Barbara J. Bowers

Point mutant mice with hypersensitive α4 nicotinic receptors show dopaminergic deficits and increased anxiety

Knock-in mice were generated that harbored a leucine-to-serine mutation in the α4 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 Parkinsons disease.

Performance of α7 nicotinic receptor null mutants is impaired in appetitive learning measured in a signaled nose poke task

Wild-type and mutant mice lacking expression of alpha5, alpha7, beta2, beta3, or beta4 neuronal nicotinic cholinergic receptors (nAChRs) were compared on a signaled nose poke task, a multi-phased task used to measure appetitive learning and impulsivity. In the early phases of training, mutants of all nicotinic lines did not differ compared to wild types in the days to reach criterion when mice were required to nose poke for a sucrose reward on FR1 or FR3 schedules, or in their ability to respond to an auditory clicker to receive a sucrose reward. However, mutants lacking alpha7 nAChRs, but not lines lacking other nAChRs, showed impairments when task difficulty was increased such that an auditory stimulus was presented on a variable schedule and mice were required to withhold their responses until the presentation of the auditory cue to obtain a reward. alpha7 mutants were impaired compared to wild types in appetitive learning as measured by the percentage of conditioned responses but overcame their deficits with extensive training for 10 days. However, when efficiency ratios were used to measure impulsivity, alpha7 mutants exhibited lower efficiency ratios even after 10 days of training. These results support a role of the alpha7 nicotinic receptor in mediating appetitive learning and suggest a potential role for the alpha7 nAChRs in the regulation of behavioral disinhibition.

Mice Lacking PKCγ Exhibit Decreased Anxiety

To investigate the contribution of the PKCγ isoform of protein kinase C (PKC) in neurochemical pathways regulating anxiety, mice lacking the gene encoding PKCγ 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 PKCγ 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.

Deletion of the α7 Nicotinic Receptor Subunit Gene Results in Increased Sensitivity to Several Behavioral Effects Produced by Alcohol

BACKGROUND The finding that most people with alcoholism are also heavy smokers prompted several research groups to evaluate the effects of ethanol on neuronal nicotinic acetylcholine receptor (nAChR) function. Data collected in vitro indicate that physiologically relevant concentrations of ethanol inhibit the functional activation of homomeric alpha7 nAChRs, which are one of the most abundant nAChR subtypes expressed in the mammalian brain. The studies outlined here used alpha7 gene knockout (null mutant) mice to evaluate the potential role of alpha7 nAChRs in modulating selected behavioral and physiological effects produced by ethanol. METHODS Current evidence indicates that many responses to ethanol are not genetically correlated. Therefore, the authors measured the effects of acute administration of ethanol on several behaviors that are altered by both ethanol and nicotine: two tests of locomotor activity, acoustic startle, prepulse inhibition of acoustic startle, and body temperature. Ethanol-induced durations of loss of righting reflex and ethanol elimination rates were also determined. These studies used null mutant (alpha7(-/-)) and wild-type (alpha7(-/-)) mice. RESULTS Relative to alpha7(+/+) mice, alpha7(-/-) mice were more sensitive to the activating effects of ethanol on open-field activity, ethanol-induced hypothermia, and duration of loss of the righting response. Deletion of the alpha7 gene did not influence the effects of ethanol on Y-maze crossing or rearing activities, acoustic startle, or prepulse inhibition of startle. Gene deletion did not alter ethanol metabolism. CONCLUSIONS These results indicate that some but not all of the behavioral effects of ethanol are mediated in part by effects on nAChRs that include the alpha7 subunit and may help to explain the robust association between alcohol consumption and the use of tobacco.

Protein and Molecular Characterization of Hippocampal Protein Kinase C in C57BL/6 and DBA/2 Mice

Abstract: Measures of protein kinase C (PKC) in C57BL/6 and DBA/2 mice using [3H]phorbol 12,13‐dibutyrate binding to tissue homogenates and brain slices demonstrated that levels of activated, membrane‐bound PKC were greater in C57BL hippocampus than in DBA hippocampus. Western analysis of α‐, βI‐, βII‐, γ‐, δ‐, and ɛ‐PKC using isozyme‐specific antibodies indicated that the increase observed in C57BL hippocampus was due primarily to the γ‐PKC protein, whose immunoreactivity was greater in the membrane‐bound fraction in C57BL mice. Characterization of α‐, βI,II‐, and γ‐PKC hippocampal mRNA using northern analysis and isozyme‐specific nucleic acid probes did not reveal differences between the strains in levels of gene expression. Restriction fragment length polymorphisms (RFLP) were found in the α‐ and γ‐, but not β‐PKC genomic DNA. The RFLPs appeared to be located in noncoding, nonregulatory regions of the gene. These findings suggest that the γ‐PKC isozyme is largely responsible for the PKC activity difference in C57BL and DBA hippocampus that has been reported previously and may be closely associated with differences in learning ability observed in these strains.

