RESUMEN
Establishing and maintaining the appropriate number of GABA synapses is key for balancing excitation and inhibition in the nervous system, though we have only a limited understanding of the mechanisms controlling GABA circuit connectivity. Here, we show that disrupting cholinergic innervation of GABAergic neurons in the C. elegans motor circuit alters GABAergic neuron synaptic connectivity. These changes are accompanied by reduced frequency and increased amplitude of GABAergic synaptic events. Acute genetic disruption in early development, during the integration of post-embryonic-born GABAergic neurons into the circuit, produces irreversible effects on GABAergic synaptic connectivity that mimic those produced by chronic manipulations. In contrast, acute genetic disruption of cholinergic signaling in the adult circuit does not reproduce these effects. Our findings reveal that GABAergic signaling is regulated by cholinergic neuronal activity, probably through distinct mechanisms in the developing and mature nervous system.
Asunto(s)
Caenorhabditis elegans/fisiología , Neuronas Colinérgicas/fisiología , Neuronas GABAérgicas/fisiología , Neuronas Motoras/fisiología , Red Nerviosa/fisiología , Sinapsis/fisiología , Transmisión Sináptica , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Encéfalo/fisiología , Caenorhabditis elegans/citología , Neuronas Colinérgicas/citología , Neuronas Motoras/citología , Red Nerviosa/citología , Neurogénesis/fisiología , Unión Neuromuscular/citología , Unión Neuromuscular/fisiología , Transducción de Señal/fisiologíaRESUMEN
An organism's ability to thrive in changing environmental conditions requires the capacity for making flexible behavioral responses. Here we show that, in the nematode Caenorhabditis elegans, foraging responses to changes in food availability require nlp-12, a homolog of the mammalian neuropeptide cholecystokinin (CCK). nlp-12 expression is limited to a single interneuron (DVA) that is postsynaptic to dopaminergic neurons involved in food-sensing, and presynaptic to locomotory control neurons. NLP-12 release from DVA is regulated through the D1-like dopamine receptor DOP-1, and both nlp-12 and dop-1 are required for normal local food searching responses. nlp-12/CCK overexpression recapitulates characteristics of local food searching, and DVA ablation or mutations disrupting muscle acetylcholine receptor function attenuate these effects. Conversely, nlp-12 deletion reverses behavioral and functional changes associated with genetically enhanced muscle acetylcholine receptor activity. Thus, our data suggest that dopamine-mediated sensory information about food availability shapes foraging in a context-dependent manner through peptide modulation of locomotory output.
Asunto(s)
Conducta Animal , Proteínas de Caenorhabditis elegans/genética , Colecistoquinina/metabolismo , Dopamina/metabolismo , Receptores de Dopamina D1/genética , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Colecistoquinina/genética , Dopamina/genética , Neuronas Dopaminérgicas , Mutación , Receptores Dopaminérgicos , Receptores de Dopamina D1/metabolismo , Transducción de Señal/genética , Transmisión SinápticaRESUMEN
Regulation of both excitatory and inhibitory synaptic transmission is critical for proper nervous system function. Aberrant synaptic signaling, including altered excitatory to inhibitory balance, is observed in numerous neurological diseases. The ubiquitin enzyme system controls the abundance of many synaptic proteins and thus plays a key role in regulating synaptic transmission. The Anaphase-Promoting Complex (APC) is a multi-subunit ubiquitin ligase that was originally discovered as a key regulator of protein turnover during the cell cycle. More recently, the APC has been shown to function in postmitotic neurons, where it regulates diverse processes such as synapse development and synaptic transmission at glutamatergic synapses. Here we report that the APC regulates synaptic GABA signaling by acting in motor neurons to control the balance of excitatory (acetylcholine) to inhibitory (GABA) transmission at the Caenorhabditis elegans neuromuscular junction (NMJ). Loss-of-function mutants in multiple APC subunits have increased muscle excitation at the NMJ; this phenotype is rescued by expression of the missing subunit in GABA neurons. Quantitative imaging and electrophysiological analyses indicate that APC mutants have decreased GABA release but normal cholinergic transmission. Consistent with this, APC mutants exhibit convulsions in a seizure assay sensitive to reductions in GABA signaling. Previous studies in other systems showed that the APC can negatively regulate the levels of the active zone protein SYD-2 Liprin-α. Similarly, we found that SYD-2 accumulates in APC mutants at GABAergic presynaptic sites. Finally, we found that the APC subunit EMB-27 CDC16 can localize to presynapses in GABA neurons. Together, our data suggest a model in which the APC acts at GABAergic presynapses to promote GABA release and inhibit muscle excitation. These findings are the first evidence that the APC regulates transmission at inhibitory synapses and have implications for understanding nervous system pathologies, such as epilepsy, that are characterized by misregulated GABA signaling.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Caenorhabditis elegans/metabolismo , Neuronas GABAérgicas/metabolismo , Unión Neuromuscular/metabolismo , Transmisión Sináptica , Ácido gamma-Aminobutírico/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/genética , Animales , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Neuronas GABAérgicas/fisiología , Péptidos y Proteínas de Señalización Intercelular , Neuronas Motoras/metabolismo , Neuronas Motoras/fisiología , Mutación , Unión Neuromuscular/fisiología , Fosfoproteínas/metabolismo , Transporte de ProteínasRESUMEN
