Jill Bettinger (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Ethanol and Neuronal Plasticity"

Jill Bettinger

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

My lab uses C. elegans genetics to address questions of neurobiology related to synaptic transmission and behavior, specifically as they relate to the physiological effects of alcohol. Alcohol abuse is a devastating and pervasive problem throughout the world. Current treatments are inadequate, and a primary difficulty with the development of novel treatments is that the molecular nature of the interaction of the nervous system with the drug is incompletely understood. Alcohol is a small, easily diffusible molecule that probably interacts with a large number of proteins in all neurons. A cardinal challenge for researchers is to determine which interactions are important for altering nervous system function, and for the development of addiction. I am particularly interested in questions of synaptic plasticity, and we have found that ethanol induces at least two types of plasticity in the worm that are also observed in humans, the development of acute tolerance and state-dependency of learned behaviors. My research focuses on understanding the molecular mechanisms of these behaviors as tractable models of neuronal plasticity. When animals (including worms and humans) are exposed to a constant concentration of ethanol for an extended period of time, they undergo a neural adaptation referred to as acute tolerance, the result of which is that animals appear to be less intoxicated over time. We have found that NPR-1 controls the rate at which animals can develop acute tolerance (Davies et al, 2004). I have devised a genetic screen to identify mutations that confer slow development of acute tolerance to ethanol. The genes affected by these mutations should be required to establish acute tolerance. I am also investigating the effects of ethanol on a more complex behavior, state-dependent learning. By pairing alcohol administration with olfactory adaptation, I demonstrated a form of state-dependent learning in worms (Bettinger and McIntire, 2004). Ethanol does not interfere with olfactory adaptation, however, worms exposed to an odorant while being treated with ethanol will only show subsequent adaptation to the odorant if ethanol is again administered during chemotaxis testing. The effect requires dopaminergic function. This provides an opportunity to pursue a molecular understanding of state-dependent learning and the molecular mechanisms of associative learning in general. I have begun to use the state-dependent learning paradigm as a genetic screening tool.

Jill C Bettinger et al. (2005) International Worm Meeting "The genetics of acute adaptation to ethanol in worms."

Steven L McIntire, Jill C Bettinger, Andrew D Goldsmith, Alfred P Kaye, Armando R De La Torre

[International Worm Meeting, 2005]

We are interested the molecular bases of ethanol response in C. elegans. Natural variation in the acute ethanol response in humans is strongly correlated with the propensity to abuse alcohol such that people with relatively higher innate tolerance are more likely to become alcoholic (Schuckit, 2002). Worms get drunk at similar concentrations of ethanol to those that intoxicate humans, suggesting that there is a conserved mechanism for the effects of ethanol on the nervous system, and that worms can be a good model for studying ethanol responses. Worms become progressively more uncoordinated, slow and Egl as the ethanol dose increases (Davies et al., 2003). In response to exposure to a constant ethanol concentration, worms undergo an acute adaptation (acute tolerance) to the presence of ethanol that does not reflect a decrease in tissue ethanol concentration. There is substantial natural variation in the rate of development of acute tolerance between the wild strains N2 and CB4856. This difference between the behavior of the wild strains is due to the previously described allelic variation at the npr-1 locus (de Bono and Bargmann, 1998; Davies et al., 2004). Genetic analysis indicates that NPR-1 activity antagonizes the development of acute tolerance. To better understand the molecular mechanisms of this development of acute tolerance, we have performed a screen for suppressors of the npr-1(ky13) rapid development of acute tolerance phenotype. To date, we have screened approximately 4000 haploid genomes and have recovered 10 suppressors from at least 6 complementation groups. Most of our mutations do not suppress npr-1-mediated clumping, indicating that they identify components that are specific to the acute ethanol tolerance pathway, rather than general suppressors of npr-1. None of the mutations has an obvious phenotype in the absence of ethanol. We have mapped and cloned the first of these suppressors, eg613, which is a null allele of the gene F49E10.5. Analysis of a transcriptional reporter construct reveals that F49E10.5 is expressed in a small subset of neurons. We are pursuing identification of these neurons and exploring the molecular mechanisms by which this molecule affects development of acute ethanol tolerance.

