Steven L. McIntire

Identification and characterization of the vesicular GABA transporter.

Synaptic transmission involves the regulated exocytosis of vesicles filled with neurotransmitter. Classical transmitters are synthesized in the cytoplasm, and so must be transported into synaptic vesicles. Although the vesicular transporters for monoamines and acetylcholine have been identified, the proteins responsible for packaging the primary inhibitory and excitatory transmitters, γ-aminobutyric acid (GABA) and glutamate remain unknown,. Studies in the nematode Caenorhabditis elegans have implicated the gene unc-47 in the release of GABA. Here we show that the sequence of unc-47 predicts a protein with ten transmembrane domains, that the gene is expressed by GABA neurons, and that the protein colocalizes with synaptic vesicles. Further, a rat homologue of unc-47 is expressed by central GABA neurons and confers vesicular GABA transport in transfected cells with kinetics and substrate specificity similar to those previously reported for synaptic vesicles from the brain. Comparison of this vesicular GABA transporter (VGAT) with a vesicular transporter for monoamines shows that there are differences in the bioenergetic dependence of transport, and these presumably account for the differences in structure. Thus VGAT is the first of a new family of neurotransmitter transporters.

A Central Role of the BK Potassium Channel in Behavioral Responses to Ethanol in C. elegans

The activities of many neuronal proteins are modulated by ethanol, but the fundamental mechanisms underlying behavioral effects of ethanol remain unclear. To identify mechanisms responsible for intoxication, we screened for Caenorhabditis elegans mutants with altered behavioral responses to ethanol. We found that slo-1 mutants, which were previously recognized as having slightly uncoordinated movement, are highly resistant to ethanol in two behavioral assays. Numerous loss-of-function slo-1 alleles emerged from our screens, indicating that slo-1 has a central role in ethanol responses. slo-1 encodes the BK potassium channel. Electrophysiological analysis shows that ethanol activates the channel in vivo, which would inhibit neuronal activity. Moreover, behaviors of slo-1 gain-of-function mutants resemble those of ethanol-intoxicated animals. These results demonstrate that selective activation of BK channels is responsible for acute intoxicating effects of ethanol in C. elegans. BK channel activation may explain a variety of behavioral responses to ethanol in invertebrate and vertebrate systems.

Genes necessary for directed axonal elongation or fasciculation in C. elegans

The outgrowth of single axons through different cellular environments requires distinct sets of genes in the nematode C. elegans. Three genes are required for the pioneering circumferential outgrowth of identified motor neuron axons between the lateral hypodermal cell membrane and the basal lamina. Three other genes are required for the longitudinal outgrowth of these axons along preexisting axon bundles as well as for the fasciculation of axons within these neuron bundles. Five additional genes are required for circumferential outgrowth, longitudinal outgrowth, and fasciculation; mutations in three of these genes disrupt axon ultrastructure, suggesting that they function in axon formation rather than in axon guidance.

Regulation of body size and behavioral state of C. elegans by sensory perception and the egl-4 cGMP-dependent protein kinase

The growth and behavior of higher organisms depend on the accurate perception and integration of sensory stimuli by the nervous system. We show that defects in sensory perception in C. elegans result in abnormalities in the growth of the animal and in the expression of alternative behavioral states. Our analysis suggests that sensory neurons modulate neural or neuroendocrine functions, regulating both bodily growth and behavioral state. We identify genes likely to be required for these functions downstream of sensory inputs. Here, we characterize one of these genes as egl-4, which we show encodes a cGMP-dependent protein kinase. We demonstrate that this cGMP-dependent kinase functions in neurons of C. elegans to regulate multiple developmental and behavioral processes including the orchestrated growth of the animal and the expression of particular behavioral states.

Genetic analysis of crawling and swimming locomotory patterns in C. elegans

Alternative patterns of neural activity drive different rhythmic locomotory patterns in both invertebrates and mammals. The neuro-molecular mechanisms responsible for the expression of rhythmic behavioral patterns are poorly understood. Here we show that Caenorhabditis elegans switches between distinct forms of locomotion, or crawling versus swimming, when transitioning between solid and liquid environments. These forms of locomotion are distinguished by distinct kinematics and different underlying patterns of neuromuscular activity, as determined by in vivo calcium imaging. The expression of swimming versus crawling rhythms is regulated by sensory input. In a screen for mutants that are defective in transitioning between crawl and swim behavior, we identified unc-79 and unc-80, two mutants known to be defective in NCA ion channel stabilization. Genetic and behavioral analyses suggest that the NCA channels enable the transition to rapid rhythmic behaviors in C. elegans. unc-79, unc-80, and the NCA channels represent a conserved set of genes critical for behavioral pattern generation.

