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Journal of Autism and Developmental Disorders
https://doi.org/10.1007/s10803-022-05684-y
ORIGINAL PAPER
Altered Developmental Trajectory inMale andFemale Rats
inaPrenatal Valproic Acid Exposure Model ofAutism Spectrum
Disorder
KumariAnshu1,4· AjayKumarNair1,3· ShobaSrinath2· T.RaoLaxmi1
Accepted: 13 July 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022
Abstract
Early motor and sensory developmental delays precede Autism Spectrum Disorder (ASD) diagnosis and may serve as early
indicators of ASD. The literature on sensorimotor development in animal models is sparse, male centered, and has mixed
findings. We characterized early development in a prenatal valproic acid (VPA) model of ASD and found sex-specific devel-
opmental delays in VPA rats. We created a developmental composite score combining 15 test readouts, yielding a reliable
gestalt measure spanning physical, sensory, and motor development, that effectively discriminated between VPA and control
groups. Considering the heterogeneity in ASD phenotype, the developmental composite offers a robust metric that can enable
comparison across different animal models of ASD and can serve as an outcome measure for early intervention studies.
Keywords Autism· Developmental milestones· Sex differences· VPA· Animal models· Composite score
The brain undergoes a series of complex, critical devel-
opmental stages over a long period of time starting from
the embryonic period, through puberty, into young adult-
hood. The initial developmental period (birth to 5 years)
is especially crucial as the child starts to interact with the
environment and rapidly develops sensory, motor, social,
linguistic, and cognitive skills (Moodie etal., 2014). The
temporal sequence in which these skills appear is predict-
able, making it possible to identify normative milestones in
the early developmental journey of a child’s growth. Timely
acquisition of these milestones indicates healthy develop-
ment of a child, and deviation from this normal trajectory
signals potential for neurodevelopmental disorders such as
autism spectrum disorder (ASD), and other adverse behav-
ioral consequences (Davidovitch etal., 2018; R. J. Landa
etal., 2013; Sheldrick etal., 2019).
Retrospective studies in children with ASD using parent
reports and old home-videos have reported deviations from
normal developmental trajectories (Grace T Baranek, 1999;
Barbaro & Dissanayake, 2009; Osterling & Dawson, 1994).
Similarly, prospective studies in children at high risk for
ASD (younger siblings of ASD diagnosed children) have
demonstrated delayed skill acquisition in sensory, motor,
and social domains (Davidovitch etal., 2018; Landa &
Garrett-Mayer, 2006; Ozonoff etal., 2010; Zwaigenbaum
etal., 2005). Both gross and fine motor skills have been
found to be delayed in children later diagnosed with ASD
(Bolton etal., 2012; Lemcke etal., 2013). During early
development, atypical responsiveness to sensory inputs
across auditory, visual, and somatosensory modalities have
been observed in children with ASD or at high risk for ASD
(Grace T Baranek, 1999; Leekam etal., 2007; McCormick
etal., 2016; Wolff etal., 2019; Zwaigenbaum etal., 2005).
* T. Rao Laxmi
laxmir@gmail.com
Kumari Anshu
anshu.dna@gmail.com
Ajay Kumar Nair
ajay.nimhans@gmail.com
Shoba Srinath
shobasrinath@gmail.com
1 Department ofNeurophysiology, National Institute
ofMental Health andNeurosciences (NIMHANS), Hosur
Main Road, Bengaluru, Karnataka560029, India
2 Department ofChild andAdolescent Psychiatry, National
Institute ofMental Health andNeurosciences (NIMHANS),
Hosur Main Road, Bengaluru, Karnataka560029, India
3 Present Address: Center forHealthy Minds, University
ofWisconsin-Madison, Madison53703, WI, USA
4 Present Address: Waisman Center, University
ofWisconsin-Madison, Madison53705, WI, USA
Journal of Autism and Developmental Disorders
1 3
Furthermore, delayed acquisition of early motor and sensory
milestones have been shown to predict subsequent cogni-
tive and behavioral outcomes in children with ASD (G. T.
Baranek etal., 2013; Bedford etal.,2016b; Gernsbacher
etal., 2008; Thye etal., 2018; Uljarević etal., 2017). Impor-
tantly, intervention programs for children with ASD are
likely to be more effective when started early in the develop-
mental period (Dawson, 2008; Rebecca J. Landa, 2018). In
summary, there is mounting evidence for the need to study
early developmental trajectory in ASD, i.e., to understand
how ASD unfolds from birth towards the appearance of core
symptoms (Jones etal., 2014; Shen & Piven, 2017).
ASD has high clinical and biological heterogeneity that
arises, at least in part, due to the large number of genetic,
environmental, and epigenetic factors and their interactions
that may contribute to ASD etiology (Banerjee etal., 2014;
Bölte etal., 2019; Cheroni etal., 2020; Kim & Leventhal,
2015). Animal models are typically based on experimental
manipulation of one of the known genetic and environmen-
tal risk factors and thus provide opportunities to delineate
specific pathways in ASD pathogenesis (Ergaz etal., 2016;
Halladay etal., 2009). For example, mutations of genes
related to synaptic scaffolding and neuronal cell adhesion
such as SHANK3, CNTNAP2, and NLGN3 genes affect syn-
aptic development and impact excitatory-inhibitory neuro-
transmission. Mutations in PTEN, and TSC1/ TSC2 inhibit
activation of the mechanistic target of rapamycin complex
and thus impact neuronal protein synthesis and growth
regulation(Chen etal., 2015; Möhrle etal., 2020). Among
environmental risk factors, maternal immune activation
due to polyinosine:cytosine (poly(I:C), viral mimic) and
lipopolysaccharide (LPS, bacterial mimic), triggers micro-
glial activation, elevations in pro-inflammatory cytokines
and transcriptome dysregulation (Knuesel etal., 2014; Lom-
bardo etal., 2018; Patterson, 2011). Prenatal exposure to
the drug valproate (VPA, a GABA agonist that also acts as
a histone deacetylase inhibitor) acts via epigenetic mecha-
nisms to influence the expression profile of different genes
involved in cell proliferation and differentiation (Favre etal.,
2013; Kataoka etal., 2013; Nicolini & Fahnestock, 2018).
Behavioral phenotyping of these animal models using a bat-
tery of assays can link these causal mechanisms to specific
domains of dysfunction found in ASD and facilitate the
development of targeted therapeutic strategies (Möhrle etal.,
2020; Michela Servadio etal., 2015; Silverman etal., 2022).
Further, characterization of early developmental trajectories
in these animal models can provide clues towards a mecha-
nistic understanding of the emergence of ASD phenotype.
In the present study, we assessed early developmental
trajectory in the VPA model. The VPA model mimics ASD
at multiple levels, starting from the behavioral alterations
seen in ASD, but also ranging from the molecular and cel-
lular levels to anatomical and circuit level atypicalities. The
VPA model has been well-validated for core ASD features—
reduced social interaction, increased stereotyped behavior—
as well as additional features such as presence of hypersensi-
tivity, enhanced anxiety, inattention, hyperactivity, and sleep
disturbances (Chaliha etal., 2020; Cusmano & Mong, 2014;
K. Markram, Rinaldi, Mendola, Sandi, & Markram, 2008;
Schneider & Przewłocki, 2005). At the molecular level, pre-
natal VPA exposure leads to reduced expression of neuroli-
gin (NLGN, a key ASD risk gene) mRNA in somatosensory
cortex and hippocampus (Kolozsi etal., 2009). Neuroana-
tomical hallmarks of VPA exposure in rodents include lower
Purkinje cell counts in the cerebellum (widely observed in
postmortem ASD brains), as well as changes in neuronal
density and neuronal cell count in prefrontal, somatosensory,
and motor cortices, regions impacted in ASD (Ingram etal.,
2000; Nicolini & Fahnestock, 2018; Roullet etal., 2013; R.
