Anthony van den Pol - US grants

The purpose of this proposal is to continue our investigation of the intrinsic neural organization of three nuclei in the medial hypothalamus. Specifically, we will focus our attention on the suprachiasmatic, paraventricular, and arcuate nuclei. We plan to use a combination of Golgi impregnations and immunocytochemistry to study the dendritic arbors, axonal ramifications, and synaptic communication between neurons resident in this part of the brain. Our first step will be to examine with computer-assistance the frequency and orientation of immunostained axons labeled with antibodies directed against peptides endodgenous to this part of the hypothalamus. Secondly, we will compare Golgi impregnated and immunocytochemically stained dendritic trees in different parts of each nucleus. Third, we will use double immunostaining at the ultrastructural level to determine what cells communicate with which other cells. Finally, we will use a combination of retrograde dye transport (fast blue) with immunocytochemistry to determine the location and specific peptide synthesized of cells which project to the neurohypophysis and median eminence. These studies are critical for our understanding the morphological substrates of hypothalamic function.

Studies will continue to focus on the intrinsic organization of the mediobasal hypothalamus. We plan to use a combination of light and electron microscopy to study Golgi impregnated and immunocytochemically stained tissue from the rat medial hypothalamus, concentrating on those areas which play an important role in neuroendocrine regulation. Using several double ultrastructural immunostaining methods developed in our lab, particular attention will be paid to characterizing the transmitters, peptides, and pituitary tropins found in both pre-synaptic and post-synaptic neurons in this part of the brain. Combinations of pre-embedding immunostaining with peroxidase and silver-intensified colloidal gold and post-embedding immunostaining with colloidal gold will be used to localize and characterize neurons and axonal endings containing various neuroactive substances. The question of subcellular localization of several neuroactive substances will be addressed with a quantitative evaluation of post-embedding immunostaining with colloidal gold particles. Cells which send efferent axonal projections to the median eminence will be identified with retrograde transport of cholera toxin conjugated to horseradish peroxidase; the pituitary tropin contained in these cells will be identified with post-embedding immunocytochemistry using 5 nm colloidal gold adsorbed to Protein A or a secondary immunoglobulin as a marker, and the neurotransmitters of synaptic terminals on identified cells will be labeled with 10 nm colloidal gold. This should allow a precise characterization of the neuroactive substances which are in direct synaptic contact with neurons with identified pituitary tropins which project to the median eminence. Golgi silver chromate impregnations and horseradish peroxidase intracellular filling will be combined with ultrastructural and immunocytochemical analyses to study the dendritic trees of immunocytochemically identified neurons; afferent boutons which are in synaptic contact with peroxidase- or Golgi- labeled dendritic trees will be immunocytochemically identified, and the relative synaptic efficacy of identified boutons will be estimated on the basis of a computer-assisted correlation between light and electron microscopy of this material. The hypothesis that estrogen-induced pituitary prolactin cell tumors may result from insult to hypothalamic dopamine neurons will be examined immunocytochemically.

A group of seven laboratories in clinical and basic neuroscience are requesting funds to obtain a Philips CM 12 Scanning/ Transmission Electron Microscope. Thirty five scientists and support staff associated with these labs are involved in electron microscopy. The Philips CM 12 will in part serve as a replacement for a 15 year old Philips EM 201. Many of the critical features of the newer microscope are not available for the older one; these features include a goniometer stage, scanning electron microscopy for both backscattered and secondary electrons, video output, computer-assisted video intensification and storage, and online quantitation. Principal investigators in the seven labs all are funded by NIH to study neuroscience questions of which all or part are based on ultrastructural analysis. This equipment will allow us to achieve better quality electron microscopy. It will also facilitate new scientific directions based on the specialized features of the Philips CM 12, and because of the more reliable operation and video storage will aid in the accommodation of the growing number of personnel here involved in ultrastructural work.

Recent data from this lab together with emerging results from other labs suggest that glutamate plays an important and widespread role in hypothalamic and neuroendocrine regulation. Despite the importance of hypothalamic glutamate, it has received relatively little attention compared with the attention given to other neuroactive substances also found in the medial hypothalamus. The proposed experiments focus on the excitatory amino acid glutamate, its receptors, and cellular responses to glutamate in the hypothalamus, using the rat as an experimental model. We will use immunocytochemistry and in situ hybridization to study the expression of glutamate receptor genes in the hypothalamus, using cDNA coding for AMPA and kainate types of glutamate receptors, and antisera against the receptor protein in tissue culture and in ultrathin sections from the hypothalamus. The excitotoxic role of glutamate will be examined with a silver stain which demonstrates the cell body and dendritic arbors of neurons soon after injury by glutamate. Digital Ca2+ imaging will be used to examine long-lasting intracellular Ca2+ rises after stimulation with glutamate agonists, particularly relative to possible cytotoxic injury. To test the hypothesis that the presence of glutamate during development of the neuroendocrine hypothalamus influences the subtype of receptor expressed, we will use in situ hybridization and patch clamp recording. To examine glutamate involvement in local circuitry of the hypothalamus, we will combine intracellular electrophysiology, dye-filling, and ultrastructural immunocytochemistry with glutamate antisera. These experiments should provide a better understanding of glutamate involvement in hypothalamic function. They will also address some basic questions from a cellular level, and will examine some clinically relevant experiments with glutamate toxicity. The hypothalamus is involved in the regulation of endocrine and metabolic events and glutamate may play a critical role in the health and disease of these and other systems.

