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Behavioral and neuroimmune vulnerability to withdrawal from chronic alcohol varies with age. The relation of anxiety-like behavior to amygdalar CCL2 responses following stress after withdrawal from chronic intermittent alcohol (CIA) was investigated in adolescent and adult rats.

Background The role of neuroimmune activation in withdrawal from chronic alcohol (ethanol) has been established in both adolescent and adult models, but direct comparisons across age are sparse. Studies need to elucidate age-dependent neuroimmune effects of alcohol and to focus research attention on age-dependent mechanisms and outcomes. Methods Adult and adolescent rats from 2 commonly used strains, Wistar and Sprague Dawley (SD), were maintained on continuous 7%, 5.35%, 4.5% alcohol diet (CAD) or cycled 7% w/v alcohol diet for 15 days. Cortical tissue was collected at 0, 8, 16, and 24 hours postwithdrawal followed by measurement of chemokine (C-C motif) ligand 2 (CCL2), tumor necrosis factor alpha, and interleukin 1 beta mRNA with quantitative real-time polymerase chain reaction. Results Both age groups and strains showed a strong cytokine mRNA response at 7% CAD. Further, a greater increase in CCL2 mRNA was observed in the cortex of adolescents at 7% CAD, which correlated with higher blood alcohol levels (BALs). Adolescents exposed to 5.35% CAD exhibited similar blood levels and cytokine responses as adults exposed to 7% CAD. Substantial variability in CCL2 mRNA responses was found only in adolescent rats exposed to 7% CAD. In this group, data could be segregated into high-responding and low-responding groups. Moreover, the data from the high-responding group were associated with seizures. Conclusions Relative to other cytokine mRNAs, CCL2 exhibits a unique response profile during withdrawal from CAD. This profile is shown in adolescents, where CCL2 is uniquely influenced by the effects of seizures. Additionally, this profile is shown by the fact that only CCL2 expression correlated with BAL that transcended age groups. These data emphasize the importance of BALs and treatment regimen on developmental neuroimmune responses and suggest that select components of the neuroimmune system are more responsive to CAD withdrawal and that neurobiological mechanisms differentiating these responses should be further explored.

Stress has been shown to facilitate ethanol withdrawal-induced anxiety. Defining neurobiological mechanisms through which stress has such actions is important given the associated risk of relapse. While CRF has long been implicated in the action of stress, current results show that stress elevates the cytokine TNFα in the rat brain and thereby implicates cytokines in stress effects. In support of this view, prior TNFα microinjection into the central amygdala (CeA) of rats facilitated ethanol withdrawal-induced anxiety-a response that could not be attributed to an increase in plasma corticosterone. To test for a possible interaction between cytokines and CRF, a CRF1-receptor antagonist (SSR125543) administered prior to the repeated administration of TNFα or MCP-1/CCL2 reduced the magnitude of the withdrawal-induced anxiety. This finding provided evidence for cytokine action being dependent upon CRF. Additionally, the sensitizing effect of stress on withdrawal-induced anxiety was reduced by treating the repeated stress exposure prior to ethanol with the MEK inhibitor SL327. Consistent with cytokines having a neuromediator function distinct from a neuroimmune action, TNFα increased firing rate and GABA release from CeA neurons. Thus, an interaction of glial and neuronal function is proposed to contribute to the interaction of stress and chronic ethanol. Interrupting this potential glial-neuronal interaction could provide a novel means by which to alter the development of emotional states induced by stress that predict relapse in the alcoholic.

Nerve growth factor, a neurotrophin, may have other functions, including a role in depressive disorders. The present study sought to determine whether NGF would (1) have antidepressant-like effects and (2) behave similarly to or differently from other well-recognized antidepressants. Over a broad dose-range, NGF reduced the exaggerated swim test immobility exhibited by the Flinders Sensitive Line (FSL) rats, but at a standard dose of 40 ng/ml, it was not as effective as desipramine (DMI, 5 mg/kg). The low social interaction behavior and locomotor activity of the FSL rats were less affected by NGF than was the immobility. Acute treatment with NGF did not induce c-fos expression in brain regions known to be activated by other acute antidepressants. The fact that chronic treatment with DMI blunted the corticosterone response to fluoxetine was replicated in this study. However, chronic treatment with NGF did not alter this response. Similarly, chronic treatment with fluoxetine blunted 5-HT 1A and 5-HT 2A receptor-mediated responses, whereas chronic treatment with NGF was without effect. Thus, NGF has antidepressant-like effects but does not appear to have biochemical actions typical of other antidepressants.

CVT-10216 is a highly selective, reversible inhibitor of ALDH-2 that reduces excessive alcohol drinking. Anxiety plays a role in alcoholism. The present study asks whether CVT-10216 has anxiolytic properties, as reflected in social interaction behavior in four unrelated rodent models: endogenous anxiety-like behavior in naïve Fawn-Hooded rats, repeated alcohol-withdrawal-induced anxiety, restraint stress-induced anxiety and drug-induced anxiety. CVT-10216 counteracted anxiety in all models except that produced by the 5-HT(2C) agonist, mCPP. CVT-10216 exhibited both acute and prophylactic inhibitions of repeated alcohol-withdrawal-induced anxiety. Importantly, anxiogenic behavior induced by the benzodiazepine receptor inverse agonist, DMCM, was counteracted dose-dependently by CVT-10216. Thus, a non-addictive selective inhibitor of ALDH-2 has both anxiolytic and antidipsotropic properties, which may be dependent, in part on the involvement of the GABA-benzodiazepine system.

