25168499
Regulation of Neurotransmitter Vesicles by the Homeodomain Protein UNC-4 and Its Transcriptional Corepressor UNC-37/Groucho inCaenorhabditis elegans Cholinergic Motor Neurons
Motor neuron function depends on neurotransmitter release from synaptic vesicles (SVs). Here we show that the UNC-4 homeoprotein and its transcriptional corepressor protein UNC-37 regulate SV protein levels in specific Caenorhabditis elegans motor neurons. UNC-4 is expressed in four classes (DA, VA, VC, and SAB) of cholinergic motor neurons. Antibody staining reveals that five different vesicular proteins (UNC-17, choline acetyltransferase, Synaptotagmin, Synaptobrevin, and RAB-3) are substantially reduced in unc-4 and unc-37mutants in these cells; nonvesicular neuronal proteins (Syntaxin, UNC-18, and UNC-11) are not affected, however. Ultrastructural analysis of VA motor neurons in the mutant unc-4(e120) confirms that SV number in the presynaptic zone is reduced (∼40%) whereas axonal diameter and synaptic morphology are not visibly altered. Because the UNC-4–UNC-37 complex has been shown to mediate transcriptional repression, we propose that these effects are performed via an intermediate gene. Our results are consistent with a model in which this unc-4 target gene (“gene-x”) functions at a post-transcriptional level as a negative regulator of SV biogenesis or stability. Experiments with a temperature-sensitive unc-4 mutant show that the adult level of SV proteins strictly depends on unc-4 function during a critical period of motor neuron differentiation.unc-4 activity during this sensitive larval stage is also required for the creation of proper synaptic inputs to VA motor neurons. The temporal correlation of these events may mean that a common unc-4-dependent mechanism controls both the specificity of synaptic inputs as well as the strength of synaptic outputs for these motor neurons.
19387822
Dominant unc-37 mutations suppress the movement defect of a homeodomain mutation in unc-4, a neural specificity gene in Caenorhabditis elegans.
The unc-4 gene of Caenorhabditis elegans encodes a homeodomain protein that defines synaptic input to ventral cord motor neurons. unc-4 mutants are unable to crawl backward because VA motor neurons are miswired with synaptic connections normally reserved for their sister cells, the VB motor neurons. These changes in connectivity are not accompanied by any visible effects upon neuronal morphology, which suggests that unc-4 regulates synaptic specificity but not axonal guidance or outgrowth. In an effort to identify other genes in the unc-4 pathway, we have devised a selection scheme for rare mutations that suppress the Unc-4 phenotype. We have isolated four, dominant, extragenic, allele-specific suppressors of unc-4(e2322ts), a temperature sensitive allele with a point mutation in the unc-4 homeodomain. Our data indicate that these suppressors are gain-of-function mutations in the previously identified unc-37 gene. We show that the loss-of-function mutation unc-37(e262) phenocopies the Unc-4 movement defect but does not prevent unc-4 expression or alter VA motor neuron morphology. These findings suggest that unc-37 functions with unc-4 to specify synaptic input to the VA motor neurons. We propose that unc-37 may be regulated by unc-4. Alternatively, unc-37 may encode a gene product that interacts with the unc-4 homeodomain.
16937490
The Groucho-like transcription factor UNC-37 functions with the neural specificity gene unc-4 to govern motor neuron identity in C. elegans.
Groucho and Tup1 are members of a conserved family of WD repeat proteins that interact with specific transcription factors to repress target genes. Here we show that mutations in WD domains of the Groucho-like protein, UNC-37, affect a motor neuron trait that also depends on UNC-4, a homeodomain protein that controls neuronal specificity in Caenorhabditis elegans. In unc-4 mutants, VA motor neurons assume the pattern of synaptic input normally reserved for their lineal sister cells, the VB motor neurons; the loss of normal input to the VAs produces a distinctive backward movement defect. Substitution of a conserved residue (H to Y) in the fifth WD repeat in unc-37(e262) phenocopies the Unc-4 movement defect. Conversely, an amino acid change (E to K) in the sixth WD repeat of UNC-37 is a strong suppressor of unc-37(e262) and of specific unc-4 missense mutations. We have previously shown that UNC-4 expression in the VA motor neurons specifies the wild-type pattern of presynaptic input. Here we demonstrate that UNC-37 is also expressed in the VAs and that unc-37 activity in these neurons is sufficient to restore normal movement to unc-37(e262) animals. We propose that UNC-37 and UNC-4 function together to prevent expression of genes that define the VB pattern of synaptic inputs and thereby generate connections specific to the VA motor neurons. In addition, we show that the WD repeat domains of UNC-37 and of the human homolog, TLE1, are functionally interchangeable in VA motor neurons which suggests that this highly conserved protein domain may also specify motor neuron identity and synaptic choice in more complex nervous systems.
