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Apoptosis, programmed cell death, is a critical component of neurodevelopment occurring in temporal, spatial, and at times, sex-specific, patterns across the cortex during the early postnatal period. During this time, the brain is particularly susceptible to environmental influences that are often used in animal models of neurodevelopmental disorders. In the present study, the timing of peak cell death was assessed by the presence of pyknotic cells in the male and female rat medial prefrontal cortex (mPFC), a cortical region that in humans, is often involved in developmental disorders. One male and one female rat per litter were sacrificed at the following ages: Postnatal day (P)2, 4, 6, 8, 10, 12, 14, 16, 18, and 25. The mPFC was Nissl-stained, the densities of pyknotic cells and live neurons were stereologically collected, and the number of pyknotic cells per 100 live neurons, pyknotic cell density, and neuron density were analyzed. Males and females showed a significant peak in the ratio of pyknotic to live neurons on P8, and in females, this elevation persisted through P12. Likewise, the density of pyknotic cells peaked on P8 in both sexes and persisted through P12 in females. The timing of cell death within the rat mPFC will inform study design in experiments that employ early environmental manipulations that might disrupt this process.
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IBRO Neuroscience Reports 10 (2021) 186–190
Available online 27 March 2021
2667-2421/© 2021 Published by Elsevier Ltd on behalf of International Brain Research Organization. This is an open access article under the CC BY-NC-ND
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Cell death in the male and female rat medial prefrontal cortex during early
postnatal development
Elli P. Sellinger
a
, Carly M. Drzewiecki
a
, Jari Willing
b
, Janice M. Juraska
b
,
*
a
Program in Neuroscience, University of Illinois at Urbana-Champaign, Champaign, IL 61801, United States
b
Department of Psychology, University of Illinois at Urbana-Champaign, 603 E Daniel St, Champaign, IL 61820, United States
ARTICLE INFO
Keywords:
Apoptosis
Pyknosis
MPFC
Neurodevelopment
ABSTRACT
Apoptosis, programmed cell death, is a critical component of neurodevelopment occurring in temporal, spatial,
and at times, sex-specic, patterns across the cortex during the early postnatal period. During this time, the brain
is particularly susceptible to environmental inuences that are often used in animal models of neuro-
developmental disorders. In the present study, the timing of peak cell death was assessed by the presence of
pyknotic cells in the male and female rat medial prefrontal cortex (mPFC), a cortical region that in humans, is
often involved in developmental disorders. One male and one female rat per litter were sacriced at the following
ages: postnatal day (P)2, 4, 6, 8, 10, 12, 14, 16, 18, and 25. The mPFC was Nissl-stained, the densities of pyknotic
cells and live neurons were stereologically collected, and the number of pyknotic cells per 100 live neurons,
pyknotic cell density, and neuron density were analyzed. Males and females showed a signicant peak in the
ratio of pyknotic to live neurons on P8, and in females, this elevation persisted through P12. Likewise, the density
of pyknotic cells peaked on P8 in both sexes and persisted through P12 in females. The timing of cell death within
the rat mPFC will inform study design in experiments that employ early environmental manipulations that might
disrupt this process.
1. Introduction
Apoptosis, a process of regulated cell death, is a critical component of
early neurodevelopment in both rodents (Buss et al., 2006) and humans
(Rakic and Zecevic, 2000) that occurs in spatially and temporally
distinct patterns across the cortex. Evidence from rodent studies sug-
gests two distinct waves of apoptosis (Blaschke et al., 1996). The rst
occurs embryonically, with the highest density of apoptotic cells
observed on embryonic day 16, specically within proliferative zones
(Thomaidou et al., 1997). The second surge in apoptosis occurs just after
birth during the rst two postnatal weeks (Ferrer et al., 1990; Mosley
et al., 2017; Nu˜
nez et al., 2001; Spreaco et al., 1995; Verney et al.,
2000). As an important developmental process, postnatal cortical
apoptosis contributes to neural circuit formation (Forger, 2009) and
disrupting this process, for example, as a result of maternal separation
stress results in altered neuron number and behaviors observed in
adolescence (Majcher-Ma´
slanka et al., 2019). Apoptosis in the cortex
can also be inuenced by gonadal hormones (Nu˜
nez et al., 2000). This
has been well-established in hypothalamic nuclei where differing rates
of apoptosis between the sexes, inuenced by perinatal gonadal hor-
mone environment, lead to sexual dimorphisms (Forger, 2009).
