Rare human nicotinic acetylcholine receptor α4 subunit (CHRNA4) variants affect expression and function of high-affinity nicotinic acetylcholine receptors.

Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). α4ß2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the α4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (α4R336C, α4P451L, and α4R487Q) to the common variant to determine their effects on α4ß2 nAChR pharmacology. We examined [(3)H]epibatidine binding, interacting proteins, and phosphorylation of the α4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the α4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified α4ß2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these α4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human α4ß2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common α4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.

Reduced locomotor responses to cocaine in ghrelin-deficient mice.

Ghrelin, an orexigenic hormone produced by the stomach, increases food intake and enhances the locomotor and rewarding effects of cocaine. Consistent with these behavioral effects, ghrelin increases dopamine cell activity in the mesolimbic system resulting in elevated levels of dopamine release and turnover in target regions such as the ventral striatum. In the current study, we examined the psychostimulant effects of acute and daily cocaine in mice with targeted deletion of the ghrelin gene (ghrelin knockout; KO) and that of their wild-type (WT) littermates. We hypothesized that ghrelin-KO mice would be hyporesponsive to the effects of cocaine as reflected in attenuated locomotor activity following both acute and chronic injections, and that this would be correlated with striatal dopamine and dopamine metabolite concentrations. Results show that the locomotor stimulating effect of cocaine (10 mg/kg) was decreased in ghrelin-KO mice as compared with their WT littermates. In addition, repeated daily injection of cocaine resulted in gradual increases in locomotor activity in WT mice, an effect that was attenuated in ghrelin-KO mice. These behavioral effects were correlated with changes in dopamine utilization in the striatum of WT mice that were not seen in ghrelin-KO mice unless these were pretreated with ghrelin. These data suggest that ghrelin is important for normal function of the mesolimbic dopaminergic system, potentially modulating both dopamine release and reuptake.

Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation.

Galanin and addiction.

There has been increasing interest in the ability of neuropeptides involved in feeding to modulate circuits important for responses to drugs of abuse. A number of peptides with effects on hypothalamic function also modulate the mesolimbic dopamine system (ventral tegmental area and nucleus accumbens). Similarly, common stress-related pathways can modulate food intake, drug reward and symptoms of drug withdrawal. Galanin promotes food intake and the analgesic properties of opiates; thus it initially seemed possible that galanin might potentiate opiate reinforcement. Instead, galanin agonists decrease opiate reward, measured by conditioned place preference, and opiate withdrawal signs, whereas opiate reward and withdrawal are increased in knock-out mice lacking galanin. This is consistent with studies showing that galanin decreases activity-evoked dopamine release in striatal slices and decreases the firing rate of noradrenergic neurons in locus coeruleus, areas involved in drug reward and withdrawal, respectively. These data suggest that polymorphisms in genes encoding galanin or galanin receptors might be associated with susceptibility to opiate abuse. Further, galanin receptors might be potential targets for development of novel treatments for addiction.

Prenatal cocaine exposure enhances responsivity of locus coeruleus norepinephrine neurons: role of autoreceptors.

Children exposed to cocaine during gestation have a higher incidence of neurobehavioral deficits. The neurochemical bases of these deficits have not been determined, but the pharmacology of cocaine and the nature of the abnormalities suggest that disruptions in catecholaminergic systems may be involved. In the current study, we used a rat model of prenatal cocaine exposure to examine the impact that this exposure has on the locus coeruleus (LC) noradrenergic system in offspring. Pregnant rats received twice-daily i.v. injections of cocaine (3 mg/kg) or saline between gestational days 10 and 20, and progeny were tested as juveniles. Exposure to a mild stressor elevated an index of norepinephrine turnover in the prefrontal cortex and also increased Fos expression in tyrosine hydroxylase-positive LC neurons in rats exposed to prenatal cocaine but not in rats exposed to prenatal saline. No change in the number of tyrosine hydroxylase-positive neurons in the LC was observed between the two prenatal treatment groups. Specific binding of [125I]-para-iodoclonidine, a radioligand with specificity for high affinity alpha2A-adrenergic receptors, was decreased in the LC of rats exposed to prenatal cocaine compared with prenatal saline controls. As alpha2-adrenergic receptors on LC norepinephrine neurons function as autoreceptors, their down-regulation by prenatal cocaine exposure provides a plausible mechanism for the observed heightened reactivity of norepinephrine neurons in these animals. These data indicate that prenatal cocaine exposure results in lasting changes to the regulation and responsivity of rat LC norepinephrine neurons. A similar dysregulation of LC norepinephrine neurons may occur in children exposed to cocaine during gestation, and this may explain, at least partly, the increased incidence of cognitive deficits that have been observed in these subjects.