Jill C Bettinger et al. (2001) International C. elegans Meeting "State-Dependency of Olfactory Adaptation"

Jill C Bettinger, Steven L McIntire, Catharine L Eastman

[International C. elegans Meeting, 2001]

Many learned responses are dependent on the context in which the learning occurs. In vertebrate learning studies, effective memory retrieval can depend on the similarity of the external context or internal physiological state of the organism present during testing to that present during learning. State-dependent learning is thought to drive aspects of addictive behavior, particularly those associated with alcholism. Worms become intoxicated by ethanol in a manner similar to that of most other organisms tested (see abstracts by A. Davies and H. Kim, this meeting). We have sought to study the mechanisms of ethanol-induced state-dependent learning in worms. Olfactory adaptation in C. elegans is a decrease in the chemotaxis response to an odorant as a result of prior exposure to the odorant (Colbert and Bargmann (1995) Neuron 14 803). We demonstrated a form of state-dependency in worms by pairing olfactory adaptation and ethanol administration: Worms exposed to an odorant while intoxicated by ethanol will only show subsequent adaptation to the odorant if ethanol is again administered during chemotaxis testing. If the odorant is presented without ethanol during testing, the animals behave as na&iuml;ve animals and fail to alter their behavior based on their previous experience or prior exposure to the odorant. Further, we have demonstrated that the state-dependent effects of ethanol require normal dopaminergic function. The dopamine-defective cat-1 and cat-2 mutants are able to adapt to volatile odorants, however, they do not show state-dependency when they are adapted to volatile odorants while intoxicated by ethanol. These results suggest that there is a conserved role of dopamine in the modulation of behavioral responses to ethanol. Other mutations tested so far, including glr-1 , an AMPA-type glutamate receptor, have not disrupted the state-dependency of olfactory adaptation. We have begun to screen for animals that are unable to pair olfactory adaptation and ethanol intoxication. From a small pilot screen (<1000 haploid genomes), we have isolated a single mutant, eg160 , that adapts to benzaldehyde in an ethanol-independent manner. We have begun to characterize this mutation, and have found that eg160 mutant animals are normal for dopaminergic function, indicating that eg160 disrupts a novel component of the pathway. The eg160 mutation is completely recessive, and has no visible phenotype. Using the snipSNP method of mapping (thanks to Wicks and Plasterk), we have tentatively assigned eg160 to the left arm of Chromosome I, and are continuing to identify recombinants in order to map it more finely. Our success thus far in finding and mapping eg160 suggests that we will be able to use this assay to identify proteins required for this form of learning in C. elegans . Further screens are underway.

Jill C Bettinger et al. (2003) International Worm Meeting "Towards Understanding Allelic Variation in Ethanol Sensitivity"

Steven L McIntire, Andrew D Goldsmith, Jill C Bettinger, Stephanie M Power

[International Worm Meeting, 2003]

Ethanol is the most widely used and abused drug by humans, yet physiological responses to ethanol are poorly understood. Ethanol causes acute intoxication in worms, resulting in depression of locomotion, pumping and egg-laying. By identifying mutations that confer hypersensitivity to ethanol in worms, we expect to identify pathways that are important in mediating behavioral responses to ethanol. npr-1 loss-of-function mutations display a more rapid recovery from intoxication during a single ethanol exposure than do N2 animals. We have used the sensitized background of npr-1(ky13) animals to screen for mutations that confer hypersensitivity to ethanol intoxication. We have screened approximately 1500 genomes and recovered four mutations (eg613, eg614, eg626 and eg642) that produce ethanol-hypersensitivity phenotypes (in an npr-1 background). None of these mutations is a simple suppressor of npr-1 as each of these strains still clumps and borders, and none has an obvious phenotype in the absence of ethanol. We have pursued molecular characterization of eg613 and eg614. Both are completely recessive. We could use CB4856 to map these mutations using SNPs because the reduced-function allele of npr-1 in the Hawaiian strain produces rapid tolerance to ethanol intoxication. In the course of mapping these mutations we encountered a technical problem with its own interesting connotation for ethanol sensitivity. We discovered that, in both crosses, approximately 1/4 of our hypersensitive F2s gave mapping data that conflicted with the preponderance of evidence. For one of these crosses, we determined by complementation testing with the original mutation (eg613) that these confusing F2s were not homozygous for eg613. We hypothesized that some combination of N2 and CB4856 alleles might assort in the progeny to alter ethanol sensitivity, even in the absence of an induced hypersensitivity mutation. We crossed npr-1(ky13) (N2 background) and CB4856 and asked if we could recover ethanol-hypersensitive animals from the cross of these two ethanol-tolerant parents, and found that we could recover such animals. None of these hypersensitive lines suppresses the npr-1 clumping and bordering, indicating that they are not simply suppressing npr-1 loss of function. Given the numbers of F2s with this hypersensitivity, we presume that there are two alleles that are recessive and independently assorting that combine to confer hypersensitivity. We are pursuing mapping of these two naturally occurring alleles. Allelic variation at multiple loci is thought to be an important determinant of alcohol sensitivity in humans; sensitivity is a predictor of propensity to alcohol dependence in humans and other vertebrates (Shuckit, 1994).

