Brain Research 1789 (2022) 147946

Contents lists available at ScienceDirect

Brain Research
journal homepage: www.elsevier.com/locate/brainres

Whisker trimming induces anti-anxiety like status via activation of

dorsomedial hypothalamus nucleus in mice
Jiangling Wang a, b, c, Weiran Shan a, Xinzhong Chen b, Zhiyi Zuo a, *
a
Department of Anesthesiology, University of Virginia, Charlottesville, VA 22908, USA
b
Department of Anesthesiology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310006, China
c
Department of Anesthesiology, Cancer Hospital of the University of Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China

A R T I C L E I N F O A B S T R A C T

Keywords: Whiskers are highly developed tactile organs in mice. Here, we showed that mice with whisker trimming had a
Anxious behavior decreased anxiety behavior and activation of dorsomedial hypothalamus compared to control mice. Inhibition or
Dorsomedial hypothalamus damage of dorsomedial hypothalamus reversed the decrease of anxiety level induced by whisker trimming. These
Mice
results expand the role of whiskers in regulating mouse behaviors to anxiety and suggest a novel function of
Whiskers
dorsomedial hypothalamus. These findings indicate importance of normal sensory functions in modulating an­
imal behavior.

1. Introduction
Touch is a vital sense for object recognition, social communication,
decision making and motor control in many mammalian species (Wang
et al., 2020; McGlone et al., 2014; Moore et al., 2016). Parental touch
raises trust and reduces social anxiety in children (Brummelman et al.,
2019). Studies have shown that neonatal tactile enrichment reduces
anxiety levels in adult rats while tactile deprivation impairs the devel­
opment of anxiety-related emotional system in the brain of mice (Baldini
et al., 2013; Soumiya et al., 2016). Whisker is the most important and
highly developed tactile organ in mice, through which mice can rapidly
locate and recognize objects in their environment. These functions help
them modulate movement and make proper decisions (Bosman et al.,
2011). Whisker-related signal is integrated in the thalamus and pro­
jected to barrel cortex where the sensory signals are decoded and
encoded (Harrell et al., 2020). Hypothalamus is a regulator of basic
brain functions including emotions (Blackshaw et al., 2010; Sevoz-
Couche et al., 2013). For example, dorsomedial hypothalamus (DMH)
regulates stress-related cardiovascular responses in rodents (Sevoz-
Couche et al., 2013). DMH also regulates anxiety (Shekhar, 1993),
projects to the thalamus (Kagan et al., 2013) and receives input from
cerebral cortex (Dampney, 2015). Although tactile functions and anxi­
ety have been well studied, little is known about the relationships be­
tween anxiety behavior and tactile impairment and the brain regions
that are critical for this relationship. Here, a tactile impairment model
induced by whisker trimming was used to investigate this relationship.
The role of DMH in the anxiety behavior was determined by using a
chemogenetic approach.
2. Results
Six- to eight-week old CD-1 male mice had whisker trimmed to the
skin every day. The sham mice had whisker root manipulation similar to
that of whisker trimming but without receiving whisker trimming. The
anxiety levels of these mice were assessed by the open field and elevated
plus maze tests after whisker trimming for 14 days. There was a differ­
ence in the center time [F(2,112) = 4.919, P = 0.009], corner time [F
(2,112) = 5.482, P = 0.0054] and number of corner entries [H(3) =
22.26, P < 0.0001] among the groups of mice with or without whisker
trimming. Mice with whisker trimming stayed longer in the center re­
gion and had fewer numbers of entries into the corner region than naïve
control mice and sham mice in the open field test. The mice with whisker
trimming also spent less time in the corner area than control mice
(Fig. 1A and 1B). There was also a difference in the open arm time [H(3)
= 11.37, P = 0.0034] among the groups of mice. Mice with whisker
trimming spent more time in the open arms than naïve control mice and
sham mice but the total amount of time spent in the closed arms were
not different among naïve control mice, sham mice and mice with

* Corresponding author at: Department of Anesthesiology, University of Virginia Health System, 1 Hospital Drive, PO Box 800710, Charlottesville, VA 22908-0710,
USA.
E-mail addresses: wangjl@zjcc.org.cn (J. Wang), sw8gm@virginia.edu (W. Shan), chenxinz@zju.edu.cn (X. Chen), zz3c@virginia.edu (Z. Zuo).

