NEUROSCIENCE

RESEARCH ARTICLE
P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96

Long-lasting Postnatal Sensory Deprivation Alters Dendritic

Morphology of Pyramidal Neurons in the Rat Hippocampus: Behavioral
Correlates
Parisa Yarmohammadi-Samani, Hamid Taghipourbibalan y and Jafar Vatanparast *
Department of Biology, School of Science, Shiraz University, Iran

Abstract—The role of normal sensory inputs in the development of sensory cortices is well known, however, their
impacts on the hippocampus, an integrator of sensory modalities with important roles in cognitive functions, has
received much less attention. Here, we applied a long-term sensory deprivation paradigm by trimming the rats’
whiskers bilaterally, from postnatal day 3 to 59. Female sensory-deprived (SD) rats showed more on-wall rearing
and visits to the center of the open-field box, shorter periods of grooming, less defecation and less anxiety-like
behaviors in the elevated plus-maze compared to controls, who had their intact whiskers brushed. Passive avoid-
ance memory retention was sex-dependently impaired in the female SD rats. In the radial arm maze, however, ref-
erence spatial memory was impaired only in the male SD rats. Nonetheless, working memory errors increased in
both sexes of SD rats. Besides depletion of CA1 and CA3 pyramidal neurons in SD rats, Sholl analysis of Golgi-
Cox stained neurons revealed that prolonged sensory deprivation has retracted the arborization of CA1 basal
dendrites in SD group, while solely female SD rats had diminished CA1 apical dendrites. Sholl analysis of CA3
neurons in SD animals also disclosed significantly more branched apical dendrites in males and basal dendrites
in females. Sensory deprivation also led to a considerable spine loss and variation of different spine types in a
sex-dependent manner. Our findings suggest that experience-dependent structural plasticity is capable of
spreading far beyond the manipulated sensory zones and the inevitable functional alterations can be expressed
in a multifactorial sex-dependent manner. Ó 2021 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: barrel-cortex sensory deprivation, hippocampus, pyramidal neurons, dendritic morphology, memory, anxiety.

INTRODUCTION tional deficits. These alterations are not restricted to the

An essential and basic feature of the neural system is its
plasticity, the ability to change the structure and/or
function of neurons in response to experience and
learning. Early life sensory experiences are especially
necessary for the normal development of the brain due
to their crucial role in the structural and functional
plasticity of neurons that determine the proper formation
of neuronal circuitries and activity (Wiesel and Hubel,
1963; Breton and Stuart, 2009; Chen et al., 2015; Ueno
et al., 2015; Zhang et al., 2016). Sensory deprivation dur-
ing early life can induce lasting behavioral impairments.
Alterations in the structure and function of cortical neu-
rons have been implicated in such cognitive and emo-
*Corresponding author. Address: Department of Biology, School of
Science, University of Shiraz, Shiraz, Iran.
E-mail addresses: vatanparast@shirazu.ac.ir, jvatanparast@gmail.
com (J. Vatanparast).
y various cognitive and emotional changes, also support
Present address: Behavioral and Translational Neuroscience
Group, Department of Psychology, Faculty of Health Sciences, UiT
The Arctic University of Norway, Tromsø, Norway.
Abbreviations: ANOVA, analysis of variance; fSTL, first step-through
latency; PND, postnatal day; SD, sensory-deprived.
deprived cerebral cortex but have also been traced in
the non-deprived cortical areas involved in other senses,
a phenomenon called cross-modal neuroplasticity. A
compensatory increase in the performance of cortical
regions processing the remaining intact senses and the
recruitment of the deprived region for other spared
senses through rewiring of the brain connections are con-
sidered as the best-known features of cross-modal plas-
ticity (Rauschecker, 1995; Merabet and Pascual-Leone,
2010). In spite of numerous reports on the effects of sen-
sory deprivation on the structure and function of neurons
within sensory regions, either deprived or non-deprived,
very little is known about the effects on the neurons
beyond the sensory regions. If sensory deprivation could
have indirect effects on non-deprived cortical regions,
brain areas beyond sensory cortices that indirectly
receive sensory information can be affected by sensory
deprivation. The fact that sensory deprivation induces
the assumption that the structural and functional alter-
ations induced by sensory deprivation may extend to
non-sensory areas of the brain.

https://doi.org/10.1016/j.neuroscience.2021.11.011
0306-4522/Ó 2021 IBRO. Published by Elsevier Ltd. All rights reserved.

