Functional role for suppression of the insular-striatal circuit in modulating interoceptive effects of alcohol

Abstract

The insular cortex (IC) is a region proposed to modulate, in part, interoceptive states and motivated behavior. Interestingly, IC dysfunction and deficits in interoceptive processing are often found among individuals with substance-use disorders. Furthermore, the IC projects to the nucleus accumbens core (AcbC), a region known to modulate the discriminative stimulus/interoceptive effects of alcohol and other drug-related behaviors. Therefore, the goal of the present work was to investigate the possible role of the IC ➔ AcbC circuit in modulating the interoceptive effects of alcohol. Thus, we utilized a chemogenetic technique (hM4D_i designer receptor activation by designer drugs) to silence neuronal activity in the IC of rats trained to discriminate alcohol (1 g/kg, IG) versus water using an operant or Pavlovian alcohol discrimination procedure. Chemogenetic silencing of the IC or IC ➔ AcbC neuronal projections resulted in potentiated sensitivity to the interoceptive effects of alcohol in both the operant and Pavlovian tasks. Together, these data provide critical evidence for the nature of the complex IC circuitry and, specifically, suppression of the insular-striatal circuit in modulating behavior under a drug stimulus control.

Keywords: accumbens; drug discrimination; insula.

Figure 1. hM4D-DREADD validation for chemogenetic silencing of IC

(a) Representative m-Cherry immunofluorescence in IC (2X, 2 mm scale bar) and (b) IC neurons (10X, 100 μm scale bar) following stereotaxic injection of AAV-hSyn-DIO-hM4D_i-mCherry+Cre into IC. (c) Decreased membrane potential in IC neurons, (d) demonstrated by representative traces of neuronal firing and (e) neuronal silencing following bath application of clozapine-n-oxide (CNO; 10 μM). (f) Co-localization of mCherry with neuronal marker NeuN demonstrates dense co-localization (marked by white circles), and (g) no co-localization with the glial marker GFAP (40X, 100 μm scale bar).

Figure 2. Chemogenetic silencing of IC increases sensitivity to the interoceptive effects of alcohol in an operant alcohol discrimination task

(a) Schematic diagram of test session. (b) Representative intra-IC hM4D-mCherry expression (2X, 1 mm scale bar) (c) with schematic demonstrating individual bilateral expression in discrimination-trained rats. (d) Silencing of IC, by CNO, increased the percentage of alcohol-appropriate responses, with partial substitution (>40%) for the alcohol training dose observed at the lowest alcohol dose (0.1 g/kg) in the hM4D group. (e) CNO did not affect response rate (responses/min). Dashed line (>80%) represents full expression of the discriminative stimulus effects of alcohol. *Significant main effect of CNO treatment (three-way ANOVA, p≤0.05). Values on graphs represent mean ± S.E.M.

Figure 3. Chemogenetic silencing of IC→AcbC projections increases sensitivity to low alcohol doses in an operant alcohol discrimination task

(a) Schematic diagram of test. (b) Representative intra-IC hM4D-mCherry expression and (c) intra-IC mCherry-Control expression (2X, 1 mm scale bar) with schematics demonstrating individual bilateral expression in discrimination-trained rats, respectively. (d) Representative bilateral AcbC injector tip placements from individual discrimination-trained rats in hM4D (depicted as red circles) or Control (depicted as blue circles) groups. (e–f) Corresponding photomicrographs (4X, 200 μm scale bar) showing an injector tip (arrow) for each group, respectively. (g) Infusion of CNO into AcbC increased percentage of alcohol-appropriate responses following 0.3 and 0.5 g/kg alcohol in the hM4D group. (h) Response rate (responses/min) was unaffected by CNO or alcohol dose. Dashed line (>80%) represents full expression of the discriminative stimulus effects of alcohol. *Significant difference from Vehicle in hM4D group (p<0.05). Values on graphs represent mean ± S.E.M.

Figure 4. Chemogenetic silencing of IC substitutes for the interoceptive effects of alcohol in a Pavlovian alcohol discrimination task

(a) Schematic diagram of test. (b) Representative intra-IC hM4D-mCherry expression and (c) intra-IC mCherry-Control expression (2X, 1 mm scale bar) with schematics demonstrating individual bilateral expression in discrimination-trained rats, respectively. (d) Silencing of IC, by CNO, following water increased the discrimination score (head entries into the liquid receptacle during the 15-s light CS minus head entries 15 s before light onset) in the hM4D group. (e) Locomotor rate (beam breaks/min) was increased with alcohol dose. *Significant difference from Vehicle in hM4D group (p<0.05), #Significant main effect of alcohol (three-way ANOVA, p<0.05). Values on graphs represent mean ± S.E.M.

Figure 5. Chemogenetic silencing of IC→AcbC projections substitutes for the interoceptive stimulus effects of alcohol in a Pavlovian alcohol discrimination task

(a) Schematic diagram of test. (b) Representative bilateral AcbC injector tip placements from individual discrimination-trained rats in hM4D (depicted as red circles) or Control (depicted as blue circles) groups. (c–d) Corresponding photomicrographs (4X, 200 μm scale bar) showing an injector tip (arrow) for each group, respectively. (e) Infusion of CNO into AcbC following 0.1 g/kg alcohol, increased the discrimination score (head entries into the liquid receptacle during the 15-s light CS minus head entries 15 s before light onset) in the hM4D group. (f) Locomotor rate (beam breaks/min) was increased with alcohol dose. *Significant difference from Vehicle in hM4D group, #Significant main effect of alcohol (three-way ANOVA, p<0.05). Values on graphs represent mean ± S.E.M.