IBN-9511262 Van den Pol, Anthony The hypothalamus is a primary regulatory center in the brain, controlling everything from the endocrine milieu to body temperature, circadian rhythms, and food and water intake. Emerging results suggest that glutamate, a fast-acting excitatory chemical messenger, plays an important role in neuroendocrine regulation by acting within the hypothalamus. A member of the peptide class of messenger molecules called neuropeptide Y (NPY) acts more slowly than glutamate. NPY appears to be involved in numerous hypothalamic functions including food intake, reproduction, and circadian rhythms. A great deal is known about the effects of NPY, but little is known about the mechanisms used by NPY to generate these effects. Dr. Van den Pol proposes to use several different experimental systems to determine the mechanisms by which NPY modulates glutamate function in the hypothalamus. He will investigate the electrophysiological effects of glutamate and the modulatory actions of NPY on hypothalamic neurons. Dr. Van den Pol will use anatomical procedures to identify the projections of NPY and glutamate containing neurons within the hypothalamus revealing unique anatomical features of these cells. The modulation of a fast amino acid transmitter by a peptide, while examined in the hypothalamus, has broad ramifications throughout neuroscience. The proposed studies will increase our understanding of the basic cellular and molecular mechanisms of NPY action and ultimately, will aid our understanding and treatment of a wide range of neurological disorders.

DESCRIPTION (Investigator's Abstract): This is a proposal to test the general hypothesis that GABA plays an excitatory role in the developing hypothalamus of the rat, in contrast to its accepted role as an inhibitory neurotransmitter in the adult. Converging approaches utilizing fura-2 digital calcium imaging, immunocytochemistry and whole cell patch clamp recording will test five specific hypotheses using cultured neurons or hypothalamic slices. First, GABA is hypothesized to depolarize the membrane, increase neuronal activity, and increase intracellular calcium early, but not late, in development. Secondly, it is proposed that glutamate-mediated synaptic activity accelerates the maturation of the inhibitory actions of GABA seen in adults. Thirdly, the PI will test whether GABA is synthesized and released during early hypothalamic development. Fourthly, GABA is proposed to promote cell survival, increase synaptogenesis, influence cell motility, and increase neurite outgrowth in early development. Finally, do the inhibitory actions of GABA in the adult revert to inhibitory actions following neuronal injury? This reversion may have important consequences for how neurons respond to calcium-mediated toxicity and how they respond to GABA in general. Since many hypothalamic circuits are inhibitory and mediated by GABA in the adult, GABA's early excitatory actions may be crucial for normal development.

In the adult brain, glutamate is the principal excitatory transmitter that acts at central synapses. Despite the importance of glutamate, relatively little work has examined the release of and response to glutamate in early neuronal development, and the functional role of glutamate in early development. During this period, glutamate may play a key role in modulating synaptogenesis, neuronal architecture, cellular migration, gene expression, and neuronal survival. We will address the central hypothesis that glutamate plays a significant role as an intercellular messenger in early brain development, even before synapse formation. To determine if axonal growth cones release glutamate, thereby influencing potential postsynaptic target cells we will use outside-out and whole cell patch-clamp electrical recordings; membrane patches, taken from glutamate receptor-containing rat hippocampal neurons, or whole neurons, will be placed close to an axonal growth cone as an ultrasensitive approach to detect glutamate release in vitro. Parallel ultrastructural immunocytochemical experiments with colloidal gold will compare glutamate immunoreactivity in growing and differentiating axons, dendrites, and synapses. We will examine th ehypothesis that glutamate modulates rapid changes in dendritic morphology, possibly as a prelude to synapse formation, through a number of glutamate receptor mechznisms including ionotropic and metabotropic receptors, and voltage activated calcium channels. We will also examine membrane turnover and transmitter vesicle exocytosis in presumptive glutamatergic neurons with the dye FM1-43, studied with confocal microscopy to test the hypothesis thta membranes of growing axons increase exocytosis on contact with a postsynaptic neuron, and that exocytosis in developing neurons is modulated by glutamate stimulation. Using Northern blots and Western blots, we will test the hypothesis that glutamate released during development modulates the expression of specific glutamate receptor subtypes by culturing neurons with specific glutamate receptor blockers. This project addresses the fundamental importance of glutamate during neuronal development using converging morphological, immunocytochemical, physiological, and molecular approaches. Glutamate~s triple role as transmitter, toxin, and tropin makes it a crucial player both in normal development and in a number of human disorders. Glutamate dysfunction has been postulated to be a factor in learning disabilities, schizophrenia, epilepsy, secondary response to brain injury or high fever, ischemia, hypoxia, AIDS-related dementia, cerebral palsy, and Rasmussen~s encephalitis.

Spinal cord injury is a devastating clinical problem, and can lead to permanent motor and sensory loss. At the cellular level, the most debilitating consequences of cord injury are not generally associated withy loss of spinal cord neurons per se, but rather from loss of axons that carry crucial motor and sensory information up and down the cord and to the brain. Olfactory ensheathing cells have been postulated to provide a high degree of axonal elongation, based on tests of only a few types of neurons. We propose to test the hypothesis that olfactory ensheathing cells will enhance axonal growth and synapse formation of additional cell types that send axons up and down the spinal cord. We will use an immortalized ensheathing cell line to test the hypothesis that, after infection into the cord, ensheathing cells will enhance axonal growth, but will not lead to further problems associated with unchecked cell division. We will test the hypothesis that the mechanisms by which ensheathing cells act is mediated both by cell surface molecules that enhance axonal extension, and by the release of trophic factors that enhance neuronal growth and survival. As the ultimate goal of these types of experiments is to provide a milieu for axonal growth in the human, we will continue our work studying interspecies facilitation of neurite elongation, and will examine axonal growth of human neurons of rodent olfactory ensheathing cells. Using time lapse digital imaging, we will also test the hypothesis that ensheathing cells can growth into cellular environments that are generally inhospitable to axonal growth, and then accompany axons across them. In order to study axonal regrowth after injury, we have developed a new model utilizing a transgene mouse that expresses the jellyfish green fluorescent protein in a restricted subset of neurons and their axons. These neurons therefore synthesize their own axonal marker, and can be identified in living and fixed cells in the cord and in culture. We will use their transgenic mouse to study the attributes of olfactory ensheathing cells in enhancing axonal recovery after cord injury.