While the presence of postnatal apoptosis has been established in the
cortex, the rates and timing of this process can vary by region. For
example, programmed cell death in the rat somatosensory cortex rea-
ches the highest levels between postnatal days (P)5-8 (Ferrer et al.,
1990; Spreaco et al., 1995), and a similar rise during the rst postnatal
week is seen in mice (Verney et al., 2000). Our laboratory has quantied
apoptosis in the visual cortex of rats of both sexes, nding distinct timing
of elevated levels in males compared to females (Nu˜
nez et al., 2001). To
quantify apoptosis, both the ratio of pyknotic cells, cells displaying
condensed nuclear chromatin, and TUNEL-labeled cells, which are
marked by fragmented DNA, to the number of live neurons were used
and showed a similar pattern. Both measures were highest in males at P7
while females showed a smaller peak at P7, and additional peaks at P11
and P25. Moreover, this developmental process is promoted, at least in
part, by androgens, not estrogen (Nu˜
nez et al., 2000). It is therefore
important to assess patterns of cortical cell death in both sexes.
The timing of cell death in the postnatal medial prefrontal cortex
* Corresponding author.
E-mail addresses: ellenps2@illinois.edu (E.P. Sellinger), carly.drzewiecki@gmail.com (C.M. Drzewiecki), jwillin@bgsu.edu (J. Willing), jjuraska@illinois.edu
(J.M. Juraska).
Contents lists available at ScienceDirect
IBRO Neuroscience Reports
journal homepage: www.sciencedirect.com/journal/IBRO-Neuroscience-Reports
https://doi.org/10.1016/j.ibneur.2021.03.002
Received 23 December 2020; Received in revised form 21 March 2021; Accepted 23 March 2021
IBRO Neuroscience Reports 10 (2021) 186–190
187
(mPFC) is unknown. The rodent mPFC is analogous to the primate
dorsolateral prefrontal cortex (Uylings et al., 2003) and this region un-
dergoes protracted development compared to other cortical regions in
both rats (van Eden et al., 1991) and humans (Gogtay et al., 2004). The
prefrontal cortex is involved in high-level cognition including
decision-making, working memory, cognitive exibility, and impulse
control in both rodents and humans (Dalley et al., 2004; Euston et al.,
2012; Uylings et al., 2003), and aberrant development of the prefrontal
cortex is implicated in several psychiatric disorders in humans
(Gunaydin and Kreitzer, 2016). Therefore, rodent models employing
environmental manipulations during early postnatal development are
commonly used to address the underlying neural changes that might
underlie psychopathology. An understanding of the patterns of cell
death during early development is critical for effective experimental
design as manipulations (i.e. exposure to stress or toxicants) may pro-
duce their effects by acting on this process. Therefore, the present study
quantied the number of pyknotic cells in the male and female rat mPFC
across early postnatal development, between P2 and P25, to identify the
periods of peak cell death.
2. Materials and methods
2.1. Animals
Male and female Long Evans rats were bred in the vivarium of the
Psychology Department at the University of Illinois, and offspring were
used as experimental subjects. Subjects were kept on a 12:12 h light-
dark cycle and housed with the dam for the duration of the experi-
ment. Dams were given ad libitum access to food (Harlan 2020x; Teklad
Diets, Madison, WI) and water. All procedures adhered to the National
Institute of Health guidelines on the ethical use of animals and were
approved by the University of Illinois Institutional Animal Care and Use
Committee.
2.2. Tissue collection
Day of birth was recorded as P0. One male and one female pup per
litter were sacriced at the following timepoints: P2, 4, 6, 8, 10, 12, 14,
16, 18, and 25. A total of 13 litters and 86 subjects were used resulting in
35 subjects per sex at each age (Table 1). On the appropriate postnatal
day between 10:00 a.m. and 2:00 p.m., subjects were deeply anes-
thetized with sodium pentobarbital and then intracardially perfused
with 0.1 M phosphate-buffered saline (PBS) followed by a xative so-
lution composed of 4% paraformaldehyde in PBS. Brains were extracted,
left in the xative solution for 24 h, transferred to a 30% sucrose solution
in PBS for 3 days, and then cut into 40-micron coronal sections on a
freezing microtome.
