6/8 NADIA U01 Adolescent Alcohol and Prefrontal Cortical Function in the Adult
Research Component 6/8: Adolescent Alcohol and Prefrontal Cortical Function in the Adult
Principal Investigator: Judson Chandler, Ph.D., Medical University of South Carolina
The consumption and abuse of alcohol during adolescence is a serious public health problem. In this age group, alcohol is often consumed in large quantities within repeated binge-like episodes that result in high levels of intoxication. In addition to legal ramifications and concerns with physical safety, these patterns of alcohol consumption appear to adversely impact continued brain and behavioral maturation during the transition from adolescence to adulthood. The prefrontal cortex (PFC) controls higher-order cognitive functions such as working-memory and behavioral flexibility. Adolescence represents a critical period of refinement of PFC neurocircuitry that supports maturation of executive cognitive functioning. This research component of the NADIA consortium will test the overarching hypothesis that AIE-induced deficits in cognitive control in adulthood are associated with alterations in DA neurotransmission in the PFC. This hypothesis is based upon previous studies demonstrating AIE-induced deficits in PFC-mediated behaviors and alterations in expression and function of prefrontal DA. The proposed studies are designed to establish a direct link between AIE-induced altered DA signaling and behavioral impairments, and further test the hypothesis that epigenetic alterations in gene expression are a primary mechanism underlying AIE-induced cognitive deficits. The proposed studies involve the following four specific aims: Aim 1 will test the hypothesis that alterations in activity of DA D1 receptor-expressing neurons in the mPFC contribute to AIE-induced deficits in behavioral flexibility. Aim 2 will test the hypothesis that DNA hypermethylation in the mPFC underlies AIE-induced cognitive deficits. Aim 3 will test the hypothesis that normalization of DNA hypermethylation will reverse AIE-induced alterations in structural plasticity in the mPFC. Aim 4 will test the hypothesis that AIE disrupts the in-growth of VTA-DA axons from the nucleus accumbens to the mPFC. These studies involve an innovative and multidisciplinary set of experiments that utilize state-of-the-art methodologies and procedures. Together, these studies will yield novel and exciting new findings and will significantly advance our understanding of the effect of adolescent alcohol exposure on cognitive function and behavioral control in the adult, and identify novel therapeutic approaches for treatment.
Adolescence is a critical period of continued brain development that is especially important for the maturation of higher-order cognitive functions that depend upon the prefrontal cortex (PFC). Evidence suggests the delayed developmental trajectory of the PFC may render it particularly vulnerable to adverse environmental insults during adolescence, including those associated with repeated binge-like episodes of alcohol intoxication. In support of this, studies carried out across the NADIA consortium using animal models have revealed that adolescent intermittent ethanol (AIE) exposure results in impaired cognitive abilities in adulthood. The mesocortical dopamine (DA) system is unique among neurotransmitters and neuromodulators in that DA axonal innervation and receptor expression in the medial PFC (mPFC) is delayed and does not reach peak levels until adolescence, at which time it then undergoes dramatic refinement in parallel with maturation of the executive function of the PFC. Importantly, disruption of this refinement in the mPFC during adolescence results in impaired cognitive control in adulthood that is characteristic of a number of neurodevelopment disorders, such as schizophrenia. We have shown that adult rats that had been subjected to AIE exposure exhibit alterations in DA neurotransmission and cognitive flexibility. Studies outlined in this component of the NADIA consortium will test the overarching hypothesis that AIE-induced deficits in cognitive control in adulthood result from alterations of DA neurotransmission in the mPFC. The research approach will investigate hypermethylation as an epigenetic mechanism linking long-term changes in prefrontal function with adolescent alcohol abuse and involves collaborative integration across multiple NADIA components and the Epigenetic Core. The overarching hypothesis of this proposal will be tested through the following set of specific aims.
SPECIFIC AIM 1: TEST THE HYPOTHESIS THAT ALTERATIONS IN ACTIVITY OF DA D1 RECEPTOR-EXPRESSING NEURONS
IN THE MPFC CONTRIBUTE TO AIE-INDUCED DEFICITS IN BEHAVIORAL FLEXIBILITY. We have previously demonstrated that AIE exposure results in alterations in intrinsic excitability and D1 receptor modulation of excitability and synaptic responses of layer 5 pyramidal cells. New preliminary data involving patch-clamp recordings from retrograde bead labeled neurons suggests that these AIE-induced alterations are projection-specific. Studies under this aim will utilize a D1-cre driver line of rats to determine the cell-type and projection-specific effects of AIE exposure on the adult mPFC. The experimental approach involves in-situ patch-clamp slice electrophysiology and projection-specific in-vivo chemogenetic manipulation of neuronal activity to modulate cognitive function of the mPFC.
SPECIFIC AIM 2: TEST THE HYPOTHESIS THAT DNA HYPERMETHYLATION IN THE MPFC UNDERLIES AIE-INDUCED COGNITIVE DEFICITS. Previous observations by NADIA investigators have shown that AIE exposure induces hypermethylation of the promotor region of a number of genes that appear to play key roles in AIE-induced alterations of behavior. The first set of studies under this aim will examine whether treatment of AIE-exposed adult rats with an inhibitor of DNA methylation can reverse the AIE-induced deficits in PFC mediated behaviors. A second set of studies involves a collaboration with the Epigenetic Core to determine the role of COMT promotor methylation in AIE-induced alterations in PFC DA and cognition. For these studies, the Epigenetic Core will use CRISPR/dCas9 technology to produce viral vectors directed at bidirectionally modulating COMT promoter methylation. A third set of complimentary collaborative studies with the Epigenetic Core involve carrying out ATAC-sequencing to identify hub genes and signaling pathways in the mPFC that are epigenetically altered by AIE.
SPECIFIC AIM 3: TEST THE HYPOTHESIS THAT NORMALIZATION OF DNA HYPERMETHYLATION WILL REVERSE AIEINDUCED ALTERATIONS IN STRUCTURAL PLASTICITY IN THE MPFC. It is becoming increasingly clear that glial cells play a critical role in modulating synaptic formation and remodeling in adulthood. Studies under this aim will employ super-resolution confocal imaging and advanced 3D analysis to examine the AIE-induced changes in morphology of microglia and astroglial cells in the mPFC, and assess changes in the physical association of astroglia processes with dendritic spines of D1 neurons. An additional set of studies will examine whether AIE exposure alters the physical association of astroglia process and perineuronal nets with parvalbumin interneurons in the mPFC. Lastly, these studies will determine whether inhibition of DNA methylation can normalize the AIE-induced alterations in structural plasticity.
SPECIFIC AIM 4: TEST THE HYPOTHESIS THAT AIE DISRUPTS THE IN-GROWTH OF VTA-DA AXONS FROM THE NUCLEUS ACCUMBENS TO THE MPFC. Recent studies conducted in mice described a unique population of VTA DA neurons whose axons initially innervate the nucleus accumbens (NAc) during early development, and then grow to the PFC during adolescence. Importantly, this PFC in-growth is critical for adolescent maturation of the executive function, and manipulations to disrupt in-growth result in cognitive deficits similar to those observed following AIE. Our preliminary data demonstrates that rats also undergo a similar adolescent period in-growth of VTA DA axons from the NAc to the mPFC. Studies under this aim will investigate whether AIE exposure alters the adolescent in-growth of the VTA-NAc axon terminals into the mPFC, and determine the impact of chemogenetic manipulations of this projection on AIE-induced cognitive deficits.