30743774
UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans.
The UNC-4 homeoprotein and the Groucho-like corepressor UNC-37 specify synaptic choice in the Caenorhabditis elegans motor neuron circuit. In unc-4 mutants, VA motor neurons are miswired with inputs from interneurons normally reserved for their lineal sisters, the VB motor neurons. Here we show that UNC-4 and UNC-37 function together in VA motor neurons to repress VB-specific genes and that this activity depends on physical contact between UNC-37 and a conserved Engrailed-like repressor domain (eh1) in UNC-4. Missense mutations in the UNC-4 eh1 domain disrupt interactions between UNC-4 and UNC-37 and result in the loss of UNC-4-dependent repressor activity in vivo. A compensatory amino acid substitution in UNC-37 suppresses specific unc-4 alleles by restoring physical interactions with UNC-4 as well as UNC-4-dependent repression of VB-specific genes. We propose that repression of VB-specific genes by UNC-4 and UNC-37 is necessary for the creation of wild-type inputs to VA motor neurons. The existence of mammalian homologs of UNC-4 and UNC-37 indicates that a similar mechanism could regulate synaptic choice in the vertebrate spinal cord.
46493885
Synaptic vesicle biogenesis, docking, and fusion: a molecular description.
Secretion of neurotransmitter is the primary means of intercellular communication within the nervous system. This process is regulated by a highly orchestrated cycle of membrane trafficking within the presynaptic nerve terminal. Characterization of proteins localized to the synaptic vesicle and the subsequent studies of their properties have led to a model for the biochemical pathway that underlies vesicle docking, activation, and fusion. The proteins found to function in the synapse are related to those in yeast and other organisms, demonstrating that the mechanisms that mediate vesicle trafficking are conserved in all eukaryotic species.
18071556
Expression of synaptophysin during the prenatal development of the rat spinal cord: Correlation with basic differentiation processes of neurons
The development of the spinal cord involves the proliferation of neurons, their migration to well-defined areas, fiber outgrowth and synapse formation. The present study was designed to correlate the spatiotemporal pattern of expression of synaptophysin, an integral membrane protein of small synaptic vesicles, with these basic processes occurring during the embryonic development of the rat spinal cord. Thoracic segments of spinal cords from embryonic days 12, 14, 16, 18, 20 and of adult spinal cords were studied. S1 nuclease protection assays and immunoblots revealed minute amounts of specific mRNA and synaptophysin at embryonic day 12. There was a steep increase of mRNA between embryonic days 14 and 16, after which levels reached a plateau. A rise in the amount of synaptophysin in the spinal cord occurred between embryonic days 12 and 14, and the levels changed only slightly until the end of embryonic development. Even higher levels of synaptophysin, found in the adult spinal cord, may indicate that its biosynthesis continued after birth. In situ hybridization histochemistry revealed the localization of specific synaptophysin mRNA in the neuroepithelium. However, immunocytochemistry failed to detect synaptophysin in the neuroepithelial cells. Following migration of the neuroblasts, synaptophysins was found in neurons concomitantly with the onset of fiber outgrowth. Thus, already at embryonic day 12, outgrowing fibers of the dorsal root sensory neurons and of motoneurons were synaptophysin positive. From embryonic day 14 throughout the prenatal period, strong synaptophysin immunoreactivity was seen in the ventrolateral and dorsal parts of the marginal layer. Most likely this staining pattern indicates transient functional synaptic contacts because, in the adult spinal cord, the corresponding region, the white matter, exhibited only faint synaptophysin immunoreactivity. In the intermediate layer of the embryonic spinal cord, which corresponds to the gray matter of the adult spinal cord, synaptophysin-positive fibers were observed prior to the formation of functional synapses. The latter are most likely permanent, since synaptophysin in the adult spinal cord is mainly confined to the gray matter. Our data (i) show transcription and translation of synaptophysin within the neurons of the spinal cord and correlate these processes with proliferation, migration, fiber outgrowth and the formation of transient or permanent synapses, and (ii) prove that synaptophysin is a marker for fiber outgrowth in addition to synapse formation.
