Patrick L. Sheets, Ph.D. - US grants

DESCRIPTION (provided by applicant): Neuropathology affecting primary motor cortex (M1) causes major neurological impairment including paralysis/paraplegia, epilepsy, and movement disorders including amyotrophic lateral sclerosis (ALS), a disease affecting upper motor neurons. Primary motor cortex (M1) is centrally involved in voluntary movement and other aspects of motor control (Denny-Brown, 1966;Phillips and Porter, 1977;Asanuma, 1989;Fetz et al., 2000;Sanes and Donoghue, 2000;Rizzolatti and Luppino, 2001;Schieber, 2001;Georgopoulos, 2002;Capaday, 2004;Curtis and Kleinfeld, 2006;Graziano, 2006;Kleinfeld et al., 2006). Local circuits of corticospinal neurons -- neurons projecting from cortex to the spinal cord -- in M1 mediate the key output pathway controlling movement of the body. Although the output of corticospinal neurons has been studied extensively, little is known about the local inputs to corticospinal neurons in any species. Hyperpolarization- activated cyclic nucleotide-gated (HCN) channels activate upon hyperpolarization, generating h-current (Ih) which can be regulated by multiple signal transduction pathways (Wahl-Schott &Biel, 2009). Ih has been observed in L5 neurons of somatosensory cortex (Spain et al., 1987: Berger et al., 2003), visual cortex (Soloman &Nerbonne, 1993b), and entorhinal cortex (Richter et al., 2000;Hamam et al., 2002). Evaluation of Ih and its effects on local circuit properties in corticospinal neurons of M1 has yet to be done. Since Ih resists changes in membrane potential, a narrow spatio-temporal integration window would be expected for local inputs to corticospinal neurons. Elucidating this will be critical for a detailed understanding of how M1 controls movement of the body, and holds the potential to reveal new therapeutic targets in the many neurological diseases that affect motor control such as ALS. ALS is a late onset neurodegenerative disease that results in progressive paralysis leading to premature death. Cortical hyperexcitability has been shown in ALS patients (Vucic et al., 2008, 2009), however, little is known about corticospinal circuits in ALS. The overall goal of these studies is to investigate in vitro circuit-level properties of mouse corticospinal neurons using use high-resolution laser scanning photostimulation (LSPS), slice electrophysiology, pharmacology, and anatomical labeling strategies. Preliminary data suggests that mouse corticospinal circuits (1) receive strong excitatory synaptic input from layer 2/3, (2) have intrinsic electrophysiology that is dominated by high expression of hyperpolarization-activated current (Ih), and (3) become altered in superoxide dismutase 1 (SOD1-693A) mutant mice, a mouse model of ALS. Thus the hypothesis for this proposal is that Ih produces class-specific synaptic integration properties in corticospinal neurons, and a corollary is that these properties are disrupted in a mouse model of ALS. The results of the proposed research will reveal fundamental mechanisms of local synaptic circuit physiology in M1 corticospinal neurons, providing a new basis for future studies of cortical dysfunction in disorders of motor control. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disease affecting corticospinal neurons that results in progressive paralysis leading to premature death. Local circuits of corticospinal neurons -- neurons projecting from cortex to the spinal cord -- mediate the key output pathway controlling movement of the body by integrating diverse inputs from multiple sensory and motor-related systems. The results of the proposed research will reveal fundamental mechanisms of local synaptic circuit physiology in M1 corticospinal neurons, providing a new basis for future studies of cortical dysfunction in ALS.

? DESCRIPTION (provided by applicant) Pain is more than a sensation processed by the peripheral nervous system. Pain engages the central nervous system via multiple cortical and subcortical networks. Neuropathic pain is a chronic pain resulting from damage to the somatosensory nervous system which can cause detrimental changes to physiological, psychological, and behavioral aspects of life. Neuropathic pain alters the structure, chemistry, and connectivity of medial prefrontal cortex (mPFC) in humans. Importantly, circuits comprising the mPFC are essential in processing emotional components of our everyday experiences, and therefore, are implicated in the affective component, or unpleasantness, of pain. A comprehensive understanding is lacking regarding the functionality of pain- relevant circuitry in the mPFC under normal conditions and in animal models of neuropathic pain. Therefore, this proposal seeks to understand the organization and function of both intracortical (local) and subcortical (long-range) synaptic inputs to a major population of mPFC neurons with specific targeting to a key subcortical pain structure: the periaqueductal gray (PAG). The PAG is a link in the primary pain-modulating network essential for endogenous analgesia and autonomic response to pain. Our objectives are 1) to elucidate the local and long-range circuitry of cortico-PAG neurons in the mPFC and 2) to assess the specific mechanisms by which the neural elements of cortico-PAG circuitry are altered by neuropathic pain. To accomplish these goals, we will develop a multifaceted approach involving retrograde labeling, electrophysiology, circuit mapping, optogenetic and behavioral techniques. The rationale for the proposed research is that identifying the neural mechanisms through which neuropathic pain alters circuit function in cognitive and emotional networks of the brain (specifically mPFC-PAG) will produce critical knowledge regarding the affective dimension of pain. Such an understanding can lead to novel strategies for therapeutic intervention. Our findings will contribute new and important insights into cellular and circuit mechanisms for how neuropathic pain alters specific cortical networks essential in the perception and emotional relevance of pain.

