George R. Breese - US grants

Recent clinical studies indicate that patients' symptoms disappear gradually during continuous antidepressant (AD) treatment. Data from our laboratory indicate there are regional brain differences in the rate of adrenergic receptor adaptation during chronic AD administration. These data may provide evidence that the gradual improvement in the symptoms of depressive illness during chronic AD treatment is due to the recruitment of successive brain areas where receptor adaptation has occurred. Consequently, one hypothesis to be tested in this grant is that regional brain differences in the rate of adrenergic receptor adaptation are common to AD treatments. Data relevant to this hypothesis will be obtained by examining effects of ADs from different chemical classes on beta- and alpha-adrenergic receptor (AR) adaptation at several different times during chronic AD treatment. This biochemical study will be complemented by an electrophysiological study evaluating sensitivity of alpha- and beta-ARs. A second seminal finding by our laboratory is that stress facilitates receptor adaptation produced by ADs while chronic AD treatment attenuates specific cellular responses to stress. Thus, the second hypothesis proposed is that the facilitation of AD action by stress and the antagonism of specific functional responses to stress by chronic AD treatment are also common to ADs. To test the first part of this hypothesis, we will determine whether combining AD treatment with forced swim will accelerate AD-induced adaptation of beta-ARs in regions where receptor adaptation is delayed. If facilitation is found, this would suggest that a component of stress can facilitate neural adaptation produced by ADs. The second portion of this hypothesis will extend our observation that chronic imipramine (IMP) treatment reduces the number of cells exhibiting c-fos-like immunoreactivity (c-FLI) in PVN and medial prefrontal cortex after forced swim. We will determine if ADs from several classes share this action with IMP on the swim stress-induced changes in c-FLI. Swim stress can also increase spontaneous LC activity. Consequently, we also will determine whether chronic AD administration will affect this stress-induced increase in LC firing as well as define neural activity in the PVN and the medial prefrontal cortex after stress. This multidisciplinary grant provides a new theoretical position concerning neural adaptation caused by chronic AD treatment that depends on an integration of time, brain region, and the specific AD administered, as well as the ability of ADs to modify responses to stress. Completion o these studies will provide specific data relevant to the two major hypotheses proposed to explain AD action and will expand our knowledge about the specific neuroanatomical sites at which ADs influence CNS function.

The Lesch-Nyhan syndrome (LNS) is characterized by a deficiency of the enzyme, HGPRT, motor dysfunction, mental retardation, hyperuricemia, self-mutilation behavior (SMB) and a loss of dopaminergic neurons. In rats, neonatal destruction of dopaminergic neurons with 6-hydroxy-dopamine (6-OHDA) increases susceptibility for SMB when D-1 dopamine receptors are activated, suggesting that neonatal-6-OHDA treatment is a model of the central dopamine deficiency of LNS. The present grant explores how this enzyme and altered purine mechanisms may interact with dopaminergic function. We plan to determine the relative amount of HGPRT in dopamine-containing (or other) neurons by measuring HGPRT activity after neurotoxin treatment. We will also determine if purine compounds, which accumulate in LNS, influence the function of dopaminergic neurons in developing rats. Recent studies have revealed that an adenosine agonist, NECA, will antagonize SMB produced by the dopamine agonist, L-DOPA, after 6-OHDA treatment. Therefore, the ability of relatively specific adenosine agonists to block L-DOPA-induced behaviors as well as those produced by D-1 and D-2 dopamine receptor agonists will be investigated. We will explore if antagonism of adenosine receptors will produce SMB in neonatally-6-OHDA-treated rats or increase the effectiveness of dopamine agonists to induce this and other behaviors. In vitro experiments are proposed in 6-OHDA-lesioned rats to measure striatal adenosine receptor characteristics and the ability of adenosine agonists to interact with D-1 dopamine agonist activation of striatal adenylate cyclase. Possible involvement of GTP in the supersensitivity of D-1 dopamine receptors in neo-natally-6-OHDA-treated rats will also be examined. Electrophysiological studies are planned to characterize interactions of adenosine and a selected dopamine agonist at the cellular level in striatum of control and lesioned animals. This multidisciplinary evaluation of dopamine-purine interactions should allow us: (1) to assess if HGPRT is contained in dopaminergic neurons and if purine compounds influence development of dopaminergic neurons; (2) to determine if 6-OHDA treatment alters adenosine receptors, their function, or action of GTP on adenylate cyclase activity; and (3) to define how alterations in an adenosine receptor subtype will influence specific dopamine receptor subtype mediated responses. Data collected in these three areas will increase our basic understanding of purine-dopamine interactions and could provide new strategies for treating SMB and other symptoms in LNS.

