Endocannabinoid control of the insular-bed nucleus of the stria terminalis circuit regulates negative affective behavior associated with alcohol abstinence

Abstract

Negative affect is a core symptom domain associated with an array of neurological and psychiatric disorders and is only partially targeted by current therapies, highlighting the need for better, more targeted treatment options. This study focuses on negative affective symptoms associated with prolonged alcohol abstinence, one of the leading causes of relapse. Using a mouse model of chronic alcohol consumption followed by forced abstinence (CDFA), prolonged alcohol abstinence increased c-fos expression and spontaneous glutamatergic neurotransmission in the dorsal bed nucleus of the stria terminalis (dBNST), a region heavily implicated in negative affect in both humans and rodents. Further, pharmacologically enhancing endogenous cannabinoids (eCB) with JZL184 prevents abstinence-induced increases in dBNST neuronal activity, underscoring the therapeutic potential of drugs targeting the brain's eCB system. Next, we used a channelrhodopsin-assisted mapping strategy to identify excitatory inputs to the dBNST that could contribute to CDFA-induced negative affect. We identified the insular cortex (insula), a region involved in regulating interoception, as a dense, functional, eCB-sensitive input to the dBNST. Using a chemogenetic strategy to locally mimic eCB signaling, we demonstrate that the insula strongly influences the CDFA behavioral phenotype and dBNST neuronal activity. Lastly, we used an anterograde strategy for transynaptic targeting of Cre expression in combination with a G_q-DREADD to selectively recruit dBNST neurons receiving insula projections. Chemogenetic recruitment of these neurons mimicked behavioral and c-fos responses observed in CDFA. Collectively, this study supports a role for the insula-BNST neural circuit in negative affective disturbances and highlights the therapeutic potential of the eCB system for treating negative affective disorders.

Fig. 1

Chronic-drinking followed by forced abstinence increases neuronal activity in the dBNST. a Chronic-drinking forced abstinence (CDFA) model. b Male and female C57BL/6J mice maintain a preference for ethanol over water throughout CDFA. c Female mice consume significantly more ethanol (g/kg/day) than male mice throughout CDFA (repeated measures two-way ANOVA, Sidak’s post hoc **p < 0.01, ***p < 0.001, ****p < 0.0001; n = 6–11/group; data presented as mean ± SEM). d Representative image of c-fos protein staining in the dBNST in male and female mice. e Male and female mice have increased c-fos expression in the dBNST 15-days into forced abstinence (two-way ANOVA, Sidak’s post hoc *p < 0.05). Data presented as cells per 10× dBNST image per hemisphere and averaged per animal (presented as mean ± SEM). f Representative RNAScope® image showing DAPI nuclear stain (gray), Fos transcripts (green), and Crh transcripts (red). Left: Crh+/Fos− dBNST neuron. Middle: Crh−/Fos− dBNST neuron. Right: Crh+/Fos+ dBNST neuron. White arrows represent Fos transcripts localized on BNST^CRF neurons. g The percentage of BNST^CRF neurons that express the Fos transcript is increased in CDFA mice 15-days into forced abstinence (Tukey’s multiple comparison post hoc **p < 0.01). Data presented as cells per 3 paneled 63× BNST images per hemisphere in one slice and averaged per animal (presented as individual data points and mean ± SEM). h The total number of BNST^CRF neurons is similar in both the water and ethanol groups. i Representative electrophysiology trace in ethanol-naive mice and CDFA-mice 15-days into forced abstinence. j sEPSC frequency in all BNST neurons recorded from. BNST cells from ethanol-exposed mice have a significantly higher sEPSC frequency relative to controls (Tukey’s multiple comparison post hoc **p < 0.01). Data presented individual data points with mean ± SEM. k sEPSC frequency in BNST^CRF neurons. CRF cells were identified from CRF-tomato mice. BNST^CRF cells from ethanol-exposed mice have a significantly higher sEPSC frequency relative to controls (Tukey’s multiple comparison post hoc **p < 0.01). Data presented individual data points with mean ± SEM