DESCRIPTION (Verbatim from the Applicant's Abstract): In adult neurons, GABA acts as the primary inhibitory transmitter. In contrast, in the developing hypothalamus GABA can be excitatory by depolarizing the membrane potential, raising cytosolic calcium, and evoking action potentials. The present proposal focuses on early synapse formation in GABAergic neurons. Converging approaches utilizing fura-2 digital calcium imaging, immunocytochemistry, Northern blot mRNA analysis, and whole cell patch clamp recording with gramididin perforations address five hypotheses. Each set of experiments tests a specific hypothesis regarding GABA's early excitatory role, using both cultured hypothalamic neurons and hypothalamic slices from rats or mice. The first set of experiments addresses the hypothesis that GABA is released from axonal growth cones prior to synapse formation, and that this release is modulated by other transmitters receptors on the growing axon. We test the hypothesis that trophic factors, specifically NT-3 and BDNF, exert rapid physiological effects at the GABA developing synapse in culture and slice. This rapid action will enhance GABA release during the period when GABA is excitatory, but this enhancing effect will disappear in older neurons. The hypothesis that activity-related release of GABA will strengthen developing GABAergic synapses by a long-lasting increase in the evoked response will be tested in cultured neurons; identification of GABAergic neurons will be aided by the use of transgenic mice that express the jellyfish gene for GFP in cultured hypothalamic GABAergic cells. The hypothesis that synaptic release of GABA enhances the expression of genes coding for synaptic proteins and transcription factors in developing, but not mature, synaptic coupled neurons will be tested with Northern blot analysis. The final set of experiments pursues our earlier work showing that GABA reverts from its adult inhibitory action to an excitatory one after neuronal injury in culture. This will be extended to test the hypothesis that injury directly to the brain will result in depolarizing actions of GABA. The hypothalamus controls body temperature, the endocrine system, circadian rhythms, the autonomic nervous system, gender differentiation, energy homeostasis, and water balance, and many of the synapses involved in these functions release GABA. GABA's excitatory actions during development are not restricted to the hypothalamus, but rather are widespread throughout the brain. Thus, what we learn from our experiments on hypothalamic neurons should have general applicability to other CNS neurons.

Neurons of the mediobasal hypothalamus act to control the endocrine system. By secreting factors into the median eminence that are carried to the anterior pituitary gland, these neurons regulate hormone release form the thyroid, ovary, testis and adrenal gland. Many different neurotransmitters are present in the mediobasal hypothalamus, several of them were first discovered in this important brain area. Neuronal communication to these neuroendocrine neurons is therefore critical in conveying information from the rest of the brain to this final common pathway regulating hormonal balances. Of the dozens of neurotransmitters found here, two amino acid transmitters, glutamate and GABA, appear to be responsible for almost all actual synaptic activity, and when their receptors are blocked, synaptic activity is generally eliminated. A number of hypothalamic peptides have been shown to play a very important role in endocrine regulation. The general scientific point addressed here is that neuroactive peptides in the mediobasal hypothalamus act primarily to modulate the activity of the amino acid neurotransmitters.
Dr. van den Pol will study a new hypothalamic peptide neurotransmitter called hypocretin, which increases neuronal activity. Hypocretin is synthesized only by neurons in the dorsolateral hypothalamus and perifornical area, a part of the brain intimately involved in the regulation of body weight and energy balance. Nerve cells that make hypocretin send long axons to the mediobasal hypothalamus, suggesting they may signal this brain region. Dr. Van den Pol's laboratory will use rat brain slices in vitro and synaptically coupled cultures to study the mediobasal hypothalamic cells that are influenced by this new transmitter and its mechanism of action. To accomplish this, they will use digital imaging and electrophysiology. To test the hypothesis that hypocretin acts by modulating GABA and glutamate neurons of the arcuate neurons, other transmitter actions will be blocked and the effect of hypocretin on GABA and glutamate mediated electrical activity will be determined. They will also investigate if hypocretin's mechanism of action involves intracellular calcium.
Results obtained from these studies could contribute to elucidate the role of hypocretin in feeding, maintenance of weight and body temperature and in the regulation of the endocrine and autonomic nervous system. It will also advance our knowledge on how neurotransmitters work.

