Social Instability in Adolescent Mice Leads to Lasting Cognitive Deficits with Reduction of Intra-Hippocampal Functional Connectivity and Parvalbumin-Containing Interneurons with Perineuronal Nets.

Social Instability in Adolescent Mice Leads to Lasting Cognitive Deficits with Reduction of Intra-Hippocampal Functional Connectivity and Parvalbumin-Containing Interneurons with Perineuronal Nets. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Social Instability in Adolescent Mice Leads to Lasting Cognitive Deficits with Reduction of Intra-Hippocampal Functional Connectivity and Parvalbumin-Containing Interneurons with Perineuronal Nets. Steven Siegel, Lindsey Crown, Krishna Parkeh, Daniel Gray, Samuel Guillemette, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9118580/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background Adolescent social stress can be detrimental to developing hippocampal-prefrontal circuits and is associated with adverse cognitive outcomes. We previously found that adolescent social instability stress (SIS) in mice resulted in later-life memory retrieval impairment as measured with novel object recognition (NOR). Methods Mice were behaviorally assessed using novel object recognition (NOR), social interaction, and the elevated plus maze. We analyzed local-field potential data during NOR. We assessed the 1/f slope of the power spectral density within the dCA1, dCA3, vCA1, and mPFC and analyzed coherence between the ventral hippocampus and medial prefrontal cortex (mPFC) and dorsal CA3 to CA1 as animals were near objects during NOR. Additionally we measured serum corticosterone levels immediately following the termination of the adolescent SIS manipulation, and used immunohistochemistry to quantify perineuronal nets (PNN) around parvalbumin (PV)-positive neurons using antibodies for PV and Wisteria floribunda agglutinin (WFA), which labels PNNs. Results NOR deficits in SIS mice were replicated. No significant changes to social interactions, elevated plus maze or the aperiodic component of spectral parameterization were observed. Intra-hippocampal gamma coherence was reduced in SIS animals around the novel object relative to controls. PNN density was reduced in the mPFC of stressed animals. Corticosterone was lower in SIS mice relative to controls. Conclusions Data support the hypothesis that altered social interactions during adolescence can result in structural, electrophysiological, and cognitive deficits that persist well into adulthood. Biological sciences/Neuroscience/Learning and memory/Hippocampus Biological sciences/Physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cognitive deficits that arise in adulthood emerge from complex interactions between a person’s unique genetic predispositions and life experience. Exposure to early life stress (ELS) can dramatically alter an individual’s life trajectory depending on the nature of the stressor and the developmental phase in which it is encountered (i.e. infancy vs. childhood vs. adolescence). Over the past decade there has been a disproportionate increase in adolescent ELS due to a number of cultural factors including the emergence of social media and increased accessibility of information. Adolescence is a particularly critical time for the development of numerous aspects of cognition including executive function, cognitive control, and social development. This is due in large part to the protracted maturation of prefrontal cortex connectivity with the hippocampus and other brain structures critical to cognition 1 , 2 , as well as the relatively late development of inhibitory neural circuits across the forebrain that mediate information processing within these circuits. These developmental reorganizations result in drastic changes in network-level activity patterns that mediate executive control and memory function during adolescence 3 – 5 . Previous studies in primates demonstrate structural changes to hippocampal cytoskeletal proteins and dentate gyrus morphology following severe social deprivation 6 . Studies in rodents have shown that, like in humans, exposure to ELS can lead to later-life cognitive impairment 7 – 10 . However, most of these studies introduce stressors at very early postnatal time points using models of maternal separation, limited bedding and nesting, and early immune activation. In a previous study from our group, we demonstrated that mice exposed to adolescent social instability stress (SIS), where animals are unable to form social hierarchies due to a constantly changing social environment, exhibit cognitive impairment at 30 weeks of age (~ 7 months; Featherstone et al., 2022). Critically, the observed cognitive deficits were domain specific, with adult SIS-exposed mice exhibiting impairment in novel object recognition (NOR) memory in the absence of measurable social withdrawal, anxiety-like or depressive phenotypes. Moreover, hippocampal expression of genes with roles in regulating cellular stress responses, were elevated in SIS-exposed mice at 66 weeks of age (~ 15 months) and correlated with poorer recognition memory 11 . Together, these observations indicate that ecologically-relevant social stress experienced in adolescence can cause persistent decrements in memory that may be mediated by moderators of the stress cascade. It has long been understood that NOR memory relies on activity within medial temporal lobe structures, primarily the hippocampus and perirhinal cortex 12 – 14 . More recent work has shown that oscillatory coupling between the hippocampus and medial prefrontal cortex (mPFC) occurs during recognition memory retrieval for novelty discrimination, and that optogenetic silencing of hippocampal inputs to the mPFC results in NOR performance deficits 15 . Oscillatory dynamics within the hippocampus and mPFC have also been shown to change across the adult lifespan in ways that impact memory function in rodents 16 , 17 . However, the extent to which exposure to early life adversity alters oscillatory activity in the adult hippocampus and mPFC has not been well studied. Oscillatory dynamics across the brain are potently regulated by inhibitory neuron activity 18 – 20 Dysfunction of inhibitory interneurons in the hippocampus and mPFC has been linked to declining memory function in the context of psychiatric disorders, normative aging, and in neurodegenerative disease conditions 21 – 24 . The maturation of a specialized extracellular matrix structures called the perineuronal net (PNN) is critical in the development of inhibitory dynamics. In cortex and the hippocampus, the PNN preferentially accumulates around fast-spiking parvalbumin(PV)-expressing interneurons 25 – 27 , which form perisomatic synaptic contacts with excitatory neurons, allowing them to potently regulate neuronal firing patterns, oscillatory activity, and inter-regional communication 28 . Thus, perturbations in the abundance and composition of the PNN could have profound impacts on memory-circuit function and cognition 29 , 30 . While the mechanisms that regulate PNN assembly and remodeling are just beginning to be elucidated, microglia, the primary immune-like cells of the central nervous system, are thought to be critical in this process. Microglia synthesize and secrete numerous degradative enzymes that can modulate PNN composition, and pharmacological depletion of microglia results in profound increases in proteoglycan deposition across the cerebral cortex 31 . The present study combines adolescent SIS stress with assessments of adult electrophysiological and behavioral function, as well as a subset of their proposed relationships to morphologic and molecular mechanisms of persistent consequences of early life experience. In doing so, we test the following five overarching hypotheses: Adolescent SIS leads to persistent impairment of episodic memory as evidenced by decreased NOR. These impairments will not be accompanied by increased anxiety or social withdrawal. Memory impairments in adulthood are accompanied by a reduction of hippocampal-PFC functional connectivity as assessed by interregional coherence during performance of NOR. Electrophysiological changes in hippocampal-PFC connectivity will be associated with concomitant changes to inhibitory networks, measured by PV expression and perineuronal net integrity. Adolescent SIS will lead to persistent changes in stress hormones in adult animals. We do not anticipate that these alterations will vary with sex. Methods Animals Subjects were C57BL/6J (B6) male and female mice obtained at 3 weeks of age from Jackson Laboratories. A total of 144 mice were used in the experiment, half as control animals that did not change cage mates, and half that underwent SIS. At postnatal day 27, all animals were microchipped (Bio Medic Data Systems). Animals were housed in a 12-hour light/dark cycle and were given food ad libitum. All behavioral tests and surgeries were conducted during the light cycle. All experiments were performed following the guidelines of the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals using protocols approved by the University of Southern California Institutional Animal Care and Use Committee. Social Instability Stress Mice were housed in same-sex groups of 4. Beginning at postnatal day 28 all mice were placed in clean cages twice per week for 7 weeks. SIS mice were placed in cages with new, randomly-assigned cage-mates at each cage change 11 ( Fig. 1 A ) . Control animals remained with the same group of animals for the duration of the experiment but received the same amount of handling and cage changes as SIS animals. At the end of the SIS period, a subgroup of animals were immediately sacrificed and used for immunohistochemistry ( Fig. 1 B ) . Behavior Behavioral conditions and handling Prior to behavior, all animals were gently handled for ~ 5 minutes and acclimated to the experimental room and electrophysiological recording system to minimize the animal stress during recordings and encourage movement and engagement during behavioral assays. Novel Object Recognition Novel object recognition (NOR) took place in a box (30.5 x 40.6 x 40.6 cm 3 ) over two days in a low-light room. On the first day, mice were allowed to habituate to the empty box for 5 minutes. Mice were then removed and placed back into their cages for 10 minutes. During this time, the box was cleaned with 70% EtOH and two identical objects (lego towers or glass flasks) were placed into the box, diagonally from each other. On the second day the process of habituation and training was repeated followed by the testing phase 10 minutes later. The mice were placed back into the box with one familiar object from the training and a novel object. Mice were allowed to explore for 5 minutes before being removed ( Fig. 2 A ) . Electrophysiological recordings took place during NOR. Behavior was recorded via an overhead video camera and scored by hand by an individual that was blind to mouse condition (SIS or control). NOR discrimination index was calculated as: (time with novel object)/(time with familiar object + novel object). Only the first 10 seconds of interaction was scored because this period has been previously indicated to best reflect true object exploration as opposed to general locomotor activity 32 . If a mouse failed to reach the 10 seconds threshold, its data was excluded from analysis. Elevated Plus Maze Elevated plus maze (EPM) was conducted in a brightly lit room in an elevated plus maze apparatus. The apparatus has 2 closed arms covered by walls (closed arms 30.5 cm x 6.4 cm 2 ; walls: 15.2 cm in height) and 2 open arms (30.5 x 5.1 cm 2 ) and was elevated above the ground at 18 inches. The animal was placed in the center of the maze and allowed to explore all parts of the maze for 5 minutes. The anxiety levels were measured as (time in the open arms)/(time in the closed arms and open). Videos were recorded and scored using Topscan Lite (Clever Sys Inc., Reston, VA, USA) ( Fig. 2 B ) . Social Interaction Social interaction was assessed using a three-chambered box (53 x 27 x 23cm 3 ). A perforated cylinder was placed at both ends of the box. Subject mice were allowed to explore the box for 5 minutes. Mice were then removed and placed back into cages. A juvenile mouse (3–4 weeks of age) of the same sex (stimulus mouse) was then placed under one cylinder and a ping-pong ball was placed under the other. Subject mice were then placed back into the box and given 5 minutes to explore. The cylinder allowed subject mice to initiate social interaction and the perforation allowed mice to both see and smell the stimulus mice. Stimulus mice were confined to the cylinder and were thus not able to initiate social interaction ( Fig. 2 C ) . Social interaction scores were measured as a ratio of (time interacting with stimulus mouse)/(time interacting with both stimulus mouse and ping-pong ball). Testing was recorded and scored using Topscan Lite (Clever Sys Inc., Reston, VA, USA). Interaction with either the mouse or ball was defined as the subject mouse being next to the cylinder, facing the cylinder, and with its nose oriented towards the cylinder. 