Both pyknotic cells and neurons can be visualized using a Nissl stain,
which labels cell bodies and nuclear chromatin. Per subject, 23 sections
of the mPFC between Bregma 4.2 mm and 3.00 mm (Paxinos and Wat-
son, 2006) were selected. Sections were washed in 0.1 M PBS, mounted
on electrostatically charged slides, and washed in 70% followed by 50%
ethanol for 2 min each. Sections were then washed in 0.2 M PBS (7
mins), followed by periodic acid (5 mins) and stained with Methylene
Blue/Azure II (3 mins) to label cell bodies (Lillie, 1997). Slides were
rinsed with 0.2 M PBS (5 mins), 50% ethanol (2 mins), 70% ethanol (20
s), 95% ethanol (2 mins), and 100% ethanol (10 s) before being placed in
CitriSolv (2 mins) and coverslipped with Permount.
2.3. Quantication
Apoptotic cells undergoing pyknosis, a stage of cell death when the
nuclear chromatin becomes condensed, appear as small, symmetric,
dark spheres with sharp boundaries in Nissl-stained tissue that can be
readily identied by an experimenter (Ferrer et al., 1990) (Fig. 1). While
apoptotic cells can also be visualized with terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) staining, which labels the
3-hydroxyl end of nuclear DNA that forms when DNA fragmentation
occurs in the nal stage of apoptosis, both methods reveal similar pat-
terns when quantifying cell death on the same cell populations (Ferrer
et al., 1994; Nu˜
nez et al., 2001). A Nissl stain allows for simultaneous
quantication of neurons in the same tissue sections.
Neurons and pyknotic cells within the mPFC were counted using
similar methods to those previously described by our laboratory
(Drzewiecki et al., 2020; Kougias et al., 2018; Willing and Juraska,
2015) using the StereoInvestigator optical disector, which allows for
unbiased stereology. In the mature mPFC, the dorsal border is distin-
guished by a thinning layer I and increased cell density in layer III while
the ventral is marked by a blurring of lamina borders. However, at most
ages examined here, the lamina and boundaries are not clearly dened
(van Eden and Uylings, 1985). Therefore, a conservative area was traced
around the mPFC to include layers 26 of both infralimbic and prelimbic
cortices. White matter was used as a guide in that parcellated regions did
not extend above or below the dorsal and ventral edges of the white
matter. Then Stereoinvestigator randomly and uniformly laid counting
frames (40 µm×40 µm×10 µm depth) across the parcellated region.
The counting frame had two inclusionedges and two exclusion
edges. Neurons and pyknotic cells that came into focus and fell within
the boundaries of the counting frame were counted, while those that fell
outside the counting frame, touched the exclusion edges, or did not
come into focus within the 10 µm depth were not. Both the number of
pyknotic cells and number of live neurons per unit volume
(40 µm×40 µm×10 µm×number of counting sites) were quantied
and are reported here as pyknotic cell density and neuron density
respectively. Because the density of all cells in the region are changing as
cells die and dendrites grow, the ratio of pyknotic cells to 100 live cells
was calculated (Nu˜
nez et al., 2001).
2.4. Statistical analysis
Data were analyzed using RStudio statistical software. The sexes
were analyzed separately in all measures. The densities of neurons and
pyknotic cells as well as the ratio of pyknotic cells to live neurons were
analyzed in an ANOVA on a mixed linear model (using the lmer
package) using Satterthwaites method to estimate degrees of freedom
and setting age as a xed factor. As two experimenters performed the
counts of pyknotic cells, the experimenter was included as a random
factor along with litter. Signicant main effects were investigated with
post hoc pairwise-comparisons in which neighboring ages were
compared and p values were adjusted using a Bonferroni correction (9
comparisons).
3. Results
There was a main effect of age on the ratio of pyknotic cells per 100
live neurons, in males [F
(9, 25.3)
=6.08, p <0.0001] (Fig. 2A) and fe-
males [F
(9, 26.7)
=3.15, p =0.01] (Fig. 2B). P8 appeared as a peak,
dened as a signicant difference between ages, in both sexes as post
Table 1
Number of subjects across age groups.
