Julie A. Kauer - US grants

DESCRIPTION(Adapted from applicant's abstract): Drugs of abuse, including amphetamine and cocaine, produce long-lasting behavioral sensitization to subsequent exposures to drug. This is manifested as increased locomotion in animals, and in humans, may underlie a progressive augmentation in the rewarding properties of the drug related to drug craving. Sensitization is initiated in the ventral tegmental area (VTA) of the midbrain, because direct injection of psychostimulants into the VTA causes behavioral sensitization like that seen with systemically administered drug, and because sensitization to peripherally administered stimulant is prevented when specific antagonists are delivered directly into the VTA. Several lines of evidence support the idea that modification of glutamatergic synapses on dopamine neurons in the VTA is required to initiate sensitization. Most compelling is the finding that sensitization is blocked by antagonists of the NMDA subclass of glutamate receptor, delivered either systemically or directly into the VTA. Given the established role of NMDA receptors in synaptic plasticity elsewhere in the brain, these data suggest the hypothesis that during behavioral sensitization an NMDA receptor-dependent form of synaptic plasticity occurs in VTA dopamine neurons that results in strengthened glutamatergic synaptic transmission. This application will test the hypothesis that synaptic plasticity in the VTA is modified by psychostimulants, and that this modification represents the onset of sensitization. We will continue using electrophysiological methods to examine the effects of amphetamine on neurons in the VTA slice preparation.

The hippocampus is a brain region known to be necessary for normal learning and memory in laboratory animals and in humans. Inhibitory interneurons exert powerful control over the electrical output of the hippocampus. The interneurons feed forward and feed back to inhibit both the cell body and dendrites of the excitatory pyramidal cells. Each interneuron synapses onto over one hundred pyramidal cells, and excitation or inhibition of small numbers of interneurons can therefore control excitability in large areas of the hippocampus. The interneurons may thus act as a gate, permitting shifts from one output pattern to another. Modulation of subgroups of interneurons (e. g., those responsible for inhibition of dendritic regions) could cause not only gross inhibition or disinhibition, but a variety of fine-tuned alterations in hippocampal output. The hippocampus is innervated by afferents from subcortical and midbrain nuclei that trigger various behavioral states in vivo. The primary function of this innervation in the hippocampus may be modulation of interneurons. A particularly well-studied example of a change in overall hippocampal excitability is the shift from desynchronized activity to theta rhythm observed in hippocampal EEG recordings. The theta rhythm is turned on by cholinergic afferents from the septum and serotonergic afferents from the raphe nucleus, which have been shown to make synaptic contacts on hippocampal interneurons. The proposed experiments will study the modulation of interneurons by acetylcholine and serotonin, as a test case of the idea that a shift from one state of hippocampal activity to another can be accomplished by modulation of interneurons. This work will define the physiological and pharmacological differences between populations of hippocampal interneurons, and will suggest ways in which distinct interneuron groups may interact to control hippocampal output. An understanding of the effects of acetylcholine and serotonin on this largely unstudied subgroup of neurons is essential for understanding changes that may occur in the hippocampus under a variety of pathological conditions, including epileptiform activity, Alzheimers disease, and drug therapies used in psychiatric treatments that antagonize cholinergic or serotonergic function. Experiments will utilize whole cell recordings of interneurons in two regions of the rat hippocampal slice. Interneurons both in stratum oriens and in stratum lacunosum/moleculare will be recorded from during application of acetylcholine and serotonin. Stimulation of regions of the slice likely to contain axons originating from septum or raphe will be used to study effects of endogenously released transmitter. The ionic basis and the receptor subtypes responsible for any observed effects will be defined using pharmacological tools. After recording, cells will be labelled with biocytin and reacted with avidin-HRP and anti-glutamate decarboxylase antibody to confirm their morphology, and GABAergic identity.

DESCRIPTION (provided by applicant): The goal of this application is to further our understanding of the role of synaptic plasticity of glutamate synapses on dopamine neurons in long-term modifications of the brain that occur with drugs of abuse. Our hypothesis is that excitatory synapses in the dopaminergic reward pathway are pathologically activated in the presence of highly addictive substances such as amphetamine and opiates, and that this activation represents an early cellular contribution to the development of addiction. Over the past three years, my laboratory has used the brain slice preparation to study cellular mechanisms of synaptic depression in the ventral tegmental area (VTA), a region necessary for the development of addictive behaviors. We have found support for our hypothesis that drugs of abuse interact with synaptic plasticity, as amphetamine entirely abolishes long-term depression at glutamatergic synapses. The block of this form of synaptic plasticity is analogous to the removal of a normal brake on excitation of the dopamine system; the long-term block induced by amphetamine continuously present for long periods of time is expected to promote abnormally enhanced firing of dopamine neurons, a cellular response characteristic of the great majority of addictive substances likely to promote the development of addiction. Further examination of the mechanisms normally underlying long-term synaptic depression and the mechanisms by which amphetamine interferes with this plasticity is an important area for further research. However, before embarking on more detailed work at the cellular level, I have increasingly felt it is necessary to verify our work in the intact brain. To date no one has examined synaptic plasticity at excitatory synapses in the VTA in vivo. Thus, it is not known whether long-term depression occurs here in the intact brain, nor whether amphetamine blocks tong-term depression. The purpose of this small grant application is to allow my lab to use an entirely new technique for us, in vivo recordings from the VTA, to verify our basic in vitro findings. We expect that the proposed studies will confirm our in vitro work, and provide important new leads to use in that work.