8295821
Coordinate and noncoordinate regulation of synaptic vesicle protein genes during embryonic development.
Because the formation of a mature nerve terminal requires the accumulation of large quantities of synaptic vesicles, the expression of synaptic vesicle proteins would be expected to correlate with synaptogenesis. However, previous studies have provided conflicting evidence on this point. We have examined the developmental pattern of expression of mRNA and protein for three RNAs derived from two genes coding for synaptic vesicle membrane proteins. For these experiments, we cloned a chick p65 (synaptotagmin) cDNA using a reduced stringency screen with a rat p65 cDNA probe. We examined p65 expression in chick forebrain in conjunction with that of synaptophysin II. RNase protection assays for p65 and the two isoforms of synaptophysin II (Bixby, 1992) show essentially coordinate increases of these three mRNAs in embryonic forebrain during the peak period of synaptogenesis (E17 to E20). However, each of the three mRNAs has a distinct temporal pattern of expression during the early stages of embryogenesis. In the ciliary ganglion, upregulation of synaptophysin II mRNA correlates very well with synaptogenesis. Our results suggest that the regulation of expression of vesicle membrane protein mRNA can serve as a marker for synaptogenesis, despite temporal differences in early expression patterns. In contrast to mRNA expression, assays for vesicle protein expression show a relatively steady rise in both p65 and synaptophysin II throughout the embryonic period, without a sharp increase corresponding to that seen in message levels. These results suggest that the expression both of the p65 and of the synaptophysin II proteins is post-transcriptionally regulated.
24929433
Dynamics of synapsin I gene expression during the establishment and restoration of functional synapses in the rat hippocampus
Synapse development and injury-induced reorganization have been extensively characterized morphologically, yet relatively little is known about the underlying molecular and biochemical events. To examine molecular mechanisms of synaptic development and rearrangement, we looked at the developmental pattern of expression of the neuron-specific gene synapsin I in granule cell neurons of the dentate gyrus and their accompanying mossy fibers during the main period of synaptogenic differentiation in the rat hippocampus. We found a significant difference between the temporal expression of synapsin I messenger RNA in dentate granule somata and the appearance of protein in their mossy fiber terminals during the postnatal development of these neurons. Next, to investigate the regulation of neuron-specific gene expression during the restoration of synaptic contacts in the central nervous system, we examined the expression of the synapsin I gene following lesions of hippocampal circuitry. These studies show marked changes in the pattern and intensity of synapsin I immunoreactivity in the dendritic fields of dentate granule cell neurons following perforant pathway transection. In contrast, changes in synapsin I messenger RNA expression in target neurons, and in those neurons responsible for the reinnervation of this region of the hippocampus, were not found to accompany new synapse formation. On a molecular level, both developmental and lesion data suggest that the expression of the synapsin I gene is tightly regulated in the central nervous system, and that considerable changes in synapsin I protein may occur in neurons without concomitant changes in the levels of its messenger RNA. Finally, our results suggest that the appearance of detectable levels of synapsin I protein in in developing and sprouting synapses coincides with the acquisition of function by those central synapses.