Jill C Bettinger et al. (2004) West Coast Worm Meeting "The genetics of acute adaptation to ethanol intoxication in worms."

Steven L McIntire, Jill C Bettinger, Armando R De La Torre, Andrew D Goldsmith

[West Coast Worm Meeting, 2004]

We have been working to unravel the molecular basis of variation in ethanol sensitivity in C. elegans . Natural variation in ethanol sensitivity in humans is strongly correlated with the propensity to abuse alcohol; people with relatively higher innate tolerance are relatively more likely to become alcoholic (Schuckit, 2002). Ethanol is intoxicating to worms and humans at similar tissue concentrations. Drunk worms become progressively more uncoordinated, slow and Egl as ethanol concentration increases (Davies et al. , 2003). This response is not static; over time worms undergo an acute adaptation (acute tolerance) to the presence of ethanol that does not reflect a decrease in internal ethanol concentration. We noted that there is substantial natural variation in ethanol response, particularly in the rate of development of acute tolerance, between the wild strains N2 and CB4856. This difference is due to the previously described allelic variation at the npr-1 locus (de Bono and Bargmann, 1998; Davies et al. , 2004). Genetic analysis indicates that NPR-1 activity antagonizes the development of acute tolerance. We have performed a genetic screen for molecules involved in the development of acute tolerance by screening for suppressors of the npr-1(ky13) rapid development of acute tolerance phenotype. We have screened approximately 4000 haploid genomes and have isolated 10 suppressors, identifying at least 6 complementation groups. Only a small subset of these mutations (two) suppresses npr-1 -mediated clumping. Thus, most of our mutants presumably identify components that are more specific to the acute ethanol tolerance pathway, rather than general suppressors of npr-1 . None of the mutations has an obvious phenotype in the absence of ethanol. We are pursuing molecular characterization of two of these suppressors, eg613 and eg614 . Both are completely recessive, and neither has a detectable ethanol-sensitivity phenotype in the absence of the npr-1 mutation.

Bettinger, Jill et al. (2009) International Worm Meeting "The Molecular Basis of Ethanol Response in C. elegans."

Bettinger, Jill, Davies, Andrew, Gardner, Jennifer, Smail, Emily, Bolling, Mia

[International Worm Meeting, 2009]

We study acute responses to ethanol intoxication, with the goal of identifying the mechanisms by which neurons are affected by and respond to ethanol. Initial (naive) sensitivity to ethanol is widely variable in humans, and has been correlated with lifetime propensity to abuse alcohol. Initial sensitivity is a complex phenotype that results from at least two different components; the instantaneous responsiveness of neurons to ethanol, and the development of acute functional tolerance, which is an immediate (within minutes) adaptation to the effects of the drug. Worms are a good model for the effects of ethanol on neurons; they respond to similar doses as those that intoxicate humans, and they display both components of initial sensitivity. Our laboratory has taken a two-pronged approach to identifying the molecules that render neurons responsive to ethanol. First, we have performed forward genetic screens for animals with altered development of acute functional tolerance. We have identified at least 10 complementation groups of genes that disrupt acute tolerance. One of the mutations was in the putative transcription factor ctbp-1, which is expressed in a small number of neurons, suggesting that we can identify an anatomical locus for the development of acute tolerance. Second, we are screening mutations in candidate neuronal genes for effects on initial sensitivity to ethanol. We are performing an exhaustive characterization of all genes known to be involved in synaptic transmission, a suspected target of ethanol''s action.