https://doi.org/10.1016/j.brainres.2022.147946
Received 21 October 2021; Received in revised form 27 April 2022; Accepted 15 May 2022
Available online 18 May 2022
0006-8993/© 2022 Elsevier B.V. All rights reserved.
J. Wang et al.
whisker trimming in the elevated plus maze test (Fig. 1C). These results
suggest that whisker trimming reduces the anxiety levels.
To identify brain regions that may be involved in the effects of
whisker trimming on anxiety, brain sections were stained for c-Fos
protein, an indicator of neuronal activation (Zeng et al., 2021). Amyg­
dala, a brain region that is involved in fear and anxiety behavior
(Ressler, 2010), had the highest number of c-Fos positive cells among all
brain regions in mice with whisker trimming (Fig. 2A and Fig. 3). DMH
had the second highest increase in c-Fos positive cells in the mice with
whisker trimming compared with sham mice or control mice (Fig. 2B
and 2C). Interestingly, certain brain regions, such as retrosplenial area,
had c-Fos positive cells in the whisker trimming and sham mice but not
in the control mice (Fig. 2D). DMH was selected to be studied because it
receives input from amygdala (Myers et al., 2014; Calhoon and Tye,
2015) and its role in anxiety behavior is less established than amygdala.
To determine the role of DMH in reducing the anxiety of mice with
whisker trimming, ibotenic acid, a neurotoxin (11), was injected into the
DMH. This injection led to the injury of neurons in the DMH when the
assessment was performed 5 days later (Fig. 4A). Although there was no
difference in the performance in the open field test (Fig. 4 B and C), there
was a difference in the amount of time spent in the open arm among the
groups with or without the injury to the DMH neurons [F(2,31) = 4.357,
P = 0.0215]. The injection of ibotenic acid reduced the time in the open
arms in the elevated plus maze test but the injection of saline into DMH
did not have any effects in mice with whisker trimming (Fig. 4D). These
results suggest that DMH is important for the decreased anxiety level of
whiskerless mice. To supplement the approach of damaging DMH by
ibotenic acid, neurons in the DMH was inhibited by a chemogenetic
approach. The injected viruses pAAV2-hSyn-hM4D(Gi)-mCherry and
pAAV2-hSyn-mCherry (control virus) were mainly in the DMH (Fig. 5A).
There was a difference in the number of entries to center area [F(3,36) =
3.669, P = 0.021] and time in the corner area [F(3,36) = 3.322, P =
0.0304] among the groups. Compared with whiskerless mice, whisker­
less mice receiving injection of pAAV2-hSyn-hM4D(Gi)-mCherry into
their DMH and then intraperitoneal injection of compound 21 had a
decrease in the number of entries to the center area and an increased
time in the corner area in the open field test (Fig. 5B and 5C). In addi­
tion, there was a difference among the groups in the amount of time
2
Brain Research 1789 (2022) 147946
spent in the closed arm of the elevated plus maze test [F(3,36) = 2.998,
P = 0.0433]. Whiskerless mice receiving injection of pAAV2-hSyn-
hM4D(Gi)-mCherry into their DMH and then intraperitoneal injection
of compound 21 spent more time in the closed arms than whiskerless
mice but the total amount of time in the open arms was not different
among the groups in the elevated plus maze (Fig. 5D). However, mice
receiving injection of pAAV2-hSyn-hM4D(Gi)-mCherry into their DMH
and then intraperitoneal injection of saline or injection of pAAV2-hSyn-
mCherry (control virus) into the DMH and then intraperitoneal injection
of compound 21 did not have alterations in their behavior in the open
field and elevated plus maze (Fig. 5B to 5D). These results from che­
mogenetic approach support the idea that DMH plays an important role
in the decreased anxiety in mice with whisker trimming. Interestingly,
there was a difference in the travel distance among the naïve control,
sham mice and whiskerless in the open field [F(2,115) = 4.931, P =
0.0088] or elevated plus maze [H(3) = 6.331, P = 0.0422]. Mice with
whiskerless traveled less distance in the open field and elevated plus
maze than the naïve control or sham mice (Fig. 6A). DMH damage by
ibotenic acid did not affect the travel distance in the open field and
elevated plus maze test (Fig. 6B). There was a difference in the travel
distance in the open field tests among the whiskerless mice with various
treatments with viral injection [H(4) = 8.523, P = 0.0364]. Inhibition of
the DMH neurons reduced the travel distance of whiskerless mice in the
open field (Fig. 6C). However, there was no difference in the travel
distance in the elevated plus maze among the whiskerless mice with or
without DMH neuron inhibition (Fig. 6C). These results suggest a
decreased locomotor function of whiskerless mice, which may not
explain the presentation of a decreased anxiety level in the whiskerless
mice. In addition, there was no consistent change in the locomotor
functions of whiskerless mice with DMH damage or inhibition. How­
ever, the damage or inhibition of DMH neurons reversed the anti-anxiety
status in the whiskerless mice. Thus, the locomotor activity changes in
mice with various treatments may not be a cause for the alternation of
anxiety levels in them.
3. Discussion
Neural circuits in human and animal brains undergo dynamic
Fig. 1. Whisker trimming reduced anxiety
behavior. (A) time spent in the center area
and the number of entries into the center
area in the open field test. (B) time spent in
the corner area and the number of entries
into the corner area in the open field test. (C)
time spent in the open arms and closed arms
of the elevated plus maze. Data were pre­
sented as means ± SEM for the left panels of
panels A and B and the top panel of panel C
(n = 37 – 39 for panels A and B, 40 for panel
C) in a bar graph with the presentation of
data of individual animal. Data in the right
panels of panels A and B and bottom panel of
panel C were in box plot with the presenta­
tion of data of individual animal. Between
lines: 95th percentile of the data; inside
boxes: 25th to 75th percentile including the
median of the data. Statistical analysis was
performed by one-way ANOVA (the left
panels of panels A and B and the top panel of
panel C) or one-way ANOVA on rank (right
panels of panels A and B and bottom panel of
panel C) followed by Tukey test.
J. Wang et al. Brain Research 1789 (2022) 147946

