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

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Abstract

Tests of ethanol effects in PKCγ null mutant mice have indicated that PKCγ plays a role in initial sensitivity to ethanol-induced sedation, hypothermia, and GABAA 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. PKCγ 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, PKCγ 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 PKCγ 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.

Introduction

Genetic research on anxiety using animal models and human populations has consistently shown that anxiety is a polygenic trait representing a broad range of behaviors. The identification of possible candidate genes mediating anxiety has been made easier using single gene mutation techniques in mouse. For example, null mutations of the serotonin-1A receptor, the corticotropin releasing hormone-1 and -2 receptors, the GABAA-γ2 receptor subunit, glutamic decarboxylase 65-kDa enzyme, and α-calmodulin kinase II enzyme are representative of several mutant lines that have produced mice exhibiting changes in baseline anxiety-related behaviors compared to wild-type control mice Bale et al., 2000, Chen et al., 1994, Crestani et al., 1999, Heisler et al., 1998, Kash et al., 1999, Kishimoto et al., 2000, Parks et al., 1998, Ramboz et al., 1998, Smith et al., 1998. Recently we have shown that mice lacking the neuronal-specific gamma isoform of protein kinase C (PKCγ) exhibit decreased anxiety in three behavioral tests designed to measure several aspects of anxiety, the elevated plus-maze, the black/white box, and the mirrored chamber aversion test (Bowers et al., 2000).

The relationship between alcohol use and anxiety in humans is complex. It is a common belief that some individuals drink alcohol to relieve anxiety or stress. However, individual differences have been reported concerning alcohol's effects as an anxiolytic. Some experimentally controlled studies suggest that particular personality types, such as aggressive, impulsive, and antisocial individuals or those who have high levels of trait anxiety may experience a greater anxiety/stress reduction effect from alcohol Eddy, 1979, Kushner et al., 1996, Pohorecky, 1991, Sher and Levenson, 1982 and that these personality types are associated with problem drinking. In contrast, other studies did not find appreciable anxiety reduction after alcohol administration in subjects diagnosed with anxiety disorders Kushner et al., 1996, Pohorecky, 1991. The genetics of anxiety and alcohol sensitivity most likely explains some of these individual differences as well as the possibility that certain types of anxiety and personality disorders are more amenable to alcohol's anxiolytic effects (Kushner et al., 1990). In rodents, alcohol's efficacy as an anxiolytic also appears to be dependent on several factors including the nature of the behavioral task, the dose of ethanol, and genotype Cao et al., 1993, Durcan and Lister, 1988, Stewart et al., 1993, Stinchcomb et al., 1989. To our knowledge, null mutant mouse lines that exhibit altered baseline anxiety-related behaviors have not been tested for ethanol's anxiolytic effects. Results from such studies could identify genes/proteins that explain some of the variation in alcohol's effects to reduce anxiety in humans and rodents.

To determine whether PKCγ mediates the pharmacological actions of ethanol as an anxiolytic, the present study evaluated the behaviors of PKCγ null mutant mice and wild-type control littermates in the elevated plus-maze, the black/white box, and the mirrored chamber after ethanol administration. These mazes were selected because, although they all represent approach-avoidance tasks, certain variables of the individual tests measure different aspects of anxiety, some of which may be more sensitive to ethanol's anxiolytic effects than others. Principal component and factor analyses of tests commonly used to measure anxiety in mice including the elevated-plus maze and the light/dark choice test, have shown that depending on which aspect of the test is measured, different components of anxiety are represented Belzung and Le Pape, 1994, File, 1996. For example, the primary fear-related factor in the elevated plus-maze is the animal's behavioral and physiological responses to the open arms that lack side walls; therefore, the fear of open spaces can be considered one variable specific to this maze compared to the black/white box and the mirrored chamber (Lister, 1990). Anxiety-related behaviors in the black/white box arise from the conflict between exploring a novel, brightly lit environment and escaping to a dark, protected environment (Lister, 1990). The mirrored chamber represents a unique test for measuring anxiety-related behaviors. The six-sided mirrored chamber may produce an artificial social stimulus due to the multiple reflections of “additional mice.” Therefore, one component of this maze may be anxiety associated with social interaction (Gallup, 1968).

Earlier studies demonstrated that PKCγ mutants exhibit decreased initial sensitivity to the sedative and hypothermic effects of ethanol Bowers et al., 1999, Harris et al., 1995. Neurochemical assays indicated that PKCγ's modulation of these ethanol-induced responses most likely occur via the GABAA receptor (Harris et al., 1995). Microsacs prepared from the cortex and cerebellum in mutant and wild-type control mice were assayed for muscimol-stimulated Cl flux in the presence of ethanol. As was observed with the behavioral responses, ethanol stimulation of Cl flux was greatly reduced in the mutant mice. The decreased sensitivity was specific to ethanol, as tests of flunitrazepam (FNZ)- and pentobarbital-induced changes in behavior and GABAergic receptor function were not changed in the null mutant mice. Therefore, in the present study a comparison of ethanol's effects with those of FNZ was also done to (1) determine whether the decreased initial sensitivity specific to ethanol that was observed in the previous studies generalizes to the anxiolytic effects of ethanol and (2) to determine if PKCγ regulates the neurochemical pathways mediating the anxiolytic effects of both ethanol and FNZ in a similar manner.

Section snippets

Animals

Male and female mice were 60–100 days of age at the time of testing and were housed in like-sex groups of two to five. Mice were allowed food (Harlan/Teklad) and water ad libitum and were maintained on a 12 h light/dark cycle (lights on at 0700) in the animal housing facility at the Institute for Behavioral Genetics (IBG), Boulder, CO. All animal use procedures were performed in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the University of Colorado

Ethanol

Previous results from this laboratory demonstrated significant baseline anxiety differences among the genotypes in naı̈ve mice in this maze (Bowers et al., 2000). Therefore, data from saline-treated mice were analyzed to determine if the same genotypic differences would be observed. A Student's t test comparison of the two genotypes indicated that the percent entrances into, percent time spent in the open arms of the maze, and total number of entries into all arms were significantly different

Discussion

The purpose of this investigation was to characterize the anxiolytic effects of ethanol in PKCγ null mutant mice, which have previously demonstrated decreased ethanol sensitivity in behavioral and biochemical assays, as well as exhibiting reduced baseline anxiety Bowers et al., 1999, Bowers et al., 2000, Harris et al., 1995. The decreased ethanol sensitivity in the null mutants observed in earlier studies was specific to ethanol and did not generalize to two other GABAergic drugs, FNZ and

Acknowledgements

This work was supported by AA-11275 to BJB and JMW, and AA-00141, an RCA to JMW.

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