Clinical studies indicate that prolonged ethanol abuse by alcoholics results in an increasing incidence of seizure susceptibility until the majority of alcoholics have this symptom. A kindling-like phenomenon produced by repetitive withdrawal from alcohol has been proposed to explain this observation. Recently, the inferior colliculus has been implicated in the genesis of audiogenic seizures following withdrawal from chronic ethanol. Since chronic electrical stimulation of the inferior collicus also produces a kindling-like phenomenon, we propose that, like repeated electrical stimulation, repeated withdrawal episodes from chronic ethanol "kindles" the inferior colliculus or other sites. To test this hypothesis, we will determine if repeated withdrawal episodes from chronic ethanol exposure increases the sensitivity of the inferior colliculus to electrically elicited seizures after rats have recovered from the acute withdrawal. These results will be compared to studies of the amygdala, a classic site for kindling, as more than one site may be involved in the kindling process. Conversely, we will determine a chronic stimulation of the inferior colliculus or amygdala will increase seizure susceptibility of chronic ethanol-treated rats upon withdrawal. Since the inferior colliculus is a crucial component for audiogenic seizures in rats treated chronically with ethanol, the neural pathways involved in both the ethanol withdrawal seizures and electrically elicited seizures from the inferior colliculus will be mapped using site injection of local anesthetics. ln addition, pharmacological interventions are planned to determine if the seizure process induced by these treatments can be attenuated. An in vivo dialysis technique will be used to define the release of excitatory and inhibitors neuroactive amino acids in the inferior colliculus following acute or chronic ethanol treatment to allow a better understanding of the neurochemical changes induced be ethanol. Investigation will explore also the role of chloride channels in ethanol's actions and will compare the biochemical consequences of kindling with changes induced by repeated ethanol withdrawal. Electrophysiological investigations in the inferior colliculus will define changes in cell responsiveness of excitatory and inhibitory transmitters after acute or chronic ethanol. These multidisciplinary investigations should provide new information concerning the genesis of seizures induced by chronic ethanol consumption and allow us to examine the neurochemical basis for the acute actions of ethanol on CNS function as well as the associated adaptation with continued ethanol exposure.

dopamine receptor; self destructive behavior; dopamine; Lesch Nyhan syndrome; mental retardation;

The present proposal will extend our previous studies of the neuroanatomic basis of action of anti-depressant (AD) drugs in order to resolve some outstanding issues concerning the pharmacology of AD drug action. Based on data suggesting that not all AD drugs have common sties of action, one outstanding issue concerns where in brain drugs act. This issue will be pursued using autoradiographic localization of AD binding sires, autoradiography of sites where adaptive changes in receptor binding occur following chronic AD administration, and delineation of sites where microinjections of AD drugs produce behavioral responses in the forced swim test. These three approaches will permit us to test the hypothesis that AD drugs with different pharmacological profiles act at distinct sites in brain. The results from work testing these hypotheses will permit us to formulate a theoretical anatomical network through which we can describe how AD drugs with differing neural mechanisms can induce common behavioral and adaptive receptor changes. Another outstanding issue is the lack of congruence between biochemical measures of monoamine receptor adaptation and measures of electrophysiological responses to monoamines receptors adaptation treatment. Two hypotheses will be tested to resolve this issue. First, studies will examine the hypotheses that anatomical specificity of receptor adaptation is responsible for the lack of congruence. It is anticipated that consistent changes in electrophysiological responses to monoamines will be observed only at cellular sites where receptor adaptation occurs. The second hypothesis, that receptor-receptor interactions are responsible for the lack of congruence between measures of receptor adaptation and electrophysiological responses, will involve electrophysiological examination of neurotransmitter interactions observed in biochemical studies. This work is expected to resolve the question of why down-regulation of a receptor subtype for a neurotransmitter by AD drugs can lead to an enhancement of the effect of a neurotransmitter on neural activity. This multidisciplinary grant provides new strategies to explore the pharmacological effects of AD drugs which will advance our knowledge of AD drugs action and lead to the development of new hypotheses to guide future research.

conflict; neuropharmacology; drug tolerance; alcoholic beverage consumption; alcoholism /alcohol abuse; GABA receptor; neuroanatomy; genetics; drug withdrawal; genetic strain; alcoholism /alcohol abuse chemotherapy; alcoholism antagonist; molecular site; benzodiazepine receptor; anxiety; histology; laboratory rat; electrophysiology; microinjections;

