Daniel S. McGehee - US grants

DESCRIPTION: The precise execution of complex movements results from the convergence of direct input from primary afferent neurons and corticospinal projections onto motoneurons, as well as indirect inputs from spinal cord interneurons. Presynaptic modulation of synaptic transmission is an important component of this circuitry. Preliminary studies in the hemisected spinal cord indicate that presynaptic nicotinic acetylcholine receptors (nAChRs) enhance excitatory synaptic inputs to spinal motoneurons. The goal of this proposal is to examine the biophysical mechanisms and receptor components that underlie nAChR-mediated enhancement of synaptic transmission in the spinal cord. The primary assay of presynaptic nAChRs will assess changes in the spontaneous and evoked synaptic transmission in recordings from motoneurons. In initial studies, the modulation of synaptic transmission by nAChRs will be examined in two semi-intact preparations, transverse slices and hemisected spinal cord, under conditions that closely mimic the native environment. Characterization of the effects of nAChR activation on evoked transmission will enable identification of the class(es) of afferent inputs that express these receptors. These findings will then guide the making of cultures of dissociated motoneurons that are innervated by explants of the appropriate afferent inputs. Using these cocultures, more detailed molecular, pharmacological and biophysical analyses will be made of the presynaptic nAChRs that modulate each type of afferent inputs to motoneurons. This proposal will also examine the influence of endogenously released ACh on presynaptic nAChRs and synaptic efficacy. Endogenous activation of presynaptic nAChRs will be achieved by stimulating the motoneuron under study to evoke a local release of ACh. Finally, the effects of inhibitors of acetylcholine esterase, the enzyme that is responsible for eliminating ACh after its release, will also be examined. Collectively, these studies will provide a detailed pharmacological, molecular, and biophysical analysis of the receptor subtypes involved in nAChR-mediated presynaptic enhancement of excitatory transmission in the spinal cord. These studies will significantly advance our understanding of the factors that influence motoneuron excitability and hopefully identify new approaches to alleviating the debilitating symptoms associated with degenerative diseases of motor and sensory neurons.

[unreadable] DESCRIPTION (provided by applicant): Nicotinic acetylcholine receptors (nAChRs) can modify synaptic transmission in many brain regions. Our recent studies show that nAChRs contribute to midbrain dopamine (DA) neuron excitability through modulation of both inhibitory GABAergic and excitatory glutamatergic inputs. The nAChR enhancement of glutamate inputs can contribute to LTP induction at this synapse. Interestingly, the nicotinic modulation of GABA transmission exhibits a transient enhancement, followed by a depression of activity. Apparently, desensitization of the nAChRs on GABA neurons inhibits endogenous cholinergic input to these cells, thus leading to a 'disinhibition' of the DA neurons. These studies were carried out in neonatal rats, primarily for technical reasons. Experiments in this proposal will extend these tests to tissue slices from adult rats that have undergone behavioral and pharmacological testing. The activity that an animal displays in a novel environment can predict nicotine self-administration in rats. The advantage of this screen is that animals predisposed to nicotine self-administration can be identified without nicotine exposure, which is known to alter sensitivity. Our preliminary results indicate differences in nAChR expression between high and low responders to novelty. We will extend these observations to test the differences in cellular and synaptic effects of nAChR activation associated with the predisposition to nicotine self-administration. The activity response to novelty has also been correlated with differences in stress hormone levels between individuals, leading to the suggestion that stress hormones contribute to the predisposition to drug-taking. Preliminary data indicate that stress hormones inhibit nAChRs through a direct interaction. We will test the hypothesis that this interaction upregulates the expression of nAChRs within the reward area, strengthens the cellular response to nicotine and thus, enhances the motivating effects of the drug. Nicotine exposure also enhances the acquisition of self-administration behavior, presumably through upregulation of nAChR expression. We will test nAChR effects on DA neuron excitability from animals that have been pre-exposed to nicotine by passive injection and self-administration testing. These studies of nAChR function within the brain reward center will provide important insights into the cellular basis of addiction. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Nicotinic acetylcholine receptors (nAChRs) can modify synaptic transmission in many brain regions. Our recent studies show that nAChRs contribute to midbrain dopamine (DA) neuron excitability through modulation of both inhibitory GABAergic and excitatory glutamatergic inputs. The nAChR enhancement of glutamate inputs can contribute to LTP induction at this synapse. Interestingly, the nicotinic modulation of GABA transmission exhibits a transient enhancement, followed by a depression of activity. Apparently, desensitization of the nAChRs on GABA neurons inhibits endogenous cholinergic input to these cells, thus leading to a 'disinhibition' of the DA neurons. These studies were carried out in neonatal rats, primarily for technical reasons. Experiments in this proposal will extend these tests to tissue slices from adult rats that have undergone behavioral and pharmacological testing. The activity that an animal displays in a novel environment can predict nicotine self-administration in rats. The advantage of this screen is that animals predisposed to nicotine self-administration can be identified without nicotine exposure, which is known to alter sensitivity. Our preliminary results indicate differences in nAChR expression between high and low responders to novelty. We will extend these observations to test the differences in cellular and synaptic effects of nAChR activation associated with the predisposition to nicotine self-administration. The activity response to novelty has also been correlated with differences in stress hormone levels between individuals, leading to the suggestion that stress hormones contribute to the predisposition to drug-taking. Preliminary data indicate that stress hormones inhibit nAChRs through a direct interaction. We will test the hypothesis that this interaction upregulates the expression of nAChRs within the reward area, strengthens the cellular response to nicotine and thus, enhances the motivating effects of the drug. Nicotine exposure also enhances the acquisition of self-administration behavior, presumably through upregulation of nAChR expression. We will test nAChR effects on DA neuron excitability from animals that have been pre-exposed to nicotine by passive injection and self-administration testing. These studies of nAChR function within the brain reward center will provide important insights into the cellular basis of addiction. [unreadable] [unreadable]

