Igor Spigelman - US grants

The long-term objectives of this proposal are: a) to characterize the electrical membrane properties and morphology of lamina I neurons in the subnucleus caudalis of the trigeminal sensory nuclear complex, b) to determine the synaptic responses of these neurons to activation of trigeminal afferent inputs, c) to examine how selective interference with inhibitory neurotransmitter receptors alters the synaptic responses of lamina I neurons. To achieve these objectives, various intracellular recording and histochemical techniques will be used to study the membrane properties, synaptic responses and morphology of trigeminal neurons in the horizontal slice preparation of the guinea pig brainstem. Hypotheses to be tested include: 1) high frequency stimulation of trigeminal afferents produces slow, neuropeptide-mediated excitatory potentials in subnucleus caudalis lamina I neurons 2) the substance P-mediated slow excitatory potentials are evoked via the NK1 tachykinin receptor subtype 3) paroxysmal discharges may be evoked in lamina I neurons following blockade of the GABA(Beta)-receptor subtype. The proposed studies will serve as a basis for the in depth analysis of signal transmission in the trigeminal system. The obtained knowledge can subsequently be used to provide a scientific basis for the complete understanding and treatment of debilitating conditions such as myofacial pain and trigeminal neuralgia which are of major clinical significance in dentistry.

9531251 Spigelman One of the parts of the brain important for learning in mammals is called the hippocampus, lying deep beneath the cererbral hemispheres. The hippocampus contains a curled formation called the dentate gyrus, which has three layers of nerve cells, or neurons. These neurons have characteristic shapes and some of their connections are known to form particular local circuits. The basket cells, granule cells, mossy cells and mossy fibers form circuits that are believed to underlie the functions of spatial learning. The postnatal development of the functional connections within these circuits are important to know if we are to understand the role of the hippocampus in early learning. This collaborative work by an electron microscopist and a neurophysiologist examines the ultrastructure of synapses (the functional cellular connections) between particular classes of neurons and the membrane properties of those neurons. Documenting changes in these structural and functional features during development will allow a better understanding of how the hippocampus begins to function during early learning. The results will be important not only for understanding brain development, but also for psychological studies on learning.

DESCRIPTION (provided by applicant): Alcohol abuse represents a significant problem in our society. Chronic intermittent ethanol (CIE) treatment of rats (60 doses of intermittent intoxication and withdrawal), encompasses all of the major characteristics of human alcoholism, including anxiety, lowered seizure thresholds, and enhanced alcohol preference after withdrawal. At least some of these symptoms may be explained by the measured reduction in the function of the 3-aminobutyric acid (A) receptor (GABAAR) and altered sensitivity to its allosteric modulators. GABAARs mediating synaptic (phasic) and extrasynaptic (tonic) inhibition appear to be altered differently. Thus, tolerance develops to acute ethanol (EtOH) potentiation of hippocampal extrasynaptic GABAARs, while synaptic GABAARs develop high sensitivity to EtOH. Such paradoxical changes in EtOH sensitivity are proposed to underlie both the development and persistence of alcoholism. Preliminary studies suggest that altered subunit composition and localization of GABAARs may account for the observed alterations in GABAAR function within the hippocampus. However, it is unknown whether other key brain areas implicated in symptoms of alcohol withdrawal and dependence experience similar neuroadaptations. It is also unknown whether EtOH-induced neurodegeneration may account for these neuroadaptations. Underscoring the persistence of CIE-induced changes is a new observation of fundamental importance: a single intoxicating dose of EtOH results in GABAAR changes similar to those seen after CIE treatment, but recovery is seen by 1-2 weeks after this single dose. The specific aims of this proposal were designed to address key hypotheses regarding GABAAR involvement in mechanisms of alcohol withdrawal and dependence by: 1) determining the dose-, duration-, and frequency-dependence of CIE treatment to produce long-lasting symptoms of EtOH dependence;2) studying changes in GABAAR subunit composition and function within nucleus accumbens and basolateral nucleus of the amygdala (brain areas known to be of major importance for the mechanisms of reward and dependence) and relating them to the behavioral measures of withdrawal from single or multiple EtOH treatments;and 3) determining whether neurodegeneration plays a role in altered GABAergic inhibition after EtOH intoxication through the use of histochemical and stereological techniques. The knowledge acquired from the proposed experiments will increase our understanding of the alcohol-induced alterations in GABAAR function, which has profound effects on various emotional and intellectual aspects of brain activity. This knowledge will also be useful to the development of therapeutics targeting the GABAergic system for the treatment of alcoholism.