There has been conflicting data concerning the effects of ethanol on GABA/A receptor function. While biochemical and behavioral investigations have been supportive of ethanol influencing GABA responses, electrophysiological studies have provided mixed results with some investigations observing an enhancement of GABA inhibition by ethanol and others reporting no effect. Recently, our laboratory observed that ethanol enhanced GABA responses in the medial septum but not in the lateral septum. To explain the differences at these sites and the literature controversy, we propose that the cellular action of ethanol on GABA/A responses depends upon a specific molecular composition of the GABA/A receptor complex which varies at different sites in brain. Three specific aims are proposed to explore this hypothesis. The first Specific Aim will identify the specific subunit composition of the GABA receptor complexes in the lateral and medial septum. This will be accomplished by measuring mRNA for the GABA/A subunits at these brain sites. The Second Aim will determine the cellular location of sites having mRNA for the GABA/A receptor like that for the medial or lateral septum. In situ hybridization of the appropriate mRNA will be used to make this determination. Finally, the third Specific Aim will characterize electrophysiologically the effect of ethanol on responses to iontophoretically applied GABA at sites defined with in-situ hybridization as having GABA/A receptors like either the medial or lateral septum. This latter study will provide a direct test of our hypothesis. To complement the electrophysiological studies, various ligands binding to the GABA complex will be tested at the sites investigated electrophysiologically. This autoradiographic evaluation will provide an alternative approach by which ethanol's actions on GABA/A receptors can be predicted. This work is expected to clarify the actions of ethanol on CNS function.

Evidence from our laboratory indicates that ethanol has a regionally specific action in brain to antagonize NMDA-induced excitation of neural activity. The NMDA receptor is composed of multiple protein subunits which form a functional ion channel. These NMDA receptor subunits have differing neuroanatomical distributions, thereby providing evidence for heterogeneity of NMDA receptors in mammalian brain. The purpose of this grant is to test the hypothesis that the regionally specific effect of ethanol on responses to NMDA in brain is linked to the expression of different combination of NMDA receptor subunits. Based upon our present observation of ethanol- induced regional inhibition of NMDA (i.e., ethanol-sensitive and ethanol- insensitive brain regions), the first Specific Aim of this proposal will determine the regional localization of the mRNA for the subunits cloned for the NMDA receptor using PCR to identify NMDAR-1 splice variants and the NMDAR-2a,b,c,&d subunits found in micropunches from brain regions shown electrophysiologically to be either insensitive or sensitive to the action of ethanol to antagonize NMDA-induced excitation. In Specific Aim II, we will determine the sensitivity of dissociated neurons to ethanol's antagonism of NMDA using fluorescent measures of increased intracellular calcium flux induced by NMDA. Subsequently, the subunit mRNA composition of the NMDA receptors in dissociated cells with proven sensitivity to ethanol will be determined with PCR amplification. In Specific Aim III, patch-clamp electrophysiology as well as changes in fluorescence will be used to compare the structural components of NMDA isoreceptors within individual neurons with their differing sensitivities to ethanol. Additionally, receptor components will be related to phosphorylation drug actions and to ethanol's inhibition of NMDA. Once the subunit composition for an NMDA receptor in individual neurons sensitive to ethanol has been documented in Specific Aim III, Specific Aim IV will map the NMDA subunits important for ethanol's action throughout brain using in situ hybridization. Following this, extracellular electrophysiological studies will be performed to test additional brain sites predicted from the mapping study to be sensitive to ethanol. Thus, with this multidisciplinary effort, it will be determined whether specific structural components making up NMDA isoreceptors predict ethanol's action on NMDA responses and will delineate 'new" sites where ethanol does or does not affect responses to NMDA. Such data will define the neurobiological and molecular basis of ethanol's regional action on this ligand-gated ion channel.

Symptoms of withdrawal are believed to contribute significantly to sustaining alcoholism. Pharmacological evidence has been obtained that endogenous compounds, including a benzodiazepine (BZD)-inverse agonist, CRF, and glutamate, play a major role in withdrawal-induced anxiety, a symptom observed during withdrawal from chronic ethanol treatment (withdrawal). Further withdrawal produces changes in metabolic activity in specific regions of brain with are proposed to be due to release of endogenous transmitters which contribute to anxiety. In addition, comparison of male and female rats have revealed gender differences when rats were performing conflict tasks during withdrawal. From such data, we suggest the anxiety and increased metabolic activity observed during withdrawal are cause by an orchestrated release of these endogenous compounds that act on central BZD receptors (CBRs), peripheral BZD receptors (PBRs), CRF, and glutamate receptors and that this increased release during withdrawal is influence by gender. To gain support of this view, Specific Aim I will identify changes in metabolic activity using 2- deoxyglucose (2-DG) accumulation and Fos expression to identify brain areas involved in the anxiety response to a challenge with air puff or elevated-plus maze in male and female rats in the presence and absence of withdrawal. Subsequently, we will use antagonists of CRF, CBRs, PBRs and glutamate receptors and antisense deoxynucleotides for the peptides to see how specific metabolic events and the anxiety induced by ethanol withdrawal are affected. In specific Aim II, we will confirm and extend observations that the endogenous inverse agonist, diazepam binding inhibitor (DBI), which acts on CBRs and PBRs, as well as the CRF peptide is altered after chronic ethanol, during withdrawal, and when male and female rats are challenged with tasks reflecting anxiety during and in the absence of withdrawal. This determination will be made by measuring content of the peptides with radioimmunoassay or quantitative immunohistochemistry. The determination of the peptides will be coupled to measurement of their levels of mRNA within specific regions of brain using in situ hybridization. Specific Aim III will test the hypothesis that treatment with antagonists of the endogenous compounds, which blocked anxiety and central metabolic activity caused by a single withdrawal, will prevent sensitization of symptoms resulting from multiple withdrawals from chronic ethanol treatment. Once completed, these aims are expected to support the hypothesis that endogenous compounds acting on CBRs, PBRs, glutamate, or CRF receptors in specific regions of brain contribute to metabolic changes and to the anxiety to differing degrees in males and females during withdrawal from chronic ethanol exposure and that repeated withdrawals accentuate these withdrawal-induced changes.