P2 P4 P6 P8 P10 P12 P14 P16 P18 P25
Males 4 4 4 5 5 5 4 5 3 4
Females 4 4 4 5 5 5 4 5 3 4
E.P. Sellinger et al.
IBRO Neuroscience Reports 10 (2021) 186–190
188
hoc tests showed a signicantly higher pyknotic cell ratio on P8
compared to P6 (p <0.0001 in males; p =0.049 in females). Females
showed a prolonged peak through at P12 where the ratio of pyknotic
cells to live neurons was signicantly higher than that seen on P14
(p =0.009).
Pyknotic cell density (number of cells per unit volume) followed a
similar pattern to that seen in the ratio of pyknotic cells to live neurons,
with a main effect of age in males [F
(9, 25.3)
=8.18, p <0.001] (Fig. 2C)
and females [F
(9, 26.7)
=4.17, p =0.002] (Fig. 2D). In males, P8 again
appeared as a peak with post hoc tests showing a signicantly higher
density of pyknotic cells compared to that seen on neighboring ages, P6
(p <0.0001) and P10 (p =0.004). In females, a longer relative peak
again emerged where pyknotic cell density was signicantly higher on
P8 compared to P6 (p =0.003) with no signicant change between P8
and P10 nor between P10 and P12 before decreasing signicantly be-
tween P12 and P14 (p =0.007).
Neuron density decreased steadily from a maximum at P2 through
P25, revealing a signicant effect of age in both males [F
(9, 32.1)
=18.5,
p<0.001] and females [F
(9, 33)
=34.6, p <0.001] (Fig. 3). Post hoc
tests conrm a signicant decrease in neuron density between P4 and P6
in both males (p <0.001) and females (p <0.001). Females showed an
additional signicant drop between P2 and P4 (p =0.03).
4. Discussion
We found elevated levels of pyknotic cells in both the male and fe-
male mPFC during the rst two postnatal weeks, similar to levels of cell
death seen in other cortical regions. We expect the pattern of pyknotic
cell density represents apoptosis occurring in the cortex at this time as
several studies have shown similar patterns of cell death using Nissl-
stained pyknotic cell detection, as we do here, compared to TUNEL
detection methods (Ferrer et al., 1994; Nu˜
nez et al., 2001). Both the
number of pyknotic cells per 100 live neurons, which accounts for
changing neuron density during this time, and the pyknotic cell density
peaked at P8 in both sexes. In males, these measures fell two days later,
on P10, whereas females displayed a continued elevation through P12.
The observed decrease in neuron density in both sexes across this early
postnatal period is driven primarily by extensive dendritic growth and
synapse formation, which has been previously reported in the visual
cortex during the rst two postnatal weeks (Juraska and Fifkov´
a, 1979;
Miller, 1981).
The pattern of cell death observed in the mPFC closely aligned with
that previously reported by our laboratory in the visual cortex, where
one main peak in apoptosis appears in males on P7 (Nu˜
nez et al., 2001).
In the female visual cortex, however, two relative peaks are seen during
the rst two postnatal weeks on P7 and P11. Comparatively, in the
mPFC, we did not observe two distinct peaks in the ratio of pyknotic cells
to live neurons but instead saw an elevation on P8 that persisted until
P12, after which levels dropped. Additionally, unlike the visual cortex
however, there did not appear to be a relative peak in cell death on P25
in the female mPFC. While it appears that testosterone, not estrogen,
mediates the rst peak in cell death in the male visual cortex, it is un-
known whether this mechanism generalizes to other cortical regions,
like the mPFC, given the regional difference in androgen receptor
expression (Nu˜
nez et al., 2003). Additionally, the mechanism behind the
later peaks seen in females is not known.
In order to model human neurodevelopmental disorders including
those linked to early life experience (such as exposure to environmental
chemicals or stress), knowledge of the rate and timing of critical pro-
cesses like apoptosis is important (Majcher-Ma´
slanka et al., 2019).
While data from human cortical tissue is limited, examination of frontal
cortex indicates that apoptosis continues through gestational week 32,
the oldest age assessed (Chan and Yew, 1998). By examining neuronal
density and estimating approximate cortical volume (Rabinowicz et al.,
1996), calculations showed that there were more cells in the human
cerebral cortex at weeks 2832 of gestation than at postnatal weeks 013
by about 70%, indicating considerable cell death at the end of gestation.
