Michael J. Beckstead - US grants

Many of the depressant effects caused by alcohol are believed to stem from an enhancement of inhibitory neurotransmission in the central nervous system, specifically the enhancement of GABAA and glycine receptor function. Glycine receptor modulators include a wide variety of chemical compounds including drugs of abuse like alcohol and inhalants, as well as clinically used drugs like volatile anesthetics and pentobarbital. While it is known that these modulators enhance glycine receptor-mediated currents, more specific molecular mechanisms have eluded us. By studying a glycine receptor that activates in the absence of neurotransmitter but retains its ability to be modulated by alcohol, new insights into activation mechanisms could be discovered. A better understanding of ligand-gated ion channel receptor mechanisms could lead to therapeutic agents such as alcoholism treatment regimens and anesthetics with fewer side effects. The work proposed may not only shed insights into the molecular mechanisms of ethanol and other receptor modulators, but also provide a fuller understanding of basic receptor/channel processes.

DESCRIPTION (provided by applicant): Dopamine cells in the ventral midbrain serve a role in a number of critical functions, including motor processes, focused attention, reward and incentive learning. It is not surprising then that the same cells participate as neural substrates of human diseases such as drug addiction, Parkinson's disease, and likely schizophrenia and attention deficit hyperactivity disorder. Dopamine cells exert their main effects distally through projections to brain regions involved in decision making, movement, and emotion. Small amounts of dopamine are also released dendritically near the midbrain nuclei of the ventral tegmental area (VTA) and substantia nigra (SN). Recently it was discovered that this dendritic release may occur through reversal of the dopamine transporter, a widely-distributed membrane-bound protein responsible for the majority of free dopamine uptake from the extracellular space. Dopamine released by this mechanism can activate D2 dopamine receptors on the dopamine cell bodies, inhibiting firing, and thus the release of dopamine in distal brain regions implicated in behavioral processes. While the existence of dopamine transporters and D2 receptors in the VTA and SN suggest a physiological role for dendritic release, the precise synaptic mechanisms have yet to be investigated. This proposal addresses this issue by investigating the mechanisms of dendrodendritic transmission in the VTA. Whole-cell patch clamp electrophysiological technique will be used to record from dopamine neurons in rat midbrain slices. Of particular interest is the elucidation of the role of metabotropic glutamate receptors in the dopamine-induced inhibition of dopamine cell firing. Amphetamine, a drug of abuse known to act on dopaminergic cells, will also be used to attempt to gain and understanding of the importance and role of the dendritic release of dopamine.

DESCRIPTION (provided by applicant): This Mentored Research Scientist Development Award (K01) proposes to investigate the neurophysiological consequences of methamphetamine self-administration while providing the candidate (Dr. Michael Beckstead) with training in behavioral neuroscience. Dr. Beckstead's immediate goals include learning behavioral techniques including stereotaxic surgeries, brain site-specific injections, methamphetamine self administration procedures, and experimental design and interpretation of models of psychostimulant administration. In addition to the scientific aims of the studies presented, the ultimate goal of the proposal is to train the investigator with behavioral techniques that, along with established expertise in electrophysiology, will form the basis for a career as an independent investigator. The career development plan involves mentorship in behavioral techniques from an established behavioral neuroscientist (Dr. Gregory Mark) while utilizing the resources and expertise of one of the top behavioral departments in the country. The experimental procedures will investigate the hypothesized relationship between methamphetamine self-administration, intracellular calcium levels, and dendrodendritic dopamine transmission in the ventral tegmental area of mice. Methamphetamine induces synaptic plasticity and D2 dopamine autoreceptor subsensitivity, but the mechanisms involved are poorly understood. Furthermore, no study has investigated the role of synaptic plasticity on methamphetamine self-administration. Using recently discovered tools including a synaptic potential mediated by dopamine and a novel form of plasticity at dopamine synapses, the experiments will determine if dopamine plasticity plays a role in methamphetamine self-administration. Relevance: Determining the neurophysiological consequences of methamphetamine self-administration is the first step in identifying intervention strategies to prevent and treat human drug abuse. A better understanding of the neural adaptations associated with drug use and administration may help to explain why some methamphetamine users become addicted to the drug, and may lead to treatments to help patients break the difficult cycle of addiction.