This Research Scientist Award is based upon current work being performed to understand the molecular basis of ethanol's specificity of action on ligand-gated ion channels. This proposal, tests the hypothesis that the selectivity of ethanol to affect responses to NMDA and GABA from some, but not all, neurons is due to the presence of specific NMDA and GABAA receptor subtypes. The model approach proposed to study the action of ethanol on NMDA and GABAA receptors is an update of progress made on R01s AA09122 and AA10025, grants defining the molecular mechanism(s) responsible for ethanol's specificity on specific receptor subtypes. In respect to ethanol's action on GABAA receptor subtypes, we have demonstrated zolpidem predicts one GABAA receptor subtype sensitive to ethanol. Likewise, we have shown a close relationship between ifenprodil and ethanol antagonism of NMDA responses. In this proposal, each cultured and dissociated neurons cell will be pharmacologically characterized with patch-clamp electrophysiology, the cytoplasm will be extracted from the cell, and mRNAs for GABAA and NMDA receptor subunits within each individual neuron will be identified with RT/PCR. In Specific Aim I, work will initially focus on the hypothesis that the alpha1beta2gamma2 subunit combination is one GABAA receptor subtype sensitive to ethanol and zolpidem. Due to present uncertainty, particular emphasis is being placed on which gamma2 variants in neurons with this subunit combination are sensitive to ethanol. A second portion of work on GABAA receptors relates to defining the molecular mechanism by which ethanol's action is dependent upon secondary neural inputs to a cell. Finally, we will determine if diazepam-insensitive receptors, containing alpha4 or alpha6 subunits, are sensitive to ethanol. Specific Aim II will identify the mRNAs for NMDA receptor subunits within individual neurons sensitive to ethanol antagonism of NMDA. Current results suggest that some, but not all, NMDA receptor subtypes sensitive to ifenprodil are sensitive to ethanol. Since ifenprodil binds to NMDA receptors purportedly containing the NMDAR-2b subunit, it can assumed that the receptor subtype sensitive to ethanol will contain this NMDA receptor subunit, as well as an appropriate NMDAR-1 variant. Studies are underway to confirm this hypothesis. The model proposed to define the molecular basis of ethanol's action to affect GABAA and NMDA receptor subtypes, when applied to all ligand- gated ion channels, will provide a means to identify neuroanatomical sites at which ethanol has the greatest probability of affecting brain function. Further, based upon structural analysis of ethanol's action on protein-protein inter-actions for the receptors, results may ultimately allow us to predict other protein-protein interactions influenced by ethanol.

DESCRIPTION: (Adapted from the Investigator's Abstract) Previous in vivo electrophysiological studies across differing brain regions demonstrated ethanol enhancement of GABA responses from some, but not all, neurons. This differential effect of ethanol was attributed to a variable action on diverse GABAA receptor subtypes. In contrast to ethanol's consistent enhancement of GABA function in vivo, GABA responses from isolated neurons in vitro were rarely enhanced by ethanol (Preliminary Data). Based upon the contrast of our in vivo and in vitro results, it was proposed that a factor, not available to isolated neurons in vitro, allowed ethanol's action in vivo. Based upon the presence of auxiliary binding sites on GABAA receptors, it was hypothesized that endogenous compounds acting on one or more of the auxiliary sites were important for ethanol to alter the action of GABA responsiveness in vitro. In support of this explanation, our laboratory recently discovered that ethanol enhanced GABA responses from most dissociated substantia nigra reticulata (SNR) neurons in the presence of a neurosteroid agonist, alphaxalone, while having no effect on GABA responses in the absence of this neurosteroid (Preliminary Data). Therefore, the present proposal will test the hypothesis that activation of specific neurosteroid sites on GABAA receptors facilitates ethanol enhancement of GABA responses from isolated neurons. Four Specific Aims will test this view. In Specific Aim I, several classes of neurosteroids will be examined for their ability to allow ethanol enhancement of GABA activity from isolated SNR cells. It is assumed that the differing neurosteroids will not have identical effects on the action of ethanol to enhance GABA responses. Specific Aim II will determine if a GABA-enhancing neurosteroid will result in ethanol having a differential influence on responsiveness to GABA from neurons isolated from selected brain regions where ethanol in vivo has varying effects on GABA function. Specific Aim III will determine the mRNAs for the GABAA receptor subunits in individual neurons sensitive and insensitive to ethanol's action to enhance GABA responses in the presence of neurosteroid. From the mRNAs in individual neurons, "candidate" GABAA receptors responsible for the differential actions of ethanol will be identified with correlational analysis. This will allow further testing of the hypothesis that selected GABAA receptor subtypes account for the variable actions of ethanol on GABA responsiveness in the presence of a neurosteroid. For Specific Aim IV, cDNAs for the "candidate" receptor subunits will be transfected into modified HEK cells to validate that neurosteroids will result in the expected response pattern to ethanol on GABA function observed from neurons. Additionally, neurons and cells with a preponderance of type-l-BZD receptors will be transduced with selected GABAA receptor subunit mutants to explore the potential molecular sites responsible for the neurosteroid influence on ethanol's action. Completion of the Aims will allow critical new information to emerge concerning the molecular basis of neurosteroid action on GABAA receptor function that allows ethanol to enhance GABA function in vitro.