The developmental timeline differs between rats and humans with rats
born signicantly more immature, so the timing of neurodevelopment
occurring during the third trimester in humans roughly corresponds to
the rst 10 postnatal days in the rat (Semple et al., 2013). Therefore, the
postnatal rise in cortical apoptosis observed in the rat in the present and
past studies (Ferrer et al., 1994; Nu˜
nez et al., 2001; Spreaco et al.,
1995) may represent a comparable event to that observed in humans
during late gestation.
4.1. Conclusions
In this study, we quantied pyknotic cell and neuron density in the
mPFC and demonstrated elevated levels of cell death during the rst two
postnatal weeks in male and female rats. These ndings ultimately
contribute to our understanding of neurodevelopment as the processes
involved in brain maturation can show distinct regional and temporal
patterns. The data presented here can be applied to the design of future
Fig. 1. Pyknotic cells. A Nissl-stained section of layers 2/3 in the mPFC on P8 (A) and P25 (B) under 63x objective where the solid arrows point to neurons and the
dashed arrows denote pyknotic cells. The scale bar denotes 10 micrometers.
E.P. Sellinger et al.
IBRO Neuroscience Reports 10 (2021) 186–190
189
Fig. 2. Ratio of pyknotic cells to live neurons and pyknotic cell density. The ratio of the number of pyknotic cells per 100 live neurons, in male (A) and female (B) rats
and the pyknotic cell density (number of pyknotic cells per unit volume (µm
3
×10
6
)) in male (C) and female (D) rats across postnatal days 225. *p <0.05;
**p <0.01; *** p <0.001.
Fig. 3. Neuron density. Neuron density as the number of neurons per unit volume (µm
3
×10
6
) in the male (A) and female (B) mPFC from ages P2 through P25.
*p <0.05; ***p <0.001.
E.P. Sellinger et al.
IBRO Neuroscience Reports 10 (2021) 186–190
190
studies involving the early environmental impact or other perinatal
insult, such as hypoxic-ischemic lesions, on mPFC development or those
modeling neurodevelopmental disorders implicating apoptosis.
Funding
E.P Sellinger and J. Willing were supported by NIH T32 ES007326,
and R21 ES026896 to J.M. Juraska.
Compliance with ethical standards
The authors certify that they were carried out in accordance with the
National Institute of Health Guide for the Care and Use of Laboratory
Animals (NIH Publications No. 80-23) revised 1996. The authors also
certify that formal approval to conduct the experiments described has
been obtained from the animal subjects review board of their institution
and could be provided upon request. The authors further attest that all
efforts were made to minimize the number of animals used and their
suffering.
CRediT authorship contribution statement
Elli Sellinger: Investigation, Formal analysis, Writing - original
draft, Visualization. Carly Drzewiecki: Conceptualization, Methodol-
ogy. Jari Willing: Investigation, Conceptualization, Methodology.
Janice Juraska: Conceptualization, Funding acquisition, Writing - re-
view & editing.
Conicts of Interest
None.
Acknowledgments
We thank the Beckman Institute Imaging Technology Group for use
of the stereology microscopy suite.
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E.P. Sellinger et al.
... We have found that exposure to BPA during gestation through postnatal day (P) 10 results in a modest increase in the number of neurons in the medial prefrontal cortex (mPFC) in male, but not in female, rats (Sadowski et al., 2014). Apoptosis occurs in the rat mPFC from late in gestation through P18 with high rates of apoptosis occurring between P8-12 (Sellinger et al., 2021). Thus it is possible that BPA interferes with naturally occurring cell death. ...
... Thus, a more targeted exposure paradigm would allow a better understanding of the mechanisms by which BPA disrupts the brain and behavior. Here, we exposed male and female rat pups to BPA for three postnatal days when high levels of apoptosis is known to occur (Sellinger et al., 2021), and then examined the immediate effects on cortical apoptosis. We also investigated the subsequent impacts on social preference behavior and cortical gene expression. ...
... Those in the early-dose group were orally administered the BPA solutions on P6, P7 and P8, while subjects in the late-dose group were orally administered the same solutions on P10, P11 and P12. These timeframes encompass two waves of high postnatal apoptosis that occur in the developing rat mPFC (Sellinger et al., 2021), with the late dose extending beyond the day when BPA was administered in our previous work (Sadowski et al., 2014). We tried two exposure time frames to see which one would give the larger effect. ...