PROJECT SUMMARY Neurons in the central amygdala (CeA) contribute to pain modulation. However, their contribution to the sensory- discriminative and/or emotional-affective dimensions of chronic pain, nor their neurochemical modulation, are understood. Our preliminary data using slice recordings and behavior provide a compelling premise for the idea that drugs targeting the receptors for the bioactive lysophospholipid, sphingosine-1-phosphate (S1P) act within the CeA to inhibit inflammatory and neuropathic pain. This sets the stage for our long-term goal to understand how lipid signaling controls the supraspinal control of acute and chronic pain. The objectives of this proposal are to: determine neurophysiological changes to molecular specific CeA neurons in multiple models of pain (Aim 1), elucidate the effects of S1P signaling on the intrinsic and synaptic excitability of defined subpopulations of CeA neurons (Aim 2), and determine if S1PR agonism in the CeA is analgesic in models of inflammatory and neuropathic pain (Aim 3). In Aim 1, we use transgenic mouse lines, electrophysiology, and optogenetics to test the hypotheses that tissue or nerve injury reduces excitability of specific subclasses of CeA neurons based on their molecular profile. In Aim 2, we test the hypotheses that activation of S1P signaling increases the excitability and synaptic connectivity within a population of molecularly distinct CeA neurons. In Aim 3, we use intracranial drug infusions and chemogenetics to test the hypothesis that activation of S1P receptors in the CeA attenuates inflammatory and neuropathic pain via a specific subtype of CeA neuron. Experimental support of these concepts will facilitate the development of existing (e.g. FDA-approved fingolimod) and novel S1PR compounds for the treatment of chronic pain.

Project Summary: A wealth of data exists describing how alcohol influences neuronal function in brain regions necessary for complex cognitive functions such prefrontal cortex (PFC). These data have proven critical to form hypotheses regarding how alcohol consumption might lead to altered behavioral states (e.g. intoxication) and the associated negative outcomes (e.g. risky behavior, violence). However, these data have been exclusively collected in reduced preparations, such as anesthetized animals or ex vivo slice preparations, and therefore may not adequately model how neural function is impaired by alcohol consumption in behaving subjects. Published and preliminary data from our group indicate that the broad reductions in neural firing previously observed in reduced preparations are not observed in awake behaving animals. This calls into question current explanations of how alcohol affects neural function, indicating that there is a critical need for additional experiments that demonstrate specifically how alcohol consumption affects neural function in the PFC of awake behaving animals. The long-term goal of this work is to create a unified model of how alcohol consumption alters the computational properties of PFC and leads to impaired behavioral control. Towards this goal, a series of experiments are proposed in rat models to test the overarching hypothesis that alcohol consumption leads to reductions in functional connectivity in PFC networks. In Specific Aim 1, a series of in vivo approaches will determine how oral consumption of alcohol influences neural activity in medial PFC (mPFC) networks. A combination of microdialysis, large-scale neural recordings, and optogenetics will be performed to determine the cell type- and layer-specific effects of alcohol consumption on neural activity. Experiments will be performed to determine which changes in neural activity evoked by oral consumption are conserved following local application of physiologically-relevant concentrations of alcohol within mPFC. Specific Aim 2 will determine which changes in neural activity observed in vivo are conserved ex vivo. In this Aim we will trace the local effects of alcohol on mPFC from the single cell to the microcircuit level. Glutamate uncaging will be combined with patch-clamp electrophysiology to assess network function and single cell function. These approaches will synergize with those in Specific Aim 1 to determine which changes in neural activity following alcohol consumption are conserved across levels of neural function and in each preparation. Collectively, these data will facilitate future studies that will integrate how the influence of consumed alcohol on cellular function translates into altered network properties and, ultimately, impaired behavioral states. Future studies will also assess changes in neural function in brain-wide networks and if/how the observations herein are altered following extended alcohol use. In sum, this proposal seeks to clarify a long held misconception in the alcohol field and answer a fundamental question on how alcohol effects neural activity.