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, gamma-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.

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.

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.

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.neural rhythms ͉ neurogenetics ͉ sodium leak channel D ifferent forms of rhythmic neural output are ubiquitously observed in motor behaviors such as locomotion, respiration, and feeding (1-4). Extensive research has revealed that a neural network can switch among alternate rhythms by altering the properties of specific intrinsic membrane currents and synapses (5). Consistent with this framework, some proteins appear to contribute more to the generation of one rhythm than other rhythms. For instance, channels that carry the persistent sodium current appear to be important for gasping but not the normal respiratory rhythm when studied in vitro (6). Physiological approaches are sometimes limited when trying to identify specific proteins involved in certain rhythms, however, because of the availability and selectivity of compounds that act on the relevant molecules. With the advent of reverse genetics, these limitations are beginning to be overcome by knocking out or modifying specific genes (7, 8), but both pharmacological and gene manipulation approaches are still limited by the a priori hypotheses on which molecules to target. In contrast, because forward genetic studies are unbiased, they can lead to the identification of novel or uncharacterized proteins that contribute to rhythmic neural output. We have therefore pursued a forward genetic approach to identify neural proteins that contribute more to the generation of one form of rhythmic locomotion (i.e., swimming) than another (i.e., crawling) in the nematode Caenorhabditis elegans.C. elegans moves by generating waves of dorsal-ventral (DV) bends along its body. Prior genetic studies have focused on the molecular mechanisms responsible for crawling over a solid agar substrate (9, 10), whereas the motion C. elegans displays in liquid has only begun to be characterized (11). Although C. elegans encounters water in its natural environment (12), it has been unclear whether its motion i...

gamma-Aminobutyric acid (GABA) neurotransmission is widespread in vertebrate and invertebrate nervous systems. Here we use a genetic approach to identify molecules specific to GABA function. On the basis of the known in vivo roles of GABAergic neurons in controlling behaviour of the nematode Caenorhabditis elegans, we identified mutants defective in GABA-mediated behaviours. Five genes are necessary either for GABAergic neuronal differentiation or for pre- or postsynaptic GABAergic function. The gene unc-30 is required for the differentiation of a specific type of GABAergic neuron, the type-D inhibitory motor neuron. The gene unc-25 is necessary for GABA expression and probably encodes the GABA biosynthetic enzyme glutamic acid decarboxylase. The genes unc-46 and unc-47 seem to be required for normal GABA release. Finally, the gene unc-49 is apparently necessary postsynaptically for the inhibitory effect of GABA on the body muscles and might encode a protein needed for the function of a GABAA-like receptor. Some of these genes are likely to encode previously unidentified proteins required for GABA function.

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. In the nematode Caenorhabditis elegans, age-dependent morphological changes are widespread in somatic tissues (2-4). However, there is little evidence for neuronal aging in C. elegans (3). The observations by Herndon et al. (3) suggest that neuronal loss or axon guidance defects do not occur in the aging C. elegans nervous system. It was also shown that whereas nuclear membranes of other somatic cell nuclei undergo drastic age-dependent deterioration, those of neuronal nuclei remain relatively intact in aging animals (4).There is, however, a clear age-dependent behavioral decline in C. elegans, including decrease in pharyngeal pumping, locomotion and chemotaxis (5). Evidence suggests that failure in neuronal activity could play a direct role in age-dependent behavioral deterioration. Cai and Sesti (6) showed that age-dependent oxidation of the C. elegans potassium channel KVS-1 causes sensory loss and that protection of neuronal KVS-1 from oxidation rescues agedependent decline in chemotaxis behavior. Electrical activity has been shown to be important for neuronal development (7) and was recently implicated in the survival or maintenance of adult mammalian and Drosophila neurons (8, 9). However, it is unclear how electrical activity promotes the integrity of adult neurons.In this paper, we address whether more subtle, subcellular changes occur in the aging C. elegans nervous system. Our results indicate that C. elegans neurons do develop age-dependent changes. Moreover, we show that electrical activity and normal attachment to the neighboring epithelial cells are required for the maintenance of adult touch receptor neurons.

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.

Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1-7) that are related to the neuronal ␥-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 system h 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.Caenorhabditis elegans UNC-47 (1) and the vertebrate homologues from rat (VGAT, 1) and mouse (VIAAT, 2) are synaptic vesicular transporters that are expressed exclusively in inhibitory neurons (see also Ref. 3) and are specific for the neurotransmitters ␥-aminobutyric acid (GABA) 1 and glycine. These proteins differ in sequence, structure, and bioenergetics from the previously characterized family of vesicular transporters that package monoamines and acetylcholine (for current reviews, see Refs. 4 and 5). Common to all of these transporters is the fact that the movement of substrate from the cytosol into synaptic vesicles is driven by a proton electrochemical gradient that is generated by the action of a vacuolar-type H ϩ -ATPase and involves the exchange of lumenal protons.A search for other vertebrate proteins related to the GABAglycine vesicular transporters has led recently to the isolation and expression of cDNAs that, surprisingly, code for Na ϩ