Stress has been implicated in precipitating relapse in alcoholics (Breese et al., 2005; Sinha, 2001), but the relationship between stress and alcohol intake in both animals and humans has been and remains controversial (e.g., Champagne and Kirouac, 1987; Lynch et al., 1999; Pohorecky, 1990, 1991) despite the apparent simplicity of the tension reduction hypothesis (e.g., Kalodner et al., 1989; Young et al., 1990). Some of these discrepancies may relate to differences in animal strains, types of stressors, or other procedural variables. For example, Lynch et al. (1999) reported that restraint stress resulted in increased voluntary alcohol consumption in Wistar rats, while Chester et al., 2004) reported a decrease in voluntary alcohol drinking in the alcohol-preferring P rats or the high alcohol drinking (HAD) rats during the application of stress. Both groups reported increased drinking after termination of stress (Chester et al., 2004; Lynch et al., 1999). Thus, stress may have biphasic effects on alcohol intake depending upon the time at which measurements are made. Footshock-induced stress resulted in a reduction in alcohol drinking in adolescent Sprague–Dawley (SD) rats (Brunell and Spear, 2005), but increased alcohol intake in a variety of alcohol-preferring rat lines, including the alcohol-preferring (P) and HAD rats (Vengeliene et al., 2003). On the other hand, repeated swim stress increased alcohol intake only in Wistar rats, not the alcohol-preferring rat lines (Vengeliene et al., 2003). Of particular relevance to the present study is the report by Funk et al. (2004). They applied repeated footshock or social defeat during 2-week deprivation periods and reported that the elevated alcohol drinking associated with the alcohol-deprivation effect (ADE) was enhanced in the stress groups. The present study shows that acute restraint stress during withdrawal from alcohol can have a similar effect in P rats. Stress has also been implicated in the induction of anxiety-like behavior (Breese et al., 2004; Valdez et al., 2002). Indeed, it has been shown that exposure to restraint stress for 60 min is comparable to an episode of withdrawal from 5 days exposure to alcohol, with both procedures sensitizing rats to the anxiety-like behavior observed during withdrawal from a final episode of alcohol exposure (Breese et al., 2004; Overstreet et al., 2002). These studies have been carried out in SD rats on a forced alcohol diet. Because these rats do not drink substantial amounts of alcohol voluntarily, it was not possible to explore the relationship between stress, voluntary alcohol drinking, and subsequent withdrawal-induced anxiety-like behavior. By using a modified repeated withdrawal protocol (Overstreet et al., 2002, 2003, 2004) in the alcohol-preferring P rats, it was possible to explore the relationship between these variables, as reported in the current communication and described briefly in a previous report (Breese et al., 2004). Another well-characterized phenomenon in the P rat is the ADE, where the rats exhibit increased drinking of and preference for alcohol following a period of deprivation (Holter et al., 1998; McKinzie et al., 1998; Rodd-Henricks et al., 2000a,b; Sinclair and Li, 1989; Spanagel and Holter, 1999, 2000). These studies often employ long-term exposure to water and alcohol solutions and have relatively long (2 weeks) deprivation periods. The present communication includes P rats that have been subjected to quite brief (5-day) exposures to water and alcohol and even briefer (2 days) deprivation (withdrawal) periods. The elevated alcohol intake and withdrawal-induced anxiety under these conditions are quite remarkable (see Breese et al., 2004). While others have reported both an increase in alcohol drinking and anxiety-like behavior in out-bred rats, the rats had access to alcohol for nearly 1 year (Holter et al., 1998; Spanagel and Holter, 1999), not just 15 days. The increase in withdrawal-induced anxiety-like behavior in SD rats has been blocked or reduced by treatments with specific pharmacological actions during the early withdrawal periods (Breese et al., 2004; Knapp et al., 2005; Overstreet et al., 2003, 2004) despite the fact that the drug treatments were given nearly a week before the behavioral test. Among the compounds that counteracted the withdrawal-induced anxiety were flumazenil, a benzodiazepine (BZD) receptor antagonist, buspirone, a 5-HT1A receptor partial agonist, and CP154,526, a corticotropin-releasing factor type 1 (CRF1) receptor antagonist. These compounds were, therefore, examined in the P rats subjected to multiple cycles of alcohol exposure and withdrawal/deprivation and stress. In addition, the following drugs known to affect alcohol intake or the ADE were tested: naloxone, an opiate receptor antagonist, reduces alcohol intake (Overstreet et al., 1999), while haloperidol, a dopamine receptor antagonist, reduces the ADE (Salimov et al., 2000) and olanzapine, an atypical antipsychotic that blocks both dopamine and serotonin receptors, reduces alcohol consumption and craving in humans (Hutchison et al., 2006). The findings of this study, which greatly extend the results previously reported (Breese et al., 2004), suggest a close but imperfect link between the elevated voluntary drinking induced by stress and the withdrawal-induced anxiety-like behavior.

Previous investigations demonstrated that repeated stresses before an ethanol exposure sensitize ethanol withdrawal-induced anxiety-like behavior ('anxiety'). In addition to activating the hypothalamic-pituitary-adrenal axis, acute stress also elevates cytokines in brain. Initially, to test possible cytokine involvement in this stress/withdrawal protocol, cytokines were increased in brain with 2 weekly repeated lipopolysaccharide (LPS) administrations (1000 microg/kg) [corrected] (LPS/withdrawal protocol) or with twice weekly intracerebroventricular (i.c.v.) administrations of the cytokines IL-1 beta, CCL2 (MCP-1) or TNFalpha (cytokine/withdrawal protocol) before exposure and withdrawal from a 5-day cycle of chronic ethanol diet. Both protocols sensitized withdrawal-induced anxiety and confirm cytokine involvement in the sensitized anxiety response. Testing of various doses of LPS (16-1000 microg/kg) and TNFalpha (3-100 ng, i.c.v.) demonstrated the dose-related nature of these protocols to sensitize withdrawal-induced anxiety. The sensitized anxiety was not produced by a single 5-day ethanol diet cycle or by repeated LPS or cytokine treatments alone. Likewise, sensitized anxiety in these protocols could not be attributed to differences in ethanol ingestion. When challenged with a subsequent re-exposure to a 5-day ethanol diet cycle 16 days after completion of the LPS/withdrawal or cytokine/withdrawal protocols, an increase in withdrawal-induced anxiety was observed-an indication of induction of an underlying persistent adaptive change. Finally, just as found previously with the stress/withdrawal protocol, administration of the benzodiazepine receptor antagonist flumazenil before the LPS or TNF treatments prevented anxiety sensitization. Together, these findings indicate that increased cytokine activity induces adaptive change that supports sensitization of ethanol withdrawal-induced anxiety that may be linked to GABA(A)-receptor function.

The benzodiazepine receptor antagonist flumazenil reduces anxiety-like behavior and sensitization of anxiety-like behavior in various models of ethanol withdrawal in rodents. The mechanism and brain region(s) that account for this action of flumazenil remain unknown. This investigation explored the potential role of several brain regions (amygdala, raphe, inferior colliculus, nucleus accumbens, and paraventricular hypothalamus) for these actions of flumazenil.Rats were surgically implanted with guide cannulae directed over the brain region of interest and then treated with an ethanol diet for three 7-day dietary cycles (5 days on ethanol diet followed by 2 days on control diet). At approximately 4 hours, flumazenil was administered intracranially into each of the first 2 withdrawals. Examinations of anxiety-like behavior followed 1 week later during a third withdrawal. In other animals, restraint stress sessions or intra-amygdala DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) injections, preceded by intraperitoneal flumazenil injections, were substituted for the first 2 ethanol treatment cycles to assess the potential anxiety-sensitizing action of stress or a benzodiazepine receptor inverse agonist, respectively.Flumazenil treatment of the amygdala during the first 2 withdrawals blocked the development of sensitized anxiety seen during a third withdrawal. Similar actions of flumazenil were found when stress sessions substituted for the first 2 cycles of ethanol exposure and withdrawal. Amygdala treatment with DMCM magnified the anxiety response to the single subthreshold chronic ethanol treatment, and prophylactic flumazenil blocked this effect.Intra-amygdala flumazenil inhibits the development of anxiety sensitized by repeated ethanol withdrawal, stress/ethanol withdrawal, or DMCM/ethanol withdrawal. These actions suggest that site-specific and persistent effects of flumazenil on gamma-aminobutyric acid-modulatory processes in this brain region are relevant to sensitized behavioral effects seen in alcoholism.

