Decreased locomotor activity in mice expressing tTA under control of the CaMKII alpha promoter.

Transgenic mice in which the tetracycline transactivator (tTA) is driven by the forebrain-specific calcium-calmodulin-dependent kinase II alpha promoter (CaMKII alpha-tTA mice) are used to study the molecular genetics of many behaviors. These mice can be crossed with other transgenic mice carrying a transgene of interest coupled to the tetracycline-responsive promoter element to produce mice with forebrain-specific expression of the transgene under investigation. The value of using CaMKII alpha-tTA mice to study behavior, however, is dependent on the CaMKII alpha-tTA mice themselves lacking a behavioral phenotype with respect to the behaviors being studied. Here we present data that suggest CaMKII alpha-tTA mice have a behavioral phenotype distinct from that of their wild-type (WT) littermates. Most strikingly, we find that CaMKII alpha-tTA mice, both those with a C57BL/6NTac genetic background (B6-tTA) and those with a 129S6B6F1/Tac hybrid genetic background (F1-tTA), exhibit decreased locomotor activity compared with WT littermates that could be misinterpreted as altered anxiety-like behavior. Despite this impairment, neither B6-tTA nor F1-tTA mice perform differently than their WT littermates in two commonly used learning and memory paradigms - Pavlovian fear conditioning and Morris water maze. Additionally, we find data regarding motor coordination and balance to be mixed: B6-tTA mice, but not F1-tTA mice, exhibit impaired performance on the accelerating rotarod and both perform as well as their WT littermates on the balance beam.

Convergent analysis of cDNA and short oligomer microarrays, mouse null mutants and bioinformatics resources to study complex traits.

Gene expression data sets have recently been exploited to study genetic factors that modulate complex traits. However, it has been challenging to establish a direct link between variation in patterns of gene expression and variation in higher order traits such as neuropharmacological responses and patterns of behavior. Here we illustrate an approach that combines gene expression data with new bioinformatics resources to discover genes that potentially modulate behavior. We have exploited three complementary genetic models to obtain convergent evidence that differential expression of a subset of genes and molecular pathways influences ethanol-induced conditioned taste aversion (CTA). As a first step, cDNA microarrays were used to compare gene expression profiles of two null mutant mouse lines with difference in ethanol-induced aversion. Mice lacking a functional copy of G protein-gated potassium channel subunit 2 (Girk2) show a decrease in the aversive effects of ethanol, whereas preproenkephalin (Penk) null mutant mice show the opposite response. We hypothesize that these behavioral differences are generated in part by alterations in expression downstream of the null alleles. We then exploited the WebQTL databases to examine the genetic covariance between mRNA expression levels and measurements of ethanol-induced CTA in BXD recombinant inbred (RI) strains. Finally, we identified a subset of genes and functional groups associated with ethanol-induced CTA in both null mutant lines and BXD RI strains. Collectively, these approaches highlight the phosphatidylinositol signaling pathway and identify several genes including protein kinase C beta isoform and preproenkephalin in regulation of ethanol- induced conditioned taste aversion. Our results point to the increasing potential of the convergent approach and biological databases to investigate genetic mechanisms of complex traits.

Sensitivity and tolerance to ethanol-induced incoordination and hypothermia in HAFT and LAFT mice.

Acute functional tolerance (AFT) manifests as rapid adaptation during a single ethanol exposure, leading to a decrease in the behavioral response to ethanol. In order to investigate the genetic and environmental components of the development of AFT, mice were selectively bred in replicate from HS/Ibg mice. High (HAFT) and low (LAFT) acute functional tolerance selected lines were bred to differ in the rate of development and magnitude of AFT to ethanol's intoxicating effects using a static dowel-balancing task. In the present set of experiments, HAFT and LAFT mice were tested for development of AFT on a fixed-speed rotarod using a protocol similar to that for which they were selected. HAFT mice developed greater AFT to ethanol than did LAFT mice. In a separate experiment, other mice from these lines were tested for initial sensitivity and the development of chronic tolerance to ethanol-induced hypothermia, and ethanol-induced incoordination in the grid test. Previous research has detected possible common genetic control of these phenotypes. No differences between lines were found in initial sensitivity to ethanol or in the development or magnitude of chronic tolerance in either test. These experiments show that genetic factors influencing the development of acute tolerance to ethanol-induced intoxication are at least partially distinct from those influencing initial sensitivity and the development of chronic tolerance to ethanol-induced hypothermia and incoordination. Furthermore, these experiments show that AFT measured by the stationary dowel generalizes to AFT measured by the fixed-speed rotarod.

Ethanol-regulated gene expression of neuroendocrine specific protein in mice: brain region and genotype specificity.

