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BACKGROUND: Arginine vasopressin (VP) has been implicated in a number of neuropsychiatric disorders with an emphasis on situations were stress increased the severity of the disorder. Based on this hypothesized role for VP in neuropsychiatric disorders, much research is currently being undertaken in humans and animals to test VP as a target for treatment of a number of these disorders including alcohol abuse. OBJECTIVES: To provide a summary of the literature regarding the role of VP in alcohol and stress related behaviors including the use of drugs that target VP in clinical trials. RESULTS: Changes in various components of the VP system occur with alcohol and stress. Manipulating VP or its receptors can alter alcohol and stress related behaviors including tolerance to alcohol, alcohol drinking, and anxiety-like behavior. Finally, the HPA axis response to alcohol is also altered by manipulating the VP system. However, clinical trials of VP antagonists have had mixed results. CONCLUSIONS: A review of VP’s involvement in alcohol’s actions demonstrates that there is much to be learned about brain regions involved in VP-mediated effects on behavior. Thus, future work should focus on elucidating relevant brain regions. By using previous knowledge of the actions of VP and determining the brain regions and/or systems involved in its different behavioral effects, it may be possible to identify a specific receptor subtype target, drug treatment combination, or specific clinical contexts that may point toward a more successful treatment.

Patients with depression and rodent models of depression show increased cytokines and activated microglia. Fawn Hooded (FH/Wjd) rats have long been used as a model of depression based on their depressive-like behaviors, high basal corticosterone levels and altered serotonergic levels, but little is known about the neuroimmune function in this model. To test whether depressive-like behaviors relate to dysfunction of the neuroimmune system, depressive-like behaviors in the forced swim test (FST) and corticosterone (CORT) response to the swim test were compared in male Fawn Hooded versus Wistar rats, and cytokine levels in plasma and brain and plasma CORT in response to lipopolysaccharide (LPS, an endotoxin that activates the neuroimmune system) or 1 h restraint were measured. Fawn Hooded rats had more depressive-like behaviors in the FST (decreased swim time and increased immobility) and increased overall plasma CORT compared with Wistar rats. Additionally, Fawn Hooded rats exhibited blunted brain and plasma cytokine response to LPS compared with Wistar rats, an effect that might be related to the blunted plasma CORT response to LPS. No strain differences were found on these measures in response to restraint stress. These results suggest that Fawn Hooded rats have a depressive-like phenotype potentially more closely associated with serotonin dysregulation and a dysregulated HPA axis and remain a relevant model for further defining the role of these systems in depressive conditions.

Neuroimmune cytokines are increased with alcohol withdrawal and may mediate clinical responses associated with alcoholism. Because minocycline regulates the level of cytokines, it has been suggested as a therapeutic for disorders associated with alcohol. Male Wistar rats were exposed to chronic intermittent alcohol (CIA) comprising three 5-day cycles of ethanol liquid diet separated by 2 days of withdrawal. Rats were tested on social interaction, a measure of anxiety-like behavior, followed immediately by collection of amygdala tissue to measure CCL2 and TNFα or collection of the blood to measure corticosterone (CORT). One group received a single minocycline injection 3 h into the final CIA withdrawal and was tested 2 h later. A second group received injections during each of the three withdrawals and was similarly tested during the final acute withdrawal. A third group received a single injection at 23 h into withdrawal (extended withdrawal) and was tested 6 h later. Results showed that CIA withdrawal increased anxiety-like behavior. A single injection of minocycline during the final acute withdrawal increased anxiety-like behavior in rats that consumed liquid diet with or without alcohol, but this effect disappeared with repeated injections of minocycline. Differences in alcohol intake, blood alcohol level, and plasma CORT levels did not explain results. Only repeated injections of minocycline decreased TNFα mRNA levels in rats that consumed liquid diet with or without alcohol. When a single injection of minocycline was given during extended withdrawal, it decreased CCL2 mRNA levels, but did not reverse the elevation of CCL2 protein. These results suggest that minocycline has actions in brain and on behavior, but minocycline does not significantly impact these actions in relation to alcohol withdrawal.

Anxiety-like responses to stress are accompanied by elevation of brain cytokine-mRNAs. Because cytokines microinjected into central-amygdala (CeA) substitute for stress in a behavioral paradigm, the possibility was raised that cytokines increased by stress influence behavior by affecting CeA-neural activity. Previously, cytokines increased firing-rate of CeA-neurons comparable to that induced by corticotropin-releasing factor (CRF). In this investigation, tumor-necrosis-factor-α (TNFα) increased amplitude, but not frequency of mEPSCs from CeA-neurons. Additionally, TNFα decreased the threshold for triggering action potentials from CeA-neurons without altering membrane-properties during current-clamp recording. Glutamate-receptor-antagonist blockade of mEPSCs and the TNFα-induced reduction in firing threshold implicated glutamate in these changes. A phosphatidyl-inositol-3-kinase-antagonist prevented the TNFα-induced increased in amplitude of mEPSCs, documenting a TNFα intracellular influence. Additionally, TNFα increased frequency, but not amplitude of mIPSCs. CRF-receptor-antagonists were found to prevent the TNFα-induced increase in mIPSC-frequency, without altering the TNFα-induced amplitude increase in mEPSCs or the reduced threshold for action-potentials by TNFα. To clarify how TNFα was increasing CRF-release in the presence of tetrodotoxin, the possibility tested was whether preventing glial-activation would prevent this elevated mIPSC-frequency blocked by CRF-receptor antagonists. Minocycline, which blocks glial activation, prevented the TNFα-induced increase in mIPSC-frequency - a finding consistent with glia contributing to the CRF-involvement in this TNFα action. To fully understand the means by which a CRF1-receptor-antagonist and minocycline prevent TNFα from increasing mIPSC-frequency will require further clarification. Nonetheless, these data provide convincing evidence that release of TNFα by stress could alter neural activity of CeA-neurons by influencing GABA-and glutamate function.

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.

Chronic pain conditions are often co-morbid with alcohol abuse. “Self-medication” with alcohol introduces a host of problems associated with the abuse of alcohol which over time has the potential of exacerbating the painful condition. Despite the prevalence of chronic pain being associated with alcohol abuse, rodent models which mimic the co-morbid conditions are lacking. In the present study, we model osteoarthritis (OA) in C57BL/6J mice by surgically destabilizing the medial meniscus (DMM). Sham operated mice served as controls. Thirteen weeks after surgery, DMM but not sham operated mice exhibited pronounced incapacitance of the surgically-manipulated hindlimb compared to the non-surgically manipulated hindlimb. At this time, the mice were exposed to the two-bottle ethanol choice, beginning with 2.5% with a gradual increasing to 20%. Compared to sham controls, DMM mice consumed more EtOH and preferred EtOH over water at the 20% EtOH concentration. Histological analysis verified that the DMM mice exhibited significant damage to the articular cartilage and osteophyte growth compared to sham controls and these measures of the severity of OA correlated with the amount of ethanol intake. Thus, the combination of the DMM model of OA with the enhanced two-bottle ethanol choice is a potential preclinical approach in mice by which the basis of the co-morbid association of alcohol abuse and chronic pain conditions can be explored.

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. Adolescent and adult Wistar rats were exposed to CIA (three 5-day blocks of dietary alcohol separated by 2 days of withdrawal) at concentrations that created similar blood alcohol levels across age. Twenty-four hours into the final withdrawal, half of the rats were exposed to 1 h of restraint stress. Four hours post-stress, rats were used for behavior or tissue assays. Anxiety-like behavior was increased versus controls by CIA in adolescents and by CIA + stress in adults. CCL2 mRNA was increased versus controls by CIA in adolescents and by CIA and CIA + stress in adults. CCL2 co-localization with neuronal marker NeuN was decreased versus controls by CIA in adolescents and by CIA + stress in adults. CCL2 co-localization with astrocytic marker GFAP was decreased versus controls by CIA and CIA + stress in adolescents, but experimental groups did not differ from controls in adults. CCL2 co-localization with microglial marker Iba1 was decreased versus controls by stress alone in adolescents and by CIA + stress in adults. Changes in CCL2 protein might control behavior at either age but are particularly associated with CIA alone in adolescents and with CIA + stress in adults. That the number of CeA neurons expressing CCL2 was altered after CIA and stress is consistent with CCL2 involvement in neural function.

Lesch-Nyhan disease is a neurologically, metabolically, and behaviorally devastating condition that has eluded complete characterization and adequate treatment. While it is known that the disease is intimately associated with dysfunction of the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene that codes for an enzyme of purine metabolism (hypoxanthine-guanine phosphoribosyltransferase) and is associated with neurological, behavioral, as well as metabolic dysfunction, the mechanisms of the neurobehavioral manifestations are as yet unclear. However, discoveries over the past few decades not only have created useful novel animal models (e.g., the HPRT-deficient mouse and the serendipitously discovered perinatal 6-hydroxydopamine (6-OHDA lesion model), but also have expanded into epigenetic, genomic, and proteomic approaches to better understand the mechanisms underlying this disease. The perinatal 6-OHDA model, in addition to modeling self-injury and dopamine depletion in the clinical condition, also underscores the profound importance of development in the differential course of maladaptive progression in the face of a common/single neurotoxic insult at different ages. Recent developments from clinical and basic science efforts attest to the fact that while the disease would seem to have a simple single gene defect at its core, the manifestations of this defect are profound and unexpectedly diverse. Future efforts employing the 6-OHDA model and others in the context of the novel technologies of genome editing, chemo- and opto-genetics, epigenetics, and further studies on the mechanisms of stress-induced maladaptations in brain all hold promise in taking our understanding of this disease to the next level.