24907479
The Human Synapsin II Gene Promoter
Synapsin II is a neuron-specific phosphoprotein that selectively binds to small synaptic vesicles in the presynaptic nerve terminal. Here we report the cloning and sequencing of the 5′-flanking region of the human synapsin II gene. This sequence is very GC-rich and lacks a TATA or CAAT box. Two major transcriptional start sites were mapped. A hybrid gene consisting of the Escherichia coli chloramphenicol acetyltransferase gene under the control of 837 base pairs of the synapsin II 5′-upstream region was transfected into neuronal and non-neuronal cells. While reporter gene expression was low in neuroblastoma and non-neuronal cells, high chloramphenicol acetyltransferase activities were monitored in PC12 pheochromocytoma cells. However, there was no correlation between reporter gene expression in the transfected cells and endogenous synapsin II immunoreactivity. Using DNA-protein binding assays we showed that the transcription factors zif268/egr-1, polyoma enhancer activator 3 (PEA3), and AP2 specifically contact the synapsin II promoter DNA in vitro. Moreover, the zif268/egr-1 protein as well as PEA3 were shown to stimulate transcription of a reporter gene containing synapsin II promoter sequences. In the nervous system, zif268/egr-1 functions as a “third messenger” with a potential role in synaptic plasticity. PEA3 is expressed in the brain and its activity is regulated by proteins encoded from non-nuclear oncogenes. We postulate that zif268/egr-1 and PEA3 couple extracellular signals to long-term responses by regulating synapsin II gene expression.
25106525
The Brn-3a transcription factor induces neuronal process outgrowth and the coordinate expression of genes encoding synaptic proteins
The Brn-3a POU family transcription factor is expressed only in posmitotic neurons in the central nervous system and identifies the first differentiated neurons to appear in the midbrain, hindbrain, and spinal cord during development. This factor is also induced when undifferentiated proliferating ND7 cells cease dividing and differentiate to a mature neuronal-like phenotype bearing numerous neurite processes. We show that overexpression of Brn-3a in undifferentiated ND7 cells induces a mature neuronal phenotype characterized by process outgrowth and the induction of genes encoding synaptic proteins, although the cells continue to proliferate. In contrast, the closely related factors Brn-3b and Brn-3c do not have this effect. Although the N-terminal activation domain of Brn-3a is required for maximum induction of neurite outgrowth and gene expression, these effects are primarily dependent on the DNA binding POU domain, which also acts as an activation domain. Overexpression of the isolated POU domain of Brn-3a is sufficient to induce neurite outgrowth, while the ability of full-length Brn-3a to do so is abolished by mutating a single amino acid in the Brn-3a POU homeodomain to its equivalent in Brn-3b. Thus, Brn-3a appears to play a critical role in the specification of the mature neuronal phenotype, acting by stimulating the expression of genes whose products are required for process outgrowth and synapse formation.
5006466
The structure of the nervous system of the nematode Caenorhabditis elegans.
The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections. The hermaphrodite nervous system has a total complement of 302 neurons, which are arranged in an essentially invariant structure. Neurons with similar morphologies and connectivities have been grouped together into classes; there are 118 such classes. Neurons have simple morphologies with few, if any, branches. Processes from neurons run in defined positions within bundles of parallel processes, synaptic connections being made en passant. Process bundles are arranged longitudinally and circumferentially and are often adjacent to ridges of hypodermis. Neurons are generally highly locally connected, making synaptic connections with many of their neighbours. Muscle cells have arms that run out to process bundles containing motoneuron axons. Here they receive their synaptic input in defined regions along the surface of the bundles, where motoneuron axons reside. Most of the morphologically identifiable synaptic connections in a typical animal are described. These consist of about 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions.
22929841
Ultrastructure of neuromuscular junctions in Drosophila: comparison of wild type and mutants with increased excitability.
The ventral longitudinal muscles of the Drosophila larval body wall are innervated by at least four types of synaptic terminals that can be distinguished on morphological grounds at the light microscopical level. The innervation of these muscles has been previously shown to be regulated by neuronal activity. In this report we investigate the ultrastructural basis for synaptic bouton differences by using serial sections, and examine the structure of synaptic terminals in mutants with increased excitability. We report that individual identifiable muscle fibers are innervated by terminals containing two to three types of synaptic boutons that can be distinguished in terms of synaptic vesicle population, presynaptic and postsynaptic specialization, and general shape. We propose a model to account for the bouton types observed at the light microscopical level. We find that in the hyperexcitable mutant eag Sh, there are dramatic ultrastructural alterations at synaptic boutons. These alterations include a partial depletion of two types of synaptic vesicles and a change in appearance of a third type, changes in number and appearance of synaptic densities, and the presence of multivesicular bodies. Our results show that an increase in neuronal excitability produces profound effects in synaptic terminal structure.