8 mice were excluded from analysis due to problems occurring during data acquisition. Electrophysiological Recordings Surgery and Electrophysiological recordings Animals were anesthetized with 3% isoflurane mixed with O2 carrier gas at a flow rate of .75 L/min. Once anesthetized, animals were placed on a stereotactic frame (David Kopf, Tujunga, CA, USA) and maintained under isoflurane anesthesia between 1–2%. A midline incision was made to expose the skull and burr holes were made over the left mPFC (AP: 1.7, ML: 0.5, DV: − 2.5), left ventral hippocampus (AP: -3.16, ML: -3.0, DV: -4.0), right dorsal CA1 (AP: -2.44, ML: 2.25, DV: -1.55) and right dorsal CA3 (AP: -1.7, ML: -2.1, DV: − 2.0) (Fig. 3 A). 4 additional holes were made for skull screws to serve as anchors. Electrodes were lowered and glue was applied to fix them to the skull. Electrodes were connected to the Neuralynx EIB-16-QC (Neuralynx, Boseman, MT, USA) electronic interface board (EIB) via stainless steel wire. A skull screw was placed over the cerebellum and served as ground and reference. Dental acrylic was used to secure the electrodes and EIB to the skull. Animals were given 1.5 weeks to recover before behavioral testing and recording began. Data were acquired using the NeuraLynx Digital Lynx SX and HS-16QC-LED headstage and behavior was tracked using the Cheetah video tracking system and an overhead camera (Neuralynx, Boseman, MT, USA). Analysis Electrophysiological data were analyzed from 37 mice (11 control male, 7 control female, 10 SIS male, and 9 SIS female). Analysis of time series data was performed in MATLAB (R2022a, MathWorks) using custom scripts. Electrodes that were not determined to be in the correct location or that were too noisy were excluded from analysis (n = 8); a noise threshold was set based on visual inspection of the recording data from each electrode for each day for each animal. This was performed blind to animal identity. As a result, the sample size for electrophysiological measures varies by task and measure because of the need to exclude certain electrodes. To examine the extent to which changes to intra-hippocampal- or hippocampal-prefrontal network activity underlie the observed NOR memory impairment in adult mice exposed to adolescent SIS, local field potentials were simultaneously recorded from dCA3, dCA1, vCA1, and mPFC in control and SIS-exposed mice (electrode locations, Fig. 3 A-E). Because increasing attention is being paid to the importance of distinguishing between true oscillatory (periodic) activity and general increases in aperiodic power spectral density when parameterizing neural power spectra 33 – 35 , we used the FOOOF algorithm (Fitting Oscillations & One-Over F) to specifically quantify this aperiodic component 34 . The aperiodic component measures the slope of the power spectrum and is most simply defined as the 1/f x , where x is the aperiodic exponent. The aperiodic component is thought to reflect the balance between excitatory and inhibitory signaling 34 , 36 – 39 . We hypothesized that SIS would alter inhibitory networks and therefore, would change the E-I balance within the hippocampus or mPFC ( Fig. 3 F-I ) . Novel object recognition has been associated with two distinct memory-related pathways, the intra-hippocampal pathway (dCA3 to dCA1) and the hippocampal-prefrontal pathway (vCA1- mPFC). Within the intra-hippocampal pathway (dCA3-dCA1) it has been proposed that this is primarily mediated by gamma frequency oscillations 40 – 42 . Alternatively, within the vCA1-mPFC pathways, lower frequency rhythms, particularly within the theta band, are associated with recognition memory (Wang et al., 2021). Therefore, we recorded local field potential activity during the recall phase of the NOR test and compared activity when animals were near the novel versus the familiar object. Figure 4 ) . Analysis was performed using Monte Carlo cluster-based permutation testing that allowed significantly modulated frequencies to be identified regardless of canonical frequency bands 43 . Corticosterone ELISA A previous study from our group demonstrated that exposure to adolescent SIS results in elevated mRNA expression of genes critical for the cellular stress response, including FK506 binding protein 5 (FKBP5) and Corticotropin Releasing Hormone Receptor 2 (CRHR2), that correlates with recognition memory deficits 11 . To determine how these previous findings align with the abundance of systemic corticosterone, we used a competitive ELISA assay to measure corticosterone levels in SIS and control mice immediately following the termination of the adolescent SIS manipulation. Prior to sacrificing, animals were acclimated to a low light room for 30 minutes. Animals were sacrificed and cardiac punctures were administered before perfusions between 9:30 am and 11 am to control for circadian effects on corticosterone levels. Blood was collected, left at room temperature for 10 minutes and then centrifuged. Serum was collected and stored at -80 degrees until testing. Corticosterone levels were measured via competitive ELISA using corticosterone parameter assay kits (Catalog #: KGE009, R&D Systems, Minneapolis, MN, USA). Immunohistochemistry Previous reports have shown that certain types of ELS prolong the developmental timeframe of the PNN into adulthood, mimicking phenotypes observed in mouse models of schizophrenia 30 , 44 . To assess whether exposure to adolescent SIS also changes the development of the PNN, brain sections from SIS-exposed and control mice were immunohistochemically labelled with antibodies directed against parvalbumin (PV; inhibitory neurons) and the wisteria floribunda agglutinin (WFA; perineuronal net) lectin. These sections were imaged with confocal microscopy, and the number of PV cells, number of PNNs, and the proportion of PV cells surrounded by the PNN were quantified ( Fig. 5 A ) . Mice were anesthetized with isoflurane and perfused intracardially with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were incubated in PFA for ~ 24 hours and then stored in PBS in a 4-degree refrigerator. Brains were sliced at 60µm on a vibratome (Leica VT1200, Wetzlar, Germany). Prior to immunohistochemistry slices were placed in blocking/permeability solution of 5% normal donkey serum (NDS) (MilliporeSigma, Burlington, MA, USA) and 0.3% Triton-X in PBS for 2 hours on a shaker. Slices were then incubated in a primary antibody solution containing a 1:1000 concentration of rabbit anti parvalbumin (PV 27, Swant, Switzerland) and 1:250 concentration of anti-wisteria floribunda agglutinin (WFA) (B-1355-2, Vector Laboratories, Newark, CA, USA), in bocking/permeability solution. Slices were then left to incubate overnight on a shaker at 4 degrees. The next day slices were washed with PBS 4 times for 10 minutes per wash on shaker. They were then placed in a secondary antibody solution containing 1:500 DyLight488 (ImmunoReagents Inc, Raleigh, NC, USA) to label PV in green and 1:500 Alex Fluor 647 (JacksonImmuno Research, West Grove, PA, USA) to label WFA in far-red. Tissue was left to incubate on a shaker for 2 hours before being washed again 4 times for 10 minutes each wash on a shaker. Tissue was then mounted on slides, cover-slipped with permount and sealed with nail polish. To quantify the number of PV+ cells, PNNs, and PV+ cells with PNNs, numbers were manually counted by an experimenter blinded to animal condition. PV+ cells, PNNs and PNNs around PV cells were counted using the Cell Counter plug in for Image J 45,46 . Results Adolescent SIS results in sustained recognition memory impairments in adulthood but no changes to social interaction or anxiety-like behavior. Our group previously demonstrated that exposing adolescent mice to SIS results in adult deficits in novel object recognition memory, but no change in social interactions or anxiety-like behavior 11 . In the current study, adult mice exposed to adolescent SIS- exhibited a lower discrimination index on the NOR task relative to controls (F(1,22) = 6.518, p = 0.018; Fig. 2 A). Adult SIS-exposed mice were not different from controls on an elevated plus maze test of anxiety-like behavior (F(1,31) = 0.004, p = 0.934; Fig. 2 B) or on a test of social preference (F(1,25) = 0.492), p = 0.490; Fig. 2 C). No effects of biological sex were observed in any behavioral test (NOR: F(1,22) = 6.518, p = 0.873; EPM: F(1,31) = 0.007, p = 0.951; Social Interaction: F(1,31) = 0.007, p = 0.951). These observations replicate previous findings suggesting that adolescent SIS results in sustained, domain-specific cognitive impairments that implicate the hippocampus and mPFC. Adolescent SIS did not alter the 1/f component of the power spectral density. FOOOF exponents were not significantly difference between SIS and control mice (dCA1 p = 0.07; dCA3 p = 0.26, mPFC p = 0.43, vCA2 p = 0.69 after Bonferroni-Holm correction for multiple comparisons; Fig. 3 F-J). Adolescent SIS reduces intra-hippocampal gamma coherence when animals are near the novel object. We found that within the intra-hippocampal pathways, control mice had greater high gamma coherence around the novel, but not familiar object (85-90Hz; p < 0.02; Fig. 4 C,E); while there were no significant differences in coherence between vCA1 and the mPFC ( Fig. 4 D,F). Within the intra-hippocampal pathway, males had greater coherence between 3-6Hz around the novel object (p = 0.016), with no differences in the vCA1-mPFC pathway (Fig. 4 G,H). Around the familiar object, females had greater coherence in the delta (1-4Hz, p = 0.002) and beta range (20-24Hz, p = 0.006) within the vCA1-mPFC pathway (Fig. 4 I,J) and no differences in intra-hippocampal coherence relative to males. Adolescent SIS reduces PNN accumulation around inhibitory neurons in the mPFC Exposure to adolescent SIS did not change PV neuron numbers in the hippocampus or mPFC (dCA1: p = 0.59, dCA3 p = 0.36, mPFC: p = 0.40); Fig. 5 D). However, SIS mice did exhibit a decreased fraction of PV cells surrounded by the PNN (WFA). (p = 0.004, corrected for multiple comparisons; Fig. 5 E), but not in the hippocampus (dCA1: p = 0.43, dCA3: p = 0.26, Fig. 5 E). This pattern within the mFPC was made more significant when controlling for the number of PV cells (p = 0.005, Fig. 5 F). Together, these histological observations indicate that adolescent SIS results in a reduction of extracellular matrix surrounding inhibitory neurons, specifically in the mPFC. Sample sizes were to low to evaluate these effects by sex. Adolescent SIS reduces systemic corticosterone Relative to control mice, SIS-exposed mice had significantly lower levels of corticosterone (F(1,66) = 7.577, p = 0.0076; Fig. 5 G). Moreover, female control and SIS mice had significantly higher systemic corticosterone levels compared to males (F(1,66) = 27.13, p < 0.0001), although no stress-by-sex interaction effects were observed. Discussion Overarching Summary The current study demonstrates that subtle changes in the ability to establish stable social bonds during adolescence can lead to lasting functional and physiological changes into adulthood. Specifically, mice which were reared using a social instability protocol replicated previous findings of a decreased episodic memory using the novel object recognition task 11 . Further, we now show that these cognitive deficits are accompanied by both physiological and anatomical changes, consistent with known structures and cellular elements that are critical for recognition and recall. These changes include a reduction in intra-hippocampal high-frequency gamma-band coherence between CA1 and CA3 in proximity to the novel object, as well as a reduction in perineuronal nets adjacent to parvalbumin-positive interneurons in mPFC. Taken together, data indicate that disruptions of normal, stable social networks during adolescence have the capacity to negatively impact formation of perineuronal nets around parvalbumin positive neurons, with concomitant changes in high frequency oscillations during a cognitive load, leaving lasting deficits. As such, the impact of recent trends (e.g. social media) and events (e.g. COVID pandemic) that reduce the formation of lasting social networks in adolescents may have negative implications for affected populations well into their adult lives. Hypothesis 1 Adolescent SIS leads to persistent impairment of episodic memory as evidenced by decreased NOR. These impairments will not be accompanied by increased anxiety or social withdrawal. This hypothesis was supported, suggesting that prior findings of impaired cognitive performance in the absence of either an anxiety phenotype or social deficit is both reliable and replicable. Recent data showing reduced proficiency in school performance among children who were in secondary school during the pandemic suggests that these findings may also