Shane T. Hentges, Ph.D - US grants

While there is considerable knowledge about many factors involved in regulating energy homeostasis, a gap remains in our understanding of local circuitry within the hypothalamus and in the cellular processes underlying these circuits. POMC neurons located in the arcuate nucleus of the hypothalamus are a major target for leptin action, and play a crucial role in regulating energy homeostasis. Thus far there is only limited information about the intrinsic physiology of POMC neurons because of the difficulties in identifying them in brain slices or primary cultures and isolating direct effects from indirect effects in these systems. Recently, our laboratory has developed a technique to identify and study isolated fluorescently abeled POMC neurons in culture. Preliminary evidence shows that in addition to expressing POMC peptides, these neurons also release and respond to GABA indicating a previously unexplored action and "nodulation of POMC neurons. In addition to studying the GABAergic nature of POMC neurons, the aims in the current proposal are also designed to 1) address the pre- and post-synaptic effects of leptin, alpha-MSH, beta-endorphin, and NPY directly on POMC neurons 2) characterize the actions of opioids 3n POMC neurons and to determine whether POMC neurons desensitize to opioids 3) determine whether interactions between POMC peptides, NPY, occur at the cellular level in POMC neurons to regulate their activity and 4) to determine if there is acute desensitization of leptin receptors in POMC neurons. Understanding the physiology of these neurons and the cellular consequences of leptin resistance in Lhese neurons will further the basic understanding of hypothalamic control of energy balance and perhaps identify potential sites for future therapeutic approaches to treat obesity.

[unreadable] DESCRIPTION (provided by applicant): Approximately two-thirds of adults in the United States are overweight or obese and therefore at increased risk for developing diabetes, coronary heart disease, stroke, some forms of cancer and other health serious problems. A better understanding of the physiological mechanisms that regulate energy balance is a key step towards developing therapeutic approaches to prevent and treat obesity. Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus release multiple neuropeptides that are necessary for the maintenance of normal energy balance. POMC neurons also release classical neurotransmitters, however the role of synaptic transmitters in POMC neurons has not been well-studied. The goal of the proposed work is to test the overall hypothesis that subsets of POMC neurons release different classical neurotransmitters and that this heterogeneity among POMC neurons is important in the regulation of energy balance. The following specific hypotheses will be tested: 1) That subpopulations of POMC neurons can be defined by the presence and release of GABA or glutamate. Transgenic mice with co-labeled POMC, GABAergic and glutamatergic neurons will be used to examine the neurotransmitter phenotype of subsets of POMC neurons and the effects that obesity and leptin treatment have on the rapid transmitter phenotype of POMC neurons will be determined. 2) That there is differential regulation of subsets of POMC neurons. The presynaptic regulation and basal activity of subsets of POMC neurons will be studied using electrophysiological recordings and the effects that leptin and fasting have on these parameters in each subpopulation will be studied. 3) That GABAergic and glutamatergic POMC neurons project to distinct target sites. Retrograde labeling experiments will be performed in transgenic mice to identify where specific populations of POMC neurons project. The results of these studies will provide a clearer picture of how POMC neurons function in the CNS circuitry controlling energy balance. 7. PUBLIC HEALTH RELEVANCE: Obesity contributes to about 112,000 deaths per year and is the primary reason for over $75 billion in heath care expenditures in the United States. A better understanding of the physiological mechanisms that regulate energy balance is a key step towards developing therapeutic approaches to prevent and treat obesity. This study will determine how specific neurons in the brain that are critical for the maintenance of normal energy balance are regulated. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Opioid receptor agonists are highly effective at producing analgesia, but their clinical use is hindered by the development of tolerance and high abuse liability resulting from activation of the neural reward circuitry. Therefore, understanding how opioids regulate reward circuits and how opioid receptor signaling changes with opioid exposure are key steps towards overcoming current clinical limitations of opioid therapy. One important reward pathway that releases and responds to opioids includes the hypothalamic proopiomelanocortin (POMC) neurons. Recent data show that POMC neurons are regulated by opioids acting on POMC neurons and also by opioids acting on terminals presynaptic to POMC neurons. Interestingly, while postsynaptic mu opioid receptors on POMC neurons undergo desensitization within minutes, presynaptic mu opioid receptors that mediate the inhibition of transmitter release do not readily desensitize. The goals of the present proposal are to 1) determine the mechanisms underlying the presynaptic resistance to desensitization and 2) to determine the consequence of differential pre- and postsynaptic opioid receptor regulation. These goals will be achieved using electrophysiologic, optogenetic and imaging approaches in brain slices. The results may provide insight into ways to prevent postsynaptic receptor desensitization and will thoroughly define how opioids affect neuron activity in a reward circuit via pre- and postsynaptic receptors during acute and chronic exposure to opioids.

Project Summary According to the CDC more that 35% of U.S. adults are obese and therefore are at risk of life-threatening diseases including diabetes, stroke, heart disease and cancers. Further, obesity has huge economic consequences in medical care and lost wages. Studies on obesity clearly indicate important roles for hypothalamic peptide transmitters in regulating aspects of energy homeostasis. Recently, we have shown that in addition to peptide transmitters, hypothalamic neurons expressing the proopiomelanocortin (POMC) gene, which potently affect energy balance, also release the fast-acting amino acid transmitters GABA and glutamate. The primary goal of this proposal is to test the hypothesis that amino acid transmitters released from POMC neurons play a key role in the regulation of metabolism, not by altering food intake, but by affecting glucose regulation and energy expenditure. In testing this hypothesis, we will 1) determine where these additional transmitters act in the brain, 2) determine the resulting consequences when these transmitters are disturbed and 3) determine if the release of these transmitters is sensitive to food intake or select peptides that signal energy state. Ultimately, understanding the neural consequences and regulation of amino acid transmitter release from POMC neurons will allow for a more comprehensive view of how these neurons exert their physiologic functions and could reveal additional levels of dysfunction leading to altered metabolism and may indicate better intervention strategies for energy balance disorders.