DESCRIPTION: (Applicant's abstract) Hippocampal epilepsy is characterized by synchronized hyperexcitable neurons. Many of the neurons in the hippocampus release the excitatory transmitter glutamate which contributes to the excitation. The general hypothesis that endogenous neuromodulators may influence levels of excitability in the hippocampus will be examined. Specifically, we will focus on neuropeptide Y (NPY) inhibition of excitatory neurons in the epileptic human hippocampus, and in a rat model of epilepsy. NPY is found throughout the hippocampus in both neurons and in presynaptic axons. It has been suggested as one of the brain's natural anti-seizure transmitters, and its expression and distribution appears to change with epilepsy. The presynaptic role of NPY in reducing glutamate actions will be studied with whole cell recordings in slices of the human hippocampus. Using a simplified model of hyperexcitability consisting of a single self-innervating rat hippocampal neuron, we will examine the effect of NPY and specific receptor agonists to test the hypothesis that NPY acts by a presynaptic mechanism via Y2 and Y5 receptors to reduce glutamate release in neurons showing epileptiform activity. In parallel we will directly test the hypothesis that NPY blocks glutamate release presynaptically by using the dye FM1-43 to study transmitter vesicle exocytosis in glutamatergic neurons. The hypothesis that NPY is found in GABAergic neurons will be tested with dual ultrastructural immunocytochemistry. The hypothesis that changes in neuronal activity mediated by glutamate will alter levels of expression of NPY and NPY receptor Y1-Y5 mRNA will be tested with cDNA-PCR and Northern blot analyses in parallel studies of different regions of the epileptic human and rat hippocampus and in a tissue culture model of hyperexcitable rat hippocampal neurons. NPY is potentially of great interest because its primary action in the normal hippocampus appears to be one of depressing hyperexcited neurons, without a substantial effect on normal neurotransmission.

DESCRIPTION (provided by applicant): Within the brain, cytomegalovirus (CMV) is the leading viral cause of congenital disease, often producing serious neurological deficits. By attacking the developing CNS, CMV causes serious brain disorders that include microencephaly, epilepsy, deafness, microgyria, mental retardation, sensory loss, motor problems, and psychiatric disturbances. CMV is also a clinically important opportunistic virus that can lead to serious neurological disease in AIDS patients. Despite the clinical importance of CMV infections of the brain, relatively little experimental work has been done in this area, and many basic questions remained unanswered. The present application addresses basic mechanisms of viral spread into and within the brain, and the repercussions of this infection. A recombinant mouse CMV expressing GFP will be used to identify infected cells and track virus dispersal. CMV shows rapid dispersal in developing but not mature brain;the hypothesis that CMV can be spread through axonal transport, but only in the developing axon, will be tested. The hypothesis that interferons alpha/beta and gamma can reduce or eliminate CMV from the brain will be addressed with in vitro and in vivo experiments. Parallel experiments will test the hypothesis that CMV activates interferon pathways in the mature brain, but not in the developing brain. Mice lacking interferon receptors will be used to test further that CMV effects are mediated by these receptors and not by a non-specific action of CMV. An ultrastructural analysis will assess differential virus binding to immature and mature neurons, and determine where on the neuron surface CMV binds. In the immunocompromised CNS, the olfactory system shows the greatest levels of infection;we will address the hypothesis that the olfactory system is a weak link in the brain's protection against virus in SCID mice. The hypothesis that neurons that recover from CMV still show physiological dysfunction will be tested with whole cell patch clamp recording using current and voltage clamp and with fura-2 calcium digital imaging. CMV can remain latent for long periods. We will test the hypothesis that after interferon or gangciclovir treatment and recovery from infection, CMV can escape from neuronal latency, and establish a new round of productive infectious virus. These experiments will help us understand the mechanisms associated with CMV-induced neurological dysfunction, and how to combat the virus within the brain with minimal complications.

Hypocretin (orexin) is a neuropeptide synthesized by lateral hypothalamic neurons that project widely throughout the CMS, including to regions of the brain that have been linked to the addictive actions of opioids and nicotine. Hypocretin plays a key role in activation of many brain circuits related to attention and arousal. Absence of hypocretin or its receptor results in narcolepsy, a neurological disease characterized by continued bouts of intense sleepiness during the day, both in animals and humans. Hypocretin neurons also play a role in addiction to opiates. Despite a growing interest in the role of these neurons in substance abuse, almost nothing is known about the cellular physiology of the responses of hypocretin cells to opiates. To facilitate the study of hypocretin neuron neurophysiology, transgenic mice that express GFP selectively in hypocretin neurons will be used. Whole cell patch clamp recording will be used to understand the role of dynorphin, an opioid neuropeptide cosynthesized with hypocretin. The hypothesis that hypocretin neurons are inhibited by dynorphin will be tested. Electron microscopy and immunocytochemistry will be used to study the subcellular localization of the dynorphin neuropeptide, and converging electrophysiological experiments will be used to address the hypothesis that dendrites and axons contain and release the opioid peptide, and that identified neurons postsynaptic to hypocretin axons show diverging responses to hypocretin and dynorphin. We will test the hypothesis that dynorphin receptors desensitize before hypocretin receptors, freeing postsynaptic neurons from opioid inhibition. Long term effects of the opiate morphine will be studied in brain slices after chronic morphine exposure in vitro and in vivo. The most common addictive substance is nicotine, found in tobacco. Nicotine increases general arousal, similar to the actions of hypocretin neurons. The hypothesis that nicotine has a direct action on hypocretin cells, and that long-term exposure will alter the physiology of these cells, will be tested. These experiments will provide a better understanding of the role hypocretin neurons play in the common lack of arousal and lethargic state shown by opiate users, and the arousal response to nicotine. Hypocretin neurons may thus serve as a future target for treatment of addictive drugs that alter the arousal state. Understanding the role of the endogenous opioid in hypocretin cells should also facilitate the understanding and treatment of narcolepsy.