be generalizable to humans who had a disruption in their ability to interact with other children during critical developmental periods 47 . Additionally, data highlight the specificity of the effect on recognition and recall, such that reduced episodic memory cannot be explained by either a generalized increase in anxiety or decreased motivation for interactions with the environment. Although counter-intuitive, mice reared with constant change in their social hierarchy did not have either increased anxiety or decreased interest in other mice when given the opportunity to avoid open arms and other mice respectively. Hypothesis 2 Memory impairments in adulthood are accompanied by a reduction of hippocampal-PFC functional connectivity as assessed by interregional coherence during performance of NOR. The shape of the power spectral density within each region was not altered in SIS animals relative to controls. These data indicate that SIS did not alter the ability of specific cell types to the extent that the overall distribution of frequencies generated was altered. Specifically, each region in isolation (dCA1, v CA1, vCA3 and mPFC) has the ability to form the full spectrum of electrical activity. However, memory impairments in adult SIS mice were accompanied by an increase of high gamma coherence between CA1 and CA3 during performance of NOR. Previous studies indicate that high gamma CA3–CA1 synchrony significantly increased when rats explored a novel object or novel location compared to familiar object or place presentation 48 . Thus, the current finding is consistent with the idea that SIS mice are experiencing the NOR task as both a novel objects and locations despite having seen them previously. Alternatively, there was no difference between SIS and control mice for Hipp-mPFC coherence. Previous data suggest that increased task difficulty or complexity triggers higher mPFC activity and potentially increased coherence to maintain attention and memory performance 49 . Thus, the current study may have employed a task that was sufficiently easy that mPFC-hippocampal coherence was not altered. The fact that the SIS mice performed less well than the controls argue against this interpretation. Additionally, prior studies indicate that interactions between mPFC and CA1 intensify remote memory recall, where prefrontal theta oscillations modulate CA1 gamma power and unit spiking to enhance contextual representations 50 , 51 . It is possible that the NOR task as performed relied on more recent memory and therefore did not engage or rely upon mPFC-hippocampal modulation the extent it engaged intra-hippocampal circuits. In such a case, one would expect changes within the hippocampus, but not necessarily between mPFC and hippocampus. Hypothesis 3 Electrophysiological changes in hippocampal-PFC connectivity will be associated with concomitant changes to inhibitory networks, measured by PV expression and perineuronal net integrity . Contrary to our hypothesis, there was no change in PV-interneuron counts. However, PNN density was reduced in the mPFC of SIS animals, supporting the idea that SIS-induced stress in adolescent mice may be affecting adult excitability by altering the proteoglycan deposition and pruning of PV-positive cells. One limitation for this interpretation stems from the observation that gamma coherence was altered within hippocampus, while changes in PV interneuron-associated PNNs occurred in mPFC. However, it remains possible that changes measured in mPFC reflect a larger process, and that the high degree of connectivity between various structures leads to a complex interplay such that structural changes in one region may be manifest (detected) as functional changes in another. Hypothesis 4 Adolescent SIS will lead to persistent changes in stress hormones in adult animals. SIS animals lower basal corticosterone levels than controls . The current study indicates that SIS mice had significantly lower levels of the stress hormone corticosterone when tested immediately after the SIS period. This differs from our prior study in which no differences were found for 1 year following cessation of the SIS protocol. As such, data indicate early alterations in modulation of the stress cascade either recover or are attenuated over the course of time. Furthermore, the observation of lower corticosterone in SIS mice may suggest that previously observed increases in the hippocampal expression of genes associated with cellular stress responses such as CRHR2, FKBP5 and SLC6A4 may emerge as a compensatory response to reduced systemic corticosterone levels. Hypothesis 5 We do not anticipate that these alterations will vary with sex. Contrary to expectations, the current study indicates that there are sex differences in both intra-Hipp and Hipp-mPFC connectivity, but no sex diff in performance of any of the behavioral tasks following SIS or control rearing. Female mice had greater beta coherence in between the vCA1 and mPFC, while males had greater coherence in the low theta band within the CA3-CA1 pathway. Similarly, females demonstrated higher levels of corticosterone, which was unrelated to rearing status. Prior studies suggest that corticosterone levels are higher in females under both basal and stressed conditions, consistent with current findings and the hypothesis that corticosterone response to SIS would not vary by sex 52 . Conclusions and Future Directions The current study, in concert with our previous work, strongly suggest that even subtle alterations in the stability of adolescent mice to establish and retain social networks and hierarchies can lead to structural, molecular, physiological, and cognitive deficits that last well into adulthood. As such, the impact of the COVID pandemic, and concomitant increase in social media use with diminution of interpersonal contact, may continue to impact the current generation of individuals who are already showing signs of poor academic performance. Future studies will focus on mediators of altered PNN, such as microglial proteins, as well as strategies to improve intrahippocampal dynamics and connectivity. Declarations Acknowledgment: This work was supported by a generous gift from the Larian family. References Simmonds, D. J., Hallquist, M. N., Asato, M. & Luna, B. Developmental stages and sex differences of white matter and behavioral development through adolescence: A longitudinal diffusion tensor imaging (DTI) study. NeuroImage 92, 356–368 (2014). Calabro, F. J., Murty, V. P., Jalbrzikowski, M., Tervo-Clemmens, B. & Luna, B. Development of Hippocampal–Prefrontal Cortex Interactions through Adolescence. Cerebral Cortex 30, 1548–1558 (2020). Borsini, A., Giacobbe, J., Mandal, G. & Boldrini, M. Acute and long-term effects of adolescence stress exposure on rodent adult hippocampal neurogenesis, cognition, and behaviour. Mol Psychiatry 28, 4124–4137 (2023). Brunson, K. L. et al. Mechanisms of Late-Onset Cognitive Decline after Early-Life Stress. J Neurosci 25, 9328–9338 (2005). Pöpplau, J. A. et al. Reorganization of adolescent prefrontal cortex circuitry is required for mouse cognitive maturation. Neuron 112, 421–440.e7 (2024). Siegel, S. J. et al. Effects of social deprivation in prepubescent rhesus monkeys: immunohistochemical analysis of the neurofilament protein triplet in the hippocampal formation. Brain Res 619, 299–305 (1993). Brunson, K. L. et al. Mechanisms of Late-Onset Cognitive Decline after Early-Life Stress. J. Neurosci. 25, 9328–9338 (2005). Pechtel, P. & Pizzagalli, D. A. Effects of early life stress on cognitive and affective function: an integrated review of human literature. Psychopharmacology 214, 55–70 (2011). Naninck, E. F. G. et al. Chronic early life stress alters developmental and adult neurogenesis and impairs cognitive function in mice. Hippocampus 25, 309–328 (2015). Yajima, H. et al. Early-life stress induces cognitive disorder in middle-aged mice. Neurobiology of Aging 64, 139–146 (2018). Featherstone, R. E. et al. Early life social instability stress causes lasting cognitive decrement and elevated hippocampal stress-related gene expression. Exp Neurol 354, 114099 (2022). Clark, R. E., Zola, S. M. & Squire, L. R. Impaired Recognition Memory in Rats after Damage to the Hippocampus. J. Neurosci. 20, 8853–8860 (2000). Eichenbaum, H. A cortical–hippocampal system for declarative memory. Nat Rev Neurosci 1, 41–50 (2000). Cohen, S. J. et al. The Rodent Hippocampus Is Essential for Nonspatial Object Memory. Current Biology 23, 1685–1690 (2013). Wang, C. et al. Hippocampus–Prefrontal Coupling Regulates Recognition Memory for Novelty Discrimination. J. Neurosci. 41, 9617–9632 (2021). Insel, N. et al. Reduced Gamma Frequency in the Medial Frontal Cortex of Aged Rats during Behavior and Rest: Implications for Age-Related Behavioral Slowing. J. Neurosci. 32, 16331–16344 (2012). Crown, L. M., Gray, D. T., Schimanski, L. A., Barnes, C. A. & Cowen, S. L. Aged Rats Exhibit Altered Behavior-Induced Oscillatory Activity, Place Cell Firing Rates, and Spatial Information Content in the CA1 Region of the Hippocampus. J. Neurosci. 42, 4505–4516 (2022). He, X. et al. Gating of hippocampal rhythms and memory by synaptic plasticity in inhibitory interneurons. Neuron 0, (2021). Buzsáki, G. & Draguhn, A. Neuronal Oscillations in Cortical Networks. Science 304, 1926–1929 (2004). Buzsáki, G. & Wang, X.-J. Mechanisms of Gamma Oscillations. Annual Review of Neuroscience 35, 203–225 (2012). Lewis, D. A., Curley, A. A., Glausier, J. & Volk, D. W. Cortical Parvalbumin Interneurons and Cognitive Dysfunction in Schizophrenia. Trends Neurosci 35, 57–67 (2012). Spiegel, A. M., Koh, M. T., Vogt, N. M., Rapp, P. R. & Gallagher, M. Hilar interneuron vulnerability distinguishes aged rats with memory impairment. Journal of Comparative Neurology 521, 3508–3523 (2013). Bañuelos, C. et al. Prefrontal Cortical GABAergic Dysfunction Contributes to Age-Related Working Memory Impairment. J. Neurosci. 34, 3457–3466 (2014). Ferguson, B. R. & Gao, W.-J. PV Interneurons: Critical Regulators of E/I Balance for Prefrontal Cortex-Dependent Behavior and Psychiatric Disorders. Front Neural Circuits 12, 37 (2018). Cabungcal, J.-H. et al. Perineuronal nets protect fast-spiking interneurons against oxidative stress. Proceedings of the National Academy of Sciences 110, 9130–9135 (2013). Druga, R., Salaj, M. & Al-Redouan, A. Parvalbumin - Positive Neurons in the Neocortex: A Review. Physiol Res 72, S173–S191 (2023). Fawcett, J. W., Oohashi, T. & Pizzorusso, T. The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function. Nat Rev Neurosci 20, 451–465 (2019). Hijazi, S., Smit, A. B. & van Kesteren, R. E. Fast-spiking parvalbumin-positive interneurons in brain physiology and Alzheimer’s disease. Mol Psychiatry 28, 4954–4967 (2023). Romberg, C. et al. Depletion of Perineuronal Nets Enhances Recognition Memory and Long-Term Depression in the Perirhinal Cortex. J. Neurosci. 33, 7057–7065 (2013). Sultana, R., Brooks, C. B., Shrestha, A., Ogundele, O. M. & Lee, C. C. Perineuronal Nets in the Prefrontal Cortex of a Schizophrenia Mouse Model: Assessment of Neuroanatomical, Electrophysiological, and Behavioral Contributions. International Journal of Molecular Sciences 22, 11140 (2021). Crapser, J. D. et al. Microglia facilitate loss of perineuronal nets in the Alzheimer’s disease brain. EBioMedicine 58, 102919 (2020). Traschütz, A., Kummer, M. P., Schwartz, S. & Heneka, M. T. Variability and temporal dynamics of novel object recognition in aging male C57BL/6 mice. Behav Processes 157, 711–716 (2018). Wen, H. & Liu, Z. Separating Fractal and Oscillatory Components in the Power Spectrum of Neurophysiological Signal. Brain Topogr 29, 13–26 (2016). Donoghue, T. et al. Parameterizing neural power spectra into periodic and aperiodic components. Nature Neuroscience 23, 1655–1665 (2020). Brake, N. et al. A neurophysiological basis for aperiodic EEG and the background spectral trend. Nat Commun 15, 1514 (2024). Buzsáki, G., Logothetis, N. & Singer, W. Scaling Brain Size, Keeping Timing: Evolutionary Preservation of Brain Rhythms. Neuron 80, 751–764 (2013). Voytek, B. et al. Age-Related Changes in 1/f Neural Electrophysiological Noise. Journal of Neuroscience https://doi.org/10.1523/JNEUROSCI.2332-14.2015 (2015) doi:10.1523/JNEUROSCI.2332-14.2015. Thuwal, K., Banerjee, A. & Roy, D. Aperiodic and Periodic Components of Ongoing Oscillatory Brain Dynamics Link Distinct Functional Aspects of Cognition across Adult Lifespan. eNeuro 8, ENEURO.0224-21.2021 (2021). Krystecka, K., Stanczyk, M., Magnuski, M., Szelag, E. & Szymaszek, A. Aperiodic activity differences in individuals with high and low temporal processing efficiency. Brain Research Bulletin 215, 111010 (2024). Bieri, K. W., Bobbitt, K. N. & Colgin, L. L. Slow and Fast Gamma Rhythms Coordinate Different Spatial Coding Modes in Hippocampal Place Cells. Neuron 82, 670–681 (2014). Colgin, L. L. Rhythms of the hippocampal network. Nature Reviews Neuroscience 17, 239–249. Colgin, L. L. et al. Frequency of gamma oscillations routes flow of information in the hippocampus. Nature 462, 353–357 (2009). Maris, E. & Oostenveld, R. Nonparametric statistical testing of EEG- and MEG-data. J Neurosci Methods 164, 177–190 (2007). Jakovljevic, A. et al. The impact of early life maternal deprivation on the perineuronal nets in the prefrontal cortex and hippocampus of young adult rats. Front Cell Dev Biol 10, 982663 (2022). Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9, 671–675 (2012). O’Brien, J., Hayder, H. & Peng, C. Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins. J Vis Exp 54719 (2016) doi: 10.3791/54719 . Cortés-Albornoz, M. C., Ramírez-Guerrero, S., García-Guáqueta, D. P., Vélez-Van-Meerbeke, A. & Talero-Gutiérrez, C. Effects of remote learning during COVID-19 lockdown on children’s learning abilities and school performance: A systematic review. Int J Educ Dev 101, 102835 (2023). Zheng, C., Bieri, K. W., Hwaun, E. & Colgin, L. L. Fast Gamma Rhythms in the Hippocampus Promote Encoding of Novel Object-Place Pairings. eNeuro 3, ENEURO.0001-16.2016 (2016). Tamura, M., Spellman, T. J., Rosen, A. M., Gogos, J. A. & Gordon, J. A. Hippocampal-prefrontal theta-gamma coupling during performance of a spatial working memory task. Nat Commun 8, 2182 (2017). Euston, D. R., Gruber, A. J. & Mcnaughton, B. L. The Role of Medial Prefrontal Cortex in Memory and Decision Making. Neuron 76, 1057–1070 (2013). Fujisawa, S. & Buzsáki, G. A 4 Hz oscillation adaptively synchronizes prefrontal, VTA, and hippocampal activities. Neuron 72, 153–165 (2011). Aoki, M., Shimozuru, M., Kikusui, T., Takeuchi, Y. & Mori, Y. Sex differences in behavioral and corticosterone responses to mild stressors in ICR mice are altered by ovariectomy in peripubertal period. Zoolog Sci 27, 783–789 (2010). Additional Declarations The authors have declared there is NO conflict of interest to disclose Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 07 Apr, 2026 Review # 2 received at journal 03 Apr, 2026 Review # 1 received at journal 26 Mar, 2026 Review # 3 received at journal 20 Mar, 2026 Reviewer # 3 agreed at journal 20 Mar, 2026 Reviewer # 2 agreed at journal 20 Mar, 2026 Reviewer # 1 agreed at journal 20 Mar, 2026 Reviewers invited by journal 19 Mar, 2026 Editor assigned by journal 17 Mar, 2026 Submission checks completed at journal 17 Mar, 2026 First submitted to journal 16 Mar, 2026 Unknown event 16 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9118580","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":609203949,"identity":"9533c76b-20da-4c24-994f-91837748904e","order_by":0,"name":"Steven Siegel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYDADNhDxoAJIHIAIMDbgVQqjE86QogUMEtuI0CI/v/mYxMcdDPZ87O0XHyTO2ybHd4DH+MMPBhvZDQewazE4xpYmOfMM0HyeM8UGidtuG0se4DGT7GFIM8aphY3H2Ji3jSGBTSInTQKoJXEDUAszA8PhRFxa5Nv4Pxv/bWOwZ5N/k/4jcc7teqAW488MDP9xamE4xsP4mLGNgbFNgv0YQ2LD7QSDAzwG0sBAwKnF4Fia4cPeNgmgX3KYJRKO3TaceZitTLLHINl4Ji6HNR9+cOBnm429fPvxhx8+1NyW5zvevPnDjwo72T5cDoMACSDmMYCwmcG241UOA+wPiFI2CkbBKBgFIw8AAEjlXhNNqipRAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8058-9713","institution":"University of Southern California","correspondingAuthor":true,"prefix":"","firstName":"Steven","middleName":"","lastName":"Siegel","suffix":""},{"id":609203950,"identity":"49f773f5-5a3d-4b32-b7e1-2a85a02309f5","order_by":1,"name":"Lindsey Crown","email":"","orcid":"","institution":"University of Southern California","correspondingAuthor":false,"prefix":"","firstName":"Lindsey","middleName":"","lastName":"Crown","suffix":""},{"id":609203951,"identity":"4d1930e2-bc09-4c32-9961-49cac116150e","order_by":2,"name":"Krishna Parkeh","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Krishna","middleName":"","lastName":"Parkeh","suffix":""},{"id":609203952,"identity":"872301e2-7da6-4b91-bb59-2df2cc138b83","order_by":3,"name":"Daniel Gray","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Gray","suffix":""},{"id":609203953,"identity":"c806df0d-7717-44ad-8ab9-6da8826286f1","order_by":4,"name":"Samuel Guillemette","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Guillemette","suffix":""},{"id":609203954,"identity":"87f1c3d0-cfe3-4920-9ae3-7a36113038e1","order_by":5,"name":"Anastasiya Demenko","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Anastasiya","middleName":"","lastName":"Demenko","suffix":""},{"id":609203955,"identity":"cfedd6da-1ddc-4f93-86f8-4ccfe969a41e","order_by":6,"name":"Madeline Kim","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Madeline","middleName":"","lastName":"Kim","suffix":""},{"id":609203956,"identity":"2d6ed5bf-68ff-4246-8990-6bb30b6ee0b0","order_by":7,"name":"Joseph Pincini","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Pincini","suffix":""},{"id":609203957,"identity":"77e12ab3-a0b5-4c28-a767-f6b259610822","order_by":8,"name":"Kaili Ogi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kaili","middleName":"","lastName":"Ogi","suffix":""},{"id":609203958,"identity":"eb376493-a907-47d1-8e05-f28ed06e9834","order_by":9,"name":"Robert Featherstone","email":"","orcid":"","institution":"Keck school of medicine","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Featherstone","suffix":""},{"id":609203959,"identity":"6916abfc-fee6-458e-b134-87d3fe8ccde6","order_by":10,"name":"Linsay Biase","email":"","orcid":"","institution":"UCLA, USA","correspondingAuthor":false,"prefix":"","firstName":"Linsay","middleName":"","lastName":"Biase","suffix":""}],"badges":[],"createdAt":"2026-03-14 00:00:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9118580/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9118580/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105309418,"identity":"c4147d5b-88b6-43d6-b68d-034656f1bebd","added_by":"auto","created_at":"2026-03-24 15:07:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":591960,"visible":true,"origin":"","legend":"\u003cp\u003eSIS procedure. A) Mice were socially-housed with a new group of conspecifics twice a week for 7 weeks. B) Timeline of SIS experiment, Following SIS, a group of mice were scarified for immunohistochemistry. The remaining mice were used for LFP recordings and behavioral assessment.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9118580/v1/86c2d5d35bee73af2ef1835b.png"},{"id":105309437,"identity":"3d870921-209b-42ba-8980-3c7ee22c47ed","added_by":"auto","created_at":"2026-03-24 15:07:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1150510,"visible":true,"origin":"","legend":"\u003cp\u003eSIS animals showed NOR deficits but no differences from controls in the EPM or SI. A) Novel object recognition. SIS animals showed less preference for the novel object than control mice (F(1,22) = 6.518, p = 0.018) B) SIS and control mice showed a similar preference for the open arm of the elevated plus maze (F(1,31) = 0.004, p = 0.934). C) SIS and control mice showed a similar preference for the chamber containing the conspecific F(1,25) = 0.492), p = 0.490).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9118580/v1/9e38f2e07651f044aed66470.png"},{"id":105309415,"identity":"9ebd25be-389e-4021-b871-ab142aaa68aa","added_by":"auto","created_at":"2026-03-24 15:07:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4220145,"visible":true,"origin":"","legend":"\u003cp\u003eNo region-specific electrophysiological deficits were observed. A) mice were implanted with 4 LFP electrodes all referenced to a cerebellar skull screw. B) Histological verification of dorsal CA1 LFP electrode. C) Histological verification of dorsal CA3 electrode. D) Histological verification of mPFC electrode. E) Histological verification of ventral CA1 electrode. F) Power spectrum during recall phase of NOR for SIS and control animals. G) Power spectrum of CA3 electrode during recall phase of NOR. H) Power spectrum of MPFC electrode during recall phase of NOR for SIS and control animals. I) Power spectrum of vCA1 electrode during recall phase of NOR for SIS and control animals. J) The aperiodic component (1/fx) was not different between SIS and control animals for any region (dCA1 p=0.07; dCA3 p= 0.26, mPFC p= 0.43, vCA2 p = 0.69) and did not differ by sex.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9118580/v1/5fc090b5691280dc37726353.png"},{"id":105309413,"identity":"6eaf6c20-54c9-4e60-a291-396d0e8b6867","added_by":"auto","created_at":"2026-03-24 15:07:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6581486,"visible":true,"origin":"","legend":"\u003cp\u003eSIS animals had reduced high-gamma coherence relative to control animals. A) A coherogram from a representative mouse shows coherence during NOR between dCA3 and dCA1. B) A coherogram between the vCA1 and mPFC during the same task in the same mouse. C) Overall coherence between CA3 and dCA1 when the animal was around the novel object was significantly different above 85Hz (p=0.02). D) Coherence between the vCA1 and mPFC when the animal is near the novel object was not different. E) Overall coherence between CA3 and dCA1 when the mouse is near the familiar object was not different. F) Coherence between the vCA1 and mPFC with the animal is near the familiar object was also not different. G) dCA3 and dCA1 coherence in males around the novel object was greater in the delta/low theta range (3-6Hz) than for female mice (p=0.016). H) There were no sex differences for vCA1-mPFC coherence when the animal was near the novel object. I) There were no sex differences in intra-hippocampal coherence around the familiar object. J) Delta and beta coherence was greater for female mice than male mice when animals were around the familiar object (1-4Hz, p=0.002; 20-24Hz, p=0.006).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9118580/v1/58458a61cbf459c8c7ab9a07.png"},{"id":105309411,"identity":"e4c864ed-d4ac-4b3b-ad31-09d59937a271","added_by":"auto","created_at":"2026-03-24 15:07:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2761694,"visible":true,"origin":"","legend":"\u003cp\u003eSIS animals had less PV cells surrounded by the perineuronal net in the mPFC. A) Slices were stained for parvalbumin. B) Slices were stained for WFA to image the perineuronal net. C) Merging the two stains reveals the PV cells surrounded by the perineuronal net. D) No differences were observed between SIS and control mice in total PV cells (dCA1: p= 0.59, dCA3 p=0.36, mPFC: p= 0.40). E) There were less PV cells with nets in the mPFC of SIS animals (dCA1: p= 0.43, dCA3: p= 0.26, mPFC=0.004, corrected for multiple comparisons). F) Normalizing this by the total amount of PV cells also shows that SIS animals had reduced PV cells surrounded by the perineuronal net in the mPFC (dCA1: p= 0.43, dCA3: p= 0.26, mPFC=0.005, corrected for multiple comparisons). G) Females had greater serum corticosterone than males (F(1,66) = 27.13, p\u0026lt; 0.0001); SIS animals had less corticosterone than controls (F(1,66) = 7.577, p = 0.0076).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9118580/v1/16ce1fc1801f62ebd0146dbe.png"},{"id":105565061,"identity":"deb1917d-7214-4614-a0c8-113ecf7f2019","added_by":"auto","created_at":"2026-03-27 12:51:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18173314,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9118580/v1/3e97b05b-19cb-4803-8b27-139fe5a00a72.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Social Instability in Adolescent Mice Leads to Lasting Cognitive Deficits with Reduction of Intra-Hippocampal Functional Connectivity and Parvalbumin-Containing Interneurons with Perineuronal Nets.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCognitive deficits that arise in adulthood emerge from complex interactions between a person\u0026rsquo;s unique genetic predispositions and life experience. Exposure to early life stress (ELS) can dramatically alter an individual\u0026rsquo;s life trajectory depending on the nature of the stressor and the developmental phase in which it is encountered (i.e. infancy vs. childhood vs. adolescence). Over the past decade there has been a disproportionate increase in adolescent ELS due to a number of cultural factors including the emergence of social media and increased accessibility of information. Adolescence is a particularly critical time for the development of numerous aspects of cognition including executive function, cognitive control, and social development. This is due in large part to the protracted maturation of prefrontal cortex connectivity with the hippocampus and other brain structures critical to cognition \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, as well as the relatively late development of inhibitory neural circuits across the forebrain that mediate information processing within these circuits. These developmental reorganizations result in drastic changes in network-level activity patterns that mediate executive control and memory function during adolescence \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies in primates demonstrate structural changes to hippocampal cytoskeletal proteins and dentate gyrus morphology following severe social deprivation \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Studies in rodents have shown that, like in humans, exposure to ELS can lead to later-life cognitive impairment \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, most of these studies introduce stressors at very early postnatal time points using models of maternal separation, limited bedding and nesting, and early immune activation. In a previous study from our group, we demonstrated that mice exposed to adolescent social instability stress (SIS), where animals are unable to form social hierarchies due to a constantly changing social environment, exhibit cognitive impairment at 30 weeks of age (~\u0026thinsp;7 months; Featherstone et al., 2022). Critically, the observed cognitive deficits were domain specific, with adult SIS-exposed mice exhibiting impairment in novel object recognition (NOR) memory in the absence of measurable social withdrawal, anxiety-like or depressive phenotypes. Moreover, hippocampal expression of genes with roles in regulating cellular stress responses, were elevated in SIS-exposed mice at 66 weeks of age (~\u0026thinsp;15 months) and correlated with poorer recognition memory \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Together, these observations indicate that ecologically-relevant social stress experienced in adolescence can cause persistent decrements in memory that may be mediated by moderators of the stress cascade.