IN VIVO STUDIES in urethane-anesthetized rats have generally shown an inhibition of neural activity by either systemic (Givens and Breese, 1990a) or locally applied ethanol (Freund et al., 1993). However, when other anesthetics or no anesthetic were used, systemic administration of ethanol produced a mixed action on cerebellar Purkinje neurons, with an increased rate occurring in some neurons and a decreased rate occurring in others (Rogers et al., 1980). Similarly, both increases and decreases in rate have been observed in recordings from Purkinje neurons in culture (Franklin and Gruol, 1987; Urrutia and Gruol, 1992) and in the cerebellar slice (Bloom and Siggins, 1987; Siggins et al., 1987). These changes in rate are often accompanied by a change in the pattern of firing of the Purkinje neurons (Bloom and Siggins, 1987; Franklin and Gruol, 1987; Siggins et al., 1987). Ethanol acts on a number of neurotransmitter-gated and G-protein-coupled ion channels (Crews et al., 1996). Additionally, ethanol can either increase (Ariwodola and Weiner, 2004; Crowder et al., 2002; Roberto et al., 2003; Ziskind-Conhaim et al., 2003) or decrease (Ziskind-Conhaim et al., 2003) neurotransmitter release. Purkinje neurons receive both excitatory and inhibitory synaptic inputs (Satake et al., 2004) and have an intrinsic pacemaker activity (Edgerton and Reinhart, 2003). Therefore, the differing effects of ethanol on these factors may be responsible for the mixed action of ethanol on Purkinje neuronal firing. Because the basis of such integration for the varied effects of ethanol on cerebellar Purkinje neuronal activity has not yet been investigated, the present study used both whole-cell voltage-clamp and extracellular recording to examine the potential basis of the variable actions of ethanol on cerebellar Purkinje neuronal activity in a slice preparation. This work included assessment of ethanol action on γ-aminobutyric acid (GABA) release, on the involvement of glutamate input, and on the intrinsic firing of cerebellar Purkinje neurons.

This article summarizes the proceedings of a symposium held at the 2005 Research Society on Alcoholism meeting. The initial presentation by Dr. Wallner provided evidence that selected GABA(A) receptors containing the delta subunit display sensitivity to low intoxicating ethanol concentrations and this sensitivity is further increased by a mutation in the cerebellar alpha6 subunit, found in alcohol-hypersensitive rats. Dr. Mameli reported that ethanol affects gamma-aminobutyric acid (GABA) function by affecting neural circuits that influence GABA release. Dr. Parsons presented data from electrophysiological and microdialysis investigations that ethanol is capable of releasing GABA from presynaptic terminals. Dr. Morrow demonstrated that systemic ethanol increases neuroactive steroids in brain, the absence of which alters various functional responses to ethanol. Dr. Criswell presented evidence that the ability of ethanol to increase GABA was apparent in some, but not all, brain regions indicative of regional specificity. Further, Dr. Criswell demonstrated that neurosteroids alone and when synthesized locally by ethanol act postsynaptically to enhance the effect of GABA released by ethanol in a region specific manner. Collectively, this series of reports support the GABAmimetic profile of acutely administered ethanol being dependent on several specific mechanisms distinct from a direct effect on the major synaptic isoforms of GABA(A) receptors.

Anxiety-like behavior resulting from repeated withdrawals from chronic ethanol diets is counteracted by systemic administration of a 5-HT2C receptor antagonist or a 5-HT1A receptor partial agonist.This study investigated whether prior treatment with these agents into the amygdala, dorsal raphe nucleus, nucleus accumbens, or paraventricular nucleus during early withdrawals would ameliorate the social interaction deficits observed after a subsequent withdrawal.Sprague-Dawley rats were exposed to three cycles of 5 days of forced ethanol diet (4.5%, w/v), with 2 days of control diet after the first and second cycles. Drugs were administered into one of four brain sites 4 h after removal of ethanol on the first and 2nd cycles but not the third. The social interaction test was performed 5 h after removal of ethanol on the third cycle. Drugs tested included SB-243213, a 5-HT2C receptor inverse agonist; buspirone, a 5-HT1A receptor partial agonist; and Ro 60 1075, a 5-HT2C receptor agonist.Only SB-243213 (at 3 microg, but not at 1 and 0.3 microg) counteracted the social interaction deficits after injections into the amygdala, while buspirone (at 0.3 and 1 microg but not at 0.1 microg) reduced deficits only when given into the dorsal raphe nucleus. In contrast, the 5-HT2C receptor agonist, Ro 60 1075, accentuated the behavioral deficit after two weekly injections into the amygdala.These results are consistent with the involvement of 5-HT2C receptors in the amygdala and 5-HT1A autoreceptors in the dorsal raphe nucleus in repeated ethanol withdrawal-induced sensitization of anxiety-like behavior.