Zhang etal., 2018). There is robust evidence implicating
excitatory-inhibitory (E/I) imbalance in ASD pathogenesis
and have been documented in the VPA model in terms of
both increased glutamatergic as well as decreased GABAe-
rgic signaling (Gogolla etal., 2009; Rinaldi etal., 2007;
Rubenstein & Merzenich, 2003). Finally, at the circuit level,
altered connectivity and excitability in cortical microcircuits
have been reported in the VPA model (Rinaldi etal., 2008a,
2008b).
Animal models can never recapitulate the full spectrum
of human condition, let alone span the heterogeneity found
in ASD. Nevertheless, the VPA model mimics ASD charac-
teristics remarkably well across many different levels, mak-
ing it an excellent model for studying ASD pathophysiology
(Mabunga etal., 2015; H. Markram, Rinaldi, & Markram,
2007; Roullet etal., 2013).
In the early development literature however, very few
studies have carried out a detailed examination of early
development in the VPA model (Hou etal., 2018) as most
studies have checked a few isolated developmental mile-
stones as part of a larger body of work (for example, Al
Sagheer etal., 2018; Dobrovolsky etal., 2019; Scheggi etal.,
2020). Another limitation is that most of these studies have
focused exclusively on development in VPA males. ASD is
more prevalent among males and there are studies sugges-
tive of sex-specific differences in the acquisition of early
developmental milestones (Carter etal., 2007; Harrop etal.,
2021; Messinger etal., 2015).
We carried out a detailed evaluation of the ontogeny of
developmental milestones in both male and female VPA
rats. We systematically characterized the early develop-
mental trajectory (from postnatal day 4 till weaning) of
VPA and control rats using a comprehensive test battery
spanning physical, sensory, and motor developmental
milestones. Finally, considering the heterogeneity of the
ASD phenotype, we developed a rodent developmental
composite score by incorporating all test readouts into
Journal of Autism and Developmental Disorders
1 3
a single score and evaluated the efficacy of this overall
developmental profile in discriminating between VPA and
control rats.
Methods
Experimental Animals andPrenatal VPA Rat Model
ofAutism
Experimental protocols were carried out with approval
from the Institutional Animal Ethics Committee, at the
National Institute of Mental Health and Neurosciences
(NIMHANS), Bengaluru. Adult Sprague Dawley female
and male rats (2–3months old) were procured from the
Central Animal Research Facility (CARF), NIMHANS.
These rats were housed in standard home cages with corn-
cob bedding and were maintained on 12:12h light/dark
cycle with lights on at 7:00 AM in the morning. Standard
chow (Special Diets Services) and water were available
adlibitum. Every effort was made to minimize the num-
ber of animals used, and to minimize their suffering.
Female rats were examined for their estrous phase
by visual inspection of external genitalia prior to mat-
ing (Byers etal., 2012). Female rats, found to be ready
or receptive for the mating (in the estrous or proes-
trous phase), were kept for overnight mating (1:2 male
to female ratio), and pregnancy was determined by the
presence of a vaginal plug on the following morning
(designated as embryonic day E1). Weight gain in dams
was checked throughout the gestation period to con-
firm the pregnancy. On E12.5, prenatal valproate (VPA)
exposure was provided to treated dams (n = 8) using a
single intraperitoneal (IP) injection of VPA (450mg/kg
sodium salt of valproic acid (NaVPA, Sigma), dissolved
in 0.9% saline solution at a concentration of 100mg/ml)
while control dams (n = 8) received a single IP injection
of saline solution (SAL). We have previously used the
above protocol (VPA dose and treatment time window)
and reported core ASD features such as reduced social
interaction as well as additional features such as impaired
sensorimotor gating and attentional atypicalities, in VPA
rats (Anshu etal., 2017). Four female dams were housed
together in a standard cage until E18 and subsequently,
dams were housed individually in standard home cages
and left undisturbed to raise their own litters until wean-
ing. No additional enrichment was provided. Sex of rat
pups was determined by examining the anogenital dis-
tance, which is larger in males as compared to females.
Experimental Plan
Experiments were carried out on male and female offspring
of the VPA treated and control dams described above. We
found complete fetal reabsorption (zero pups born) in 50%
(4/8) VPA injected dams, and 0% (0/8) SAL injected dams.
Complete fetal reabsorption has been reported to occur in
the VPA model (Favre etal., 2013). All offspring from
the four VPA injected dams that gave birth were used in
the study. Accordingly, offspring from four SAL injected
dams, with similar litter sizes, were used as controls. There
was no difference in average litter size for the VPA and
SAL groups used in the present study (t6 = 0, p = 0.873;
SAL (n = 4): 6.5 ± 0.866; VPA (n = 4): 6.5 ± 0.957). See
supplementary information (SI section1.2 and Figure S1)
for additional details of pregnancy outcomes in SAL and
VPA rats. Overall, eight litters were used in the study,
with a total of 52 offspring (12 SAL male, 14 SAL female,
18 VPA male, and 8 VPA female). Day of birth was con-
sidered postnatal day 1 (P1). Developmental assays were
performed in SAL and VPA pups between postnatal day 4
(P4) and postnatal day 21 (P21, weaning) or until all pups
achieved developmental milestones. To assess the overall
growth of rat pups, body weight was measured daily dur-
ing this period. Finally, locomotor activity was assessed in
these pups using an open field task (Smirnov & Sitnikova,
2019; Subhadeep etal., 2020) on P21. See Fig.1 for the
experimental timeline.
Developmental Milestones Testing
Physical maturity and the ontogeny of sensory and motor
milestones were assessed (Fig.2) using an extensive
rodent developmental test battery. All tests were con-
ducted in the lights-on period and all behavioral assess-
ments were performed by the first author. Each day, at the
time of testing, rat pups from the same litter were taken
out from their home-cage and transferred to a heated hold-
ing chamber. To avoid any order effect, for each test, rat
pups were randomly removed one by one from the holding
chamber, assessed for developmental milestones, and then
returned to the holding chamber. Once all pups from a lit-
ter were tested, they were returned to their home-cage at
the same time. There is a natural progression in terms of
the age (postnatal day) at which different developmental
milestones are achieved. To estimate the approximate time
of onset, prior to actual data acquisition, developmental
testing was performed on 3 control rat litters. To avoid
unnecessary testing and to minimize the time of separa-
tion of pups from their mother, testing for each milestones
began two days prior to the day of expected milestone
achievement.
Physical Maturation
Rat pups are immature at birth, i.e., their eyes and ears are
closed, and they are devoid of their fur. Appearance of these
Journal of Autism and Developmental Disorders
1 3
physical milestones reflects the physical maturity of pups.
Development of the following physical characteristics was
checked: opening of both eyes (eye opening), detachment of
both pinnae from the cranium (pinnae detachment), open-
ing of the ear canal (ear opening), protrusion of the upper
incisors (incisor eruption), and appearance of body hair
(fur development). The day of first appearance was noted as
achievement of each milestone.