1423367
Ultrastructural differences among afferent synapses on cochlear hair cells: Correlations with spontaneous discharge rate
The major class of cochlear afferent fibers, the type‐I or radial‐fiber (RF) population, has been subdivided into three functional groups according to spontaneous discharge rate (SR): those with low SR have the highest acoustic thresholds, high SR fibers have the lowest thresholds and medium SR fibers are of intermediate sensitivity (Liberman [1978] J. Acoust. Soc. Amer. 63:442–455). Existing evidence from intracellular labeling studies at the light microscopic level (Liberman [1982a] Science 216:1239–1241) suggests that a single cochlear inner hair cell makes synaptic contact with representatives of all three functional groups; however, low and medium SR fibers are spatially segregated from high SR fibers around the hair cell circumference, and low and medium SR fibers are smaller in caliber than those with high SR. The present study extends to the ultrastructural level the structure‐function correlations available via intracellular labeling. Analysis is based on serial section reconstruction of the synaptic contacts between 11 radial fibers of known SR and their target hair cells. Results suggest systematic differences in synaptic ultrastructure among fibers of the three SR groups: with decreasing SR, the size and complexity of the synaptic body (a presynaptic specialization characteristic of the peripheral afferent synapses in all hair cell systems and some other peripheral receptors) tend to increase, as does the associated number of synaptic vesicles. The possible functional significance of these trends is discussed in the context of other known structural and functional differences among the three SR groups. © 1996 Wiley‐Liss, Inc.
8323902
The Caenorhabditis elegans Gene unc-25Encodes Glutamic Acid Decarboxylase and Is Required for Synaptic Transmission But Not Synaptic Development
The neurotransmitter GABA has been proposed to play a role during nervous system development. We show that theCaenorhabditis elegans gene unc-25encodes glutamic acid decarboxylase (GAD), the GABA biosynthetic enzyme. unc-25 is expressed specifically in GABAergic neurons. Null mutations in unc-25 eliminate the UNC-25 protein or alter amino acids conserved in all known GADs, result in a complete lack of GABA, and cause defects in all GABA-mediated behaviors. In unc-25 mutants the GABAergic neurons have normal axonal trajectories and synaptic connectivity, and the size and shape of synaptic vesicles are normal. The number of synaptic vesicles at GABAergic neuromuscular junctions is slightly increased. Cholinergic ventral nerve cord neurons, which innervate the same muscles as GABAergic ventral cord neurons, have normal morphology, connectivity, and synaptic vesicles. We conclude that GAD activity and GABA are not necessary for the development or maintenance of neuromuscular junctions in C. elegans.
4984055
A Genome-Wide Search for Synaptic Vesicle Cycle Proteins in Drosophila
The recent completion of the Drosophila genome sequence opens new avenues for neurobiology research. We screened the fly genome sequence for homologs of mammalian genes implicated directly or indirectly in exocytosis and endocytosis of synaptic vesicles. We identified fly homologs for 93% of the vertebrate genes that were screened. These are on average 60% identical and 74% similar to their vertebrate counterparts. This high degree of conservation suggests that little protein diversification has been tolerated in the evolution of synaptic transmission. Finally, and perhaps most exciting for Drosophila neurobiologists, the genomic sequence allows us to identify P element transposon insertions in or near genes, thereby allowing rapid isolation of mutations in genes of interest. Analysis of the phenotypes of these mutants should accelerate our understanding of the role of numerous proteins implicated in synaptic transmission.