Bettinger, Jill C. et al. (2011) International Worm Meeting "Membrane lipid environment is important for the development of acute functional tolerance to ethanol."

Leung, Kapo, Alaimo, Joseph T., Bettinger, Jill C., Bolling, Mia H., Davies, Andrew G.

[International Worm Meeting, 2011]

There is substantial evidence from studies in humans that genetic variation that contributes to an individual's naive level of response to ethanol strongly impacts their susceptibility to become alcoholic. Our laboratory uses worms to study the genetic influences on the level of acute ethanol response. There are at least two components to the level of response to ethanol, initial sensitivity (measured at 10 minutes exposure), and a robust development of acute functional tolerance (AFT, measured at 30 and 50 minutes). We have used a forward genetic screen to isolate mutants that fail to develop AFT, and have recovered 16 recessive mutations that identify at least 14 complementation groups. We cloned the first of these, eg613, which is a mutation in the gene ctbp-1, a transcriptional repressor. CTBP-1 acts with PAG-3 and ZAG-1 to repress transcription (Nicholas, H. et al., J Mol Biol 375:1-11). We also recovered alleles of pag-3 and zag-1 from our screen, demonstrating that we are able to identify genetic pathways using this approach. The gene encoding the triacylglycerol (TAG) lipase LIPS-7 has been shown to be inappropriately upregulated in a ctbp-1 mutant strain, resulting in decreased TAG levels (Chen, S. et al., PNAS 106(5): 1496-501). We demonstrated that dysregulation of lips-7 is at least partially responsible for the failure of ctbp-1(eg613) to develop AFT, and that loss of function of lips-7 results in fast development of AFT. TAGs are used as fat stores in the worm, so we tested if other mutations that alter fat levels also impact AFT, and found that there was no correlation between overall fat levels and AFT. We hypothesized that lips-7 might also be altering membrane composition and/or structure, which has been shown in worms and other systems to influence the function of membrane-bound proteins. Such modulation of function may represent a mechanism for developing tolerance to the depressive effects of ethanol. Supporting this hypothesis, we found that worms reared on cholesterol-depleted media were unable to develop AFT. We also found that altering lips-7 levels modulated the function of two gain-of-function mutations in the ethanol-sensitive BK channel, SLO-1. Together, these data suggest a model in which the lipid bilayer plays an important role in the ability of proteins to modulate their function in response to ethanol treatment.

Bettinger, Jill C. et al. (2017) International Worm Meeting "SWI/SNF chromatin remodeling complexes are required for normal behavioral responses to acute ethanol exposure."

Mathies, Laura D., Davies, Andrew G., Bettinger, Jill C., Blackwell, GinaMari G.

[International Worm Meeting, 2017]

Fifteen million adults in the United States have an alcohol use disorder (AUD). There are very few effective pharmacological interventions for AUD, and one reason that has slowed the rational development of pharmacotherapies has been difficulty in identifying relevant targets of ethanol's action. We use C. elegans to model the acute effects of ethanol on neuronal function to identify the molecular targets of ethanol and determine the mechanisms of the development of tolerance to those effects. A human genome-wide association study identified variation in two different members of the SWI/SNF chromatin remodeling complex as being associated with alcohol use disorder. We examined the role of the SWI/SNF complex in acute behavioral responses to ethanol in worms. There are different SWI/SNF complexes that share the core enzymatic subunits but differ in their accessory subunits. We found that different SWI/SNF complexes regulate different components of the acute response to ethanol: SWI/SNF BAF complexes are required for the initial depressive effects of ethanol, whereas SWI/SNF PBAF complexes are required for the development of acute ethanol tolerance. Although SWI/SNF complexes play important and diverse roles in many tissues, we found that they are required in adults and in neurons for normal ethanol responses. We predict that SWI/SNF complexes regulate the expression of genes that are important for determining the level of acute sensitivity to ethanol and the development of tolerance to ethanol's effects. We have focused first on the development of tolerance to ethanol. We have localized a function of PBAF complexes in ethanol responses to GABAergic neurons. To identify the mediators of the response to ethanol, we have undertaken gene expression studies in which we are comparing several strains to identify PBAF-regulated genes specifically in adult neurons. We have found that among the differentially regulated genes, there is an over representation of genes involved in lipid metabolism. We are testing a model in which lipid effects on membrane microarchitecture, and the proteins contained within, modulate the behavioral effects of ethanol, and SWI/SNF modifies ethanol responses through regulation of these lipid mediators.