DESCRIPTION (provided by applicant): Work in our laboratory has demonstrated that repeated stresses and withdrawals leads to sensitization of the withdrawal-induced anxiety to a brief exposure to chronic ethanol, in further support of the Ballenger and Post (1978)"kindling" hypothesis. A major finding is that flumazenil, a benzodiazepine (BZD) antagonist, minimizes the sensitization of anxiety following the repeated withdrawal protocol. Based upon these data with flumazenil, it is presumed that an endogenous substance having a negative effect on GABAA receptors is responsible for the sensitization of anxiety induced by the repeated stress sensitization of anxiety. In support of this hypothesis, two doses of DMCM, a BZD-inverse agonist, given instead of withdrawal results in sensitization of anxiety during a final withdrawal. The purpose of the present investigation is to test specific hypotheses to account for these findings. Specific Aim 1 will determine whether repeated stress sensitization of withdrawal-induced anxiety will be antagonized by flumazenil and whether this treatment results in an increase in mRNA expression for diazepam binding inhibitor (DBI), an endogenous BZD-inverse agonist, allowing identification of brain sites where adaptive change in DBI could be contributing to withdrawal-induced anxiety. Specific Aim 2 will test whether the repeated stresses or exposure to a BZD-inverse agonist will persist in inducing sensitization of withdrawal-induce anxiety upon later re-exposure to chronic ethanol, as occurs with repeated withdrawals. Additionally, we will determine if exposure to stress or a BZD-inverse agonist during multiple withdrawals will extend the duration during which later re-exposure to chronic ethanol will continue result in sensitization of anxiety. Specific Aim 3 will determine if microinjection of flumazenil into brain regions identified in Specific Aim 1 will block sensitization of withdrawal-induced anxiety following multiple stresses and whether microinjection of a BZD-inverse agonist into the selected sites will sensitize withdrawal-induced anxiety. Specific Aim 4 will test whether GABA(A) and BZD binding is affected by repeated stresses and a single withdrawal and whether responsiveness to a BZD-inverse agonist is increased by the repeated stresses. This will be complemented by experiments to identify whether an endogenous compound sensitive to flumazenil increases with repeated withdrawals. Defining the basis of the pathological adaptive mechanism(s) responsible for "kindling" of withdrawal-induced anxiety associated with repeated stresses may provide greater insight into alcohol abuse and provide new avenues for treatment.

DESCRIPTION (provided by applicant): The anxiety of withdrawal from chronic ethanol exposure has been implicated in sustaining alcohol abuse. Work in our laboratory during the previous granting period demonstrated that repeated withdrawal from chronic ethanol exposure led to a sensitization of anxiety that accompanied withdrawal. Serotonin.a major neurotransmitter in brain.and the 5-HT2C and 5-HT1A receptor subtypes were implicated in this sensitization process associated with repeated withdrawals. The purpose of the present series of nvestigations is to extend these data to test specific hypotheses that define specific brain regions where the 5-HT2C and 5-HT1A receptors contribute to the sensitization of anxiety and that account for the neurobiological basis of the sensitization of the anxiety. Specific Aim 1 will test the hypothesis that release of serotonin in the amygdala is responsible for the sensitization of anxiety induced by multiple withdrawals from chronic ethanol exposures. This work will include microinjection studies to establish the importance of 5-HT2C receptors at this site in the withdrawal-induced anxiety. Additionally, studies will explore whether the raphe has a role in the release of serotonin during withdrawal from the multiple ethanol exposures that results in a sensitization of anxiety. Specific Aim 2 will test the hypothesis that 5-HT2C and 5-HT1A receptor number or sensitivity will be enhanced in selected brain regions by repeated withdrawals as contributing factors in the sensitization of anxiety following repeated withdrawals. Specific Aim 3 of this proposal will test the hypothesis that specific proteins in the second messenger cascade down-stream from the 5-HT2C and 5-HT1A receptors are persistently altered and are critical for sustaining the persistent anxiety induced by repeated withdrawals. Emphasis will be on the G-protein coupled pathways linked to PLC and adenylyl cyclase. Complementing these biochemical measures will be the use of "knock outs" of specific genes associated with proteins in the 5-HT2C and 5-HT1A receptor second messenger cascades to test in vivo the importance of specific messenger proteins in the pathways in sustaining the sensitization of anxiety observed with repeated withdrawals from chronic ethanol.