Fig. 2

Insula inputs onto dBNST^CRF neurons are endocannabinoid sensitive. a sEPSC frequency in dBNST neurons in the presence of vehicle (0.1% (v/v) DMSO), JZL184 (1 µM), or JZL184+rimonabant (5 µM). Pretreating slices with JZL184 for at least 1 h significantly decreased sEPSC frequency (one-way ANOVA treatment effect, *p < 0.05; Tukey’s multiple comparison post hoc *p < 0.05) and JZL184+rimonabant pretreatment had no effect on sEPSC frequency (Tukey’s multiple comparison post hoc p = 0.975). Data presented individual data points with mean ± SEM. b Neither JZL184 alone nor JZL184+rimonabant had an effect on sEPSC amplitude (one-way ANOVA; p = 0.284). Data presented individual data points with mean ± SEM. c Representative electrophysiology trace from ethanol naive mice and CDFA mice 15 days into forced abstinence. Slices were treated with either vehicle (0.1% (v/v) DMSO) or JZL184 (1 µM). d Pretreating BNST slices for at least 1 h in JZL184 decreases sEPSC frequency in ethanol-naive mice (two-way ANOVA treatment effect, ***p < 0.001; Tukey’s multiple comparison post hoc, **p < 0.01) and prevents CDFA-induced increase in sEPSC frequency (drug effect, ***p < 0.001; Tukey’s multiple comparison post hoc **p < 0.01). Data in gray is presented and described in Fig. 1. Data presented individual data points with mean ± SEM. e Pretreating BNST slices from CRF-tomato mice in JZL184 decreases sEPSC frequency in ethanol-naive mice (two-way ANOVA treatment effect, ***p < 0.001; Tukey’s multiple comparison post hoc, **p < 0.001) and prevents CDFA-induced increase in sEPSC frequency (dark green line; drug effect, ****p < 0.0001, Tukey’s multiple comparison post hoc ***p < 0.001). Data in gray is presented and described in Fig. 1. Data presented individual data points with mean ± SEM. f Schematic for CRF-tomato mouse breeding, ChR2 injection in the insula, and ChR2-evoked optical EPSCs in the BNST. Atlas image from Allen Brain Mouse Atlas [73]. g Representative image of CRF cells in the dBNST (red) and ChR2-labeled insula fibers in the dBNST (green). h Blue light stimulation evoked an optical EPSC in dBNST^CRF neurons that was significantly decreased after 10-min bath application of WIN55,212-2 (4 µM; Student’s paired t-test, ***p < 0.001). Data presented as individual cells before and after 10 min of WIN55,212-2. i Representative 10× image of c-fos protein staining in the caudal-anterior insula from water mice and ethanol mice 15-days into abstinence. Atlas image from Allen Brain Mouse Atlas [73]. j Mice 15-days into abstinence exhibit increased caudal-posterior insula c-fos expression relative to water control mice (Student’s unpaired t-test, **p < 0.01). Data presented as cells per 10× caudal-posterior insula image per hemisphere and averaged per animal (presented as individual data points and mean ± SEM)

