1985 |
Siggins, George Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Morphine-Like Brain Peptides: Cellular Neurobiology @ Scripps Clinic and Research Foundation
Multi-disciplinary methods of cellular neurobiology will be used to determine the brain circuitry and functional properties of brain endorphin and enkephalin peptides and their role in neurophysiological and behavioral phenomena. Immunocytochemical studies, together with lesions, manipulations of axoplasmic transport, and retrograde axonal transport will be employed to define endorphin peptide circuits in avian, rodent and primate brain and in invertebrate nervous systems. Radio-immunoassays, immunocytochemistry, explant cell culture, and in vitro brain slices will be used to characterize points of presumptive endorphinergic transmission by electrophysiological methods of extracellular recording, interacellular recording, micro-iontophoresis and micro-pressure adimistration of peptides and related drugs. Electroencephalography will be employed to examine the pharmacology, receptor specificity, and transmitter interactions underlying the basis of epileptiform limbic seizure produced by injection of endorphin peptides into cerebrospinal fluid. Experiments in awake unrestrained normal or morphine-dependent animals will determine the role of specific endorphin- or enkephalin peptide-mediated circuits in responsiveness to noxious stimuli, and in control of specific hypothalamic and thalamic functions including general somatic sensitivity, body temperature, feeding, drinking, affective components of animal responses, and in general behavioral mechanisms. Chronic implanted stimulating electrodes will be used to simulate human models of endorphin-mediated pain suppression. By defining how and where endogenous opiate peptides function normally, and by determining their characteristic receptor mechanisms, these experiments will help to provide fundamental details of how opiates act to reduce pain and how opioid peptide-mediated cellular mechanisms are altered by phenomena of narcotic abuse.
|
0.945 |
1986 — 1997 |
Siggins, George Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Morphine-Like Brain Peptides--Cellular Neurobiology @ Scripps Research Institute
This application proposes to continue and extend cellular neurophysiological studies on opioid peptides and opiates in a brain region, the nucleus accumbens septi (NAcc), containing proenkephalin- and prodynorphin-derived peptides. This region was chosen because of its reported role in heroin self-administration, thus to be developed as a cellular model for opiate reward or dependence, with the ultimate goal of testing therapeutic interventions. The overall objective is to determine the physiological role of central opioid peptide-containing neurons in normative and opiate seeking behavior, using intracellular (current- and voltage-clamp) and whole-cell clamp recording in brain slices in vitro. Our previous studies have confirmed an opiate reduction of synaptic transmission in the NAcc, similar to that produced by dopamine (DA) and ethanol. Studies in other labs have noted interactions of opiates with DA and glutamate. Therefore, our specific aims are to: 1) Study possible opiate and glutamate (especially NMDA) interactions in NAcc neurons, since recent reports show that NMDA antagonists can block opiate dependence and tolerance, and since ethanol also blocks NMDA currents; 2) Continue to examine the role of DA in the responses of NAcc neurons to opiates and opioid peptides using exogenous DA, DA antagonists and DA uptake blockers. We will seek interactions of DA and opiates and alterations of opiate effects by DA antagonists and uptake blockers. 3) Test opiate effects on voltage-dependent Ca++ and K+ (e.g., M and inward rectifying) currents in NAcc neurons using intracellular and whole-cell clamp methods. If such effects are found, we will test for possible mediation by second messengers or 0-proteins; as such K+ and Ca++-current effects of opiates in other neurons or cell lines appear to involve such mediators. 4) Repeat specific aims #1-3 in slices from animals that have been self- administering heroin for various periods of time under different reinforcement schedules. These animals will be provided by the George Koob and Steven Henriksen groups and will have a well-defined dossier of behaviors for statistical comparison and correlation to the electrophysiological and pharmacological (opiate and transmitter sensitivity) data we will provide. We will be especially alert for possible 'switch-points' in the transition from non-dependent to dependent states. In these specific aims we will look for the possibility of different cell types in NAcc contributing to heterogeneity in electrophysiological and pharmacological responses, using the infrared DIC videomicroscopic method. These studies should help more clearly specify the role of opioid peptides and their receptors in normal, opiate-seeking and opiate addictive-tolerant behavior, and help to provide a cellular basis for therapeusis of "craving."
