Nigel S. Atkinson

Evolution and divergence of sodium channel genes in vertebrates

Invertebrate species possess one or two Na+ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na+ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na+ channel gene followed by tandem duplications of Na+ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na+ channel genes in a teleost fish. Using an approach designed to clone all of the Na+ channel genes in a genome, we found six Na+ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na+ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na+ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na+ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na+ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than {D[A(BC)]}.

Drug-Induced Epigenetic Changes Produce Drug Tolerance

Tolerance to drugs that affect neural activity is mediated, in part, by adaptive mechanisms that attempt to restore normal neural excitability. Changes in the expression of ion channel genes are thought to play an important role in these neural adaptations. The slo gene encodes the pore-forming subunit of BK-type Ca2+-activated K+ channels, which regulate many aspects of neural activity. Given that induction of slo gene expression plays an important role in the acquisition of tolerance to sedating drugs, we investigated the molecular mechanism of gene induction. Using chromatin immunoprecipitation followed by real-time PCR, we show that a single brief sedation with the anesthetic benzyl alcohol generates a spatiotemporal pattern of histone H4 acetylation across the slo promoter region. Inducing histone acetylation with a histone deacetylase inhibitor yields a similar pattern of changes in histone acetylation, up-regulates slo expression, and phenocopies tolerance in a slo-dependent manner. The cAMP response element binding protein (CREB) is an important transcription factor mediating experience-based neuroadaptations. The slo promoter region contains putative binding sites for the CREB transcription factor. Chromatin immunoprecipitation assays show that benzyl alcohol sedation enhances CREB binding within the slo promoter region. Furthermore, activation of a CREB dominant-negative transgene blocks benzyl alcohol–induced changes in histone acetylation within the slo promoter region, slo induction, and behavioral tolerance caused by benzyl alcohol sedation. These findings provide unique evidence that links molecular epigenetic histone modifications and transcriptional induction of an ion channel gene with a single behavioral event.

Molecular Separation of Two Behavioral Phenotypes by a Mutation Affecting the Promoters of a Ca-Activated K Channel

The Drosophila slowpoke gene encodes a BK-type calcium-activated potassium channel. Null mutations inslowpoke perturb the signaling properties of neurons and muscles and cause behavioral defects. The animals fly very poorly compared with wild-type strains and, after exposure to a bright but cool light or a heat pulse, exhibit a “sticky-feet” phenotype. Expression of slowpoke arises from five transcriptional promoters that express the gene in neural, muscle, and epithelial tissues. A chromosomal deletion (ash218) has been identified that removes the neuronal promoters but not the muscle–tracheal cell promoter. This deletion complements the flight defect ofslowpoke null mutants but not the sticky-feet phenotype. Electrophysiological assays confirm that theash218 chromosome restores normal electrical properties to the flight muscle. This suggests that the flight defect arises from a lack of slowpoke expression in muscle, whereas the sticky-feet phenotype arises from a lack of expression in nervous tissue.

Impaired clock output by altered connectivity in the circadian network

Substantial progress has been made in elucidating the molecular processes that impart a temporal control to physiology and behavior in most eukaryotes. In Drosophila, dorsal and ventral neuronal networks act in concert to convey rhythmicity. Recently, the hierarchical organization among the different circadian clusters has been addressed, but how molecular oscillations translate into rhythmic behavior remains unclear. The small ventral lateral neurons can synchronize certain dorsal oscillators likely through the release of pigment dispersing factor (PDF), a neuropeptide central to the control of rhythmic rest-activity cycles. In the present study, we have taken advantage of flies exhibiting a distinctive arrhythmic phenotype due to mutation of the potassium channel slowpoke (slo) to examine the relevance of specific neuronal populations involved in the circadian control of behavior. We show that altered neuronal function associated with the null mutation specifically impaired PDF accumulation in the dorsal protocerebrum and, in turn, desynchronized molecular oscillations in the dorsal clusters. However, molecular oscillations in the small ventral lateral neurons are properly running in the null mutant, indicating that slo is acting downstream of these core pacemaker cells, most likely in the output pathway. Surprisingly, disrupted PDF signaling by slo dysfunction directly affects the structure of the underlying circuit. Our observations demonstrate that subtle structural changes within the circadian network are responsible for behavioral arrhythmicity.

Susceptibility to ethanol withdrawal seizures is produced by BK channel gene expression.

Alcohol withdrawal seizures are part of the symptomatology of severe alcohol dependence and are believed to originate from long‐term neural adaptations that counter the central nervous system depressant effects of alcohol. Upon alcohol withdrawal, however, the increased neural excitability that was adaptive in the presence of alcohol becomes counter‐adaptive and produces an imbalanced hyperactive nervous system. For some individuals, the uncovering of this imbalance by alcohol abstention can be sufficient to generate a seizure. Using the Drosophila model organism, we demonstrate a central role for the BK‐type Ca2+‐activated K+ channel gene slo in the production of alcohol withdrawal seizures.