Neuropeptide Y (NPY) and Protein Kinase A (PKA) have been implicated in neurobiological responses to ethanol. We have previously reported that mutant mice lacking normal production of the RIIβ subunit of PKA (RIIβ−/− mice) show enhanced sensitivity to the locomotor stimulant effects of ethanol and increased behavioral sensitization relative to littermate wild-type RIIβ+/+ mice. We now report that RIIβ−/− mice also show increased NPY immunoreactivity in the nucleus accumbens (NAc) core and the ventral striatum relative to RIIβ+/+ mice. These observations suggest that elevated NPY signaling in the NAc and/or striatum may contribute to the increased sensitivity to ethanol-induced behavioral sensitization that is characteristic of RIIβ−/− mice. Consistently, NPY−/− mice failed to display ethanol-induced behavioral sensitization that was evident in littermate NPY+/+ mice. To more directly examine the role of NPY in the locomotor stimulant effects of ethanol, we infused a recombinant adeno-associated virus (rAAV) into the region of the NAc core of DBA/2J mice. The rAAV-FIB-NPY13-36 vector expresses and constitutively secretes the NPY fragment NPY13-36 (a selective Y2 receptor agonist) from infected cells in vivo. Mice treated with the rAAV-FIB-NPY13-36 vector exhibited reduced expression of ethanol-induced behavioral sensitization compared to mice treated with a control vector. Taken together, the current data provide the first evidence that NPY signaling in the NAc core and the Y2 receptor modulate ethanol-induced behavioral sensitization.

Stress may elevate ethanol drinking and anxiety associated with ethanol drinking. Studies to identify relevant neurobiological substrates are needed.To assess roles of brain regions in corticotrophin releasing factor (CRF) effects on stressor-enhanced, ethanol deprivation-induced drinking and anxiety-like behavior.Ethanol-preferring rats (P rats) were exposed to three cycles of a two-bottle choice paradigm with two 2-day deprivation periods that included 1 h exposure to a restraint stressor. To assess the role of CRF and to identify relevant brain regions, a CRF-1 receptor antagonist (SSR125543; 10 ug) was injected into the nucleus accumbens (NAC), amygdala (Amyg), or dorsal raphe nucleus (DRN) prior to exposure to the restraint stressor. In a second study, CRF (0.5 ug) was injected into one of these regions, or the ventral tegmental area (VTA), or paraventricular nucleus of the hypothalamus (PVN).Applying the restraint stressor during deprivation increased voluntary intake and sensitized anxiety-like behavior. Antagonist injection into the NAC prevented increased drinking without affecting anxiety-like behavior, whereas injection into the Amyg or DRN prevented the anxiety-like behavior without affecting drinking. To confirm CRF actions in the stressor effect, CRF was injected into selected brain regions. NAC injections (but not the VTA, Amyg, DRN, or PVN) facilitated drinking but did not change anxiety-like behavior. Injections into the DRN or Amyg (but not PVN or VTA) enhanced anxiety-like behavior.Results emphasize that a restraint stressor elevates ethanol intake and sensitizes ethanol deprivation-induced anxiety-like behavior through CRF1 receptors in the NAC and Amyg/DRN, respectively.

Adolescence is known to be a time of increased use of alcohol, and this use is the strongest predictor of alcohol dependence in adulthood (Grant, 1998; Grant and Dawson, 1997). Additionally, the dependency course (time between initial use and dependence) is more rapid in adolescents than in adults (Clark et al., 1998). Further, it has been suggested that stress during adolescence is also a contributing factor for the development of alcohol dependence (Arnsten and Shansky, 2004; Enoch, 2006; Wagner, 1993). Studies in adolescent rodents have generated mixed findings regarding the consequences of stress on ethanol consumption (Brunell and Spear, 2005; Chester et al., 2008; Doremus et al., 2005). While these studies have demonstrated that the interactions between stress and ethanol intake during adolescence are likely complex, minimal work has been performed to evaluate how stress impacts ethanol withdrawal-related behaviors (i.e., anxiety). Studies in rodents showed that repeated episodes of ethanol withdrawal lead to increased sensitivity for seizure sensitization or “kindling” following repeated ethanol withdrawals (Becker and Hale, 1993; Kokka et al., 1993; McCown and Breese, 1990). Further investigations demonstrated that other symptoms of withdrawal (i.e., anxiety) could also undergo a kindling-like process in adolescent and adult rodents (Breese et al., 2004; Overstreet et al., 2002; Wills et al., 2009). In these experiments, it was shown that withdrawal from three 5-day cycles of ethanol diet (see Fig. 1A) induced anxiety-like behavior (measured by a reduction in social interaction), which was not produced by withdrawal from 15 continuous days of ethanol exposure (Overstreet et al., 2002; Wills et al., 2009). This anxiety-like behavior only following repeated ethanol withdrawals illustrated that the pattern of ethanol exposure was important in sensitizing anxiety-like behavior (Breese et al., 2005a). Later work set out to determine whether there were sensitivity differences between adolescent and adult rats in the anxiety produced from repeated ethanol withdrawals. While there were no differences between ages in the amount of anxiety (% decrease in SI from controls) 5 hours into withdrawal, there were differences in the duration of this anxiety (length of time present after final withdrawal). In adolescent rats, anxiety-like behavior was present 7 days after the final withdrawal, while in adult rats anxiety had returned to baseline with 2 days. These data indicated increased sensitivity to the effects of repeated withdrawals in adolescent rats. Fig. 1 Procedure for repeated ethanol withdrawal, stress/withdrawal, and CRF/withdrawal paradigms (Panels A–C). (A) In the repeated ethanol withdrawal paradigm, rats are given three 5-day cycles of ethanol diet separated by two 2-day withdrawal period ... Other studies (mainly in adults) have also established an interaction between stress and CRF in the development of this anxiety-like behavior during ethanol withdrawal. Initial work found pretreatment with a CRF1 receptor antagonist during repeated ethanol withdrawals prevented the development of anxiety-like behavior in both adolescent and adult rats (Breese et al., 2004; Knapp et al., 2004; Wills et al., 2009). Additional work in adult rats has shown that two weekly 1-hour restraint stress sessions followed by a single 5-day cycle of ethanol diet (not anxiogenic alone) could sensitize anxiety-like behavior (see Fig. 1B; Breese et al., 2004). Further work in adult rats found that CRF administered intraventricularly also substituted for either withdrawals or stress (see Fig. 1C) to produce anxiety-like behavior (Overstreet et al., 2004a). Because adolescent rats were shown to be more sensitive than adults to the effects of repeated ethanol withdrawals, the present work determined whether adolescent rats were also more sensitive to the effects of stress and CRF on ethanol withdrawal-induced anxiety-like. First, it was evaluated whether stress can substitute for withdrawals (stress/withdrawal paradigm) to produce anxiety-like behavior in adolescent rats, as it has been shown to do in adult rats. Further, it was evaluated whether the duration of this anxiety-like behavior (measured as a reduction in social interaction) following stress/withdrawal differs between adolescents and adults, as this metric revealed a sensitivity differences in the repeated withdrawal paradigm. Additional studies were performed to determine whether CRF substituted for withdrawals or stress to produce anxiety-like behavior in adolescent rats and whether the effective doses were similar to those used in adult rats. Finally, experiments were performed to assess the levels of CRF in stress- and anxiety-responsive nuclei (the central nucleus of the amygdale; CeA and paraventricular nucleus of the hypothalamus; PVN) following stress/withdrawal and repeated withdrawal paradigms in both adolescent and adult rats.

Research on the actions of ethanol at the GABAergic synapse has traditionally focused on postsynaptic mechanisms, but recent data demonstrate that ethanol also increases both evoked and spontaneous GABA release in many brain regions. Using whole-cell voltage-clamp recordings, we previously showed that ethanol increases spontaneous GABA release at the rat interneuron-Purkinje cell synapse. This presynaptic ethanol effect is dependent on calcium release from internal stores, possibly through activation of inositol 1,4,5-trisphosphate receptors (IP(3)Rs). After confirming that ethanol targets vesicular GABA release, in the present study we used electron microscopic immunohistochemistry to demonstrate that IP(3)Rs are located in presynaptic terminals of cerebellar interneurons. Activation of IP(3)Rs requires binding of IP(3), generated through activation of phospholipase C (PLC). We find that the PLC antagonist edelfosine prevents ethanol from increasing spontaneous GABA release. Diacylglycerol generated by PLC and calcium released by activation of the IP(3)R activate protein kinase C (PKC). Ethanol-enhanced GABA release was blocked by two PKC antagonists, chelerythrine and calphostin C. When a membrane impermeable PKC antagonist, PKC (19-36), was delivered intracellularly to the postsynaptic neuron, ethanol continued to increase spontaneous GABA release. Overall, these results suggest that activation of the PLC/IP(3)R/PKC pathway is necessary for ethanol to increase spontaneous GABA release from presynaptic terminals onto Purkinje cells.