DESCRIPTION (provided by applicant): Excitatory synaptic transmission provides the basis of normal CNS function, and abnormalities of excitatory neurotransmission contribute to neurological and psychiatric disorders as wide-ranging as epilepsy, schizophrenia, and ischemia. Considerable progress has been made in understanding the processes that underlie normal excitatory synaptic transmission and synaptic plasticity at dendritic spine synapses between CA3 and CA1 pyramidal hippocampal neurons in the in vitro slice preparation. In this application, we propose to use electrophysiological recording to examine excitatory synapses made by the same afferents from CA3 pyramidal cells onto a different target population, GABAergic interneurons. These synapses have markedly different properties from those on their pyramidal cell neighbors. The interneurons are almost free of spines, so excitatory synapses are found nearly exclusively on dendritic shafts. Synapses on interneurons also are reported to lack the AMPAR subunit, GluR2. Furthermore, when high-frequency stimulation is delivered to CA3 afferents, synapses they make upon pyramidal cells undergo LTP; in contrast, our earlier work demonstrates that the same high-frequency stimulation to CA3 afferents triggers LTD at synapses onto interneurons. The fact that the same presynaptic afferents make synapses with distinct properties onto these two targets affords a unique opportunity to compare synaptic function, plasticity, and modulation in a defined and well-understood system in mature brain tissue. This work will provide insight into the proteins and signaling molecules required for regulation of normal synaptic transmission and plasticity at CNS excitatory synapses.

This proposal will continue our work examining the role of synaptic plasticity in the response of ventral tegmental area (VTA) neurons to drugs of abuse, using electrophysiological and pharmacological tools. Our studies will contribute at two levels. First, the proposed experiments will define the basic synaptic and circuit properties of this brain region, essential for processing rewarding and aversive stimuli under physiological conditions. Second, we will link these synaptic and circuit properties with responses to ^-opioids in vitro and in vivo. We are especially interested in defining alterations in VTA function that may be candidate mechanisms contributing to their addictive qualities. In this application, I will explore the role of inhibitory VTA synapses in responses to ^-opioids. About 20- 30% of VTA neurons are GABAergic, and opioids and ethanol interact almost exclusively with GABAergic neurons and GABA receptor function. GABA agonists or antagonists delivered into the VTA are rewarding and powerfully influence behavioral responses to addictive drugs. Furthermore, repeated exposure of rats to either cocaine or morphine alters synaptic function at GABAB receptor synapses on VTA neurons, and a single exposure to ethanol alters GABAA receptor synaptic transmission onto VTA dopamine neurons. Chronic treatment with opioids alters the way the VTA circuit is activated by rewarding stimuli, and this is correlated with changes in GABAergic receptor function in the VTA. First we will compare synapses on dopamine neurons with those on GABAergic neurons. We will then examine rapid effects of opioids on GABAergic transmission at relevant VTA synapses, and finally, we will treat animals with morphine in vivo and then test GABAergic synaptic transmission in brain slices to see if changes in GABAA synaptic transmission have occurred. We will also define the properties of the novel LTP of GABAergic synaptic transmission we have recently discovered, and test the effects on IPSC LTP of acute or in vivo treatment with ^-opioids. I expect our work to elucidate the cellular mechanisms by which opioid compounds alter the normal function of this brain area essential for normal reward processing.