In further support of Ballenger and Post (1978) that a kindling process occurs with repeated chronic ethanol exposures, work in our laboratory demonstrated that repeated withdrawals from chronic ethanol results in withdrawal-induced sensitization of anxiety-like behavior [i.e., a decrease in social interaction &deficit in the elevated plus-maze] in P-rats. Like multiple withdrawals, recent data also indicate that repeated stresses prior to a single withdrawal from chronic ethanol exposure results in sensitization of withdrawal-induced anxiety-like behavior in the P-rats. Additionally, stress during multiple withdrawals increases voluntary drinking of ethanol in P-rats. Preliminary data have implicated corticotropin releasing factor (CRF)--a major neurotransmitter peptide in brain-- and the CRF-1 receptor subtype in the sensitization of anxiety-like behavior associated with the repeated withdrawal and stresses/withdrawal protocols, as well as in the increased drinking induced by stress during repeated withdrawals. The purpose of the present series of investigations is to define the neuroanatomical and neurobiological basis of the CRF contribution to the sensitization of the withdrawal-induced anxiety-like behavior induced by repeated withdrawals and the stresses in P-rats. Based upon a CRF-1 receptor antagonist blocking sensitization of anxiety-like behavior induced by the repeated withdrawal as well as by the repeated stress/withdrawal protocols, Specific Aim 1 will test the hypothesis that a CRF-1 receptor antagonist microinjected into amygdala or other selected brain sites with CRF receptors will block the persistent consequence of repeated stress and multiple withdrawal sensitization of anxiety-like behavior. While focus will be on defining the site where a CRF- 1 receptor antagonist blocks sensitization of anxiety-like behavior by stress and repeated withdrawals, microinjection of a CRF-2 antagonist into specific sites will determine if this CRF receptor subtype can contribute to this sensitization. Specific Aim 2 will test the hypothesis that multiple stresses and withdrawals that induce increased drinking linked to the alcohol deprivation effect is dependent upon CRF at brain sites identified in Aim 1 that related to sensitization of anxiety-like behavior. Finally, Specific Aim 3 will test the hypothesis that increased CRF release, altered CRF receptor number, or an increased responsiveness to CRF is critical for the sensitization of withdrawal-induced anxiety-like behavior and the increased voluntary ethanol drinking at the brain sites identified in Specific Aims 1 &2 in the P-rats. Thus, this proposal tests the overall hypothesis that multiple withdrawal and the stress/withdrawal protocols that sensitize anxiety-like behavior and the increased voluntary ethanol drinking are dependent upon activation of adaptive change(s) in CRF mechanisms within specific brain sites of ethanol preferring rats. Since both stress and withdrawal symptoms from chronic ethanol exposure have been implicated in sustaining alcohol abuse, this basic effort can be expected to provide data that will assist in defining the pathological adaptive processes that contribute to withdrawal symptoms and craving during abstinence and that facilitate loss of control upon relapse in the alcoholic. New treatment strategies could emerge from such knowledge

21+ years old; 4H-Imidazo(1,5-a)(1,4)benzodiazepine-3-carboxylic acid, 8-fluoro-5,6-dihydro-5-methyl-6-oxo-, ethyl ester; Absolute ethanol; Adolescent; Adolescent Youth; Adult; Alcohol abuse; Alcohol withdrawal syndrome; Alcohol, Ethyl; Alcoholic; Alcoholism; Anxiety; Behavior; Behavioral; Boozer; Brain; CCL2; CCL2 gene; Chronic; Common Rat Strains; Data; Dependent drinker; Diet; Dose; Drugs; ETOH; Encephalon; Encephalons; Ethanol; Exposure to; Flumazenil; Flumazepil; Foundations; Future; GDCF-2; GDCF-2 HC11; Grain Alcohol; HC11; Human; Human, Adult; Human, General; MCAF; MCP-1; MCP1; MGC9434; Mammals, Rats; Man (Taxonomy); Man, Modern; Medication; Membrane; Methylcarbinol; Nature; Nerve Transmitter Substances; Nervous; Nervous System, Brain; Neurochemistry; Neurotransmitters; Pathology; Peptides; Pharmaceutic Preparations; Pharmaceutical Preparations; Predisposition; Probability; Protocol; Protocols documentation; Rat; Rattus; Receptor Protein; Relapse; Research; SCYA2; SMC-CF; Science of neurochemistry; Social Support System; Standards; Standards of Weights and Measures; Stress; Support System; Susceptibility; System; System, LOINC Axis 4; Testing; Withdrawal; Work; Youth Drinking; adolescent alcohol use; adolescent drinking; adult human (21+); alcohol problem; alcohol withdrawal; base; caN protocol; cytokine; day; drinking; drug/agent; ethanol abuse; ethanol withdrawal; hazardous alcohol use; inhibitor; inhibitor/antagonist; insight; intervention development; juvenile; juvenile human; membrane structure; neural; neural mechanism; neurochemistry; neuromechanism; novel; prevent; preventing; problem drinker; problem drinking; receptor; relating to nervous system; teen drinking; teenage drinking; therapy development; treatment development; underage drinking; withdrawal from alcohol