Sensory andMotor Development
Sensory and motor reflexes were tested daily based on ear-
lier protocols (Altman & Sudarshan, 1975; Heyser, 2004;
Nguyen, Armstrong, & Yager, 2017) until the first positive
response was observed and that day was considered as day
of onset for that milestone. These reflexes appear at differ-
ent stages of development, resulting in variable end dates
for the tests.
• Righting reflex—surface righting: The pup was gently
held and placed on a padded horizontal surface in supine
position for 5s and then released to check if the pup was
able to turn over onto its belly, i.e., the prone position.
If the pup took longer than 60s to turn, the test was
stopped, and the pup was turned to its normal prone posi-
tion.
• Negative geotaxis—turn: The pup was placed head fac-
ing downwards, on the center of a 35cm long inclined
board (30 degrees from horizontal plane). The inclined
board had a rough surface (i.e., enough friction) to pre-
vent sliding and to support the pups when they tried to
turn around. A pile of cotton was kept below the board
to protect pups from injury in case of a fall during unsuc-
cessful trials. The ability of the pup to successfully turn
around 180° (to change its orientation from face down-
wards to face upwards on the inclined plane) within 60s
was recorded. This successful postural reaction is con-
sidered a negative geotaxis-turn.
• Negative geotaxis—climb: After turning is achieved on
the negative geotaxis test, pups usually begin climbing up
the inclined plane. If the pup successfully reached the top
of the board within 60s after the turn, it was considered
a negative geotaxis-climb.
• Cliff avoidance: The ‘cliff’ was a cardboard platform
set at a height of 30cm above a table. A pile of cotton
was kept at the bottom of the cliff as a precautionary
safety measure. The pup was placed at the edge of the
cliff such that its snout and forepaws were placed beyond
the edge. The cliff avoidance response depends on the
vestibular system and does not depend on visual informa-
tion and thus can be present before the eyes are open. It
was recorded if the pup successfully avoided the cliff by
crawling away from the edge of the platform by moving
backwards or by turning towards either side (maximum
time 30s).
• Hind limb placing response: The pup was held gently
around the trunk and suspended in air. A thin wooden
stick was used to gently touch the dorsum of the hind paw
and it was checked if the pup withdrew that paw, raised it,
and then placed it on the wooden stick. If both hind limbs
showed the placing response, the milestone was recorded
as achieved.
• Vibrissae placing response: The pup was held gently
around the trunk and suspended in air. Vibrissae were
slowly stroked once using the blunt end of a wooden
Fig. 1 Experimental Design. A schematic presentation to show the
timeline of experiments performed on rat pups prenatally exposed to
VPA. This is for illustration purposes and the distance between the
two points on the developmental age line does not represent the actual
age gap in days. E = Embryonic day, P = Postnatal day. Developmen-
tal milestones were tested between P4-P20. Body weights of pups
were recorded from P4-P21. On P21, pups were tested in open field to
assess their locomotor activity
Journal of Autism and Developmental Disorders
1 3
Fig. 2 Images of development milestones testing. Physical milestones
(A–E), Vibrissa placing (F), Cliff avoidance (G), Negative geotaxis
turn (H), Limb Placing (I), Surface righting reflex (J), Grasp reflex
(K), Bar holding test (L), Vertical screen test (M), Horizontal screen
test (N), Negative geotaxis climb (O)
Journal of Autism and Developmental Disorders
1 3
toothpick. It was noted if the pup moved its head away
and extended its forelimbs to grasp the stick.
• Grasp reflex: The pup was gently held by the nape of its
neck making the pup immobile and relaxed. Each fore-
limb paw was gently stroked once with the blunt end of a
wooden toothpick and the presence or absence of grasp-
ing response, i.e., whether the pup was able to grasp the
toothpick, was recorded.
• Bar holding: Each pup was gently lifted by the nape of
its neck and brought close to a thin wooden bar (3mm
diameter) suspended 30cm above a table surface. The
pup was positioned to allow it to grab the wooden bar
with its forepaws. Once the pup held the bar tightly, the
pup was released such that it was hanging from the bar. If
the pup was able to hang onto the bar by holding it with
both forepaws for 10s, the test was marked as successful.
If the pup fell immediately, two more trials were given.
To prevent injury to the pups during fall, piles of cotton
were kept on the table surface.
• Horizontal screen test (also called level screen test): The
pup was placed on a horizontal metal wire screen (16
mesh) and its tail was gently pulled to drag it backwards.
The ability of the pup to hold on to the horizontal screen
and not get dragged behind was noted.
• Vertical screen test: Each pup was placed on the hori-
zontal screen (16 mesh) and allowed to hold the mesh
properly. The screen was then slowly rotated by 90° to
make it vertical such that the pup’s head was facing up
due to the rotation. The climbing response of the pup on
the vertical screen was checked for 60s. To prevent pups
from injury during fall, piles of cotton were kept on the
table surface.
Test ofLocomotor Activity
To determine the effect of prenatal VPA exposure on loco-
motor activity, SAL and VPA pups were tested in the open
field test on P21 (Smirnov & Sitnikova, 2019). Each pup was
gently placed in the center of an evenly illuminated (30lx)
rectangular open field apparatus (80cm × 40cm × 40cm)
and allowed to freely explore the apparatus for 5min. At
the end of the test, the pup was immediately removed from
the apparatus and returned to its home-cage. Open field
apparatus was cleaned with 70% alcohol and allowed to dry
after each experiment. All sessions were recorded with a top
mounted video camera and videos were tracked and ana-
lyzed offline for locomotor activity by using “animal tracker”
software (Image J plugin). Total distance covered, and mean
velocity were calculated to evaluate locomotor functions.
Additionally, center and periphery regions of the appara-
tus were defined during offline tracking and time spent in
periphery and time spent in center were calculated. If the
pup covered less than 2cm in one second, it was considered
immobile. Total immobility time was also recorded.
Developmental Composite Score
We combined the results of all 15 tests spanning physical,
sensory, and motor domains in our developmental test bat-
tery into a single developmental composite score. First, each
test readout was converted into a Z-score to standardize it
to a common scale with mean 0 and standard deviation of 1
(El-Kordi etal., 2013). To facilitate comparisons, standardi-
zation was done across all rats (both sexes and groups) by
subtracting the mean and dividing by the standard deviation
for that test readout. Then, the Z-scores for all tests were
averaged to arrive at the developmental composite score for
each rat. Additionally, Z-scores from the tests belonging to
physical, sensory, and motor domains were averaged sepa-
rately to arrive at domain specific composite scores— see
SI Sect.1.1 for details. A higher value on the developmen-
tal composite score denotes greater developmental delay.
Thus, a developmental composite score of 0 indicates that
the development milestones for that rat was representative of
the overall group. A rat with a score of -1 would indicate that
the rat had earlier development than the group mean, and a
rat with a score of 1 indicates that the milestones for this rat
were one standard deviation later than the mean for the over-
all group. Cronbach’s alpha was used to assess the reliability
(internal consistency) of the developmental composite score.
Binary logistic regression was used (El-Kordi etal.,
2013) to determine if the developmental composite score
could reliably discriminate between SAL and VPA rats. The
data for each sex were separated and further split into a train-
ing set (80%) and a validation (or ‘hold out’) set (20%) using
the caret package in R (R Core Team, 2019). The training
set was used to fit a logistic regression model and fivefold
cross-validation was carried out with five repeats to arrive
at the cross-validated accuracy scores. Finally, the model
performance was assessed using the validation sets for both
males and females.