DESCRIPTION (provided by applicant): TRPV1 channels are ligand- and heat-gated ion channels that have a calcium permeability comparable to NMDAR channels. First identified and cloned in primary sensory afferent neurons in the peripheral nervous system, mounting evidence supports the expression and activity of these channels in brain. My laboratory recently published the first functional evidence that TRPV1 channels are present on hippocampal pyramidal cells. Using electrophysiological methods in brain slices, we found that TRPV1 channels are essential for a form of hippocampal synaptic plasticity, the first evidence that any TRP channel is required for synaptic plasticity. The hippocampus is a brain region required for normal formation of new long-term memories. The discovery of a new, highly Ca2+permeable cation channel in hippocampal neurons and its ability to trigger synaptic changes has important implications for hippocampal information processing. Furthermore, the hippocampus is nearly always involved in temporal lobe epilepsy. Our evidence suggests that activation of hippocampal TRPV1 channels will both depolarize the excitatory pyramidal neurons directly, and persistently depress excitatory drive to inhibitory interneurons. This combination of effects is relevant to the development of epilepsy because it is expected to drive the hippocampus into a more excitable state. In this proposal I plan to define the conditions under which hippocampal TRPV1 channels are activated, using as electrophysiological assays both excitability changes in pyramidal neurons and alterations of synaptic function produced by TRPV1 activation. In the peripheral nervous system, TRPV1 has been characterized as a heat-activated ion channel. Although the brain is generally protected from large temperature changes, our preliminary data suggest that hippocampal TRPV1 channels may contribute to heat-activated seizure activity. We will begin to explore the idea that TRPV1 could play a role in febrile seizures, seizures generated in young children during fever for which there is currently no effective treatment. In the proposed experiments, we will test activation of TRPV1 channels by heat and endogenous ligands to begin to identify the conditions under which TRPV1 channels alter hippocampal function. PUBLIC HEALTH RELEVANCE: We have identified for the first time in the hippocampus, a region required for normal learning and memory and frequently implicated in human seizure disorders, a protein called TRPV1 that is activated by heat and natural compounds found in the brain. The heat-sensitivity of TRPV1 suggests that this protein could contribute to fever-induced seizures in young children, a disorder that can progress to epilepsy and that currently has no effective treatment. Here we will explore the novel hypothesis that TRPV1 contributes to hippocampal excitability, and could represent a novel therapeutic target for epileptic seizures.

DESCRIPTION (provided by applicant): In this application, we will characterize the modulation of synaptic plasticity in response to acute stress. In particular, we will use electrophysiological and pharmacological tools and knockout mice to examine long-term potentiation of glutamatergic and GABAergic synapses in the ventral tegmental area (VTA), a key region required for processing rewarding and aversive stimuli under physiological conditions, and also required for addiction to drugs of abuse. I expect our studies to contribute at two levels. First, we will define alterations in the basic synaptic and circuit properties of this brain region after stress. Second, we will link these alterations to stress-induced reinstatement of drug-seeking. In this application, I will focus on the effects of stress on VTA synapses. First we will identify the cellular changes in the VTA that follow a brief stressful stimulus. We will compare different forms of stress and test the time course of stress effects on synapses. Our preliminary data suggest that stress modifies VTA synapses by releasing endogenous opioid peptides, and we will explore this idea. Finally, we will use the molecular information gained in these experiments to promote or block synaptic plasticity in the VTA in vivo while assessing the ability of a brief stress to trigger reinstatement of drug-seeking. These experiments will test for the first time the hypothesis that synaptic changes occurring in the VTA are essential for stressful stimuli to elicit drug-seeking behavior. If my hypothesis is correct, our work will suggest novel molecular targets for therapeutics designed to interfere with the neuroadaptations caused by stress.

DESCRIPTION (provided by applicant): Following peripheral injury, changes in tactile perception develop, including primary and secondary hyperalgesia and allodynia. A loss of GABAergic or glycinergic inhibition is one mechanism that can cause these changes in pain perception. Inhibitory neurons in the dorsal horn play a key role in controlling the flow of nociceptive information through ascending pathways to the brain where it is perceived as painful. Interleukin-1beta (IL-1beta) is an inflammatory cytokine released in the spinal cord during injury and is known to promote pain and cause hyperalgesia when introduced into the spinal cord. Using electrophysiological methods in spinal cord slices, we have found that IL-1beta upregulates inhibitory glycine receptors on inhibitory neurons in the dorsal horn. The rapid inhibition of inhibitory neurons is expected to promote the transmission of pain signals to the brain, and thus may explain how IL-1beta causes pain. Our preliminary results indicate that inflammation in vivo potentiates glycinergic synapses similarly to IL-1beta potentiation observed in vitro. IL-1beta is also released during neuropathic pain, and here we propose to test whether glycinergic neurotransmission on inhibitory interneurons in the pain pathway is upregulated in a model of neuropathic pain. Our work could suggest novel drug targets for the treatment of persistent pain conditions that are currently difficult to treat effectively.