\u003c/p\u003e \u003cp\u003eIt has long been understood that NOR memory relies on activity within medial temporal lobe structures, primarily the hippocampus and perirhinal cortex \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. More recent work has shown that oscillatory coupling between the hippocampus and medial prefrontal cortex (mPFC) occurs during recognition memory retrieval for novelty discrimination, and that optogenetic silencing of hippocampal inputs to the mPFC results in NOR performance deficits\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Oscillatory dynamics within the hippocampus and mPFC have also been shown to change across the adult lifespan in ways that impact memory function in rodents \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, the extent to which exposure to early life adversity alters oscillatory activity in the adult hippocampus and mPFC has not been well studied.\u003c/p\u003e \u003cp\u003eOscillatory dynamics across the brain are potently regulated by inhibitory neuron activity \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Dysfunction of inhibitory interneurons in the hippocampus and mPFC has been linked to declining memory function in the context of psychiatric disorders, normative aging, and in neurodegenerative disease conditions \u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The maturation of a specialized extracellular matrix structures called the perineuronal net (PNN) is critical in the development of inhibitory dynamics. In cortex and the hippocampus, the PNN preferentially accumulates around fast-spiking parvalbumin(PV)-expressing interneurons \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, which form perisomatic synaptic contacts with excitatory neurons, allowing them to potently regulate neuronal firing patterns, oscillatory activity, and inter-regional communication \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Thus, perturbations in the abundance and composition of the PNN could have profound impacts on memory-circuit function and cognition\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. While the mechanisms that regulate PNN assembly and remodeling are just beginning to be elucidated, microglia, the primary immune-like cells of the central nervous system, are thought to be critical in this process. Microglia synthesize and secrete numerous degradative enzymes that can modulate PNN composition, and pharmacological depletion of microglia results in profound increases in proteoglycan deposition across the cerebral cortex \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present study combines adolescent SIS stress with assessments of adult electrophysiological and behavioral function, as well as a subset of their proposed relationships to morphologic and molecular mechanisms of persistent consequences of early life experience. In doing so, we test the following five overarching hypotheses:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAdolescent SIS leads to persistent impairment of episodic memory as evidenced by decreased NOR. These impairments will not be accompanied by increased anxiety or social withdrawal.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMemory impairments in adulthood are accompanied by a reduction of hippocampal-PFC functional connectivity as assessed by interregional coherence during performance of NOR.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eElectrophysiological changes in hippocampal-PFC connectivity will be associated with concomitant changes to inhibitory networks, measured by PV expression and perineuronal net integrity.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAdolescent SIS will lead to persistent changes in stress hormones in adult animals.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eWe do not anticipate that these alterations will vary with sex.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSubjects were C57BL/6J (B6) male and female mice obtained at 3 weeks of age from Jackson Laboratories. A total of 144 mice were used in the experiment, half as control animals that did not change cage mates, and half that underwent SIS. At postnatal day 27, all animals were microchipped (Bio Medic Data Systems). Animals were housed in a 12-hour light/dark cycle and were given food ad libitum. All behavioral tests and surgeries were conducted during the light cycle.\u003c/p\u003e \u003cp\u003eAll experiments were performed following the guidelines of the United States National Institutes of Health \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e using protocols approved by the University of Southern California Institutional Animal Care and Use Committee.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSocial Instability Stress\u003c/h3\u003e\n\u003cp\u003eMice were housed in same-sex groups of 4. Beginning at postnatal day 28 all mice were placed in clean cages twice per week for 7 weeks. SIS mice were placed in cages with new, randomly-assigned cage-mates at each cage change\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Control animals remained with the same group of animals for the duration of the experiment but received the same amount of handling and cage changes as SIS animals. At the end of the SIS period, a subgroup of animals were immediately sacrificed and used for immunohistochemistry \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eBehavior\u003c/h3\u003e\n\u003cp\u003e \u003cstrong\u003eBehavioral conditions and handling\u003c/strong\u003e \u003cp\u003ePrior to behavior, all animals were gently handled for ~\u0026thinsp;5 minutes and acclimated to the experimental room and electrophysiological recording system to minimize the animal stress during recordings and encourage movement and engagement during behavioral assays.\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eNovel Object Recognition\u003c/h3\u003e\n\u003cp\u003eNovel object recognition (NOR) took place in a box (30.5 x 40.6 x 40.6 cm\u003csup\u003e3\u003c/sup\u003e) over two days in a low-light room. On the first day, mice were allowed to habituate to the empty box for 5 minutes. Mice were then removed and placed back into their cages for 10 minutes. During this time, the box was cleaned with 70% EtOH and two identical objects (lego towers or glass flasks) were placed into the box, diagonally from each other. On the second day the process of habituation and training was repeated followed by the testing phase 10 minutes later. The mice were placed back into the box with one familiar object from the training and a novel object. Mice were allowed to explore for 5 minutes before being removed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eElectrophysiological recordings took place during NOR. Behavior was recorded via an overhead video camera and scored by hand by an individual that was blind to mouse condition (SIS or control). NOR discrimination index was calculated as: (time with novel object)/(time with familiar object\u0026thinsp;+\u0026thinsp;novel object). Only the first 10 seconds of interaction was scored because this period has been previously indicated to best reflect true object exploration as opposed to general locomotor activity \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. If a mouse failed to reach the 10 seconds threshold, its data was excluded from analysis.\u003c/p\u003e\n\u003ch3\u003eElevated Plus Maze\u003c/h3\u003e\n\u003cp\u003eElevated plus maze (EPM) was conducted in a brightly lit room in an elevated plus maze apparatus. The apparatus has 2 closed arms covered by walls (closed arms 30.5 cm x 6.4 cm\u003csup\u003e2\u003c/sup\u003e; walls: 15.2 cm in height) and 2 open arms (30.5 x 5.1 cm\u003csup\u003e2\u003c/sup\u003e) and was elevated above the ground at 18 inches. The animal was placed in the center of the maze and allowed to explore all parts of the maze for 5 minutes. The anxiety levels were measured as (time in the open arms)/(time in the closed arms and open). Videos were recorded and scored using Topscan Lite (Clever Sys Inc., Reston, VA, USA) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSocial Interaction\u003c/h2\u003e \u003cp\u003eSocial interaction was assessed using a three-chambered box (53 x 27 x 23cm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e). A perforated cylinder was placed at both ends of the box. Subject mice were allowed to explore the box for 5 minutes. Mice were then removed and placed back into cages. A juvenile mouse (3\u0026ndash;4 weeks of age) of the same sex (stimulus mouse) was then placed under one cylinder and a ping-pong ball was placed under the other. Subject mice were then placed back into the box and given 5 minutes to explore. The cylinder allowed subject mice to initiate social interaction and the perforation allowed mice to both see and smell the stimulus mice. Stimulus mice were confined to the cylinder and were thus not able to initiate social interaction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSocial interaction scores were measured as a ratio of (time interacting with stimulus mouse)/(time interacting with both stimulus mouse and ping-pong ball). Testing was recorded and scored using Topscan Lite (Clever Sys Inc., Reston, VA, USA). Interaction with either the mouse or ball was defined as the subject mouse being next to the cylinder, facing the cylinder, and with its nose oriented towards the cylinder. 8 mice were excluded from analysis due to problems occurring during data acquisition.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eElectrophysiological Recordings\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSurgery and Electrophysiological recordings\u003c/h2\u003e \u003cp\u003eAnimals were anesthetized with 3% isoflurane mixed with O2 carrier gas at a flow rate of .75 L/min. Once anesthetized, animals were placed on a stereotactic frame (David Kopf, Tujunga, CA, USA) and maintained under isoflurane anesthesia between 1\u0026ndash;2%. A midline incision was made to expose the skull and burr holes were made over the left mPFC (AP: 1.7, ML: 0.5, DV: \u0026minus;\u0026thinsp;2.5), left ventral hippocampus (AP: -3.16, ML: -3.0, DV: -4.0), right dorsal CA1 (AP: -2.44, ML: 2.25, DV: -1.55) and right dorsal CA3 (AP: -1.7, ML: -2.1, DV: \u0026minus;\u0026thinsp;2.0) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). 4 additional holes were made for skull screws to serve as anchors. Electrodes were lowered and glue was applied to fix them to the skull. Electrodes were connected to the Neuralynx EIB-16-QC (Neuralynx, Boseman, MT, USA) electronic interface board (EIB) via stainless steel wire. A skull screw was placed over the cerebellum and served as ground and reference. Dental acrylic was used to secure the electrodes and EIB to the skull. Animals were given 1.5 weeks to recover before behavioral testing and recording began. Data were acquired using the NeuraLynx Digital Lynx SX and HS-16QC-LED headstage and behavior was tracked using the Cheetah video tracking system and an overhead camera (Neuralynx, Boseman, MT, USA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis\u003c/h2\u003e \u003cp\u003eElectrophysiological data were analyzed from 37 mice (11 control male, 7 control female, 10 SIS male, and 9 SIS female). Analysis of time series data was performed in MATLAB (R2022a, MathWorks) using custom scripts. Electrodes that were not determined to be in the correct location or that were too noisy were excluded from analysis (n\u0026thinsp;=\u0026thinsp;8); a noise threshold was set based on visual inspection of the recording data from each electrode for each day for each animal.