|
1 |
1988 — 1997 |
Siggins, George Robert |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Somatostatin and Brain Function @ Scripps Research Institute
The long-range objectives of this proposal are to determine the physiological effects and mechanisms of action of the neuropeptide family of prosomatostatin-derived peptides (SSs) and the functional role of endogenous somatostatins. Other major objectives are to investigate possible interactions of the SSs with other transmitter candidates and to correlate neuronal SS responsivity with immunohistochemical indices of SSergic innervation. Preliminary electrophysiological studies of the SSs suggest that the K+ conductance known as the M-current is enhanced by SS14 and SS28, leading to the suggestion that SSs may function to clamp the neuronal membrane potential at resting levels. Thus, SS may play a major role in reducing responses to excitatory amino acids (glutamate and NMDA) and therefore could be involved in certain brain phenomena such as long-term potentiation (LTP; a model of learning), hyperexcitability and excitotoxicity. The SS potentiation of cholinergic effects (seen previously) could also be significant with respect to memory precesses and also to Alzheimer's dementia. The specific aims of this proposal are therefore to: 1) characterize the physiological mechanisms of action of SS; 2) further characterize SS interactions with other transmitters; 3) seek anatomical correlates of SS responses; 4) determine if SSs, with or without ACh, alter LTP in the hippocampus; 5) determine if SSs alter low-Mg++ induced neuronal hyperexcitability. To achieve these aims, intracellular current-clamp and single electrode voltage-clamp recording of neurons in several in vitro brain slice preparations will be used, including those from the hippocampus CA1, dentate gyrus, complex of the solitary tract, and cerebral cortex. SSs, acetylcholine, GABA, glutamate, NMDA, CRF and other drugs will be applied by superfusion or locally via pipette. Pathway stimulation and/or treatment with a SS antagonist, cysteamine or antibodies to SS will be used to determine the role of endogenous SSs. Intracellular injection of Lucifer Yellow and immunohistochemical staining of SS- containing fibers will provide anatomical correlates. These studies will help to clarify the sites and mechanisms of action of the SS peptides, and perhaps lead to therapeutic use of synthetic SS peptides in clinical disease states.
|
1 |
1996 — 2000 |
Siggins, George Robert |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Core--Functional Assessment @ Scripps Research Institute
NeuroAIDS is a significant aspect of HIV infection in which the pathological and toxic changes induced by the virus affect central nervous system structure and function. HIV-Associated Cognitive and Motor Complex may affect up to 30% of patients in the early stages of immune suppression and up to 50% of symptomatic patients, while about 10-20% of those with AIDS develop frank dementia. With recent treatment-induced increases in survival time for AIDS patients, the large and growing numbers of surviving AIDS patients likely to experience NeuroAIDS obliges us to understand and treat the bases of this complex. Animal models of NeuroAIDS have been developed which mimic the overall pathogenesis of immunodeficiency infection (FIV,SIV) or focus on selected potential candidates for virus- derived or host-derived factors involved in HIV neuropathogenesis (transgenic mice). The utility of the animal models is augmented by demonstration that the neurologic disease evident in these models is sufficient to produce CNS functional changes reminiscent of those associated with HIV infection in humans. The goal of the Functional Assessment Core is to document alterations of CNS functions that develop in the various mouse, cat and monkey models of NeuroAIDS, and to determine if such functional alterations can be reversed or arrested by therapeutic means. This Core is divided into 2 aspects: electrophysiological and behavioral assessment. Electrophysiological assessment will directly measure neuronal function as electrical excitability and responsivity to synaptic input or neurotransmitters, before and after viral infection or cytokine expression, using four in vivo and in vitro assays: 1) Extracellularly recorded evoked field potentials and single unit activity (in vivo and in vitro; a regional assay of circuit functionality); 2) Intracellularly recorded membrane properties of single neurons in vitro (i.e., a cellular assay of neuronal function); 3) Intracellularly- or path-clamp-recorded responses to synaptic input, transmitters, cytokines and viral fragments (i.e., a receptor/molecular assay); 4) Infrared videomicroscopic evaluation of global changes in neuronal excitability in large brain regions (in vitro); Neurobehavioral assessment will be conducted from birth through adulthood, in order to study the consequences of transgene protein production on neurobehavioral maturation and behavior in adults. Targeted behaviors include those known to be impaired in patients with NeuroAIDS and behaviors associated with brain regions, affected in AIDS patients and recapitulated in the mouse models. In adult mice, the neurobehavioral sequelae of transgenic expression will be probed in tasks related to motor ability, reactivity to stressors and novelty/exploration, and cognition. Thus, the Functional Assessment Core will help to integrate the overall purpose of the Center by correlating disruptions in brain function, measured by electrophysiological and behavioral means, with virological, morphological, and immunopathological findings.