DESCRIPTION (provided by applicant): The amino acid GABA acts as the primary inhibitory transmitter in the adult brain. In contrast, in the developing hypothalamus GABA can be excitatory by depolarizing the membrane potential, raising cytosolic calcium, and evoking action potentials. The present proposal focuses on the excitatory actions of GABA in developing hypothalamic neurons. Converging approaches utilize fura-2 calcium digital imaging, gene chips, immunocytochemistry, electron microscopy, and whole cell patch clamp recording with conventional and gramicidin access. Each set of experiments tests a specific hypothesis regarding GABA's early excitatory role, using both cultured hypothalamic neurons and hypothalamic slices from mice. Hypothalamic slices containing the lateral hypothalamus/perifornical area will be used to examine early excitatory actions of GABA, and to study timing events related to depolarizing excitation or shunting. The hypothesis that spike-dependent release of GABA will strengthen developing GABAergic synapses by a long-lasting increase in the evoked response will be tested in a model system of a single autaptic neuron in vitro, focusing on a single type of GABA neuron that synthesizes the peptide MCH, and is identified by transfection with dsRed or GFP reporter genes driven by the MCH promoter. The hypothesis that synaptic actions of GABA, when excitatory, increase neuronal growth will be studied in hypothalamic MCH neurons, using time-lapse imaging. Gene arrays will be used to test the hypothesis that excitatory synaptic actions of GABA enhance the expression of specific genes coding for synaptic proteins, trophic and transcription factors, and CI- transporters in developing, but not mature, hypothalamic neurons; and that neuron trauma depresses outward CI- transporter expression and recapitulates the excitatory actions of GABA on gene expression. The hypothalamus controls body temperature, the endocrine system, circadian rhythms, the autonomic nervous system, gender differentiation, energy homeostasis, and water balance, and many of the synapses involved in these critical functions release GABA. GABA's excitatory actions during development are widespread throughout the brain. Thus, what we learn from our experiments on hypothalamic neurons should have general applicability to other CNS neurons.

DESCRIPTION (provided by applicant): The neurons of the hypothalamus that synthesize melanin concentrating hormone (MCH) have been postulated to play a key role in the initiation of feeding and in energy homeostasis. MCH application in the brain increases feeding and weight gain, whereas loss of the MCH gene causes a decrease. Despite a large amount of fascinating data on the general actions of MCH, almost nothing is known about the cellular physiology of these cells. The primary focus of this proposal is to study the physiological characteristics, and the responses to neurotransmitters and neuropeptides postulated to play a role in energy homeostasis, and to examine the response of these neurons to metabolic signals. Whole cell patch clamp recording will be done in transgenic mouse hypothalamic slices containing MCH neurons identifiable by selective expression of GFP. First, we will study the passive and active membrane properties of the MCH cells to provide a foundation for subsequent work. Neuropeptide Y, an orexigenic peptide, has been suggested to increase feeding by activation of the MCH neurons. In contrast, we postulate that NPY exerts inhibitory actions on MCH neurons, consistent with previous electrophysiological data from other regions of the hypothalamus; parallel experiments will test the hypothesis that MCH cells are excited by melanocortin peptides such as alpha-MSH, postulated to inhibit food intake. The hypothesis that MCH neurons respond to metabolic signals relating to energy homeostasis will be tested, focusing on synaptic and cellular response to glucose, ghrelin, and leptin. To study mechanisms of release and modulation of presynaptic MCH axons, we will use an in vitro model where MCH axons terminate on the cell of origin; we will test the corroborating hypothesis that MCH cells synthesize and release the inhibitory transmitter GABA using dual ultrastructural immunocytochemistry and whole cell recording. The hypothesis that MCH inhibits the release of GABA, potentially leading to disinhibition, will be tested. Together, theses studies address questions relating to the cellular characteristics and responses of these unique hypothalamic neurons. Obesity has become a major health problem leading to increases in heart disease, strokes, mortality and other risks. Studies of the cellular physiology of MCH neurons should provide a better understanding of the role of this system in regulating energy homeostasis and obesity.

[unreadable] DESCRIPTION (provided by applicant): Glioblastoma are aggressive and invasive brain tumors that generally lead to death within a year of diagnosis. No cure is available, and current treatments prolong life by only a few months. In the current application, we propose to study the potential use of vesicular stomatitis virus (VSV) to selectively infect and kill these brain tumors. We have developed recombinant VSVs that selectively infect human glioblastoma and not control cells in vitro, and target glioblastoma cells transplanted into the mouse brain. As these viruses are replication-competent, after killing the first round of glioblastoma, progeny viruses are released which cytolytically infect more glioblastoma cells. Experiments are based on in vitro use of human and mouse glioblastoma and control cells, and on human or mouse tumor cells transplanted into the mouse brain. Four sets of experiments are planned. In the first set of experiments, recombinant VSVs will be used to test the hypothesis that enhancing the cellular interferon response or slowing replication will selectively protect normal, but not brain tumor, cells. VSVs that selectively infect glioblastoma will then be used with in vivo experiments in which human glioblastoma cells that express a red reporter gene are transplanted into the brains of SCID mice, or red mouse glioblastoma is transplanted into the normal immunocompetent mouse brain. VSV will be administered either directly into the brain tumor, or by systemic injection into the tail vein to test the hypothesis that the virus will selectively infect and kill the glioblastoma cells within the brain with little spread to the normal brain tissue. To increase the safety profile related to oncolytic viruses in the brain, a third set of in vitro and in vivo experiments will test several antiviral drugs (interferons, ribavirin, aspirin, polylC, IL-12) that have been reported to be effective in controlling viral infections to determine which is the most efficacious in blocking possible VSV infections of normal brain. The hypothesis that peripheral VSV immunization will attenuate a later cerebral infection by recombinant VSV will be tested. A final set of experiments will test the hypothesis that recombinant VSV will show no, or relatively little infection and replication in non-tumor slices of the human brain in vitro. The primary goal of these studies is to test the efficacy and safety of variant oncolytic VSVs as the first step as a potential treatment for currently incurable human brain tumors. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant): Modern drug-discovery research over the last two decades aimed at improving the pharmacological treatment of epileptic seizures has resulted in more than a dozen new anti- epileptic drugs (AEDs). Although these new AEDs may have reduced side effects and drug- drug interactions, the percentage of epileptic patients who continue to have seizures while treated with the new AEDs has remained at 30-40% for >20 years. In this proposal, we aim to develop a gene-therapy approach to treat pharmacoresistant epilepsy through the implementation of a novel designer receptor system that should not affect normal brain function, and therefore is less likely to have significant side-effects. This system will utilize adeno-associated viral (AAV) delivery of a modified human ¿1glycine receptor with reduced glycine sensitivity and enhanced sensitivity to the FDA-approved drug, ivermectin. Recombinant AAVs will be genetically optimized for expression in epileptic neurons and tested for efficacy in animal models of epilepsy. Ivermectin, which has minimal effects on the normal brain at the very low concentrations necessary to activate the modified receptor, will then act as an AED specifically at the site of generation of the epileptic seizures, thus suppressing epileptic seizures without affecting normal brain function. These studies are intended to lead directly to clinical trials. The use of a human receptor and an FDA approved drug should facilitate this advancement from bench side to clinical treatment so that by the end of the 4 year grant, we will have a gene transfer vector in hand that could be considered for clinical trials in pharmacoresistant epilepsy patients.