The dystrophin-associated protein complex (DAPC) consists of several transmembrane and intracellular scaffolding elements that have been implicated in maintaining the structure and morphology of the vertebrate neuromuscular junction (NMJ). Genetic linkage analysis has identified loss-of-function mutations in DAPC genes that give rise to degenerative muscular dystrophies. Although much is known about the involvement of the DAPC in maintaining muscle integrity, less is known about the precise contribution of the DAPC in cell signaling events. To better characterize the functional role of the DAPC at the NMJ, we used electrophysiology, immunohistochemistry, and fluorescent labeling to directly assess cholinergic synaptic transmission, ion channel localization, and muscle excitability in loss-of-function (lf) mutants of Caenorhabditis elegans DAPC homologues. We found that all DAPC mutants consistently display mislocalization of the Ca(2+)-gated K(+) channel, SLO-1, in muscle cells, while ionotropic acetylcholine receptor (AChR) expression and localization at the NMJ remained unaltered. Synaptic cholinergic signaling was also not significantly impacted across DAPC(lf) mutants. Consistent with these findings and the postsynaptic mislocalization of SLO-1, we observed an increase in muscle excitability downstream of cholinergic signaling. Based on our results, we conclude that the DAPC is not involved in regulating AChR architecture at the NMJ, but rather functions to control muscle excitability, in an activity-dependent manner, through the proper localization of SLO-1 channels.
Asunto(s)
Potenciales de Acción/fisiología , Caenorhabditis elegans/fisiología , Calcio/metabolismo , Complejo de Proteínas Asociado a la Distrofina/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Músculos/fisiología , Alelos , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Colina/metabolismo , Complejo de Proteínas Asociado a la Distrofina/genética , Genes de Helminto/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Musculares/metabolismo , Mutación/genética , Unión Neuromuscular/metabolismo , Neuronas/metabolismo , Transporte de Proteínas , Receptores Colinérgicos/metabolismo , Transducción de SeñalRESUMEN
Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium. Here, we discover the conserved ion channel CLHM-1 as a ciliary EV cargo. Imaging of EVs released from sensory neuron cilia of Caenorhabditis elegans expressing fluorescently tagged CLHM-1 and TRP polycystin-2 channel PKD-2 shows enrichment of these cargoes in distinct EV subpopulations that are differentially shed in response to mating partner availability. PKD-2 alone is present in EVs shed from the cilium distal tip, whereas CLHM-1 EVs bud from a secondary site(s), including the ciliary base. Heterotrimeric and homodimeric kinesin-2 motors have discrete impacts on PKD-2 and CLHM-1 colocalization in both cilia and EVs. Total loss of kinesin-2 activity decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde intraflagellar transport is required for selective enrichment of protein cargoes into heterogeneous EVs with different signaling potentials.
RESUMEN
Neuromodulators promote adaptive behaviors that are often complex and involve concerted activity changes across circuits that are often not physically connected. It is not well understood how neuromodulatory systems accomplish these tasks. Here, we show that the Caenorhabditis elegans NLP-12 neuropeptide system shapes responses to food availability by modulating the activity of head and body wall motor neurons through alternate G-protein coupled receptor (GPCR) targets, CKR-1 and CKR-2. We show ckr-2 deletion reduces body bend depth during movement under basal conditions. We demonstrate CKR-1 is a functional NLP-12 receptor and define its expression in the nervous system. In contrast to basal locomotion, biased CKR-1 GPCR stimulation of head motor neurons promotes turning during local searching. Deletion of ckr-1 reduces head neuron activity and diminishes turning while specific ckr-1 overexpression or head neuron activation promote turning. Thus, our studies suggest locomotor responses to changing food availability are regulated through conditional NLP-12 stimulation of head or body wall motor circuits.
Asunto(s)
Adaptación Psicológica , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Neuropéptidos/genética , Receptores Acoplados a Proteínas G/fisiología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Locomoción/genética , Neuropéptidos/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMEN
With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end-point PCR in Caenorhabditis elegans Each SS primer consists of a 5' "anchor" that hybridizes to the template, followed by a noncomplementary "bridge," and a "foot" corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3' nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude C. elegans lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems.