DESCRIPTION (provided by applicant): Methamphetamine (METH) addiction currently presents an enormous public health issue, and yet no therapeutic agent is currently approved for its treatment. Psychostimulant addiction is a chronic, progressive disease driven by numerous persistent neurophysiological adaptations. METH self-administration increases input of the neuropeptide neurotensin onto dopamine (DA) neurons in the ventral tegmental area (VTA), which are extensively implicated in drug reward processes. While literature evidence and our preliminary results suggest that neurotensin decreases DA autoreceptor-mediated signaling, the role of DA D2 autoreceptors in drug self-administration has not been described. We have recently identified what had been a missing tool in the study of DA autoreceptors and DA-mediated synaptic transmission: an inhibitory postsynaptic current (or IPSC) mediated directly by dendrodendritic DA neurotransmission in the VTA. The identification of the DA IPSC allows us, for the first time, to directly address synaptic questions concerning the relationship between METH abuse and DA neurotransmission. The goal of this application is to determine key synaptic adaptations at the level of the DA cell body that are responsible for escalating METH self-administration. Our central hypothesis is that METH use decreases D2 autoreceptor signaling in VTA DA neurons through a neurotensin-dependent rise in intracellular calcium, producing an escalation of METH self-administration behavior. We will test this by combining patch clamp electrophysiology in brain slices with intravenous METH self-administration and VTA site-specific drug microinjections in mice. The studies in Aim 1 will determine the mechanisms responsible for long-term depression of the DA IPSC. The hypothesis to be tested is that that long term depression of DA IPSCs is produced by a neurotensin receptor-dependent rise in intracellular calcium producing the activation of protein phosphatase 3. The studies in Aim 2 will determine the changes in the DA IPSC produced by METH self-administration. The hypothesis to be tested is that in vivo contingent METH self-administration decreases autoreceptor signaling through a neurotensin-dependent mechanism. The studies in Aim 3 will determine the role of DA autoreceptor-mediated neurotransmission on the escalation of METH self-administration. The hypothesis to be tested is that autoreceptor signaling directly limits METH intake, and that neurotensin-induced depression of this signal contributes to the escalation of self-administration observed with prolonged access to the drug. The results of these studies will identify key cellular mechanisms responsible for decreased autoreceptor signaling, and will determine how this decrease in dendrodendritic DA neurotransmission produces escalation of METH self-administration. These findings will provide a detailed understanding of the relationship between neurotensin, DA neuron activity and METH self-administration and will lay the foundation for therapeutics targeting neurotensin- and autoreceptor-mediated signaling.

Aging is associated with a decrease in movement and cognition, and understanding the neurophysiological bases of these behavioral deficits will be key to increasing healthspan. Dopamine neurons in the substantia nigra are key central mediators of voluntary movement and reward-related behavior, and the function of these neurons is known to decline with age. Unfortunately, little is currently known about how normal aging affects the specific intrinsic channels and synaptic inputs that are responsible for dopamine neuron excitability and function. Our lab has developed reliable methodology for making electrophysiological recordings of dopamine neurons in brain slices from mice of advanced age, thus a comprehensive investigation into the effects of aging on ion channel physiology in these neurons is now possible. We will combine electrophysiology, behavior, gene expression analysis, and optogenetics to elucidate the effects of aging on ion channel signaling and dopamine-mediated behaviors. Furthermore, we will use dietary restriction (an established healthspan- increasing intervention) to identify the ionic mechanisms that counteract age-related deficits in behavior. Our central hypothesis is that age-related deficits in movement and reward-related behaviors can be attributed to specific ion channel conductances in substantia nigra dopamine neurons, and that these deficits can be attenuated by dietary restriction. Aim 1 is to determine the relationship between age-related deficits in specific ion channel currents and cell firing. We will also relate (in individual mice) our electrophysiological findings in single neurons to previously obtained locomotor behavioral data. Aim 2 is to determine the effects of aging on excitatory inputs from the hypothalamus to dopamine neurons as well as reward-related behavior. For this aim we will employ optogenetics to study identified glutamate inputs in a brain region-specific manner. Aim 3 is to determine the effects of aging on dopamine autoreceptor-mediated neurotransmission. Aim 4 is to determine the effects of dietary restriction in aging mice as an intervention that can counteract age-related decline of ion conductances in dopamine neurons and related behaviors. These studies will provide the first detailed understanding of the relationship between aging, dopamine neuron activity, and dopamine-mediated behaviors, and determine the protective effects of dietary restriction on these parameters.