DESCRIPTION (provided by applicant): The prognosis for survival in patients with metastatic melanoma has not changed in the last 20 years and remains dismal despite advances in tumor detection and the development of melanoma-specific systemic therapies. Because of the failure of current chemotherapeutic and immunologically- based treatments to eradicate melanoma, we propose a new approach. In this proposal, we test a replication-competent oncolytic virus generated in this lab at Yale, VSVrp30. In preliminary tests VSVrp30 shows considerable promise in the potential treatment of melanoma. In vitro and in vivo experiments show that the virus selectively and rapidly infects and destroys human metastatic melanoma, with relatively little or no infection of normal human melanocytes. We seek funding to pursue experiments to determine if the virus can target and destroy melanoma cells in multiple conditions in animal and in vitro models. We will first test the oncolytic actions of the virus with a series of in vitro experiments on a large number of human melanomas and normal melanocytes available at Yale. Another set of experiments will employ human melanoma that is stably transfected with a coral reporter gene that generates a red fluorescence in the cancer cells. These human cells will be transplanted into SCID mice, both as a solid tumor, and as dispersed metastatic-like cancer cells. Virus will be given intratumorally and intravenously to test the hypothesis that the virus wll target and kill the red tumor cells with minimal infection of normal cells. Infected cells can be readily detected by the expression of a GFP reporter incorporated into the viral genome. A third set of parallel experiments will be done using the mouse melanoma line B16 in syngeneic C57Bl/6 mice with a normal immune system, allowing us to test the hypothesis that the virus can selectively detect and destroy melanoma in the presence of a normal immune system, and prolong mouse survival from melanoma; if the virus does not completely eliminate the melanoma cells, we will test the secondary hypothesis that temporarily depressing the systemic or innate immune systems with immunosuppressant drugs will enhance the oncolytic actions of the virus. A fourth set of experiments will examine the genetic mechanisms underlying the increased viral infection of melanoma cells, using an extensive series of human melanomas in which the exomes have been sequenced. These experiments will be complemented by experiments to test the hypothesis that specific induced gene mutations involving BRAF, PTEN, and CDKN2A that are common to melanoma, directly increase virus infection. A final series of experiments will test the hypothesis that the virus can cross the blood brain barrier and selectively destroy melanoma in the mouse brain, and that pre-immunization, potentially followed by transient immunosuppression, will enhance oncolysis and provide another layer of protection to the brain. If we detect collateral damage to normal brain, then we will test a new virus, 1,2-VSV, that we recently generated which is the most attenuated of any VSV we have worked with, yet still targets melanoma. Its highly attenuated nature reduces concerns relating to infection of normal brain tissue. If these experiments are successful, they will form a major advance toward clinical trials for metastatic melanoma in humans.