Fig. 2. Whisker trimming altered c-Fos expression in selected brain regions. Mouse brain was harvested for immunofluorescent staining 1 h after the completion of
elevated plus maze test. (A) Heat map of the number of c-Fos positive cells in different brain regions in naïve control, sham control and whiskerless mice. (B) Heat
map of the comparisons of the number of c-Fos positive cells between sham control and naïve control mice, whiskerless and naïve control mice or whiskerless and
sham control mice. (C) Immunofluorescent images of c-Fos positive cells in the DMH. Scale bar = 100 µm. (D) Heat map of the number of c-Fos positive cells in brain
regions where naïve control mice had no c-Fos positive cells. Data were presented as means (n = 6) and analyzed by t-test or rank sum test. Abbreviations for brain
regions: Amyg: amygdala; Arc: arcuate hypothalamic nucleus; BL: basolateral amygdala nuclei; Cpu: caudate-putamen (neostriatum); DMH: dorsomedial hypo­
thalamus; Ect: ectorhinal cortex; GrDG: granular layer of the dentate gyrus of hippocampus; LaDL: dorsolateral division of the lateral amygdala nucleus; LHb: lateral
habenular nuclei; MD: mediodorsal thalamus nucleus; Me: medial nucleus of the amygdaloid complex; PoDG: polymorphic cell layer of the dentate gyrus of hip­
pocampus; PRh: perirhinal cortex; PVT: paraventricular thalamus; RSP: retrosplenial area; VMH: ventromedial hypothalamic nucleus.

Fig. 3. Whiskerless mice had increased expression of c-Fos in selected brain regions. (A) Naïve control mice. (B) sham control mice. (C) mice with whisker trimming.
Scale bar = 50 µm. Amyg: amygdala; PoDG: polymorphic cell layer of the dentate gyrus of hippocampus; PRh: perirhinal cortex; PVT: paraventricular thalamus.

reorganizations in response to environmental or sensory changes, which
can help them to develop proper behavior including anxiety behavior
(Calhoon and Tye, 2015; Guo et al., 2014). Tactile sensory circuit is one
of the most important circuits in the central nervous system. Whiskers
are the major tactile organs in rodents and critical tools for them to
detect stimuli, distinguish texture, locate objects, communicate and
navigate (Staiger and Petersen, 2021; Iwasato and Erzurumlu, 2018; Liu
et al., 2018). Our results provide initial evidence for the involvement of
whiskers in regulating anxiety behavior and the role of DMH in this
involvement.

3
J. Wang et al. Brain Research 1789 (2022) 147946

Fig. 4. Neuron injury in the DMH increased anxious behavior in mice with whisker trimming. (A) Representatives of cresyl violet images showing the injury to the
DMH whose area is indicated by white dash line in mice with ibotenic acid injection. Scale bar = 100 μm in the top panel and = 50 μm in the bottom panel. (B) time
spent in the center area and the number of entries into the center area in the open field test. (C) time spent in the corner area and the number of entries into the corner
area in the open field test. (D) time spent in the open arms and closed arms of the elevated plus maze. Data were presented as means ± SEM (n = 9–14) in a bar graph
with the presentation of data of individual animal. Statistical analysis was performed by one-way ANOVA followed by Tukey test.