In abstinent alcoholics, stress induces negative affect—a response linked to craving and relapse. In rats, repeated stresses at weekly intervals before 5-day ethanol diet sensitize withdrawal-induced anxiety-like behavior (“anxiety”) that is blocked by a corticotrophin-releasing factor 1 (CRF-1)-receptor antagonist. Current experiments were performed to identify brain sites that support CRF involvement in stress sensitization of ethanol withdrawal-induced anxiety-like behavior. First, different doses of CRF microinjected weekly into the central amygdala (CeA) before ethanol exposure produced a dose-related sensitization of anxiety during ethanol withdrawal. Subsequently, CRF microinjection into the basolateral amygdala, dorsal raphe nucleus (DRN), or dorsal bed nucleus of the stria terminalis (d-BNST) also sensitized ethanol withdrawal-induced anxiety. In contrast, sensitization of ethanol withdrawal-induced anxiety was not observed after weekly CRF administration into the ventral-BNST, CA1-hippocampal region, or hypothalamic-paraventricular nucleus. Then, experiments documented the CRF receptor subtype responsible for CRF and stress sensitization of withdrawal-induced anxiety. Systemic administration of a CRF-1 receptor antagonist before CRF microinjection into the CeA, DRN, or d-BNST prevented CRF-induced sensitization of anxiety during ethanol withdrawal. Furthermore, repeated microinjections of urocortin-3, a CRF-2 receptor agonist, into the CRF-positive sites did not sensitize anxiety after withdrawal from ethanol. Finally, microinjection of a CRF-1 receptor antagonist into the CeA, DRN, or d-BNST before stress blocked sensitization of anxiety-like behavior induced by the repeated stress/ethanol withdrawal protocol. These results indicate that CRF released by stress acts on CRF-1 receptors within specific brain regions to produce a cumulative adaptation that sensitizes anxiety-like behavior during withdrawal from chronic ethanol exposure.

Resting and CO2 stimulated respiration were measured by means of a whole-body plethysmograph in rats lightly anaesthetized with halothane. Rats pretreated neonatally with intracisternal 5,7-dihydroxytryptamine (5,7-DHT) to destruct permanently central serotonergic neurones had significantly lower resting and CO2 stimulated respiratory frequency (RF) and minute volume (VM) than naive rats. In the 5,7-DHT pretreated rats, but not in naive rats, the monoamine oxidase inhibitors clorgyline and pargyline further reduced both resting and CO2 stimulated RF and Vm, whereas 1-deprenyl stimulated respiration. The results provide additional evidence that monoaminergic mechanisms are involved in central modulation of respiration in which activation of a serotonergic neuronal system depresses, and dopaminergic activation stimulates respiration.

Background: The alcohol deprivation effect (ADE) is characterized by transient excessive alcohol consumption upon reinstatement of ethanol following a period of ethanol deprivation. While this phenomenon has been observed in rats using both bottle drinking (consummatory behavior) and operant self-administration (consummatory and appetitive “ethanol-seeking” behavior) procedures, ADE studies in mice have primarily relied on bottle drinking measures. Furthermore, the neurochemical pathways that modulate the ADE are not well understood. Therefore, we determined whether the ADE can be observed in C57BL/6J mice using operant self-administration procedures and if expression of the ADE is modulated by the corticotropin releasing factor-1 (CRF-1) receptor. Methods: C57BL/6J mice were trained in a 2-hour operant self-administration paradigm to lever press for 10% ethanol or water on separate response keys. Between operant sessions, mice had access to ethanol in their homecage. Once stable responding occurred, mice were deprived of ethanol for 4 days and were then retested with ethanol in the operant paradigm for 3 consecutive days. Next, to assess the role of the CRF-1 receptor, mice were given intraperitoneal (i.p.) injection (0, 10, or 20 mg/kg) of the CRF-1 receptor antagonist CP-154,526 30 minutes before ADE testing. Additional experiments assessed (i) ADE responding in which the alternate response lever was inactive, (ii) the effects of CP-154,526 on self-administration of a 1% sucrose solution following 4 days of deprivation, and (iii) ADE responding in which mice did not received i.p. injections throughout the experiment. Results: Mice exhibited a significant increase in postdeprivation lever responding for ethanol with either a water reinforced or inactive alternate lever. Interestingly, i.p. injection of a 10 mg/kg dose of CP-154,526 protected against the ADE while not affecting lever responding for a sucrose solution. Finally, baseline and deprivation-induced increases of ethanol reinforced lever responding were greater in mice not given i.p. injections. Conclusions: The ADE in C57BL/6J mice can be modeled using the operant self-administration paradigm and increased ethanol self-administration associated with the ADE is modulated by CRF-1 receptor signaling.

Rats lesioned with 6-hydroxydopamine (6-OHDA) as neonates exhibit behavioral and neurochemical abnormalities in adulthood that mimic Lesch-Nyhan disease, schizophrenia and other developmental disorders of frontostriatal circuit dysfunction. In these animals, a latent sensitivity to D1 agonists is maximally exposed by repeated administration of dopamine agonists in the post-pubertal period (D1 priming). In neonate-lesioned, adult rats primed with SKF-38393, we found selective, persistent alterations in the morphology of pyramidal neuron apical dendrites in the prelimbic area of the medial prefrontal cortex (mPFC). In these animals, dendrite bundling patterns and the typically straight trajectories of primary dendritic shafts were disrupted, whereas the diameter of higher-order oblique branches was increased. Although not present in neonate-lesioned rats treated with saline, these morphological changes persisted at least 21 days after repeated dosing with SKF-38393, and were not accompanied by markers of neurodegenerative change. A sustained increase in phospho-ERK immunoreactivity in wavy dendritic shafts over the same period suggested a relationship between prolonged ERK phosphorylation and dendritic remodeling in D1-primed rats. In support of this hypothesis, pretreatment with the MEK1/2-ERK1/2 pathway inhibitors PD98059 or SL327, prior to each priming dose of SKF-38393, prevented the morphological changes associated with D1 priming. Together, these findings demonstrate that repeated stimulation of D1 receptors in adulthood interacts with the developmental loss of dopamine to profoundly and persistently modify neuronal signaling and dendrite morphology in the mature prefrontal cortex. Furthermore, sustained elevation of ERK activity in mPFC pyramidal neurons may play a role in guiding these morphological changes in vivo.

Adult rats exhibit increased anxiety-like behavior after exposure to repeated cycles of chronic ethanol and withdrawal. While adolescent rats have differential responses to both acute and chronic ethanol treatments, the potential differences in the effects of repeated withdrawals in this population have yet to be determined.Male adult and adolescent rats received three 5-day cycles of either a 4.5% or 7% ethanol diet (ED) separated by two 2-day withdrawal periods. Five hours into the final withdrawal, rats were tested for social interaction (SI) deficits (an index of anxiety-like behavior) and then assessed for seizure thresholds (audiogenic and bicuculline-induced). Ethanol intake was monitored throughout, and blood ethanol concentrations (BEC) were obtained from a separate group of rats.Adolescent rats have reduced SI during the final withdrawal from either ED and exhibit a greater reduction in SI compared to adult rats when exposed to a 7%ED. Audiogenic seizures were not increased during withdrawal from either ED in adult rats, but adolescent rats that received 7%ED displayed increased seizures. The bicuculline seizure thresholds were decreased in both ages exposed to a 7%ED, but only adolescent rats showed this decreased threshold after 4.5%ED. Ethanol intakes and BECs were higher in adolescent rats compared to similarly treated adults. However, ethanol intakes and BECs were comparable between 4.5%ED-treated adolescent and 7%ED-treated adult rats.Behavioral results from the 7%ED-treated groups suggested that adolescent rats may be more vulnerable to repeated withdrawals from ethanol than adults; however, differences in ethanol intake and BECs may be at least in part responsible. When ethanol intakes and BECs were similar between 4.5%ED-treated adolescent and 7%ED-treated adult rats, behavioral effects were not different. Importantly, these data illustrated that adolescent rats can exhibit anxiety and reduced seizure thresholds following this repeated withdrawal paradigm.