DESCRIPTION (provided by applicant): Glioblastomas are aggressive and invasive brain tumors that generally lead to death within a year of diagnosis. No cure is available, and current treatments prolong life by only a few months, often at the expense of quality of life. A number of parvoviruses have been reported to show oncolytic potential against cancer cells, and a rat parvovirus, H-1, is currently in clinical trials to treat glioma patients. In our initial work screning a large number of parvoviruses, we found one relatively obscure parvovirus, LuIII, that performed substantially better than any of the others tested, including H-1, and was the only parvovirus tested that successfully killed multiple human gliomas. LuIII appears safe in the brain, and does not target or kill neurons and shows minimal infection of normal glia. Here we test a number of hypotheses related to the ability of LuIII to selectively infect, replicate in, an kill human glioma. In the first set of experiments, we test the hypothesis that LuIII will successfully target and kill glioblastoma cells that are transplanted into the brain, with relativey little toxicity to the normal brain, both after an intratumoral virus injection, and after intravenus inoculation. Tumors are detected by expression of a red fluorescent reporter, and virally infected cells are detected by green immunofluorescent staining for the LuIII viral antigen NS1. A critical clinical problem with glioma is their recurrence after surgical or radiation treatment; here we tes the hypothesis that LuIII may maintain an asymptomatic low level of infection in normal human glial cells, and that LuIII arising from normal cells will thereby attenuate or block glioma cell expansion or recurrence. We will corroborate our preliminary findings and test the hypothesis that LuIII does not generate an anti-viral interferon (IFN) response, and that LuIII infection is nt attenuated by IFN. This independence from the IFN system sets LuIII apart from a number of other oncolytic viruses used in the brain which are sensitive to IFN. LuIII's insensitivity to IFN would allow a co-treatment with LuIII and IFN (IFN is a partially effective anti-tumor treatment in a subset of brain tumors), or co-treatment with LuIII together with an IFN-sensitive oncolytic virus. We will employ deep whole exome genetic sequencing, and sequencing of the mRNA transcriptome, to search for gene mutations in glioma that correlate with high levels of LuIII infection. This genetic information is useful both to understand the mechanisms underlying LuIII's selective infection of gliomas, and also may prove useful as a diagnostic predictor of which tumor-related mutations are most likely to be associated with a high LuIII infection. Finally, our preliminary data suggest that LuIII is effective at not only targeting and killing glioblastoma, but also infects and kills other cancers that invade the brain, including melanoma and lung cancer. Lung cancer metastasis is the most common problem involving secondary cancer in the brain. We will test the hypothesis that LuIII targets lung cancer cells in the brain n experiments parallel to those above involving glioma. Our central goal is to test the potential of LuIII as a safe and effective means of substantially attenuating or destroying brain tumors in humans.

? DESCRIPTION (provided by applicant): Glioblastomas are aggressive and invasive brain tumors that generally lead to death within a year of diagnosis. No cure is available. Current treatments prolong life by only a few months, often at the expense of quality of life. Here we test the general hypothesis that a novel recombinant chimeric virus will target and kill gliomas with no detectable adverse effect to the brain. Of the large number of viruses we have tested, vesicular stomatitis virus (VSV) appears to be one of the most effective for targeting and destroying brain tumors. However, VSV has the potential unwanted side effect of infecting neurons, and half of our efforts in the last few years have focused on reducing or controlling potential neurotoxicity of VSV. To avoid complications and toxicity of the VSV G-protein, particularly its binding to neurons, we have compared a number of recombinant chimeric viruses in which the VSV G-protein gene was replaced by genes coding for binding glycoproteins of non-related viruses including rabies, lymphocytic choriomeningitis, Marburg, Ebola, and Lassa viruses. Of these chimeric viruses tested, one stood out as a clear superlative safe oncolytic candidate: a chimeric virus consisting of a gene coding for the Lassa glycoprotein together with genes coding for the VSV N,P,M, and L proteins, and a GFP reporter gene which further attenuates the virus. Lassa-VSV is safe, both in rodents and primates. Of considerable importance, our direct injection of Lassa-VSV into the brains of normal mice or rats, or even into the brains of immunodeficient mice has not resulted in any detectable adverse effects, whereas injections of native or other attenuated VSVs generated neurological complications sometimes resulting in death. Equally important, in our preliminary experiments in vitro and in vivo, Lassa-VSV targets and destroys gliomas completely without damage to the host brain, and extends survival of tumor-bearing mice indefinitely. In the first set of experiments, we test the hypothess that Lassa-VSV successfully targets and kills glioblastoma cells that are transplanted into the brain, both after an intratumoral virus injection, and after intravenous inoculation. We use both glioma cell lines and primary human gliomas transplanted into immunodeficient mice. We also test syngeneic mouse glioma implanted into immunocompetent mouse host brains. Tumors are detected by expression of a red fluorescent reporter and virally infected cells are detected by expression of a reporter gene coding for green fluorescent protein. In the second Aim, we address the hypothesis that the mechanism behind the safety and selectivity of Lassa-VSV in the brain is that the virus either does not bind to receptors on neurons or normal glia, or is not internalized, whereas binding and internalization in glioma is robust. The lack of virus infection of neurons is studied by blocking or enhancing various steps in the infectious pathway coupled with reverse transcriptase quantitative PCR, and corroborated with electron microscopy, and in additional species and in human brain slices. A key hypothesis we test in Aim 3 is that Lassa-VSV initiates an attack by the systemic immune system, particularly by CD8+ T cells, on the glioma that continues even after the virus is eliminated, thereby preventing the recurrence of tumor. This is tested by infection of glioma in the brain; after the virus is eliminated, we examin the potential of newly implanted glioma to grow in the presence of the enhanced immune response. Immune targeting is complemented with adoptive transfer, CD8-T cell elimination, and immunocytochemistry to detect immune cells recruited to the infected tumor. Lassa-VSV is remarkable in that it can completely kill glioma with no detectable adverse side effects in the brain or elsewhere. If our experiments are successful, we think this virus would be a top priority candidate for clinical trials.