22972757
Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin
Synaptotagmin is an abundant synaptic vesicle-associated protein proposed to be involved in calcium-mediated neurotransmitter release. Our molecular and genetic results demonstrate that, although synaptotagmin is required for the proper function of the presynaptic nerve terminal in C. elegans, some neurotransmitter release persists in synaptogamin mutants. In C. elegans neurons, synaptotagmin is localized to regions known to be rich in synapses and appears to be associated with synaptic vesicles. Mutants defective in the synaptotagmin gene, called snt-1, exhibit severe behavioral abnormalities that are characteristic of deficiencies in synaptic function, including severe locomotion, feeding, and defecation defects. The mutants are defective in exocytosis, since they accumulate acetylcholine, and are resistant to cholinesterase inhibitors, but they nevertheless remain sensitive to cholinergic receptor agonists. In spite of these exocytic defects, snt-1 mutants are capable of coordinated motor movements, indicating that the mutants do not have a complete block of neurotransmitter release.
7849796
Caenorhabditis elegans rab-3 Mutant Synapses Exhibit Impaired Function and Are Partially Depleted of Vesicles
Rab molecules regulate vesicular trafficking in many different exocytic and endocytic transport pathways in eukaryotic cells. In neurons, rab3 has been proposed to play a crucial role in regulating synaptic vesicle release. To elucidate the role of rab3 in synaptic transmission, we isolated and characterizedCaenorhabditis elegans rab-3 mutants. Similar to the mouse rab3A mutants, these mutants survived and exhibited only mild behavioral abnormalities. In contrast to the mouse mutants, synaptic transmission was perturbed in these animals. Extracellular electrophysiological recordings revealed that synaptic transmission in the pharyngeal nervous system was impaired. Furthermore,rab-3 animals were resistant to the acetylcholinesterase inhibitor aldicarb, suggesting that cholinergic transmission was generally depressed. Last, synaptic vesicle populations were redistributed in rab-3 mutants. In motor neurons, vesicle populations at synapses were depleted to 40% of normal levels, whereas in intersynaptic regions of the axon, vesicle populations were elevated. On the basis of the morphological defects at neuromuscular junctions, we postulate that RAB-3 may regulate recruitment of vesicles to the active zone or sequestration of vesicles near release sites.
8293836
A genetic selection for Caenorhabditis elegans synaptic transmission mutants.
We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.
8227340
Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.
The number of nongonadal nuclei in the free-living soil nematode Caenorhabditis elegans increases from about 550 in the newly hatched larva to about 810 in the mature hermaphrodite and to about 970 in the mature male. The pattern of cell divisions which leads to this increase is essentially invariant among individuals; rigidly determined cell lineages generate a fixed number of progeny cells of strictly specified fates. These lineages range in length from one to eight sequential divisions and lead to significant developmental changes in the neuronal, muscular, hypodermal, and digestive systems. Frequently, several blast cells follow the same asymmetric program of divisions; lineally equivalent progeny of such cells generally differentiate into functionally equivalent cells. We have determined these cell lineages by direct observation of the divisions, migrations, and deaths of individual cells in living nematodes. Many of the cell lineages are involved in sexual maturation. At hatching, the hermaphrodite and male are almost identical morphologically; by the adult stage, gross anatomical differences are obvious. Some of these sexual differences arise from blast cells whose division patterns are initially identical in the male and in the hermaphrodite but later diverge. In the hermaphrodite, these cells produce structures used in egg-laying and mating, whereas, in the male, they produce morphologically different structures which function before and during copulation. In addition, development of the male involves a number of lineages derived from cells which do not divide in the hermaphrodite. Similar postembryonic developmental events occur in other nematode species.
21834487
The structure of the ventral nerve cord of Caenorhabditis elegans.
The nervous system of Caenorhabditis elegans is arranged as a series of fibre bundles which run along internal hypodermal ridges. Most of the sensory integration takes place in a ring of nerve fibres which is wrapped round the pharynx in the head. The body muscles in the head are innervated by motor neurones in this nerve ring while those in the lower part of the body are innervated by a set of motor neurones in a longitudinal fibre bundle which joins the nerve ring, the ventral cord. These motor neurones can be put into five classes on the basis of their morphology and synaptic input. At any one point along the cord only one member from each class has neuromuscular junctions. Members of a given class are arranged in a regular linear sequence in the cord and have non-overlapping fields of motor synaptic activity, the transition between fields of adjacent neurones being sharp and well defined. Members of a given class form gap junctions with neighbouring members of the same class but never to motor neurones of another class. Three of the motor neurone classes receive their synaptic input from a set of interneurones coming from the nerve ring. These interneurones can in turn be grouped into four classes and each of three motor neurone classes receives its synaptic input from a unique combination of interneurone classes. The possible developmental and functional significance of these observations is discussed.
