Michael C. Salling, Ph.D. - US grants

DESCRIPTION (provided by applicant): Alcohol addiction is a disease that can negatively affect people's relationships, productivity and can lead to major health problems including death. It is estimated that 17.6 million Americans suffer from alcohol abuse or alcoholism yet there are very few effective treatments available signifying the need to better understand how alcohol affects the mammalian brain. This goal of this proposal is to better understand the molecular mechanisms that mediate alcohol consumption and relapse. It is widely accepted that alcohol consumption leads to neuroadaptive changes in intracellular signaling cascades that regulate neuroplasticity. Brain circuits that contribute to alcohol seeking behaviors are particularly vulnerable and their dysregulation may mediate behavior pathologies associated with alcohol addiction. One brain region of interest is the amygdala, which regulates alcohol reinforcement and reward- related learning. Preliminary data identified the alpha subunit of calcium/calmodulin kinase II (CAMKII) as a calcium-signaling protein kinase that is upregulated in the amygdala following chronic alcohol self- administration in C57BL/6J mice. CAMKII modulates receptor activity and initiates multiple transcription factors that control neural plasticity, yet the functional involvement of CAMKII in alcohol self-administration remains unknown. This project seeks to test the overall hypothesis that CAMKII is increased following alcohol self-administration and that modulating CAMKII activity functionally regulates alcohol-seeking behaviors in three separate but integrated specific aims: 1) to characterize the effect of chronic alcohol self- administration on the CAMKII pathway using western blot and immunohistochemical analysis;2) to examine the effect of operant alcohol self-administration on CAMKII activity and determine if CAMKII functionally regulates the reinforcing effects of alcohol in mice using site-specific infusions of a pharmacological CAMKII inhibitor into the amygdala;and 3) to determine if CAMKII in the amygdala functionally regulates cue- induced reinstatement of alcohol-seeking, a behavioral model of relapse, using site-specific infusions of the CAMKII inhibitor. These preclinical experiments have the potential to reveal a novel molecular mechanism that underlies alcohol reinforcement and relapse, behavioral pathologies that characterize alcohol addiction.

DESCRIPTION (provided by applicant): The consequences of alcohol abuse on the American public are profound, both in terms of individual well-being and impact on the family structure, as well as the enormous cost to society in terms of lost productivity and associated health care expenses. Despite increasing efforts, our understanding of the neurobiological mechanisms that underlie the effects of alcohol and the development of alcohol use disorders remains incomplete. Epidemiological research has pointed to adolescence as a critical period in the development of alcohol disorders. One area of the brain that appears especially vulnerable to the effects of alcohol is the prefrontal cortex (PFC), which remains immature at the onset of adolescence. The PFC is involved in decision- making, memory and impulse control, behaviors that are often disrupted following consumption of alcohol and are believed to underlie long-term addiction. In the PFC, pharmacologically relevant doses of alcohol are known to depress neuronal activity (Tu et al. 2007), but very little is known of its interactions with the inhibitor receptors, GABA and glycine, within the PFC. This is despite the expression of GABA-A receptor subtypes in the PFC (Hoestgaard-Jensen et al. 2010) that mediate tonic inhibition in other brain regions and demonstrate high sensitivity to alcohol (Wei et al. 2004, Jia et al. 2008). In addition, we note that functional glycine receptors are also present in PFC (Ye et al. 2011) but have received little to no attention. The goal of this research proposal is to examine the pharmacological actions of alcohol on neuronal excitability and on GABA and glycine-mediated synaptic and tonic inhibition in the adolescent PFC, and to determine if chronic alcohol drinking during adolescence leads to specific alterations of inhibition that affect neuronal excitability. Tis information could enhance our understanding of the negative effects of alcohol drinking during adolescence and an increased focus on cellular mechanisms involved can help lead to the development of therapeutic strategies to treat alcohol use disorders. PUBLIC HEALTH RELEVANCE: Drinking alcohol during adolescence is a major public health concern as it often leads to alcohol and substance abuse disorders later in life. The goal of this proposal is to better understand the effects of alcohol on the adolescent brain which will be accomplished by measuring changes in brain cell activity in animals that drink alcohol during adolescence. Findings from this work will raise awareness on alcohol vulnerability during adolescence and could lead to the development of medicines that treat alcohol use disorders.

PROJECT SUMMARY Excessive alcohol consumption has widespread personal and societal consequences, negatively affecting individual health while creating a significant economic and legal burden. Despite increasing efforts over the last few decades to identify genetic influences and neuroadaptations that are associated with the development of alcohol use disorders (AUDs), progress has been limited by the complexity of the underlying neuroanatomy. It is clear that the experimental resolution needs to be improved to the level of identifying adaptations in specific neural circuits that underlie dissociable behaviors related to AUD pathology. Thus, the goal of my research is to identify the neuroadaptations resulting from adolescent binge drinking that perpetuate heavy drinking and cognitive deficits in adulthood. I have focused my research on the prefrontal cortex (PFC), as this region continues to develop during adolescence and may be vulnerable to heavy alcohol consumption. For instance, PFC dysfunction is observed in binge drinkers and likely contributes to compulsive alcohol drinking and cognitive deficits observed in AUDs. In mice, I have found that binge drinking during adolescence disrupts performance on a PFC-dependent working memory task, increases alcohol consumption in adulthood, and significantly alters the intrinsic excitability of PFC pyramidal neurons. Discerning the mechanisms underlying these effects requires the use of tools capable of detecting physiological changes in specific neural circuits, as well as the ability to modulate their activity during behavioral analyses. In the mentored phase (K99) of this proposal, I will learn to use viral genetic strategies to visualize PFC projections affected by binge drinking and characterize the circuit-specific changes in excitability following adolescent binge drinking using ex vivo electrophysiology. Further, I will be trained to modulate PFC activity during behavior using chemogenetics, toward the goal of determining the specific role of these prefrontal pathways in binge-drinking and working memory. In the R00 phase, I will utilize a newly developed system for gaining permanent genetic access to neuronal ensembles that are active during defined behaviors (FosTRAP). In combination with the techniques learned in the mentored phase, I will use this technique to identify, characterize and modulate neuronal ensembles engaged in binge-like alcohol consumption. Taken together, the experiments in this proposal were designed to test the overarching hypothesis that adolescent binge drinking differentially affects the intrinsic excitability of medial PFC pyramidal neuron subpopulations and that these subpopulations play separate roles in binge alcohol consumption and working memory. The proposed experiments will integrate my previous training in behavioral pharmacology, immunohistochemistry and electrophysiology with new viral genetic strategies to identify and manipulate neural circuits. This training plan, in combination with guidance provided by my mentors, will greatly improve my ability to answer important research questions regarding the neurobiology of alcoholism while promoting my transition into an independent research scientist.