Fig. 5. Inhibition of neurons in the DMH increased anxious behavior in mice with whisker trimming. (A) Representatives of immunofluorescent images showing the
accurate injection of viruses into the DMH whose area is indicated by white dash line. Scale bar = 50 μm. (B) time spent in the center area and the number of entries
into the center area in the open field test. (C) time spent in the corner area and the number of entries into the corner area in the open field test. (D) time spent in the
open arms and closed arms of the elevated plus maze. Data in panels B, left panel of panel C and right panel of panel D were presented as means ± SEM (n = 9–11) in
a bar graph with the presentation of data of individual animal. Data in the right panel of panel C and left panel of panel D were in box plot with the presentation of
data of individual animal. Between lines: 95th percentile of the data; inside boxes: 25th to 75th percentile including the median of the data. Statistical analysis was
performed by one-way ANOVA (panel B, left panel of panel C and right panel of panel D) or one-way ANOVA on rank (right panel of panel C and left panel of panel D)
followed by Tukey test.

DMH receives inputs from cerebral cortex, amygdala and other brain
regions that regulate respiration, food intake and energy state (Damp­
ney, 2015; Rau and Hentges, 2019). DMH may be involved in cardio­
vascular response to emotional stress (Fontes et al., 2014). Our results
have identified an additional function of DMH by showing its role in
modulating anxiety behavior in mice with somatosensory impairment.
Our results showed that mice with the activation of DMH neurons had a
decreased anxiety level. Consistent with our finding, activation of GABA
receptors in the DMH neurons induces an anxiolytic effect (Shekhar,
1993).
Vibrissal primary somatosensory cortex, also called “barrel cortex”,
is the most studied cortex related to whisker tactile input (Guo et al.,
2014; Brecht, 2007). This input involves thalamus (Guo et al., 2014;
Petreanu et al., 2009; Hooks et al., 2013; Mao et al., 2011). Thalamus
receives projects from hypothalamus (Kagan et al., 2013; Hsu and Price,
2009). Signals from the hypothalamus can be transmitted to the down-

4
J. Wang et al. Brain Research 1789 (2022) 147946

Fig. 6. Neuronal injury or inhibition of neurons in the DMH did not affect travel distance of mice with whiskerless. (A) travel distance of naïve control, sham control
and whiskerless mice in the open field test and elevated plus maze test. (B) travel distance of mice with or without neuronal injury in the DMH in the open field test
and elevated plus maze test. (C) travel distance of mice with or without inhibition of neurons in the DMH in the open field test and elevated plus maze test. Data in
panel B, left panel of panel A and bottom panel of panel C were presented as means ± SEM (n = 9–11) in a bar graph with the presentation of data of individual
animal. Data in the right panel of panel A and top panel of panel C were in box plot with the presentation of data of individual animal. Between lines: 95th percentile
of the data; inside boxes: 25th to 75th percentile including the median of the data. Statistical analysis was performed by one-way ANOVA (panel B, left panel of panel
A and bottom panel of panel C) or one-way ANOVA on rank (right panel of panel A and top panel of panel C) followed by Tukey test. EPM: Elevated plus maze, OF:
Open field.