79
80 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96
Hippocampus is an allocortical structure with crucial
roles in spatial representation and consolidation of
declarative memories (Eichenbaum and Cohen, 2014).
It indirectly receives inputs from different sensory cortices,
including the somatosensory cortex, mainly through its
tight connections to the entorhinal cortex (Liu et al.,
2018). The fact that hippocampal place cells fire only
when animal receives multi-modal sensory cues of a par-
ticular place is a well-known example that shows sensory
inputs are critical for the function of hippocampus
(O’Keefe, 1976). Environmental enrichment also
increases the dendritic complexity and spine density in
hippocampal neurons (Rampon et al., 2000; Van Praag
et al., 2000; Faherty et al., 2003). CA1 and CA3 pyramidal
neurons are the main principal neurons of the hippocam-
pus and also the source of major hippocampal outputs
(Graves et al., 2012). In many studies strong correlation
between structural alterations in CA1 and CA3 subfields
and behavioral deficits have been reported (Soetanto
et al., 2010; Morales-Medina et al., 2013; Bencsik et al.,
2019). Despite several reports on the influences of
enhanced sensory inputs on the structure of neurons in
the hippocampus, surprisingly, there is no comprehen-
sively decisive report about the effects of sensory depriva-
tion on neuronal structures in the hippocampus, and their
potential correlations with behavioral outcomes. These
issues are the main focus of the current study.
EXPERIMENTAL PROCEDURES
Animals
All procedures were performed in accordance with the
National Institute of Health Guide for the Care and Use
of Laboratory Animals and were approved by the local
animal ethics committee at Shiraz University. All efforts
were made to minimize the number of animals used and
their suffering. Male and female Wistar rats from our
breeding colony were kept in a well-ventilated room at
22 ± 2 °C with a 12 h light–dark cycle and free access
to food and water, unless otherwise mentioned. On the
day after birth (postnatal day1, PND1), litters were
culled to seven or eight pups with an equal number of
males and females, whenever possible. Litters were
randomly allocated into the treatment and control
groups. In the treatment group, the whiskers were
trimmed bilaterally to less than 1 mm every other day
from PND 3 to PND 59. Animals in the control group
were similarly handled and their whiskers were gently
brushed with scissors to mimic the stimulation that rats
in the treatment group received during whisker trimming.
Animals were weaned on PND 23, separated by sex on
PND 40 and maintained three to four per cage until the
day of experiment. All animals within a litter were
assigned to the same group but no more than one male
and one female rat from each litter were used per
experimental group. Other animals in each litter were
used for other experiments (for either this or other
projects). The number of animals for each experiment
was determined according to the estimate of the
standard deviation of the results. Therefore, the sample
size is generally larger for behavioral experiments than
for morphological experiments. Only one data point per
animal was obtained in each experiment and used for
statistical analysis.
Behavioral tests
From PND 67 rats were subjected to a battery of
behavioral tests including open field, elevated plus
maze, eight arms radial maze and passive avoidance
(Fig. 1). The equipment used for behavioral tests were
cleaned with a damp paper towel after each trial to
remove residual odors. All behavioral tests were
conducted during the light phase between 10:00–14:00.
The rats were moved to the adjacent test room 30 min
before the experiment starts. To reduce the behaviors
driven by olfactory cues, the floor and the walls of the
testing devices were cleaned by mild detergent between
trials for rats from the same homecage and wiped with
70% ethanol for rats of different sex.
Open field
The Open-Field chamber was a custom-made
40  40  40 cm opaque plexiglas box evenly
illuminated from the top and a camera was installed
above the chamber on a tripod to record the animal’s
activity for subsequent manual analysis. The floor of the
chamber was divided into four equal squares by distinct
marker lines. The center of the arena was determined
as the ¼ inner part of the floor also marked with dashes
representing a smaller square. Rat were individually
placed at a designated corner of the box and their
activity was recoded for 5 min.
The total number of line crossings (square crossing)
was considered as the locomotor activity. Each time all
four paws of the animal crossed the lines as the animal
entered the adjacent square either horizontally or
diagonally, one movement was registered. A rearing
gesture was scored whenever the rat took a fully upright
posture by standing on its hind limbs. This behavioral
activity was further featured by on-wall rearing in which
the rat stood fully upright with taking the aid of the walls,
and off-wall rearing in which the rat took an upright
positon in the open air without any wall support. Time
spent in the central square, either by standing still or
passing through, and the total time spent on grooming
were registered by a stopwatch. The animal was
considered in a grooming state if stood still showing
stereotypical behaviors such as licking paws or body
and stroking face and ears. The number of fecal boli
was counted at the end of each trial.
Elevated plus maze
The elevated plus maze consisted of two opposite closed
arms (50 cm long  10 cm wide  40 cm height)
alternating at right angles with two open arms (50 cm
long  10 cm wide) made of gray PVC and was kept
60 cm above the floor by a metal pedestal (Borj Sanat
Co., Tehran, Iran). Illumination was provided by a red-
tinted bulb lamp positioned about 1.5 m above and
oblique to the maze to provide a dim light not directly
 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96
illuminating the closed arms and not exposing the open
arms with high lux intensity. Each rat was placed in the
central platform facing an open arm and allowed to
freely explore the maze for 5 min. The trial was
recorded by an overhead camera and the number of
entrances and the time spent in the closed and open
arms were scored off-line.
Radial arm maze
The radial maze consisted of eight arms (65 cm
long  10 cm wide with 17 cm side walls) extending
radially from an octagonal central hub (32 cm in
diameter). The apparatus floor was made of opaque
gray PVC and the side walls were made of transparent
plexiglas. The maze was supported by a metal frame
40 cm above the floor in a quiet room containing several
visual cues external to the maze. A food cup
(5 cm  5 cm and 2.5 cm deep) was placed near the
distal end of each arm and peanut halves were used as
food reward. The task was performed as described
earlier (Alipour et al., 2019). In brief, the diet of rats was
reduced for a week to keep their body weight at approxi-
mately 90% of free-feeding level. The test was performed
in shaping and training phases. Two sessions of social
shaping and two sessions of individual shaping were con-
ducted in four consecutive days to familiarize rats with the
apparatus and the food reward. During social shaping,
three rats were placed in the maze and gained access
to arms and peanut halves, which were scattered
throughout all arms. Animals were removed as soon as
peanuts were eaten or 10 min had elapsed. In individual
shaping sessions, each rat was allowed 5 min to explore
the maze with all eight arms baited. In each day of shap-
ing, peanuts were placed further distal into each arm until
they were finally placed at food cups. Rats were then
trained for 12 sessions, once per day. At the beginning
of each training session four arms were baited and the
other four arms were left unbaited. The pattern of baited
and unbaited arms was consistent among rats and
throughout testing and no more than two consecutive
arms were left unbaited. Each trial began by placing the
rat in the opaque cylinder on the central platform for
10 s. The cylinder was then removed and the rat was
allowed to explore the maze and to eat peanuts. The trial
was terminated and the animal was removed as soon as
all peanut halves were eaten or 5 min had elapsed. Con-
sidering the involvement of the cholinergic system in sen-
sory processing (Eggermann et al., 2014), learning/
memory and attention (Teles-Grilo Ruivo and Mellor,
2013), and previous reports addressing the interactions
of sensory deprivation and cholinergic modulation on the
structural plasticity on neurons (Schliebs et al., 1982;
Maurer and Williams, 2017), following the 12 sessions
of radial maze training, rats were trained for four more
days in which they were challenged with two doses of
muscarinic acetylcholine receptor antagonist, scopo-
lamine. Rats received i.p. injections of scopolamine (0.1
and 0.2 mg/kg) or 0.9% saline as vehicle (1 ml/kg)
30 min before the start of each radial arm maze session.
Two non-drugged (vehicle) days were inserted between
drugged sessions for drug washout. The training sessions
81
were recorded by a video camera from the side of the
maze for subsequent analysis. An arm entry was
recorded when all paws entered an arm. The first entry
into an unbaited arm was recorded as a reference mem-
ory error. Secondary efforts to an unbaited arm and sub-
sequent entries into already visited baited arms during a
training session were defined as working memory error.
Passive avoidance test
The passive avoidance apparatus consisted of an
illuminated and a dark compartment of the same size
(20 cm  20 cm  30 cm) adjoining each other through
a guillotine door. Floors were constructed of 3 mm
stainless steel rods spaced 1 cm apart. The floor of the
dark chamber was connected to a stimulator so that
could be electrified by intermittent electric pulses.
Illumination inside the lighted chamber was provided by
a 60 W lamp 40 cm above the chamber.
The passive avoidance test was conducted as
described earlier (Vatanparast et al., 2013). In brief, rats
were given two trials to habituate to the apparatus. Each
animal was gently placed in the illuminated compartment
facing away from the dark compartment. After 10 s the
guillotine door was raised and the animal was allowed
to enter the dark compartment. Entrance latency into
the dark compartment (first step-through latency, fSTL)
was recorded when the animal had placed all four paws
into the dark compartment. If the animal waited for more
than100s to enter the dark compartment, it was excluded
from the experiment. Upon entering the dark compart-
ment, the door was closed and 30 s later the rats were
taken from the dark compartment into their home cage.
The trial was repeated after 30 min and followed after
the same interval by the acquisition trial during which a
0.6 mA, 50 Hz unavoidable foot-shock was applied for
2.5 s when the rat entered into the dark compartment after
the animal had entered the dark compartment and the
guillotine door was closed. After 30 s the animal was
removed from the apparatus and placed into the home
cage. Two minutes later, the animal was placed again
inside the illuminated compartment, and if the animal
refrained entering the dark compartment during the
120 s of the session it was considered to reach the learn-
ing criterion. Otherwise, by entering the dark compart-
ment (before120s), the door was closed and the animal
received the same shock again. The acquisition of PA
was tested again after 2 min and animal received a foot-
shock each time entered the dark compartment. The
training was terminated when the rat remained in the light
compartment for 120 consecutive seconds. The number
of trials (entries into the dark chamber) was recorded as
an index of task acquisition and short-term memory.
The retention test was performed twenty-four hours
after the training session. Each animal was placed in