? DESCRIPTION (provided by applicant): Obesity, which often leads to secondary health complications including heart disease, diabetes, stroke, cancer, and early death, has become one of the primary health concerns in the US. Many neurotransmitters and neuromodulators in the CNS participate in the regulation of energy homeostasis. Dopamine is a great interest in this regard because it promotes food intake, but its role in energy homeostasis is complex and it is often considered as part of a CNS reward system. Here we test the general hypothesis that dopaminergic axons innervate the hypothalamic arcuate nucleus and directly modulate food intake and body weight by regulating the activity of two key neuron types here, but in opposite directions. The arcuate nucleus plays a key role in physiological homeostasis, and particularly energy homeostasis. Two key types of arcuate nucleus neurons are the anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP)/neuropeptide Y (NPY) neurons. The studies proposed below are supported by our preliminary data showing strong innervation of POMC and AgRP neurons by dopamine axons, and by a robust dopaminemediated inhibition of POMC neurons, but excitation of neighboring AgRP neurons. The first Aim examines the structural substrates for interaction between dopamine axons and the POMC and AgRP neurons. We test the hypothesis that dopamine axons make direct synaptic connections with POMC or AgRP neurons using confocal scanning laser microscopy and dual immunolabel electron microscopy with antisera against dopamine to identify dopamine axons. To test the hypothesis that dopamine projections to the ARC arise from the ventral tegmental area (VTA) or other dopaminergic populations, we will use a combination of transgenic mice expressing Cre recombinase under control of the tyrosine hydroxylase or dopamine transporter promoter, coupled with focused intracerebral injections of viral vector containing floxed-stop GFP or tdTomato reporter genes to study efferent projections to the arcuate nucleus from dopaminergic cells. We will also employ injection into the arcuate nucleus of a Cre recombinase-dependent Brainbow-type pseudorabies virus; after retrograde axonal transport the PRV normally expresses a red fluorescent reporter in infected cells, but in dopamine cells expressing Cre, changes color to yellow or blue. Aim 2 tests the hypothesis that dopamine exerts opposing actions on POMC and AgRP neurons, inhibiting POMC cells, but exciting AgRP neurons, both actions enhancing food intake. We will employ transgenic mice expressing various reporter genes, and whole cell voltage- and current clamp recording in hypothalamic brain slices. Whole cell recording will allow us to address different mechanisms of dopamine actions on the POMC and AgRP cells. In Aim 3, a final set of experiments employs optogenetics and transgenic mice expressing Cre recombinase in dopamine neurons, coupled with viral vectors expressing floxed-stop channelrhodopsin-2 (ChR2) or ChIEF. Blue light activates a ChR2- or ChIEF-mediated inward current, allowing us to photostimulate selectively the dopamine axons within the arcuate nucleus. This approach will be used in brain slices to test the hypothesis that light-activated release of transmitter from dopamine axons will exert opposite effects on POMC and AgRP neurons. In mouse in vivo experiments, we test the hypothesis that light-activated transmitter release from dopamine axons will enhance food intake. Food intake, body weight, and activity will be monitored during periods of blue light stimulation of the dopamine axons, and during control periods. Together, these experiments will examine in detail with converging structural, tracing, electrophysiological, and behavioral analyses the hypothesis that dopamine axons innervating arcuate POMC and AgRP neurons play a positive role in energy homeostasis. With the health problems associated with the growing levels of obesity in this country, by some estimates reaching 30% of the adult population, and the associated health complications, knowing and understanding the brain cells involved will help to identify novel approaches to reducing the trend toward obesity.

Title: Zona incerta GABA neurons modulate energy homeostasis Abstract. Obesity, which often leads to secondary health complications including heart disease, diabetes, stroke, cancer, and early death, has become a major health concern in the US. Here we test the general hypothesis that neurons in the rostromedial zona incerta (ZI), and particularly inhibitory GABA neurons, play an unexpectedly profound orexigenic role in increasing food intake and body weight. Most of the work done on the neuronal regulation of energy homeostasis has previously focused on neurons in other brain regions. The first Aim examines the structural substrates for interaction between ZI GABA axons and their postsynaptic targets. Using confocal scanning laser microscopy and dual immunolabel electron microscopy coupled with cre recombinase-dependent AAV and rabies virus tracers, we test the hypothesis that ZI axons project to a number of sites, including the paraventricular thalamus (PVT) and hypothalamic ventromedial nucleus (VMH) where direct synaptic connections are made with excitatory neurons. We use multiple transgenic mouse lines expressing Cre recombinase under control of various neuron-selective promoters including mice that express Cre in GABA neurons driven by a vGAT promoter. These will be coupled with intracerebral microinjections of AAV viral vectors containing floxed GFP or tdTomato reporter genes to study ZI GABA neuron efferent and afferent axon projections. We will also employ injection of a Cre recombinase-dependent Brainbow-type pseudorabies virus into the ZI; after retrograde axonal transport this PRV expresses a red reporter in wild-type cells, but in Cre-expressing GABA cells, reporter expression changes to yellow or cyan, helping define the cell of interest. Aim 2 tests the hypothesis that ZI GABA cells respond to long distance signals of energy homeostasis including ghrelin and leptin, and also to axonally released neuropeptide modulators of food intake. Whole cell recording allows us to test different mechanisms of action on ZI GABA cells produced by neuromodulator signals from other neurons involved in energy homeostasis. C-fos expression will be examined after food deprivation to test the hypothesis that ZI GABA neurons are more active during reduced food availability. Optogenetics is used in brain slices to test the hypothesis that release of transmitter from ZI GABA axons will exert similar inhibitory effects on PVT and VMH neurons; neighboring ZI dopamine cells are also tested. In Aim 3, we examine the role that rostromedial ZI GABA neurons play in ongoing energy homeostasis by cell silencing (using Gi-DREADDs and caspase) to test the hypothesis that body weight and food intake is reduced. Optogenetic activation with ChR2 variant ChIEF will test the hypothesis that stimulation of ZI GABA axons in different terminal zones will each enhance food intake and body weight. We also test the hypothesis that ZI GABA neuron activation provides a positive emotional valence. Together, these experiments examine converging structural, electrophysiological, and behavioral analyses, focusing on the role of the GABA ZI neurons in energy homeostasis. With the growing levels of obesity in this country approaching 30% of the adult population, and the associated health complications, identifying and understanding the brain cells that control and sense energy homeostasis will help to identify novel approaches to reducing the trend toward obesity.