A family of yeast proteins mediating bidirectional vacuolar amino acid transport.

Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1–7) that are related to the neuronal γ-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport systemh described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles.

Natural Variation in the npr-1 Gene Modifies Ethanol Responses of Wild Strains of C. elegans

Variation in the acute response to ethanol between individuals has a significant impact on determining susceptibility to alcoholism. The degree to which genetics contributes to this variation is of great interest. Here we show that allelic variation that alters the functional level of NPR-1, a neuropeptide Y (NPY) receptor-like protein, can account for natural variation in the acute response to ethanol in wild strains of Caenorhabditis elegans. NPR-1 negatively regulates the development of acute tolerance to ethanol, a neuroadaptive process that compensates for effects of ethanol. Furthermore, dynamic changes in the NPR-1 pathway provide a mechanism for ethanol tolerance in C. elegans. This suggests an explanation for the conserved function of NPY-related pathways in ethanol responses across diverse species. Moreover, these data indicate that genetic variation in the level of NPR-1 function determines much of the phenotypic variation in adaptive behavioral responses to ethanol that are observed in natural populations.

Genetic analysis of age-dependent defects of the Caenorhabditis elegans touch receptor neurons.

Although many genes have been implicated in the pathogenesis of common neurodegenerative diseases, the genetic and cellular mechanisms that maintain neuronal integrity during normal aging remain elusive. Here we show that Caenorhabditis elegans touch receptor and cholinergic neurons display age-dependent morphological defects, including cytoskeletal disorganization, axon beading, and defasciculation. Progression of neuronal aging is regulated by DAF-2 and DAF-16 signaling, which also modulate adult life span. Mutations that disrupt touch-evoked sensory activity or reduce membrane excitability trigger accelerated neuronal aging, indicating that electrical activity is critical for adult neuronal integrity. Disrupting touch neuron attachment to the epithelial cells induces distinct neurodegenerative phenotypes. These results provide a detailed description of the age-dependent morphological defects that occur in identified neurons of C. elegans, demonstrate that the age of onset of these defects is regulated by specific genes, and offer experimental evidence for the importance of normal levels of neural activity in delaying neuronal aging.

Loss of RAB‐3/A in Caenorhabditis elegans and the mouse affects behavioral response to ethanol

The mechanisms by which ethanol induces changes in behavior are not well understood. Here, we show that Caenorhabditis elegans loss‐of‐function mutations in the synaptic vesicle‐associated RAB‐3 protein and its guanosine triphosphate exchange factor AEX‐3 confer resistance to the acute locomotor effects of ethanol. Similarly, mice lacking one or both copies of Rab3A are resistant to the ataxic and sedative effects of ethanol, and Rab3A haploinsufficiency increases voluntary ethanol consumption. These data suggest a conserved role of RAB‐3‐/RAB3A‐regulated neurotransmitter release in ethanol‐related behaviors.

The G-Protein-Coupled Serotonin Receptor SER-1 Regulates Egg Laying and Male Mating Behaviors in Caenorhabditis elegans

Serotonin (5-HT) is a neuromodulator that regulates many aspects of animal behavior, including mood, aggression, sex drive, and sleep. In vertebrates, most of the behavioral effects of 5-HT appear to be mediated by G-protein-coupled receptors (GPCRs). Here, we show that SER-1 is the 5-HT GPCR responsible for the stimulatory effects of exogenous 5-HT in two sexually dimorphic behaviors of Caenorhabditis elegans, egg laying and male ventral tail curling. Loss of ser-1 function leads to decreased egg laying in hermaphrodites and defects in the turning step of mating behavior in males. ser-1 is expressed in muscles that are postsynaptic to serotonergic neurons and are known to be required for these behaviors. Analysis of the ser-1 mutant also reveals an inhibitory effect of 5-HT on egg laying that is normally masked by SER-1-dependent stimulation. This inhibition of egg laying requires MOD-1, a 5-HT-gated chloride channel. Loss of mod-1 function in males also produces defects in ventral tail curling and enhances the turning defects in ser-1 mutant males. Sustained elevations in 5-HT levels result in behavioral adaptation to both the stimulatory and inhibitory actions of the neurotransmitter, indicating that both SER-1 and MOD-1 signaling can be modulated. Removal of wild-type animals from high levels of exogenous 5-HT produces a SER-1-dependent withdrawal response in which egg laying is significantly decreased. These studies provide insight into the role of 5-HT in behavior and the regulation of 5-HT2 receptor function.