Accumulating evidence indicates that adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is involved in the neurobiological responses to ethanol. Previous reports indicate that mice lacking the RIIbeta subunit of PKA (RIIbeta(-/-)) voluntarily consume more ethanol than wild-type controls (RIIbeta(+/+)) using 2-bottle testing procedures. Although such procedures primarily measure consummatory behavior, operant self-administration procedures allow analysis of consummatory as well as appetitive or "ethanol-seeking" behavior (i.e., lever pressing is required to gain access to the ethanol solution). Therefore, we determined whether the high ethanol consumption characteristic of RIIbeta(-/-) mice would be complemented by increased appetitive ethanol-seeking behavior in an operant paradigm.RIIbeta(-/-) (n=8) and RIIbeta(+/+) (n=8) mice were initially sucrose-faded until they were lever responding for nonsweetened ethanol (10, 14, and 18%). Following the self-administration testing, RIIbeta(+/+) and RIIbeta(-/-) mice were given access to 2 bottles, one containing water and the other ethanol to replicate the voluntary ethanol drinking data previously from our laboratory. Finally, immediately after voluntary consumption all mice were again tested for self-administration of 10% ethanol. Alterations in the reinforcement schedule were also explored as RIIbeta(+/+) and RIIbeta(-/-) mice were tested for self-administration of 10% ethanol at FR-3 and FR-5 schedules.The RIIbeta(-/-) mice displayed lower operant responding for ethanol and food reinforcement compared with RIIbeta(+/+) controls. However, this effect was driven by a significant increase in lever responses made by female RIIbeta(+/+) mice. When the excessive lever responses of the female RIIbeta(+/+) mice are accounted for, the RIIbeta(-/-) mice show ethanol lever responses comparable to controls. Following operant self-administration testing, RIIbeta(-/-) mice of both sexes consumed more ethanol solution compared with RIIbeta(+/+) mice during 2-bottle testing.Increased ingestion of ethanol by RIIbeta(-/-) mice is likely the result of altered PKA activity within neuronal pathways that control ethanol-consummatory behaviors. Conversely, the RIIbeta subunit of PKA appears not to play a critical role in neuronal pathways that regulate appetitive behaviors directed at obtaining ethanol. Finally, increased operant self-administration of food and ethanol by female wild-type mice was absent in female RIIbeta(-/-) mice, suggesting that normal PKA signaling may be part of a general, and sex-dependent, mechanism involved with reinforcement-seeking behavior.

NEUROPEPTIDE Y (NPY) is a 36 amino acid neuro-modulator belonging to the PP-fold family of peptides (Berglund et al., 2003; Colmer and Wahlestedt, 1993; Dumont et al., 1992). NPY immunoreactivity (IR) has been noted in brain regions such as the amygdala, cerebral cortex, caudate putamen, hippocampus, hypothalamus, and nucleus of the solitary tract (Gray and Morley, 1986) and has been shown to be involved in the regulation of a wide-ranging group of biological functions. It has been implicated in the control of food intake (Clark et al., 1984; Levine and Morley, 1984), cardiovascular homeostasis (Pedrazzini et al., 1998), integration of emotional behavior (Heilig et al., 1993; Heilig and Widerlov, 1995), neuronal development (Hansel et al., 2001a,b), seizure activity (Woldbye et al., 1996, 1997), pain modulation (Shi et al., 1999, 2001), thermogenesis (Lopez-Valpuesta et al., 1996), circadian rhythms (Biello et al., 1997; Golombek et al., 1996; Gribkoff et al., 1998; Harrington and Schak, 2000), and reproduction (Kalra et al., 1998; Kasuya et al., 1998). In recent years, evidence has emerged showing that NPY is also involved with neurobiological responses to ethanol (Pandey et al., 2003a; Thiele and Badia-Elder, 2003; Thiele et al., 2003, 2004b). Specifically, administration of ethanol and ethanol withdrawal alter central NPY expression in rodents (Bison and Crews, 2003; Clark et al., 1998; Ehlers et al., 1998; Kinoshita et al., 2000; Roy and Pandey, 2002; Thiele et al., 2000a). Further, NPY has been implicated in the modulation of ethanol consumption in rats selectively bred for ethanol preference. A genetic linkage analysis conducted in F2 intercross progenies of selectively bred alcohol-preferring (P) and -nonpreferring (NP) rat lines identified a chromosomal region that includes the gene for the NPY precursor (Bice et al., 1998; Carr et al., 1998; Foroud et al., 2000). P rats and the high alcohol drinking (HAD) rats have low levels of NPY in the amygdala when compared with controls (Ehlers et al., 1998; Hwang et al., 1999), and ventricular administration of NPY reduces ethanol drinking in P and HAD rats but not in NP rats, low alcohol drinking (LAD) rats, or outbred Wistar rats (Badia-Elder et al., 2003; Badia-Elder et al., 2001; Gilpin et al., 2003; Katner et al., 2002a,b; Slawecki et al., 2000). Interestingly, in genetically modified mice, voluntary ethanol consumption and resistance to the intoxicating effects of ethanol are inversely related to NPY levels (Thiele et al., 1998, 2000b, 2002, 2004a). Corticotropin releasing factor (CRF) is a 41 amino acid polypeptide that is expressed in many of the same brain regions as NPY, including the hypothalamus and amygdala (Swanson et al., 1983). Interestingly, NPY and CRF often produce opposing neurobiological actions. Thus, after ethanol withdrawal, there are low levels of NPY (Pandey et al., 2003b; Roy and Pandey, 2002) and increased levels of CRF (Merlo Pich et al., 1995) in the amygdala. Furthermore, central infusion of NPY (Pandey et al., 2003b) or CRF receptor antagonists (Breese et al., 2004; Knapp et al., 2004; Overstreet et al., 2004; Rassnick et al., 1993) reverses the anxiogenic effect of ethanol withdrawal. Of critical interest, while NPY decreases ethanol consumption in high ethanol drinking models (Badia-Elder et al., 2003; Badia-Elder et al., 2001; Gilpin et al., 2003), central infusion of the CRF receptor antagonist D-Phe-CRF(1,2-14) eliminates excessive ethanol drinking by rats made dependent with chronic exposure to ethanol vapor (Valdez et al., 2002). Because of their opposing actions, it has been suggested that CRF and NPY exert a reciprocal regulation of drug self-administration through allostatic interactions within the extended amygdala (Koob, 2003; Koob and Le Moal, 2001). The goal of the present study was twofold. First, we determined if ethanol consumption was inversely related to NPY levels in two inbred strains of mice that differ dramatically in their voluntary consumption of ethanol (Belknap et al., 1993; Le et al., 1994; Meliska et al., 1995; Mittleman et al., 2003; Risinger et al., 1998). C57BL/6J mice voluntary consume 10–12 g/kg/day of ethanol when offered a 10% (v/v) solution, while DBA/2J mice consume approximately 1.0 g/kg/day (Belknap et al., 1993). Second, because of the proposed relationship between NPY and CRF, we determined if C57BL/6J and DBA/2J mice also differed in central CRF levels. Immunohistochemistry procedures were used to determine NPY and CRF levels in critical brain regions within the hypothalamus and the extended amygdala. On the basis of observations utilizing genetically altered mice and selectively bred rats, we predicted that C57BL/6J mice would have low NPY levels in specific brain regions when compared with DBA/2J mice. Furthermore, because excessive ethanol drinking may be modulated by enhanced CRF receptor signaling (Valdez et al., 2002), we predicted that the C57BL/6J mice would also differ from the DBA/2J mice in brain CRF levels.