Aging is associated with a decrease in movement and cognition, and understanding the neurophysiological bases of these behavioral deficits will be key to increasing healthspan. Dopamine neurons in the substantia nigra are key central mediators of voluntary movement and reward-related behavior, and the function of these neurons is known to decline with age. Unfortunately, little is currently known about how normal aging affects the specific intrinsic channels and synaptic inputs that are responsible for dopamine neuron excitability and function. Our lab has developed reliable methodology for making electrophysiological recordings of dopamine neurons in brain slices from mice of advanced age, thus a comprehensive investigation into the effects of aging on ion channel physiology in these neurons is now possible. We will combine electrophysiology, behavior, gene expression analysis, and optogenetics to elucidate the effects of aging on ion channel signaling and dopamine-mediated behaviors. Furthermore, we will use dietary restriction (an established healthspan- increasing intervention) to identify the ionic mechanisms that counteract age-related deficits in behavior. Our central hypothesis is that age-related deficits in movement and reward-related behaviors can be attributed to specific ion channel conductances in substantia nigra dopamine neurons, and that these deficits can be attenuated by dietary restriction. Aim 1 is to determine the relationship between age-related deficits in specific ion channel currents and cell firing. We will also relate (in individual mice) our electrophysiological findings in single neurons to previously obtained locomotor behavioral data. Aim 2 is to determine the effects of aging on excitatory inputs from the hypothalamus to dopamine neurons as well as reward-related behavior. For this aim we will employ optogenetics to study identified glutamate inputs in a brain region-specific manner. Aim 3 is to determine the effects of aging on dopamine autoreceptor-mediated neurotransmission. Aim 4 is to determine the effects of dietary restriction in aging mice as an intervention that can counteract age-related decline of ion conductances in dopamine neurons and related behaviors. These studies will provide the first detailed understanding of the relationship between aging, dopamine neuron activity, and dopamine-mediated behaviors, and determine the protective effects of dietary restriction on these parameters.

Alzheimer's disease (AD) is most commonly diagnosed subsequent to memory deficits, but co-morbidities include a slew of cognitive and non-cognitive impairments including depression, apathy, and movement disorders. While most work on AD has thus far focused on effects in the cortex and hippocampus, the pathophysiology associated with AD is found throughout the brain, and many of the secondary symptoms suggest involvement of the midbrain dopaminergic system. A recent major study as well as preliminary data suggest that dopamine neurons of the ventral tegmental area (VTA) may be affected in mouse models of AD prior to the formation of amyloid plaques and neurofibrillary tangles, suggesting a possible role for the VTA in the prodromal phase of the disease. This field is currently limited by the lack of information on the structural and functional deficits that develop in single dopamine neurons in the early stages of AD, as well as their relationship with decrements in hedonic and reward learning behavior. The experiments in this proposal will focus on two established mouse models of AD. APP/PS1 mice express a mutated human amyloid precursor protein and a deletion of presenilin 1, while triple transgenic 3xTg-AD mice also express a transgene for a human mutant tau. The general strategy will be to measure deficits in dopamine-mediated behavior (sucrose preference, an operant learning task, and locomotor assays) in mutant mice aged 3, 6, and 10 months, and non-transgenic controls, followed by sacrifice for electrophysiology and RNA sequencing of single VTA dopamine neurons. Aim 1 will focus on both intrinsic and synaptic conductances that affect dopamine neuron firing and have been identified in preliminary studies as possibly being affected in AD. Aim 2 will test if a commonly used anti-aging intervention, dietary restriction, can mitigate some or all decrements in ion channel physiology, behavior, and neuronal morphology. As the prevalence of ADRD continues to increase, we are in desperate need of AD treatments that not merely alleviate symptoms but slow or halt the progression of the disease. These treatments do not currently exist because we lack a fundamental understanding of the decrements in cellular and circuit function that occur during the early stages of pathology. The work in this proposal will advance the field toward better treatments by establishing the involvement of dopaminergic processes in AD mouse models. Delineating the sequence of pathological events will allow for the identification of molecular targets for intervention in early AD.