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Bisphenol A (BPA) is an endocrine disruptor found in polycarbonate plastics and exposure in humans is nearly ubiquitous and it has widespread effects on cognitive, emotional, and reproductive behaviors in both humans and animal models. In our laboratory we previously found that perinatal BPA exposure results in a higher number of neurons in the adult male rat prefrontal cortex (PFC) and less play in adolescents of both sexes. Here we examine changes in the rate of postnatal apoptosis in the rat prefrontal cortex and its timing with brief BPA exposure. Because an increased number of neurons in the PFC is a characteristic of a subtype of autism spectrum disorder, we tested social preference following brief BPA exposure and also expression of a small group of genes. Males and females were exposed to BPA from postnatal days (P) 6 through 8 or from P10 through 12. Both exposures significantly decreased indicators of cell death in the developing medial prefrontal cortex in male subjects only. Additionally, males exposed to BPA from P6 – 8 showed decreased social preference and decreased cortical expression of Shank3 and Homer1, two synaptic scaffolding genes that have been implicated in social deficits. There were no significant effects of BPA in the female subjects. These results draw attention to the negative consequences following brief exposure to BPA during early development.
... In this study, we noted a decrease in neuronal density in the hippocampus and PFC of both rat strains from birth to PND7. Our findings are in line with the data of Sellinger and coworkers [36], who demonstrated that neuronal density in the PFC of male rats diminishes from PND2 to PND6 and then increases by PND8. Sellinger and coworkers have linked the decrease in neuron density in the PFC throughout the early postnatal period with extensive dendritic growth and synapse formation [36]. ...
... Our findings are in line with the data of Sellinger and coworkers [36], who demonstrated that neuronal density in the PFC of male rats diminishes from PND2 to PND6 and then increases by PND8. Sellinger and coworkers have linked the decrease in neuron density in the PFC throughout the early postnatal period with extensive dendritic growth and synapse formation [36]. We can speculate that the same is the case for the PFC of Wistar and OXYS rats. ...
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Sporadic Alzheimer’s disease (AD) is a severe disorder of unknown etiology with no definite time frame of onset. Recent studies suggest that middle age is a critical period for the relevant pathological processes of AD. Nonetheless, sufficient data have accumulated supporting the hypothesis of “neurodevelopmental origin of neurodegenerative disorders”: prerequisites for neurodegeneration may occur during early brain development. Therefore, we investigated the development of the most AD-affected brain structures (hippocampus and prefrontal cortex) using an immunohistochemical approach in senescence-accelerated OXYS rats, which are considered a suitable model of the most common—sporadic—type of AD. We noticed an additional peak of neurogenesis, which coincides in time with the peak of apoptosis in the hippocampus of OXYS rats on postnatal day three. Besides, we showed signs of delayed migration of neurons to the prefrontal cortex as well as disturbances in astrocytic and microglial support of the hippocampus and prefrontal cortex during the first postnatal week. Altogether, our results point to dysmaturation during early development of the brain—especially insufficient glial support—as a possible “first hit” leading to neurodegenerative processes and AD pathology manifestation later in life.
... Также в результате митохондриальной дисфункции повышается производство активных форм кислорода, что усугубляет нейрональный апоптоз [6]. В перинатальном периоде апоптотическая NMDA опосредованная гибель нейронов максимальна в стволе мозга, сразу после рождения -в таламусе и других подкорковых областях, а в первые 2 недели постнатальной жизни -в области коры головного мозга [7]. ...
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The prefrontal cortex (PFC) is a late developing region of the cortex, and its protracted maturation during adolescence may confer a period of plasticity. Closure of critical, or sensitive, periods in sensory cortices coincides with perineuronal net (PNN) expression, leading to enhanced inhibitory function and synaptic stabilization. PNN density has been found to increase across adolescence in the male rat medial PFC (mPFC). Here, we examined both male and female rats at four time points spanning adolescent development to stereologically quantify the number and intensity of PNNs in the mPFC. Additionally, because puberty coincides with broad behavioral and neuroanatomical changes, we collected tissue from age-matched pre- and post-pubertal siblings within a litter. Results indicate that both males and females show an increase in the total number and intensity of mPFC PNNs between postnatal day (P) 30 and P60. As we have previously found, white matter under the mPFC also increased at the same time. Male puberty did not affect PNNs, while female pubertal onset led to an abrupt decrease in the total number of PNNs that persisted through mid-adolescence before increasing at P60. Despite the change in PNN number, the intensity of female PNNs was not affected by puberty. Thus, though males and females show increases in mPFC PNNs during adolescence, the pubertal decrease in the number of PNNs in female rats may indicate a difference in the pattern of maximal plasticity between the sexes during adolescence.