The rationale for proposing the "kindling"/stress hypothesis is to provide a conceptual basis for the insidious development and maintenance of alcohol abuse.An objective of the hypothesis is to emphasize how continued alcohol abuse is linked to progressive neural adaptation. Work has shown that repeated withdrawals from chronic low levels of alcohol sensitize ("kindle") anxiety-like behavior ("anxiety") in rats, a finding consistent with multiple withdrawal kindling of seizure activity. Additionally, stress substitutes for initial cycles of the multiple withdrawal protocol to sensitize withdrawal-induced anxiety, which is indicative that stress is capable of facilitating neuroadaptive processes related to withdrawal. The persistence of adaptation caused by stress and multiple withdrawals is revealed by the appearance of withdrawal-induced anxiety following a future re-exposure to a single 5-day period of alcohol. This persisting adaptation also permits stress to induce anxiety during a period of abstinence--a response not observed in animals without previous exposure to alcohol. Furthermore, stress interacts with repeated withdrawals to enhance voluntary alcohol drinking. Results of other preclinical and clinical studies reported in the literature are integrated with these investigations in support of the proposed hypothesis.The "kindling"/stress hypothesis is based on the premise that repeated withdrawals from cycles of chronic alcohol exposure contribute to a progressive development of persisting adaptive change that sensitizes withdrawal-induced anxiety and allows stress to evoke symptoms associated with negative affect during abstinence. Thus, these consequences of repeated withdrawals account for the evolution of major characteristics of alcoholism, which include worsened acute withdrawal symptoms and increased stress-induced negative affect during abstinence, both of which enhance the likelihood of relapse--and with relapse an inability to limit an abusive pattern of alcohol intake. The "kindling"/stress hypothesis provides a clear strategy for future studies to explore the advancing neural adaptation proposed to contribute to the pathogenesis of alcoholism.

Previous research indicated that male rats exhibited anxiety-like behavior following withdrawal from chronic ethanol exposure. This behavior, as well as other symptoms of withdrawal such as seizure susceptibility, may be sensitized in male rats repeatedly withdrawn from chronic ethanol exposure. Because there are sex differences in some alcohol effects, the present study explored whether male and female rats would respond differently to a course of repeated ethanol withdrawals. Similarly aged male and female rats were exposed to a control liquid diet or a diet containing ethanol (7% w/v). Ethanol-exposed rats had only one 5-day cycle of exposure or three cycles, with 2 days of control diet (CD) between cycles. At 5 h after the final ethanol was removed, the rats were placed as same-sexed pairs in the social interaction test; approximately half of the rats were tested later in the elevated plus maze. Rats withdrawn from ethanol after three cycles exhibited reduced the time spent in social interaction and general activity in the social interaction test and reduced the open and closed arm entries in the elevated plus maze. There were no sex differences in these effects. However, male rats exhibited a small anxiety-like response after one cycle of 5 days’ exposure to ethanol and female rats did not. Thus, there are no sex differences in the three-cycle multiple-withdrawal paradigm, but there may be differences after briefer exposures.

Anxiety-like behaviors are integral features of withdrawal from chronic ethanol exposure. In the experiments in the current study, we tested the hypothesis that anxiety can be regulated independently of other withdrawal signs and thus may be responsive to selective pharmacological agents. For 17 days, rats were fed ethanol (8-12 g/kg/day) in a liquid diet. Between 5 and 6 h after cessation of ethanol treatment, rats were tested in either the social interaction or plus-maze test of anxiety-like behavior after treatment with drugs hypothesized to have anxiolytic action. SB242084, flumazenil, and CRA1000-antagonists for 5-hydroxytryptamine (serotonin) (5-HT) 2C (5-HT(2C)), benzodiazepine, and corticotropin-releasing factor type 1 (CRF(1)) receptors, respectively-attenuated decreased social interaction without concomitant effects on activity measures. In contrast, ifenprodil, MDL 72222, and zolpidem-antagonists for N-methyl-d-aspartate (NMDA) and 5-HT(3) receptors, and agonist for benzodiazepine type 1 receptors, respectively-did not share this effect. Results for SB242084, flumazenil, and ifenprodil in the elevated plus-maze test were comparable to those in the social interaction test. These results support the suggestion that multiple neuronal systems (CRF(1), 5-HT(2C), and benzodiazepine receptors) contribute to the ethanol withdrawal sign of decreased social interaction. Furthermore, the selective effects of pharmacological agents on social interaction seem to indicate that this behavior can be dissociated from other signs. Because anxiety may be a complicating factor in alcohol withdrawal and relapse, future studies of this type are needed to provide focus for the effort to define selective and novel antianxiety agents for these disorders.