Stress may be a key contributor to the development of ethanol dependence and relapse (Breese et al., 2005; Koob, 2003). Stressful life events, such as those underlying post-traumatic stress disorder, are comorbid with ethanol abuse disorders and human laboratory studies show that stress increases the self-report of craving in abstinent alcoholics (Back et al., 2006; Breslau et al., 2003; Fox et al., 2007). Clinical research implicates stress in the relapse to pathological ethanol use in formerly abstinent alcoholics, perhaps as a means to self-medicate heightened anxiety and negative affect associated with withdrawal and abstinence from alcohol (Brady and Sonne, 1999; Breese et al., 2005; Kushner et al., 1994; Sinha, 2001). Recent investigations show that stress can also impact ethanol consumption in animal models (Chester et al., 2004; Croft et al., 2005; Le et al., 2000; Little et al., 1999; Liu and Weiss, 2002; Sillaber et al., 2002). Various stress paradigms reliably elicit stress-induced increases in ethanol consumption, especially among low ethanol consuming animals (Chester et al., 2004; Croft et al., 2005; Little et al., 1999). For example, selectively bred ethanol non-preferring NP rats exposed to 10 days of restraint stress showed significant and enduring increases in ethanol consumption beginning approximately 2 weeks following the stress procedure, while ethanol preferring P rats showed only transient stress-induced increases in ethanol drinking immediately after the stress procedure (Chester et al., 2004). Additionally, 3 weeks of stress induced by daily saline injections (Little et al., 1999) or 5 consecutive days of social defeat stress (Croft et al., 2005), significantly increased ethanol consumption approximately 2 weeks after the stress procedure among C57BL/10 mice displaying initially low preference for ethanol. An interesting commonality among many animal studies that assess the effects of stress on ethanol intake is that the effects of stress on ethanol drinking are delayed, typically occurring weeks after stress exposure (Chester et al., 2004; Croft et al., 2005; Little et al., 1999). Both ethanol and stress activate the hypothalamic-pituitary-adrenal (HPA) axis by inducing the release of corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and glucocorticoids (Brady and Sonne, 1999). The relationship between ethanol and the HPA-axis appears to be bidirectional, as exogenous administration of CRF, ACTH, and glucocorticoids alter ethanol consumption (Bell et al., 1998; O’Callaghan et al., 2002; Thorsell et al., 2005). Given that neurobiological responses to both stress and ethanol exposure involve HPA-axis signaling, it is possible that the neurochemicals and hormones associated with the HPA-axis modulate stress-induced increases of ethanol consumption. One such candidate is CRF, a 41 amino acid polypeptide that integrates both neuroendocrine and behavioral responses to stress (Smith et al., 1998). CRF-containing neurons are expressed throughout the brain, including in regions implicated in neurobiological responses to ethanol such as the bed nucleus of the stria terminalis, the amygdala, and the lateral hypothalamus (Koob, 2003). Of the two G protein-coupled receptors, the CRF1 receptor (CRF1R) appears to be involved with the integrate emotional behavior while the CRF2 receptor (CRF2R) may modulate ingestive behaviors (Koob, 2003; Zorrilla and Koob, 2004; Zorrilla et al., 2004). Corticotropin-releasing factor receptor signaling has been implicated in a variety of neurobiological responses to ethanol. For example, CRF receptor antagonists attenuate the anxiogenic effect of ethanol withdrawal (Breese et al., 2004; Knapp et al., 2004; Overstreet et al., 2004; Rassnick et al., 1993), prevent excessive ethanol self-administration in dependent animals (Funk et al., 2007; Valdez et al., 2002), and block foot shock-induced reinstatement of ethanol-seeking behavior (Liu and Weiss, 2002). The CRF1R also appears to be involved in stress-induced increases in ethanol consumption. Mutant mice lacking normal production of the CRF1R displayed significantly greater ethanol consumption beginning approximately 2 weeks after a social defeat stress procedure, an effect that was not evident in normal wild-type mice. Subsequent exposure to forced swim stress further augmented ethanol consumption in CRF1R knockout mice (Sillaber et al., 2002). While the Sillaber et al. (2002) study provides genetic evidence suggesting a role for the CRF1R in modulating stress-induced increases in ethanol consumption, the goal of the present experiment was to use a pharmacological approach to determine if pretreatment with the selective CRF1R antagonist, CP-154,526, would buffer the effects of stress and thus attenuate the development of stress-induced increases in ethanol intake in BALB/cJ mice. Therefore, we predicted that (1) ethanol consumption would increase among animals with a history of stress exposure and (2) pretreatment with CP-154,526 would attenuate stress-induced increases in ethanol consumption among animals with a history of stress. BALB/cJ mice were chosen because this strain has been shown to have high sensitivity to the effects of stress on both behavioral and neurobiological measures (Crawley et al., 1997) and drinks low levels of ethanol (Belknap et al., 1993). We also assessed the effects of stress exposure on ethanol consumption by C57BL/6N mice, a strain that voluntarily consumes high amounts of ethanol (Belknap et al., 1993). Here we show that 5 consecutive days of exposure to a 5-minute forced swim stress procedure caused significant and delayed increases in voluntary ethanol consumption in BALB/cJ mice, an effect which was attenuated by pretreatments with the CRF1R antagonist before each stress session. On the other hand, stress exposure did not alter ethanol intake by C57BL/6N mice.

Lesch-Nyhan disease is a neurologically, metabolically, and behaviorally devastating condition that has eluded complete characterization and adequate treatment. While it is known that the disease is intimately associated with dysfunction of the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene that codes for an enzyme of purine metabolism (hypoxanthine-guanine phosphoribosyltransferase) and is associated with neurological, behavioral, as well as metabolic dysfunction, the mechanisms of the neurobehavioral manifestations are as yet unclear. However, discoveries over the past few decades not only have created useful novel animal models (e.g., the HPRT-deficient mouse and the serendipitously discovered perinatal 6-hydroxydopamine (6-OHDA lesion model), but also have expanded into epigenetic, genomic, and proteomic approaches to better understand the mechanisms underlying this disease. The perinatal 6-OHDA model, in addition to modeling self-injury and dopamine depletion in the clinical condition, also underscores the profound importance of development in the differential course of maladaptive progression in the face of a common/single neurotoxic insult at different ages. Recent developments from clinical and basic science efforts attest to the fact that while the disease would seem to have a simple single gene defect at its core, the manifestations of this defect are profound and unexpectedly diverse. Future efforts employing the 6-OHDA model and others in the context of the novel technologies of genome editing, chemo- and opto-genetics, epigenetics, and further studies on the mechanisms of stress-induced maladaptations in brain all hold promise in taking our understanding of this disease to the next level.

Self-injurious behavior is a common problem in many developmental disorders. The neurobiology of this behavior is not well understood, but the differing behavioral manifestations and associations with different disorders suggest that the underlying biological mechanisms are heterogeneous. The behavioral and biological heterogeneity is also evident in several animal models, where different manifestations can be provoked under different experimental conditions. Identifying commonalities among the different mechanisms is likely to be helpful in the design of treatments useful for the broadest populations of patients. The current studies reveal that nifedipine suppresses self-injurious behavior in 4 unrelated animal models: acute administration of high doses of ±BayK 8644 or methamphetamine in mice, dopamine agonist treatment in rats with lesions of dopamine pathways during early development and repeated administration of pemoline in rats. The effect of nifedipine does not appear to be due to nonspecific mechanisms, such as sedation, since other classes of behaviors are unaffected or exaggerated. These results suggest that nifedipine may target a common biological mechanism in the expression of self-injurious behavior, and they suggest it should be considered in the treatment of self-injury in humans.

Mutant mice lacking the RIIbeta subunit of protein kinase A (regulatory subunit II beta(-/-)) show increased ethanol preference. Recent evidence suggests a relationship between heightened ethanol preference and susceptibility to ethanol-induced locomotor sensitization. It is currently unknown if protein kinase A signaling modulates the stimulant effects and/or behavioral sensitization caused by ethanol administration. To address this question, we examined the effects of repeated ethanol administration on locomotor activity RIIbeta(-/-) and littermate wild-type (RIIbeta(+/+)) mice on multiple genetic backgrounds.Over three consecutive days, mice were given single i.p. saline injections and immediately placed in a locomotor activity apparatus to establish a composite baseline for locomotor activity. Next, mice maintained on a hybrid 129/SvEvxC57BL/6J or pure C57BL/6J genetic background were given 10 i.p. ethanol injections before being placed in the activity apparatus. Each ethanol injection was separated by 3-4 days. To determine if changes in behavior were specific to ethanol injection, naïve mice were tested following repeated daily saline injections. The effects of ethanol injection on locomotor behavior were also assessed using an alternate paradigm in which mice were given repeated ethanol injections in their home cage environment.Relative to RIIbeta(+/+) mice, RIIbeta(-/-) mice, regardless of genetic background, consistently showed significantly greater ethanol-induced locomotor activation. RIIbeta(-/-) mice also showed increased sensitivity to ethanol-induced locomotor sensitization resulting from repeated administration, an effect that was dependent on genetic background and testing paradigm. Increased locomotor activity by RIIbeta(-/-) mice was specific to ethanol injections, and was not related to altered blood ethanol levels.These data provide novel evidence implicating an influence of protein kinase A signaling on ethanol-induced locomotor activity and behavioral sensitization. The observation that RIIbeta(-/-) mice are more sensitive to the effects of repeated ethanol administration suggests that normal protein kinase A signaling limits, or is protective against, the stimulant effects of ethanol and the plastic alterations that underlie behavioral sensitization.

This article represents the proceedings of a symposium at the 2004 annual meeting of the International Society for Biomedical Research on Alcoholism in Heidelberg, Germany. The symposium was organized by Zachary A. Rodd and Giancarlo Colombo. The presentations were (1) Pharmacological reversal of cycled withdrawal-sensitized or stress-sensitized withdrawal anxiety and enhanced ethanol drinking, by Darin J. Knapp and George R. Breese, (2) Alcohol craving and relapse in rats genetically selected for high alcohol preference, by Zachary A. Rodd and Richard L. Bell, (3) Exposure to stress increases dopaminergic burst firing in awake rats, by Kristin Anstrom and Donald J. Woodward, (4) Involvement of cannabinoid CB1 and GABAB receptors in the control of relapse-like drinking in alcohol-preferring Sardinian alcohol-preferring rats by Giancarlo Colombo and Salvatore Serra, and (5) Stress-induced ethanol drinking in CB1−/−, POMC, and PENK knockout mice, by Idiko Racz and Andreas Zimmer.