Statistical Analyses
Shapiro-Wilks test was used to assess normality in the data.
As milestones data did not pass the normality test, we used
the nonparametric Mann–Whitney U test to assess any sig-
nificant differences in developmental milestones between
SAL and VPA treatment groups for males and females sep-
arately. Locomotor activity, and developmental composite
score data met the normality assumption and accordingly,
two-way ANOVA followed by Tukey’s post hoc tests were
carried out. A linear mixed model (LMM) was used for
body weight-gain comparisons across the developmental
time course. Subject was taken as a random effect parameter
Journal of Autism and Developmental Disorders
1 3
and Sex, Treatment and Postnatal Day were taken as fixed
effects. We did not control for litter effects in our analyses.
Data were checked for outliers (1.5 times the inter-quartile
range or IQR) and extreme outliers (3 times the IQR). All
reported findings were consistent with or without removal
of outliers (if any), therefore, all results reported in the main
text are based on the complete dataset. See SI Sect.1.7 for
additional details. R software version 3.6.0 (R Core Team,
2019) and the packages tidyverse, psych, ggpubr, caret,
rstatix, nlme, phia, emmeans, Hmisc, kableExtra, corrplot
were used for statistical analyses. Descriptive results were
represented as mean ± SEM and statistical significance was
set at p < 0.05. p < 0.1 was considered to be an indicator of
a trend (Thiese etal., 2016).
Results
Body Weight
All rats progressively gained weight (Fig.3) throughout the
early developmental period (from age P4-P22). VPA rats
did not show any difference in body weight during the entire
early developmental period as compared to control rats.
However, there was a clear sex difference in body weight
gain as females from both VPA and control groups were
lighter than males from P16 onwards. Please SI Sect.1.3
for statistical details.
Physical Landmarks ofDevelopment
Major physical landmarks of rodent development such as eye
and ear canal openings, pinnae detachment, fur appearance,
and incisor eruption were evaluated in the rat pups (Fig.4).
Comparisons between treatment groups revealed that
as compared to controls, incisor eruption was delayed
in VPA males (9.0 ± 0 days versus 8.41 ± 0.15 days,
U = 45, p < 0.001) as well as females (9.0 ± 0 days
versus 8.35 ± 0.13 days, U = 20, p = 0.004). Similarly,
pinnae detachment was also delayed in both VPA males
(6.11 ± 0.30days versus 5.0 ± 0days, U = 48, p = 0.002)
and females (6.63 ± 0.53days versus 5.0 ± 0days, U = 21,
p = 0.001). There were no differences between SAL and
VPA rats in the onset of other physical milestones such as
eye opening (males: U = 129, p = 0.315; females: U = 59.5,
p = 0.813), ear canal opening (males: U = 105, p = 0.901;
females: U = 50, p = 0.642) and fur appearance (males:
U = 105, p = 0.901; females: U = 50, p = 0.642).
Early Sensory andMotor Milestones
VPA pups showed significant developmental delays when
tested for sensory and motor reflexes crucial for over-
all growth and development (Fig.5 and Fig.6). Among
sensory milestones, as compared to control males, VPA
males showed delayed ontogeny of negative geotaxis-turn
(8.16 ± 0.27days versus 6.41 ± 0.14days, U = 15, p < 0.001),
and hind limb placing response (6.5 ± 0.33 days versus
5.0 ± 0days, U = 36, p < 0.001) but not vibrissa placing
response (8.44 ± 0.39days versus 7.83 ± 0.27days, U = 106,
p = 0.946). On the other hand, female VPA pups showed
delayed onset of all the above reflexes (negative geotaxis-
turn: 8.75 ± 0.49 days versus 7.35 ± 0.19 days, U = 24,
p < 0.001; hind limb placing response: 6.62 ± 0.53 days
versus 5.0 ± 0 days, U = 14, p < 0.001; vibrissa placing
response: 9.37 ± 0.62days versus 6.78 ± 0.39days, U = 18,
p = 0.008) in comparison to control females. Within treat-
ment groups, comparisons showed a sex dependent differ-
ence in the ontogeny of negative geotaxis-turn as control
females took more days to develop this reflex as compared
to control males (7.35 ± 0.199 versus 6.41 ± 0.149days,
U = 140, p = 0.002) whereas VPA females and males did not
show this difference (8.75 ± 0.491 versus 8.16 ± 0.271days,
U = 89, p = 0.334). Additionally, control females showed a
Fig. 3 Body weight across early
postnatal developmental period.
After first two postnatal weeks,
females were lighter than
males in both groups. There
was no effect of prenatal VPA
treatment on the body weight
of pups. F: Female, M: Male.
Data expressed as mean ± SEM.
n = 18 VPA male pups, 8 VPA
female pups, 12 SAL male pups
and 14 SAL female pups. Sex
differences within each treat-
ment group (Male vs. Female):
###p < .001, ## p< .01, #p< .05,
^p < .1
Journal of Autism and Developmental Disorders
1 3
trend towards earlier onset of vibrissa-placing as compared
to control males (6.79 ± 0.395 versus 7.83 ± 0.271days,
U = 51, p = 0.074) but VPA females and males did not show
this difference (9.38 ± 0.625 versus 8.44 ± 0.398 days,
U = 95.5, p = 0.175).
Among motor milestones, VPA males showed
delayed onset of grasp reflex (9.27 ± 0.28 days versus
8.0 ± 0days, U = 36, p < 0.001), bar holding performance
(13.33 ± 0.36days versus 11.33 ± 0.51days, U = 43.5,
p = 0.005), and horizontal screen (13.22 ± 0.12days ver-
sus 12.08 ± 0.41days, U = 59.5, p = 0.026) as compared
to control males. VPA females had delayed onset of
grasp reflex (9.75 ± 0.49days versus 8.0 ± 0days, U = 14,
p < 0.001) and horizontal screen (13.25 ± 0.49days ver-
sus 12.64 ± 0.19days, U = 28, p = 0.048) but not bar hold-
ing performance as compared to female controls. Thus,
there was a sex-specific difference in terms of bar-holding
performance where VPA males showed a developmental
delay, but VPA females did not. Other motor milestones
such as onset of surface righting reflex, vertical screen
and negative geotaxis-climb were unaffected in male and
female VPA pups. Additionally, within treatment groups,
comparisons showed a sex dependent ontogeny of verti-
cal screen performance, as control males took less time
to develop this motor milestone than control females
(12.83 ± 0.423days versus 14.00 ± 0.182days, U = 123,
Fig. 4 Development of Physical Milestones. Both male and female
VPA pups were late in pinnae detachment and incisors eruption
as compared to sex matched SAL pups. F: Female, M: Male. Data
expressed as box and whisker plots. n = 18 VPA male pups, 8 VPA
female pups, 12 SAL male pups and 14 SAL female pups. Differences
between treatment groups (VPA vs. SAL): ***p < .001, **p < .01,
*p < .05
Journal of Autism and Developmental Disorders
1 3
p = 0.03) but this differential development was not seen
in VPA male and female pups (13.77 ± 0.173days versus
13.50 ± 0.189days, U = 58, p = 0.409). In summary, both
male and female VPA rats demonstrated a delayed devel-
opmental trajectory in terms of physical growth and the
ontogeny of sensory and motor milestones.
