Prelimbic cortex perineuronal net expression and social behavior: Impact of adolescent intermittent ethanol exposure

Abstract

Adolescent intermittent ethanol (AIE) exposure in rats leads to social deficits. Parvalbumin (PV) expressing fast-spiking interneurons in the prelimbic cortex (PrL) contribute to social behavior, and perineuronal nets (PNNs) within the PrL preferentially encompass and regulate PV interneurons. AIE exposure increases PNNs, but it is unknown if this upregulation contributes to AIE-induced social impairments. The current study was designed to determine the effect of AIE exposure on PNN expression in the PrL and to assess whether PNN dysregulation contributes to social deficits elicited by AIE. cFos-LacZ male and female rats were exposed every other day to tap water or ethanol (4 g/kg, 25% w/v) via intragastric gavage between postnatal day (P) 25-45. We evaluated neuronal activation by β-galactosidase expression and PNN levels either at the end of the exposure regimen on P45 and/or in adulthood on P70. In addition, we used Chondroitinase ABC (ChABC) to deplete PNNs following adolescent exposure (P48) and allowed for PNN restoration before social testing in adulthhod. AIE exposure increased PNN expression in the PrL of adult males, but decreased PNNs immediately following AIE. Vesicular glutamate transporter 2 (vGlut2) and vesicular GABA transporter (vGat) near PNNs were downregulated only in AIE-exposed females. Gene expression of PNN components was largely unaffected by AIE exposure. Removal and reestablishment of PrL PNNs by ChABC led to upregulation of PNNs and social impairments in males, regardless of adolescent exposure. These data suggest that AIE exposure in males upregulates PrL PNNs that likely contribute to social impairments induced by AIE.

Keywords: Adolescence; Ethanol; Perineuronal nets; Sex-differences.

Figure 1.

AIE increases PNN expression and their colocalization with activated neurons in cFos-LacZ transgenic rats. A. Timeline of adolescent water or ethanol (AIE) exposure and social testing in adulthood. B-D. Representative images of WFA-labeled PNNs, β-gal, and co-labeling of WFA and β-gal, respectively. E. AIE exposure led to an increase in WFA+ cells in the PrL compared to water-exposed males and females of both exposure groups (marked with *). F. No differences were found for the number of β-gal+ cells in the PrL of socially tested rats. G. A larger percentage of WFA+ cells were activated in AIE-exposed males relative to water-exposed controls. * p < 0.05 and ** p < 0.01.

Figure 2.

Females, but not not males, display changes in synaptic puncta on PNN expressing cells following AIE. A. Timeline of adolescent exposure and tissue collection for histology. B-D. Representative images of labeling PNNs and synaptic puncta (dapi (grey), WFA (green), vGlut2 (red), and vGat (blue)), PNN volume alone, and rendering of synaptic puncta within 1 μm of the PNNs, respectively. E. Vglut2 expressing synapses were decreased in AIE-exposed females relative to water-exposed females. F-G. No differences in Vgat labeling or excitatory/inhibitory ratio. * p < 0.05 indicates difference between water- and AIE-exposed females. # p < 0.05 indicates main effect of exposure group. Scale bar represents 40 μm.

Figure 3.

Reductions in PNNs are evident immediately following the final AIE exposure. A. Male and female rats exposed to water or AIE then euthanized for WFA labeling PNNs following the final exposure on P45. B. AIE-exposed males had lower WFA labeling than water-exposed controls at the end of the exposure regimen. * p < 0.05 indicates difference from AIE-exposed P45 group.

Figure 4.

PNN gene expression is selectively altered following AIE exposure. A. Subjects were exposed to water or AIE and brains extracted for gene expression analysis either 30 minutes following the final exposure on P45 or in adulthood on P70. D. Aggrecan gene expression was elevated in AIE-exposed males on P45 (immediately following the end of AIE) but not after 25 days of abstinence. E. Neurocan gene expression was significantly lower in adults, regardless of adolescent exposure. * p < 0.05 and ** p < 0.01 indicate difference from AIE-exposed P45 group. $ $ p < 0.01 indicates difference between ages.

Figure 5.

ChABC degradation results in a delayed increases in PNNs. A-C. Representative images of PrL WFA labeling in animals one, two, and three weeks following ChABC injection, respectively. D. Labeling of PNNs with WFA was the lowest in animals one-week post-ChABC, with gradual increases evident two and three weeks post-ChABC injection. Dotted line represents PNN levels in subjects without ChABC. ** p < 0.01 and **** p < 0.0001 indicate difference between weeks post-ChABC. Scale bar represents 100 μm.

Figure 6.

PrL PNN depletion during late adolescence in water controls replicates AIE-induced decreases in social behavior in adult rats. A. PNNs were degraded with ChABC two days after the end of AIE (or water) exposure and allowed to regrow prior to social testing in adulthood. B-C. Social investigation and social preference were decreased following ChABC-induced depletion of PNNs in males specifically. Dotted line represents social investigation values in that did not receive a surgical manipulation during late adolescence (Towner et al., 2023). D. AIE-exposed males displayed greater locomotor behavior, regardless of PNN degradation. E-G. Social behavior of females was unaltered following PNN degradation in the PrL. $ p < 0.05 and $ $ p < 0.01 indicate differences between vehicle and ChABC injected subjects.

Figure 7.

PrL PNN depletion during late adolescence increases PNN levels in adult rats. A-B. Representative images of WFA labeling in animals injected with vehicle and ChABC respectively. C. Males injected with ChABC 28 days before behavior displayed an increase in WFA labeling in the PrL compared with vehicle injected rats. D. No difference in β-gal expression was found between groups. E. In females, ChABC injected rats had greater WFA labeling, regardless of adolescent exposure. F. Neuronal activation, indicated by β-gal expression, was not affected by PNN degradation in females. $$ p < 0.01 indicates difference between vehicle and ChABC injected subjects. Scale bar represents 100 μm.