\u003c/p\u003e \u003cp\u003eThis was performed blind to animal identity. As a result, the sample size for electrophysiological measures varies by task and measure because of the need to exclude certain electrodes. To examine the extent to which changes to intra-hippocampal- or hippocampal-prefrontal network activity underlie the observed NOR memory impairment in adult mice exposed to adolescent SIS, local field potentials were simultaneously recorded from dCA3, dCA1, vCA1, and mPFC in control and SIS-exposed mice (electrode locations, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E).\u003c/p\u003e \u003cp\u003eBecause increasing attention is being paid to the importance of distinguishing between true oscillatory (periodic) activity and general increases in aperiodic power spectral density when parameterizing neural power spectra \u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, we used the FOOOF algorithm (Fitting Oscillations \u0026amp; One-Over F) to specifically quantify this aperiodic component \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The aperiodic component measures the slope of the power spectrum and is most simply defined as the 1/f\u003csup\u003ex\u003c/sup\u003e, where x is the aperiodic exponent. The aperiodic component is thought to reflect the balance between excitatory and inhibitory signaling \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. We hypothesized that SIS would alter inhibitory networks and therefore, would change the E-I balance within the hippocampus or mPFC \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-I\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eNovel object recognition has been associated with two distinct memory-related pathways, the intra-hippocampal pathway (dCA3 to dCA1) and the hippocampal-prefrontal pathway (vCA1- mPFC). Within the intra-hippocampal pathway (dCA3-dCA1) it has been proposed that this is primarily mediated by gamma frequency oscillations \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Alternatively, within the vCA1-mPFC pathways, lower frequency rhythms, particularly within the theta band, are associated with recognition memory (Wang et al., 2021). Therefore, we recorded local field potential activity during the recall phase of the NOR test and compared activity when animals were near the novel versus the familiar object. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Analysis was performed using Monte Carlo cluster-based permutation testing that allowed significantly modulated frequencies to be identified regardless of canonical frequency bands \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCorticosterone ELISA\u003c/h2\u003e \u003cp\u003eA previous study from our group demonstrated that exposure to adolescent SIS results in elevated mRNA expression of genes critical for the cellular stress response, including FK506 binding protein 5 (FKBP5) and Corticotropin Releasing Hormone Receptor 2 (CRHR2), that correlates with recognition memory deficits \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. To determine how these previous findings align with the abundance of systemic corticosterone, we used a competitive ELISA assay to measure corticosterone levels in SIS and control mice immediately following the termination of the adolescent SIS manipulation. Prior to sacrificing, animals were acclimated to a low light room for 30 minutes. Animals were sacrificed and cardiac punctures were administered before perfusions between 9:30 am and 11 am to control for circadian effects on corticosterone levels. Blood was collected, left at room temperature for 10 minutes and then centrifuged. Serum was collected and stored at -80 degrees until testing. Corticosterone levels were measured via competitive ELISA using corticosterone parameter assay kits (Catalog #: KGE009, R\u0026amp;D Systems, Minneapolis, MN, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003ePrevious reports have shown that certain types of ELS prolong the developmental timeframe of the PNN into adulthood, mimicking phenotypes observed in mouse models of schizophrenia \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. To assess whether exposure to adolescent SIS also changes the development of the PNN, brain sections from SIS-exposed and control mice were immunohistochemically labelled with antibodies directed against parvalbumin (PV; inhibitory neurons) and the \u003cem\u003ewisteria floribunda agglutinin\u003c/em\u003e (WFA; perineuronal net) lectin. These sections were imaged with confocal microscopy, and the number of PV cells, number of PNNs, and the proportion of PV cells surrounded by the PNN were quantified \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMice were anesthetized with isoflurane and perfused intracardially with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were incubated in PFA for ~\u0026thinsp;24 hours and then stored in PBS in a 4-degree refrigerator. Brains were sliced at 60\u0026micro;m on a vibratome (Leica VT1200, Wetzlar, Germany).\u003c/p\u003e \u003cp\u003ePrior to immunohistochemistry slices were placed in blocking/permeability solution of 5% normal donkey serum (NDS) (MilliporeSigma, Burlington, MA, USA) and 0.3% Triton-X in PBS for 2 hours on a shaker. Slices were then incubated in a primary antibody solution containing a 1:1000 concentration of rabbit anti parvalbumin (PV 27, Swant, Switzerland) and 1:250 concentration of anti-wisteria floribunda agglutinin (WFA) (B-1355-2, Vector Laboratories, Newark, CA, USA), in bocking/permeability solution. Slices were then left to incubate overnight on a shaker at 4 degrees.\u003c/p\u003e \u003cp\u003eThe next day slices were washed with PBS 4 times for 10 minutes per wash on shaker. They were then placed in a secondary antibody solution containing 1:500 DyLight488 (ImmunoReagents Inc, Raleigh, NC, USA) to label PV in green and 1:500 Alex Fluor 647 (JacksonImmuno Research, West Grove, PA, USA) to label WFA in far-red. Tissue was left to incubate on a shaker for 2 hours before being washed again 4 times for 10 minutes each wash on a shaker. Tissue was then mounted on slides, cover-slipped with permount and sealed with nail polish.\u003c/p\u003e \u003cp\u003eTo quantify the number of PV+ cells, PNNs, and PV+ cells with PNNs, numbers were manually counted by an experimenter blinded to animal condition. PV+ cells, PNNs and PNNs around PV cells were counted using the Cell Counter plug in for Image J \u003csup\u003e45,46\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eAdolescent SIS results in sustained recognition memory impairments in adulthood but no changes to social interaction or anxiety-like behavior.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eOur group previously demonstrated that exposing adolescent mice to SIS results in adult deficits in novel object recognition memory, but no change in social interactions or anxiety-like behavior \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In the current study, adult mice exposed to adolescent SIS- exhibited a lower discrimination index on the NOR task relative to controls (F(1,22)\u0026thinsp;=\u0026thinsp;6.518, p\u0026thinsp;=\u0026thinsp;0.018; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Adult SIS-exposed mice were not different from controls on an elevated plus maze test of anxiety-like behavior (F(1,31)\u0026thinsp;=\u0026thinsp;0.004, p\u0026thinsp;=\u0026thinsp;0.934; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) or on a test of social preference (F(1,25)\u0026thinsp;=\u0026thinsp;0.492), p\u0026thinsp;=\u0026thinsp;0.490; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). No effects of biological sex were observed in any behavioral test (NOR: F(1,22)\u0026thinsp;=\u0026thinsp;6.518, p\u0026thinsp;=\u0026thinsp;0.873; EPM: F(1,31)\u0026thinsp;=\u0026thinsp;0.007, p\u0026thinsp;=\u0026thinsp;0.951; Social Interaction: F(1,31)\u0026thinsp;=\u0026thinsp;0.007, p\u0026thinsp;=\u0026thinsp;0.951). These observations replicate previous findings suggesting that adolescent SIS results in sustained, domain-specific cognitive impairments that implicate the hippocampus and mPFC.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalicUnderline\" class=\"BoldItalicUnderline\" name=\"Emphasis\"\u003eAdolescent SIS did not alter the 1/f component of the power spectral density.\u003c/span\u003e \u003c/p\u003e \u003cp\u003eFOOOF exponents were not significantly difference between SIS and control mice (dCA1 p\u0026thinsp;=\u0026thinsp;0.07; dCA3 p\u0026thinsp;=\u0026thinsp;0.26, mPFC p\u0026thinsp;=\u0026thinsp;0.43, vCA2 p\u0026thinsp;=\u0026thinsp;0.69 after Bonferroni-Holm correction for multiple comparisons; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-J).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdolescent SIS reduces intra-hippocampal gamma coherence when animals are near the novel object.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe found that within the intra-hippocampal pathways, control mice had greater high gamma coherence around the novel, but not familiar object (85-90Hz; p\u0026thinsp;\u0026lt;\u0026thinsp;0.02; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,E); while there were no significant differences in coherence between vCA1 and the mPFC \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD,F). Within the intra-hippocampal pathway, males had greater coherence between 3-6Hz around the novel object (p\u0026thinsp;=\u0026thinsp;0.016), with no differences in the vCA1-mPFC pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG,H). Around the familiar object, females had greater coherence in the delta (1-4Hz, p\u0026thinsp;=\u0026thinsp;0.002) and beta range (20-24Hz, p\u0026thinsp;=\u0026thinsp;0.006) within the vCA1-mPFC pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI,J) and no differences in intra-hippocampal coherence relative to males.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAdolescent SIS reduces PNN accumulation around inhibitory neurons in the mPFC\u003c/h2\u003e \u003cp\u003eExposure to adolescent SIS did not change PV neuron numbers in the hippocampus or mPFC (dCA1: p\u0026thinsp;=\u0026thinsp;0.59, dCA3 p\u0026thinsp;=\u0026thinsp;0.36, mPFC: p\u0026thinsp;=\u0026thinsp;0.40); Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). However, SIS mice did exhibit a decreased fraction of PV cells surrounded by the PNN (WFA). (p\u0026thinsp;=\u0026thinsp;0.004, corrected for multiple comparisons; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), but not in the hippocampus (dCA1: p\u0026thinsp;=\u0026thinsp;0.43, dCA3: p\u0026thinsp;=\u0026thinsp;0.26, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). This pattern within the mFPC was made more significant when controlling for the number of PV cells (p\u0026thinsp;=\u0026thinsp;0.005, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Together, these histological observations indicate that adolescent SIS results in a reduction of extracellular matrix surrounding inhibitory neurons, specifically in the mPFC. Sample sizes were to low to evaluate these effects by sex.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAdolescent SIS reduces systemic corticosterone\u003c/h2\u003e \u003cp\u003eRelative to control mice, SIS-exposed mice had significantly lower levels of corticosterone (F(1,66)\u0026thinsp;=\u0026thinsp;7.577, p\u0026thinsp;=\u0026thinsp;0.0076; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Moreover, female control and SIS mice had significantly higher systemic corticosterone levels compared to males (F(1,66)\u0026thinsp;=\u0026thinsp;27.13, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), although no stress-by-sex interaction effects were observed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cstrong\u003eOverarching Summary\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eThe current study demonstrates that subtle changes in the ability to establish stable social bonds during adolescence can lead to lasting functional and physiological changes into adulthood. Specifically, mice which were reared using a social instability protocol replicated previous findings of a decreased episodic memory using the novel object recognition task \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Further, we now show that these cognitive deficits are accompanied by both physiological and anatomical changes, consistent with known structures and cellular elements that are critical for recognition and recall. These changes include a reduction in intra-hippocampal high-frequency gamma-band coherence between CA1 and CA3 in proximity to the novel object, as well as a reduction in perineuronal nets adjacent to parvalbumin-positive interneurons in mPFC. Taken together, data indicate that disruptions of normal, stable social networks during adolescence have the capacity to negatively impact formation of perineuronal nets around parvalbumin positive neurons, with concomitant changes in high frequency oscillations during a cognitive load, leaving lasting deficits. As such, the impact of recent trends (e.g. social media) and events (e.g. COVID pandemic) that reduce the formation of lasting social networks in adolescents may have negative implications for affected populations well into their adult lives.