stream limbic targets, such as amygdalae (Otis et al., 2019), and lateral
septum-bed nucleus of the stria terminalis, structures that are known to
regulate anxiety behavior (Johnson et al., 2008). Our results showed
that neurons in the paraventricular thalamic nucleus and DMH were
activated after whisker trimming, suggesting that tactile deficiency may
affect the activation of neurons in the hypothalamus and thalamus
circuits.
Our results showed that DMH neurons were highly activated in
whisker trimmed mice and moderately activated in sham whisker
trimmed mice but were not active in control mice (Fig. 2). Neuronal
activation in sham whisker trimmed mice may be due to the mechanical
stimulus of whiskers by sham trimming procedure.
Our results showed mice with whiskerless spent more time in the
center area during the open field test, suggesting that they have more
exploratory behaviors than control mice. It is unclear whether these
mice are more exploratory or have a higher non-purposeful move due to
the tactile impairment. However, mice with whiskerless had a shorter
travel distance, suggesting that the increased time in the center area is
not due to an increase in non-purposeful move of these mice. Our results
also showed that mice with tactile impairment have a reduced anxiety
level. It is difficult to know the biological implication of this finding.
However, one can speculate that the decreased anxiety level in mice
with tactile impairment may have help maintain their locomotor ac­
tivity at a reasonable level.
Our study has limitations. First, only male mice were used in the
study. Future studies may investigate whether our findings here are
applicable to female mice. Additional brain regions, such as lateral
habenular nucleus that had an increased number of c-Fos positive cells
in the mice with whiskerless, can be investigated for their role in the
decreased anxiety of these mice. Finally, our study has only determined
the baseline anxiety levels of mice with and without whiskers. It has not
been determined whether the reduced anxiety in mice with whiskerless
remains if a stimulus to elicit anxiety is applied.
4. Conclusions
Our results suggest a role of whiskers, a structure that is primarily
involved in tactile sense, in modulating anxiety behavior. The neural
circuit for this modulation includes DMH.
5. Experimental procedures
The animal protocol was approved by the Institutional Animal Care
and Use Committee of the University of Virginia (Charlottesville, VA,
USA). All animal experiments were carried out in accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory
Animals (NIH publications number 80–23) revised in 2011 and reported
according to the ARRIVE guidelines.
5.1. Animals
Six- to eight-week old CD-1 male mice (weighing 30–36 g) from
Charles River Laboratories International Inc. (Wilmington, MA, USA)
were used. The mice were group-housed in cages (5 mice/cage) on a 12-
h light/dark cycle with free access to water and food. All experimental
procedures or behavior tests were performed during the light cycle.