THE MELANOCORTIN (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Central MC peptides are produced by neurons within the hypothalamic arcuate nucleus and the medulla (Dores et al., 1986; Jacobowitz and O’Donohue, 1978; O’Donohue and Dorsa, 1982). These peptides include adrenocorticotropic hormone, α-melanocyte–stimulating hormone (α-MSH), β-MSH, and γ-MSH (Hadley and Haskell-Luevano, 1999). Genetic and pharmacological evidence reveals that MC receptor (MCR) signaling is involved in grooming behavior (Gispen et al., 1975), antipyretic (Murphy et al., 1983) and anti-inflammatory (Macaluso et al., 1994) responses, learning (Zhao et al., 1995), reproductive function (Schioth and Watanobe, 2002), and regulation of appetite and energy homeostasis (Chen et al., 2000; Fan et al., 1997; Huszar et al., 1997; Marsh et al., 1999; Schwartz and Wisse, 2000). There are several observations that suggest that the MC system is a prime candidate for regulating drug-seeking behavior. MCRs are expressed in brain regions thought to mediate drug self-administration, including the nucleus accumbens (NAcc), the hypothalamus, and the ventral tegmental area (VTA) (Alvaro et al., 1996; Griffon et al., 1994; Mountjoy et al., 1994; Roselli-Rehfuss et al., 1993). Interestingly, α-MSH administered into the VTA increases dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the NAcc (Lindblom et al., 2001), and chronic intracerebroventricular (ICV) infusion of the nonselective MCR agonist melanotan II (MTII) to rats increases dopamine D1 receptor binding in the NAcc and dopamine D2 receptor binding in the VTA, suggesting that MTII alters dopamine signaling in these regions (Lindblom et al., 2002a). Chronic treatment of a high dose of morphine decreases MC4 receptor (MC4R) mRNA expression in the NAcc, the periaqueductal gray, and neostriatum (Alvaro et al., 1996), brain regions that modulate drug reward, opiate tolerance, and psychomotor stimulation, respectively (Kalivas and Stewart, 1991; Koob and Bloom, 1988; Wise and Bozarth, 1987). On the other hand, chronic treatment with low doses of morphine or cocaine increases MC4R mRNA in the striatum and hippocampus (Alvaro et al., 2003). Consistent with a role in drug self-administration, central infusion of an MCR agonist decreases the acquisition of heroin self-administration in rats (van Ree et al., 1981). Ethanol has direct effects on central POMC and α-MSH activity (Angelogianni and Gianoulakis, 1993; Rainero et al., 1990). It is therefore possible that MC neuropeptides modulate neurobiological responses to ethanol and participate in the control of voluntary ethanol consumption. Several observations are consistent with this hypothesis. First, α-MSH is expressed in brain regions involved with neurobiological responses to ethanol, including the striatum, NAcc, VTA, amygdala, hippocampus, and hypothalamus (Bloch et al., 1979; Dube et al., 1978; Jacobowitz and O’Donohue, 1978; O’Donohue and Jacobowitz, 1980; O’Donohue et al., 1979; Yamazoe et al., 1984). Second, rats selectively bred for high ethanol drinking (AA [Alko, alcohol]) have low levels of MC3R in the shell of the NAcc but have high levels of MC3R and MC4R in various regions of the hypothalamus, when compared with low–ethanol-drinking rats (Lindblom et al., 2002b). Third, central infusion of MTII significantly reduces voluntary ethanol drinking in AA rats with an established history of ethanol intake (Ploj et al., 2002). Recently, MTII-induced reduction of ethanol consumption was shown to be receptor mediated and not associated with alterations of ethanol metabolism in C57BL/6 mice (Navarro et al., 2003). One objective of the present report was to study the role of selected MCRs in the modulation of MCR agonist–induced reduction of ethanol intake. In rodents, MC peptides act through five receptors (MC1R–MC5R) (Hadley and Haskell-Luevano, 1999). MC3R and MC4R are expressed at high levels in the brain (Alvaro et al., 1997), whereas MC1R and MC5R are detected at low levels and in only limited brain regions (Adan and Gispen, 1997; Barrett et al., 1994; Xia et al., 1995). MTII binds, with varying affinity, to all centrally expressed MCRs (Haskell-Luevano et al., 1997; Schioth et al., 1997). Thus, it is unclear which MCRs are important for modulating MTII-induced reductions of ethanol consumption (Navarro et al., 2003; Ploj et al., 2002). To assess the contribution of MC3R, we examined MTII-induced alteration of ethanol drinking in Mc3r–knock-out (Mc3r−/−) and wild-type (Mc3r+/+) mice. To assess the role of MC4R, we studied ethanol intake by C57BL/6J mice after central infusion of the highly selective MC4R agonist cyclo(NH-CH2-CH2-CO-His-d-Phe-Arg-Trp-Glu)-NH2. A second objective was to determine whether central administration of AgRP-(83-132) would increase ethanol consumption by mice. In vivo, AgRP-(83-132) is a potent and nonselective MCR antagonist (Quillan et al., 1998). Such results would strengthen the argument that MCR signaling plays an important role in the modulation of ethanol self-administration. Because MCR agonists reduce and MCR antagonists increase feeding (Hagan et al., 2000; Hohmann et al., 2000; Hollopeter et al., 1998; Marsh et al., 1999; Thiele et al., 1998), food intake was concurrently assessed in most studies described in the present report.

Early behavioral investigations supported the contention that systemic ethanol displays a GABAmimetic profile. Microinjection of GABA agonists into brain and in vivo electrophysiological studies implicated a regionally specific action of ethanol on GABA function. While selectivity of ethanol to enhance the effect of GABA was initially attributed an effect on type-I-benzodiazepine (BZD)-GABA(A) receptors, a lack of ethanol's effect on GABA responsiveness from isolated neurons with this receptor subtype discounted this contention. Nonetheless, subsequent work identified GABA(A) receptor subtypes, with limited distribution in brain, sensitive to enhancement of GABA at relevant ethanol concentrations. In view of these data, it is hypothesized that the GABAmimetic profile for ethanol is due to activation of mechanisms associated with GABA function, distinct from a direct action on the majority of postsynaptic GABA(A) receptors. The primary action proposed to account for ethanol's regional specificity on GABA transmission is its ability to release GABA from some, but not all, presynaptic GABAergic terminals. As systemic administration of ethanol increases neuroactive steroids, which can enhance GABA responsiveness, this elevated level of neurosteroids is proposed to magnify the effect of GABA released by ethanol. Additional factors contributing to the degree to which ethanol interacts with GABA function include an involvement of GABA(B) and other receptors that influence ethanol-induced GABA release, an effect of phosphorylation on GABA responsiveness, and a regional reduction of glutamatergic tone. Thus, an integration of these consequences induced by ethanol is proposed to provide a logical basis for its in vivo GABAmimetic profile.

This report of the proceedings of a symposium presented at the 2004 Research Society on Alcoholism Meeting provides evidence linking stress during sobriety to craving that increases the risk for relapse. The initial presentation by Rajita Sinha summarized clinical evidence for the hypothesis that there is an increased sensitivity to stress-induced craving in alcoholics. During early abstinence, alcoholics who were confronted with stressful circumstances showed increased susceptibility for relapse. George Breese presented data demonstrating that stress could substitute for repeated withdrawals from chronic ethanol to induce anxiety-like behavior. This persistent adaptive change induced by multiple withdrawals allowed stress to induce an anxiety-like response that was absent in animals that were not previously exposed to chronic ethanol. Subsequently, Amanda Roberts reviewed evidence that increased drinking induced by stress was dependent on corticotropin-releasing factor (CRF). In addition, rats that were stressed during protracted abstinence exhibited anxiety-like behavior that was also dependent on CRF. Christopher Dayas indicated that stress increases the reinstatement of an alcohol-related cue. Moreover, this effect was enhanced by previous alcohol dependence. These interactive effects between stress and alcohol-related environmental stimuli depended on concurrent activation of endogenous opioid and CRF systems. A.D. Lê covered information that indicated that stress facilitated reinstatement to alcohol responding and summarized the influence of multiple deprivations on this interaction. David Overstreet provided evidence that restraint stress during repeated alcohol deprivations increases voluntary drinking in alcohol-preferring (P) rats that results in withdrawal-induced anxiety that is not observed in the absence of stress. Testing of drugs on the stress-induced voluntary drinking implicated serotonin and CRF involvement in the sensitized response. Collectively, the presentations provided convincing support for an involvement of stress in the cause of relapse and continuing alcohol abuse and suggested novel pharmacological approaches for treating relapse induced by stress.