the light chamber for 10 s, the guillotine door was
raised, and the STL and the time spent in the dark
compartment (TDC) were recorded up to 300 s. If the
rat did not enter the dark compartment within 300 s, the
retention test was terminated and a ceiling score of
300 s was assigned to STL. During the retention
sessions, no electric shock was applied.
82 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96
Golgi-cox staining
Golgi-Cox staining was performed according to the
protocol suggested by (Das et al., 2013) and (Ranjan
et al., 2014) with slight modifications. Deeply anesthetized
rats were perfused with normal saline and the brains were
removed and each brain was cut into 5–8 mm coronal
blocks containing the dorsal hippocampus. Brain blocks
were placed in dark colored bottles containing freshly pre-
pared Golgi-Cox (5% potassium dichromate solution, 5%
mercuric chloride, 5% potassium chromate and distilled
water in the ratio 5:5:4:10) and stored in dark at 37 °C
for 24 h. The blocks were then transferred to 30% sucrose
solution at 4 °C for 8 h, and then placed in fresh 30 %
sucrose solution at 4 °C for 3–7 days. 120 lm thick coro-
nal sections were cut using vibratome, collected in 6%
sucrose solution and transferred to gelatin-coated slides.
Slides were then washed in distilled water, immersed in
75% ammonium hydroxide for 10 min, washed and then
immersed in 1% sodium thiosulfate for 10 min in dark.
Slides were washed and dehydrated with serial dilutions
of alcohol and finally kept in a solution of chloroform,
xylene and 100% alcohol (ratio 1:1:1) for 4 min. The sec-
tions were cleaned for 4 min with xylene and left in fresh
xylene for 1 h in the dark. Slides were then mounted in
Entellan, and visualized under a bright field microscope
at 10x/0.25 and 100x/1.25 oil-immersion objective (Nikon
Eclipse 80i microscope connected to a Nikon DS-Fi1 dig-
ital camera) for studying dendrite branching and dendritic-
spine morphology, respectively.
Imaging, Sholl and spine analysis
Pyramidal neurons of the dorsal CA1 and CA3
hippocampal subregions (plates 57–60 of Paxinos and
Watson, 2006) were selected for this study. The following
criteria were used to select pyramidal neurons in the hip-
pocampus: (1) their triangle-shaped soma, (2) complete
dendrites without apparent truncation, (3) the presence
of at least one apical and two primary basilar dendrites
that branched at least once, (4) the cell soma location
must be in the dorsal CA3 and CA1 hippocampal subre-
gions. Estimates of total dendritic length, branch order
and soma area were obtained from a total of 271 (137
CA1 and 134 CA3) neurons in the dorsal hippocampus
(6–8 animals per sex in each group and 7–10 neurons
per animal). Images of the neurons were captured using
a Nikon Eclipse 80i microscope connected to a Nikon
DS-Fi1 digital camera. Analysis of the images was per-
formed using the NeuronJ plugin in ImageJ software
(Rasband, W.S., ImageJ, US National Institutes of Health,
Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–
2012), NeuronStudio, and Bonfire scripts in MATLAB soft-
ware (Kutzing et al., 2010; Langhammer et al., 2010).
For spine analysis, 3–5 neuron and 3 apical and 3
basal second- or third-order segments per rat (with 3–5
rat from every sex per group) that were 20 lm in length
and originated at least 40 lm from the soma were
traced in bright-field images. We used the ImageJ
software for obtaining a detailed morphometric profile of
spines. Then, analysis of images was performed using
the semi-automated Spine Classifier of NeuronStudio
(http://research.ssm.edu/cnic/tools-ns.html), and spines
were classified as mushroom, stubby and thin,
according to (Peters and Kaiserman-Abramof, 1970)
and were manually corrected.
Cresyl Violet staining
Rats were deeply anesthetized and perfused with normal
saline followed by 4% paraformaldehyde in phosphate-
buffered saline. The brain tissue was post-fixed at 4 °C
in the same fixative. Paraffin-embedded blocks of brain
were serially sectioned in coronal plane (5-mm-thick)
using a microtome. The sections were deparaffinized,
stained with Cresyl Violet, dehydrated, cleaned and
mounted on slides. The stained pyramidal neurons were
counted in hippocampal CA1 and CA3 subfields by
using a ruled graticule eyepiece in a total area of
0.063 mm2. Digitized images of hippocampal neurons in
CA1 and CA3 regions were captured using a Nikon light
microscope.
Statistical analysis
The Shapiro–Wilk normality test was used to assess the
normal distribution of data. Homogeneity of variances
was confirmed by Leven test. Two-way fixed effect
analysis of variance (ANOVA) followed by Tukey’s post
hoc test was used to compare groups with normally
distributed variables. When data were not normally
distributed, Kruskal-Wallis test followed by Dunn’s test
was used. Following Tukey or Dunn tests, p values
were adjusted for multiple comparisons. To detect the
possible interactions between treatment and sex, an
initial ANOVA was run. When a significant interaction
was found between treatment and sex, the data set was
divided by sex and the analysis was done separately on
the subdivisions. Otherwise, the data for males and
females were combined and comparisons between
control and SD groups were done using unpaired t-test
or Mann-Whitney U test depending on the normality of
the distribution.
The accepted level of significance for main treatment
effects was p < 0.05 and for interactions was p < 0.1.
RESULTS
Open field test
Whisker trimming during PND 3-PND 59 significantly
increased the time spent in the center zone of the maze
(unpaired t-test; t94 = 2.74, p < 0.01), but did not affect
line crossing score and off-wall rearing. These
parameters did not show significant interactions with
sex, so data from male and female rats were pooled
within each group. Kruskal-Wallis test revealed
significant difference between groups in on-wall rearing
(H3 = 21.8, p < 0.001), time spent in self-grooming
(H3 = 26.9, p < 0.001) and number of fecal boli
(H3 = 8.5, p < 0.05). Post hoc pairwise comparisons
showed more frequent on-wall rearing (Dunn’s test;
Z = 2.76, p < 0.05), reduced fecal boli counts (Dunn’s
test; Z = 2.7, p < 0.05) and the time spent on self-
grooming (Dunn’s test; Z = 2.65, p < 0.05) in SD
 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96
Fig. 1. Schematic of the experimental timeline. At the end of these
experiments, the remaining animals were used for other studies (not
presented here).
female rats compared to control female rats but not in the
male rats. While the time spent on self-grooming was not
different between male and female control rats, this time
was dramatically longer in SD females compared to SD
male rats (Dunn’s test; Z = 5.11, p < 0.001) (Fig. 2).
Elevated plus maze
Analysis of data obtained on the elevated plus maze using
ANOVA and Kruskal-Wallis tests showed significant
difference between groups in percentage of open arm
entries (H3 = 12.3, p < 0.01), percentage of time spent
in the open arms (H3 = 11.9, p < 0.01), number of
closed arm returns (H3 = 16.2, p < 0.01) and the time
spent in the center square (F3,87 = 3.4, p < 0.05). Post
hoc multiple comparisons showed that postnatal whisker
deprivation did not significantly affect the performance of
male rats in the elevated plus maze. On the other hand,
female rats in the SD group showed higher percentage
of open arm entries (Dunn’s test; Z = 2.65, p < 0.05),
percentage of time spent in the open arms (Dunn’s test;
Z = 2.74, p < 0.05), number of closed arm returns
(Dunn’s test; Z = 3.25, p < 0.01) and the time spent in
the center square (Tukey’s post hoc test; q = 4.44,
p < 0.05) compared to the sex-matched controls (Fig. 3).
Passive avoidance test
During the acquisition trial, the number of shocks required
for learning the task was not significantly different
between sensory deprived and control rats (Mann-
Whitney U test; U44,48 = 582, p = 0.45). On the other
hand, Kruskal-Wallis test revealed significant difference
between groups in STL (H3 = 22.8, p < 0.001). Post
hoc pairwise comparisons showed that female rats in
the SD group represent significantly shorter STLs
compared to control females (Dunn’s test; Z = 2.76,
p < 0.05). In control rats, the mean STL was not
significantly different between male and female rats, but
SD female rats showed significantly shorter STLs
compared to SD male rats (Dunn’s test; Z = 4.59,
p < 0.001) (Fig. 4).
Spatial memory in the radial arm maze
Control animals showed sex differences in the radial
maze task, with males committing fewer reference and
working memory errors (Figs. 5C, 6D). Sensory
deprivation did not significantly affect the number of
reference memory errors in female rats, but increased
reference memory errors in males. Prolonged postnatal
sensory deprivation eliminated the difference between
83
male and female rats in the reference memory that
normally expected in the radial arm maze test. Sensory
deprivation also increased working memory errors in
both male and female rats, but this effect was more
pronounced in female rats and, as a result, exaggerated
the difference in the working memory between male and
female rats (Fig. 6).
Analysis of data obtained on the drug challenge
experiments using Kruskal-Wallis tests showed
significant difference between groups in reference
memory errors (H11 = 68.2, p < 0.001) and working
memory errors (H11 = 102.4, p < 0.001). Post hoc
pairwise comparisons showed that higher dose of
scopolamine (0.2 mg/kg) significantly increased the
number of reference memory errors in male (Dunn’s
test; Z = 3.5, p < 0.05) and female (Dunn’s test;
Z = 4.32, p < 0.01) control rats. In sensory-deprived
rats, the lower dose also significantly increased
reference memory errors in male rats compared to their
sex matched controls that received the same dose of
scopolamine (Dunn’s test; Z = 3.4, p < 0.05).
Following the injection of the higher dose of
scopolamine, the number of reference memory errors in
the sensory-deprived female rats was significantly lower
than control female rats that received the same drug
(Dunn’s test; Z = 3.38, p < 0.05). The lower dose of
scopolamine significantly increased working memory
errors only in the control male rats (Dunn’s test;
Z = 3.38, p < 0.05), while the dose of scopolamine
increased working memory errors in both male (Dunn’s
test; Z = 3.64, p < 0.05) and female control rats, with
a more pronounced effect in female rats (Dunn’s test;
Z = 4.7, p < 0.001). Both doses of scopolamine
increased working memory errors in a dose dependent
manner in male SD rats, but in female SD rats only the
higher dose showed a significant effect. The effects of
scopolamine on the number of working memory errors
were not significantly different between control and SD
rats in either males or females (Fig. 7).
Neuronal morphology, dendritic spine properties and
density of pyramidal neurons in CA1 and CA3
hippocampal subfields
Golgi staining was used to visualize individual pyramidal
neurons in the CA1 and CA3 subfields of the
hippocampus. The dendritic morphology of the neurons
of adult rats (PND 60) from control and sensory
deprived groups were quantified using Sholl analysis.
Whisker deprivation did not affect apical dendrites of
CA1 neurons in male rats but significantly reduced total
dendritic length, number of branch points and terminal
points of basal dendrites. Statistical analysis also
revealed a significant decrease of Sholl intersections in
proximal (<60 lm from the soma periphery),
intermediate (60–120 lm from the soma periphery) and
distal (>120 lm from the soma periphery) segments of
the basal dendrites of CA1 neurons in male SD rats
compared to naive male rats (Fig. 8). In female rats, the
total length of apical dendrites of CA1 neurons was
shorter than males in both control and SD groups, but
there was not a significant difference between control
84 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96

