Impact of Perineuronal Net Removal in the Rat Medial Prefrontal Cortex on Parvalbumin Interneurons After Reinstatement of Cocaine Conditioned Place Preference

Abstract

Parvalbumin (PV)-positive cells are GABAergic fast-spiking interneurons that modulate the activity of pyramidal neurons in the medial prefrontal cortex (mPFC) and their output to brain areas associated with learning and memory. The majority of PV cells within the mPFC are surrounded by a specialized extracellular matrix structure called the perineuronal net (PNN). We have shown that removal of PNNs with the enzyme chondroitinase-ABC (Ch-ABC) in the mPFC prevents the consolidation and reconsolidation of cocaine-associated conditioned place preference (CPP) memories. Here we examined the extent to which retrieval of a CPP memory during cocaine-primed reinstatement altered the levels and function of PV neurons and their surrounding PNNs during the reconsolidation period. We further determined the extent to which PNN removal prior to reinstatement altered PV intensity levels and PV cell function. Male Sprague-Dawley rats were trained for cocaine-induced conditioned place preference (CPP) followed by extinction training, microinjection of Ch-ABC in the prelimbic PFC, and cocaine-induced reinstatement. Rats were sacrificed immediately prior to reinstatement or at 2 h, 6 h, or 48 h after reinstatement for immunohistochemistry or 2 h later for electrophysiology. Our findings indicate that PNN removal only partially diminished reinstatement. Cocaine-primed reinstatement produced only minor changes in PNN or PV intensity in vehicle controls. However, after PNN removal, the intensity of remaining PNN-surrounded PV cells was decreased at all times except at 2 h post-reinstatement, at which time cocaine increased PV intensity. Consistent with this, in vehicle controls, PV neurons naturally devoid of PNNs showed a similar pattern to Ch-ABC-treated rats prior to and after cocaine reinstatement, suggesting a protective effect of PNNs on cocaine-induced changes in PV intensity. Using whole-cell patch-clamp, cocaine-primed reinstatement in Ch-ABC-treated rats decreased the number of elicited action potentials but increased excitatory synaptic transmission, which may have been compensatory. These findings suggest that without PNNs, cocaine-induced reinstatement produces rapid changes in PV intensity and PV cell excitability, which may in turn regulate output of the mPFC post-memory retrieval and diminish the maintenance of cocaine memory during reconsolidation.

Keywords: addiction; cocaine; conditioned place preference; memory; parvalbumin; perineuronal nets; prefrontal cortex; substance use disorder.

Figure 1

PNN removal in the mPFC has minimal impact on cocaine-primed reinstatement. (A) Experimental timeline for cocaine-induced CPP training, testing, and reinstatement. (B) (Top panels) Representative images of WFA-stained coronal sections of mPFC of vehicle and Ch-ABC-treated rat. White line = 100 μm. Upper right panel: WFA/PV double-labeled cells in vehicle-treated rat; Lower right panel: WFA/PV double-labeled cells in Ch-ABC-treated rat; note lack of WFA surrounding PV cells. (C) Schematic diagram (Paxinos and Watson, 2007) of mPFC coronal rat brain sections depicting stereotaxic coordinates of vehicle and Ch-ABC microinjections; purple, vehicle; teal, Ch-ABC. (D) There were no differences in treatment within subjects on initial preference (Initial Pref) or test day. Both treatment groups showed cocaine CPP on test day compared to their initial preference day within subjects. Both treatment groups also showed cocaine-induced reinstatement compared to their last extinction day (Last Ext). Vehicle subjects showed increased preference on reinstatement day (Reinstate) compared to their initial preference day. Data are mean ± SEM. For vehicle, n = 40; Ch-ABC n = 31 rats. ⁺P < 0.05 compared with initial preference day; ^#P < 0.05, compared with last extinction day. Created with BioRender.com.

Figure 2

Cocaine-primed reinstatement does not alter PV or WFA intensity in vehicle controls, while Ch-ABC treatment in the mPFC increases PV intensity 2 h post-reinstatement. (A) Intensity of total labeled PV cells in prelimbic PFC; PV intensity of both vehicle and Ch-ABC-treated rats decreased at 48 h compared within group to intensity at pre-reinstatement (pre-reinstate); intensity of Ch-ABC-treated animals increased at 2 h compared to vehicle-treated animals. (B) Intensity of total labeled WFA cells in prelimbic PFC; WFA intensity in Ch-ABC-treated animals decreased at 2, 6, and 48 h compared to vehicle treated animals; intensity of WFA in vehicle-treated animals at 48 h decreased compared to their pre-reinstatement levels. (C) Intensity of PV cells containing WFA in prelimbic PFC; intensity of PV cells in Ch-ABC-treated animals was decreased at pre-reinstatement and at 6 and 48 h compared to vehicle-treated animals; intensity of PV cells in Ch-ABC-treated animals increased at 2 h compared to pre-reinstatement levels in Ch-ABC-treated animals. (D) Intensity of WFA cells in prelimbic PFC containing PV; intensity of WFA in Ch-ABC-treated animals decreased at 6 h compared to vehicle-treated animals. (E) Intensity of PV with or without WFA in prelimbic PFC in vehicle groups; intensity of PV devoid of PNNs (without WFA) decreased at pre-reinstatement and at 6 h compared to PV cells with PNNs (with WFA). (F) Intensity of WFA with or without co-labeled PV in prelimbic PFC. Data are mean ± SEM. All values were normalized to vehicle pre-reinstatement values. For vehicle: pre-reinstate n = 10; 2 h n = 7; 6 h n = 6; 48 h n = 5. For Ch-ABC: pre-reinstate n = 6; 2 h n = 6; 6 h n = 5; 48 h n = 2. ^$P < 0.05, compared within-group to Pre-reinstate; *P < 0.05 compared to vehicle group at the same time point.

Figure 3

Cocaine-primed reinstatement does not alter the number of PV or WFA cells in the prelimbic PFC. (A) Number of PV cells in prelimbic PFC. (B) Number of WFA cells in prelimbic PFC; WFA cell number decreased in Ch-ABC treated animals at baseline, 2, 6, and 48 h compared to vehicle. (C) Number of PV/WFA double labeled cells in prelimbic PFC; PV/WFA cell number decreased at baseline, 2 h, and 6 h compared to vehicle. Bars show mean ± SEM, and individual data points depict the mean number of cells per rat across four images; N-sizes are as in Figure 2. *P < 0.05 compared to vehicle group at the same time point.

Figure 4

Ch-ABC treatment prior to cocaine-primed reinstatement decreases the firing rate of PNN surrounded FSIs within the prelimbic PFC and changes mEPSC frequency and amplitude onto FSIs. (A) Decrease in average frequency of action potentials across range of current injections; shown are representative traces of action potentials evoked by 500 pA current injection. Scale bars represents 20 ms, 20 mV; (B) Resting membrane potential; (C) Decrease in action potential threshold; (D) Rise time; (E) Increase in action potential half-width; (F) Membrane capacitance; (A–F): Vehicle, n = 6 rats, Ch-ABC = 8 rats. (G) Decrease in mEPSC amplitude; (H) Increase in mEPSC frequency; Data are mean ± SEM (bars) with individual cells (circles). (G,H): Vehicle n = 4 rats, Ch-ABC n = 5 rats; Mann-Whitney, *P < 0.05.