|
1 |
1998 — 2002 |
Siggins, George Robert |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Cellular Models of Dependence Using Brain Slices @ Scripps Research Institute
This project proposes to continue cellular studies of the role of synaptic transmitters and their receptors in alcohol intoxication and addiction. The rationale behind this approach is the considerable literature showing the synapse as the most sensitive site of ethanol action, and particularly those mediated by glutamate and GABA release. Our past studies lead us to hypothesize a role for presynaptic opiate and GABA/B receptors and postsynaptic NMDA and GABA/A receptors in ethanol intoxication and dependence. Our rationale is also based on behavioral findings with abused drugs, including alcohol, suggesting that the nucleus accumbens (NAcc) and amygdala are key areas in the reinforcing properties of these drugs, and that these properties may involve the same transmitter candidates, among others. We propose four sets of experiments: 1) To examine the effects of chronic ethanol treatment (via the ethanol vapor inhalation method) and withdrawal on membrane and synaptic properties in NAcc core and shell and amygdala neurons; 2) To examine synaptic and membrane properties in NAcc core and shell and amygdala neurons in an animal model of 'relapse' or protracted abstinence, whereby rats are treated chronically with ethanol vapor and then withdrawn for 7-14 days (at a time when 'craving' would be maximal); 3) To continue studies of the effect of the 'anti-alcoholism' drug acamprosate on NMDA and GABA/b receptors in NA/cc neurons, but now in the relapse model of specific aim #2; 4) To initiate normative in vitro studies of acute and chronic ethanol on membrane and synaptic properties of accumbens neurons in the mouse, for later studies of murine 'relapse' and genetic models. These studies will use hippocampal, NA/cc and amygdala brain slices and involve standard intracellular and "patch-slice" whole- cell clamp methods. We will use the infrared videomicroscopic method to distinguish morphologically different cell types for comparison to electrophysiological properties. We will examine evoked, pharmaco- logically-isolated monosynaptic events, responses to exogenous transmitters, paired-pulse inhibition and spontaneous synaptic events, to test the specificity and site of ethanol action. We believe these studies will provide important new information on possible sequelae of ethanol dependence at the cellular level, and--by virtue of comparisons of ethanol and acamprosate actions in control and protracted abstinence models--will also provide clues as to the cellular and ion channel correlates of alcohol seeking behavior.