5
J. Wang et al.
5.2. Whisker trimming
Mice in the whisker trimming group had whiskers trimmed to skin
every day for 2 weeks as described previously (Wang et al., 2020). Mice
in the sham control group received manipulation to the roots of the
whiskers, which mimicked the whisker trimming. Mice in the naïve
control group did not receive any stimulations.
5.3. Open field test
Mice were placed in an open field apparatus made of black polyvinyl
chloride. The time and distance traveled in different zone and total
distance traveled were recorded for 5 min and analyzed by the ANY-
maze system (SD Instruments, San Diego, CA).
5.4. Elevated plus maze test
Mice were subjected to the elevated plus maze 2 days after the open
field test. The elevated plus maze was made of black polyvinyl chloride
with two opposing open arms (30 × 5 × 0.5 cm) and two opposing
enclosed arms (30 × 5 × 15 cm) connected by a central platform (5 × 5
cm). Mice were placed in the central area in this maze as previously
described (Wang et al., 2020; Zeng et al., 2021). As they freely explored
the maze, their behavior was recorded for 5 min by a video camera
mounted above the maze and analyzed using the ANY-maze system. The
duration and number of entries into the open arms or closed arms were
calculated to measure anxiety-like behavior.
5.5. Injection of viruses or chemicals into dorsomedial hypothalamus
The pAAV2-hSyn-hM4D(Gi)-mCherry virus (catalogue number:
50475-AAV2, Addgene) or pAAV2-hSyn-mCherry virus (catalogue
number: 114472-AAV2, Addgene) was injected into DMH as described
previously (Zeng et al., 2021; Gautron et al., 2010). Briefly, mice were
anesthetized with ketamine HCl/xylazine HCl (60 – 75 mg/kg, i.p.) and
placed in a stereotaxic apparatus. A small hole was drilled into the skull
under aseptic conditions. The viruses were administered slowly (250 nl
of 7.6 × 1012 particles/ml solution for each side) into the DMH with the
following coordinates: − 1.88 mm from bregma, 0.43 mm lateral to the
midline and 5.3 mm depth from the surface of the skull. These co­
ordinates were chosen for the injection into the ventral part of the DMH.
Both sides of the DMH were injected. Ibotenic acid (catalogue number:
ab146670, Abcam) was injected into DMH at a dosage of 2.0 µg (10 µg/
µl in distilled water, 200 nl) per side for bilateral injection. Only one
injection of the virus or ibotenic acid solution to each side of the DMH
was performed. The incision of injection site was closed with surgical
sutures. Behavior tests were performed 4 weeks later after virus injec­
tion as a previous study has demonstrated that AAV-mediated delivery
of protein expression is identical between 2 and 12 weeks after surgery
(Chamberlin et al., 1998). To induce inhibition of neurons, intraperi­
toneal injections of compound 21 at 3 mg/kg were performed 30 min
before mice were subjected to open field or elevated maze tests. Saline
was injected as control solution.
5.6. Immunofluorescent staining
Immunofluorescent labeling was performed to detect c-Fos positive
cells in the brain. One hour after the elevated plus maze test, mice were
anesthetized with ketamine/xylazine (60–75 mg/kg) and transcardially
perfused with 0.1 M phosphate-buffered saline (PBS), followed by 4%
paraformaldehyde in 0.1 M PBS. Brains were harvested. One hour after
the elevated plus maze test was chosen to harvest the brain because c-
Fos protein expression has reached a peak in rodents after various
stimuli in our previous study and other studies (Zeng et al., 2021; Zhong
et al., 2014; Barros et al., 2015). Frozen coronal sections (25 μm thick) of
the brain were cut from the whole brain sequentially in an anterior to
6
Brain Research 1789 (2022) 147946
posterior direction. One section from every 10 sections was kept for
staining c-Fos and DAPI. Antigen retrieval with sodium citrate buffer
(10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed at
95–100 ◦ C for 20 min. After being washed in Tris-buffered saline (TBS)
containing 0.1% triton-X 100, sections were blocked with 10% donkey
serum plus 1% bovine serum albumin in TBS for 2 h at room temperature
and then incubated at 4 ◦ C overnight with the mouse monoclonal anti-c-
Fos (1:1000 dilution; catalogue number: ab190289, Abcam) as primary
antibody. Sections were rinsed in TBS with 0.1% triton-X 100 and then
incubated with donkey anti-mouse IgG antibody conjugated with Alexa
Fluor 594 (1:200 dilution; catalogue number: A21203, Invitrogen) for 1
h at room temperature in the dark. The slides were then rinsed in TBS
three times for 5 min each, co-stained with 4′ ,6-diamidino-2-phenyl­
indole (DAPI) and cover-slipped with VECTASHIELD HardSet mounting
medium. The heat map of the number of c-Fos positive cells in the brain
was constructed as we reported before (Zeng et al., 2021). Briefly, three