Hayden, Ryan et al. (2015) International Worm Meeting "Determining the function of long-chain polyunsaturated fatty acids in the behavioral response to ethanol."

Davies, Andrew, Mathies, Laura, Hayden, Ryan, Jill, Bettinger

[International Worm Meeting, 2015]

We are interested in the genetic and environmental influences on the physiological response to ethanol. In humans, sensitivity to the intoxicating effects of ethanol is an extremely strong predictor of the liability to develop an alcohol use disorder. Ethanol intoxicates worms and depresses several behaviors. We have used worm locomotion to model two aspects of the level of response to ethanol, initial sensitivity, defined as the most severe degree of acute intoxication, and the development of acute functional tolerance to ethanol (AFT), which is a recovery of speed and coordination of locomotion over the course of an ethanol exposure despite a gradual increase in tissue ethanol concentration.Here, we have explored the role of long-chain polyunsaturated fatty acids (LC-PUFAs), the levels of which are influenced both by genetics and diet, on the physiological response to ethanol. We have previously found that there are distinct requirements for particular LC-PUFAs in the acute behavioral response to ethanol. Loss of eicosapentaenoic acid (EPA) eliminates the development of AFT, and dietary supplementation of EPA enhances the development of AFT in wild-type animals, but EPA levels do not affect initial sensitivity to ethanol. In contrast, AA is not required for AFT, but dietary supplementation of AA increases initial sensitivity to ethanol (1). These LC-PUFAs have several known biological roles; they can act as signaling molecules, they are precursors for the generation of eicosanoids, and they are components of specialized microdomains in the plasma membrane. We are testing each of these functions for both AA and EPA to determine which is/are involved in the physiological response to ethanol.(1) Raabe et al. (2014). PLoS One. 2014 Aug 27;9(8):e105999.

Severson AF et al. (2000) West Coast Worm Meeting "The formin protein CYK-1 acts in parallel to an aurora-like kinase/MKLP-1 pathway to execute cytokinesis in early Caenorhabditis elegans embryos"

Bowerman BA, Hamill DR, Severson AF

[West Coast Worm Meeting, 2000]

The C. elegans Formin Homology protein CYK-1 localizes to cleavage furrows in dividing embryonic cells and, based on an analysis of partial loss-of-function mutations, is required late in cytokinesis (Swan et al. 1998). Analysis of a more severe allele indicates that CYK-1 also is required early in cytokinesis for contractile ring assembly or function. In addition to CYK-1, embryonic cytokinesis in C. elegans requires the Aurora-like kinase AIR-2 and the mitotic kinesin-like protein ZEN-4. Genetic interactions involving these loci suggest that an AIR-2/ZEN-4 mitotic spindle pathway functions in parallel to a contractile ring pathway that includes CYK-1. We have identified temperature-sensitive alleles of both air-2 and zen-4. A temporal analysis of their function suggests that AIR-2 acts in metaphase or early anaphase, to localize ZEN-4 to the spindle interzone, while ZEN-4 acts in cytokinesis, during late anaphase or telophase. Intriguingly, ZEN-4 may also be required well after the apparent completion of cytokinesis, to maintain the separation of daughter cells. We are currently using the yeast two-hybrid system to determine if AIR-2 and ZEN-4 interact directly. Additionally, we are collaborating with Dr. Jill Schumacher (University of Texas) to determine if AIR-2 and ZEN-4 associate in stable complexes that can be immunprecipitated. Our analysis provides genetic evidence that separable, parallel pathways coordinate microfilament and microtubule functions during cytokinesis in an animal embryo.