|
1 |
1999 — 2003 |
Siggins, George Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Morphine Like Brain Peptides--Cellular Neurobiology @ Scripps Research Institute
DESCRIPTION (Adapted From The Applicant's Abstract): The purpose of the application is to continue and extend cellular neurophysiological studies on opioid peptides and opiates in two brain regions, the hippocampus and the nucleus accumbens, that contain proenkephalin- and prodynorphin-derived peptides. These regions were chosen because of their suggested roles in opiate reinforcement, as cellular models for opiate reward or dependence. The overall objective is to determine the physiological role of central opioid peptide-containing neurons in normal (Naive) subjects and those chronically-treated with opiates, using intracellular (current and voltage-clamp) and whole-cell clamp recording in rat brain slices in vitro. Our hippocampal studies during the last funding period have confirmed a dynorphin-induced augmentation of the M-current, a voltage-dependent K conductance, and have found that nociceptin (orphanin FQ), the agonist for orphan' receptor (ORL1), also augments IM via an opiate receptor mechanism and elicits another non-opiate receptor-mediated action likely to involve a K conductance. In addition, our studies in slices of nucleus accumbens core confirmed the opiate reduction of synaptic transmission in the nucleus accumbens and found pronounced interaction of opiates with glutamate receptor agonists, and especially with those acting on non-NMDA and NMDA receptor-mediated neurotransmission, in neurons from rats treated chronically with morphine. Therefore, our specific aims for the proposed funding period are to: 1) determine the second messenger(s) mediating the dynorphin and nociceptin effects on Im in CA1 hippocampal neurons. 2) Examine the effects of opioid on pharmacologically-isolated EPSCs and responses to NMDA, AMPA and kainate in CA1 of hippocampus. 3) Perform a battery of pharmacological tests to determine the site of action of chronic morphine treatment and possible changes in the postsynaptic NMDA receptor subunit composition in rat nucleus accumbens neurons. We also will use immunohistochemical methods, with selective antibodies to the NMAR2 subunits, and single-cell reverse transcriptase PCR, to determine if chronic morphine treatment causes a cellular and subcellular re- distribution of NMDA receptor subunits. These models will allow later test of several hypotheses, including those concerning neuroadaptative mechanisms following chronic opiate treatment. These studies should help more clearly specify the role of opioid and nociceptin peptides and their receptor in normal, opiate- seeking and opiate addictive-tolerant behavior, and help to provide a cellular basis for therapeusis of opiate craving'.
|
1 |
1999 — 2003 |
Siggins, George Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Somatostatin Related Peptides and Brain Function @ Scripps Research Institute
DESCRIPTION: (Applicant's Abstract) The long-range objectives of this application are to determine the physiological effects, mechanisms of action and neuronal function of the family of prosomatostatin-derived and related peptides (SSTs and cortistatin). Despite decades of research, the function of SST in extrahypothalamic brain remains unknown. The new studies we plan are based on our findings that: 1) SSTs hyperpolarize CA1 hippocampal neurons (HPNs) by augmenting two K+ conductances: the M-current (Im) and the leak current (Ilk); muscarinic agonists block both these currents; 2) arachidonic acid (AA) metabolites mediate these SST K+ channel effects; 3) SST reduces excitatory (but not inhibitory) postsynaptic currents (EPSCs), and especially polysynaptic or hyperexcitable neurotransmission, in both CA1 and CA3 hippocampal pyramidal neurons (HPNs); 4) cortical regions contain a novel SST-like peptide, cortistatin (CST), resulting from a different gene but exerting hippocampal effects qualitatively similar to those of SST. We hypothesize from these findings that these two 'statin' systems function in a concerted way (perhaps with GABA, with which they are often co-localized) as a 'brake' to reduce excitability in the hyperexcitable or epileptic hippocampus. Therefore, the specific aims of this renewal application are to: 1) Continue studies to determine whether the synaptic effects of SST and CST on EPSCs are exerted pre- or postsynaptically; 2) Examine the effects of CST, in both CA1 and dentate, on synaptic plasticity (STP, LTP and LTD) and 3) on the hyperexcitability and epileptiform activity produced by superfusion of bicuculline or low Mg++ concentrations; 4) Begin comparative studies of membrane and synaptic properties, and their responses to SST and CST receptor agonists, in HPNs of several murine genetic models, including those containing knockouts (null mutations) for the CST and SST peptides and for SST receptors, and those overexpressing neuronal CST. Bigenic crosses of these knockouts and CST overexpression will also be studied. These models will allow tests of several hypotheses on the function of CST and SST in brain, including the idea that endogenous CST and SST combine to reduce or prevent feedforward synaptic hyperexcitability and epileptiform activity in hippocampus. To achieve these aims, intracellular and whole-cell voltage-clamp ('patch-slice') neuronal recording will be applied to in vitro slice preparations of hippocampus. CST, SST and other drugs will be applied by superfusion or locally from pipettes. We believe these studies will help to clarify the sites and mechanisms of action of the endogenous SST and CST peptides and their possible role in hyperexcitability and learning and memory, and will help to characterize possible CST- or SST-related dysfunctions in certain disease states such as epilepsy and Alzheimer's dementia.
