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. 2024 May 7;15(1):39.
doi: 10.1186/s13293-024-00616-0.

Early adversity causes sex-specific deficits in perforant pathway connectivity and contextual memory in adolescent mice

Rafiad Islam  1 , Jordon D White  1 , Tanzil M Arefin  2   3 , Sameet Mehta  4 , Xinran Liu  5   6 , Baruh Polis  1 , Lauryn Giuliano  1 , Sahabuddin Ahmed  1 , Christian Bowers  1 , Jiangyang Zhang  2 , Arie Kaffman  7
Affiliations

Early adversity causes sex-specific deficits in perforant pathway connectivity and contextual memory in adolescent mice

Rafiad Islam et al. Biol Sex Differ. .

Abstract

Background: Early life adversity impairs hippocampal development and function across diverse species. While initial evidence indicated potential variations between males and females, further research is required to validate these observations and better understand the underlying mechanisms contributing to these sex differences. Furthermore, most of the preclinical work in rodents was performed in adult males, with only few studies examining sex differences during adolescence when such differences appear more pronounced. To address these concerns, we investigated the impact of limited bedding (LB), a mouse model of early adversity, on hippocampal development in prepubescent and adolescent male and female mice.

Methods: RNA sequencing, confocal microscopy, and electron microscopy were used to evaluate the impact of LB and sex on hippocampal development in prepubescent postnatal day 17 (P17) mice. Additional studies were conducted on adolescent mice aged P29-36, which included contextual fear conditioning, retrograde tracing, and ex vivo diffusion magnetic resonance imaging (dMRI).

Results: More severe deficits in axonal innervation and myelination were found in the perforant pathway of prepubescent and adolescent LB males compared to LB female littermates. These sex differences were due to a failure of reelin-positive neurons located in the lateral entorhinal cortex (LEC) to innervate the dorsal hippocampus via the perforant pathway in males, but not LB females, and were strongly correlated with deficits in contextual fear conditioning.

Conclusions: LB impairs the capacity of reelin-positive cells located in the LEC to project and innervate the dorsal hippocampus in LB males but not female LB littermates. Given the critical role that these projections play in supporting normal hippocampal function, a failure to establish proper connectivity between the LEC and the dorsal hippocampus provides a compelling and novel mechanism to explain the more severe deficits in myelination and contextual freezing found in adolescent LB males.

Keywords: Cortical thinning; Early adversity; Limited bedding and nesting; Myelination; Perforant pathway; Reelin.

Plain language summary

Childhood adversity, such as severe deprivation and neglect, leads to structural changes in human brain development that are associated with learning deficits and behavioral difficulties. Some of the most consistent findings in individuals exposed to childhood adversity are reduced hippocampal volume and abnormal hippocampal function. This is important because the hippocampus is necessary for learning and memory, and it plays a crucial role in depression and anxiety. Although initial studies suggested more pronounced hippocampal deficits in men, additional research is needed to confirm these findings and to elucidate the mechanisms responsible for these sex differences. We found that male and female mice exposed to early impoverishment and deprivation exhibit similar structural changes to those observed in deprived children. Interestingly, adolescent male mice, but not females, display severe deficits in their ability to freeze when placed back in a box where they were previously shocked. The ability to associate “shock/danger” with a “box/place” is referred to as contextual fear conditioning and requires normal connections between the entorhinal cortex and the hippocampus. We found that these connections did not form properly in male mice exposed to impoverished conditions, but they were only minimally affected in females. These findings appear to explain why exposure to impoverished conditions impairs contextual fear conditioning in male mice but not in female mice. Additional work is needed to determine whether similar sex-specific changes in these connections are also observed in adolescents exposed to neglect and deprivation.

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Conflict of interest statement

The authors have no competing interests to declare.

