Perineuronal nets in the lateral hypothalamus area regulate cue-induced reinstatement of cocaine-seeking behavior

Abstract

We previously reported that a small, circumscribed region of the lateral hypothalamus, the anterior dorsal region (LHAad), stains heavily for PNNs and dense extracellular matrix (PNNs/ECM) with Wisteria floribunda agglutinin (WFA), and critically contributes to the acquisition of cocaine-induced conditioned place preference and cocaine self-administration. Here we tested the role of LHAad PNNs/ECM in cue-induced reinstatement in cocaine self-administering (SA) rats and identified how it is embedded in the circuitry of motivated behavior and drug reward. Degradation of PNNs/ECM in the LHAad using chondroitinase ABC (Ch-ABC) blocked the expression of cue-induced reinstatement of cocaine- but not sucrose-seeking behavior. We also identified for the first time the phenotype of LHAad PNN/ECM-surrounded neurons. LHAad neurons co-localized mainly with parvalbumin (PV+) and GABA. Predominant co-localization of WFA with VGLUT2 and GABA but not with GAD65/67 or glutamate indicates that the PNN/ECM-rich LHAad is predominantly GABAergic and receives dense glutamatergic input. The LHAad did not express significant amounts of melanin-concentrating hormone (MCH), orexin, or galanin; neuropeptides that regulate both food-induced and cocaine-induced behavior. In addition, retrobead injections demonstrated that the LHAad receives robust prelimbic prefrontal cortex (PFC) input and provides moderate input to the prelimbic PFC and ventral tegmental area (VTA), with no apparent input to the nucleus accumbens. In summary, the dense PNN/ECM zone in the LHAad embedded within the circuitry associated with reward pinpoints a novel region that controls the expression of cocaine-seeking behavior.

Fig. 1

LHAad PNN/ECM degradation blocks cue-induced reinstatement of cocaine seeking. a Timeline of the experiment. b Depiction of the targeted region characterized by robust PNN/ECM expression using WFA. This area is the lateral hypothalamic area (LHA), anterior region (LHAa), dorsal (LHAd) zone (LHAad) ventromedial to the internal capsule (int). c Strong PNN/ECM expression in the LHAad is abolished by Ch-ABC administration. d SA active lever presses, e SA reinforced lever presses, and f EXT active lever presses. g SA inactive lever presses, and h EXT inactive lever presses. i Active lever presses in the first 30-min of the last extinction session compared to the 30 min cue-induced reinstatement session, and j inactive lever presses during the 30 min cue-induced reinstatement session (Data are mean ± SEM). Differences were considered significant when *p < 0.05. k Injection sites in cocaine trained animals that received intra-LHAad microinjection of vehicle (black circles; n = 7) and Ch-ABC (gray circles; n = 9)

Fig. 2

LHAad PNN/ECM degradation does not block cue-induced reinstatement of sucrose seeking. a Timeline of the experiment. b SA active lever presses, c SA reinforced lever presses, d EXT active lever presses, e SA inactive lever presses, and f EXT inactive lever presses. g Active lever presses in the first 30 min of the last extinction session compared to the 30 min cue-induced reinstatement session, and h inactive lever presses during the 30 min cue-induced reinstatement session (Data are mean ± SEM). Differences were considered significant when *p < 0.05. i Injection sites in sucrose trained animals that received intra-LHAad microinjection of vehicle (black circles; n = 7) and Ch-ABC (gray circles; n = 8)

Fig. 3

LHAad neurons are GABAergic PV⁺ neurons, receive dense glutamatergic input, and do not co-localize with neuropeptides involved in feeding. Positive control regions were the a anterior hypothalamic nucleus (AHN), ventral juxtaventromedial LHA (LHAjvv), paraventricular hypothalamus (PVH), and b juxtadorsomedial LHA (LHAjd). c WFA colabels with parvalbumin (PV), d WFA does not colabel with GAD65/67, e WFA colabels with GABA, f WFA colabels with VGLUT2, g WFA only colabels with glutamate near the ventral tier of the LHAad, h WFA does not colabel with CaMKIIα, i LHAjvv CaMKIIα positive control, j minimal orexin expression in the LHAad, k LHAjd orexin positive control, l minimal MCH expression in the LHAad, m AHN MCH positive control, n minimal galanin expression in the LHAad, o PVH galanin positive control (n = 11 total: 3 galanin/orexin/MCH; 4 GABA, glutamate, CaMKIIα; 4 VGLUT2/PV/GAD65/67)

Fig. 4

Neurocircuitry of the LHAad using retrobead injections. a Injections into the LHAad resulted in dense retrobead expression in the prelimbic PFC (b, c). Injections into the VTA (d) resulted in moderate expression in the LHAad (e). Injections into the (f) prelimbic PFC resulted in moderate expression in the LHAad (g). Injections into the h nucleus accumbens i resulted in minimal expression in the LHAad. j Quantification of the density of RB+ cells/mm². k Summary of LHAad neurocircuitry receiving robust prelimbic PFC (thicker arrow) inputs, while providing moderate outputs (thin arrows) to the prelimbic PFC and VTA (N = 17 total: 6 VTA; 4 LHAad; 3 prelimbic PFC; 4 NA)