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.

Open field activity and EtOH activation of gamma-PKC null mutants.

Null mutants of the neural-specific gamma-isotype of protein kinase C (gamma-PKC) have demonstrated differential responses to acute administration of ethanol in comparison with wild-type animals. Previous studies have shown that the mutants are less sensitive to ethanol-induced loss of righting response. Null mutants also consume more ethanol and exhibit less behavioral inhibition. In order to determine if these sensitivity differences extend to ethanol activation of locomotor activity in an open-field arena, baseline activity and the effect of two low doses of ethanol were assessed in gamma-PKC null mutants and wild-type littermates. Null mutants demonstrated higher levels of baseline activity than did their wild-type counterparts. Further analysis revealed that a 1.0 g/kg dose of ethanol increased locomotor activity in males and females of both genotypes, whereas only null mutant males were activated by a 1.25 g/kg ethanol dose. The current study demonstrates that male gamma-PKC null mutants exhibit increased sensitivity to activating doses of ethanol in contrast to previous findings of decreased sensitivity to higher, depressive doses. This reflects the pleiotropic effects of the gamma-PKC null mutation on the behavioral effects of ethanol.

Learning deficits in forebrain-restricted brain-derived neurotrophic factor mutant mice.

Brain-derived neurotrophic factor (BDNF) participates in synaptic plasticity and the adaptive changes in the strength of communication between neurons thought to underlie aspects of behavioral adaptation. By selectively deleting BDNF from the forebrain of mice using the Cre site-specific DNA recombinase, we were able to study the requirements for BDNF in behaviors such as learning and anxiety. Early-onset forebrain-restricted BDNF mutant mice (Emx-BDNF(KO)) that develop in the absence of BDNF in the dorsal cortex, hippocampus, and parts of the ventral cortex and amygdala failed to learn the Morris Water Maze task, a hippocampal-dependent visuo-spatial learning task. Freezing during all phases of cued-contextual fear conditioning, a behavioral task designed to study hippocampal-dependent associative learning, was enhanced. These mice learned a brightness discrimination task well but were impaired in a more difficult pattern discrimination task. Emx-BDNF(KO) mice did not exhibit altered sensory processing and gating, as measured by the acoustic startle response or prepulse inhibition of the startle response. Although they were less active in an open-field arena, they did not show alterations in anxiety, as measured in the elevated-plus maze, black-white chamber or mirrored chamber tasks. Combined, these data indicate that although an absence of forebrain BDNF does not disrupt acoustic sensory processing or alter baseline anxiety, specific forms of learning are severely impaired.

Mouse molecular genetic technologies: promise for psychiatric research.

Recent advances in mammalian genomics are providing unprecedented opportunities to identify genes that influence neural systems relevant to psychiatric illnesses. As a genetically tractable mammalian species in which complex behaviors may be modeled, mice have been the focus of much attention for examining relationships between genes and behavior. Many investigators are pursuing experimental strategies in which the functions of known genes are examined by studying the impact of their manipulation in mice. These studies are providing important information regarding genetic influences on behavior, as well as animal models relevant to human disease processes. Additional powerful genetic strategies have recently been initiated to search broadly for genes that influence particular clinically relevant behavioral traits. These approaches promise to uncover a large number of novel genetic influences on neuronal pathways that regulate behavior. In this review, mouse molecular genetic techniques are described and illustrative examples of their application to neurobehavioral processes relevant to clinical disorders are provided. Future directions in technology development that promise to further enhance the utility of these approaches for translational research are also described.

Ethanol consumption and behavioral impulsivity are increased in protein kinase Cgamma null mutant mice.

Etiological factors influencing the development of alcoholism are complex and, at a minimum, include an interaction between polygenic factors and personality and biological traits. Human and animal studies suggest that some genes may regulate both the traits associated with alcohol abuse, such as decreased sensitivity or anxiety, and vulnerability to alcoholism. The identification of these genes could elucidate neurochemical pathways that are important in the development of alcohol abuse. Results from the present study indicate that the gene encoding the neuronal-specific gamma subtype of protein kinase C (PKCgamma) influences both ethanol consumption and behavioral impulsivity, a personality characteristic associated with Type II alcoholics, in a pleiotropic manner. Mice lacking PKCgamma consume more ethanol in a two-bottle choice paradigm and also demonstrate increased behavioral impulsivity in an appetitive-signaled nosepoke task when compared with wild-type littermate control mice. Therefore, PKCgamma may be an important mechanism within the cell that mediates one or more neurochemical pathways relevant to an increased predisposition to alcoholism.

Quantitative genetics and mouse behavior.