DESCRIPTION (provided by applicant): Drinking during adolescence enhances the probability of alcoholism upon reaching adulthood. Likewise, stress has been demonstrated to have an important role in sustaining alcohol abuse. In rats, repeated stresses prior to chronic ethanol sensitized withdrawal-induced anxiety. Based upon stress increasing cytokines in brain in adolescent and adult rats, preliminary research in adolescent and adult rats demonstrated that repeated weekly lipopolysaccharide (LPS) dosing to increase cytokines in brain followed by 5 days of ethanol (LPS/withdrawal protocol) sensitized withdrawal- induced anxiety and increased the 14-GABA(A) receptor subunit in brain 3 days after withdrawal. This effort supports the conclusion that cytokines contribute to the action of stress to enhance adaptive change induced by ethanol. Therefore, studies in adolescent rats will first characterize the time course of the 14 subunit change in cortex during and after the LPS/withdrawal protocol. Subsequently, it will be determine if this increase in the 14 subunit is accompanied by changes in 11, 15, 32, & 4 GABA(A) receptor subunits. To assess if this adaptive change has regional specificity, these assessments will be made in other regions of brain including the hippocampus, thalamus, and amygdala. To examine GABA(A) receptor function, electrophysiological studies will be performed to test if changes in synaptic and extrasynaptic GABA function occur for an extended period after the LPS/withdrawal protocol. Additionally, pharmacological studies will be carried out to identify selected GABA(A) receptor subunits at these cellular sites in chosen brain regions. Finally, studies will be undertaken to determine if blocking sensitization of ethanol withdrawal-induced anxiety induced by the LPS/withdrawal protocol will prevent the adaptive change in GABA(A) receptor subunits and diminish electrophysiological changes at synaptic and extrasynaptic sites. This latter strategy will be accomplished by preventing the LPS/withdrawal protocol behavioral sensitization of ethanol withdrawal-induced anxiety by administering drugs that block this sensitization when given prior to each of the weekly LPS doses injected before ethanol exposure. This work will test the hypothesis that the LPS/withdrawal protocol induction of persistent adaptation in GABA(A) receptor function and functional changes at synaptic and extrasynaptic sites in adolescent rats will correlate with the sensitization of withdrawal-induced anxiety induced by this protocol. The data collected should provide a foundation for understanding the role cytokines contribute to stress support of the functional pathology that increases adolescent susceptibility to continued alcohol abuse as adults.

