.1016/j.jneuroim.2009.04.013 Integrative and Comparative Physiology, 279, R141–R147. https://
Deltheil, T., Tanaka, K., Reperant, C., Hen, R., David, D. J., & Gardier, doi.org/ 10.1152/ajpregu.2000.279.1.R141
A. M. (2009). Synergistic neurochemical and behavioural effects Huang, Y. Y., Oquendo, M. A., Friedman, J. M., Greenhill, L. L., Brodsky,
of acute intrahippocampal injection of brain‐derived neurotrophic B., Malone, K. M., … Mann, J. J. (2003). Substance abuse disorder
factor and antidepressants in adult mice. International Journal of and major depression are associated with the human 5‐HT1B receptor
Neuropsychopharmacology, 12, 905–915. https://doi.org/10.1017/ gene (HTR1B) G861C polymorphism. Neuropsychopharmacology,
S1461145709000017 28, 163–169. https://doi.org/10.1038/sj.npp.1300000
Egeland, M., Warner‐Schmidt, J., Greengard, P., & Svenningsson, P. Kiianmaa, K., & Fuxe, K. (1977). The effects of 5,7‐dihydroxytrypt-
(2011). Co‐expression of serotonin 5‐HT(1B) and 5‐HT(4) recep- amine‐induced lesions of the ascending 5‐hydroxytryptamine path-
tors in p11 containing cells in cerebral cortex, hippocampus, cau- ways on the sleep wakefulness cycle. Brain Research, 131, 287–301.
date‐putamen and cerebellum. Neuropharmacology, 61, 442–450. Kong, Y., Ruan, L., Qian, L., Liu, X., & Le, Y. (2010). Norepinephrine
https://doi.org/10.1016/j.neuropharm.2011.01.046 promotes microglia to uptake and degrade amyloid beta peptide
File, S. E., Hyde, J. R., & MacLeod, N. K. (1979). 5,7‐dihydroxytrypt- through upregulation of mouse formyl peptide receptor 2 and in-
amine lesions of dorsal and median raphé nuclei and performance in duction of insulin‐degrading enzyme. Journal of Neuroscience, 30,
the social interaction test of anxiety and in a home‐cage aggression 11848–11857. https://doi.org/10.1523/JNEUROSCI.2985-10.2010
test. Journal of Affective Disorders, 1, 115–122. Krahl, S. E., Senanayake, S. S., Pekary, A. E., & Sattin, A. (2014). Vagus
Follesa, P., Biggio, F., Gorini, G., Caria, S., Talani, G., Dazzi, L., … nerve stimulation (VNS) is effective in a rat model of antidepressant
Biggio, G. (2007). Vagus nerve stimulation increases norepineph- action. Journal of Psychiatric Research, 38, 237–240. https://doi.
rine concentration and the gene expression of BDNF and bFGF in org/10.1016/j.jpsychires.2003.11.005
the rat brain. Brain Research, 1179, 28–34. https://doi.org/10.1016/j. Manta, S., Dong, J., Debonnel, G., & Blier, P. (2009). Enhancement
brainres.2007.08.045 of the function of rat serotonin and norepinephrine neurons by
Furmaga, H., Sadhu, M., & Frazer, A. (2012). Comparison of ΔFosB sustained vagus nerve stimulation. Journal of Psychiatry and
immunoreactivity induced by vagal nerve stimulation with that Neuroscience, 34, 272–280.
 |

14609568, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ejn.14368 by UFES - Universidade Federal do Espirito Santo, Wiley Online Library on [01/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1830    SHIN et al.

Mendez, J., Kadia, T. M., Somayazula, R. K., El‐Badawi, K. I., & receptor function by p11 in depression‐like states. Science, 311,
Cowen, D. S. (1999). Differential coupling of serotonin 5‐HT1A 77–80. https://doi.org/ 10.1126/science.1117571
and 5‐HT1B receptors to activation of ERK2 and inhibition of ade- Svenningsson, P., Kim, Y., Warner‐Schmidt, J., Oh, Y. S., & Greengard,
nylyl cyclase in transfected CHO cells. Journal of Neurochemistry, P. (2013). p11 and its role in depression and therapeutic responses to
73, 162–168. antidepressants. Nature Reviews Neuroscience, 14, 673–680. https://
Nestler, E. J., Barrot, M., DiLeone, R. J., Eisch, A. J., Gold, S. J., & doi.org/10.1038/nrn3564
Monteggia, L. M. (2002). Neurobiology of depression. Neuron, 34, Toth, I., & Neumann, I. D. (2013). Animal models of social avoidance
13–25. and social fear. Cell and Tissue Research, 354, 107–118. https://doi.