Quantitative differences are observed for most complex behavioral and pharmacological traits within any population. Both environmental and genetic influences regulate such individual differences. The mouse has proven to be a superb model in which to investigate the genetic basis for quantitative differences in complex behaviors. Genetically defined populations of mice, including inbred strains, heterogeneous stocks, and selected lines, have been used effectively to document these genetic differences. Recently, quantitative trait loci methods have been applied to map the chromosomal regions that regulate variation with the goal of eventually identifying the gene polymorphisms that reside in these regions.

Differential sensitivity to the anxiolytic effects of ethanol and flunitrazepam in PKCgamma null mutant mice.

Tests of ethanol effects in PKCgamma null mutant mice have indicated that PKCgamma plays a role in initial sensitivity to ethanol-induced sedation, hypothermia, and GABA(A) receptor function and impacts neurochemical pathways mediating anxiety. The present study was undertaken to evaluate whether the decreased sensitivity to ethanol previously observed in these mice generalized to the anxiolytic effects of ethanol. PKCgamma null mutant mice and wild-type controls were tested in the elevated-plus maze, the black/white box, and the mirrored chamber after ethanol (0, 1.0, 1.25, 1.5 g/kg) or flunitrazepam (FNZ) (0, 0.015, 0.03, 0.06 mg/kg). Results indicated that although both genotypes exhibited anxiolytic responses to ethanol in the elevated plus-maze, null mutant mice were less sensitive than wild-type control mice; however, in the black/white box, PKCgamma null mutants were more sensitive than controls to the anxiolytic effects of FNZ. Neither ethanol nor FNZ produced anxiolytic responses in the mirrored chamber for either genotype. These results suggest that PKCgamma differentially mediates anxiolytic responses to ethanol and FNZ and that this relationship interacts with each drug's efficacy in reducing anxiety-related behaviors specific to each of the three mazes.

Nicotine enhances latent inhibition and ameliorates ethanol-induced deficits in latent inhibition.

Alcohol and nicotine are drugs of abuse that are used frequently together. One possible explanation for this co-administration is that nicotine prevents or lessens alcohol-associated impairments. The present study examined the dose-dependent effects of acute administration of nicotine, alcohol, or alcohol plus nicotine on latent inhibition as measured by lick suppression in C57BL/6 mice. Alterations in a lick suppression ratio were measured by assessing the effects of 10 pre-exposures to an auditory conditioned stimulus (CS) on formation of subsequent CS-shock unconditioned stimulus (US) associations. Mice pre-exposed to the CS were expected to develop a weaker CS-US association. Nicotine administered prior to pre-exposure to the CS produced increased suppression ratios, ethanol given prior to pre-exposure to the CS decreased suppression ratios, and nicotine reversed the effects of ethanol when the two drugs were co-administered. These opposing actions of nicotine and ethanol may have relevance to the high incidence of smoking and drinking in humans.

The role of RNA editing of kainate receptors in synaptic plasticity and seizures.

The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.

Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety.

Knock-in mice were generated that harbored a leucine-to-serine mutation in the alpha4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.

GABA(A)-receptor delta subunit knockout mice have multiple defects in behavioral responses to ethanol.

BACKGROUND: The gamma-aminobutyric acid type A receptors (GABARs) are involved in mediating some of the behavioral effects of beverage alcohol (ethanol). However, the unique pharmacological and behavioral responses conferred by each of the various receptor subunits are not well understood. METHODS: To address the role of the GABAR delta subunit in mediating ethanol responses, gene knockout mice that lack this subunit were tested for a variety of ethanol-induced behavioral responses. RESULTS: Our results indicate that, compared with controls, delta-deficient mice (delta-/-) have (1) reduced ethanol consumption, (2) attenuated withdrawal from chronic ethanol exposure, and (3) reduced anticonvulsant (seizure-protective) effects of ethanol. These mice demonstrate a normal anxiolytic response to ethanol and a normal hypothermic response to ethanol, and they develop both chronic and acute tolerance. CONCLUSIONS: These results further establish the link between GABARs and specific behavioral responses to ethanol and begin to reveal the role of the delta subunit in these responses.

Confirmation of contextual fear conditioning QTLs by short-term selection.

A short-term selection study for contextual fear conditioning was conducted as a confirmational strategy to analyze the chromosomal locations of five previously mapped contextual fear conditioning quantitative trait loci (QTLs). The founding population was a C57BL/6 (B6) x DBA/2 (D2) F2 intercross. High and low lines were selected for three generations based on contextual fear conditioning scores. Fear conditioning was quantified as the percentage of time spent in a "frozen" posture when placed back into the chamber, where a mild footshock and a tone had been paired with exposure to the context 24 h earlier. Allele frequencies of at least three SSLP DNA markers linked to each of the five QTLs were determined in each generation. As the selection progressed, the frequency of D2 alleles decreased in the low line and increased in the high line for chromosomes 1 and 16, while the opposite was observed in chromosomes 2, 3, and 10. The direction of divergence for alleles on these five chromosomes is consistent with the original QTL mapping study. Differences between the lines in D2 allele frequencies were found to be significant for markers on chromosomes 2, 3, and 16 but did not reach significance on chromosomes 1 or 10. In general, the results are in good agreement with our original fear conditioning QTL mapping study and provide further evidence that these QTLs regulate variation in contextual fear conditioning in crosses of B6 and D2 mice.