The nicotinic antagonist mecamylamine has antidepressant-like effects in wild-type but not beta2- or alpha7-nicotinic acetylcholine receptor subunit knockout mice.

RATIONALE: Increases in cholinergic transmission are linked to depression in human subjects and animal models. We therefore examined the effect of decreasing nicotinic acetylcholine receptor (nAChR) activity in tests of antidepressant efficacy using C57BL/6J mice. OBJECTIVES: We determined whether the noncompetitive nAChR antagonist mecamylamine had antidepressant-like effects in the forced swim test (FST) and tail suspension test (TST). These experiments were repeated in mice lacking either the beta2- or alpha7-nAChR subunits to identify the nAChR subunits involved in mediating the antidepressant response to mecamylamine. MATERIALS AND METHODS: Adult mice on the C57BL/6J background were acutely administered mecamylamine i.p. 30 min before testing in the FST or TST. RESULTS: A dose-response study showed that mecamylamine significantly decreased immobility time in the TST at the 1.0-mg/kg dose but did not alter baseline locomotor activity. The competitive nAChR antagonist dihydro-beta-erythroidine, but not the blood-brain barrier impermeant antagonist hexamethonium, also decreased immobility in the TST. One milligram per kilogram of mecamylamine also significantly decreased time immobile in the FST whereas both beta2- and alpha7-knockout mice were insensitive to the effects of mecamylamine in the FST. CONCLUSIONS: Decreased activity of central nAChRs has antidepressant-like effects in both the TST and FST and these effects are dependent on both beta2 and alpha7 subunits. Therefore, compounds that decrease nAChR activity may be attractive new candidates for development as antidepressants in humans.

Expression of ezrin in glial tubes in the adult subventricular zone and rostral migratory stream.

Ezrin is a member of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal linking proteins. ERM proteins are involved in a wide variety of cellular functions including cell motility, signal transduction, cell-cell interaction and cell-matrix recognition. A recent in situ hybridization study showed that the mRNA encoding ezrin is expressed in neurogenic regions of the mature brain including the subventricular zone (SVZ) and rostral migratory stream (RMS); however, the specific cell types expressing ezrin and their relationship to migrating and proliferating cells in these regions have not been characterized previously. In this study, we used immunocytochemistry to perform double labeling with a variety of cell-type specific markers to characterize the expression of ezrin in the SVZ and RMS of adult mice. Ezrin was expressed at high levels in both the SVZ and RMS where ezrin-immunopositive processes formed a trabecular network surrounding the proliferating and migrating cells. Ezrin-positive cells co-labeled with the glial makers S100beta and GFAP (glial fibrillary acidic protein), but only minimally with the early neuronal markers beta III tubulin and polysialylated form of neural cell adhesion molecule 1 (PSA-NCAM), indicating that ezrin was expressed primarily in the glial tube cells. Ezrin positive cells also expressed beta-catenin, a membrane-complex protein previously implicated in the regulation of stem-cell proliferation and neuronal migration. Glial tube cells act as both precursors of, and a physical channel for, migrating neuroblasts. Bi-directional signals between glial tube cells and migrating neuroblasts have been shown to regulate the rates of both proliferation of the precursor cells and migration of the newly generated neuroblasts. Our finding that ezrin and beta-catenin are both present at the cell membrane of the glial tube cells suggests that these proteins may be involved in those signaling processes.

Galanin: a novel therapeutic target for depression, anxiety disorders and drug addiction?

Galanin is a neuropeptide synthesized in many neuronal types including brainstem norepinephrine-producing cells of the locus coeruleus and the serotonin-producing neurons of the dorsal raphe nucleus. Galanin inhibits the firing of rodent norepinephrine, serotonin and dopamine neurons and reduces release of these neurotransmitters in forebrain target regions. The distribution of galanin and its receptors and its actions on monoamine signaling has fostered interest in this neuropeptide in the field of behavioral pharmacology and the potential role of galanin in the pathophysiology of neurological diseases such as Alzheimer's disease, epilepsy, stroke, and in psychiatric disorders such as anxiety, depression, and drug addiction, particularly withdrawal. In rodent models, expression of galanin in brain is altered by various stressors, while administration of galanin can modulate anxiety-like responses to stress. Emerging evidence further supports a role for galanin in the mediation of depression-related behaviors in rodents. Recently, galanin agonists have been shown to decrease behavioral signs of opiate withdrawal, which are thought to result from hyperactivation of brain stress pathways. Studies using genetically modified mice suggest that galanin normally plays a protective role against opiate reinforcement and withdrawal. The present article reviews current evidence on a potential role for galanin in modulating stress-related neural pathways and behaviors, and speculates on the therapeutic potential of targeting this galanin system for emotional disorders and opiate addiction.