A D 100 Sholl intersections was detected

50 in the proximal (<120 lm from
the soma periphery) apical
###
40 dendrites of CA1 neurons in SD
Line crossings

Grooming (s)
female rats, but intermediate
(120–240 lm from the soma
30 periphery) and distal (>240 lm
50 from the soma periphery) apical
20 dendrites were not significantly
affected by sensory deprivation

10 * (Fig. 8C). Significant reduction of

Sholl intersections was observed
in proximal and intermediate basal
0 0 dendrites of CA1 neurons of SD
Control SD Male Female female rats, but this effect did not
B E reach significance level for the
80 6 majority of distal portions of basal
Control dendrites. Male control rats had
Time in the center (s)

** SD more Sholl intersections in the

distal segment of apical and
60
Fecal boli

4 intermediate segment of basal

dendrites of CA1 neurons
40 compared to female control rats.
In the SD group, the lower
2 dendritic arborization of female
rats compared to males was
20
* mainly in the intermediate range.
The difference between male and
female rats in the number of Sholl
0 0 intersections was reduced in the
Control SD Male Female
SD compared to the control
C F group, which was more evident in
##
30 40 the intermediate segment
(Fig. 8C, D).
* Representative Golgi-stained
Off-wall rearing

On-wall rearing

30 CA3 neurons of male and female

rats from control and SD rats
20
have been shown in Fig. 9A.
Apical dendrites of CA3 neurons
20
in female control rats showed
* significantly more branch points
10 compared to control males, while
10 total dendritic length and number
of branch points in the basal
dendrite of CA3 neurons in female
0 0 control rats were significantly
Control SD Male Female
lower than those in male rats
Fig. 2. Effects of sensory deprivation on the performance in the open field test. Total line crossing (A), (Fig. 9B). Long-term postnatal
time spent in the central area (B), number of off-wall rearing (C), time spent on self-grooming (D), the whisker deprivation increased
number of fecal boli (E) and number of on-wall rearing (F). Box diagrams show interquartile range, apical dendrites of CA3 neurons
horizontal lines are median and whiskers represent 5th and 95th percentiles. Outliers are data outside in young adult male rats, but not
5th to 95th percentile. In (A–C) the interaction with sex was not significant and data were pooled for
both sexes (n = 46–50 per group of both sexes). In (D–F) significant interaction of treatment  sex
in the SD female rats. Male rats
was detected and data were analyzed separately for each sex (n = 22–27 per sex in each group). from control group showed more
*p < 0.05 compared to control female rats, **p < 0.01 compared to control group. ##p < 0.01 and dendritic intersection in the
###p < 0.001 compared to male rats in the same group. SD: sensory-deprived. proximal range of CA3 apical
dendrites while female control rats
had more dendritic arborization in
and SD female rats for this variable. SD female rats also
the intermediate dendrite segments (Fig. 9C). Basal
had significantly fewer branch points and terminal points
dendrites of CA3 neurons in male SD rats showed
in the apical dendrites of CA1 neurons compared to the
reduced Sholl intersections in the proximal and
sex-matched controls (Fig. 8B). Significant decrease of
 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96 85

A C intermediate ranges, while in

60 Control 30 female SD rats, basal dendrites of
Time in open arms (%)

SD

Closed arm returens

CA3 neurons showed increased
Sholl intersections in all dendritic
segments, especially in distal
40 * 20 ** portion. In the control group, male
rats had CA3 neurons with more
intermediate and distal basal
dendrite arborization compared to
20 10
females, but the sex difference
was not detected between CA3
neurons of male and female rats
0 0 in the SD group (Fig. 9D).
Male Female Male Female Basal and apical dendritic
B D spine densities were measured in
Entries into open arms (%)

60 100 CA1 and CA3 pyramidal neurons

of control and sensory-deprived
80
* rats. Our results showed that
Center time (s)

40
* whisker deprivation led to a
significant decrease in spine
60
density on both apical and basal
dendrites of CA1 and CA3
40 pyramidal neurons in female rats.
20
Sensory deprivation also
20 significantly decreased the density
of dendritic spines on the apical
0 0 dendrites of CA3 pyramidal
Male Female Male Female neurons and on the basal
dendrites of CA1 pyramidal
Fig. 3. Effects of sensory deprivation on the performance in elevated plus maze test. The time spent neurons of male rats. Examining
in the open arms (A), number of entries into open arms (B), number of returns into closed arms (C) the types of dendritic spines
and time spent in the center of maze (D). Box diagrams show interquartile range, horizontal lines are
median and whiskers represent 5th and 95th percentiles. Outliers are data outside 5th to 95th revealed that in the sensory-
percentile (n = 21–25 per sex in each group). *p < 0.05, **p < 0.01 compared to control female rats. deprived male rats, the thin
SD: sensory-deprived. dendritic spines were the most
reduced type, especially on the
basal dendrites of CA1 pyramidal
neurons. In female rats,
mushroom and thin spines were
A Control B most affected in apical and basal
4 SD 400 ### dendrites of CA1 pyramidal
Trials in acquisition session