DESCRIPTION (provided by applicant): Synthetic and naturally occurring cannabinoids are a focus of strong social, legal and medical controversy concerning their therapeutic utility, yet studies show that cannabinoids reduce the hyperalgesia and allodynia associated with persistent pain of neuropathic origin in humans and animals. Moreover, cannabinoids are effective in alleviating neuropathic pain symptoms after repeated treatment, unlike opioids, which have only limited effectiveness. However, the usefulness of existing cannabinoid-based analgesics is limited by their profile of psychotropic side-effects. The targets of cannabinoids may be defined by the distribution of two cloned subtypes of cannabinoid receptors, CB1R and CB2R. Both are members of the superfamily of G protein- coupled receptors of which CB1Ris expressed at high levels in the hippocampus, cortex, cerebellum and basal ganglia, whereas CB2R is primarily expressed in immunocompetent cells outside the central nervous system (CNS). CB1Rs are also found outside the CNS, especially in primary sensory neurons which are common sources of hyperexcitability thought responsible for the painful symptoms of many neuropathies. In this Exploratory and Developmental Research Project we seek to (Aim 1) develop peripherally-acting ligands at CB1Rs by designing analogs that are unable to cross the blood-brain barrier by virtue of their being charged compounds. Following their synthesis, these compounds will be compared with their parent brain-permeable CB1R ligands for their ability to activate CB1Rs using well established in vitro CB1R screening assays (Aim 2). Promising ligands will be screened for blood-brain barrier permeability in another in vitro assay to estimate the likely extent of the compounds'CNS penetration. These will be followed by (Aim 3) studying the effectiveness of peripherally-acting ligands in reducing the pain symptoms in a rat model of peripheral neuropathy. The most promising compounds will also be examined for potential CNS side effects in a modified tetrad assay. The data obtained from this pilot project are expected to prove useful to the development of new analgesics for the treatment of chronic pain of neuropathic origin. Our strategy may also contribute to the understanding of cannabinoid receptor pharmacology and to introduce a novel set of tools that can help study the physiological and pathophysiological importance of peripheral cannabinoid receptors. PUBLIC HEALTH RELEVANCE Neuropathic pain is extremely difficult to treat, in part because available drugs carry a high burden of central nervous system side-effects. Here we propose to design and test analogs of cannabinoid receptor ligands that are unable to cross the blood-brain barrier by virtue of their being charged compounds. As a result, we expect these drugs to exhibit analgesic properties with minimal psychotropic side-effects.

DESCRIPTION (provided by applicant): Chronic pain affects >50 million adults in the U.S. and represents a major socioeconomic and clinical challenge, in part because even the most efficacious of the current medications are limited by their side-effects profile. Similarly, alcoholism is a major problem with clinical treatments showing limited efficacy in reducing craving, relapse and abstinence rates. Clinical studies revealed a complicated association between chronic pain and alcohol dependence. Thus, chronic pain states significantly affect alcohol use patterns and promote the development of alcoholism, while long-term alcohol dependence induces symptoms of hyperalgesia and may exacerbate chronic pain arising from other sources. Alcohol's acute analgesic effects and chronic alcohol's hyperalgesic effects have been studied in animal models. However, the precise relationship between chronic pain and development of alcohol dependence has yet to be examined in animals, in part due to the lack of robust models that reflect the complex relationship between chronic pain and alcohol dependence in humans. In this exploratory project we propose 2 related specific aims designed to develop a robust animal model which reflects findings in humans and test specific hypotheses regarding the precise relationship between chronic pain and alcohol dependence. SA1 examines the dual consequences of alcohol dependence and chronic (neuropathic) pain development on voluntary alcohol drinking. This aim will be achieved through the combined use of three well-characterized rat models of: a) alcohol dependence induced by forced chronic intermittent ethanol (CIE) administration, b) peripheral neuropathy induced by sciatic nerve entrapment (SNE) and c) voluntary (2-bottle choice, 2BC) regimen of ethanol (EtOH) consumption. SA2 examines if chronic pain- induced increases in voluntary EtOH intake is due to the analgesic effects of EtOH. This aim will be achieved by first exposing rats to voluntary (2BC) regimen of EtOH consumption followed by EtOH withdrawal, SNE neuropathy induction and subsequent re-introduction of 2BC EtOH consumption. Upon 2BC EtOH re- introduction rats are provided with relief of neuropathy symptoms using unique novel analgesics without central nervous system side effects and their EtOH intake compared to that of vehicle-injected rats. In total, the experiments outlined in SA1 and SA2 will provide the much needed data on the role that chronic pain may play in the development and maintenance of alcohol dependence in robust, validated animal models. Future studies could make use of these models to further unravel the complex interactions between chronic pain and alcohol dependence, as well as test novel targeted treatments for these disorders.