11532651
Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans
unc-104 encodes a novel kinesin paralog that may act as a microtubule-based motor in the nervous system. Neuronal cell lineages and axonogenesis are normal in unc-104 null mutants, but axons have few synaptic vesicles and make only a few small synapses. By contrast, neuron cell bodies have surfeits of similar vesicles tethered together within the cytoplasm. Based on behavioral and cellular phenotypes, we suggest that UNC-104 is a neuron-specific motor used for anterograde translocation of synaptic vesicles along axonal microtubules. Other membrane-bounded organelles are transported normally.
8932037
The Caenorhabditis elegans unc-17 gene: a putative vesicular acetylcholine transporter.
Mutations in the unc-17 gene of the nematode Caenorhabditis elegans produce deficits in neuromuscular function. This gene was cloned and complementary DNAs were sequenced. On the basis of sequence similarity to mammalian vesicular transporters of biogenic amines and of localization to synaptic vesicles of cholinergic neurons in C. elegans, unc-17 likely encodes the vesicular transporter of acetylcholine. Mutations that eliminated all unc-17 gene function were lethal, suggesting that the acetylcholine transporter is essential. Molecular analysis of unc-17 mutations will allow the correlation of specific parts of the gene (and the protein) with observed functional defects. The mutants will also be useful for the isolation of extragenic suppressors, which could identify genes encoding proteins that interact with UNC-17.
42983584
Functional Properties of the unc-64 Gene Encoding aCaenorhabditis elegans Syntaxin*
Phenotypes of Caenorhabditis elegans unc-18 and unc-64 gene mutations are similar. Whileunc-18 is known to be essential for normal synaptic transmission (Hosono, R., Hekimi, S., Kamiya, Y., Sassa, T., Murakami, S., Nishiwaki, S., Miwa, J., Taketo, A., and Kodaira, K.-I. (1992)J. Neurochem. 58, 1517–1525), the function ofunc-64 remains unclear. Here we describe the cloning, and the molecular and genetic characterization of the unc-64gene, especially in relation to unc-18. unc-64 encodes a protein (C. elegans syntaxin) showing sequence and structural similarities to mammalian syntaxin 1A. Fromunc-64, at least three types of poly(A)+ RNA are transcribed, which encode two types of syntaxin that differ in the deduced transmembrane domain. In gene expression, unc-64closely resembles unc-18, that is, both are expressed in neural cells, especially in motor neurons and neurons constituting head ganglions. C. elegans syntaxin binds to UNC-18 with high affinity. The unc-64 (e246) mutation producing a mild phenotype causes an Ala → Val conversion in the conserved COOH-terminal region in mammalian syntaxin 1A or Drosophilasyntaxin-1A whose site is included in three types of transcripts. The binding of the mutant C. elegans syntaxin to UNC-18 is greatly reduced, indicating the mutation site contributes to the binding.
12124034
The Caenorhabditis elegans unc-64 locus encodes a syntaxin that interacts genetically with synaptobrevin.
We describe the molecular cloning and characterization of the unc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63-64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those of Drosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.
30965606
The unc‐18 Gene Encodes a Novel Protein Affecting the Kinetics of Acetylcholine Metabolism in the Nematode Caenorhabditis elegans
Abstract: Genes affecting acetylcholine (ACh) levels without influencing choline acetyltransferase activity have been identified in Caenorhabditis elegans. We have examined one such gene, unc‐18. We isolated a transposon‐insertion allele for unc‐18 and used it to clone a genomic region containing the unc‐18 locus. The unc‐18 location within this region was determined by rescuing the unc‐18 mutant phenotype in a germline transformation experiment and identifying transcripts affected by four independent unc‐18 mutations. A single‐sized poly(A)+ RNA was synthesized from the gene. Expression of the transcript appears to be stage specific: The transcript is found in abundance at the early larval stage but in decreased amounts at the fourth larval and the adult stages. These results show that the unc‐18 gene plays a role in development as well as in the kinetics of ACh metabolism.