Role of neuronal nicotinic receptors in the effects of nicotine and ethanol on contextual fear conditioning.

Nicotine can enhance contextual learning while ethanol impairs some forms of learning. Nicotine can overcome some of the impairing effects of ethanol when the two drugs are co-administered. The specific brain nicotinic acetylcholine receptors (nAChRs) that mediate nicotine's effects on contextual learning and whether any of ethanol's actions are mediated by nAChRs are unknown. The potential roles of nAChRs in contextual and cued fear conditioning as well as the effects of nicotine, ethanol, or co-administration of nicotine and ethanol were examined in wild type and homozygous null mutant mice from alpha7, beta2, beta3, and beta4 mouse lines at 24 h after training. Nicotine was given prior to training and testing, whereas ethanol was given only before training. Nicotine enhanced contextual learning in both alpha7 wild types and mutants when mice were trained at 0.17 mA, but not 0.35 mA. Mutants lacking the alpha7 subunit were less sensitive to the memory impairing effects of ethanol trained at 0.35 mA. beta2 Null mutants receiving saline showed a small, but significant, impairment in contextual learning compared with wild type littermates when the shock stimulus was 0.35 mA. Beta2 Null mutant mice also did not respond to the cognitive enhancing effects of nicotine alone, or after ethanol administration. beta3 and beta4 null mutants did not differ from wild types either after saline or any of drug treatments. These results show that beta2-containing nAChRs, but not beta3- or beta4-containing receptors, mediate the enhancing effects of nicotine on contextual learning and confirm previous studies implicating beta2 in other forms of learning. A new role for alpha7 nAChRs in regulating sensitivity to the cognitive disrupting effects of ethanol is proposed.

Sex differences in response to oral amitriptyline in three animal models of depression in C57BL/6J mice.

RATIONALE: Knockout and transgenic mice provide a tool for assessing the mechanisms of action of antidepressants. The effectiveness of oral administration of the tricyclic antidepressant amitriptyline (AMI) was assessed in C57BL/6J (B6) mice, a common genetic background on which knockout and transgenic mice are maintained. OBJECTIVES: We determined whether oral AMI would have antidepressant-like effects in B6 mice and whether these effects varied according to sex, duration of treatment, and the depression model utilized. METHODS: Male and female B6 mice were administered AMI (200 microg/ml) in the drinking water as the sole source of fluid, along with 2% saccharin to increase palatability. Control mice were administered 2% saccharin alone. Mice were assessed for responsiveness to AMI in the tail suspension test (TST), the forced swim test (FST), and the learned helplessness (LH) paradigm. RESULTS: In the TST, AMI decreased immobility time regardless of sex or duration of treatment. AMI also decreased immobility time in the FST, but chronic treatment was necessary for full efficacy in both sexes. In the LH paradigm, both subchronic and chronic AMI treatment decreased escape latencies in female mice, but AMI was effective only after chronic treatment in males. The antidepressant-like effects of AMI could not be explained by differences in locomotor activity because activity levels were not altered by antidepressant treatment. CONCLUSIONS: Overall, oral AMI administration provides a valid model for behavioral assessment of antidepressant-like effects in knockout and transgenic mice maintained on a B6 background, but the effectiveness of oral AMI varies depending on sex, duration of treatment, and the depression model used.

Acute and long-term changes in the mesolimbic dopamine pathway after systemic or local single nicotine injections.

