Enhanced fear extinction through infralimbic perineuronal net digestion: The modulatory role of adolescent alcohol exposure

Abstract

Perineuronal nets (PNNs) are specialized components of the extracellular matrix that play a critical role in learning and memory. In a Pavlovian fear conditioning paradigm, degradation of PNNs affects the formation and storage of fear memories. This study examined the impact of adolescent intermittent ethanol (AIE) exposure by vapor inhalation on the expression of PNNs in the adult rat prelimbic (PrL) and infralimbic (IfL) subregions of the medial prefrontal cortex. Results indicated that following AIE, the total number of PNN positive cells in the PrL cortex increased in layer II/III but did not change in layer V. Conversely, in the IfL cortex, the number of PNN positive cells decreased in layer V, with no change in layer II/III. In addition, the intensity of PNN staining was significantly altered by AIE exposure, which narrowed the distribution of signal intensity, reducing the number of high and low intensity PNNs. Given these changes in PNNs, the next experiment assessed the effects of AIE and PNN digestion on extinction of a conditioned fear memory. In Air control rats, digestion of PNNs by bilateral infusion of Chondroitinase ABC (ChABC) into the IfL cortex enhanced fear extinction and reduced contextual fear renewal. In contrast, both fear extinction learning and contextual fear renewal remained unchanged following PNN digestion in AIE exposed rats. These results highlight the sensitivity of prefrontal PNNs to adolescent alcohol exposure and suggest that ChABC-induced plasticity is reduced in the IfL cortex following AIE exposure.

Keywords: adolescent alcohol; extinction; fear; infralimbic cortex; medial prefrontal cortex; perineuronal nets.

Figure 1.. Experimental details and timeline.

(A) Experimental timeline for perineuronal net (PNN) expression experiments. The inset image details the AIE vapor exposure paradigm. (B) Schema displaying the age of rats at each phase of the fear learning and extinction experiments. The inset image details the fear paradigm and timing of microinfusions.

Figure 2.. Representative Images of cortical perineuronal net staining in air and adolescent intermittent ethanol exposed animals.

(A) Representative confocal images of Air (control) animals. A section of tissue within one hemisphere in the prelimbic (PrL) cortex is highlighted with a white hashed rectangle. A section of infralimbic (IfL) cortex is highlighted with a yellow hashed rectangle. Also included is an indication of cortical layer in a white box. Below the 10X magnification image depicting both hemispheres, inset panels are shown representing a single hemisphere of each cortical subregion, shown at higher magnification. The inset panels include corresponding white (PrL) and yellow (IfL) hashed outlines (B) Representative confocal images of AIE animals are organized and depicted as in (A).

Figure 3.. Adolescent intermittent ethanol exposure bidirectionally modulated perineuronal net numbers in the prelimbic and infralimbic cortex.

(A) Adult male rats exposed to adolescent intermittent ethanol (AIE) displayed increased numbers of perineuronal net (PNN)-positive neurons in the prelimbic (PrL) cortex. (B) Layer-specific analyses revealed that AIE-exposure led to a significant increase in PNN numbers in layer II/III, while layer V showed a non-significant trend toward increased PNN numbers. (C) In the infralimbic (IfL) cortex, there was no significant difference in PNN counts between AIE exposed rats and Air control rats. (D) Within the IfL, AIE did not significantly affect the number of PNN-positive cells in layer II/III, however, in layer V AIE decreased the number of these cells. * denotes p < 0.05. n = 5 rats/group with 5–6 replicates/animal.

Figure 4.. Adolescent intermittent ethanol exposure shifted the distribution of perineuronal net staining intensity in the prelimbic and infralimbic cortex.

(A) Relative frequency of Wisteria floribunda agglutinin (WFA) staining intensity of individual perineuronal nets (PNNs) in the prelimbic (PrL) cortex. Within the PrL cortex, adolescent intermittent ethanol (AIE) exposure increased the relative frequency of intermediate intensity PNNs. (B) Gaussian curves showing the decreased variability of PNN intensity and the increased frequency of intermediate intensity PNNs in the PrL cortex of AIE exposed rats. (C) Relative frequency of individual PNN staining intensity in the infralimbic (IfL) cortex. Within the IfL cortex, the distribution of PNN staining intensity was altered by AIE exposure, reflecting an AIE-induced increase in the proportion of intermediate intensity PNNs. (D) Gaussian curves displaying the decreased variability of PNN intensity and the increased frequency of intermediate intensity PNNs in the IfL cortex of AIE exposed rats. * denotes p < 0.05. For the PrL, n = 9,745 PNNs (Air) and 11,084 PNNs (AIE). For the IfL, n = 4,509 PNNs (Air) and 4,016 PNNs (AIE). There were 5 rats/group.

Figure 5.. Depletion of infralimbic perineuronal nets enhanced extinction learning: modulation by adolescent alcohol exposure.

(A) Images depicting Wisteria floribunda agglutinin (WFA) staining of perineuronal nets 24 hr, 7 days, and 21 days after microinfusion of chondroitinase ABC (ChABC). Significant digestion of PNNs is evident 24 hr after the microinfusion. At 7 days post-infusion limited recovery of PNNs is apparent, with a more complete recovery of PNN levels visible at 21 days post-infusion. (B) Depiction of the increase in freezing to the tone that occurred across three fear conditioning sessions in which the tone was paired with a foot shock. There were no significant differences between Air control and adolescent intermittent ethanol (AIE) exposed rats in acquisition of the tone-shock pairing. (C) Graphic displaying the gradual decline in freezing to the tone that occurred over 5 extinction learning sessions in which the tone was presented with an accompanying foot shock. Extinction learning was significantly enhanced in rats that received a microinfusion of ChABC into the infralimbic cortex. (D) Graphic representation of freezing in response to the tone during the final conditioned fear extinction session. Adolescent alcohol exposure prevented the enhanced extinction learning that occurred in Air control rats following the digestion of PNNs in the IfL cortex. (E) Depiction of freezing in response to a single tone presentation during the context-dependent fear renewal task. Digestion of PNNs by ChABC significantly reduced fear renewal in Air control but not AIE exposed rats. * denotes p < 0.05. n = 9 (Air + bovine serum albumin [BSA]), 4 (Air + ChABC), 4 (AIE + BSA), 5 (AIE + ChABC).