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The importance of cell death in brain development has long been appreciated, but many basic questions remain, such as what initiates or terminates the cell death period. One obstacle has been the lack of quantitative data defining exactly when cell death occurs. We recently created a "cell death atlas," using the detection of activated caspase-3 (AC3) to quantify apoptosis in the postnatal mouse ventral forebrain and hypothalamus, and found that the highest rates of cell death were seen at the earliest postnatal ages in most regions. Here we have extended these analyses to prenatal ages and additional brain regions. We quantified cell death in 16 forebrain regions across nine perinatal ages from embryonic day (E) 17 to postnatal day (P) 11 and find that cell death peaks just after birth in most regions. We find greater cell death in several regions in offspring delivered vaginally on the day of parturition compared to those of the same post-conception age but still in utero at the time of collection. We also find massive cell death in the oriens layer of the hippocampus on P1, and in regions surrounding the anterior crossing of the corpus callosum on E18, as well as the persistence of large numbers of cells in those regions in adult mice lacking the pro-death Bax gene. Together, these findings suggest that birth may be an important trigger of neuronal cell death, and identify transient cell groups that may undergo wholesale elimination perinatally. This article is protected by copyright. All rights reserved.
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Adolescence is a period of extensive brain maturation. In particular, the regions of the medial prefrontal cortex (mPFC) undergo intense structural and functional refinement during adolescence. Disturbances in mPFC maturation have been implicated in the emergence of multiple psychopathologies during adolescence. One of the essential risk factors for the development of mental illness in adolescence is early-life stress (ELS), which may interfere with brain maturation. However, knowledge of the mechanisms by which ELS affects mPFC maturation and functioning in adolescents is very limited. In the present study, we applied a maternal separation (MS) procedure in rats to model ELS and studied its effect on the number of neurons and glial cells in the prelimbic region of the mPFC (PLC) of adolescent rats. Moreover, the expression of markers of cell proliferation and apoptosis was also studied. We found that MS rats had more neurons, astrocytes, and NG2-glial cells in the PLC. In contrast, the number of microglial cells was reduced in MS rats. These changes were accompanied by the decreased expression of proapoptotic genes and the increased expression of some prosurvival genes. Concurrently, MS did not affect cell proliferation in adolescents. Moreover, MS induced anxiety-like behaviors, but not anhedonic-like behavior, in adolescents. These results suggest that ELS may disturb neurodevelopmental apoptosis of neurons and early-postnatal proliferation and/or apoptosis of different populations of glial cells in the PLC. ELS-induced aberrations in the postnatal maturation of the PLC may affect cortical network organization and functioning and determine vulnerability to psychopathologies in adolescents.
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The growth and organization of the developing brain are known to be influenced by hormones, but little is known about whether disruption of hormones affects cortical regions, such as mPFC. This region is particularly important given its involvement in executive functions and implication in the pathology of many neuropsychiatric disorders. Here, we examine the long-term effects of perinatal exposure to endocrine-disrupting compounds, the phthalates, on the mPFC and associated behavior. This investigation is pertinent as humans are ubiquitously exposed to phthalates through a variety of consumer products and phthalates can readily cross the placenta and be delivered to offspring via lactation. Pregnant dams orally consumed an environmentally relevant mixture of phthalates at 0, 200, or 1000 μg/kg/d through pregnancy and for 10 d while lactating. As adults, offspring were tested in an attentional set-shifting task, which assesses cognitive flexibility. Brains were also examined in adulthood for stereological quantification of the number of neurons, glia, and synapses within the mPFC. We found that, independent of sex, perinatal phthalate exposure at either dose resulted in a reduction in neuron number, synapse number, and size of the mPFC and a deficit in cognitive flexibility. Interestingly, the number of synapses was correlated with cognitive flexibility, such that rats with fewer synapses were less cognitively flexible than those with more synapses. These results demonstrate that perinatal phthalate exposure can have long-term effects on the cortex and behavior of both male and female rats.