DESCRIPTION (provided by applicant): Despite the great importance of glycine receptors in key areas of the nervous system with relevance for human health, remarkably little is known about the regulation of glycinergic synapse strength, and even less about glycinergic synapses in functional circuits. Glycinergic synapses comprise much of the inhibitory drive controlling networks in the spinal cord, brainstem and midbrain, regulating motor behavior, rhythm generation, somatosensory, auditory, and retinal signaling, and coordination of reflex responses. Our long-term goal is to understand how to control the strength of glycinergic synapses in the central nervous system to provide novel drug targets for disorders of spinal cord and brainstem circuits. The objective of this research proposal is to define how potentiation of glycinergic synapses is triggered and maintained, using functional studies in intact spinal cord slices from adolescent mice. Using electrophysiological recordings in spinal cord slices, we find that the inflammatory cytokine, IL-1beta, rapidly upregulates inhibitory glycine receptors on inhibitory neurons in the dorsal horn. To our knowledge, this is the first example of long-term potentiation (LTP) of glycine receptors anywhere in the CNS. The rapid inhibition of inhibitory dorsal horn neurons is expected to promote the transmission of pain signals to the brain, likely contributing to the known nociceptive effects of intrathecal IL-1beta. Our preliminary data support the hypothesis to be tested in this application: that IL-1beta released in the dorsal horn by peripheral injury activates cell adhesion molecules and intracellular protein kinase cascades, rapidly increasing synaptic glycinergic receptor numbers. The rationale for the proposed research is that by identifying the signaling cascades that normally control glycinergic synapse strength, we will provide novel therapeutic targets to treat pain and other glycine receptor-dependent disorders. Proposed experiments will elucidate the signaling pathways and receptor subtypes involved in glycinergic LTP (Aims 1 and 2), primarily relying on sensitive electrophysiological recordings in spinal cord slices. Our preliminary results also indicate that inflammation in vivo potentiates glycinergic synapses, similarly to IL-1beta potentiation observed in vitro. We will therefore identify the role of glycine receptor LTP after peripheral inflammation (Aim 3), using electrophysiological and behavioral assays. The proposed work is innovative, in our opinion, because 1) we have identified the first example of LTP at glycinergic synapses in the mammalian CNS, and 2) as synaptic plasticity can underlie pathology, delineating the underlying mechanisms offers a new way to control glycinergic synapses in disease. The contributions of this research will be the elucidation of as yet entirely unknown mechanisms underlying glycine receptor signaling and synaptic potentiation in a developed tissue setting. These contributions are significant because they constitute critical first steps towards the development of new treatments for pain, respiratory and motor disorders, and auditory disorders.

? DESCRIPTION (provided by applicant): Predoctoral Training Program in Trans-Disciplinary Pharmacological Sciences This is the resubmission of an application to continue the existing Predoctoral Training Program in Trans- Disciplinary Pharmacological Sciences (T32) within the Molecular Pharmacology and Physiology (MPP) Graduate Program at Brown University. In its first cycle (2010-2015), the training grant funded 13 predoctoral trainees. This renewal application requests funding for 4 predoctoral trainees per year for 5 years and is intended to fund trainees in their second and third years of study. The training program is designed to produce graduates capable of establishing outstanding independent research in the interdisciplinary fields contributing to modern pharmacological sciences. The T32 already has produced several major improvements in this small, developing graduate program, with positive effects extending to the Division of Biology & Medicine and the University. The training program has 33 outstanding faculty trainers drawn from several departments at Brown University and its Warren Alpert Medical School. The research productivity, funding and mentoring records of the training faculty are strong, and there are many collaborative interactions that benefit the trainees. The research areas of trainers within the program fall into five broad categories: 1) molecular structure and its role in disease; 2) neuropharmacology and neural circuit function; 3) receptor and channel pharmacology, physiology and signal transduction; 4) translational and clinical applications; and 5) chemical biology and its applications. Trainees acquire career skills and proficiency in the areas of pharmacology through coursework, lab rotations, and many mechanisms for scientific interaction with each other and with faculty trainers, as well as with scientists from outside Brown. There is extensive advising and evaluation by the Graduate Program Director, Training Grant PI, Graduate Program Committee, Thesis Advisors and Thesis Committees, as well as by Brown's Office of Graduate and Postdoctoral Studies. Trainees also are exposed to a variety of career paths through programs sponsored within and outside the MPP graduate program. We have almost no attrition, and our graduates have outstanding career outcomes. The program strives to recruit and retain students of all ethnicities and socioeconomic backgrounds, and has great success to-date -- the program typically consists of ~40% underrepresented minorities, including African-American, Hispanic and Native American. Continued funding of the Predoctoral Training Program in Trans-Disciplinary Pharmacological Sciences will allow us to continue to design new methods with the goal of graduating outstanding members of the scientific community with the skills essential to developing new drugs and therapeutics.