Domain Level Assessment ofDevelopmental Delays
We consolidated z-scores domain-wise (by averaging
z-scores from individual milestones within each domain) to
be able to examine differential patterns, if any, in develop-
mental delay. VPA females had significantly higher physi-
cal and sensory domain composite scores (Fig.7A and B)
whereas VPA males had higher sensory and motor domain
composite scores (Fig.7B and C) as compared to their
Fig. 5 Development of Sensory Milestones. Both male and female
VPA pups showed delayed ontogeny of negative geotaxis and hind
limb placing response as compared to sex matched SAL pups. Female
VPA pups also showed delayed onset of vibrissa placing response as
compared to female control pups. Additionally, SAL females were
slower than SAL males in acquiring the negative geotaxis reflex and
had a trend towards an earlier onset of the vibrissa placing response
as compared to SAL males. It is noteworthy that VPA rats did not
show these sex-differences. F: Female, M: Male. Data expressed as
box and whisker plots. n = 18 VPA male pups, 8 VPA female pups, 12
SAL male pups and 14 SAL female pups. Differences between treat-
ment groups (VPA vs. SAL): ***p < .001, **p < .01, *p < .05. Sex
differences within each treatment group (Male vs. Female): ##p < .01,
^p < .1
Journal of Autism and Developmental Disorders
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respective controls (see SI Sect.1.4 for statistical details).
Thus, there were clear sex-specific differences in develop-
mental trajectory in male and female VPA rats.
Locomotor Activity
Having observed the delay in acquisition of motor mile-
stones in VPA rats, we used open field test (OFT) to explore
if their general locomotor activity was also affected.
The locomotor activity was evaluated in terms of total
distance covered and the average velocity during the open
field activity (Fig.8A and Fig.8B). We did not find any
effect of VPA treatment in total distance covered as well as
in mean velocity in open field. Two-way ANOVA showed
no significant main effects of Treatment (F(1, 46) = 0.98,
p = 0.33), or Sex (F(1, 46) = 0.02, p = 0.88) or interaction effect
of Treatment X Sex (F(1, 46) = 0.68, p = 0.41) on total distance
covered. Similarly, there were no main effects of Treatment
(F(1, 46) = 1.32, p = 0.26), or Sex (F(1, 46) = 0.01, p = 0.95),
Fig. 6 Development of Motor Milestones. Female VPA pups showed
delayed development of grasp reflex and horizontal screen test in
comparison with female control pups. In contrast, male VPA pups
showed delayed development in grasp reflex test, bar holding test
and horizontal screen test as compared to male SAL pups, showing
widespread motor delays in male VPA pups. There was also a sex
difference as female control rats took more time to develop vertical
screen test response than control males and this difference was not
seen in VPA rats. F: Female, M: Male. Data expressed as box and
whisker plots. n = 18 VPA male pups, 8 VPA female pups, 12 SAL
male pups and 14 SAL female pups. Differences between treatment
groups (VPA vs. SAL): ***p < .001, **p < .01, *p < .05. Sex differ-
ences within each treatment group (Male vs. Female): #p < .05
Journal of Autism and Developmental Disorders
1 3
or interaction effect of Treatment X Sex (F(1, 46) = 0.55,
p = 0.46) for mean velocity. Additionally, we assessed time
spent in center and periphery of the open field chamber, as
well as total immobility time in the chamber, and did not
find any significant differences between SAL and VPA (SI
Sect.1.5, Figure S2A-C). In summary, VPA rats did not
show differences on these measures of open field test that
were assessed in our study. These results are in accordance
with previous findings of unchanged locomotor activity in
VPA rats at weaning age (Olexova etal., 2013; Dobrovolsky
etal., 2019).
Correlations Among Developmental Delays
andOpen Field Test Measures
We explored correlations among onset of developmental
milestones and the open field test measures. The num-
ber of significant correlations between pairs of individual
developmental milestones differed widely across condi-
tions (SAL males: 2; SAL females: 6; VPA females: 8 and
VPA males: 18). Among VPA males and females (but not
SAL), onset of several sensory and motor milestones were
highly correlated with each other. Among VPA males, onset
of horizontal screen milestone was positively correlated
(r = 0.76, p < 0.001) with velocity in open field chamber.
See SI Sect.1.6 for statistical details and FiguresS3A-D
and S4A-D.
Developmental Composite Score
We formulated a developmental composite score that
combined results of all fifteen tests, encompassing overall
physical and sensory-motor behavioral development of rat
pups. This approach provides an effective gestalt measure,
and thereby, a robust metric of developmental differences
between control and VPA rats. The composite score showed
high reliability (standardized Cronbach’s alpha of 0.769)
indicating high internal consistency in integrating all test
readouts into a single composite.
Two-way ANOVA on the developmental compos-
ite score revealed significant main effects of Treatment
(F(1, 48) = 38.84, p < 0.001), and Sex (F(1, 48) = 4.45, p = 0.04)
but no interaction effect of Treatment X Sex (F(1, 48) = 0.06,
p = 0.80). Post hoc comparisons showed developmental
composite scores were significantly higher in both VPA
males (p < 0.001) as well as VPA females (p < 0.001) as
compared to their sex-matched controls (Fig.9A), showing
that prenatal VPA exposure altered the overall developmen-
tal trajectory in these rats.
The frequency distributions of the developmental com-
posite score showed less overlap in male rats than female
rats (Fig.9C and Fig.9B) indicating that the distinction
between VPA and control populations was better in males
than in females. This is further revealed by the plot of
individual developmental composite scores that provides
Fig. 7 Domain-specific composite scores. Composite scores were
computed for A physical milestones, B sensory milestones and C
motor milestones, by combining z-transformed raw data from cor-
responding individual milestone tests. Data presented as box and
whisker plots. Higher values of a domain-specific composite score
represent greater delay in that domain. Differences between treatment
groups (VPA vs. SAL): ***p < .001, **p < .01, *p < .05
Fig. 8 Locomotor Activity in Open Field Test. VPA pups did not
show differences in total distance covered (A) and mean velocity (B)
in the open field test. Data expressed as box and whisker plots. n = 16
VPA male pups, 8 VPA female pups, 12 SAL male pups and 14 SAL
female pups
Journal of Autism and Developmental Disorders
1 3
a qualitative demarcation between groups among males
(Fig.9E) and females (Fig.9D). We verified this demar-
cation quantitatively using binary logistic regression
analyses. The models showed high discriminative ability
in correctly predicting the group of a given rat (VPA or
SAL) on the basis of the developmental composite score in
the cross-validated training sets for both males (accuracy
96%) and females (76.3%). The models also performed
well on the validation sets (prediction accuracy: males
100%, females 66.7%).
Discussion
The present study aimed to characterize early develop-
ment in the prenatal VPA rat model of autism. Our find-
ings demonstrate significantly deviated developmental
Fig. 9 Developmental Composite Score. A developmental compos-
ite score (A) was computed by combining the z-transformed raw
data from all 15 tests used in our developmental test battery. Data
presented as box and whisker plots. Higher values of developmental
composite score represent greater delay in overall development of
VPA rats. Plots for frequency distribution of developmental compos-
ite score (B for females and C for males) show less overlap in male
rats than female rats. Developmental composite scores of individual
rats (D for females and E for males) distinguish between control and
VPA rats. Logistic regression analysis revealed high discrimination
between VPA and control rats in both males (96% accuracy) and
females (76.3%). n = 18 VPA male pups, 8 VPA female pups, 12 SAL
male pups and 14 SAL female pups. Differences between treatment
groups (VPA vs. SAL): ***p < .001
Journal of Autism and Developmental Disorders
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trajectories in both male and female VPA rats in compari-
son to typically developing control rats.