Figures

Fig. 1
Fig. 1
LB inhibits oligodendrocyte differentiation in the hippocampus of P17 prepubescent pups. A Experimental timeline. B Heatmap showing the effect size of differentially regulated genes (DRGs) at FDR < 0.05 between CTL and LB. C Pathway analysis of the top downregulated and upregulated pathways affected by LB. D Transcription factor analysis identified changes in Myrf, Olig2 and Sox10 activity as the most dysregulated factors affected by LB. LB does not affect the expression of OPC-specific genes E but significantly decreases the expression of genes expressed in mature oligodendrocytes (F–G). N = 7–8 mice per rearing and sex group. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001
Fig. 2
Fig. 2
OPC differentiation in the SLM is impaired in P17 Prepubescent LB mice. A Low (4X) and higher (20X) magnification of myelin basic protein (MBP) staining in the SLM of P17 pups. B Representative images of PDGFRα-positive OPCs, CC1-positive mature oligodendrocytes, and MBP in the SLM. Quantification of MBP (C), PDGFRα-positive OPCs (D), CC1 mature oligodendrocytes (E) and OPC differentiation index (F) in the SLM group. N = 5–6 mice per rearing and sex group. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001
Fig. 3
Fig. 3
LB causes a more pronounced reduction in axonal staining in the SLM of Prepubescent 17-day-old LB male mice. A Representative images of axonal staining using anti-NF-H and anti-MBP antibodies in the SLM. Quantification of NF-H staining (B), MBP staining (C) and NF-H-MBP colocalization (D). Scale bars in Fig. 4A are 50 microns. N = 5 mice per rearing and sex. Error bars represent the mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001
Fig. 4
Fig. 4
Transmission electron microscopy of myelinated axons in the SLM of P17 Prepubescent CTL and LB male mice. A Left, a light microscopy image of the same toluidine-stained sample to identify the SLM; Right, TEM images of the SLM in CTL and LB mice (visible axons indicated with red dots). B Image analysis of covariance between the G-ratio and axonal diameter (C). N = 20 axons per mouse with 3 male mice per condition. Error bars represent the mean ± SEM. ****p < 0.0001
Fig. 5
Fig. 5
Adolescent P29 LB mice are hyperactive and show extensive reduction in cortical and subcortical gray matter. A Experimental timeline. Effects of rearing and sex on body weight (B), and exploratory behavior in the open filed test in P29 adolescent mice (C–E). CTL males N = 15, CTL females N = 14, LB males N = 16, LB females N = 24. F High resolution ex vivo dMRI found significant effects of rearing on volumetric changes using (minimal cluster size > 25 voxels, FDR < 0.1, p < 0.005). Areas with significant volume reduction are shown in red. N = 6 per rearing and sex. G Effects of rearing and sex on CA1 and dentate gyrus volumes. H–J The left entorhinal cortex was the only region showing a significant rearing by sex interaction (minimal cluster size > 5 voxels, FDR < 0.1, p < 0.0023). AI: Insular cortex, CB: Cerebellum, cpd: Cerebral peduncle, ENT: Entorhinal cortex, GP: Globus pallidus, HB: Hindbrain, HPF: Hippocampus, HY: HY: Hypothalamus, Int: Internal capsule, LSX: Lateral septal complex, MO: Motor cortex, OLF: Olfactory area, opt: Optic tract, OT: Olfactory tubercle, PFC: Prefrontal cortex, PTLp: Posterior parietal association cortex, RSP: Retrosplenial cortex, SC: Superior colliculus, SS: Somatosensory cortex, STR: Striatum, TH Thalamus, VIS: Visual cortex
Fig. 6
Fig. 6
LB causes more severe deficits in contextual fear conditioning in adolescent P34 male mice. A Experimental timeline. B Contextual fear conditioning. CTL male = 17, CTL females = 18, LB males = 13, LB females = 18. C, D NF-H axonal staining in the SLM of adolescent mice, N = 6 mice per rearing and sex group. E The intensity of axonal staining correlates with contextual freezing behavior. Error bars represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 7
Fig. 7
LB reduces structural connectivity between the entorhinal cortex (ENT) and the dorsal hippocampus (dHPC) in P29 mice. Representative images (A) and quantification of ENT-dHPC tractography in the left (B) and right (C) hemispheres. N = 6 mice per rearing and sex condition. Error bars represent the mean ± SEM. *p < 0.05, **p < 0.01
Fig. 8
Fig. 8
LB causes sex-specific deficits in lateral perforant pathway connectivity in P35 adolescent mice. A CTL and LB mice were injected with the retrograde tracer Alexa 555-CTB into the left SLM at P28, tested in contextual fear conditioning (P33-34), and perfused to assess the number of CTB-positive cells in the LEC at P35. B Contextual fear conditioning. C Schematic depiction of CTB injection at P28 and CTB labeling in the LEC at P35. D Number of CTB-positive cells in the LEC at P35. E The number of CTB-positive cells in the LEC correlates with contextual fear conditioning
Fig. 9
Fig. 9
LB reduces the number of reelin-positive projections in adolescent males. A Representative images of Reelin- and CTB-positive cells in the LEC (scale bars in A are 50 µm). Higher magnification of rectangular areas of merged images is shown on the right. B The number of CTB-positive cells is reduced in the LEC of male LB mice. C All CTB-positive cells are reelin-positive regardless of rearing and sex conditions. D LB reduces the number of reelin-positive cells in the LEC. E Effects of rearing and sex on the percentage of reelin-positive CTB-negative cells in the LEC
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