Significant progress has been made in the development of several nonpeptide antagonists of the type 1 receptor for corticotropin-releasing factor (CRF). The present review outlines the properties of this drug class that point to their potential as agents for depression and/or anxiety. Behavioral tests of CRF1 receptor antagonists relevant to depression have been mixed. For example, most CRF1 receptor antagonists did not lower swim test immobility in a subacute paradigm, where three injections of the drug are given in the 24 hrs between a 15-min pre-swim and a 5-min test swim. However, reliable effects in other tests of antidepressant action were observed in the chronic mild stress protocol or the Flinders Sensitive Line rat that exhibits innately high swim test immobility when the drugs were given chronically. Because these latter tests have a very good record of predicting antidepressant activity, CRF1 receptor antagonists are good candidates for clinical trials for depression. Evidence indicates that the CRF1 receptor antagonists do not exhibit changes in anxiety-like behavior under basal conditions. However, this drug class effectively blocks the anxiogenic states produced by withdrawal from chronic alcohol, by exposure to stress, or in rats selectively bred for high anxiety. CRF1 receptor antagonists also prevent the anxiety associated with multiple ethanol withdrawals when given only during the earlier withdrawals. Thus, these agents may also have clinically relevant anxiolytic effects, not only in individuals with anxiety disorders but also in individuals who have anxiety episodes during alcohol or drug withdrawal. Drug Dev. Res. 65:191–204, 2005. © 2005 Wiley-Liss, Inc.

Previous work demonstrated that rats subjected to multiple withdrawals from chronic ethanol exhibit a sensitization of anxiety-like behavior compared to animals withdrawn from treatment with an equal but continuous amount of ethanol. This study sought to examine whether corticotropin-releasing factor (CRF) could modulate this ethanol-withdrawal-induced anxiety-like behavior. Initially, rats were administered with CRF (1 microg) or vehicle intraventricularly on two occasions 5 days apart while on control diet (CD) followed by exposure to 7% ethanol diet (ED) for 5 days, with social interaction assessed 5 h into withdrawal. Social interaction was significantly reduced in the CRF-treated animals compared to vehicle-treated rats and vehicle- and CRF-treated rats maintained on CD, indicative that CRF given before ethanol exposure was capable of inducing an adaptive change that sensitized withdrawal-induced anxiety-like behavior. Next, the CRF(1) receptor antagonist CRA1000 (3 mg/kg, systemically), the CRF(2) receptor antagonist antisauvagine-30 (20 microg intraventricularly), or vehicle was injected 4 h after the ethanol was removed following the first and second cycles of chronic ethanol exposure and the effect on the multiple-withdrawal-induced anxiety-like behavior determined after the third withdrawal cycle. The CRF(1) receptor antagonist blocked the reduced social interaction behavior, whereas the CRF(2) receptor antagonist was without effect. Similar pretreatment with another CRF(1) receptor antagonist CP-154,526 (10 mg/kg systemically) during the first and second withdrawals also counteracted anxiety-like behavior. These findings indicate that the CRF system and CRF(1) receptors play key roles in the adaptive change responsible for the anxiety-like behavior induced by repeated withdrawals from chronic ethanol.

Repeated withdrawals from chronic ethanol sensitize the withdrawal-induced reduction in social interaction behaviors. This study determined whether stress might substitute for repeated withdrawals to facilitate withdrawal-induced anxiety-like behavior. When two 1-h periods of restraint stress were applied at 1-week intervals to rats fed control diet, social interaction was reduced upon withdrawal from a subsequent 5-day exposure to ethanol diet. Neither this ethanol exposure alone nor exposure to three restraint stresses alone altered this measure of anxiety. Further, the repeatedly stressed singly withdrawn rats continued to exhibit a reduction in social interaction 16 days later, upon withdrawal from re-exposure to 5 days of chronic ethanol, consistent with a persistent adaptation by the multiple-stress/withdrawal protocol. Weekly administration of corticosterone in place of stress induced no significant change in social interaction upon withdrawal from the single chronic ethanol exposure, indicative that corticoid release is not responsible for the stress-induced reduction in anxiety-like behavior during withdrawal. In the multiple-withdrawal protocol, stress applied during withdrawal from voluntary ethanol drinking by P-rats facilitated ethanol drinking sufficiently, to induce a withdrawal-induced reduction in social interaction. Administration of a CRF-1 receptor antagonist, a benzodiazepine receptor antagonist, or a 5-HT(1A) receptor agonist prior to each stress minimized sensitization of the withdrawal-induced reduction in anxiety-like behavior. Since these pharmacological consequences on the induction of anxiety-like behavior following the stress/withdrawal protocol are like those previously seen when these drug treatments were given prior to multiple withdrawals, evidence is provided that repeated stresses and multiple withdrawals sensitize the withdrawal reduction in social interaction by similar central adaptive mechanisms.

In vivo, ethanol alters the effect of N-methyl-D-aspartate (NMDA) and GABA in some brain regions but is without effect in others. To determine whether these regional differences were due to differences in the effect of ethanol on postsynaptic NMDA or GABAA receptors, we examined the effect of ethanol on NMDA- and GABA-gated currents from neurons acutely dissociated from the lateral septal nucleus, substantia nigra, thalamus, hippocampus, and cerebellum. Ethanol decreased the effect of NMDA similarly in all brain areas tested and had similar effects on Chinese hamster ovary cells expressing NR2A or NR2B subunits with an NR1-1a subunit. However, ifenprodil reduced the inhibition by ethanol of NMDA-gated currents from neurons isolated from the lateral septum without affecting neurons from the substantia nigra. In contrast to the robust effect of ethanol on NMDA-gated currents, ethanol (25-300 mM) was without effect on GABA-gated currents at all brain sites tested or on Ltk- cells stably expressing the alpha1, beta2, and gamma2L or gamma2S subunits. The neuroactive steroid alphaxalone profoundly enhanced GABA-gated currents in all brain areas and cell types tested, indicating a similar sensitivity to allosteric modulation; however, there was no interaction of alphaxalone with ethanol at any site tested. These data suggest that the regional differences in the effect of ethanol observed in vivo are not due to a differential action of ethanol at the postsynaptic NMDA or GABAA receptor subtypes.

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.

Lesch-Nyhan disease, a neurogenetic disorder caused by congenital deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase, is associated with a prominent loss of striatal dopamine. The current studies address the hypothesis that oxidant stress causes damage or dysfunction of nigrostriatal dopamine neurons in a knockout mouse model of the disease, by assessing several markers of oxidative damage and free radical scavenging systems. Some of these measures provided evidence for an increase in oxidative stress in the mutant mice (aconitase activity, oxidized glutathione, and lipid peroxides), but others did not (superoxide dismutase, protein thiol content, carbonyl protein content, total glutathione, glutathione peroxidase, catalase, and thiobarbituric reducing substances). Immunolocalization of heme-oxygenase 1 provided no evidence for oxidative stress restricted to specific elements of the striatum or midbrain in the mutants. Striatal dopamine systems of the mutant mice were more vulnerable to a challenge with the neurotoxin 6-hydroxydopamine, but they were not protected by cross-breeding the mutants with transgenic mice over-expressing superoxide dismutase. Overall, these data provide evidence for increased oxidative stress, but the failure to protect the knockout mice by over-expressing SOD1 argues that oxidative stress is not the sole process responsible for the loss of striatal dopamine.

Background: Inhibition of NMDA receptor function in brain is believed to be an important action of ethanol (EtOH). To investigate EtOH inhibition of NMDA receptor responses in vivo, the interaction of these agents in brain after different routes of administration were investigated by using transcription factor Fos protein expression to follow NMDA receptor activation and EtOH inhibition of this response. Methods: The induction of Fos-like immunoreactivity (Fos-LI) in 38 regions of the rat brain was measured 2 hr after treatment with NMDA, EtOH, or both. To determine the relative contribution of abdominal drug effects on Fos induction, rats received either intraperitoneal (ip) or intragastric (ig) EtOH and ip or intravenous (iv) NMDA. Rats received EtOH (2.5 g/kg ip or 4 g/kg ig) or vehicle 15 min before NMDA (125 mg/kg ip or 60 mg/kg iv) or vehicle. Results: For the 38 forebrain regions examined, ip and iv NMDA significantly induced Fos-LI in 13 and 32 regions, respectively. These effects occurred without elicitation of tonic-clonic seizure activity and were strong after iv NMDA in the frontal, prefrontal, and cingulate cortices, supraoptic nucleus, anterior lateral septum, and dentate gyrus. For EtOH, prominent Fos-LI induction was found in the central amygdala, dorsolateral bed nucleus of the stria terminalis, Edinger-Westphal nucleus, and paraventricular hypothalamus. Despite ip and ig EtOH induction of Fos-LI in these regions, the major effect of EtOH was to block NMDA-induced Fos-LI in 8 of 13 (ip) and 27 of 32 (ig) of the NMDA-positive regions, respectively, including retrosplenial, cingulate, and medial prefrontal cortices, central amygdala, and taenia tecta. Conclusions: These results provide new evidence for the regionally specific functional interactions of EtOH on NMDA receptors in vivo. Moreover, these results support efforts to identify brain region–specific targets for EtOH and EtOH-induced changes in gene expression.