Step through latency (s)

neurons and apical dendrites of

* CA3 pyramidal neurons, while on
3 300 the basal dendrite of CA3
pyramidal neurons, only thin spine
were prominently reduced (Fig. 10).
2 200 The soma size of CA1 and CA3
neurons did not show a significant
difference between controls and
1 100 sensory deprived young adult rats
(data not presented). We did not
detect a significant difference in the
0 0 density of pyramidal neurons in the
Control SD Male Female CA1 and CA3 subfields of the
hippocampus between male and
Fig. 4. Effects of postnatal sensory deprivation of barrel cortex on passive avoidance performance. female rats in the control group.
Sensory deprivation did not affect the number of trials to acquisition during training (data were pooled Long-term postnatal whisker
for both sexes of control, n = 43, and SD animals, n = 46) (A), but sex-dependently reduced step deprivation significantly reduced
through latency of female rats in the retention test performed 24 h after the training (n = 21–24 per
sex in each group) (B). Box diagrams show interquartile range, horizontal lines are median and neuronal density in CA1 and CA3
whiskers represent 5th and 95th percentiles. Outliers are data outside 5th to 95th percentile. regions of both male and female
*p < 0.05 compared to control female rats. ###p < 0.001 compared to SD male rats. rats (Fig. 11).
86 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96

A C
Male Control
6 Control 6 Male

Reference memory errors

Reference memory errors

5 SD 5 Female

4 4 *
* *
3 3
*
2 2

1 1

0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12

B D
Female SD
6 6 Male

Reference memory errors

Control
Reference memory errors

5 SD 5 Female

4 4

3 3

2 2

1 1

0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12
Days of testing Days of testing
Fig. 5. Effects of sensory deprivation on the spatial reference memory performance in the radial arm maze. Sensory deprivation increased
reference memory errors in male rats (A), but not in female rats (B). The normal difference that was observed between male and female rats in the
control group (C) was eliminated in the sensory-deprived (SD) rats (D). n = 17–23 per sex in each group. The data are presented as mean ± SEM.
Mean differences of all groups were analyzed using Kruskal-Wallis followed by Dunn’s multiple comparison post hoc test, but the results are
presented as pairwise comparisons in the line graphs. *p < 0.05.

DISCUSSION area of the open field is usually considered as an index

We found that chronic postnatal whisker deprivation
affects behavioral responses of young adult rats, mainly
in a sex dependent manner. The major finding of the
current study was that these enduring behavioral
alterations are associated with modifications of neuronal
morphology and density in the CA1 and CA3
hippocampal subfields, neural sites far away from the
deprived cerebral cortex, i.e. the barrel cortex, and
these modifications seem to contribute to the behavioral
outcomes.
Long-term postnatal whisker deprivation did not alter
the number of line crossing and off-wall rearing, but
increased the time spent in the center area of the open
field in both male and female rats. The number of line
crossing and rearing are mostly used as indices of
horizontal and vertical motor activity, respectively, but
these are also indicators of exploratory activity
(Zimcikova et al., 2017). The time spent in the central
of reduced anxiety and increased exploratory/risk-taking
behavior (Shieh and Yang, 2019). Given that even blind
rats with intact whiskers are capable of scoring well in a
gap-crossing test (Kleinfeld, 2008), it is apparent that
whiskers have a crucial role in generating a tactile map
of the environment, perception of the surrounding and
navigational movements. There are papers reporting
failed gap-crossing ability during adolescence after
neonatal whisker trimming (Lee et al., 2009; Chu et al.,
2013), showing that whisker trimming during the critical
neonatal period is enough to set the sensory apparatus
out of order in the course of a tactile-specific challenge.
We assume, a poor and imprecise representation of the
surrounding tactile world shaped by the blunt receptive
fields of neurons in the deprived cortex (Rema et al.,
2003) has led to a pale perceptual image of the world,
ending up with excessive explorative behaviors for gath-
ering enough information from the environment to make
a proper behavioral response. Even partial sensory
 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96 87

A C
Male Control
3 Control 5 Male
Working memory errors

Working memory errors

SD Female
4
2
3

* 2 ** ***
1
*
1

0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12

B Female D SD
5 5
Control Male
Working memory errors

4
SD Working memory errors 4
Female
** *
3 * ** *
3
***
*** *
2 2 *
*
1 1

0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12
Days of testing Days of testing
Fig. 6. Effects of sensory deprivation on the spatial working memory performance in the radial arm maze. Sensory deprivation increased working
memory errors in both male (A) and female rats (B). The normal difference that was observed between male and female rats in the control group (C)
was even more intensified in the sensory-deprived (SD) rats (D). n = 17–23 per sex in each group. The data are presented as mean ± SEM. Mean
differences of all groups were analyzed using Kruskal-Wallis followed by Dunn’s multiple comparison post hoc test, but the results are presented as
pairwise comparisons in the line graphs. *p < 0.05, **p < 0.01 and ***p < 0.001.

deprivation of barrel cortex during the first postnatal week
impairs whisker-dependent exploratory behavior in mice,
which necessitates more exploratory activity in gap-
crossing task, evidenced by more approaches to the
gap (Papaioannou et al., 2013). Our assumption can also
be verified by pondering on the explorative behaviors
observed in our open field test, where SD rats try signifi-
cantly more on-wall rearing attempts but perform alike
control rats while doing off-wall rearing. In contrast to
off-wall rearing which is mainly associated with visual
experience, on-wall rearing is performed against a sur-
face, and also involves active whisker-mediated explo-
ration. The observed increase of on-wall rearing in
sensory-deprived rats could be attributed to the blunt vib-
rissal tactile perception of the surrounding surfaces. The
impaired tactile acuity may also contribute to the aug-
mented visits to the central area of the open field, which
can also be associated with whisker-dependent explo-
ration of the surface. This should be mentioned that
Papaioannou et al. (2013) who had used both male and
female mice for their experiments and performed the
gap-crossing test on immature animals (PND31-33), but
reported no sex difference. In our study, however, the
increased activity against the walls was only significant
in female rats (Papaioannou et al., 2013). Haridas et al.,
(2018) also reported that acute whisker desensitization
of adult male mice by lidocaine increases the time spent
in the center of the open-field arena (Haridas et al.,
2018). Although it is difficult to compare our study with
the aforementioned works, due to the differences in the
paradigm and method of sensory deprivation plus the nat-
ural differences in behaviors of rats and mice, but they
also interpreted this anxiolytic effect as a consequence
of sensory insufficiency. Meanwhile, these findings can-
not discount the possible involvement of indirect effects
on areas with motor functions. The development of motor
cortex and the functional properties of its neurons are
highly dependent on early-life tactile experiences
(Donoghue and Sanes, 1988). It has been shown that
neonatal whisker deprivation reduces the size of M1 vib-
88 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96

Male Female anxiety evidenced by increased

8 10 time in the exposed arms of the

Reference memory erros

Reference memory erros

†
elevated plus maze or central area
##
of the open-field box might be an
* 8 ††
6 indicative of increased risk-taking
† * and aggressive behaviors
6 (Valvassori et al., 2017) as
#
4 reported in other sensory depriva-
4 tion studies directly measured
aggressive behaviors (Soumiya
2 et al., 2016). Whisker trimming
2
has been reported to cause distur-
bances in the regulation of
0 0 amygdala-dependent functions
saline Sc 0.1 Sc 0.2 saline Sc 0.1 Sc 0.2 (Soumiya et al., 2016). Presum-
ably, an imbalance among net-
10 Control 15 works directly or indirectly
Working memory errors
Working memory errors