DESCRIPTION (provided by applicant): Alcoholism is a chronic relapsing disorder characterized by compulsive ethanol-seeking and loss of control over alcohol intake. Alcoholism is known to be associated with a persistent dysregulation of the hypothalamic pituitary adrenal (HPA) axis and corticotropin-releasing hormone (CRH) signaling that leads to inappropriate responses to stress, thereby increasing relapse susceptibility in abstinent alcoholics. However, the cellular and molecular mechanisms responsible for the blunted HPA axis responses to stress in abstinent alcoholics have yet to be uncovered. In rats, acute restraint stress induces a CRH-dependent depression of N-methyl-D- aspartate receptor (NMDAR) function in parvocellular neurosecretory cells (PNCs) of the paraventricular nucleus of the hypothalamus (PVN) which allows for the unmasking of associative short-term synaptic potentiation (STP) following a burst of high-frequency stimulation (HFS) of excitatory inputs. This represents a cellular mechanism by which stress induces neuroadaptive responses of the HPA axis. Preliminary results in a rat model of alcohol dependence induced by chronic intermittent ethanol (CIE) exposure show that STP can be induced in PNCs of CIE rats without acute stress. By contrast, STP is impaired in PNCs from acutely stressed CIE rats. We also demonstrated long-lasting potentiation of postsynaptic NMDAR function associated with increased expression of the GluN2B subunit of NMDARs in PNCs of CIE rats. Altogether, preliminary results strongly suggest that CIE exposure modifies both basal and stress-induced synaptic plasticity in PNCs, which could be responsible for the characteristically blunted hormonal response of the HPA axis to stress in alcohol- dependent rats. Thus, the main hypothesis of this proposal is that CIE-induced impairment of STP is mediated by the long-lasting alterations in both NMDAR function and CRH signaling in the PVN. We also hypothesize that restoring this stress-induced plasticity will restore the HPA axis responsiveness to stressors. To test these hypotheses we will use a combination of behavioral, electrophysiological, biochemical and pharmacological techniques to determine: 1) the role of altered NMDAR signaling in stress-induced plasticity at glutamatergic synapses in PNCs after withdrawal from chronic EtOH exposure, and 2) the mechanisms by which chronic EtOH exposure alters CRH signaling in PNCs of the PVN during protracted withdrawal. These studies will help uncover the cellular mechanisms behind the dysregulation of the HPA axis response to stress in alcohol dependence, which will be useful in the discovery of new effective therapies for stress-induced compulsive alcohol seeking.

? DESCRIPTION (provided by applicant) Synthetic and naturally occurring cannabinoids (CBs) have demonstrated effectiveness in numerous chronic inflammatory and neuropathic disorders in humans and in animal models. However, major impediments to the widespread use of CB-based therapies are their psychotropic side-effects, mediated by the activation of central nervous system (CNS) CB1 receptors (CB1Rs). Recently, we developed a series of synthetic peripherally-restricted CBs (PRCBs), and demonstrated potent reversible and repeated suppression of chronic inflammatory and neuropathic pain symptoms in the absence of CNS-mediated side effects. The therapeutic utility of PRCBs may include many other indications where brain-permeant CBs have been shown to be effective. Cancer pain and chemotherapy-induced neuropathy are particularly attractive targets since brain-permeant CBs possess antitumorigenic properties and have been shown to ameliorate the pain symptoms of cancer and chemotherapy-induced neuropathies mainly by peripheral mechanisms. Therefore, this proposal focuses on establishing effectiveness and mechanisms of action of PRCBs against: a) human oral carcinoma proliferation, b) oral carcinoma-induced pain, and c) chemotherapy-induced peripheral neuropathies (CIPNs). We will achieve these aims through the use of innovative and validated operant assays that provide a measure of cerebral processing and orofacial function in mouse oral cancer and rat CIPN models. Gender differences in cancer and CIPN pain sensitivity and their responsiveness to PRCBs will be determined. To further characterize PRCBs we will perform pharmacokinetic studies and determine their receptor targets with tissue-specific transgenic mice. To assess potential off-target actions and peripheral side effects of PRCBs we will use a suite of invasive and non-invasive physiological tools. We will also assess the potential development of tolerance to PRCBs after chronic administration and determine if pre-treatment with PRCBs may actually prevent the onset and maintenance of CIPN symptoms. Successful completion of the proposed studies would allow us to translate pre- clinical findings to a clinical trial; this work would improve outcome for cancer patients.