We have examined several neurochemical and behavioural parameters related to the function of the mesolimbic dopamine (DA) pathway in animals treated with nicotine following three modes of drug administration, i.e. systemic intraperitoneal injection, intra-accumbens (Acb) infusion or intraventral tegmental area (intra-VTA) microinjection. The present modes of systemic, intra-Acb and intra-VTA nicotine administration elicited comparable acute increases in dialysate DA levels from the Acb. The increase in extracellular DA levels was paralleled by a significant enhancement of locomotion in a habituated environment in the case of systemic or intra-VTA nicotine administration, whereas unilateral or bilateral intra-Acb nicotine infusion was ineffective, showing that accumbal DA increase is not sufficient to elicit locomotion in this experimental paradigm. Intra-VTA, but not systemic or intra-Acb, nicotine administration caused a long-term (at least 24-h) increase in basal dialysate DA levels from the Acb. In addition, significant increases in tyrosine hydroxylase (TH) and GluR1 (but not dopamine transporter or NR1) mRNA levels in the VTA were detected 24 h after intra-VTA nicotine administration. Systemic nicotine injection caused only an increase in TH mRNA levels while intra-Acb infusion did not modify any of the mRNAs tested. The long-term increase in basal DA levels in the Acb and TH, and GluR1 mRNA levels in the VTA upon intra-VTA nicotine microinjection indicates that even a single nicotine injection can induce plastic changes of the mesolimbic DA pathway.

Nicotine receptor inactivation decreases sensitivity to cocaine.

The reinforcing properties of nicotine and psychomotor stimulants are thought to be mediated through the mesolimbic dopamine (DA) system. This study investigates the role of high affinity nicotinic acetylcholine receptors (nAChRs) in cocaine place preference and examines some neurochemical changes in the mesolimbic DA system that might account for the interaction between nicotine and cocaine. 5 mg/kg is the lowest dose of cocaine able to condition a place preference in C57Bl/6 mice. Co-treatment with the nicotinic antagonist mecamylamine (1.0 mg/kg) disrupted place preference to 5 mg/kg cocaine. In addition, mice lacking the high affinity nAChR containing the beta2 subunit showed decreased place preference to 5 mg/kg cocaine, although higher doses of cocaine could condition a place preference in these knock out animals. In contrast, co-administration of a low dose of nicotine (0.2 mg/kg) potentiated place preference to a subthreshold dose of cocaine (3 mg/kg). DA turnover was monitored in several brain regions using tissue levels of DA and its primary metabolite DOPAC as an indication of DA release. Wild type mice showed decreased DA turnover following treatment with 5 mg/kg cocaine; whereas, this response was not seen in mice lacking the beta2 subunit of the nAChR. Induction of chronic fos-related antigens by cocaine was also reduced in mutant mice as compared to their wild type siblings, implying that downstream actions of cocaine were also affected by inactivation of the high affinity nAChR. These data indicate that activation of the high affinity nAChR may contribute to cocaine reinforcement.

Effects of nicotine pretreatment on dopaminergic and behavioral responses to conditioned fear stress in rats: dissociation of biochemical and behavioral effects.

BACKGROUND: We have examined the effects of nicotine pretreatment on dopaminergic and behavioral responses to conditioned fear stress in the rat. METHODS: Rats were pretreated daily with saline or nicotine for 20 days then challenged with nicotine or saline on day 21. Animals were trained in a classical conditioned fear paradigm. Dopamine utilization in the medial prefrontal cortex and nucleus accumbens shell and conditioned fear stress-induced immobility responses were assessed. RESULTS: Saline pretreated animals rapidly acquired the conditioned fear stress response as assessed by preferential activation of mesoprefrontal dopamine metabolism and tone-elicited immobility responses. Repeated, but not acute, nicotine pretreatment significantly reduced conditioned fear stress-induced dopamine metabolism in the medial prefrontal cortex and nucleus accumbens shell. Repeated nicotine pretreatment did not modify the acquisition or expression of conditioned fear stress responses, however. CONCLUSIONS: The dissimilar effects of repeated nicotine exposure on the cortical dopamine and behavioral responses to conditioned fear stress suggest that nicotine differs from other agents with anxiolytic activity that produce coordinated changes in conditioned fear stress-induced cortical dopaminergic and behavioral responses. Furthermore, compared with results of acute footshock stress, repeated nicotine pretreatment appears to have differential effects on physical versus psychological stressors. Results are discussed within the clinical context of stress-related psychopathology syndromes and comorbid nicotine dependence.

Galanin receptor 1 gene expression is regulated by cyclic AMP through a CREB-dependent mechanism.