Asunto(s)
Caenorhabditis elegans/genética , Técnicas de Genotipaje/métodos , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Alelos , Animales , Cartilla de ADN/química , Técnicas de Genotipaje/normas , Reacción en Cadena de la Polimerasa/normas , Sensibilidad y EspecificidadRESUMEN
The syntaxin-interacting protein tomosyn is thought to be a key regulator of exocytosis, although its precise mechanism of action has yet to be elucidated. Here we examined the role of tomosyn in peptide secretion in Caenorhabditis elegans tomosyn (tom-1) mutants. Ultrastructural analysis of tom-1 mutants revealed a 50% reduction in presynaptic dense-core vesicles (DCVs) corresponding to enhanced neuropeptide release. Conversely, overexpression of TOM-1 led to an accumulation of DCVs. Together, these data provide the first in vivo evidence that TOM-1 negatively regulates DCV exocytosis. In C. elegans, neuropeptide release is promoted by the calcium-dependent activator protein for secretion (CAPS) homolog UNC-31. To test for a genetic interaction between tomosyn and CAPS, we generated tom-1;unc-31 double mutants. Loss of TOM-1 suppressed the behavioral, electrophysiological, and DCV ultrastructural phenotypes of unc-31 mutants, indicating that TOM-1 antagonizes UNC-31-dependent DCV release. Because unc-31 mutants exhibit synaptic transmission defects, we postulated that loss of DCV release in these mutants and the subsequent suppression by tom-1 mutants could simply reflect alterations in synaptic activity, rather than direct regulation of DCV release. To distinguish between these two possibilities, we analyzed C. elegans Rim mutants (unc-10), which have a comparable reduction in synaptic transmission to unc-31 mutants, specifically attributed to defects in synaptic vesicle (SV) exocytosis. Based on this analysis, we conclude that the changes in DCV release in tom-1 and unc-31 mutants reflect direct effects of TOM-1 and UNC-31 on DCV exocytosis, rather than altered SV release.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Calmodulina/metabolismo , Regulación hacia Abajo/fisiología , Sinapsis/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Calmodulina/antagonistas & inhibidores , Mutación , Péptidos/antagonistas & inhibidores , Péptidos/metabolismo , Terminales Presinápticos/metabolismo , Vesículas Secretoras/metabolismo , Sinapsis/ultraestructuraRESUMEN
The SNARE proteins, syntaxin, SNAP-25 and synaptobrevin form a tertiary complex essential for vesicle fusion. Proteins that influence SNARE complex assembly are therefore likely to be important regulators of fusion events. In this study we have focused on tomosyn, a highly conserved, neuronally enriched, syntaxin-binding protein that has been implicated in the regulation of vesicle exocytosis. To directly test the role of tomosyn in neurosecretion we analysed loss-of-function mutants in the single Caenorhabditis elegans tomosyn gene, tom-1. These mutants exhibit enhanced synaptic transmission based on electrophysiological analysis of neuromuscular junction activity. This phenotype is the result of increased synaptic vesicle priming. In addition, we present evidence that tom-1 mutants also exhibit enhanced peptide release from dense core vesicles. These results indicate that tomosyn negatively regulates secretion for both vesicle types, possibly through a common mechanism, interfering with SNARE complex formation, thereby inhibiting vesicle fusion.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/metabolismo , Unión Neuromuscular/fisiología , Neurotransmisores/metabolismo , Sinapsis/fisiología , Animales , Proteínas de Caenorhabditis elegans/genética , Unión Neuromuscular/metabolismo , Sinapsis/metabolismoRESUMEN
The Caenorhabditis elegans neuromuscular junction (NMJ) contains three pharmacologically distinct ionotropic receptors: gamma-aminobutyric acid receptors, levamisole-sensitive nicotinic receptors, and levamisole-insensitive nicotinic receptors. The subunit compositions of the gamma-aminobutyric acid- and levamisole-sensitive receptors have been elucidated, but the levamisole-insensitive acetylcholine receptor is uncharacterized. To determine which of the approximately 40 putative nicotinic receptor subunit genes in the C. elegans genome encodes the levamisole-resistant receptor, we utilized MAPCeL, a microarray profiling strategy. Of seven nicotinic receptor subunit transcripts found to be enriched in muscle, five encode the levamisole receptor subunits, leaving two candidates for the levamisole-insensitive receptor: acr-8 and acr-16. Electrophysiological analysis of the acr-16 deletion mutant showed that the levamisole-insensitive muscle acetylcholine current was eliminated, whereas deletion of acr-8 had no effect. These data suggest that ACR-16, like its closest vertebrate homolog, the nicotinic receptor alpha7-subunit, may form homomeric receptors in vivo. Genetic ablation of both the levamisole-sensitive receptor and acr-16 abolished all cholinergic synaptic currents at the NMJ and severely impaired C. elegans locomotion. Therefore, ACR-16-containing receptors account for all non-levamisole-sensitive nicotinic synaptic signaling at the C. elegans NMJ. The determination of subunit composition for all three C. elegans body wall muscle ionotropic receptors provides a critical foundation for future research at this tractable model synapse.