|
1 |
2000 — 2002 |
Siggins, George Robert |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 4 @ Scripps Research Institute
Though much research has been devoted to the characterization of METH- and HIV-1 viral-induced toxicity; very little is known about the neurophysiological effects of acute and chronic METH on the functional integrity of the CNS and the combined effects of HIV-1 like infection with METH neuropharmacology. Elucidating the cellular mechanisms responsible for these interactions is essential for understanding the sequence of neurophysiological events leading to METH and HIV-1- induced neurotoxicity and thus,, determining in a rational manner, the potential role of METH in HIV-1 viral infection. The objectives of our component are to determine: (a) whether the effects of METH on the murine hippocampus are a function of the treatment schedule and the time following the last drug administration; (b) whether METH produces additive or synergistic effects accelerating the progression of the functional deficits of NeuroAIDS; and (c) whether selected CNS pro- inflammatory cytokines (IL6, INFalpha) or the HIV-1 coat protein gp120 are essential elements in the synergy between METH neurotoxicity and NeuroAIDS pathogenesis. We will study hippocampal (CA1 and dentate gyrus) neurons using extracellular and intracellular techniques in both in vivo and in vitro preparations. We will determine their neuronal excitability, membrane, membrane and synaptic properties and in vitro preparations. We will determine their neuronal excitability, membrane and synaptic properties, local circuit interactions and synaptic plasticity. In addition, we will characterize hippocampal synaptic plasticity and the neuronal modulatory effects of afferent inputs from subcortical structures on hippocampal dentate function in vivo. These data will help determine whether basic properties of the METH treatment such as the dose of METH, the history of METH treatment, the treatment threshold for seizure activity, and on hippocampal physiology. The studies will also identify the interactions between METH and of specific isolation of specific neuronal populations and neurotransmitter systems prone to be targets of NeuroAIDS and METH neuropharmacology. By characterizing the effects of METH treatment and HIV-1-like infection on hippocampal function, these studies will contribute important information about the combined effects of HIV-1 like infection and METH neuropharmacology and will provide critical data for the future identification of beneficial approaches for the treatment and prevention of these conditions.
|
1 |
2001 — 2011 |
Siggins, George Robert |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Electrophysiology of Alcohol in Extended Amygdala @ Scripps Research Institute
DESCRIPTION (provided by applicant): We have pursued electrophysiological studies of the role of synaptic transmitters, neuropeptides and their receptors in alcohol effects, with the rationale that these elements are the most sensitive sites of ethanol action. This project is based on behavioral findings that the nucleus accumbens (Nace) and extended amygdala are key areas in the reinforcing properties of abused drugs, and that these properties also may involve several transmitters (e.g., GABA, glutamate) and neuropeptides (e.g., CRF and opioids). The amygdala has been implicated in motivated behaviors and anxiety states, and it is hypothesized that these same neuropharmacological systems within the extended amygdala mediate the increases in ethanol self-administration that occur during withdrawal from chronic ethanol. Therefore, we propose several sets of experiments: 1) To begin in vitro brain slice studies of acute and chronic ethanol effects on membrane and synaptic properties of central amygdala (CeA) neurons and NAcc neurons in the mouse for comparison to our rat data, and to prepare for studies of murine genetic models. 2) To determine the role of CRF receptors, whose expression may be responsible for excessive alcohol consumption, by examining extended amygdala network and cellular functioning in brain slices taken from mice with knockouts for brain CRF-I receptors. 3) To determine the role of opiate receptors that could be responsible for excessive alcohol consumption, by examining extended amygdala network and cellular functioning in brain slices taken from rnice with knockouts for brain mu opiate and, later, delta opiate receptors. These studies will use amygdala and NAce brain slices and involve standard intracellular (current- and voltage-clamp) and "patch-slice" whole-cell clamp methods. The infrared DIC-videoniieroseopic method will be used to identify morphologically different cells types for comparison to electrophysiological and pharmacological properties. We will record evoked, pharmacologically-isolated monosynaptic currents or potentials, and spontaneous and miniature synaptic events, to test the specificity and site of action of ethanol effects. These studies should provide important new information on possible sequelac of ethanol intoxication at the cellular level, and, by comparisons of ethanol and peptide actions in control, ethanol-withdrawn, protracted abstinence, and knockout models, will also provide clues as to the cellular and ion channel correlates of ethanol dependence.