DESCRIPTION (provided by applicant): During abstinence from alcohol, stress in alcoholics can result in negative affect and craving (1), a response which is accompanied by a change in brain functional magnetic resonance imaging (fMRI)-effects of stress not seen in social drinkers (2). To model the negative affect induced by stress during abstinence in alcoholics, an extended period of chronic intermittent alcohol (CIA) exposure was found to cause an enduring adaptation that sensitizes stress-induced negative affect during abstinence. These series of clinical and basic findings are consistent with the kindling/stress hypothesis of alcoholism. Even though the central amygdala (CeA) is known to play an important role in stress-induced negative affect after CIA exposure, unknown is the role input circuits to the CeA have in facilitating this stress-induced anxiety. In addition to CRF, other inputs that synapse on CeA neurons are glutamate (GLU) terminals from the basolateral amygdala (BLA), oxytocin (OXY) terminals from the hypothalamus, and vasopressin (VP ) terminals. Nonetheless, the influence these other inputs have on stress-induced anxiety after CIA exposure has not been identified. Likewise unidentified is whether CeA medial division (CEM) neurons that output terminals to the periaqueductal gray (PAG) and other sites are a critical component of the neural circuit that supports stress-induced anxiety associated with CIA. To guide addressing these unknowns, the hypothesis tested is that stress facilitates anxiety during abstinence from CIA exposure by influencing a neural circuit composed of terminal inputs to the lateral (CEL) and medial (CEM) divisions of the CeA that modulate CEM output. First, to permit optogenetic investigations of the potential role BLA terminals have in facilitating stress-induced anxiety after CIA exposure, an AAV5-eYFP vector with a CAMKII promoter containing the rhodopsin derivatives halorhodopsin (NpHR3.0) or channel- rhodopsin (ChR2) will be placed into the BLA to confirm localization of GLU terminals from the BLA to the lateral (CEL) and the medial (CEM) divisions of the CeA. Subsequently tested with optogenetics will be whether ChR2 activation or NpHR3.0 inhibition of BLA terminal release of GLU on CEL neurons will influence facilitation of stress-induced anxiety after CIA exposure. This latter optogenetic strategy will be complemented by determining if activation of CEL neurons with an OXY receptor agonist will block facilitation of stress-induced anxiety after CIA exposure. Upon completing investigations of CEL neural inputs, optogenetic inhibition or excitation of GLU-containing BLA terminals synapsing on CEM neurons will assess if the stress-induced anxiogenic action after CIA exposure is affected. Because VP-containing terminals synapse on CEM neurons to induce anxiety- like behavior, it is reasoned that activation of CEM neurons with VP may contribute to the anxiety induced by stress following CIA exposure. To confirm a direct involvement of CEM neurons in VP action, an AAV-eYFP vector with the NpHR3 expressed in CEM neurons will test if optogenetic inhibition of CEM neurons will alter both stress- as well as VP-induced anxiety after CIA exposure. To confirm that VP action contributes to stress-induced anxiety after CIA exposure, VP receptor subtypes will be pharmacologically antagonized in the CEM prior to stress to determine if the stress- induced anxiety after CIA exposure is prevented. Upon confirmation of CEM terminal presence in the PAG, involvement of these CEM terminals in the PAG in stress- and VP-induced anxiety observed after CIA exposure will be assessed by optogenetic inhibition or excitation of these CEM terminals- an approach to implicate both the CEM and the PAG in facilitation of the stress-induced anxiety after CIA exposure. Collectively, the proposed pharmacological and optogenetic strategies utilized can be expected to define whether involvement of specific neural inputs to the CEL and CEM, which are accompanied by CEM output to the PAG and other sites, form a circuit that influences facilitation of stress-induced negative affect after CIA exposure. This proposed circuit involving the CeA is felt to be associated with the neuropathology responsible for the facilitated negative affect observed to stress that facilitates craving in alcoholics durin abstinence. New knowledge concerning adaptations in this circuit after CIA exposure could possibly provide clues by which to minimize the barrier to developing new and improved therapeutics to treat the negative symptoms to stress observed in abstinent alcoholics.

DESCRIPTION (provided by applicant): During abstinence in alcoholics, stress induces a dysfunctional HPA axis response, an increased fMRI response accompanied by negative affect, and excess drinking upon relapse. In spite of these well documented events induced by stress in alcoholics, the neural basis of these facilitated dysfunctional responses is unknown. In this respect, even though corticotropin releasing factor (CRF) is accepted to contribute to stress induced dysfunctions after chronic alcohol (CA), an overlooked area is the possibility that the stress increase in cytokines also contributes to neuromediation of these effects of stress during abstinence. The goal of the present research is to provide support for the hypothesis that CA induces a persisting neural maladaptation that supports the stress induction of cytokines in selected brain regions associated with dysfunctional responses, facilitates stress and cytokine-induced alcohol drinking in models of relapse, alters factors that can affect stress-induced expression of brain cytokines, and intensifies cytokine responses from neurons in the central amygdala (CeA)-a brain site that supports stress-induced negative affect. The innovative strategies to be undertaken will allow testing this hypothesis. Studies will first assess if adolescent and adult rats with differing CA exposures induce a common increase in the regional distribution cytokines in brain, but differing durations of the stress-induced increase in cytokine after CA. Subsequently, to test further that cytokines have a role in stress, determinations will define whether cytokines will substitute for stress facilitation of the alcohol deprivation effect (ADE) and enhancement of operant responding for alcohol. To explore possible means by which the degree of brain cytokines is increased by stress alone or after CA, investigations will determine if the alteration in the cytokine increase during stress alone and/or after CA exposure relates to CRF activity, to an indirect involvement of an endogenous agonist on TLR4 receptors, or to induction of HPA axis dysfunction. To explore neural actions of cytokines released by stress after CA, changes in neural excitability, pre-synaptic release of GABA and/or glutamate, as well as post-synaptic changes of CeA neurons by cytokines will be explored in controls and after CA exposure. A particularly innovative component is characterization of cell-types sensitive to actions of cytokines in the lateral or medial portions of the CeA with either oxytocin or vasopressin, respectively, and exploration of whether neural actions of cytokines on CeA neurons depend upon CRF. With these important processes to resolve issues concerning cytokine neuromediation in brain, a rational basis is expected to emerge that cytokine action initiated by stress after CA exposure contributes to the negative consequences associated with stress in the abstinent alcoholic. This innovative effort should be critical for redirecting the fous of drug discovery initiatives so that therapeutic approaches for treating alcohol abuse associated with stress can be improved.