Mapping of quantitative trait loci for hypnotic sensitivity to ethanol in crosses derived from the C57BL/6 and DBA/2 mouse strains.

BACKGROUND: Acute ethanol sensitivity is thought to be a predisposing factor toward the development of alcoholism. Accumulated evidence suggests that this characteristic may be at least partly heritable. A widely accepted approach for identifying genes thought to contribute to alcoholism is to map quantitative trait loci (QTLs) for various ethanol-related behaviors in rodent models. METHODS: Ethanol sensitivity QTLs were interval-mapped in a C57BL/6 (B6) X DBA/2 (D2) F2 intercross that contained 391 mice. Sensitivity was measured as the duration of loss of righting reflex (LORR) after 4.1 g/kg ip. LORR also was evaluated in a chromosome 1 marker-assisted congenic strain that had an approximately 30 centiMorgan (cM) portion of D2 DNA from the distal end of chromosome 1 introgressed onto a B6 background. RESULTS: A suggestive QTL was mapped on chromosome 1 (LOD = 3.3; approximately 80 cM) and a provisional QTL on chromosome 5 (LOD = 2.3; approximately 26 cM). The provisional chromosome 5 QTL was found to be sex-specific (LOD = 2.5 for males; LOD < 1 for females) with the D2 allele increasing LORR. The chromosome 1 D2 allele decreased LORR. Consistent with the F2 QTL mapping, congenic mice heterozygous for the chromosome 1 interval (B6/D2) had a significantly different mean (+/- SEM) LORR of 74.0 +/- 4.9 min (n = 36) compared with 90.8 +/- 6.2 min (n = 33) for their homozygous (B6/B6) littermates (p = 0.02). Blood ethanol concentration at regain of righting reflex was 377 +/- 10 mg% for the B6/D2 and 368 +/- 10 mg% (p = NS) for the B6/B6. CONCLUSIONS: LORR results in the chromosome 1 congenic mice were consistent with and very similar to what was predicted from the QTL analysis in the B6 X D2 F2 population. These results support a suggestive LORR QTL on the distal end of mouse chromosome 1. The results also indicate that there is a provisional sex-specific LORR QTL on chromosome 5.

Mice lacking PKC gamma exhibit decreased anxiety.

To investigate the contribution of the PKC gamma isoform of protein kinase C (PKC) in neurochemical pathways regulating anxiety, mice lacking the gene encoding PKC gamma were tested with heterozygote and wild-type littermates in three approach-avoidance tests of anxiety. Null mutant mice consistently displayed a decrease in baseline anxiety-related behaviors in the elevated plus-maze, the black/white box, and the mirrored chamber. In the elevated plus-maze, mutant mice entered the open arms significantly more often and spent more time in the open arms of the maze. In the black/white box, transitions between the compartments were greatest in the null mutant mice, and in the mirrored chamber, mutant mice were markedly less anxious with significantly decreased latencies to enter and more time spent in the chamber. Indices of locomotor activity in the mazes and tests of activity in home cages indicated that the reduced anxiety observed in the mutant mice was not due to baseline locomotor activity differences among the genotypes. These results suggest that PKC gamma be considered as one factor in the etiology of anxiety, perhaps via its post-synaptic regulation of GABAA and 5-HT2 receptors, two receptors implicated in the neurobiology of anxiety.

Behavioural changes produced by transgenic overexpression of gamma2L and gamma2S subunits of the GABAA receptor.

Transgenic mice overexpressing either the mouse gamma2L or gamma2S subunit of the GABAA receptor were generated in a C57BL/6 J x DBA/2 J mixed background and expanded into transgenic lines. Transgenic mice and littermate controls were analysed with respect to altered behaviour indicative of anxiety, motor activity and acute effects of benzodiazepines and alcohol, as well as with regard to altered responses to alcohol withdrawal and acute functional tolerance to alcohol. Biochemical tests assessed flunitrazepam- and ethanol-enhanced 36Cl- flux stimulated by muscimol in cerebellar and cortical microsacs and [3H]-flunitrazepam binding to cerebellar membranes. There were no significant differences in any of these measures between the transgenic and control mice, except in tests of acute functional tolerance to acute injection of ethanol. Compared to controls, mice carrying either the gamma2L or gamma2S transgene developed significantly less tolerance to the ataxic effects of ethanol. We conclude that acute functional tolerance to ethanol is very sensitive to the amount of GABAA receptor gamma2 subunit available (regardless of whether it is gamma2L or gamma2S) but overexpression of neither subunit isoform alters other behavioural and biochemical phenotypes.