SD †† connecting barrel field in the pri-

8 ### mary somatosensory cortex
† (S1BF) and amygdala caused by
10 sensory deprivation is implicated
6 † in the aggressive or risk-taking
# behaviors registered in the ele-
4 vated plus maze and open-field
5 tests.
2 Meanwhile the data pertaining
fear-based memory in the current
0 study confirms such assumption
0 to some extent. We found that
saline Sc 0.1 Sc 0.2 saline Sc 0.1 Sc 0.2
long-term postnatal whisker
Fig. 7. The effects of two doses of scopolamine (SC), 0.1 mg/kg and 0.2 mg/kg, on the spatial deprivation also influenced the
reference and working memory of control and sensory-deprived (SD) rats in the radial arm maze. The fear-based memory of the rats.
lower dose of scopolamine induced more impairment of reference memory in SD rats than control Passive avoidance retention was
animals, while its higher concentration was less effective in SD female rats. The effect of scopolamine impaired in SD female rats, while
on working memory was not significantly different between control and SD rats. Box diagrams show
interquartile range, horizontal lines are median and whiskers represent 5th and 95th percentiles. it was not significantly affected in
Outliers are data outside 5th to 95th percentile. n = 17–23 per sex in each group. *p < 0.05 SD male rats. Amygdala not only
compared to drug-matched control rats, #p < 0.05, ##p < 0.05 and ###p < 0.001 compared to assesses fear and anxiety
saline-injected control rats, yp < 0.05 and yyp < 0.05 compared to saline-injected SD rats. attributed to stimuli but also
evaluates rewarding or aversive
consequences of a behavior,
rissa representation and alters its output, which affects
thereby strongly affecting sensory perceptions, memory
the highly integrated sensory–motor behaviors including
qualities and behavioral strategies (White and
exploratory activity (Milani et al., 1989; Huntley, 1997).
McDonald, 1993; Maren and Holmes, 2016). Several
Accordingly, the structural and functional refinements of
brain regions contribute to fear memory that include, but
motor cortices may also take part to the altered motor
are not limited to amygdala and hippocampus (Pittenger
and exploratory activity of SD rats, though the extent that
et al., 2006) that are reciprocally connected and are coor-
such possible alterations contribute to the SD-induced
dinately activated during fear learning and memory retrie-
behavioral outcomes requires further studies.
val (Pitkänen et al., 2000). Hippocampal outputs to the
The lower level of anxiety of SD rats, evidenced in
basolateral amygdala provide contextual information that
augmented visits to the central area of the open-field
are essential for fear conditioning (Maren and Fanselow,
box, was further supported by reduced defecation and
1995; Fendt and Fanselow, 1999). The activity of both
shortened time of self-grooming, particularly in female
CA1 and CA3 pyramidal neurons has been implicated in
SD rats. The behavioral performance in the open-field
the retrieval of fear memories (Hall et al., 2001; Sun
test was accordingly correlated with the observations in
et al., 2017). The retrieval of contextual memory is corre-
the elevated plus-maze; where once again, female SD
lated with selective activation of CA1 hippocampal neu-
rats spent more time in the exposed arms of the maze.
rons (Hall et al., 2001). The reactivity of CA3 neurons
Even though it is hard to interpret based on these two
during retrieval of fear memory has also been reported
tests whether the higher number of open-arm entries/
(Sun et al., 2017).
closed-arm returns as well as the increased time spent
Spatial memory performance in the radial arm maze, a
in the junction of the arms is the fated outcome of
classical hippocampus-dependent test, was also impaired
higher explorative behavior or a token of hesitant
by chronic postnatal whisker deprivation in a sex-
decision-making (Sestakova et al., 2013). The reduced
 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96 89

A Control SD C
Apical dendrites - Male Apical dendrites - Control
10

Number of intersections
10

8 Control 8 Male
SD Female
6 6
Male

4 4

2 2

0 0
0 100 200 300 400 0 100 200 300 400

Apical dendrites - Female Apical dendrites - SD

Number of intersections
8 8
Control Male
Female

6 SD 6 Female

4 4

2 2

0 0
0 100 200 300 400 0 100 200 300 400
Radial distance from soma (µm) Radial distance from soma (µm)
B
Total dendritic length (103 µm)

Apical dendrites Basal dendrites

D Basal dendrites - Male Basal dendrites - Control
4 #
4
#
Number of intersections

# ***
15 Control 15 Male
2 2 * SD Female

10 10

0 0
Male Female Male Female
5 5
Number of branch points

40

40
* 0 0
* 0 50 100 150 200 250 0 50 100 150 200 250

20 *
20 Basal dendrites - Female rats Basal dendrites - SD

15
Number of intersections

0 0 12
Male Female Male Female Control Male
Number of terminal points

10 SD Female
Control 8
SD
40 40
* **
5 4

20 20 *
0 0
0 50 100 150 200 250 0 50 100 150 200 250
0 0 Radial distance from soma (µm) Radial distance from soma (µm)
Male Female Male Female

Fig. 8. The effects of sensory deprivation on the dendritic morphology of CA1 pyramidal neurons in male and female rats. A: Representative
photomicrographs of Golgi-stained hippocampal CA1 pyramidal neurons from control and sensory-deprived male and female rats. Scale
bar = 100 mm. B: The effects of whisker deprivation on total dendritic length, branch points and terminal points in apical and basal dendrites of CA1
neurons. Box diagrams show interquartile range, horizontal lines are median and whiskers represent 5th and 95th percentiles. Outliers are data
outside 5th to 95th percentile. Sholl plots (depicted as mean ± SEM) show the number of apical (C) and basal (D) dendritic intersections at
concentric rings with increasing radii centered on the soma of CA1 neurons. The left panel represents differences between control and SD rats in
either male or female rats, the right panel shows sex differences in dendritic intersections within each group. *p < 0.05, **p < 0.01, ***p < 0.001
compared to sex-matched control, #p < 0.05, ##p < 0.01 compared to male rats in the same group.

dependent manner. Here, we reported that male SD rats memory errors in both male and female rats but this effect
had more reference memory errors compared to the sex- was more pronounced in females. Earlier studies have
matched controls. Sensory deprivation increased working reported the effects of sensory deprivation on the
90 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96

A Control SD C
Apical dendrites - Male Apical dendrites - Control

Number of intersections
10
8 Control Male
8
SD Female
6
6
Male

4 4

2 2

0 0
0 100 200 300 400 0 100 200 300 400

Apical dendrites - Female Apical dendrites - SD

Number of intersections
10

8 Control 8 Male
Female

SD Female
6 6

4 4

2 2

0 0
0 100 200 300 400 0 100 200 300 400
Radial distance from soma (µm) Radial distance from soma (µm)
B Apical dendrites Basal dendrites
Number of branch points Total dendritic length (103 µm)

4 D
Basal dendrites - Male Basal dendrites - Control
4
Number of intersections

#
2 12 12 Male
Control
2
SD Female
8 8

0 0
Male Female Male Female
4 4
30 40 Control
# SD
0 0
20
0 50 100 150 200 250 0 50 100 150 200 250
#
20

10
*** Basal dendrites - Female rats Basal dendrites - SD

15
Number of intersections

12
0 0 Control
Male Female Male Female
Male
SD Female
Number of terminal points

10
30 8

40
20 5 4

*** $$
20
10
0 0
0 50 100 150 200 250 0 50 100 150 200 250
0 0 Radial distance from soma (µm) Radial distance from soma (µm)
Male Female Male Female

Fig. 9. The effects of sensory deprivation on the dendritic morphology of CA3 pyramidal neurons in male and female rats. A: Representative photo
micrographs of Golgi-stained hippocampal CA3 pyramidal neurons from control and sensory-deprived male and female rats. Scale bar = 100 mm.
B: The effects of whisker deprivation on total dendritic length, branch points and terminal points in apical and basal dendrites of CA3 neurons.
Box diagrams show interquartile range, horizontal lines are median and whiskers represent 5th and 95th percentiles. Outliers are data outside 5th to
95th percentile. Sholl plots (depicted as mean ± SEM). Sholl plots representing the number of apical (C) and basal (D) dendritic intersections at
concentric rings with increasing radii centered on the soma of CA3 neurons. The left panel show differences between control and sensory-deprived
(SD) rats in either male or female rats, the right panel represents sex differences in dendritic intersections within each group. *p < 0.05, **p < 0.01,
***p < 0.001 compared to sex-matched control, #p < 0.05, ##p < 0.01, ###p < 0.001 compared to male rats in the same group.

