Optogenetic characterization of corticotropin-releasing factor pathways in the central nucleus of the amygdala in alcohol dependence

In alcohol-dependent animals, withdrawal from alcohol activates a neuronal ensemble in the central nucleus of the amygdala (CeA) that is responsible for the escalation of alcohol drinking. However, the neuronal phenotype and neuronal pathways that are controlled by these neurons are unknown. We investigated the cellular identity of this CeA neuronal ensemble and found that most neurons expressed corticotropin-releasing factor (CRF). Using Crh-Cre transgenic rats combined with in vivo optogenetics, we tested whether the inactivation of CeA CRF neurons prevents excessive alcohol self-administration during withdrawal. Rats were injected with AAV-DIO-NpHR-eYFP or AAV-DIO-eYFP (control) and implanted with optical fibers in the CeA. The animals were then exposed to chronic intermittent ethanol to induce alcohol dependence. The inactivation of CeA CRF neurons decreased alcohol drinking in dependent rats to levels that were observed before the animals became dependent and completely prevented activation of the entire CeA neuronal ensemble (Fos+ neurons) during withdrawal. No effect was observed in the AAV-DIO-eYFP control group or on water or saccharin self-administration in either group. In a second experiment, the rats were injected with AAV-DIO-NpHR-eYFP in the CeA, and optical fibers were implanted into downstream projection regions of CeA CRF neurons, including the bed nucleus of the stria terminalis (BNST), lateral hypothalamus (LH), parasubthalamic nucleus (pSTN), substantia innominata (SI), and parabrachial nuclei (PBN). The optogenetic inactivation of CRF terminals in the BNST recapitulated the effect on alcohol drinking that was observed with the inactivation of CeA CRF cell bodies, whereas inactivation of the LH, SI, and PBN had no effect. These results indicate that the activation of CeA CRF neurons during withdrawal is required for recruitment of the CeA neuronal ensemble that is responsible for excessive alcohol drinking in dependent rats. The CRF projection to the BNST but not LH, pSTN, SI, or PBN appears to be the main downstream pathway that is responsible for excessive alcohol drinking in dependent rats.