Alterations in hippocampal GAP-43 phosphorylation and protein level following contextual fear conditioning.

C57BL/6 (B6) mice display better contextual learning than the DBA/2 (D2) mice. The possibility that GAP-43, is differentially affected as a function of strain and learning was investigated in the present study. No basal difference between C57BL/6J (B6) and DBA/2J (D2) mice in the amount of hippocampal GAP-43 was observed, but naive D2 mice have slightly lower basal levels of GAP-43 phosphorylation than do B6 mice. Interestingly, alterations in hippocampal GAP-43 protein levels and phosphorylation state in response to training for contextual learning were observed only in B6 mice. Immediate-shocked mice, serving as nonlearning controls, showed no GAP-43 alterations, nor did D2 mice subjected to either training condition. These results suggest that modulation of hippocampal GAP-43 may be important for contextual learning and that strain-specific alterations in GAP-43 may be part of a disrupted pathway in D2 mice that is essential for learning.

Background genotype modulates the effects of γ-PKC on the development of rapid tolerance to ethanol-induced hypothermia.

The role of γ-PKC in initial sensitivity and in the development of rapid tolerance to the hypothermic effects of ethanol were investigated in γ-PKC null mutant mice. Effects of the single gene mutation were evaluated on three different genetic backgrounds. Null mutants from a C57BL/6J X 129/SvJ mixed genetic background failed to develop rapid tolerance after 4 days of i.p. ethanol injections. However, when the null mutation was introgressed onto a C57BL/6J background for six generations to create a congenic line, the expression of rapid tolerance unexpectedly reoccurred in the null mutant mice. Subsequent outcrossing of the γ-PKC null mutation to a C57BL/6J X 129/SvEvTac mixed background did not restore the no tolerance phenotype. These observations, taken together with similar results reported previously concerning the development of chronic tolerance to ethanol in these same genotypes, ¹ indicate that the gene coding for gamma-PKC has pleiotropic effects in the expression of both rapid and chronic tolerance to ethanol-induced hypothermia. However, the impact of γ-PKC is modulated by the background genotype. These results stress the necessity of understanding interactions with genetic background when interpreting the effects of single gene mutations on complex behavioral traits.

Nicotine enhancement of contextual fear conditioning.

Nicotine has been suggested to have cognitive enhancing effects. The present study examined the effects of nicotine and the nicotinic antagonist mecamylamine on contextual fear conditioning in C57BL/6 mice. The fear conditioning task was chosen because the task examines two types of learning: contextual learning, and conditioned stimulus (CS)-unconditioned stimulus (US) learning. Multiple doses of nicotine were tested and 0.5 mg/kg nicotine, given on both training and testing days, improved contextual learning but had no effect on formation of an auditory CS-US association. No effect was found at lower doses or when nicotine was given on training day only, or testing day only. The nicotinic receptor antagonist mecamylamine (1 and 2 mg/kg) did not alter contextual fear conditioning but mecamylamine did prevent the nicotine-associated increase in contextual learning. A higher dose of nicotine (1 mg/kg, training day only) interfered with contextual conditioning when the context was paired with both the CS and US, but had no effect on the auditory CS-US association. This effect of 1 mg/kg nicotine on contextual learning disappeared when mice were trained without the CS. The present results indicate that nicotine enhancement of contextual fear conditioning is dose-dependent, but the presence of nicotine is required both during training and testing.

Decreased ethanol sensitivity and tolerance development in gamma-protein kinase C null mutant mice is dependent on genetic background.

Initial sensitivity and tolerance development to the sedative-hypnotic and hypothermic effects of ethanol were investigated in gamma-protein kinase C (PKC) null mutant mice. Null mutants from a C57BL/6J x 129/SvJ mixed genetic background demonstrated decreased ethanol sensitivity and failed to develop chronic tolerance after 10 days of ethanol liquid diet. However, when the null mutation was introgressed onto a C57BL/6J background for six generations, the "no tolerance" phenotype for sedative-hypnotic and hypothermic effects of ethanol was no longer apparent Outcrossing the gamma-PKC null mutation to a C57BL/6J x 129/SvEvTac mixed background restored the "no tolerance" phenotype to ethanol-induced sedation after chronic ethanol diet; however, as measured by hypothermia, tolerance was still evident in the null mutant mice. These observations and the results of tests of chronic tolerance in the C57BL/6J, 129/SvJ, and 129/SvEvTac background inbred strains indicate that gamma-PKC plays an important role in initial sensitivity and tolerance to ethanol. However, the impact of gamma-PKC is modulated by the background genotype. These results stress the importance of including the effect of genetic background when evaluating the effects of single gene mutations on quantitative behavioral traits.