Computer automated movement detection for the analysis of behavior

Currently, measuring ethanol behaviors in flies depends on expensive image analysis software or time intensive experimental observation. We have designed an automated system for the collection and analysis of locomotor behavior data, using the IEEE 1394 acquisition program dvgrab, the image toolkit ImageMagick and the programming language Perl. In the proposed method, flies are placed in a clear container and a computer-controlled camera takes pictures at regular intervals. Digital subtraction removes the background and non-moving flies, leaving white pixels where movement has occurred. These pixels are tallied, giving a value that corresponds to the number of animals that have moved between images. Perl scripts automate these processes, allowing compatibility with high-throughput genetic screens. Four experiments demonstrate the utility of this method, the first showing heat-induced locomotor changes, the second showing tolerance to ethanol in a climbing assay, the third showing tolerance to ethanol by scoring the recovery of individual flies, and the fourth showing a mouses preference for a novel object. Our lab will use this method to conduct a genetic screen for ethanol-induced hyperactivity and sedation, however, it could also be used to analyze locomotor behavior of any organism.

CREB regulation of BK channel gene expression underlies rapid drug tolerance

Pharmacodynamic tolerance is believed to involve homeostatic mechanisms initiated to restore normal neural function. Drosophila exposed to a sedating dose of an organic solvent, such as benzyl alcohol or ethanol, acquire tolerance to subsequent sedation by that solvent. The slo gene encodes BK‐type Ca2+‐activated K+ channels and has been linked to alcohol‐ and organic solvent‐induced behavioral tolerance in mice, Caenorhabditis elegans (C. elegans) and Drosophila. The cyclic AMP response element‐binding (CREB) proteins are transcription factors that have been mechanistically linked to some behavioral changes associated with drug addiction. Here, we show that benzyl alcohol sedation alters expression of both dCREB‐A and dCREB2‐b genes to increase production of positively acting CREB isoforms and to reduce expression of negatively acting CREB variants. Using a CREB‐responsive reporter gene, we show that benzyl alcohol sedation increases CREB‐mediated transcription. Chromatin immunoprecipitation assays show that the binding of dCREB2, with a phosphorylated kinase‐inducible domain, increases immediately after benzyl alcohol sedation within the slo promoter region. Most importantly, we show that a loss‐of‐function allele of dCREB2 eliminates drug‐induced upregulation of slo expression and the production of benzyl alcohol tolerance. This unambiguously links dCREB2 transcription factors to these two benzyl alcohol‐induced phenotypes. These findings suggest that CREB positively regulates the expression of slo‐encoded BK‐type Ca2+‐activated K+ channels and that this gives rise to behavioral tolerance to benzyl alcohol sedation.

BK channels play a counter-adaptive role in drug tolerance and dependence.

Disturbance of neural activity by sedative drugs has been proposed to trigger a homeostatic response that resists unfavorable changes in net cellular excitability, leading to tolerance and dependence. The Drosophila slo gene encodes a BK-type Ca2+-activated K+ channel implicated in functional tolerance to alcohol and volatile anesthetics. We hypothesized that increased expression of BK channels induced by these drugs constitutes the homeostatic adaptation conferring resistance to sedative drugs. In contrast to the dogmatic view that BK channels act as neural depressants, we show that drug-induced slo expression enhances excitability by reducing the neuronal refractory period. Although this neuroadaptation directly counters some effects of anesthetics, it also causes long-lasting enhancement of seizure susceptibility, a common symptom of drug withdrawal. These data provide a possible mechanism for the long-standing counter-adaptive theory for drug tolerance in which homeostatic adaptations triggered by drug exposure to produce drug tolerance become counter-adaptive after drug clearance and result in symptoms of dependence.

Alcohol-Induced Histone Acetylation Reveals a Gene Network Involved in Alcohol Tolerance

Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol.

Tolerance in Drosophila

The set of genes that underlie ethanol tolerance (inducible resistance) are likely to overlap with the set of genes responsible for ethanol addiction. Whereas addiction is difficult to recognize in simple model systems, behavioral tolerance is readily identifiable and can be induced in large populations of animals. Thus, tolerance lends itself to analysis in model systems with powerful genetics. Drosophila melanogaster has been used by a variety of laboratories for the identification of genes that interfere with the acquisition of ethanol tolerance. Here, I discuss the genes identified as being important for the production of ethanol tolerance in Drosophila. Some of these genes have also been shown to be important for the production of tolerance in mammals, demonstrating that gene discovery in Drosophila has predictive value for understanding the molecular pathways that generate tolerance in mammals.