Project Summary Alcohol addiction is a chronic relapsing disorder characterized by compulsive alcohol-seeking and the emergence of a negative emotional state (allostasis) that leads to dependence on alcohol. Alcohol use disorders are associated with a persistent dysregulation of the hypothalamic pituitary adrenal (HPA) axis and corticotropin- releasing factor (CRF) signaling that leads to inappropriate responses to stress, thereby increasing relapse susceptibility in abstinent alcoholics. Although research has progressed to better understand the detrimental relationship between the allostatic HPA axis response to stress and alcohol relapse, the cellular mechanisms underlying the HPA axis allostatic response to stress in EtOH-dependence remain unclear and warrant further investigation in order to develop the much needed novel therapies to treat alcoholism and the consequent alcohol-related diseases. Stress information is processed in higher brain regions and converged onto parvocellular neurosecretory cells (PNCs) within the paraventricular nucleus of the hypothalamus (PVN) which represents the final central integrative and output step of HPA axis response. Stress induces multiple distinct forms of glutamatergic and GABAergic synaptic plasticity of PNCs which depend on different mediators of the stress-induced HPA axis response such as CRF, NMDAR, cortisol/corticosterone (CORT) and norepinephrine (NE) within the PVN. These different forms of stress-induced synaptic plasticity provide a cellular mechanism by which stress induces neuroadaptive responses of the HPA axis. Chronic intermittent ethanol (CIE) treatment alters CORT-mediated negative feedback, NE, glutamatergic, and GABAergic neurotransmissions leading to a dysregulated HPA axis function. Preliminary results show that CIE exposure increases NMDAR function and prevents CRF-induced depression of NMDAR function in PNCs. Moreover, stress-induced CRF/NMDAR- dependent glutamatergic short-term synaptic plasticity (STP) is impaired in PNCs of CIE rats but is restored after inhibition of NMDAR function. We also related the impairment of STP in PNCs to dysregulated HPA axis hormonal (CORT and ACTH) and grooming (behavioral) responses to repeated stressors. Thus, the main hypothesis of this proposal is that CIE exposure leads to the alteration of CRF-mediated NMDAR- dependent synaptic plasticity, which together with alterations in NE, CORT, and GABA signaling in the PVN is responsible for the HPA axis allostatic response to stress in EtOH dependence. To test this hypothesis we will use a combination of behavioral, electrophysiological, biochemical and pharmacological techniques to determine: 1) the role of NMDAR, 2) CORT, and 3) NE in CIE-induced neuroadaptive changes in PNCs and the maladaptive behavioral and hormonal HPA responses to repeated stress, and 4) the intra-PVN role of NMDAR, CORT, and NE signaling in excessive EtOH intake, decreased stress-induced grooming, and stress-induced increases in EtOH intake of CIE rats.

PROJECT ABSTRACT A small but significant percentage of people that experience a traumatic, life-threatening, event develop a condition called Post-Traumatic Stress Disorder (PTSD), which is characterized by heightened anxiety and disturbed fear processing. The condition is more prevalent in women. Additionally, PTSD is highly comorbid with other psychiatric conditions notably alcohol abuse. We have developed an animal model that captures many of the features of PTSD and as such should be helpful in developing an understanding of the biological changes that produce the disorder and suggesting novel treatments. Here we propose to use a milder stress than we have used in our published work because it produces enhanced fear learning in a smaller proportion of animals (18%) that better approximates the rate of PTSD in humans. The rats subjected to this stress will be tested on a number of measures of anxiety, altered fear processing and alcohol intake. We will use this as a tool to understand how PTSD symptoms group together to form subtypes and how these subtypes relate to the brain changes that cause maladaptive behavior.