ABS 027: Comparison of Neuroplasticity Between Maternal and Non- Maternal Rats: Perineuronal Nets in the Basolateral Amygdala

Chris Lin ¹, Hanna Dobson ², Brenna Hammond ³, and Joseph Lonstein ² ³.

¹ Ronald E. McNair Scholar, Trio Program, Hunter College, New York City, New York 10065, United States
² Department of Psychology, Michigan State University, East Lansing, Michigan 48824, United States
³ Neuroscience Program, Michigan State University, East Lansing, Michigan 48824, United States

The Van Wickle Journal (2026) Volume 2, ABS027

Introduction: During motherhood, the brain undergoes changes that fine-tune maternal caregiving to accommodate the behavioral needs of offspring. A key adaptation is the reduced aggregation of perineuronal nets (PNNs) that surround cortical parvalbumin+ interneurons (PV+ INs), which promotes neuroplasticity. PNNs are extracellular glycoprotein matrices that regulate synaptic plasticity by serving as physical barriers to cellular connections within the brain. While widely studied in the cortex of laboratory rodents, PNNs remain relatively unexplored in the basolateral amygdala (BLA) – a region critical for mediating pup-related and other sensory inputs to brain systems involved in the rewarding and hormonal signaling underlying maternal caregiving. During pregnancy and postpartum, PPNs in many brain areas exhibit significant structural alterations in their chondroitin sulfate proteoglycans (CSPG), leading to enhanced opportunities for synaptic neuroplasticity. In our experiment, we hypothesized that, during motherhood, BLA undergoes significant neuroplasticity, including in its PNNs. To test this, the BLA of female rats that recently gave birth, or nulliparous female rats, were studied to observe the presence and changes in levels of CSPG by using real-time quantitative polymerase chain reaction (RT-qPCR) to measure mRNA levels of CSPG components in PNNs - brevican, aggrecan, and phosphacan. We found that brevican and phosphacan were not statistically significant, while aggrecan showed an approaching significance. While not statistically significant, the differences between new mothers and the nulliparity control group in aggrecan levels may indicate changes in the capacity for neuroplasticity in the peripartum BLA that increase pup-related sensory inputs that drive maternal responses to young.

Methods: Twelve Long-Evans rats (Harlan Labs, IN), six rats in the experimental group and six rats in the virgin control group, were 60-90 days old. The two groups were housed in pairs or triplets with clear plastic cages, lined with wooden chip bedding, and provided ad libitum access to food and water (Tekland). Humidity-controlled rooms were kept at 22 - 23°C on a 12-hour light-dark cycle (lights on at 7 am). Vaginal cytology was used daily to monitor the estrous phase of the females. The experiment group was paired with a male for 24 hours, when a female rat had reached the proestrus cycle. Pregnant females were housed singly at the start of day 20 of pregnancy. and the parturition group was euthanized within 3 hours of birth. All rats were euthanized via pentobarbital overdose, and their brains were extracted and fresh frozen at -80°C for storage until sectioning and RT-qPCR analysis.

RT-qPCR
Frozen brains were sectioned coronally at 300 µm with a cryostat (Leica CM1950, Nussloch, Germany). A 1.0 mm micro punch (Harris Micropunch, Hatfield, PA) was used to collect the BLA bilaterally. After, the collected tissue was homogenized in RLT buffer (74134, Qiagen, Valencia, CA) using pulsed sonication (Fisher Scientific, Pittsburgh, PA). mRNAs were extracted using RNeasy Plus Mini Kits (74134, Qiagen, Valencia, CA). The concentration and purity of the extracted mRNAs were analyzed using a QubitTM Flex Fluorometer (Thermo Fisher Scientific, Pittsburgh, PA). mRNA samples were then converted to cDNA with a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA).

The mRNA levels of three commonly studied proteoglycans in PNNs - aggrecan, brevican, and phosphacan - were analyzed, along with the mRNA level of HPRT1, a housekeeping gene, as the control. Primers were added to convert the cDNA, along with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and samples were run in triplicate for each of the three proteoglycans of interest for the control and experimental rats. An ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) was used to amplify the sequences for quantification: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Lastly, the ΔΔCT method was used to calculate fold changes in the proteoglycan mRNA levels compared to the HPRT control gene.

Statistics
Grubbs tests were used to identify outliers, which were removed. Two-tailed independent sample T-tests were used to compare the experimental and control groups in each proteoglycan. P-values of p<0.05 were used as a criterion to assess the significant differences.

Results: In the basolateral amygdala, mRNA expression of the three proteoglycans did not significantly differ between the parturient group and the virgin group. Aggrecan expression approaches significance, though, with p = 0.063 (Fig.1A). Some studies have shown that aggrecan is involved in neuroplasticity in the cortex (Ueno et al., 2018; McRae et al., 2007) and has a possible role in closure of critical periods (Guimaraes et al., 1990).

Discussion: The mechanisms of PNNs’ abrupt degradation during parturition (Uriarte et al., 2020) are still poorly understood. Matrix metalloproteinase-9 (MMP-9) represents one potential cause, acting as a key regulator of perineuronal net (PNN) degradation and exhibiting a trend of negative correlation with aggrecan expression (Xiao et al., 2024). Notably, the female brain may experience gestational stress, which can dramatically decrease PNNs (Leuner et al., 2023). In addition to increasing oxidative stress during labor to birth (Simon‑szabo et al., 2021), it can affect the expression of PNNs. Since PNNs provide a protective barrier but are also sensitive to excess oxidative stress, their protective function likely depends on the balance between oxidative stress-induced degradation and the system's capacity to maintain them (Cabungcal et al., 2013).

While we found that PNN proteoglycan mRNA levels in the BLA did not change significantly, it is known that gene levels often do not correspond to protein levels (Pascal et al., 2008; Maier et al., 2009), so future research quantifying protein expression throughout gestation could clarify PNN fluctuations in the BLA. Furthermore, generating a PNN knockout model would enable investigation of its necessity for maternal behavior.