Title: A Subset of VTA DA Neurons Demonstrates High Sensitivity to Acute Ethanol and Enhanced Sensitivity after Adolescent Drinking
Description: Ethanol (EtOH) is a commonly used drug which exerts many of its effects by altering neurotransmission in the mesolimbic dopamine (DA) system. Although there is little debate that EtOH acts to increase the activity of DA neurons in the ventral tegmental area (VTA), and that this action is necessary for some of the reinforcing effects of EtOH, research in vitro has only been able to demonstrate an excitatory effect on VTA DA neurons in response to very high concentrations of EtOH. These concentrations, typically in the range of 50-100 mM, correspond to sedative or lethal levels for typical humans. Therefore, the significance of findings from in vitro experiments can be difficult to interpret. We sought to determine why high concentrations of EtOH are needed in vitro and whether this could be explained by simple experimental factors, including cytosolic washout from whole cell electrophysiological recordings; heterogeneity among VTA DA neurons, where previous studies may have inadvertently focused on an EtOH-insensitive population; or selection of animal population, where perhaps low EtOH response is characteristic in naïve, rather than EtOH-experienced, animals.

To achieve this, we performed cell-attached recordings on a large number of midbrain DA neurons of EtOH-naïve and experienced mice. We report evidence for a highly EtOH-responsive, medially located population of VTA DA neurons. These neurons, found within the rostral linear and interfascicular nuclei and considered “atypical” in terms of physiological criteria ascribed to DA neurons, exhibited a concentration-dependent increase of firing activity in response to EtOH, with some neurons responsive to as little as 20 mM EtOH. In contrast, DA neurons in the lateral VTA and substantia nigra were either unresponsive or responded only to 100 mM EtOH. We then examined neuronal activity following adolescent binge-like alcohol drinking in mice, to determine whether EtOH experience drives increased EtOH sensitivity of DA neurons. We find that in medial VTA DA neurons, drinking experience greatly increased firing activity driven by subsequent exposure to EtOH itself, without altering other measures of intrinsic excitability.

This enhanced sensitivity was no longer significant in the presence of glutamate receptor blockade. We attempted to further characterize the EtOH-sensitive, medially located VTA DA neurons by utilizing retrograde tracing to identify a population of nucleus accumbens medial shell-projecting neurons. We find that this population exhibits an increased sensitivity to 50 mM EtOH after adolescent drinking. As a result of these experiments, we have identified a previously uncharacterized, highly EtOH-responsive population of DA neurons in the medial VTA. This population demonstrates an excitatory response to 10 and 20 mM EtOH, concentrations which are more pharmacologically relevant than those typically tested in vitro. We further demonstrate evidence for experience-induced neural adaptations which result in enhanced sensitivity to EtOH in vitro. These adaptations are only apparent in medial VTA DA neurons, and this phenomenon only occurs in response to adolescent drinking. These data provide evidence for a novel form of plasticity in which neurons respond to a primary reinforcer, in this case EtOH, after drinking experience.

These findings provide an anatomical and pharmacological distinction between DA neuron subpopulations that will facilitate future mechanistic studies on the actions of EtOH in the VTA.