THE USE OF NULL MUTANT MICE TO STUDY COMPLEX LEARNING AND MEMORY PROCESSES

A number of neural substrates have been proposed to mediate complex learning and memory processes in mammalian organisms. One strategy for testing the involvement of a particular gene in learning and memory is to create a mouse line with a null mutation in that gene. Recently, embryonic stem cell-based gene-targeted homologous recombination techniques have been employed to create a number of such mutant mouse lines that do not express interesting candidate genes. These animals have been examined for impairments in several complex learning paradigms which are known to depend on the integrity of the hippocampus. In this review several complex learning and memory paradigms are described, the techniques to create null mutants are reviewed, and the results of recent studies with null mutants are described. Finally, the limitations for interpretation of behavioral data using null mutants are discussed.

Behavioural changes produced by transgenic overexpression of γ2L and γ2S subunits of the GABAA receptor

Transgenic mice overexpressing either the mouse γ2L or γ2S subunit of the GABAA receptor were generated in a C57BL/6 J × 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 γ2L or γ2S 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 γ2 subunit available (regardless of whether it is γ2L or γ2S) but overexpression of neither subunit isoform alters other behavioural and biochemical phenotypes.

Differential sensitivity to the anxiolytic effects of ethanol and flunitrazepam in PKCγ 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 drugs efficacy in reducing anxiety-related behaviors specific to each of the three mazes.

The effects of ethanol and Ro 15–4513 on elevated plus-maze and rotarod performance in long-sleep and short-sleep mice

The effects of ethanol and diazepam were examined in long-sleep (LS) and short-sleep (SS) mice using the elevated plus-maze. Ethanol had more pronounced effects in SS mice than in LS mice. In contrast, LS mice were more sensitive to the effects of diazepam on the elevated plus-maze. The ataxic effects of ethanol were measured by rotarod performance. SS mice were more resistant to the ataxic effects of a 2.0 g/kg dose of ethanol than LS mice. Ro 15-4513 reversed ethanols ataxic effects when administered after ethanol in both LS mice and SS mice. Pentobarbital-induced ataxia was unaffected by treatment with Ro 15-4513. Studies of competition of Ro 15-4513 on 3H-flunitrazepam binding indicated that LS and SS mice did not differ in this measure in cortex, cerebellum or hippocampus.

Biochemical and behavioral effects of steroids on GABAA receptor function in long- and short-sleep mice

The in vitro and in vivo effects of alphaxalone, a steroid anesthetic, and two physiological steroids, tetrahydrodeoxycorticosterone (THDOC) and pregnenolone sulfate (PS), on GABAA receptor function were evaluated in long-sleep (LS) and short-sleep (SS) mice. In vitro, both alphaxalone and THDOC enhanced GABAergic inhibition as measured by [3H]FNZ binding and GABA-stimulated 36Cl- flux. However, with the exception of alphaxalone potentiation of [3H]FNZ binding, which was greater in SS brain regions, LS and SS mice did not differ in their degree of enhancement. Pregnenolone sulfate produced mixed agonistic and antagonistic effects on GABAergic function, dependent upon brain region, with few differences between the lines of mice. In vivo effects of these steroids on sleep time indicated that, like other anesthetic agents, THDOC and alphaxalone induced longer sleep times in LS mice. Antagonism by PS of ethanol-induced sleep time was observed in LS mice only; however, this effect was dependent upon the dose of ethanol used and on the vehicle used to prepare the steroid. Pentobarbital-induced sleep time was not reduced by PS treatment in either line of mouse. These results demonstrate that few differences in sensitivity of the GABAergic receptor to these steroids exist between LS and SS mice. Thus, unlike the differences between LS and SS mice in GABAergic mediation of responses to ethanol and benzodiazepines, there is little genetic variability in subtypes of GABAA receptors capable of modulation by steroids in these lines of mice.