MANY NEUROTRANSMITTERS, NEUROMODULATORS, and hormones transduce their signal into cells by activating G protein–coupled receptors. After G protein–coupled receptor binding, cyclic adenosine monophosphate (cAMP) levels are either enhanced or inhibited via changes in adenylyl cyclase activation. This action leads to concomitant increases or decreases, respectively, in cAMP-dependent protein kinase A (PKA) activity. PKA is a holoenzyme that consists of a regulatory (R) subunit homodimer and two catalytic (C) subunits (Brandon et al., 1997). In the mouse, PKA includes four regulatory subunits (RIα, RIβ, RIIα, and RIIβ) and two catalytic subunits (Cα and Cβ), which are expressed in tissue-specific patterns (McKnight, 1991). A growing body of evidence from both human and animal studies suggests that variation of the cAMP/PKA system alters ethanol-seeking behavior and acute alcohol-induced physiological responses. Human alcoholics have lower platelet adenylyl cyclase activity when compared with controls (Ikeda et al., 1998; Parsian et al., 1996), and cAMP levels in lymphocytes collected from alcoholics are higher than normal under basal conditions but are lower after chronic exposure to ethanol (Nagy et al., 1988). In vitro studies indicate that NG108-15 cells exposed to ethanol show translocation of the Cα subunit from the Golgi area to the nucleus as long as ethanol is present (Dohrman et al., 1996). Furthermore, long-term (12-hr) exposure to ethanol causes translocation of both the Cα and RIIβ subunits to the nucleus (Dohrman et al., 2002). This translocation and the phosphorylation of cAMP-response element binding protein (CREB) are blocked by pretreatment with a type II PKA antagonist (Constantinescu et al., 2002). In vivo studies show that mutant drosophila lacking production of the RII subunit of PKA are resistant to the intoxicating effects of ethanol (Park et al., 2000). In agreement, we have reported that genetically altered mice lacking the RIIβ subunit of PKA (RIIβ−/−) are resistant to ethanol-induced sedation and drink significantly more ethanol than litter-mate wild-type (RIIβ+/+) mice (Thiele et al., 2000b). The goal of this research was 2-fold. First, we determined whether RIIβ−/− mice drink increased amounts of ethanol when maintained on alternate genetic backgrounds. A growing body of literature is emerging indicating that phenotypes, including neurobiological responses to ethanol, can depend on the genetic background of the knock-out model (Bowers et al., 1999; Howe et al., 2002; Kelly et al., 1998; Palmer et al., 2003; Phillips et al., 1999; Simpson et al., 1997; Thiele et al., 2000a). We have previously studied RIIβ−/− mice maintained on a mixed 129/SvJ × C57BL/6J genetic background (Thiele et al., 2000b). To extend these findings in this study, we assessed ethanol consumption by RIIβ−/− mice backcrossed over eight generations to a pure C57BL/6J inbred background and by RIIβ−/− mice maintained on a mixed 129/SvEv × C57BL/6J background. In addition to background strain (Belknap et al., 1993), sex is a factor that can influence ethanol consumption (Middaugh et al., 1999). Thus, we also determined whether the high ethanol drinking by RIIβ−/− mice is sex dependent. If both male and female RIIβ−/− mice showed increased ethanol intake on multiple genetic backgrounds, this would strengthen the argument that the RIIβ subunit of PKA is a key mediator of voluntary ethanol consumption. The second goal was to identify possible predictors of the high ethanol consumption that is characteristic of RIIβ−/− mice. One factor that has been proposed to contribute to the initiation of ethanol consumption or to continued use of this drug is high basal levels of anxiety (Bibb and Chambless, 1986; Cappell and Herman, 1972; Cornelius et al., 2003; Koob, 2003; Schuckit and Hesselbrock, 1994). Low PKA, phosphorylated CREB (p-CREB), and CREB-targeted gene activity in the central and medial amygdala—brain regions that modulate fear and anxiety (Davis, 1997)—are associated with increased anxiety-like behavior in rodents (Pandey et al., 2003a,b; Roy and Pandey, 2002). Because RIIβ−/− mice have blunted cAMP-induced PKA activity in the amygdala (Thiele et al., 2000b), we used a battery of behavioral tests to determine whether the increased ethanol drinking by RIIβ−/− mice is associated with increased basal levels of anxiety. Sex and strain are factors that influence anxiety in mice (Contet et al., 2001; Voikar et al., 2001); therefore, we studied anxiety-like behavior in male and female RIIβ−/− mice maintained on multiple genetic backgrounds. A second factor that may predispose to high ethanol consumption is an inherent resistance to the intoxicating effects of ethanol (Hodge et al., 1999; Schuckit, 1994; Thiele et al., 1998, 2000b, 2002; Wand et al., 2001; Weinshenker et al., 2000). Recent evidence indicates that low cAMP/PKA signaling promotes resistance to the intoxicating properties of ethanol (Park et al., 2000; Thiele et al., 2000b; but see Wand et al., 2001); therefore, we further characterized male and female RIIβ−/− mice to determine whether they consistently show low sensitivity to ethanol-induced sedation when maintained on alternate genetic backgrounds.

Repeated withdrawals from chronic forced ethanol exposure sensitize animals to withdrawal-induced deficits in social interaction behavior. The deficits in social interaction behavior following withdrawal from continuous ethanol exposure can be reduced following acute treatments with 5-HT(2C) antagonists or 5-HT(1A) agonists.The present study investigated whether prior treatment with these serotonergic agents during early withdrawals in rats subjected to repeated withdrawals from ethanol exposure would ameliorate the social interaction deficits observed following the final withdrawal.Sprague-Dawley rats were exposed to three cycles of 5 days forced ethanol (7%, w/v), with 2 days of control diet after the first and second cycles. Drugs were administered IP 4 h after removal of ethanol on the first and second cycles but not the third in one group and 4.5 h after removal of ethanol on the third cycle in another. The social interaction test was performed 5 h after removal of ethanol on the third cycle. Drugs tested included SB-242084, a 5-HT(2C) antagonist; buspirone, a 5-HT(1A) partial agonist; WAY-100635, a 5-HT(1A) antagonist; ketanserin, a 5-HT(2A) antagonist; ritanserin, a mixed 5-HT(2A/2C) antagonist; and Ro-601075, a 5-HT(2C) agonist.Both SB-242084 and buspirone reduced ethanol withdrawal-induced deficits in social interaction when given either acutely 30 min before the test or at 4 h after withdrawal from the first and second cycles. WAY-100635 and ketanserin were completely ineffective regardless of mode of treatment. In contrast, the 5-HT(2C) agonist, Ro-601075, accentuated the withdrawal-induced deficit in social interaction behavior in rats exposed to either 4.5 or 7% ethanol diet.These results support the utility of 5-HT(1A) agonists and 5-HT(2C) antagonists in reducing anxiety-like behavior induced by ethanol withdrawal and reducing the adaptive changes associated with repeated withdrawals.

The N-methyl-D-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1A, patch clamp studies found a significant decrease in maximal NMDA-evoked currents, indicative of a decrease in the number of NMDA receptors. Similarly, infusion of AAV-tTAK-NR1A (1 microl) into the rat temporal cortex significantly decreased NMDAR1-like immunoreactivity in layer V pyramidal cells. When AAV-tTAK-NR1A vectors were infused into the seizure-sensitive site of the rat inferior collicular cortex, the seizure sensitivity increased significantly over a period of 4 weeks. However, collicular infusion of AAV-CMV-NR1A vectors caused the opposite effect, a significant decrease in seizure sensitivity. Subsequent collicular coinfusion of vector encoding green fluorescent protein (GFP) driven by the tetracyclineoff promoter (AAV-tTAK-GFP) and vector encoding beta-galactosidase driven by the CMV promoter (AAV-CMV-LacZ) transduced distinct neuronal populations with only partial overlap. Thus, differing transduction ratios of inhibitory interneurons to primary output neurons likely account for the divergent seizure influences. Although AAV vector-derived NMDAR1 antisense can influence NMDA receptor function both in vitro and in vivo, promoter-related tropic differences dramatically alter the physiological outcome of this receptor-based gene therapy.