randomly selected and independent microscopic fields of each brain
structure of interest were obtained. Three sections from each mouse
were analyzed by image J. The 9 values of each mouse were averaged to
reflect the number of c-Fos positive cells in the brain region of the
mouse. The examiner was blind to the group assignment of the mouse.
Identification of brain anatomical regions was based on the morphology
and DAPI counter staining images. Data of 6 mice from each experi­
mental condition were used to build the c-Fos expression heat map of
brain regions.
Frozen coronal sections (25 μm thick) from − 1.6 to − 2.2 mm relative
to bregma were used to examine the location of mCherry expression
after virus injection. The slides were rinsed in TBS three times for 5 min
each, co-stained with DAPI and cover-slipped with VECTASHIELD
HardSet mounting medium.
5.7. Cresyl violet staining
Five days after the injection of ibotenic acid injection, mouse brain
was harvested for paraffin embedding. Coronal 10 μm thick sections
were used for cresyl violet staining. Briefly, sections were deparaffi­
nized, stained in 0.1% cresyl violet for 25 min at 37 ◦ C, rinsed quickly in
distilled water, differentiated in 95% ethanol for 2 min, dehydrated in
100% alcohol and cleared in xylene. Images were taken under a mi­
croscope (Olympus DP70, Olympus Corporation, Tokyo, Japan).
5.8. Statistical analysis
Results are presented as means ± S.E.M. with the presentation of
individual animal data in the bar graph (for normal distribution data),
median and 95th percentile with the presentation of individual animal
data in the box plot (for non-normal distribution data) or means ± S.E.
M. in the line plots. Data were analyzed by one-way ANOVA followed by
Tukey test if the data were normally distributed, by one-way ANOVA on
rank followed by Tukey test if the data were not normally distributed, by
t-test or rank sum test. Three ANOVA assumptions, i.e., normal distri­
bution of the data, approximately equal variance of the data among
groups and independence of individuals, were observed during the data
analysis. Significant difference was defined as P < 0.05. All statistical
analyses were performed with SigmaStat (Systat Software, Inc., Point
Richmond, CA, USA).
6. Authors’ contribution
ZZ conceived the project; JW, WS, XC and ZZ designed the studies;
JW and WS performed the experiments. JW did initial data analysis,
drafted the manuscript and prepared the figures. ZZ rewrote the
manuscript.
J. Wang et al.
Funding
This study was supported by the Robert M. Epstein Professorship
endowment (to Z Zuo), University of Virginia, Charlottesville, VA, USA.
References
Baldini, S., Restani, L., Baroncelli, L., Coltelli, M., Franco, R., Cenni, M.C., Maffei, L.,
Berardi, N., 2013. Enriched early life experiences reduce adult anxiety-like behavior
in rats: a role for insulin-like growth factor 1. J. Neurosci. 33, 11715–11723.
Barros, V.N., Mundim, M., Galindo, L.T., Bittencourt, S., Porcionatto, M., Mello, L.E.,
2015. The pattern of c-Fos expression and its refractory period in the brain of rats
and monkeys. Front. Cell. Neurosci. 9, 72.
Blackshaw, S., Scholpp, S., Placzek, M., Ingraham, H., Simerly, R., Shimogori, T., 2010.
Molecular pathways controlling development of thalamus and hypothalamus: from
neural specification to circuit formation. J. Neurosci. 30, 14925–14930.
Bosman, L.W., Houweling, A.R., Owens, C.B., Tanke, N., Shevchouk, O.T., Rahmati, N.,
Teunissen, W.H., Ju, C., Gong, W., Koekkoek, S.K., De Zeeuw, C.I., 2011. Anatomical
pathways involved in generating and sensing rhythmic whisker movements. Front.
Integr. Neurosci. 5, 53.
Brecht, M., 2007. Barrel cortex and whisker-mediated behaviors. Curr. Opin. Neurobiol.
17, 408–416.
Brummelman, E., Terburg, D., Smit, M., Bogels, S.M., Bos, P.A., 2019. Parental touch
reduces social vigilance in children. Dev. Cogn. Neurosci. 35, 87–93.
Calhoon, G.G., Tye, K.M., 2015. Resolving the neural circuits of anxiety. Nat. Neurosci.
18, 1394–1404.
Chamberlin, N.L., Du, B., de Lacalle, S., Saper, C.B., 1998. Recombinant adeno-associated
virus vector: use for transgene expression and anterograde tract tracing in the CNS.
Brain Res. 793, 169–175.
Dampney, R.A., 2015. Central mechanisms regulating coordinated cardiovascular and
respiratory function during stress and arousal. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 309, R429–R443.
Fontes, M.A., Xavier, C.H., Marins, F.R., Limborco-Filho, M., Vaz, G.C., Muller-Ribeiro, F.
C., Nalivaiko, E., 2014. Emotional stress and sympathetic activity: contribution of
dorsomedial hypothalamus to cardiac arrhythmias. Brain Res. 1554, 49–58.
Gautron, L., Lazarus, M., Scott, M.M., Saper, C.B., Elmquist, J.K., 2010. Identifying the
efferent projections of leptin-responsive neurons in the dorsomedial hypothalamus
