PROJECT SUMMARY MAIN 5 Alcohol consumption during early pregnancy is a common occurrence in our society. Unfortunately, prenatal alcohol exposure increases the risk of onset of alcohol consumption to problematic levels earlier in adolescence. Both prenatal and adolescence periods are critical windows for neurodevelopment and insults to the developing nervous system, such as alcohol exposure, may dysregulate typical neurogenesis and synapse formation, ultimately resulting in behavioral maladaptations such as augmented anxiety-like behavior and pathological pain perception. The pain and the addiction circuit overlap with many mesocortical interactions. The anterior cingulate cortex (ACC) is at the intersection of addiction and pain circuit and receives dopamine projections from the ventral tegmental area. Dopamine in the ACC contributes to the excitation/inhibition balance within layer 2/3 in the ACC and is affected by chronic ethanol exposure during adolescence in the nucleus accumbens in a sex- and age of exposure-dependent manner. Interestingly however, changes in cortical dopamine and the resulting shift in excitation/inhibition balance is unknown following prenatal ethanol exposure (PAE), adolescent intermittent ethanol exposure (AIE), or double-hit with PAE+AIE exposure. Therefore in Main Project 5, we will elucidate the effect of PAE alone, AIE alone, and the combination of the two (PAE+AIE) on the interplay between dopamine and glutamate and the resulting changes in affective behavior and mechanical nociception. Our overarching hypothesis is that PAE+AIE exposure synergistically augments anxiety-like behaviors and mechanical allodynia in male and female rats via altered interactions between glutamate and dopamine in the ACC. Specifically, in Aim 1 we will examine the excitability of glutamate neurons and dopamine terminal excitability in the ACC during protracted abstinence from PAE, AIE, and PAE+AIE exposures using electrophysiology and fast scan cyclic voltammetry (FSCV), respectively. We predict that PAE+AIE with reduce glutamate and dopamine transmission significantly more than either PAE or AIE alone. In Aim 2, we will determine the impact of subsequent ethanol exposure on overall neural activity and glutamate and dopamine transmission in ACC layer 2/3 using electrophysiology and FSCV, respectively. We expect to find a sensitized response to acute ethanol with respect to glutamate and dopamine transmission in PAE+AIE exposed rats compared to rats with single (PAE or AIE) exposure. Finally, in Aim 3 we will determine a causal link between ACC layer 2/3 neuron activity and/or dopamine and anxiety-like behavior and mechanical allodynia using chemogenetics. We predict that dysregulation in both glutamate and dopamine will contribute to the pathological pain perception and augmented anxiety-like behavior in PAE+AIE exposed rats. Collectively, this proposal will identify the impact of alcohol exposure during two developmentally critical wind...