The galanin receptor-1 (GalR1) protein belongs to a family of G protein-coupled receptors for the neuropeptide galanin (GalR1, GalR2 and GalR3) distributed throughout the central and peripheral nervous system. Activation of galanin receptors by their ligands results in increased feeding, impaired learning, enhanced opiate analgesia and decreased opiate place preference. We have shown that opiate withdrawal, which is known to increase levels of cAMP in the locus coeruleus (LC), results in an increase in the number of galanin binding sites and the level of GalR1 mRNA in the LC. We have isolated a 3.6-kb fragment 5' of the inititiation codon of the mouse GalR1 gene and generated a series of deletion mutations of this fragment driving expression of luciferase for use in transient transfection assays in PC12 and Cath.a cell lines. Treatment with forskolin, but not dideoxyforskolin, up-regulates GalR1 transcription, likely through elevation of cAMP levels. The region between - 1050 and - 700 base pairs upstream of exon one is necessary both for basal activity of the GalR1 promoter and for forskolin-mediated increases in transcription. The forskolin effect can be blocked by simultaneous mutation of a CRE-like site and a CRE/DRE-like site, but not mutation of either site alone. Gel shift and super-shift experiments demonstrate that the transcription factor CREB can bind to both sites and is likely to be responsible for the cAMP-mediated increase in GalR1 promoter activity. This study provides a molecular mechanism for the increased GalR1 expression in the LC seen following opiate withdrawal.

Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum.

Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [(3)H]choline. Agonist-stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. alpha-Conotoxin MII did not inhibit this release, while alpha-conotoxin AuIB inhibited 50% of agonist-stimulated release. Comparison of this process with [(3)H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was confirmed by assays using mice homozygous for the beta 2 subunit null mutation. The deletion of the beta 2 subunit had no effect on agonist-stimulated acetylcholine release, but abolished agonist-stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the beta 2 subunit null mutation showed decreased dopamine release evoked by L-nicotine with no apparent change in EC(50) value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.

Neuronal nicotinic acetylcholine receptor subunit knockout mice: physiological and behavioral phenotypes and possible clinical implications.

Nicotinic acetylcholine receptors (nAChRs) in the muscle, autonomic ganglia, and brain are targets for pharmacologically administered nicotine. Several of the subunits that combine to form neuronal nicotinic receptors have been deleted by knockout or mutated by knockin in mice using homologous recombination. We will review the biochemical, pharmacological, anatomical, physiological, and behavioral phenotypes of mice with genetically altered neuronal nAChR subunits. Clinically relevant mutations in nAChR genes will also be discussed. In addition, some of the signal transduction pathways activated through nAChRs will be described in order to delineate the longer-term changes that might result from persistent activation or inactivation of nAChRs. Genetically manipulated mice have greatly increased our understanding of the subunit composition and physiological properties of nAChRs in vivo. In addition, these mice have provided a model system to determine the molecular basis for many of the pharmacological actions of nicotine on neurotransmitter release and behavior. Genetic manipulations in mice have also elucidated the role of nAChR subunits in various disease states, and suggest several avenues for drug development.

Nicotinic-agonist stimulated (86)Rb(+) efflux and [(3)H]epibatidine binding of mice differing in beta2 genotype.

Nicotinic acetylcholine receptor function and binding was measured in 12 brain regions from mice differing in beta2 subunit expression. Function was measured by on-line detection of (86)Rb(+) efflux stimulated under conditions that measure two pharmacologically distinct nicotinic responses: (1) stimulation with 10 microM nicotine, a response that is relatively sensitive to inhibition by the antagonist, dihydro-beta-erythroidine (DHbetaE); and (2) stimulation with 10 microM epibatidine in the presence of 2 microM DHbetaE, a response that is relatively resistant to inhibition by DHbetaE. Deletion of the beta2 subunit profoundly reduced both DHbetaE-sensitive and -resistant (86)Rb(+) efflux in each brain region and essentially eliminated activity in regions such as cerebral cortex and thalamus. However, residual activity was observed in regions such as olfactory bulbs and inferior colliculus. [(3)H]Epibatidine binding was measured under conditions that allow estimation of both high- and low-affinity sites. High-affinity sites sensitive to inhibition by the nicotinic agonist, cytisine, were virtually eliminated in every region by the beta2 null mutation. In contrast, only a subset of the high-affinity sites insensitive to inhibition by cytisine were eliminated in beta2 null mutants, suggesting receptor heterogeniety. Similarly, low affinity [(3)H]epibatidine binding was heterogeneous in that a fraction of the sites required the beta2 subunit. Many remaining sites were sensitive to inhibition by alpha-bungarotoxin indicating that a subset of the low affinity [(3)H]epibatidine binding are of the alpha7* subtype. Distinct regional variation was observed among the 12 brain regions. These studies confirm important roles for beta2-containing receptors in mediating pharmacologically distinct functions and as components of several identifiable binding sites.