Neuroendocrine specific protein or reticulon 1 (NSP/RTN1) was identified as a putative ethanol-regulated gene using mRNA differential display in mice genetically selected for severe ethanol withdrawal (withdrawal seizure-prone, WSP). One transcript of RTN1 (3.0 kb) showed a statistically significant increase (13%) in relative abundance in whole brain of ethanol-treated WSP mice but not in mice selected for resistance to ethanol withdrawal convulsions (WSR). We hypothesized that ethanol-induced regulation of gene expression of mRTN1 is specific to mice predisposed to exhibit severe ethanol withdrawal and that the gene might be regulated differentially in specific brain regions. WSP and WSR selected lines and DBA/2J and C57BL/6J inbred strains of mice were exposed to ethanol vapor or air for 72 h. mRNA steady-state expression of RTN1 was assessed in hippocampus, cortex, and cerebellum. Results indicated that the pattern of ethanol-induced changes in gene expression was dependent upon transcript size, brain region, and genotype. Modest increases in the relative abundance of both transcripts of RTN1 were observed in the hippocampus and cortex of all ethanol-treated mice. Results from cerebellum showed a moderate decrease in expression of RTN1 (3.0 kb transcript) in WSP and DBA/2J mice, but not in the mice resistant to ethanol withdrawal (C57BL/6J and WSR). These results suggest a genotype-specific effect of chronic ethanol exposure on steady-state mRNA levels of RTN1 in the cerebellum. Overall, the results indicate a complex pattern of ethanol-induced regulation of the putative mouse homologue of RTN1 and suggest that specific brain regional changes may be involved in the expression of physical dependence.

Sensitivity to ethanol-induced motor incoordination in 5-HT(1B) receptor null mutant mice is task-dependent: implications for behavioral assessment of genetically altered mice.

Neuromuscular impairment by ethanol likely involves complex effects on balance, gait, muscle strength, and other features of motor coordination. The present experiments showed that relative sensitivity to ethanol-induced motor impairment in serotonin 1B (5-HT(1B)) null mutant and control mice was task dependent. We found that ethanol-treated null mutant mice made fewer missteps on a balance beam than did ethanol-treated wild-type mice, and confirmed a previous finding of their lesser ethanol sensitivity in the grid test. The genotypes did not differ in ethanol sensitivity as measured by the screen test, static dowel, fixed-speed rotarod, accelerating rotarod, grip strength, or loss of righting reflex tests. These experiments suggest that within a behavioral domain, alternative tests of function are not equivalent, so multiple assessment tools should be used to avoid misinterpretation of gene function.

Identification of neuroendocrine-specific protein as an ethanol-regulated gene with mRNA differential display.

Chronic ethanol exposure produces changes in behavior that may result from effects of ethanol on gene expression. To identify potentially ethanol-regulated genes, mRNA differential display was used to screen the expressed genes in whole brain of mice chronically exposed to ethanol vapors. Mice genetically selected for susceptibility (Withdrawal Seizure-Prone; WSP) or resistance (Withdrawal Seizure-Resistant; WSR) to ethanol withdrawal convulsions were exposed to either ethanol vapor (ETOH group) or air (CTL group) for 72 h. A putative ethanol-regulated product was isolated; nucleotide sequence analysis of this product revealed >85% nucleotide identity to human neuroendocrine-specific protein (NSP) gene. Northern analysis of the expression of this product revealed hybridization to two transcripts ( approximately 3.0 kb and 1.4 kb) on blots containing whole brain RNA, consistent with the transcript sizes of hNSP. Ethanol-induced regulation of mNSP was suggested in whole brain of WSP mice, but not in WSR mice, by Northern blot analysis. One transcript (3.0 kb) suggests a 26% increase in relative abundance in whole brain of ethanol-exposed WSP mice, while there was no effect of ethanol on abundance of the 1.4-kb transcript in WSP mice. No effects of ethanol were observed for WSR mice. These preliminary findings suggest that mNSP represents a novel ethanol-induced gene in mice selected for genetic susceptibility to severe ethanol withdrawal.

Sensitivity to ethanol-induced ataxia in HOT and COLD selected lines of mice.