Since ballenger and Post (1978) summarized evidence that increased symptoms after multiple withdrawals from ethanol resemble a kindling process, a number of supportive clinical and animal studies have appeared. McCown and Breese (1990) were the first to report an increase in seizure susceptibility of the inferior colliculus after repeated ethanol withdrawals, which supported the kindling hypothesis. This effort demonstrated that seizures generated by electrical stimulation of the inferior colliculus could be elicited more easily in rats withdrawn repeatedly from ethanol, even though this increased sensitivity or sensitization was not observed in the amygdala (McCown and Breese, 1990). Thus, a key observation was that the adaptive changes due to repeated ethanol withdrawal could be different in different brain regions. In further support of this concept, Veatch and Gonzalez (1996, 1999, 2000) reported that several cycles of exposure to an ethanol inhalation chamber followed by an extended withdrawal period resulted in more electrical stimulation being required to kindle seizures from the hippocampus. On the other hand, mice exposed to an ethanol vapor chamber became more dependent with repeated exposures, as revealed by a decrease in seizure threshold or an increase in handling-induced convulsions (Becker and Hale, 1993; Becker et al., 1997, 1998; Jarvis and Becker, 1998). These examples provide convincing evidence for pathologic changes in brain function with repeated exposures to and withdrawals from ethanol. Others, including some studies in humans, have reported a similar sensitization process with repeated ethanol withdrawals (Brown et al., 1988; Holter et al., 1998; Maier and Pohorecky, 1989; Malcolm et al., 2000a,b; Meert and Huysmans, 1994; Ulrichsen et al., 1996, 1998). Thus, evidence supports the presence of a sensitization process in animals subjected to multiple withdrawals from ethanol. Of particular relevance to this article is the work of Spanagel and Holter (1999). They gave outbred rats access to water and three different concentrations of ethanol over prolonged periods. Periodically, the ethanol solutions were withdrawn. These authors noted that the rats increased their drinking of ethanol during these prolonged exposures and began to prefer the bottle containing the higher concentration of ethanol. Moreover, the animals exhibited anxiety-like behavior as indexed by the elevated plus maze (Holter et al., 1998; Spanagel and Holter, 1999). Although the anxiety-like behavior increased over time, the lengthy duration of these studies precludes detailed mechanistic investigations. The present article demonstrates that anxiety-like behavior can be induced by brief (15 days) exposures to ethanol and that the behavior can be accentuated if the exposure is cycled. A key behavioral pathology associated with ethanol withdrawal is anxiety (e.g., Criswell and Breese, 1992; File et al., 1989; Kampov-Polevoy et al., 2000; Knapp et al., 1998; Moy et al., 1997, 2000). However, except for the studies involving long-term access to alcohol (Holter et al., 1998; Spanagel and Holter, 1999), previous studies have not determined whether repeated withdrawals will sensitize animals to the anxiety associated with withdrawal. The social interaction test has been used widely to study anxiety-related behavior in rodents (e.g., File, 1993; File and Hyde, 1978; File et al., 1999; Gonzalez et al., 1998), including that related to withdrawal from drugs of abuse (Andrews et al., 1997; Costall et al., 1990; File et al., 1989; Irvine et al., 2001; Kampov-Polevoy et al., 2000). Thus, the social interaction test is well suited for determining whether changes in anxiety-related behavior occur during repeated exposures to and withdrawals from ethanol. The present study summarizes the findings of a three-cycle protocol of ethanol exposure and withdrawal on anxiety-like behavior, as measured by the social interaction test.

Givens and Breese1,2 reported that systemic administration of ethanol enhanced responses to GABA from some, but not all, medial septal neurons, while having no effect on responses from lateral septal neurons. Subsequently, Simson et al.3 reported that local application of ethanol had effects similar to those of systemically administered ethanol on GABA responses from medial septal neurons (i.e., ethanol enhanced some, but not all, responses to GABA). A follow-up report by Criswell et al.4 indicated ethanol enhanced responses to GABA from most, but not all, spontaneously active neurons in the substantia nigra reticulata, inferior colliculus, and ventral pallidum. In contrast to the positive action of ethanol to enhance GABA in the former brain regions, spontaneously active neurons in the lateral septum, ventral tegmental area, and the hippocampus were not sensitive to the action of ethanol to enhance GABA.4,5 During the course of these various investigations, it was noted that a high concentration of zolpidem binding was present in brain regions where neurons were found to be sensitive to ethanol enhancement of GABA, whereas zolpidem binding was low or virtually absent in brain regions where ethanol did not enhance responses to GABA.4–7 Therefore, the action of zolpidem and ethanol on GABA responses was tested on individual neurons in various regions of the brain 4,5 A positive relationship between the action of zolpidem and ethanol to enhance GABA-induced inhibition was found, providing support for the contention that the zolpidem-sensitive GABAA benzodiazepine (BZD) receptor would respond to ethanol enhancement of GABA.8 However, there were a few neurons encountered in the medial septum where this positive relationship between the actions of ethanol and zolpidem did not occur.8 In the survey of zolpidem binding in the brain, the cerebellar Purkinje layer contained a high concentration of zolpidem binding.6,7 Earlier work noted that only a relatively small population of cerebellar Purkinje neurons were sensitive to ethanol enhancement of GABA responses.9–12 In a portion of the Purkinje neurons insensitive to ethanol enhancement of GABA, Lin et al.9,10 reported that iontophoretic application of norepinephrine or isoproterenol allowed ethanol potentiation of GABA-induced inhibition from Purkinje neurons. Furthermore, application of a β-adrenergic antagonist to cerebellar Purkinje neurons initially sensitive to ethanol enhancement of GABA reduced this action of ethanol on GABA responsiveness.11,12 Based upon these previous data that cerebellar Purkinje neurons became sensitive to ethanol enhancement of GABA when a β-adrenergic agonist was added to the cell,9–12 the present investigation determined whether those cerebellar Purkinje neurons sensitive to β-adrenergic influences on ethanol action would be sensitive to zolpidem enhancement of responses to GABA. Thus, this approach was to provide information about the GABAA receptor subtype(s) on cerebellar Purkinje neurons initially sensitive or insensitive to ethanol enhancement of GABA. Given the variable response pattern reported for ethanol enhancement of responses to GABA from cerebellar Purkinje neurons,9,10 the present investigation initially tested the degree to which ethanol sensitivity of this cell type was related to the action of zolpidem to enhance responses to GABA. This investigation allowed an assessment of whether the action of zolpidem would be predictive of the action of ethanol on responses to GABA from cerebellar Purkinje neurons.

