The basolateral amygdala (BLA) is critical to the development of alcohol use disorder (AUD). Its unique position within reward- and aversion-related circuitry allows it to regulate alcohol intake and withdrawal-induced anxiety. Glutamatergic pyramidal neurons drive these behavioral responses such that higher pyramidal neuron excitability facilitates anxiety and reward-seeking. Pyramidal neurons receive glutamatergic inputs from midline structures like the medial prefrontal cortex through the stria terminalis (ST) and more lateral cortical areas send glutamatergic inputs through the external capsule (EC). GABAergic lateral paracapsular cells (LPCs) and ‘local’ interneurons regulate pyramidal neurons. The goal of my project is to examine how chronic intermittent ethanol and withdrawal (CIE/WD) dysregulates BLA neurons projecting to reward and aversion-related regions to understand the mechanisms driving increased alcohol intake. The nucleus accumbens (NAC) is involved in reward-seeking while the bed nucleus of the stria terminalis (BNST) regulates anxiety. Therefore, my project will use BLA-NAC neurons as ‘reward’ neurons and BLA-BNST neurons as ‘aversion’ neurons. Our laboratory has shown that WD increases glutamatergic function through pre- and postsynaptic mechanisms at ST inputs and EC-BLA synapses, respectively. My preliminary data reveal that WD increases glutamate release from ST inputs onto BLA-BNST neurons regardless of sex, whereas the WD-mediated increase in postsynaptic function at EC-BLA synapses is both projection- and sex-dependent. My preliminary data also indicate that WD alters the excitability of BLA-NAC and BLA-BNST neurons when GABAergic transmission is blocked. In BLA-NAC neurons, females have higher basal excitability and only males increase excitability after CIE/WD. In BLA- BNST neurons, WD increases excitability, regardless of sex. Blocking glutamatergic and GABAergic transmission abolished the increase in excitability, emphasizing the role of glutamatergic transmission in WD- mediated hyperexcitability. This proposal will continue to investigate how WD impacts ‘reward’ and ‘aversion’ BLA neurons. Specific Aim 1 will employ electrophysiology and retrograde labeling to examine GABAergic function in synapses with BLA-NAC and BLA-BNST neurons. Laboratory data reveal that WD suppresses LPC GABA release and postsynaptic GABAergic function in ‘local’ interneuron synapses. Moreover, reduced LPC GABA release is unique to males. Specific Aim 1 will also examine structural plasticity in GABAergic synapses with BLA-NAC and BLA-BNST neurons using immunohistochemistry, confocal microscopy, and retrograde labeling. Specific Aim 2 will combine behavioral approaches and chemogenetics to inhibit specific BLA circuits and evaluate their role in post-CIE alcohol intake. These experiments will establish that ‘reward’ and ‘aversion’ BLA neurons undergo distinct neurophysiological and structural changes, leading to increased alcohol intake afte...