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHypothesis 1\u003c/strong\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eAdolescent SIS leads to persistent impairment of episodic memory as evidenced by decreased NOR.\u003c/b\u003e These impairments will not be accompanied by increased anxiety or social withdrawal. This hypothesis was supported, suggesting that prior findings of impaired cognitive performance in the absence of either an anxiety phenotype or social deficit is both reliable and replicable. Recent data showing reduced proficiency in school performance among children who were in secondary school during the pandemic suggests that these findings may also be generalizable to humans who had a disruption in their ability to interact with other children during critical developmental periods \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Additionally, data highlight the specificity of the effect on recognition and recall, such that reduced episodic memory cannot be explained by either a generalized increase in anxiety or decreased motivation for interactions with the environment. Although counter-intuitive, mice reared with constant change in their social hierarchy did not have either increased anxiety or decreased interest in other mice when given the opportunity to avoid open arms and other mice respectively.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHypothesis 2\u003c/strong\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eMemory impairments in adulthood are accompanied by a reduction of hippocampal-PFC functional connectivity as assessed by interregional coherence during performance of NOR.\u003c/b\u003e The shape of the power spectral density within each region was not altered in SIS animals relative to controls. These data indicate that SIS did not alter the ability of specific cell types to the extent that the overall distribution of frequencies generated was altered. Specifically, each region in isolation (dCA1, v CA1, vCA3 and mPFC) has the ability to form the full spectrum of electrical activity. However, memory impairments in adult SIS mice were accompanied by an increase of high gamma coherence between CA1 and CA3 during performance of NOR. Previous studies indicate that high gamma CA3–CA1 synchrony significantly increased when rats explored a novel object or novel location compared to familiar object or place presentation \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Thus, the current finding is consistent with the idea that SIS mice are experiencing the NOR task as both a novel objects and locations despite having seen them previously. Alternatively, there was no difference between SIS and control mice for Hipp-mPFC coherence. Previous data suggest that increased task difficulty or complexity triggers higher mPFC activity and potentially increased coherence to maintain attention and memory performance \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Thus, the current study may have employed a task that was sufficiently easy that mPFC-hippocampal coherence was not altered. The fact that the SIS mice performed less well than the controls argue against this interpretation. Additionally, prior studies indicate that interactions between mPFC and CA1 intensify remote memory recall, where prefrontal theta oscillations modulate CA1 gamma power and unit spiking to enhance contextual representations \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. It is possible that the NOR task as performed relied on more recent memory and therefore did not engage or rely upon mPFC-hippocampal modulation the extent it engaged intra-hippocampal circuits. In such a case, one would expect changes within the hippocampus, but not necessarily between mPFC and hippocampus.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHypothesis 3\u003c/strong\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eElectrophysiological changes in hippocampal-PFC connectivity will be associated with concomitant changes to inhibitory networks, measured by PV expression and perineuronal net integrity\u003c/b\u003e. Contrary to our hypothesis, there was no change in PV-interneuron counts. However, PNN density was reduced in the mPFC of SIS animals, supporting the idea that SIS-induced stress in adolescent mice may be affecting adult excitability by altering the proteoglycan deposition and pruning of PV-positive cells. One limitation for this interpretation stems from the observation that gamma coherence was altered within hippocampus, while changes in PV interneuron-associated PNNs occurred in mPFC. However, it remains possible that changes measured in mPFC reflect a larger process, and that the high degree of connectivity between various structures leads to a complex interplay such that structural changes in one region may be manifest (detected) as functional changes in another.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHypothesis 4\u003c/strong\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eAdolescent SIS will lead to persistent changes in stress hormones in adult animals. SIS animals lower basal corticosterone levels than controls\u003c/b\u003e. The current study indicates that SIS mice had significantly lower levels of the stress hormone corticosterone when tested immediately after the SIS period. This differs from our prior study in which no differences were found for 1 year following cessation of the SIS protocol. As such, data indicate early alterations in modulation of the stress cascade either recover or are attenuated over the course of time. Furthermore, the observation of lower corticosterone in SIS mice may suggest that previously observed increases in the hippocampal expression of genes associated with cellular stress responses such as CRHR2, FKBP5 and SLC6A4 may emerge as a compensatory response to reduced systemic corticosterone levels.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHypothesis 5\u003c/strong\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eWe do not anticipate that these alterations will vary with sex.\u003c/b\u003e Contrary to expectations, the current study indicates that there are sex differences in both intra-Hipp and Hipp-mPFC connectivity, but no sex diff in performance of any of the behavioral tasks following SIS or control rearing. Female mice had greater beta coherence in between the vCA1 and mPFC, while males had greater coherence in the low theta band within the CA3-CA1 pathway. Similarly, females demonstrated higher levels of corticosterone, which was unrelated to rearing status. Prior studies suggest that corticosterone levels are higher in females under both basal and stressed conditions, consistent with current findings and the hypothesis that corticosterone response to SIS would not vary by sex \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e"},{"header":"Conclusions and Future Directions","content":"\u003cp\u003eThe current study, in concert with our previous work, strongly suggest that even subtle alterations in the stability of adolescent mice to establish and retain social networks and hierarchies can lead to structural, molecular, physiological, and cognitive deficits that last well into adulthood. As such, the impact of the COVID pandemic, and concomitant increase in social media use with diminution of interpersonal contact, may continue to impact the current generation of individuals who are already showing signs of poor academic performance. Future studies will focus on mediators of altered PNN, such as microglial proteins, as well as strategies to improve intrahippocampal dynamics and connectivity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgment:\u003c/h2\u003e \u003cp\u003eThis work was supported by a generous gift from the Larian family.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSimmonds, D. J., Hallquist, M. N., Asato, M. \u0026amp; Luna, B. Developmental stages and sex differences of white matter and behavioral development through adolescence: A longitudinal diffusion tensor imaging (DTI) study. \u003cem\u003eNeuroImage\u003c/em\u003e 92, 356\u0026ndash;368 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalabro, F. J., Murty, V. P., Jalbrzikowski, M., Tervo-Clemmens, B. \u0026amp; Luna, B. Development of Hippocampal\u0026ndash;Prefrontal Cortex Interactions through Adolescence. \u003cem\u003eCerebral Cortex\u003c/em\u003e 30, 1548\u0026ndash;1558 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorsini, A., Giacobbe, J., Mandal, G. \u0026amp; Boldrini, M. Acute and long-term effects of adolescence stress exposure on rodent adult hippocampal neurogenesis, cognition, and behaviour. \u003cem\u003eMol Psychiatry\u003c/em\u003e 28, 4124\u0026ndash;4137 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrunson, K. L. \u003cem\u003eet al.\u003c/em\u003e Mechanisms of Late-Onset Cognitive Decline after Early-Life Stress. \u003cem\u003eJ Neurosci\u003c/em\u003e 25, 9328\u0026ndash;9338 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026ouml;pplau, J. A. \u003cem\u003eet al.\u003c/em\u003e Reorganization of adolescent prefrontal cortex circuitry is required for mouse cognitive maturation. \u003cem\u003eNeuron\u003c/em\u003e 112, 421\u0026ndash;440.e7 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegel, S. J. \u003cem\u003eet al.\u003c/em\u003e Effects of social deprivation in prepubescent rhesus monkeys: immunohistochemical analysis of the neurofilament protein triplet in the hippocampal formation. \u003cem\u003eBrain Res\u003c/em\u003e 619, 299\u0026ndash;305 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrunson, K. L. \u003cem\u003eet al.\u003c/em\u003e Mechanisms of Late-Onset Cognitive Decline after Early-Life Stress. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 25, 9328\u0026ndash;9338 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePechtel, P. \u0026amp; Pizzagalli, D. A. Effects of early life stress on cognitive and affective function: an integrated review of human literature. \u003cem\u003ePsychopharmacology\u003c/em\u003e 214, 55\u0026ndash;70 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaninck, E. F. G. \u003cem\u003eet al.\u003c/em\u003e Chronic early life stress alters developmental and adult neurogenesis and impairs cognitive function in mice. \u003cem\u003eHippocampus\u003c/em\u003e 25, 309\u0026ndash;328 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYajima, H. \u003cem\u003eet al.\u003c/em\u003e Early-life stress induces cognitive disorder in middle-aged mice. \u003cem\u003eNeurobiology of Aging\u003c/em\u003e 64, 139\u0026ndash;146 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeatherstone, R. E. \u003cem\u003eet al.\u003c/em\u003e Early life social instability stress causes lasting cognitive decrement and elevated hippocampal stress-related gene expression. \u003cem\u003eExp Neurol\u003c/em\u003e 354, 114099 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClark, R. E., Zola, S. M. \u0026amp; Squire, L. R. Impaired Recognition Memory in Rats after Damage to the Hippocampus. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 20, 8853\u0026ndash;8860 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEichenbaum, H. A cortical\u0026ndash;hippocampal system for declarative memory. \u003cem\u003eNat Rev Neurosci\u003c/em\u003e 1, 41\u0026ndash;50 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen, S. J. \u003cem\u003eet al.\u003c/em\u003e The Rodent Hippocampus Is Essential for Nonspatial Object Memory. \u003cem\u003eCurrent Biology\u003c/em\u003e 23, 1685\u0026ndash;1690 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, C. \u003cem\u003eet al.\u003c/em\u003e Hippocampus\u0026ndash;Prefrontal Coupling Regulates Recognition Memory for Novelty Discrimination. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 41, 9617\u0026ndash;9632 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInsel, N. \u003cem\u003eet al.\u003c/em\u003e Reduced Gamma Frequency in the Medial Frontal Cortex of Aged Rats during Behavior and Rest: Implications for Age-Related Behavioral Slowing. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 32, 16331\u0026ndash;16344 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrown, L. M., Gray, D. T., Schimanski, L. A., Barnes, C. A. \u0026amp; Cowen, S. L. Aged Rats Exhibit Altered Behavior-Induced Oscillatory Activity, Place Cell Firing Rates, and Spatial Information Content in the CA1 Region of the Hippocampus. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 42, 4505\u0026ndash;4516 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, X. \u003cem\u003eet al.