The chain of cause and effect of nicotine addiction starts with the interaction of this tobacco alkyloid with nicotinic acetylcholine receptors (nAChRs). This interaction leads to activation of reward centers in the CMS, including the mesoaccumbens dopamine (DA) system, which ultimately leads to behavioral reinforcement and addiction. Our recent investigations into the contribution of nAChRs to the control of midbrain dopamine (DA) neuron excitability indicate that nAChRs can modify both inhibitory and excitatory inputs to DA neurons. The enhancement of excitatory glutamatergic transmission by nAChRs can contribute to long-term potentiation of these synapses. Interestingly, the modulation of inhibitory GABAergic transmission has biphasic characteristics, where nicotine causes both an increase and then a decrease in activity, leading to disinhibition of the DA neurons. While these observations on acute responses to nicotine provide interesting connections between nAChRs and the excitability of VTA DA neurons, experiments in this proposal will extend these observations to test the impact of nicotine exposure on the sensitivity and function of nAChRs within the reward circuitry. Nicotine exposure is known to cause upregulation of nAChR function and radioligand binding. In addition, nicotine along with other drugs of abuse can induce long term potentiation (LTP) of the excitatory inputs to DA neurons. We will test the hypothesis that nAChR upregulation contributes to LTP induction within the DA system and its afferent projections. Using electrophysiology in brain slices from adult rats, we will assess the changes in nAChR properties that occur in animals that have been exposed to nicotine in one of four regimens, ranging from one to 15 days of exposure. The regimens vary in intensity and duration and are designed to represent a spectrum of nicotine exposure, mimicking to the wide variation in nicotine exposure seen in humans. We expect that the different regimens will result in a range of upregulation of nAChR function and radioligand binding. Experiments in this project will investigate functional upregulation and LTP expression following nicotine exposure. Results will be correlated with data from behavioral, biochemical, and molecular assays conducted in Projects 1 and 3. Correlating the findings with our collaborators'will provide novel insights into the nicotine-induced changes in reward circuitry that ultimately contribute to the etiology of nicotine addiction.

