The underlying health problem that this application addresses is the growing epidemic of obesity that now affects 30% of the adult population, and the resultant increase in heart disease, hypertension, diabetes, joint dysfunction, stroke, cancer, and early death that is estimated to cost upwards of 75 billion dollars per year. Many factors contribute to the obesity problem today. The hypothalamic arcuate nucleus in the brain acts like the information hub of energy balance, receiving information both from peripheral organs involved in energy storage or release, and receiving axonal information from other regions of the brain that also play important roles in CNS regulation of energy homeostasis, and sending out efferent information that regulates food intake and utilization. A focus for many years in this field has been the neuropeptides involved in energy regulation, and the hypothalamic neurons that secrete them. Most of the critical peptides involved in the regulation of energy homeostasis have been colocalized with the inhibitory transmitter GABA in the arcuate nucleus. This application focuses on what appears to be a new cellular player in the CNS regulation of energy balance that we have identified, the arcuate glutamatergic neuron, a cell that has the profile of one that reduces food intake. In most other regions of the brain glutamate is recognized as a major neurotransmitter. But in the hypothalamus, relatively little attention has been given to glutamate neurons, despite the fact that in the presence of glutamate receptor antagonists there is virtually no excitatory synaptic activity in the arcuate nucleus, or elsewhere in the hypothalamus. Prior to submitting this application, we have solved a central problem, that of recognizing these glutamate cells that exhibit no morphological difference from other hypothalamic cells, by generating a transgenic mouse that expresses the reporter GFP under the control of the vesicular glutamate transporter 2 (vGluT2) selectively in glutamate neurons. Our experiments utilize a combination of whole cell patch clamp electrophysiology, tract tracing with fluorogold and pseudorabies virus, ultrastructural immunocytochemistry, and altered gene expression in the context of challenges to whole animal energy balance. The first set of experiments address the hypothesis that the glutamate neurons show the same efferent axonal projections as the inhibitory neurons of the arcuate nucleus. This will be tested with fluorogold and recombinant pseudorabies virus microinjections into putative target regions. To test the hypothesis that arcuate glutamate cells regulate the activity of anorexigenic proopiomelanocortin (POMC) neurons, we will record from POMC neurons while stimulating local glutamate cells with the excitatory microdrop method to activate cell bodies but not axons of passage. Parallel experiments address the question of whether glutamate cells innervate each other, thereby increasing the timing and power of their output. Ultrastructural dual label immunocytochemistry will be used to test the hypothesis that local orexigenic neuropeptide Y (NPY) immunoreactive axons make synaptic contact with the glutamate cells, similar to the NPY axons that synapse with the anorexigenic POMC neurons. A second set of experiments, using whole cell patch clamp recording in hypothalamic slices, addresses the question of "What active or passive membrane characteristics make the glutamate neurons unique", compared with the GABAergic neurons of the arcuate nucleus that have received substantial attention. A third set of electrophysiological experiments tests the hypothesis that neuropeptides released from other arcuate nucleus neurons involved in the regulation of energy homeostasis modulate the activity of the arcuate glutamate neurons. In the fourth set of experiments, we ask whether arcuate glutamate neurons respond to long distance cues relating to energy homeostasis, particularly glucose and leptin. Together, these experiments will reveal the organization and cellular actions and responses of a unique and previously uncharacterized excitatory neuron in the arcuate nucleus. Understanding these glutamatergic cells should give us a better appreciation of the cellular mechanisms underlying energy homeostasis and body weight regulation, and should give us new insight into the potential treatment of obesity through those neurons that control food intake and expenditure. Many neurons in the arcuate nucleus have multiple roles;it is possible that the glutamate cell is no exception, and may play a role in other functions that this small but critical part of the brain controls, including regulation of the pituitary and other endocrine organs, reproduction and lactation, growth, metabolism, and response to stress.

Public Health Relevance

The growing epidemic of obesity, found in about 30% of the adults in the USA, has led to an increase in heart disease, hypertension, diabetes, stroke, cancer, and early death that is estimated to cost upwards of 75 billion dollars per year. Previous work on how the brain controls energy homeostasis and body weight has focused mostly on inhibitory peptidergic neurons that use the neurotransmitter GABA. In addition the hypothalamic arcuate nucleus plays a key role in pituitary control, endocrine regulation, reproduction, and metabolism. We will focus on a newly discovered neuron in the hypothalamic arcuate nucleus that is excitatory and uses glutamate as a neurotransmitter. Based on how the neuron responds to a number of stimuli studied in preliminary work, we will test the hypothesis that these cells may function to reduce body weight by local excitation of other neurons that are known to reduce food intake and body weight, and by inhibition from cells that increase food intake. Understanding this cell and how it responds to signals of food intake, and what other cells it regulates should allow a new avenue to intervene in how the brain controls body weight, and ultimately may serve to enhance our ability to reverse the obesity epidemic. Similar to other neurons of the arcuate nucleus that serve multiple homeostatic functions, the function of the excitatory arcuate glutamate neuron may include other roles in addition to that of energy homeostasis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK084052-03
Application #
8235967
Study Section
Neuroendocrinology, Neuroimmunology, and Behavior Study Section (NNB)
Program Officer
Hyde, James F
Project Start
2010-04-10
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
3
Fiscal Year
2012
Total Cost
$343,666
Indirect Cost
$136,013
Name
Yale University
Department
Neurosurgery
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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