Previous work has suggested that the therapeutic efficacy of olanzapine might be partially dependent on action at the D(1)-dopamine (DA) receptor site. Because early DA loss can lead to supersensitive D(1)-DA receptors, effects of olanzapine were investigated in adult rats given lesions to DA-containing neurons as neonates. In these animals, locomotor effects of SKF-38393 (a D(1)-DA agonist) were attenuated by olanzapine, but at doses (5 and 10 mg/kg) that decreased activity when given alone. Olanzapine prevented induction of striatal Fos protein by SKF-38393 and partially attenuated the long-term "priming" effect of repeated SKF-38393 treatment. Olanzapine also antagonized the stimulant effects of quinpirole (a D(2)-type DA agonist) in animals lesioned as young adults, at doses lower than those necessary to antagonize SKF-38393-induced activity. In addition, olanzapine antagonized apomorphine-induced self-injurious behavior in neonate-lesioned rats in a dose-related fashion. Attenuation of self-injury in this animal model suggests that olanzapine should be tested against this symptom in patient populations.

This paper summarizes a conference held at the National Institute of Child Health and Human Development on December 6-7, 1999, on self-injurious behavior [SIB] in developmental disabilities. Twenty-six of the top researchers in the U.S. from this field representing 13 different disciplines discussed environmental mechanisms, epidemiology, behavioral and pharmacological intervention strategies, neurochemical substrates, genetic syndromes in which SIB is a prominent behavioral phenotype, neurobiological and neurodevelopmental factors affecting SIB in humans as well as a variety of animal models of SIB. Findings over the last decade, especially new discoveries since 1995, were emphasized. SIB is a rapidly growing area of scientific interest to both basic and applied researchers. In many respects it is a model for the study of gene-brain-behavior relationships in developmental disabilities.

Rat lines that were selectively bred for high (high DPAT-sensitive, HDS) or low (low DPAT-sensitive, LDS) hypothermic responses to the specific 5-HT(1A) receptor agonist, 8-hydroxy-di-n-propylaminotetralin (8-OH-DPAT), differ in receptor binding and certain behaviors related to anxiety and depression. After reviewing this literature, the present communication summarizes new experiments designed to clarify and extend the nature of the pharmacological and biochemical differences between the lines. A challenge with the 5-HT(2) receptor agonist, DOI, produced similar degrees of head shakes and skin crawls in the HDS and LDS rats, suggesting similar sensitivity of 5-HT(2A) and 5-HT(2C) receptors. In contrast, DOI-induced flat body posture (FBP), which has been linked to 5-HT(1A) receptor stimulation, was observed more readily in the HDS rats. The HDS and LDS rats exhibited similar degrees of increase in 8-OH-DPAT-stimulated [35S]GTPgammaS binding in several brain regions. This result suggests that the dramatic differences in hypothermia in HDS and LDS rats cannot be related to 5-HT(1A) receptor-mediated action on G proteins. Overall, these findings indicate that the selective breeding for 5-HT(1A)-mediated hypothermia has been fairly selective, and that differences in emotionally relevant behaviors between these two rat lines can strongly be associated with an unidentified component of the 5-HT(1A) receptor signaling pathway.

BACKGROUND Electrophysiological recording reveals that only a portion of cerebellar Purkinje neurons are sensitive to ethanol enhancement of gamma-aminobutyric acid (GABA) responses. Although activation of beta-adrenergic receptors permits ethanol enhancement of GABA function from some cerebellar Purkinje neurons, other neurons remain insensitive to ethanol. These findings are consistent with the finding that other external neural inputs are required to allow ethanol enhancement of GABA responses from Purkinje neurons. Because of a high expression of GABA(B) receptors on Purkinje cells, we tested whether activation of GABA(B) receptors might modulate the action of ethanol on GABA responsiveness. METHODS Extracellular single-unit electrophysiological recording was used to investigate the effects of ethanol on responses to GABA and muscimol (a GABA(A) agonist) from cerebellar Purkinje neurons. Drugs tested were baclophen (a GABA(B) agonist) and CGP35348 (a GABA(B) antagonist). RESULTS Ethanol did not enhance responses to GABA and muscimol from all Purkinje neurons. Systemic administration of the GABA(B) agonist, baclophen (3 mg/kg intravenously), permitted ethanol to enhance GABA inhibition from approximately 75% of cerebellar Purkinje neurons not initially enhanced by ethanol. Local iontophoretic application of baclophen to Purkinje neurons also allowed ethanol to enhance GABA and muscimol responsiveness from a portion of neurons in which ethanol initially did not affect their actions. An inhibitory action of ethanol on responses to GABA and muscimol, which was also influenced by baclophen, was observed from some Purkinje neurons. From Purkinje neurons initially sensitive to ethanol enhancement of GABA and muscimol function, administration of CGP35348, a GABA(B) antagonist, diminished the effect of ethanol on the responsiveness of these agonists from the majority (9/15) of neurons. CONCLUSIONS The present findings demonstrated that baclophen allows ethanol enhancement of GABA and muscimol responsiveness from some, but not all, cerebellar Purkinje neurons initially not sensitive to ethanol. Likewise, a GABA(B) antagonist can diminish ethanol enhancement of GABA and muscimol responses from some ethanol-sensitive neurons. Thus, these results emphasize that GABA(B) receptors on a portion of Purkinje neurons act as an auxiliary neural input that allows ethanol enhancement of GABA responses. Consequently, receptor structure alone does not account for the action of ethanol on GABA(A) receptor function on this cell type.

The effect of ethanol on responses to nicotine from rat cerebellar Purkinje neurons was investigated using extracellular single-unit recording. Systemic administration of ethanol initially enhanced the nicotine-induced inhibition from 50% of the Purkinje neurons. However, irrespective of whether there was an initial enhancement, systemic administration of ethanol antagonized the response to nicotine from the majority of Purkinje neurons. When varying ethanol concentrations were electro-osmotically applied to this neuronal cell type, the responses to nicotine (6/8) were enhanced when a low concentration of ethanol (40 mM) was in the pipette, whereas the majority of nicotine responses (10/11) were antagonized when a higher concentration of ethanol (160 mM) was applied to Purkinje neurons. Thus, the concentration of ethanol presented to the neuron seemed to explain the biphasic consequence of systemically administered ethanol on responses to nicotine. In order to determine whether ethanol affected a specific nACh receptor subtype containing the alpha-7 subunit, it was initially established that the nicotinic antagonists, alpha-bungarotoxin (alpha-BTX) and methyllycaconitine (MLA), which are associated with this subunit, had identical actions on responses to nicotine from Purkinje neurons. When MLA was tested against responses to nicotine from this cell type, MLA antagonized the response to nicotine from 45% (9/20) of the neurons tested. In a direct comparison of the action of ethanol to inhibit responses to nicotine with the action of MLA on the same Purkinje neuron, ethanol inhibited responses to nicotine on all neurons sensitive to MLA. However, ethanol also affected nicotine-induced neural changes from some Purkinje neurons not sensitive to MLA antagonism of nicotine. These data support the supposition that ethanol affects a nACh receptor subtype which has an alpha-7 subunit as well as other nACh receptor subtypes without this specific subunit.

Previous research has suggested that rats tested at 28 to 30 days of age show a marked subsensitivity to the sedative effects of ethanol. In the present study, rats of different ages were tested for aerial righting following acute ethanol (3 g/kg) treatment These results were compared with the effects of the atypical benzodiazepine zolpidem (3 and 5 mg/kg) and pentobarbital (10 and 15 mg/kg). Animals tested at 25, 28, or 35 days of age were significantly less impaired by ethanol than preweanling rats (age 20 days) or older rats (age 65 to 75 days), whereas animals tested at 25 or 28 days of age were less impaired by the higher dose of zolpidem. With pentobarbital, the most distinct age-related trend was greater impairment in 20-day-old rats. Because ethanol may be active at the same type I GABA A receptor site selectively labeled by [ 3 H]zolpidem, levels of pH]zolpidem binding were determined for rats of different ages. Although some brain regions showed progressive increases in binding of [ 3 H]zolpidem across development, other regions demonstrated increased binding from day 12 or 17 to day 20, then a plateau of binding levels across days 20, 25, and 28, with further increases occurring by day 36 or day 60. This pattem was observed in the cingulate cortex, medial septal nucleus, globus pallidus, inferior colliculus, red nucleus, and cerebellum. Overall, the results indicate that the period of subsensitivity to the sedative effects of ethanol is coincident with a change in the developmental pattern of GABA A receptor sites targeted by [ 3 H]zolpidem.

