Optogenetic characterization of CeA CRF pathways in alcohol dependence

In alcohol dependent animals, withdrawal from alcohol activates a neuronal ensemble in the central nucleus of amygdala (CeA) that is responsible for escalation of alcohol drinking. However, the neuronal phenotype and neuronal pathways controlled by these neurons is unknown. We investigated the cellular identity of this CeA neuronal ensemble and found that most neurons express corticotropin releasing factor (CRF). Using Crh-Cre transgenic rats combined with in vivo optogenetics, we tested if 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. Animals were then exposed to chronic intermittent ethanol (CIE) to induce alcohol dependence. Inactivation of CeA CRF neurons decreased alcohol drinking in dependent rats to levels observed before the animals became dependent, and completely prevented the 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, 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 (pSTN), substantia innominata (SI) or parabrachial nuclei (PBN). Optogenetic inactivation of CRF terminals in the BNST recapitulated the effect on alcohol drinking observed with inactivation of CeA CRF cell bodies, whereas inactivation of the LH, SI or PBN had no effect. Taken together, these results demonstrate that activation of CeA CRF neurons during withdrawal is required for the recruitment of the CeA neuronal ensemble that is responsible for excessive alcohol drinking in dependent rats, and that the CRF projection to the BNST, but not the LH, pSTN, SI, and PBN is the main downstream pathway responsible for excessive alcohol drinking in dependent rats.