O'Keane, V., Dinan, T. G., Scott, L., & Corcoran, C. (2005). Changes org/10.1007/s00441-013-1636-4
in hypothalamic‐pituitary‐adrenal axis measures after vagus nerve Tsankova, N. M., Berton, O., Renthal, W., Kumar, A., Neve, R. L., &
stimulation therapy in chronic depression. Biological Psychiatry, Nestler, E. J. (2006). Sustained hippocampal chromatin regulation
58, 963–968. https://doi.org/10.1016/j.biopsych.2005.04.049 in a mouse model of depression and antidepressant action. Nature
Olofsson, P. S., Levine, Y. A., Caravaca, A., Chavan, S. S., Pavlov, V. A., Neuroscience, 9, 519–525. https://doi.org/10.1038/nn1659
Faltys, M., & Tracey, K. J. (2015). Single‐pulse and unidirectional Vergnes, M., & Kempf, E. (1982). Effect of hypothalamic injections
electrical activation of the cervical vagus nerve reduces tumor ne- of 5,7‐dihydroxytryptamine on elicitation of mouse‐killing in rats.
crosis factor in Endotoxemia. Bioelectronics in Medicine, 2, 37–42. Behavioral Brain Research, 5, 387–397.
Paxinos, G. A., & Watson, C. (1998). The rat brain in stereotaxic coor- Voigt, M. M., Laurie, D. J., Seeburg, P. H., & Bach, A. (1991). Molecular
dinates, 4th ed. San Diego, CA: Academic Press. cloning and characterization of a rat brain cDNA encoding a 5‐hy-
Penry, J. K., & Dean, J. C. (1990). Prevention of intractable partial sei- droxytryptamine1B receptor. EMBO Journal, 10, 4017–4023.
zures by intermittent vagal stimulation in humans: preliminary re- Wirth, A., Holst, K., & Ponimaskin, E. (2017). How serotonin re-
sults. Epilepsia, 31(Suppl. 2), S40–S43. ceptors regulate morphogenic signalling in neurons. Progress
Ramboz, S., Oosting, R., Amara, D. A., Kung, H. F., Blier, P., in Neurobiology, 151, 35–56. https://doi.org/10.1016/j.
Mendelsohn, M., … Hen, R. (1998). Serotonin receptor 1A knock- pneurobio.2016.03.007
out: an animal model of anxiety‐related disorder. Proceedings of the Xu, H., Chen, Z., He, J., Haimanot, S., Li, X., Dyck, L., & Li, X. M.
National Academy of Sciences of the United States of America, 95, (2006). Synergetic effects of quetiapine and venlafaxine in prevent-
14476–14481. ing the chronic restraint stress‐induced decrease in cell proliferation
Ruf, B. M., & Bhagwagar, Z. (2009). The 5‐HT1B receptor: a novel and BDNF expression in rat hippocampus. Hippocampus, 16, 551–
target for the pathophysiology of depression. Current Drug Targets, 559. https://doi.org/10.1002/hipo.20184
10, 1118–1138. Zabara, J. (1985a). Peripheral control of hypersynchronous discharge
Sari, Y. (2004). Serotonin1B receptors: from protein to physiological in epilepsy. Electroencephalography and Clinical Neurophysiology,
function and behavior. Neuroscience and Biobehavioral Reviews, 61s, S162.
28, 565–582. https://doi.org/10.1016/j.neubiorev.2004.08.008 Zabara, J. (1985b). Time course of seizure control to brief, repetitive
Shin, H. C., Lee, J. H., Kim, K. J., Shin, H. J., Choi, J. J., Lee, C. Y., stimuli (abstract). Epilepsia, 26, 518.
… Jung, I. C. (2017). Modulation of hippocampal neuronal activity Zhang, X., Cao, B., Yan, N., Liu, J., Wang, J., Tung, V. O., & Li, Y.
by So‐ochim‐tang‐gamibang in mice subjected to chronic restraint (2013). Vagus nerve stimulation modulates visceral pain‐related af-
stress. BMC Complementary and Alternative Medicine, 17, 456. fective memory. Behavioral Brain Research, 236, 8–15. https://doi.
https://doi.org/10.1186/s12906-017-1963-1 org/10.1016/j.bbr.2012.08.027
Shirayama, Y., Chen, A. C., Nakagawa, S., Russell, D. S., & Duman,
R. S. (2002). Brain‐derived neurotrophic factor produces anti-
depressant effects in behavioral models of depression. Journal of How to cite this article: Shin HC, Jo BG, Lee C‐Y, Lee
Neuroscience, 22, 3251–3261. K‐W, Namgung U. Hippocampal activation of 5‐HT1B
Suarez, E. C., Krishnan, R. R., & Lewis, J. G. (2003). The relation of receptors and BDNF production by vagus nerve
severity of depressive symptoms to monocyte‐associated proin-
stimulation in rats under chronic restraint stress. Eur J
flammatory cytokines and chemokines in apparently healthy men.
Psychosomatic Medicine, 65, 362–368.
Neurosci. 2019;50:1820–1830. https://doi.org/10.1111/
Svenningsson, P., Chergui, K., Rachleff, I., Flajolet, M., Zhang, X., ejn.14368
El Yacoubi, M., … Greengard, P. (2006). Alterations in 5‐HT1B