using a novel conditional tracing approach. J. Comp. Neurol. 518, 2090–2108.
Guo, Z.V., Li, N., Huber, D., Ophir, E., Gutnisky, D., Ting, J.T., Feng, G., Svoboda, K.,
2014. Flow of cortical activity underlying a tactile decision in mice. Neuron 81,
179–194.
Harrell, E.R., Goldin, M.A., Bathellier, B., Shulz, D.E., 2020. An elaborate sweep-stick
code in rat barrel cortex. Sci. Adv. 6, eabb7189.
Hooks, B.M., Mao, T., Gutnisky, D.A., Yamawaki, N., Svoboda, K., Shepherd, G.M., 2013.
Organization of cortical and thalamic input to pyramidal neurons in mouse motor
cortex. J. Neurosci. 33, 748–760.
Hsu, D.T., Price, J.L., 2009. Paraventricular thalamic nucleus: subcortical connections
and innervation by serotonin, orexin, and corticotropin-releasing hormone in
macaque monkeys. J. Comp. Neurol. 512, 825–848.
Iwasato, T., Erzurumlu, R.S., 2018. Development of tactile sensory circuits in the CNS.
Curr. Opin. Neurobiol. 53, 66–75.
Johnson, P.L., Truitt, W.A., Fitz, S.D., Lowry, C.A., Shekhar, A., 2008. Neural pathways
underlying lactate-induced panic. Neuropsychopharmacology 33, 2093–2107.
Brain Research 1789 (2022) 147946
Kagan, R., Kainz, V., Burstein, R., Noseda, R., 2013. Hypothalamic and basal ganglia
projections to the posterior thalamus: possible role in modulation of migraine
headache and photophobia. Neuroscience 248, 359–368.
Liu, Y., Latremoliere, A., Li, X., Zhang, Z., Chen, M., Wang, X., Fang, C., Zhu, J.,
Alexandre, C., Gao, Z., Chen, B., Ding, X., Zhou, J.Y., Zhang, Y., Chen, C., Wang, K.
H., Woolf, C.J., He, Z., 2018. Touch and tactile neuropathic pain sensitivity are set
by corticospinal projections. Nature 561, 547–550.
Mao, T., Kusefoglu, D., Hooks, B.M., Huber, D., Petreanu, L., Svoboda, K., 2011. Long-
range neuronal circuits underlying the interaction between sensory and motor
cortex. Neuron 72, 111–123.
McGlone, F., Wessberg, J., Olausson, H., 2014. Discriminative and affective touch:
sensing and feeling. Neuron 82, 737–755.
Moore, E.R., Bergman, N., Anderson, G.C., Medley, N., 2016. Early skin-to-skin contact
for mothers and their healthy newborn infants. Cochrane Database Syst. Rev. 11,
CD003519.
Myers, B., Mark Dolgas, C., Kasckow, J., Cullinan, W.E., Herman, J.P., 2014. Central
stress-integrative circuits: forebrain glutamatergic and GABAergic projections to the
dorsomedial hypothalamus, medial preoptic area, and bed nucleus of the stria
terminalis. Brain Struct. Funct. 219, 1287–1303.
Otis, J.M., Zhu, M., Namboodiri, V.M.K., Cook, C.A., Kosyk, O., Matan, A.M., Ying, R.,
Hashikawa, Y., Hashikawa, K., Trujillo-Pisanty, I., Guo, J., Ung, R.L., Rodriguez-
Romaguera, J., Anton, E.S., Stuber, G.D., 2019. Paraventricular thalamus projection
neurons integrate cortical and hypothalamic signals for cue-reward processing.
Neuron 103, 423–431.
Petreanu, L., Mao, T., Sternson, S.M., Svoboda, K., 2009. The subcellular organization of
neocortical excitatory connections. Nature 457, 1142–1145.
Rau, A.R., Hentges, S.T., 2019. GABAergic inputs to POMC neurons originating from the
dorsomedial hypothalamus are regulated by energy state. J. Neurosci. 39,
6449–6459.
Ressler, K.J., 2010. Amygdala activity, fear, and anxiety: modulation by stress. Biol.
Psychiatry 67, 1117–1119.
Sevoz-Couche, C., Brouillard, C., Camus, F., Laude, D., De Boer, S.F., Becker, C.,
Benoliel, J.J., 2013. Involvement of the dorsomedial hypothalamus and the nucleus
tractus solitarii in chronic cardiovascular changes associated with anxiety in rats.
J. Physiol. 591, 1871–1887.
Shekhar, A., 1993. GABA receptors in the region of the dorsomedial hypothalamus of rats
regulate anxiety in the elevated plus-maze test. I. Behavioral measures. Brain Res.
627, 9–16.
Soumiya, H., Godai, A., Araiso, H., Mori, S., Furukawa, S., Fukumitsu, H., 2016. Neonatal
whisker trimming impairs fear/anxiety-related emotional systems of the amygdala
and social behaviors in adult mice. PLoS ONE 11, e0158583.
Staiger, J.F., Petersen, C.C.H., 2021. Neuronal circuits in barrel cortex for whisker
sensory perception. Physiol. Rev. 101, 353–415.
Wang, C., Liu, H., Li, K., Wu, Z.Z., Wu, C., Yu, J.Y., Gong, Q., Fang, P., Wang, X.X.,
Duan, S.M., Wang, H., Gu, Y., Hu, J., Pan, B.X., Schmidt, M.V., Liu, Y.J., Wang, X.D.,
2020. Tactile modulation of memory and anxiety requires dentate granule cells
along the dorsoventral axis. Nat. Commun. 11, 6045.
Zeng, Q., Shan, W., Zhang, H., Yang, J., Zuo, Z., 2021. Paraventricular thalamic nucleus
plays a critical role in consolation and anxious behaviors of familiar observers
exposed to surgery mice. Theranostics 11, 3813–3829.
Zhong, J., Liang, M., Akther, S., Higashida, C., Tsuji, T., Higashida, H., 2014. c-Fos
expression in the paternal mouse brain induced by communicative interaction with
maternal mates. Mol. Brain. 7, 66.