|
1 |
2003 — 2007 |
Siggins, George Robert |
P60Activity Code Description: To support a multipurpose unit designed to bring together into a common focus divergent but related facilities within a given community. It may be based in a university or may involve other locally available resources, such as hospitals, computer facilities, regional centers, and primate colonies. It may include specialized centers, program projects and projects as integral components. Regardless of the facilities available to a program, it usually includes the following objectives: to foster biomedical research and development at both the fundamental and clinical levels; to initiate and expand community education, screening, and counseling programs; and to educate medical and allied health professionals concerning the problems of diagnosis and treatment of a specific disease. |
Cellular Neurobiology Research Project @ Scripps Research Institute
DESCRIPTION (provided by applicant): This project proposes to continue cellular studies of the role of neurotransmitters and their receptors in alcohol intoxication and addiction, with the rationale that the synapse is the most sensitive site of ethanol action, and particularly those synapses mediated by glutamate, GABA, and neuropeptide release. Our past studies led us to hypothesize a role for 'metabotropic' receptors and postsynaptic NMDA and GABAA receptors in ethanol intoxication and dependence. A second rationale is based on behavioral findings suggesting that the extended amygdala is a key system in the addictive properties of several drugs, including alcohol, and that several transmitters, neuropeptides, and endocannabinoids may be involved there. The amygdala has been implicated in motivated behaviors and anxiety states. Stressors and anxiety-provoking stimuli may trigger relapse in human alcoholics, and ethanol withdrawal is associated with increases both in anxiety-like behavior and in ethanol self-administration in rodents. Therefore, we hypothesize that the same neuropharmacological systems within the extended amygdala's circuitry mediate increases in anxiety state and in ethanol self-administration that occur during withdrawal from chronic ethanol, and we propose the following 3 sets of cellular studies to test this hypothesis: 1) Examination of the effects and interactions of acute and chronic ethanol administration, early withdrawal and protracted abstinence on CRF effects in central amygdala (CeA) neurons. 2) Examination of the effects of acute and chronic ethanol administration, early withdrawal and abstinence on neuropeptide Y (NPY) effects in CeA neurons. 3) Testing the effects of acute and chronic ethanol, early withdrawal and abstinence on endocannabinoid effects in CeA and nucleus accumbens (NAcc). In all 3 of these aims, subjects will be sham (control) male rats or those receiving chronic ethanol either via ethanol vapor inhalation and/or via self-administration, and their CeA or NAcc sliced and studied 1-12 hours or 1-2 weeks after withdrawal. We will record from amygdala and NAcc brain slices with intracellular (current- and voltageclamp) and "patch-slice" whole-cell clamp methods. The infrared DIC-videomicroscopic method will be used to identify morphologically distinct cells types for comparison of electrophysiological and pharmacological properties. We will record evoked, pharmacologically-isolated monosynaptic currents or potentials, and spontaneous and miniature synaptic events, to better test the specificity and site of action of ethanol and ligands. We believe these studies will provide important new information on possible sequelae of ethanol intoxication at the cellular level, and, by virtue of analyses of ethanol and peptide/cannabinoid interactions in control, chronic and protracted abstinence models, will also provide clues as to the cellular and ion channel correlates of ethanol dependence.