Fig. 3

Chemogenetic inhibition of insula neurons reduces CDFA-induced increase in neuronal activity. a Timeline for insula hM4Di RNAScope® experiment. b Ethanol preference for all insula hM4Di mice treated with either saline (light green) or CNO (dark green). c Ethanol consumption (g/kg/day) for all insula hM4Di mice treated with either saline (light green) or CNO (dark green). d Total number of Fos+ BNST neurons. CDFA significantly increases Fos expression in the BNST 15-days into abstinence (one-way ANOVA, **p < 0.01; Tukey’s multiple comparison post hoc, **p < 0.01). CNO (3 mg/kg) activation of the insula hM4Di significantly reduced Fos expression in the BNST 15-days into ethanol abstinence (Tukey’s multiple comparison post hoc, **p < 0.01). e CNO activation of the hM4Di in the insula significantly reduced the percentage of Fos+ BNST^CRF neurons 15-days into ethanol abstinence (one-way ANOVA, *p < 0.05; Tukey’s multiple comparison post hoc, *p < 0.05). Data in gray is presented and described in Fig. 1. f Schematic for electrophysiology experimental timeline. hM4Di was injected into both caudal-anterior insulae prior to CDFA. sEPSC were recorded in BNST neurons before and after bath application of CNO (10 µM). g sEPSC frequency is significantly increased 15-days into EtOH abstinence. Bath application of CNO significantly reduced sEPSC frequency in ethanol abstinent mice but not in water mice (two-way ANOVA; treatment effect **p < 0.01; drug effect *p < 0.05; Sidak’s multiple comparison post hoc, **p < 0.01). Data presented as individual data points before and after drug application. h CDFA had no effect on sEPSC amplitude in BNST neurons. CNO did not alter sEPSC amplitude

Fig. 4

Chemogenetic inhibition of insula neurons decreases abstinence-induced negative affect. a Experimental timeline for CDFA behavioral experiments in (d) and (f). b CNO (3 mg/kg) in the absence of hM4Di has no effect on CDFA-induced increase in latency to first bite on NSFT (two-way ANOVA treatment effect **p < 0.01, Tukey’s multiple comparison post hoc water-sal versus water-CNO, p = 0.773; ethanol-sal versus ethanol-CNO, p = 0.991). c Latency to feed on NSFT is not different between ethanol-naive mice, No-hM4Di, CNO-treated (3 mg/kg) mice (Ctl-CNO), ethanol-naive insula hM4Di, saline-treated mice (hM4Di-Sal), and ethanol-naive insula hM4Di, CNO-treated (3 mg/kg) mice (hM4Di-CNO). d CNO administered to insula hM4Di mice 15-days into the abstinence phase of CDFA results in a significant reduction in latency to first bite on NSFT (Student’s unpaired t-test, *p < 0.05). e Forced swim test immobility time is not different between ethanol-naive mice, No-hM4Di, CNO-treated mice (Ctl-CNO), ethanol-naive insula hM4Di, saline-treated mice (hM4Di-Sal), and ethanol-naive insula hM4Di, CNO-treated mice (hM4Di-CNO). f Immobility time during FST was significantly decreased in CNO-treated insula hM4Di mice relative to saline-treated mice (Student’s unpaired t-test, *p < 0.05). All data presented as individual data points with mean ± SEM

Fig. 5

Chemogenetic activation of dBNST neurons receiving projections from the insula produces a negative affect phenotype. a Anterograde transsynaptic Cre (AAV1.Cre) was injected into both caudal-anterior insulae. Cre-dependent hM3Dq (AAV5.DIO.hM3Dq) was injected into both dBNST. b Representative 4× (left) and 20× image of DIO.hM3Dq (red) and c-fos (green) expression in the dBNST. c CNO (3 mg/kg) significantly increased latency to first bite in the AAV1.Cre/hM3Dq mice relative to the no-virus control groups and AAV1.Cre/hM3Dq-saline group (Tukey’s multiple comparison post hoc, **p < 0.01). d CNO (3 mg/kg) significantly decreased 10′ post-NSFT home cage consumption in AAV1.Cre/hM3Dq mice relative to the no-virus control groups and AAV1.Cre/hM3Dq-saline group (Tukey’s multiple comparison post hoc, **p < 0.01, ****p < 0.0001). e CNO (3 mg/kg) significantly increased BNST c-fos+ neurons in the AAV1.Cre/hM3Dq mice relative to the no-virus control groups and AAV1.Cre/hM3Dq saline group (Tukey’s multiple comparison post hoc, **p < 0.01). Data presented as cells per 10× dBNST image per hemisphere and averaged per animal (presented as individual data points and mean ± SEM). f Latency to first bite on NSFT positively correlates with number of c-fos+ dBNST neurons (R² = 0.403, **p = 0.001). Data presented as linear regression best fit line with 95% confidence interval bands