VPA Pups did notShow Reduced Body Weight
During Early Postnatal Period
We checked body weight in VPA and control rats in their
early developmental period until weaning (P4-P22) and did
not find any group differences, in line with a few earlier stud-
ies (Favre etal., 2013; Servadio etal., 2018) using a similar
VPA dosage (500mg/kg or less). Studies where offspring
were prenatally exposed to higher VPA dosage (600mg/kg
or more) have found lower body weight in the VPA group
(Du etal., 2017; Zhang etal., 2018). Thus, our findings of
developmental delays in VPA rats were not affected by dif-
ferences in body weight.
Delayed Appearance ofPhysical, Sensory, andMotor
Developmental Milestones inVPA Rats
In rodents, pups are born with eyes and ears closed, with
no fur on the body, underdeveloped sensory sensitivity,
and random, uncoordinated movements. These processes
continue to develop in a temporally programmed manner
during the first 21 postnatal days. Assessment of develop-
mental milestones, such as physical landmarks and tests of
sensory and motor abilities, during this initial critical period
can indicate effects of prenatal insult on the development
of brain functions and characterize early markers for later
behavior (Heyser, 2004). Ontogeny of specific sensory and
motor developmental milestones are known to be an index
of maturation in specific brain regions or neural circuits in
animals, including humans (Rice & Barone, 2000).
In the present study, early postnatal development of VPA
rats was continuously monitored and compared with the
typical development of sex-matched control rats. VPA male
and female rats were slow to acquire some physical mile-
stones (incisor eruption and pinnae detachment), indicating
a slightly delayed physical maturation in VPA rats. Ontogeny
of physical maturity milestones are not directly relevant for
clinical populations. However, we included these tests in our
developmental test battery as onset of somatic milestones is
a crucial component of rodent neurodevelopmental trajec-
tory (Semple etal., 2013). For example, delays in physical
milestones such as eye and ear development may be indica-
tive of altered sensory development as well as subsequent
visual and auditory input-oriented behavior respectively
(Boitnott etal., 2021; Smirnov & Sitnikova, 2019). Although
we did not find delays in eye opening, VPA pups showed
delayed ear development.
Additionally, both male and female VPA rats had delayed
development of sensory systems indicated by late appear-
ance of negative geotactic and limb placing responses. VPA
females also had a delayed vibrissa placing response. These
tests mainly assess dynamic postural adjustments (induced
by sensory stimuli) involving the vestibular system for neg-
ative geotactic reaction, and the combined vestibular and
exteroceptive systems for limb placing and vibrissa plac-
ing responses (Altman & Sudarshan, 1975; Schneider &
Przewłocki, 2005). Central regulation by medullary, cerebel-
lar and sensory-motor cortical systems also plays a major
role (Altman & Sudarshan, 1975; Wagner etal., 2006; Y.
Zhang, Li, Yang, Zhang, & Yang, 2010).
Finally, we also saw evidence of delayed motor system
development due to prenatal VPA exposure. The ontogeny
of forelimb grasp reflex was affected in both males and
females. Although the grasping response is a spinal reflex,
it is regulated by the action of higher brain centers com-
prising non-primary motor areas such as premotor cortex
and supplementary motor cortex, on the spinal interneurons
(Hashimoto & Tanaka, 1998; Nguyen etal., 2017). Moreo-
ver, bar holding performance, and horizontal screen perfor-
mance that were used to assess muscle tone, limb strength,
as well as sensorimotor coordination (Deacon, 2013; Heyser,
2004; Slamberova etal., 2006), appeared late in VPA males
indicating delayed motor system development in the brain
(Feather-Schussler & Ferguson, 2016; van de Wijer etal.,
2019). VPA females also showed delayed horizontal screen
performance indicating that motor system development was
delayed in both sexes. However, VPA rats did not show any
impairment in open field test at weaning, indicating that
once motor milestones were acquired, locomotor functions
were normal.
Overall, our findings of delayed ontogeny of critical sen-
sory and motor developmental landmarks in first three weeks
after birth are indicative of prenatal VPA induced altera-
tions in the early development of the brain regions regulating
these sensorimotor functions, mainly somatosensory cortex,
motor cortex, cerebellum, and brainstem. This is supported
by previous reports of lower cell density and smaller neu-
ronal cell sizes (Al Sagheer etal., 2018; Ingram etal., 2000;
Kataoka etal., 2013; Lukose etal., 2011; Spisak etal., 2019;
Varghese etal., 2017) as well as altered excitatory-inhibitory
(E/I) balance, i.e., increased glutamatergic and decreased
GABAergic signaling, in these brain regions (Chau etal.,
2017; Hou etal., 2018; Rinaldi etal., 2007; Tartaglione
etal., 2019). It is important to note, however, that most of
these studies were carried out during adolescence and adult-
hood, and not during early development. Future studies dur-
ing the neonatal period can help elucidate the neuroanatomi-
cal and neurochemical underpinnings of the time course of
developmental trajectory alterations in the VPA rat model
of autism.
Alterations in E/I dynamics is known to impact the tim-
ing of critical periods in brain development, and eventually,
alterations in the formation of functional networks (Gogolla
Journal of Autism and Developmental Disorders
1 3
etal., 2009; D. D. Wang & Kriegstein, 2011). For exam-
ple, Wang & Kriegstein, 2011, disrupted E/I balance in
typically developing mice pups during the perinatal period
and reported developmental delays in onset of sensory and
motor milestones such as negative geotaxis and bar hold-
ing abilities. Further, Nagode and colleagues demonstrated
that prenatal VPA exposure in mice altered the development
of the earliest cortical circuits involved in sensory process-
ing via altering E/I balance (Nagode etal., 2017). Indeed,
hyper-connected microcircuits in the somatosensory cortex
as well as altered synaptic plasticity in the somatosensory
cortex and cerebellum have been found in the VPA rat model
(Iijima etal., 2016; Silva etal., 2009; R. Wang etal., 2018).
Future studies in the VPA model could try to restore E/I bal-
ance in these regions during the early developmental period
and evaluate rescue of behavioral milestones to test the pos-
sibility of a causal role of E/I imbalance in the developmen-
tal delays seen in the VPA model.
Comparison withPrevious Studies ofEarly
Development inPrenatal VPA Rodent Model
Previous studies that explored early development in prena-
tal VPA rodent model of autism provided mixed findings
for different milestones (Dobrovolsky etal., 2019; Li etal.,
2017; Schneider & Przewłocki, 2005; Wagner etal., 2006;
R. Zhang etal., 2018). There are two possibilities for this
discrepancy in the literature—first, the methodology used
to assess developmental milestones; and second, the data
analysis steps prior to statistical testing.
Most studies investigating early development in VPA rats
incorporated only few tests as part of a larger study with
a different goal. Moreover, many of these studies assessed
early development within a short postnatal time window or
on a specific postnatal day (Dobrovolsky etal., 2019; Sch-
neider & Przewłocki, 2005; Wagner etal., 2006). The litera-
ture on early development in rodents shows that milestones
occur in a temporal sequence in a pup’s early life (Heyser,
2004). For example, milestones such as incisor eruption, fur
development, surface righting, cliff avoidance, horizontal
screen, negative geotaxis appear early, whereas eye open-
ing, ear canal opening, vertical screen and bar holding are
late appearing milestones. Importantly, the age of onset of
different developmental milestones differs between species,
between strains of the same species, and between individ-
ual pups of a given species and strain. Additionally, as we
observed in our data, VPA rats had delayed appearance of
several milestones, but they did not miss achieving any mile-
stone entirely. Therefore, testing for a milestone too early
(when that specific milestone might not have appeared) or
too late (when even VPA pups have acquired that milestone)
might miss finding group differences that might be present.
