Cell death in the male and female rat medial prefrontal cortex during early postnatal development

Abstract

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.

Full text

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
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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
3–5 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, 2–3 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 2–6 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 “inclusion” edges 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 Satterthwaite’s 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 28–32 of gestation than at postnatal weeks 0–13
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 2–25. *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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