Studies with inbred strains of mice have suggested that there may be a genetic correlation between strain sensitivities to the ataxic and hypothermic responses to ethanol (EtOH), which would suggest that some genes influence both responses. To test this hypothesis, EtOH sensitivity was determined in replicate lines of mice selectively bred for sensitivity (COLD) or resistance (HOT) to acute ethanol hypothermia. Several tests were used to index ataxia, related traits such as muscle strength, and locomotor activity. The screen test yielded a dose-dependent EtOH-induced decrease in performance that did not differ between the selected lines. Based on the dose-response characteristics of this task, 2.5 g/kg of EtOH was used as the test dose for the remaining experiments. Results from the fixed-speed rotarod and the grid test of motor incoordination also indicated no significant differences between HOT and COLD mice in sensitivity to EtOH impairment. When the selected lines were tested on an accelerating rotarod, COLD mice were impaired by the acute EtOH injection, but HOT mice were unaffected. COLD mice were more sensitive to EtOH-induced decrements in grip strength and locomotor activity. Overall, the results indicated that HOT and COLD mice were only differentially sensitive to EtOH in some tasks related to ataxia, suggesting that some genes must be associated uniquely with EtOH-induced hypothermia or ataxia. The mixed results from the various tests indicate that ataxia can best be conceived as a group of related complex behaviors that cannot be assessed adequately by the use of a single task and that ataxia-related behaviors are influenced by different groups of genes.

Elevated alcohol consumption in null mutant mice lacking 5-HT1B serotonin receptors.

Substantial evidence links alcohol drinking and serotonin (5-HT) functioning in animals. Lowered central 5-HT neurotransmission has been found in a subgroup of alcoholics, possibly those with more aggressive, assaultive tendencies. Several rodent studies have also suggested that intact 5-HT systems are important determinants of sensitivity and/or tolerance to ethanol-induced ataxia and hypothermia. Null mutant mice lacking the 5-HT1B receptor gene (5-HT1B-/-) have been developed that display enhanced aggression and altered 5-HT release in slice preparations from some, but not all, brain areas. We characterized these mice for sensitivity to several effects of ethanol. Mutant mice drank twice as much ethanol as wild-type mice, and voluntarily ingested solutions containing up to 20% ethanol in water. Their intake of food and water, and of sucrose, saccharin and quinine solutions, was normal. Mutants were less sensitive than wild-types on a test of ethanol-induced ataxia and, with repeated drug administration, tended to develop tolerance more slowly. In tests of ethanol withdrawal and metabolism, mutants and wild-type mice showed equivalent responses. Our results suggest that the 5-HT1B receptor participates in the regulation of ethanol drinking, and demonstrate that serotonergic manipulations lead to reduced responsiveness to certain ataxic effects of ethanol without affecting dependence.

Regulated expression of the neurofibromin type I transcript in the developing chicken brain.

Neurofibromatosis type 1 (NF-1) is among the most common inherited diseases affecting cells of the central and peripheral nervous systems. A region of the NF-1 gene is similar in sequence to the ras-GTPase activator protein (ras-GAP), and investigations have confirmed that the NF-1 gene product (now known as neurofibromin) stimulates ras-GTPase activity in vitro and in vivo. Neurofibromin modulates the ability of ras proteins to regulate cellular proliferation and/or differentiation, suggesting a possible role in normal development. An alternative form of the neurofibromin transcript with an additional 63-bp exon inserted in the GAP-related domain (GRD) has been described recently. To determine whether differential expression of the two forms of neurofibromin GRD mRNA plays a role in embryonic development, we have isolated and characterized the corresponding chicken cDNA. The predicted amino acid sequence for the inserted exon is identical between chick and human, as are the exon-intron boundaries. RNase protection and RNA-polymerase chain reaction analyses demonstrate that most tissues express predominantly type II mRNA (which contains the insert) throughout embryonic development. In contrast, whereas type II is the major form in the brain early in development, expression of the type I transcript (without the insert) in this tissue increases dramatically at later times. Analysis of primary cultures derived from chick embryo brain indicates that the type I mRNA is enriched in neurons.

Analysis of the sequence and embryonic expression of chicken neurofibromin mRNA.

Neurofibromatosis type 1 (NF1) is a common inherited disorder that primarily affects tissues derived from the neural crest. Recent identification and characterization of the human NF1 gene has revealed that it encodes a protein (now called neurofibromin) that is similar in sequence to the ras-GTPase activator protein (or ras-GAP), suggesting that neurofibromin may be a component of cellular signal transduction pathways regulating cellular proliferation and/or differentiation. To initiate investigations on the role of the NF1 gene product in embryonic development, we have isolated a partial cDNA for chicken neurofibromin. Sequence analysis reveals that the predicted amino acid sequence is highly conserved between chick and human. The chicken cDNA hybridizes to a 12.5-kb transcript on RNA blots, a mol wt similar to that reported for the human and murine mRNAs. Ribonuclease protection assays indicate that NF1 mRNA is expressed in a variety of tissues in the chick embryo; this is confirmed by in situ hybridization analysis. NF1 mRNA expression is detectable as early as embryonic stage 18 in the neural plate. This pattern of expression may suggest a role for neurofibromin during normal development, including that of the nervous system.