learning and memory. It has been shown that whisker- (Haridas et al., 2018). Soumiya et al., (2016) reported that
related tactile information are essential for the short whisker trimming of male mice during early postnatal per-
term recognition memory in novel object recognition test iod (PND1–PND10) impairs tactile sensory performance
 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96 91

A CA1 apical dendrite B CA1 basal dendrite

Male Female Male Female

Control

SD

30 Male Female 40
Male Female Control
SD

Spines /20 µm
Spines /20 µm

30
*
20 *
20

10
* * 10 *
*
0 0
m

m
m

m
in

in
by

by
by
l

by
in

l
in
ta

ta

ta

ta
oo

oo
oo

Th

oo

Th
Th

Th
ub

ub
ub
To

To

ub
To

To
hr

hr
hr

hr
St

St
St

St
us

us
us

us
M

M
M

C CA3 apical dendrite D CA3 basal dendrite

Male Female Male Female

Control

SD

30 Male Female Male Female Control

SD
Spines /20 µm
Spines /20 µm

** 20 ***
20 *

10
10
** ** ***
0 0
m
m

in
m

by
in

by
l

l
by

in
by

l
l
in

ta

ta
ta
ta

oo
oo

oo

Th
oo

Th

Th
Th

ub

ub
To

To
ub

ub
To

To

hr
hr

hr
hr

St

St
St

St

us
us

us
us

M
M

M
M

Fig. 10. Sensory deprivation altered the density of spines on dendrites of CA1 and CA3 pyramidal neurons. Representative photomicrographs of
apical and basal dendritic segments of CA1 and CA3 neurons from control and sensory-deprived (SD) male and female rats are shown at the top of
(A–D) panels. Scale bar = 10 mm. Density of different spine types in control and sensory-deprived male and female rats are represented at the
bottom of (A–D) panels. Box diagrams show interquartile range, horizontal lines are median and whiskers represent 5th and 95th percentiles.
*p < 0.05, **p < 0.01 and ***p < 0.001 compared to control.

in gap-crossing test, 8–9 weeks after birth, while learning the hippocampal function (Morris, 1981). Zhao and col-
and memory in a whisker-cued eight-arm radial maze is leagues have reported that temporary auditory depriva-
not affected (Soumiya et al., 2016). In this type of radial tion by perforating the eardrum of male rats on PND 14
maze, what is tested is the tactile perception-based impairs spatial memory performance in Morris water
reward-driven memory, rather than visual-cued spatial maze and Y maze performed on PND 42, when the ear-
memory and animal have to memorize the relationship drum has been healed. Structural and functional studies
between a whisker cue at the entrance of the bated arms on hippocampal slices revealed decreased number of
and the food reward at the ends. This essential difference dendritic spines on CA1 neurons, reduced postsynaptic
of the maze task and the shorter period of whisker depri- densities and suppressed NMDA receptor-mediated post-
vation may underlie the different findings, compared to synaptic currents (Zhao et al., 2018). It is reasonable to
our study. The spatial memory task in a common radial interpret that the impairment of spatial memory in SD rats
maze, as the one we employed, is highly dependent on found in this study, stems from evident alteration on neu-
92 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96

A CA1 region CA3 region

Control SD Control SD
Male
Female

CA1 region CA3 region

Number of neurons/0.063mm2

Number of neurons/0.063mm2
B 100 100
Control
SD

** **
50 *** 50 **

0 0
Male Female Male Female
Fig. 11. Effects of whisker deprivation on the numbers of pyramidal neurons in CA1 and CA3 subfields of the hippocampus in males and females. A:
Representative images of Cresyl Violet stained neurons in CA1 and CA3 regions. B: Comparison of neuronal density in CA1 and CA3 regions
showed a significant reduction of density in both male and female sensory-deprived (SD) rats compared to control rats. n = 4–6 per sex in each
group. **p < 0.01 and ***p < 0.001 compared to control.