Another important consideration is that many studies,
including our own, used a binary assessment i.e., whether
milestones were present or not, tested on each postnatal
day, until the acquisition of the developmental landmarks
(Li etal., 2017; R. Wang etal., 2018). Other studies have
used a graded scoring system (no response to maximum
response) for the acquisition of each developmental mile-
stone (Al Sagheer etal., 2018). Graded scoring can be more
informative about the process of ontogeny of developmental
landmarks but is more heavily influenced by subjective bias
than the binary scoring system. We recommend that future
studies use the binary measure for all milestones to facilitate
comparison across studies, and additionally adopt the graded
system where appropriate as per study goals.
Another methodological difference in the literature
involves quantification. Several studies quantified mile-
stones such as latency to reorient in negative geotaxis and
surface righting (Gandal etal., 2010; R. Zhang etal., 2018).
Some studies have compared proportions–i.e., how many
pups in each group achieved a milestone on a postnatal day
(Reynolds etal., 2012). Considering these methodological
differences in assessment and analysis, the mixed evidence
in the literature is not surprising. Other contributing factors
include differences in VPA dose and concentration, timing,
and mode of VPA administration, species, and strains of
model animals.
In summary, there is mounting evidence that the VPA
model engenders developmental delays and that there is a
clear need to use a unified approach to quantifying these
delays in different models that vary in terms of species,
strains, VPA dose etc.
Comparisons withPrevious Studies ofEarly
Development inOther Environmental Rodent
Models ofASD
Neurodevelopmental trajectories have been examined in
other environmental insult-based models of ASDs, including
the maternal immune activation (MIA), a prominent envi-
ronmental model of ASD (Guma etal., 2019). Studies using
LPS induced MIA model did not find any major difference
in the onset of neonatal reflexes and physical milestones
(Baharnoori etal., 2012; Fernandez de Cossio etal., 2017;
Foley etal., 2014). In contrast, sensorimotor milestones such
as righting reflex, gait, and negative geotaxis were delayed
in another study using the LPS model (Rousset etal., 2013).
Similarly, Malkova etal., 2012 did not find delays in devel-
opment of negative geotaxis, grasp reflex and righting reflex
in a Poly I:C induced MIA model. On the other hand, Haida
etal., 2019 assessed onset of eye opening, negative geo-
taxis, and righting reflex using the Poly I:C model and found
delays in these milestones.
Journal of Autism and Developmental Disorders
1 3
Delays in righting reflex and negative geotaxis were found
in a prenatal Vitamin D deficiency model of ASD (Ali etal.,
2019) whereas physical milestones were not affected. Simi-
larly, delayed sensorimotor development in milestones such
as righting reflex, cliff avoidance and negative geotaxis were
found in prenatal chlorpyrifos (CPF, an insecticide) model
(Lan etal., 2017), however, physical milestones were not
examined. In a pharmacological model, perinatal fluoxetine
treated rats showed delayed onset of righting reflex, negative
geotaxis, vibrissa placing and bar holding abilities, but no
delay in eye opening, the only physical milestone that was
tested (Kroeze etal., 2016).
In summary, early postnatal development in other envi-
ronmental models show mixed findings for different mile-
stones that could be due to several factors. Differences in
protocols to develop the models, differential impact of these
different environmental risk factors on brain development,
differences in the degree of their construct and phenotypic
validity for the ASD condition, as well as methodological
differences in assessment of developmental milestones,
could be contributing factors for these mixed findings.
Detailed comprehensive studies spanning multiple physical,
sensory, and motor milestones are needed to facilitate com-
parisons between different environmental models of ASD.
Male andFemale VPA Rats—Similarities
andDifferences inEarly Development
There is limited literature on early development in females
in animal models of ASD. Most studies have focused on
males (Kazdoba etal., 2016; Wohr etal., 2013) and this is
the case in the VPA model too (Hou etal., 2018; Wang etal.,
2018). Al Sagheer etal., 2018 found significant delays in eye
opening and surface righting reflex in male as well as female
VPA mice. Similarly, Wagner etal., 2006 observed delayed
appearance of surface righting as well as negative geotaxis
in both male and female VPA mice. Another study found
delayed auditory startle reflex in female VPA mice, but no
males were studied, precluding assessment of sex differences
(Kazlauskas etal., 2016).
In the present study, we found overall delayed develop-
ment in both male and female VPA rats (developmental com-
posite score data). However, there were differences in the
pattern of developmental delays in VPA males and females.
In the motor domain, VPA exposure impacted males more
than females since only males showed delayed bar holding
performance and the noteworthy sex-dependent advantage
shown by controls males for the vertical screen was absent
among VPA males. In the sensory domain, females were dis-
proportionately impacted by VPA exposure as the vibrissa
placing response was delayed only in VPA females who also
did not show the sex-dependent advantage shown by con-
trol females for this test. However, in a different sensory
test (negative geotaxis), although both males and females
were affected, the male advantage seen among controls was
missing among VPA showing that VPA males were more
affected by the prenatal VPA exposure. These differences
may be attributed to the task-specific nuances in the sensory
domain as vibrissa placing is a tactile-stimulus dependent
test whereas the negative geotaxis test involves the vestibular
system as well as sensory-motor coordination for turning the
body by 180° on an inclined plane.
We constructed domain specific composite scores to
smooth over test specific differences and provide a consoli-
dated representation for the physical, sensory, and motor
developmental domains. Indeed, these composites provided
greater clarity into the sex-specific patterns of developmen-
tal delays in VPA. Specifically, although both VPA males
and females had delays in the sensory domain composite,
only VPA males had delays in the motor domain composite
and further, only VPA females had delays in the physical
domain composite.
Sex specific differences in developmental profiles of ASD
children are not clear. There are reports showing female
advantage in the acquisition of language, visual reception,
and fine motor milestones among ASD children (Messinger
etal., 2015). Some studies have shown better language and
motor (gross and fine) skills in autistic boys and better visual
reception in girls (Carter etal., 2007) but other studies have
also shown no sex specific differences in early sensory and
motor development of ASD children (Bedford etal., 2016a;
Reinhardt etal., 2015). It is possible that heterogeneity in
the autism phenotype as well as methodological differences
may have contributed to the inconsistencies in the literature.
Taken together, our findings suggest that prenatal VPA
exposure impacted both sexes but there were important sex-
dependent differences. The test-specific nuances seen in our
study suggests that methodological considerations could
shed light on better understanding the differential neurobi-
ology of ASD in boys and girls.
Associations Among Developmental Milestones
andLocomotor Functions
We carried out exploratory correlation analyses to exam-
ine associations among early developmental milestones and
locomotor function assessed at P22 using open field test
measures. These analyses were conducted with individual
developmental milestones as well as with domain specific
composites. Our findings indicate that for VPA rats, onset of
several milestones were highly correlated with each other.
This pattern was not observed among SAL rats. Further,
VPA males showed a positive correlation between veloc-
ity in open field and onset of a motor milestone (horizontal
screen) possibly suggestive of a compensatory increase in
motor activity with delay in motor development. Overall,
Journal of Autism and Developmental Disorders
1 3
these exploratory analyses support the possibility that pre-
natal VPA exposure provides a common underlying driver
(disrupting connectivity between brain regions during devel-
opment) leading to the observed correlated delays in mile-
stones along the developmental trajectory.