|
1 |
2004 — 2008 |
Siggins, George Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Morphine Like Brain Peptides-Cellular Neurobiology @ Scripps Research Institute
[unreadable] DESCRIPTION (provided by applicant): The overall objective of this project is to reveal the cellular mechanisms underlying opiate effects and opiate dependence, and the role of central opioid peptides in both naive and opiate-dependent subjects. We propose to study the central amygdala nucleus (CeA), thought to be critically involved in stress and drug reward and dependence, and to contain opioids, nociceptin (orphanin FQ) and their relevant receptors. In pilot electrophysiological studies we found unusual actions of nociceptin on evoked and spontaneous IPSPs in a CeA slice, and in blocking the effects of ethanol on these IPSPs. We also found a mediator role for CRF receptors, and a regulator role for mu opiate receptors, in the ethanol effects. Also, withdrawal from chronic morphine treatment increased spontaneous activity and caused depolarization shifts in CeA neurons. Thus, we propose the following 3 Specific Aims: 1) To examine the membrane and synaptic effects of mu, delta, kappa and ORL-1 selective agonists on rat CeA neurons and determine if chronic morphine treatment alters these effects or baseline properties of CeA neurons; 2) To determine if chronic morphine causes a re-composition of NMDAR subunits in rat CeA as it appears to do in nucleus accumbens, by using a battery of multidisciplinary tests of NMDARs, including electrophysiological, pharmacological, molecular and histochemical methods for profiling NR2 subunits; 3) To assess the possibility of interactions between opioids and CRF on synaptic and membrane properties of CeA neurons, and determine if chronic morphine alters the interactions. These aims will test several hypotheses, including those concerning neuroadaptative mechanisms following chronic opiate treatment, and that some actions of opiates in CeA involve CRF receptors. To accomplish these aims, we will record from CeA neurons of brain slices using intracellular or whole-cell patch methods and measure pharmacologically-isolated, evoked and spontaneous synaptic currents and responses to exogenous agonists. We also will use molecular (Western blots, quantitative RT-PCR) and histochemical (in situ hybridization) methods to determine relative levels of different NR2 subunits. These studies may help define the role of opioids and their receptors in behavior and provide rational targets for therapeusis of opiate "craving. [unreadable] [unreadable]
|
1 |
2008 — 2010 |
Siggins, George Robert |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Electrophysiology of Alcohol in Extended Amygdela @ Scripps Research Institute
This project is based on behavioral findings that the central amygdala nucleus (CeA) and locus coeruleus (LC) are key brain areas involved in stress reactions and the reinforcing properties of abused drugs, and that these behaviors may involve several transmitters (GABA, glutamate, norepinephrine) and neuropeptides (CRF, opioids and galanin). Both regions are implicated in motivated behaviors and anxiety states, and we hypothesize that these same neurochemical systems within the CeA and LC are involved in the excessive ethanol drinking seen in dependent animals. Therefore, we propose several sets of experiments: 1) To assess the role of CRF receptors in excessive drinking, by comparing the CeA cellular and network function in brain slices from control and excessively drinking mice (WID model) mice, with respect to the ethanol augmentation of GABAergic IPSCs or inhibition of glutamatergic EPSPs, combined with cytochemical localization of CRF and CRF receptors. 2) To determine the role of kappa opiate receptors (KORs) in excessive drinking, for comparison to our mu and delta receptor data, by examining CeA cellular function in brain slices from WID mice with a knockout (KO) for brain KORs. 3) To determine the role of galanin and its receptors in excessive drinking, by examining CeA and LC cellular in slices from WID mice and those with KOs for brain Gall and Gal2 receptors and with galanin over-expression, and by neurochemical and molecular biological measures in CeA and LC neurons. 4) To determine the effects on the largest WID- induced changes from the results of Specific Aims 1-3, in the HDID mice selectively bred by the Crabbe and Finn groups for high drinking in the dark versus their controls, and for SHAG vs.SLAG lines, selected for scheduled high and low alcohol consumption.The electrophysiological studies will use CeA and LC brain slices and involve standard intracellular and whole-cell clamp methods. We will use a battery of measures to assess the pre- versus postsynaptic sites of action of ethanol and peptide effects. RIA, real-time PCR and receptor binding studies will be used in the galanin studies. This project should provide important new [unreadable] information on the possible sequelae of ethanol intoxication at the cellular level, and, by comparisons of ethanol and peptide actions in control, excessively drinking, and knockout models, will also provide clues as to the synaptic, cellular and ion channel correlates of ethanol dependence.
|
1 |