DESCRIPTION (provided by applicant): Nicotine addiction remains a major health problem in the US and throughout the world. The rewarding effects of nicotine are well-documented, but higher doses of nicotine have intensely aversive effects. Initial responses to nicotine can predict future dependence (i.e., subjects whose first nicotine experiences were rewarding are more likely to become nicotine dependent relative to subjects whose first nicotine experiences were aversive). Thus, the balance between nicotine reward and aversion likely contributes to the development and maintenance of nicotine addiction. Despite considerable understanding of the effects of nicotine on reward-related circuitry, treatments for nicotine dependent populations remain limited. Understanding the mechanisms that underlie aversion to nicotine can provide novel insights into the factors that contribute to nicotine dependence. These insights have the potential to reveal novel therapeutic targets and strategies. Recently, the projection from the medial habenula (MHb) to the interpeduncular nucleus (IPN) was shown to mediate nicotine aversion. Suppressing MHb-IPN activity reduces the aversive effects of nicotine, while enhancing MHb-IPN activity enhances aversion to nicotine. Although these results implicate the MHb-IPN circuitry, the downstream post-synaptic targets of the IPN and the neurotransmitters involved remain largely uncharacterized. Some evidence suggests that this circuitry ultimately suppresses VTA dopamine (DA) neurons. Burst activity in DA neurons has been linked to motivated behaviors, and suppression of DA neuron output results in an aversive experience. While the IPN projects to several brain areas, it strongly innervates the lateral dorsal tegmental nucleus (LDTg), a brainstem cholinergic center that controls burst firing of VTA DA neurons. Activation of LDTg neurons that project to the VTA results in enhanced activity in the VTA as well as conditioned place preference, whereas inhibition of VTA DA neurons has been linked to conditioned place aversion. Therefore, inhibiting the LDTg (via IPN excitation), and consequently VTA DA neurons, may diminish the rewarding effects of nicotine. Experiments outlined in this proposal will examine the cellular mechanisms underlying the aversive effects of nicotine and how this aversion impacts reward circuitry. We will use optogenetic strategies to explore the cellular mechanisms mediating the communication between the MHb-IPN pathway and VTA DA neurons. Causal links between these pathways and nicotine-induced behaviors will be explored by optogenetic excitation or inhibition of these pathways while testing nicotine-related behaviors. Exploring the mechanisms by which the MHb-IPN circuitry mediates nicotine-aversion could yield novel therapeutic targets and treatment strategies for helping smokers quit successfully.

The therapeutic effects of L-DOPA are remarkable in early stage Parkinson's disease (PD). However, with chronic dopamine replacement therapy, motor side effects such as dyskinesia become a severe problem in advanced PD. Mechanisms underlying PD symptoms and therapy are still poorly understood. Our recent studies in animal models have for the first time demonstrated that experience-dependent aberrant motor learning (learned motor inhibition) under low dopamine conditions may play a major role in PD motor symptoms, which was supported by recent studies on PD patients and by computational models. Moreover, we have demonstrated that glutamatergic inputs in combination with dopamine signaling through the adenylyl cyclase type 5 (AC5) and the cAMP pathway contributes to corticostriatal long-term potentiation and depression (LTP and LTD) in the dopamine D2 receptor-expressing striatal medium spiny neurons (MSNs). More importantly, we have found that aberrant LTP is associated with aberrant motor learning while prevention of such aberrant LTP is associated with prevention of aberrant motor learning. Our behavioral and electrophysiological advances have set the stage for identifying and testing potential PD therapies based on preventing and/or reversing aberrant corticostriatal LTP. In this application, we propose to test the precise conditions/parameters for induction of corticostriatal LTP/LTD in D2-expressing MSNs. We then aim to establish a causal link between aberrant corticostriatal LTP and aberrant motor learning as well as to test treatments that can prevent such aberrant corticostriatal LTP and aberrant motor learning. Finally, we will test the therapeutic effects of preventing and reversing aberrant LTP in PD models.

While the percentage of US adults who smoke tobacco has been declining in recent years, smoking continues to be a leading cause of preventable death and disease worldwide. Further, nicotine administration via alternative delivery systems has increased in recent years. Chronic use of nicotine induces neuroadaptations that contribute to withdrawal symptoms upon cessation of use. The severity of withdrawal symptoms in the first week of abstinence has been associated with increased vulnerability to relapse. The goal of this proposal is 1) to demonstrate that behavioral expression nicotine withdrawal-induced aversion can be modulated through, and is dependent upon, the IPN-LDTg-VTA pathway, and 2) to identify neuroadaptations within this circuitry induced by chronic nicotine that mediate affective withdrawal. The ultimate objective is to identify novel therapeutic approaches to reduce affective nicotine withdrawal symptoms to improve success during quit attempts.