The Developmental Composite Score—
Comprehensive Measure toAssess Early
Developmental Trajectory inRodent Model ofASD
Individuals on the autism spectrum show a huge diversity
in the severity of symptom presentation. This variability in
symptoms is also found in animal models of autism, pre-
senting a problem in effectively capturing symptom severity
and comparing across models. The problem exists even in
animals containing the same autism-related mutation (Dere
etal., 2014). A useful approach is to compute a composite
score spanning multiple test readouts rather than compar-
ing groups for each individual test readout (El-Kordi etal.,
2013).
Our focus in this study was to develop a developmental
composite score consolidating all 15 milestones comprising
physical, sensory, and motor developmental domains, into a
single number snapshot, a gestalt measure, of the develop-
mental trajectory of each rat from birth to weaning. The high
reliability of the developmental composite score and overall
ability to accurately discriminate between VPA and control
rats demonstrates its value for the comprehensive assess-
ment of neurodevelopment in rat pups and might have trans-
lational value for studying developmental disorders during
early life and in early intervention studies in animal models.
Comparison withClinical Conditions
Our findings of delayed developmental milestones in VPA
rats are well in line with a large body of clinical evidence
indicating that early motor and sensory developmental
delays precede ASD diagnosis and may serve as early indi-
cators of ASD (Bhat etal., 2011; Dadalko & Travers, 2018;
Estes etal., 2015; Harris, 2017; Robertson & Baron-Cohen,
2017).
Several studies have reported delayed development of
early gross motor functions (e.g., sitting and standing with-
out support and independent walking) in children with, and
at high risk for, ASD (Davidovitch etal., 2018; R. Landa &
Garrett-Mayer, 2006; Lemcke etal., 2013). Delays in fine
motor functions such as fine motor coordination, reaching,
and grasping have also been reported in children with ASD
(Bolton etal., 2012; R. Landa & Garrett-Mayer, 2006; Lib-
ertus etal., 2014). In our study, VPA rats showed delays in
bar holding and horizontal screen tests which mainly assess
gross aspects of motor functions such as stamina and limb
strength (Brooks & Dunnett, 2009; Feather-Schussler &
Ferguson, 2016). Early fine motor skills such as involuntary
grasp response as seen in the grasp reflex test and active
grasp response with proximal stability as seen in bar holding
test (Anekar & Bordoni, 2020; Diener & Bregman, 1998;
Whishaw & Kolb, 2020) were also delayed in VPA rats.
These early fine motor skills are pre-requisites for volun-
tary reaching and grasping—fine motor skills that develop
at maturity (at least 4weeks of age in rats).
The literature on early sensory processing problems in
autistic children is mixed. However, several studies in chil-
dren with ASD, or those at high risk for ASD, have reported
atypical responsiveness to sensory inputs across auditory,
visual, and somatosensory modalities during early develop-
ment (Grace T Baranek, 1999; G. T. Baranek etal., 2013;
Baum etal., 2015; Robertson & Baron-Cohen, 2017; Wolff
etal., 2019; Zwaigenbaum etal., 2005). Thus, our find-
ings of delayed somatosensory development in VPA rats,
reflected by delays in negative geotaxis, vibrissa placing and
limb placing tests, are in concordance with the findings in
children with ASD.
It is noteworthy that the somatosensory system plays
a central role in early childhood, and that somatosensory
inputs are critical not only for the development of both gross
and fine motor skills, but also for healthy social and com-
munication development (Cascio, 2010; Metcalfe etal.,
2005; Thompson etal., 2017). Neural computations under-
lying basic sensory and motor processing are well conserved
between humans and rodents. Future studies on the struc-
tural and functional aspects of early sensorimotor develop-
ment in VPA model would be instrumental in shedding light
on the time course of ASD neurobiology.
Limitations andFuture Directions
Our study provides comprehensive evidence of devel-
opmental delay in the ontogeny of physical, sensory, and
motor milestones in the prenatal VPA exposure rat model
of autism. However, there are a few limitations that warrant
further investigation.
First, the sample size was small for VPA females. It is
unclear if the lower accuracy in discriminating between
VPA, and control females based on the developmental com-
posite scores was due to lower power or due to the higher
individual variability in female VPA rats as compared to
male VPA rats. Second, our focus was on sensory and motor
development and at the time of the study, we did not include
tests for early social development in our test battery. Future
studies should consider including social development assess-
ment, such as ultrasonic vocalizations test and social-odor
discrimination task (Potasiewicz etal., 2020; Schneider &
Przewłocki, 2005), in their early developmental test battery
for additional relevance for ASD models.
Journal of Autism and Developmental Disorders
1 3
Third, these rat pups were not followed up and tested for
core features of autism (social interaction deficits and repeti-
tive behavior) later in life. These rat pups had gone through
additional handling during their early developmental period
as part of the assessment of developmental milestones and
studies indicate that additional handling of neonatal rodents
impacts their performance in social interaction test and other
behavioral measures in adulthood (Heyser, 2004; Schnei-
der etal., 2006; Todeschin etal., 2009). To avoid this con-
found, we refrained from testing these pups later in life. We
have previously provided robust evidence of the presence
of ASD like phenotype, such as reduced social interaction,
altered attentional processing, and impaired sensorimotor
gating in a separate cohort of naïve VPA adult rats that had
not undergone detailed developmental assessment (Anshu
etal, 2017) and we replicated these results across multiple
cohorts. Nonetheless, this is an important point to consider
and it would be valuable to carry out a future study in the
VPA model that does follow up testing of core features after
detailed evaluation of developmental trajectory, and com-
pares these findings with a separate cohort of rats that have
not undergone this additional handling.
Conclusion
The present study aimed to characterize the acquisition of
different milestones during early postnatal development in
the VPA rat model of autism. Our findings demonstrate sig-
nificantly deviated developmental trajectories in both male
and female VPA rats in comparison to typically developing
control rats and are in line with clinical findings showing
delayed development in children with ASD. The evidence
of sex-specific differences in developmental milestones
in VPA rats underscore the need for carrying out autism
related studies in both sexes. Our findings suggest an altered
developmental programming of prenatal brain development
process after prenatal VPA exposure in these rats. Future
studies are needed to pinpoint the underlying mechanisms
leading to early developmental delay followed by autism
like phenotype later in life. Overall, the present study pro-
vides a detailed characterization of early development in the
prenatal VPA model of autism in both sexes and presents a
developmental composite score (combining a range of tests
for different physical, sensory, and motor developmental
landmarks) as a robust measure for evaluating early develop-
ment in different animal models of autism and as a potential
outcome measure for early intervention studies.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s10803- 022- 05684-y.
Acknowledgments This work was supported by University Grants
Commission (UGC), Government of India for the fellowship to K.A.
and National Institute of Mental Health and Neurosciences (NIM-
HANS), Bengaluru for the infrastructure facilities and support to carry
out the work. We thank Dr. U.D. Kumaresan for helpful discussions.
Author contribution KA, AKN, SS and TRL contributed to the study
conception and design. Data collection was performed by KA. Data
analysis was performed by KA and AKN. The first draft of the manu-
script was written by KA and all authors provided feedback. All authors
read and approved the final manuscript.
Declarations
Conflict of interest The authors have no conflicts of interests to de-
clare.
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