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Circuit dysfunction models of psychiatric disease posit that pathological behavior results from abnormal patterns of electrical activity in specific cells and circuits in the brain. Many psychiatric disorders are associated with abnormal activity in the prefrontal cortex and in the basal ganglia, a set of subcortical nuclei implicated in cognitive and motor control. Here we discuss the role of the basal ganglia and connected prefrontal regions in the etiology and treatment of obsessive-compulsive disorder, anxiety, and depression, emphasizing mechanistic work in rodent behavioral models to dissect causal cortico-basal ganglia circuits underlying discrete behavioral symptom domains relevant to these complex disorders. Expected final online publication date for the Annual Review of Physiology Volume 78 is February 10, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
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Adolescence is a critical period for brain maturation characterized by the reorganization of interacting neural networks. In particular the prefrontal cortex, a region involved in executive function, undergoes synaptic and neuronal pruning during this time in both humans and rats. Our laboratory has previously shown that rats lose neurons in the medial prefrontal cortex (mPFC) and there is an increase in white matter under the frontal cortex between adolescence and adulthood. Female rats lose more neurons during this period, and ovarian hormones may play a role as ovariectomy before adolescence prevents neuronal loss. However, little is known regarding the timing of neuroanatomical changes that occur between early adolescence and adulthood. In the present study, we quantified the number of neurons and glia in the male and female mPFC at multiple time points from preadolescence through adulthood (postnatal days 25, 35, 45, 60 and 90). Females, but not males, lost a significant number of neurons in the mPFC between days 35 and 45, coinciding with the onset of puberty. Counts of GABA immunoreactive cell bodies indicated that the neurons lost were not primarily GABAergic. These results suggest that in females, pubertal hormones may exert temporally specific changes in PFC anatomy. As expected, both males and females gained white matter under the prefrontal cortex throughout adolescence, though these gains in females were diminished after day 35, but not in males. The differences in cell loss in males and females may lead to differential vulnerability to external influences and dysfunctions of the prefrontal cortex that manifest in adolescence. Copyright © 2015. Published by Elsevier Ltd.
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Some have claimed that the medial prefrontal cortex (mPFC) mediates decision making. Others suggest mPFC is selectively involved in the retrieval of remote long-term memory. Yet others suggests mPFC supports memory and consolidation on time scales ranging from seconds to days. How can all these roles be reconciled? We propose that the function of the mPFC is to learn associations between context, locations, events, and corresponding adaptive responses, particularly emotional responses. Thus, the ubiquitous involvement of mPFC in both memory and decision making may be due to the fact that almost all such tasks entail the ability to recall the best action or emotional response to specific events in a particular place and time. An interaction between multiple memory systems may explain the changing importance of mPFC to different types of memories over time. In particular, mPFC likely relies on the hippocampus to support rapid learning and memory consolidation.
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Although neuroanatomical plasticity has been demonstrated in the rat visual cortex, no systematic data on the dendritic development of the area are available. In the present study, the visual cortex of hooded rats at 1, 3, 5, 7, 10 and 15 postnatal days of age (P1-P15) was impregnated with the rapid Golgi method. The cortex was divided into the superficial layers, II-IV, and the middle layer V. At P1, pyramidal neurons had apical shafts and the beginning of the apical terminal arch. Analysis of both basilar and oblique dendritic number showed that pyramidal neurons of the middle layer developed more quickly than those in the superficial layers. The number of lower order basilar dendritic branches reached asymptote over the examined time period, whereas the higher order branches were still increasing in number but at a decelerating rate by P15. Dendrites at all ages exhibited varicosities which were especially prominent on the thin dendritic branches of the earlier ages. Some thin, filamentous processes, termed protospines, were found on dendrites and cell bodies at P1 to P5. They seemed to decrease by P7, when a few mature spines appeared. Spines increased in number on days P10 and P15. A comparison of the data from this study with quantified Golgi studies in adult rats indicates that by P10 and P15 the number of basilar branches is in the range seen in the adult.