Both thyrotropin-releasing hormone (TRH) and RO 15-4513 antagonize ethanol-induced depression, but this common property does not infer that both compounds share similar mechanisms of action. In the present studies, both TRH (30 mg/kg, i.p.) and RO 15-4513 (10 mg/kg, i.p.) reversed ethanol-induced depression of locomotor activity, in accord with previous reports. However, the benzodiazepine antagonist, RO 15-1788, blocked this action of RO 15-4513, while exerting no effect on the analeptic action of TRH. Using a model of seizure activity electrically elicited from the inferior colliculus, ethanol exerted a dose-related attenuation of seizure activity. This anticonvulsant action of ethanol was not altered by TRH (30 mg/kg, i.p.), but RO 15-4513 (3 mg/kg) reversed the effect of the 0.5, but not the 1.0 g/kg, dose of ethanol. In addition, pretreatment with RO 15-4513 (1 or 3 mg/kg, i.p.), but not TRH (30 mg/kg, i.p.), caused seizure generalization into the forebrain following inferior collicular stimulation, further verifying the proconvulsant properties of RO 15-4513. In conclusion, the analeptic action of TRH appears independent of benzodiazepine activity, and in contrast to RO 15-4513, TRH does not exhibit proconvulsant properties. Furthermore, because TRH did not antagonize both depressant actions of ethanol studied, it appears unlikely that TRH directly interacts with the molecular basis of ethanol action.

Antagonism of ethanol-induced depression of locomotion was observed after intracisternal injection of thyrotropin releasing hormone (TRH) and bicuculline methiodide (BICM), as well as after microinjection of these drugs into the medial septum. The present investigation compared the behavioral and physiological consequence of administering TRH and BICM into the medial septum, inferior colliculus and substantia nigra to quantitate the similarities between these compounds. BICM produced a major increase in locomotor activity when injected into the medial septum and stereotypies when injected into the substantia nigra, suggesting that GABA-containing neurons have widespread influences on motor function. The wild running and seizure activity observed after BICM injection into the inferior colliculus was also consistent with this latter view. The marked increase in rectal temperature observed when BICM was injected into the medial septum may also implicate GABAergic mechanisms in temperature control at this brain site. TRH produced no such behavioral or physiological changes when administered into these three sites. Thus, this work strongly suggests that TRH does not exert a widespread action as a GABA antagonist because TRH did not produce the same changes induced by BICM. The actions of BICM and TRH to antagonize ethanol-induced depression when microinjected into the medial septum suggests that this brain area may be a critical site for the depressant action of ethanol.

When rats were pretreated with ethanol (3.0 g/kg, IP), subsequent microinjection of thyrotropin-releasing hormone (TRH) (500 or 1000 ng) into the medial septum, 30 minutes later, significantly shortened the time necessary for the rats to regain their righting reflex. Conversely, microinjection of TRH into the nucleus accumbens (1000 ng/side) or the area of the raphe obscurus (1000 ng) had no effect on ethanol-induced depression, although both of these structures mediate specific TRH effects in CNS. In order to determine if this antagonism was due to a specific TRH interaction, TRH Fab fragments were microinjected into the medial septum just prior to the microinjection of TRH. Under these conditions, TRH did not alter ethanol's depressant actions. Finally, this TRH antagonism of ethanol-induced depression appears attributable to a net increase in neuronal activity, because electrical stimulation (160 μA, 120 Hz, 1.5 msec duration) of the medial septum antagonized ethanol's impairment of the righting reflex. These results are discussed in relationship to a potential CNS site for the action of ethanol.

Adult rats lesioned with 6-hydroxydopamine (6-OHDA), either as neonates or as adults, demonstrated increased turning, compared to unlesioned controls, when muscimol was unilaterally microinjected into the substantia nigra reticulata (SNR). At the higher doses of muscimol, the lesioned rats were so intensely lateralized that circling was impeded. These data suggest a functional supersensitivity of receptors associated with GABA function in the SNR of 6-OHDA-lesioned rats. When 30 ng muscimol was administered bilaterally into the SNR, self-mutilation behavior (SMB) was observed in 2/11 of the control unlesioned rats, in 0/8 adult 6-OHDA-lesioned rats, and in 11/11 of the neonatally-lesioned rats tested. The ability of muscimol to produce SMB in the rats lesioned as neonates was dose related. Behavioral observations indicated that behaviors associated with SMB (self-biting and taffy pulling) were present in neonatal, but not adult lesioned rats. Behavioral responses to dopamine agonist administration were also different between rats lesioned as neonates and those lesioned as adults with 6-OHDA. These data support the view that lesions of dopaminergic neurons cause an increased functional responsiveness of receptors acted upon by muscimol in the SNR, and that the increased susceptibility for SMB in neonatally lesioned rats is determined by neurons distal to the GABA receptor complex in the SNR.

The significance of central noradrenergic, dopaminergic and serotonergic neural systems for the locomotor stimulant effects of methylphenidate was investigated in the rat. In order to study the role of brain catecholamines, rats were pretreated with reserpine (2.5 mg/kg) followed 24 hrs later by treatment with alpha-methyltyrosine (25 mg/kg) or U-14,624 (75 mg/kg), a dopamine-beta-hydroxylase inhibitor. In these experiments, methylphenidate stimulated motor activity was antagonized by alpha-methyltyrosine and enhanced after treatment with U-14,624, suggesting that release of newly synthesized dopamine is important to a locomotor stimulant action of methylphenidate. Evidence implicating brain serotonin in the actions of methylphenidate was obtained in rats pretreated with pargyline or p-chlorophenylalanine (PCPA). Administration of pargyline 1 hr prior to methylphenidate was found to reduce the locomotor activity induced by methylphenidate and this was antagonized by pretreatment with low doses of PCPA. Higher doses of PCPA caused a significant elevation of methylphenidate induced activity which could be reduced by 5-hydroxytryptophan. Destruction of serotonergic neurons with 5,7-dihydroxytryptamine also potentiated methylphenidate induced locomotion. These latter findings suggest that serotonergic fibers have an inhibitory function in brain. These results are discussed in relation to the possible mechanism by which methylphenidate may act in hyperkinesis.

Normetanephrine-H 3 injected into the cisterna magna of rats is rapidly metabolized and disappears from brain with an initial half-life of about 12 min. Monoamine oxidase inhibition prevents almost completely the conversion of normetanephrine-H 3 to other metabolites and markedly diminishes the rate of disappearance of radioactivity from brain ( T 1 2 = 2·4 hr ) . These data show that normetanephrine is normally metabolized primarily by monoamine oxidase and that unaltered normetanephrine does not readily pass out of the brain. Free 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3-methoxy-4-hydroxymandelic acid (VMA) formed from intracisternally injected normetanephrine-H 3 represent only a small fraction of the radioactivity in brain. The major metabolite was identified as the sulfate conjugate of MHPG. After intracisternal administration of norepinephrine-H 3 , 3-methoxy-4-hydroxyphenylglycol sulfate (MHPG-SO 4 ) was also found to be the major metabolite present in brain. These findings suggest that deamination, reduction, and subsequent conjugation with sulfate is the primary route of metabolism of normetanephrine in rat brain and that norepinephrine is also metabolized to this sulfate conjugate.