\u003c/em\u003e Gating of hippocampal rhythms and memory by synaptic plasticity in inhibitory interneurons. \u003cem\u003eNeuron\u003c/em\u003e 0, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuzs\u0026aacute;ki, G. \u0026amp; Draguhn, A. Neuronal Oscillations in Cortical Networks. \u003cem\u003eScience\u003c/em\u003e 304, 1926\u0026ndash;1929 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuzs\u0026aacute;ki, G. \u0026amp; Wang, X.-J. Mechanisms of Gamma Oscillations. \u003cem\u003eAnnual Review of Neuroscience\u003c/em\u003e 35, 203\u0026ndash;225 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewis, D. A., Curley, A. A., Glausier, J. \u0026amp; Volk, D. W. Cortical Parvalbumin Interneurons and Cognitive Dysfunction in Schizophrenia. \u003cem\u003eTrends Neurosci\u003c/em\u003e 35, 57\u0026ndash;67 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpiegel, A. M., Koh, M. T., Vogt, N. M., Rapp, P. R. \u0026amp; Gallagher, M. Hilar interneuron vulnerability distinguishes aged rats with memory impairment. \u003cem\u003eJournal of Comparative Neurology\u003c/em\u003e 521, 3508\u0026ndash;3523 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBa\u0026ntilde;uelos, C. \u003cem\u003eet al.\u003c/em\u003e Prefrontal Cortical GABAergic Dysfunction Contributes to Age-Related Working Memory Impairment. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 34, 3457\u0026ndash;3466 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerguson, B. R. \u0026amp; Gao, W.-J. PV Interneurons: Critical Regulators of E/I Balance for Prefrontal Cortex-Dependent Behavior and Psychiatric Disorders. \u003cem\u003eFront Neural Circuits\u003c/em\u003e 12, 37 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabungcal, J.-H. \u003cem\u003eet al.\u003c/em\u003e Perineuronal nets protect fast-spiking interneurons against oxidative stress. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 110, 9130\u0026ndash;9135 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDruga, R., Salaj, M. \u0026amp; Al-Redouan, A. Parvalbumin - Positive Neurons in the Neocortex: A Review. \u003cem\u003ePhysiol Res\u003c/em\u003e 72, S173\u0026ndash;S191 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFawcett, J. W., Oohashi, T. \u0026amp; Pizzorusso, T. The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function. \u003cem\u003eNat Rev Neurosci\u003c/em\u003e 20, 451\u0026ndash;465 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHijazi, S., Smit, A. B. \u0026amp; van Kesteren, R. E. Fast-spiking parvalbumin-positive interneurons in brain physiology and Alzheimer\u0026rsquo;s disease. \u003cem\u003eMol Psychiatry\u003c/em\u003e 28, 4954\u0026ndash;4967 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomberg, C. \u003cem\u003eet al.\u003c/em\u003e Depletion of Perineuronal Nets Enhances Recognition Memory and Long-Term Depression in the Perirhinal Cortex. \u003cem\u003eJ. Neurosci.\u003c/em\u003e 33, 7057\u0026ndash;7065 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSultana, R., Brooks, C. B., Shrestha, A., Ogundele, O. M. \u0026amp; Lee, C. C. Perineuronal Nets in the Prefrontal Cortex of a Schizophrenia Mouse Model: Assessment of Neuroanatomical, Electrophysiological, and Behavioral Contributions. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e 22, 11140 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrapser, J. D. \u003cem\u003eet al.\u003c/em\u003e Microglia facilitate loss of perineuronal nets in the Alzheimer\u0026rsquo;s disease brain. \u003cem\u003eEBioMedicine\u003c/em\u003e 58, 102919 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrasch\u0026uuml;tz, A., Kummer, M. P., Schwartz, S. \u0026amp; Heneka, M. T. Variability and temporal dynamics of novel object recognition in aging male C57BL/6 mice. \u003cem\u003eBehav Processes\u003c/em\u003e 157, 711\u0026ndash;716 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen, H. \u0026amp; Liu, Z. Separating Fractal and Oscillatory Components in the Power Spectrum of Neurophysiological Signal. \u003cem\u003eBrain Topogr\u003c/em\u003e 29, 13\u0026ndash;26 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonoghue, T. \u003cem\u003eet al.\u003c/em\u003e Parameterizing neural power spectra into periodic and aperiodic components. \u003cem\u003eNature Neuroscience\u003c/em\u003e 23, 1655\u0026ndash;1665 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrake, N. \u003cem\u003eet al.\u003c/em\u003e A neurophysiological basis for aperiodic EEG and the background spectral trend. \u003cem\u003eNat Commun\u003c/em\u003e 15, 1514 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuzs\u0026aacute;ki, G., Logothetis, N. \u0026amp; Singer, W. Scaling Brain Size, Keeping Timing: Evolutionary Preservation of Brain Rhythms. \u003cem\u003eNeuron\u003c/em\u003e 80, 751\u0026ndash;764 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVoytek, B. \u003cem\u003eet al.\u003c/em\u003e Age-Related Changes in 1/f Neural Electrophysiological Noise. \u003cem\u003eJournal of Neuroscience\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/JNEUROSCI.2332-14.2015\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.2332-14.2015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015) doi:10.1523/JNEUROSCI.2332-14.2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThuwal, K., Banerjee, A. \u0026amp; Roy, D. Aperiodic and Periodic Components of Ongoing Oscillatory Brain Dynamics Link Distinct Functional Aspects of Cognition across Adult Lifespan. \u003cem\u003eeNeuro\u003c/em\u003e 8, ENEURO.0224-21.2021 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrystecka, K., Stanczyk, M., Magnuski, M., Szelag, E. \u0026amp; Szymaszek, A. Aperiodic activity differences in individuals with high and low temporal processing efficiency. \u003cem\u003eBrain Research Bulletin\u003c/em\u003e 215, 111010 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBieri, K. W., Bobbitt, K. N. \u0026amp; Colgin, L. L. Slow and Fast Gamma Rhythms Coordinate Different Spatial Coding Modes in Hippocampal Place Cells. \u003cem\u003eNeuron\u003c/em\u003e 82, 670\u0026ndash;681 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColgin, L. L. Rhythms of the hippocampal network. \u003cem\u003eNature Reviews Neuroscience\u003c/em\u003e 17, 239\u0026ndash;249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColgin, L. L. \u003cem\u003eet al.\u003c/em\u003e Frequency of gamma oscillations routes flow of information in the hippocampus. \u003cem\u003eNature\u003c/em\u003e 462, 353\u0026ndash;357 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaris, E. \u0026amp; Oostenveld, R. Nonparametric statistical testing of EEG- and MEG-data. \u003cem\u003eJ Neurosci Methods\u003c/em\u003e 164, 177\u0026ndash;190 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJakovljevic, A. \u003cem\u003eet al.\u003c/em\u003e The impact of early life maternal deprivation on the perineuronal nets in the prefrontal cortex and hippocampus of young adult rats. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e 10, 982663 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchneider, C. A., Rasband, W. S. \u0026amp; Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. \u003cem\u003eNat Methods\u003c/em\u003e 9, 671\u0026ndash;675 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Brien, J., Hayder, H. \u0026amp; Peng, C. Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins. \u003cem\u003eJ Vis Exp\u003c/em\u003e 54719 (2016) doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3791/54719\u003c/span\u003e\u003cspan address=\"10.3791/54719\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCort\u0026eacute;s-Albornoz, M. C., Ram\u0026iacute;rez-Guerrero, S., Garc\u0026iacute;a-Gu\u0026aacute;queta, D. P., V\u0026eacute;lez-Van-Meerbeke, A. \u0026amp; Talero-Guti\u0026eacute;rrez, C. Effects of remote learning during COVID-19 lockdown on children\u0026rsquo;s learning abilities and school performance: A systematic review. \u003cem\u003eInt J Educ Dev\u003c/em\u003e 101, 102835 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, C., Bieri, K. W., Hwaun, E. \u0026amp; Colgin, L. L. Fast Gamma Rhythms in the Hippocampus Promote Encoding of Novel Object-Place Pairings. \u003cem\u003eeNeuro\u003c/em\u003e 3, ENEURO.0001-16.2016 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamura, M., Spellman, T. J., Rosen, A. M., Gogos, J. A. \u0026amp; Gordon, J. A. Hippocampal-prefrontal theta-gamma coupling during performance of a spatial working memory task. \u003cem\u003eNat Commun\u003c/em\u003e 8, 2182 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuston, D. R., Gruber, A. J. \u0026amp; Mcnaughton, B. L. The Role of Medial Prefrontal Cortex in Memory and Decision Making. \u003cem\u003eNeuron\u003c/em\u003e 76, 1057\u0026ndash;1070 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujisawa, S. \u0026amp; Buzs\u0026aacute;ki, G. A 4 Hz oscillation adaptively synchronizes prefrontal, VTA, and hippocampal activities. \u003cem\u003eNeuron\u003c/em\u003e 72, 153\u0026ndash;165 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAoki, M., Shimozuru, M., Kikusui, T., Takeuchi, Y. \u0026amp; Mori, Y. Sex differences in behavioral and corticosterone responses to mild stressors in ICR mice are altered by ovariectomy in peripubertal period. \u003cem\u003eZoolog Sci\u003c/em\u003e 27, 783\u0026ndash;789 (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9118580/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9118580/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAdolescent social stress can be detrimental to developing hippocampal-prefrontal circuits and is associated with adverse cognitive outcomes. We previously found that adolescent social instability stress (SIS) in mice resulted in later-life memory retrieval impairment as measured with novel object recognition (NOR).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMice were behaviorally assessed using novel object recognition (NOR), social interaction, and the elevated plus maze. We analyzed local-field potential data during NOR. We assessed the 1/f slope of the power spectral density within the dCA1, dCA3, vCA1, and mPFC and analyzed coherence between the ventral hippocampus and medial prefrontal cortex (mPFC) and dorsal CA3 to CA1 as animals were near objects during NOR. Additionally we measured serum corticosterone levels immediately following the termination of the adolescent SIS manipulation, and used immunohistochemistry to quantify perineuronal nets (PNN) around parvalbumin (PV)-positive neurons using antibodies for PV and Wisteria floribunda agglutinin (WFA), which labels PNNs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNOR deficits in SIS mice were replicated. No significant changes to social interactions, elevated plus maze or the aperiodic component of spectral parameterization were observed. Intra-hippocampal gamma coherence was reduced in SIS animals around the novel object relative to controls. PNN density was reduced in the mPFC of stressed animals. Corticosterone was lower in SIS mice relative to controls.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eData support the hypothesis that altered social interactions during adolescence can result in structural, electrophysiological, and cognitive deficits that persist well into adulthood.\u003c/p\u003e","manuscriptTitle":"Social Instability in Adolescent Mice Leads to Lasting Cognitive Deficits with Reduction of Intra-Hippocampal Functional Connectivity and Parvalbumin-Containing Interneurons with Perineuronal Nets.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 15:07:19","doi":"10.21203/rs.3.rs-9118580/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-04-07T08:07:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-03T09:23:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-26T12:40:35+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-20T08:46:39+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-20T08:08:37+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-20T06:50:37+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-20T05:19:45+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-03-20T03:27:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-17T16:40:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-17T16:05:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Psychiatry","date":"2026-03-16T17:31:23+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-03-16T16:37:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"85c7305f-f3b1-41dd-a118-0c3c23cf0b6d","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":64823477,"name":"Biological sciences/Neuroscience/Learning and memory/Hippocampus"},{"id":64823478,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-07T08:14:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 15:07:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9118580","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9118580","identity":"rs-9118580","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}