ronal morphology and density in the hippocampus, even
though the structural and functional alteration in other
brain circuits contributing to the task could also potentially
be implicated.
The septohippocampal cholinergic input innervates
CA1 and CA3 pyramidal neurons in their basal
dendrites. This cholinergic projection is involved in
learning/memory and attention (Teles-Grilo Ruivo and
Mellor, 2013). In the current study the lower dose
(0.1 mg/kg) of scopolamine impaired working memory in
control and SD male rats, while the reference memory
was impaired by a lower dose of scopolamine only in
SD rats. Many studies have reported that scopolamine
mainly disrupts spatial working memory but spares spatial
reference memory (Hodges Jr et al., 2009; Kay et al.,
2010). Here, the higher dose of scopolamine impaired ref-
erence memory in both control and SD male and female
rats. The distinctive action of scopolamine on the spatial
working and reference memory (usually studied in male
animals) has been attributed to the level of training; when
sufficient training reduces the level of reference memory
errors, the effect of scopolamine on this type of memory
becomes evident (Lydon and Nakajima, 1992). Interest-
ingly, here we found that the lower dose of scopolamine
was not able to induce significant working memory errors
in female control rats. In support of Lydon and Nakajima’s
assumption, the higher baseline level of working memory
errors in control female rats, compared to males, might
have masked the potential amnestic effect of the lower
dose of scopolamine on working memory in female rats.
The finding that the lower dose of scopolamine impaired
reference memory in both male and female SD rats (while
it was not able to do so in control rats) could be a result of
partial impairment of septal cholinergic inputs to the hip-
pocampus, which make this system more vulnerable to
even a slight disruption by a low dose of scopolamine.
Meanwhile, the higher dose of scopolamine impaired ref-
erence memory in male and female controls, but this
effect was much weaker in the SD rats. This finding also
supports the lower contribution of cholinergic system to
 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96
the spatial memory, as a consequence of its impairment.
The septohippocampal cholinergic innervation regulates
neuronal activity and synaptic plasticity of hippocampal
pyramidal neurons (Dannenberg et al., 2017; Maurer
and Williams, 2017) and a potential impairment of the
cholinergic system, besides the contribution to memory
impairment, may also be involved in the structural
changes of the pyramidal neurons. This assumption
requires further work to be verified.
The observed structural remodeling of neuronal
dendrites and density of dendritic spines in the
hippocampal CA1 and CA3 subregions reflects the
alterations in their connectivity with multiple input
sources that in turn could affect their processing
capacity and functional outputs. Experience-dependent
neuronal activity is required to establish and selectively
preserve synaptic connectivity; a key process for normal
wiring of neural circuits (De Roo et al., 2008; Johnson-
Venkatesh et al., 2015). Neurons in the barrel cortex
show several structural adaptations to the reduced/elimi-
nated sensory inputs started during early postnatal days.
Increased secondary branch points (Maravall et al.,
2004), reduced dendritic spine loss (Zuo et al., 2005),
impaired development and reduced density of dendritic
spine (though no longer detectable in adult animals)
(Briner et al., 2010), enlarged soma size, increased den-
dritic protrusion and expansion of basilar dendritic arbor
and retraction of apical dendrites (Chen et al., 2012;
Chen et al., 2015) are among the morphological alter-
ations reported in pyramidal neurons following sensory
deprivation of barrel cortex. In the current study we used
a very long-term tactile deprivation paradigm that was
almost unique in terms of the onset and duration besides
the brain structure that was studied, a region far higher in
the hierarchy of processing and integrating sensory infor-
mation. The activity of hippocampal pyramidal neurons is
evidently influenced by sensory experiences through tem-
poroammonic path. Sensory inputs drive the activity of
CA1 and CA3 neurons through direct and indirect tem-
poroammonic connections that are important for proper
structural and functional development of hippocampal
neurons (Hill and Best, 1981; Johnson-Venkatesh et al.,
2015; Milshtein-Parush et al., 2017). In behaving rats,
touch-guided behavior or electrical stimulation of the
infraorbital nerve, which carries sensory signals from
whiskers to the brain, evokes firing of CA1 neurons
(Pereira et al., 2007; Itskov et al., 2011; Vatanparast
et al., 2013). It has been shown that whisker deprivation
during early life reduces the activity of CA3 pyramidal
neurons in mice and facilitates their synapses to CA1 neu-
rons (Milshtein-Parush et al., 2017).
In this work, some structural alterations in the
morphology and density of CA1 and CA3 neurons were
detected that, similar to the behavioral outcomes,
showed sex-dependency. Whisker deprivation reduced
arborization and density of dendritic spines on the apical
dendrites of CA1 neurons of female rats, while the basal
dendrites of CA1 neurons showed retraction along with
reduced density of dendritic spines in both male and
female rats. CA3 pyramidal neurons showed extended
arborization of apical dendrites in SD male rats but their
93
basal dendrites were reduced. The reduction of dendritic
spines reached the significance level only for the total
density of spines of all types (not for any of the three
types), and was not affected on the basal dendrites of
CA3 neuros of SD male rats. In SD female rats, apical
dendrites of CA3 neurons were not affected but the
basal dendrites showed more arborization while the
density of dendritic spines was reduced in both apical
and basal dendrites of SD female rats. The effects of
postnatal whisker deprivation on behavioral outcomes
were sex-dependent, as in the case of memory tests.
This might be mediated by sex-dependent effects of
whisker deprivation on the complexity of apical dendrites
of CA1 neurons and both apical and basal dendrites of
CA3 neurons. We also found that thin spines were
reduced in basal and apical dendrites of CA1 neurons of
female SD rats. The reduction of mushroom spines was
also significant for these regions unless for basal
dendrite of CA3 neurons in female SD rats. In male SD
rats, only the thin spines showed significant reduction in
the basal dendrite of CA1 neurons. The thin spines are
dominated by NMDA receptors and have high structural
flexibility, enabling them to expand and stabilize or
retract. Mushroom spines are more mature and stable
spines dominated by AMPA receptors. These features
have suggested the thin spines as mediators of learning
that could be turned into the mushroom spines, as
memory spines (Bourne and Harris, 2007). The more pro-
nounced and significant reduction of the thin and mush-
room spines density in CA1 and CA3 pyramidal neurons
of SD female rats could contribute to the more severe
memory impairment, and possibly other behavioral alter-
ations observed in females following whisker deprivation.
The well-known mutual connections of the hippocampus
with cerebral cortex and amygdala (Pikkarainen et al.,
1999; Pitkänen et al., 2000), the contribution of hippocam-
pus to anxiety, spatial memory and fear based memory
(Best and Orr Jr, 1973; Bernabeu et al., 1997; Izquierdo
et al., 2008), together with our findings on the sex-
dependent alterations of neuronal morphology in the
CA1 and CA3 hippocampal subregions of SD rats may
provide further explanations for the results obtained in
the behavioral test. It should be noted that the differences
of neuronal structure between sexes do not always
‘‘cause” differences in cognitive functions and behavior,
but may also ‘‘compensate” for differentiating effects that
potentially could originate from other sources (De Vries,
2004).
Multiple mechanisms may contribute to the selective
retraction/expansion of dendritic arbor and dendritic
spine loss in SD rats. An important candidate could be
the heterogeneous source of synaptic inputs, depending
on the type of neuron (CA1 or CA3 pyramidal neurons)
as well as on the location of dendritic tree in a specific
neuronal type. The basal dendrites and proximal region
of apical dendrites of CA1 neurons mainly receive inputs
from the Schaeffer collaterals of CA3 pyramidal neurons
while the distal segments of apical dendrites receive
direct innervation from the entorhinal cortex via the
performant pathway. The apical dendrites of CA3
neurons are innervated by the axons of granule cells
94 P. Yarmohammadi-Samani et al. / Neuroscience 480 (2022) 79–96
(mossy fibers) and the basal dendrites primarily receive
recurrent inputs from other CA3 neurons (Spruston,
2008). In accord with behavioral findings, the structural
alterations of pyramidal neurons were more pronounced
in female rats. The expansion of basal dendritic arbors
of CA3 neurons, which can indicate increased local recur-
rent inputs, could be explained as an adaptive response
to compensate for the reduced indirect temporoammonic
inputs. On the other hand, a ‘‘loss-of-function” associated
with reduced direct inputs from entorhinal cortex to CA1
apical dendrites, might underlie the retraction of CA1 api-
cal dendrites in female SD rats. A major challenge to
these hypotheses is the lack of such effects in the male
SD rats. Several studies have reported sex differences
in structure and plasticity of hippocampal neurons that
underlie the basal sex-related disparities in behavioral
and cognitive functions and different responses to envi-
ronmental factors (Shors et al., 2001; Scharfman and
MacLusky, 2017; Yagi and Galea, 2019; Brandt et al.,
2020). Such sex-based disparities of hippocampal
responsiveness may have provided a ground for the dif-
ferent effects of SD on the neuronal morphology in CA1
and CA3 hippocampal subregions of male and female
rats, and the consequent outcomes in the vast
hippocampal-dependent behavioral paradigms.
At present, an important open question is the
molecular mediators that correlate the absence of
sensory inputs to the altered morphology/density of
hippocampal pyramidal neurons, and cognitive and
behavioral outcomes. Altered expression of
neurotrophins, especially brain-derived neurotrophic
factor, is among the leading candidates. Different
neurotrophins are expressed in the hippocampus, that
are important for neuronal survival, maintenance of
dendritic spines and regulation of dendritic arborization
that usually work selectively and in an activity-
dependent manner (McAllister, 2000; Kellner et al.,
2014). In the cerebral cortex, sensory experience controls
the neuronal development and function through an
activity-driven expression of neurotrophins (Rocamora
et al., 1996; Jiao et al., 2011). The altered excitability of
hippocampal CA1 and CA3 pyramidal neurons is another
critical factor that might be correlated to the structural
changes and be involved in the cognitive and behavioral
impairments as well. Further work to clarify this issue,
and also structural analysis of CA1 and CA3 neurons dur-
ing an earlier (PND 40) and later periods of development
(PND 100) are warranted.
In summary, here we reported that long-term sensory
deprivation of the barrel-cortex, as an axial sensory
processing unit in rodents, concludes in a severe
alteration in the structure of hippocampal CA1 and CA3
regions and hippocampal-dependent behaviors. Our
observations provide another line of evidence for the
idea that the behavioral impairments following to
sensory deprivation are not solely underlain by
neuroplastic changes taking place in the sensory
cortices. Instead, the activity-dependent neural
alterations could be tracked in the regions having tight
structural and functional associativity with cerebral
cortex at a higher level of the sensory information
processing hierarchy. The witnessed discrepancies in
the behavioral alterations and neuronal modifications of
male and female SD rats, could arise from differential
interaction of sensory deprivation with internal
substrates that underlie intrinsic sex differences
between male and female rats. A further progress in
discovering the true mechanisms behind our
observations, require studying the molecular mediators
governing the functional organization and architecture of
neural circuits in an activity-dependent medium.
DECLARATIONS OF INTEREST
None.
ACKNOWLEDGMENTS
This work was supported by a grant (No. 96005769) from
Iran National Science Foundation (INSF) and Cognitive
Science and Technology Council (CSTC). We thank
Mojgan Jouybar, Fatemeh Taghavi, Nima Rahaei and
Zahra Naderi for technical assistance, observing video
tracks and extracting behavioral data.
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