Subanesthetic doses of ketamine have been shown to exacerbate symptoms in schizophrenia and to induce positive, negative, and cognitive schizophrenic-like symptoms in normal subjects. The present investigation sought to define brain regions affected by subanesthetic doses of ketamine, using high resolution autoradiographic analysis of 14C-2-deoxyglucose (2-DG) uptake and immunocytochemical staining for Fos-like immunoreactivity (Fos-LI). Both functional mapping approaches were used because distinct and complementary information is often obtained with these two mapping methods. Ketamine, at a subanesthetic dose of 35 mg/kg, substantially increased 2-DG uptake in certain limbic cortical regions, including medial prefrontal, ventrolateral orbital, cingulate, and retrosplenial cortices. In the hippocampal formation, the subanesthetic dose of ketamine induced prominent increases in 2-DG uptake in the dentate gyrus, CA-3 stratum radiatum, stratum lacunosum moleculare, and presubiculum. Increased 2-DG uptake in response to 35 mg/kg ketamine was also observed in select thalamic nuclei and basolateral amygdala. Ketamine induced Fos-LI in the same limbic cortical regions that exhibited increased 2-DG uptake in response to the subanesthetic dose of the drug. However, no Fos was induced in some brain regions that showed increased 2-DG uptake, such as the hippocampal formation, anterioventral thalamic nucleus, and basolateral amygdala. Conversely, ketamine induced Fos in the paraventricular nucleus of the hypothalamus and central amygdala, although no effect of the drug on 2-DG uptake was apparent in these regions. In contrast to the increase in 2-DG uptake observed in select brain regions after the subanesthetic dose, an anesthetic dose of ketamine (100 mg/kg) produced a global suppression of 2-DG uptake. By contrast, a robust induction of Fos-LI was observed after the anesthetic dose of ketamine that was neuroanatomically identical to that produced by the subanesthetic dose. Results of the present investigation show that anesthetic and subanesthetic doses of ketamine have pronounced effects on regional brain 2-DG uptake and induction of Fos-LI. The alterations in regional brain metabolism induced by the subanesthetic dose may be relevant to effects of ketamine to induce schizophrenic-like symptoms.

The relationship between zolpidem sensitivity and GABA(A) receptor alpha subunits was studied in individual dissociated neurons from rat brain. Using whole-cell recording, similar EC50 values were demonstrated for the effect of gamma-aminobutyric acid (GABA) on gated-chloride currents from substantia nigra reticulata (SNR) and lateral septal neurons. Subsequently, many neurons from both the SNR or lateral septum were found to exhibit enhanced GABA-gated chloride currents across concentrations of zolpidem ranging from 10 to 300 nM. Some neurons exhibited a greater than 20% increase in responsiveness to GABA at 30 nM of zolpidem without further increase at higher concentrations of zolpidem. Conversely, zolpidem enhancement of GABA from another group of neurons was not observed at 30 nM zolpidem, but between 100 and 300 nM the response to GABA increased greater than 20%. Finally, a third group of neurons reached both of these criteria for zolpidem enhancement of GABA. This latter spectrum of responses to GABA after varying concentrations of zolpidem was consistent with the presence of either two GABA(A) receptors or a single receptor with differing affinities for zolpidem on an individual neuron. Following determination of the sensitivity of neurons from SNR or lateral septum to zolpidem, cytoplasm was extracted from some individual cells to allow identification of cellular mRNAs for the alpha1, alpha2 and alpha3 GABA(A) receptor subunits with RT-PCR. Those neurons that responded to the 30 nM zolpidem concentration invariably expressed the alpha1-GABA(A) receptor subunit. This result is consistent with the GABA(A) alpha1-receptor subunit being an integral part of a functional high-affinity zolpidem type 1-BZD receptor complex on neurons in brain. Those neurons which showed enhancement of GABA from 100 to 300 nM zolpidem contained mRNAs for the alpha2 and/or the alpha3 receptor subunits, a finding consistent with these alpha subunits forming type 2-BZD receptors. Some individual dissociated SNR neurons were sensitive to both low and high concentrations of zolpidem and contained mRNAs for all three alpha-receptor subunits. These latter individual neurons are proposed to have at least two functional GABA(A) receptor subtypes. Thus, the present investigation emphasizes the importance of characterizing the relationship between endogenous GABA(A) receptor function and the presence of specific structural components forming GABA(A) receptor subtypes on neurons.

To further understand the relationship between emotional state and alcohol intake in rats, the tendency to emit ultrasonic vocalizations in response to an aversive, but nonpainful, air puff stimulus was tested in several rat lines. Included in this group were Maudsley Reactive (MR) and Non-Reactive (MNR) rats, and several lines of rats with either high ethanol preference or a low ethanol preference: Preferring, (P), Alko-Alcohol (AA), and Fawn-Hooded (FH) animals; and Non-Preferring (NP), Alko-Non-Alcohol (ANA), and Flinders Resistant Line (FRL). MR rats emitted fewer ultrasonic vocalizations (USVs) and showed less preference for ethanol than did MNR animals. An overall analysis that included the P, NP, FH, FRL, AA, and ANA groups demonstrated a significant negative correlation between the total number of USVs emitted and ethanol consumption. NP, FRL, and especially ANA rats (low ethanol-preferring) emitted the most USVs--to an extent similar to that typically found for normal rats. The duration of vocalizing was higher only in the NP and the FRL rats the relative to their P and FH comparison groups, respectively. In the ethanol-preferring and nonpreferring lines, the numbers of USVs emitted correlated positively with the duration of vocalizing, but not with the latency to vocalize, which in turn did not correlate strongly with ethanol intake. The latency to vocalize did not correlate significantly with ethanol intake across all drinking lines or MR or MNR rats, but was found to be higher in FH and AA rats relative to their nondrinking comparison groups. These associations suggest that the relationship between emotional state and ethanol drinking is complex and cannot be attributed to a simple elevated state of anxiety or emotionality. Further examination of the central nervous system mechanisms mediating the difference in USVs between paired lines of ethanol-preferring and nonpreferring rats may identify neurochemical factors that predict ethanol preference.

Both Lesch-Nyhan syndrome and Parkinson's disease are associated with decreased brain dopamine, yet each disorder is characterized by a different set of motor symptoms. Lesch-Nyhan syndrome is manifested in early childhood, while parkinsonism usually does not appear until adulthood, suggesting that age at the time of dopamine loss is one determinant of the effects of neurotransmitter deficiency. Support for this view is found in studies of animals given dopamine-depleting lesions at different ages and then tested in adulthood. Animals lesioned as neonates show a supersensitivity to dopamine agonists, especially D1-dopamine receptor agonists, and to MK-801, an NMDA receptor antagonist. In addition, neonatally treated animals show a 'priming' effect following repeated exposure to D1-dopamine agonists. Animals depleted of dopamine as adults are more supersensitive to agonists acting on the D2-dopamine receptor, and do not evidence priming to dopamine agonists or an enhanced response to MK-801. These differential pharmacological profiles suggest that the changes in neurotransmitter systems following dopamine depletion are, at least in part, determined by age at the time of the lesion.

Immunohistochemical staining for Fos-like immunoreactivity (Fos-LI) was used to map functional activation in discrete brain regions of rats processed in three empirical models of anxiety: foot shock avoidance responding in a shuttle box, the elevated plus maze, and an air puff-induced ultrasonic vocalization test. The avoidance test and elevated plus maze induced prominent Fos-LI in select brain regions, including the medial prefrontal, cingulate, and ventrolateral orbital cortices, taenia tecta, nucleus accumbens, paraventricular nucleus of the hypothalamus, medial nucleus of the amygdala and lateral septum. Air puff stimuli that produced ultrasonic vocalizations induced Fos-LI to a more limited extent compared to the plus maze and avoidance test, with only the medial prefrontal cortex, medial nucleus of the amygdala, and lateral septum being significantly affected by air-puff. Even though the sensory stimuli and environmental conditions associated with the three anxiety models were markedly different, specific common forebrain regions were affected, i.e. the medial prefrontal cortex, medial amygdala, and lateral septum. It is hypothesized that these regions are components of a circuit in the rat brain related to anxiety or distress. To determine the potential relationship between generalized arousal and the observed induction of Fos-LI in the anxiety models, rats were tested in a non-aversive situation involving marked behavioral activation. Accordingly, after vigorous bar pressing behavior for reinforcement with sweetened condensed milk, induction of Fos-LI was minimal and comparable to that in unhandled control rats. These latter data indicate that the distinctive neuroanatomical patterns of Fos-LI observed in the paradigms related to anxiety were not simply due to generalized behavioral activation. In summary, select common brain regions were identified that express Fos-LI in empirical models of anxiety. These data provide a functional framework to explore neuroanatomical sites of action of psychotherapeutic drugs that influence behavioral responses in these tasks.

Safe, long-term gene expression is a primary criteria for effective gene therapy in the brain, so studies were initiated to evaluate adeno-associated virus (AAV) vector transfer of a reporter gene into specific sites of the rat brain. In the 4 day old rat, site infusions of AAV-CMV-lacZ (1 microliter; 5 x 10(4) particles) produced neuronal beta-galactosidase gene expression 3 weeks later in the hippocampus and inferior colliculus, but not in the cerebral cortex. Seven days after infusion of AAV-CMV-lacZ viral vectors (1 microliter) in the adult rat, beta-galactosidase gene expression was found in the olfactory tubercle, caudate, hippocampus, piriform cortex and inferior colliculus. primarily in multipolar neurons close to the infusion site. Three months after vector microinfusion, similar levels of gene expression remained in the olfactory tubercle and the inferior colliculus, with some reduction found in the caudate, but substantial reductions in beta-galactosidase gene expression occurred in the hippocampus and piriform cortex. In no case were obvious signs of toxicity noted. Therefore, AAV vectors can transfer foreign genes into the adult and neonatal CNS, but the pattern and longevity of gene expression depends upon the area of brain being studied.