Parkinson?s disease (PD) is a progressive neurodegenerative disease leading to motor impairment due to degeneration of nigrostriatal dopaminergic projections. Currently, L-DOPA therapy effectively relieves the motor symptoms in initial stages of PD, however long term treatment with L-DOPA results in loss of efficacy and the development of dyskinesia. Thus, there is a need for other effective therapies for PD. Dopamine in the striatum plays a critical role in modulation of the direct and indirect motor output pathways by activating D1 and D2 receptors in those pathways, respectively. Under normal conditions, dopamine facilitates movement by increasing activity of the direct pathway and decreasing activity of the indirect pathway. In patients with PD, the loss of dopamine results in decreased activity of the direct pathway and increased activity of the indirect pathway. D1 agonists can relieve PD symptoms by increasing cAMP levels and increasing activity of direct pathway medium spiny neurons (MSNs) that express the D1 dopamine receptors. Exenatide, an FDA approved pharmacotherapy commonly prescribed for the treatment of type 2 diabetes, is a glucagon like peptide-1 receptor (GLP-1R) agonist, and a recent clinical trial reported therapeutic benefits of exenatide for PD symptoms. In preclinical studies, GLP-1R agonist treatment has also been shown to protect dopamine neurons from cell death in PD models, but the acute or chronic effects of these receptors on MSN excitability have not been investigated. Our preliminary data suggest that GLP-1Rs are expressed by direct pathway MSNs, and that activation of these receptors increases the excitability of those neurons. As GLP-1R is positively coupled to adenylyl cyclase activity and cAMP levels, the improved motor control in PD patients receiving the GLP-1R agonist may be related to modulatory effects on striatal circuitry. Experiments outlined in this proposal will use a mouse model of PD to examine two aims: The first is to test whether acute GLP-1R agonist exposure can increase intrinsic excitability and corticostriatal synaptic plasticity in striatal MSNs. The second Aim will explore the effects of chronic GLP-1R agonist treatment on motor performance and corticostriatal plasticity of MSNs. These studies will test the hypothesis that GLP-1R agonists may provide alternate treatment strategies for PD symptoms through their effects on striatal circuitry and synaptic plasticity.

Chronic pain conditions plague more than 20% of adults in the United States, emphasizing the need for a more comprehensive understanding of pain control mechanisms and better pain therapies. This need is amplified further by the current opioid crisis, which killed 47,600 Americans in 2018. While opioids are remarkably effective treatments for acute and chronic pain, adverse side effects, including abuse liability and tolerance to the analgesic effects with repeated use, highlight the need for novel non-opioid pain therapies. This need motivates our investigations of pain modulation by acetylcholine (ACh) and its receptors (AChRs) in the ventrolateral periaqueductal gray (vlPAG). Ascending pain signaling from periphery to central nervous system is modulated by the descending pain pathway, including the vlPAG and its projections to rostral ventromedial medulla (RVM) and locus ceruleus (LC). This descending pain pathway is a key site of action of opioids and endogenous pain control. While much is known about this circuitry, the pain modulatory effects of cholinergic inputs to vlPAG are understudied. Our preliminary data show that optogenetic stimulation of cholinergic projections from the pedunculopontine tegmental nucleus (PPTg) to vlPAG is antinociceptive in acute pain. Using cell- and circuit-specific optogenetic approaches, we will test the effect of stimulating cholinergic projections to vlPAG on nociception in a chronic neuropathic pain model, identify the cholinergic receptors mediating these antinociceptive effects, and explore possible interactions between cholinergic signaling and the opioid system. In Aim 1, we will use optogenetic approaches to test antinociceptive effects of stimulating cholinergic PPTg neurons projecting to vlPAG in mice experiencing chronic neuropathic pain. We will also assay affective pain relief of stimulating these projections using a real-time place preference assay. In Aim 2, we will identify cholinergic receptor(s) mediating synaptic communication between PPTg and vlPAG neurons, using high- throughput fluorescence in situ hybridization assay to visualize mRNA expression of muscarinic and nicotinic AChRs on vlPAG neurons. After identifying candidate AChR(s) based on mRNA expression, we will test functional role of these receptors in synaptic communication, using slice electrophysiology and ex vivo optogenetics. Finally, we will test the causal role of identified AChRs in antinociception in vivo using optogenetics and pharmacology in neuropathic pain assays. In Aim 3, we will explore possible interactions between these cholinergic mechanisms and the opioid system, by testing the efficacy of cholinergic analgesia in morphine-tolerant mice and during naloxone precipitated withdrawal from chronic morphine treatment. These studies will employ histological, electrophysiological, optogenetic and pharmacological approaches to advance our knowledge of cholinergic modulation of descending pain pathways, to ultimately help identify novel opioid-independent targets for treating chronic pain.