Fear conditioning and latent inhibition in mice lacking the high affinity subclass of nicotinic acetylcholine receptors in the brain.

Nicotine can enhance performance in several tests of cognition but the specific nicotinic receptor subtypes mediating these effects are largely unknown. Knock out mice lacking the beta2 subunit of the nicotinic receptor were evaluated in fear conditioning and latent inhibition tasks to begin to determine which receptor subtypes mediate the cognitive effects of nicotine. Young (2-4 months) knock out and wild type mice did not differ in either contextual or tone-conditioned fear, but aged (9-20 months) knock out males were impaired in freezing to both context and tone compared to aged wild type males. No differences in fear conditioning were observed between aged knock out and wild type females. Latent inhibition of fear to a pre-exposed tone, as measured by behavioral freezing, was also assessed. Both knock out and wild type mice displayed similar levels of latent inhibition, although overall levels of freezing were lower in knock out mice. These results support a previous study showing spatial learning deficits in aged beta2 subunit knock out mice [EMBO J. 18 (1999) 1235] and suggest that performance of other cognitive tasks may not be influenced by absence of beta2 subunit-containing receptors.

Nicotinic modulation of mesoprefrontal dopamine neurons: pharmacologic and neuroanatomic characterization.

Schizophrenics have cortical dysfunction that may involve mesoprefrontal dopamine (DA) systems. Rates of nicotine dependence approach 90% in schizophrenia, and nicotine administration through cigarette smoking may ameliorate cognitive dysfunction, which may be related to cortical DA dysregulation. We have shown that repeated, but not acute, nicotine pretreatment (0.15 mg/kg daily s.c.) reduces footshock stress-induced mesoprefrontal DA metabolism and immobility responses. This effect of repeated nicotine is dependent on mecamylamine (MEC)-sensitive nicotinic acetylcholine receptor (nAChR) stimulation and endogenous opioid peptides. In the present study, we have further characterized these effects of repeated nicotine on the stress reactivity of mesoprefrontal DA neurons by using the following: 1) local infusion of MEC into cell bodies (ventral tegmental area) and terminal fields (medial prefrontal cortex) to determine the site of action of nicotine; and 2) systemic administration of selective nAChR antagonists. Results of bilateral local infusions of MEC (0.1-1.0 microgram/side) into ventral tegmental area or medial prefrontal cortex in saline- and nicotine-pretreated rats suggests a modulatory role for somatodendritic versus terminal field nAChRs on mesoprefrontal DA neurons under stress-induced states. Experiments with dihydro-beta-erythroidine (a beta2-subunit-selective blocker; 0.0-3.0 mg/kg) and methylycaconitine (an alpha7-subunit-selective blocker; 0.0-8.4 mg/kg) suggest that both alpha4beta2- and alpha7-containing nAChRs modulate mesoprefrontal DA neurons. Thus, complex regulation of mesoprefrontal DA neurons by nAChRs is suggested, which may have relevance to prefrontal cortical DA dysfunction and the high comorbid rates of nicotine dependence in schizophrenia.

Gender differences in learned helplessness behavior are influenced by genetic background.

Learned helplessness behavior was examined in female and male C57BL/6J (B6), 129/J (129) and (B6 x 129)F1 mice, common genetic backgrounds for the generation of knockout models, as well as in mice of a mixed genetic background (outbred mice). Both genotype and gender differences were observed in learned helplessness. Outbred males showed increased shuttle escape latencies following 60, 120, or 360 inescapable shocks compared to nonshocked controls, but outbred females showed no increase in escape latencies following inescapable shock pretreatment. B6 females showed increased escape latencies following 60, 120, or 360 inescapable shocks, whereas B6 males showed increased latencies only after 360 shocks. Female and male 129 and B6129F1 mice did not show an increase in escape latencies following inescapable shock, but this was most likely due to poor escape performance in nonshocked control mice. Differences in escape performance could not be explained by differences in pain thresholds between genotypes. These results support the idea that genetic background and gender are important to consider when using the learned helplessness model in genetically manipulated mice.