15043008
Control of Alternative Behavioral States by Serotonin in Caenorhabditis elegans
Serotonin has been implicated in the regulation of a wide range of brain functions involving alternative behavioral states, including the control of mood, aggression, sex, and sleep. Here, we report that in the nematode Caenorhabditis elegans, serotonin controls a switch between two distinct, on/off states of egg-laying behavior. Through quantitative analysis of the temporal pattern of egg-laying events, we determined that egg laying can be modeled as a novel random process, in which animals fluctuate between discrete behavioral states: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs are retained. Single-cell ablation experiments indicate that two pairs of motor neurons, HSNL/HSNR and VC4/VC5, can induce the active phase by releasing serotonin. These neurons also release acetylcholine, which appears to trigger individual egg-laying events within the active phase. Genetic experiments suggest that determination of the behavioral states observed for C. elegans egg laying may be mediated through protein kinase C-dependent (PKC-dependent) modulation of voltage-gated calcium channels.
15698387
Egg-laying defective mutants of the nematode Caenorhabditis elegans.
We have isolated 145 fertile mutants of C. elegans that are defective in egg laying and have characterized 59 of them genetically, behaviorally and pharmacologically. These 59 mutants define 40 new genes called egl. for egg-laying abnormal. Most of the other mutants are defective in previously identified genes. The egl mutants differ with respect to the severity of their egg-laying defects and the presence of behavioral or morphological pleiotropies. We have defined four distinct categories of mutants based on their responses to the pharmacological agents serotonin and imipramine, which stimulate egg laying by wild-type hermaphrodites. These drugs test the functioning of the vulva, the vulval and uterine muscles and the hermaphrodite-specific neurons (HSNs), which innervate the vulval muscles. Mutants representing 14 egl genes fail to respond to serotonin and to imipramine and are likely to be defective in the functioning of the vulva or the vulval and uterine muscles. Four mutants (representing four different genes) lay eggs in response to serotonin but not to imipramine and appear to be egg-laying defective because of defects in the HSNs; three of these four were selected specifically for these drug responses. Mutants representing seven egl genes lay eggs in response to serotonin and to imipramine. One egl mutant responds to imipramine but not to serotonin. The remaining egl mutants show variable or intermediate responses to the drugs. Two of the HSN-defective mutants, egl-1 and her-1(n695), lack HSN cell bodies and are likely to be expressing the normally male-specific program of HSN cell death. Whereas egl-1 animals appear to be defective specifically in HSN development, her-1(n695) animals exhibit multiple morphological pleiotropies, displaying partial transformation of the sexual phenotype of many cells and tissues. At least two of the egl mutants appear to be defective in the processing of environmental signals that modulate egg laying and may define new components of the neural circuitry that control egg laying.
33706469
Alternative splicing leads to two cholinergic proteins in Caenorhabditis elegans.
The cha-1 gene of Caenorhabditis elegans encodes choline acetyl-transferase (the acetylcholine synthetic enzyme). The C. elegans unc-17 gene encodes a synaptic vesicle-associated acetylcholine transporter. The two genes thus define sequential biochemical steps in the metabolism of the neurotransmitter acetylcholine. Cloning, sequencing, and molecular analysis of the unc-17 region indicate that cha-1 and unc-17 transcripts share a 5' untranslated exon, and the rest of the unc-17 transcript is nested within the long first intron of cha-1. Thus, two proteins with related functions but with no sequences in common are produced as a result of alternative splicing of a common mRNA precursor. The structure of this transcription unit suggests a novel type of coordinate gene expression, and a temporal processing model is proposed for the regulation of cha-1 and unc-17 expression.
5002665
The embryonic cell lineage of the nematode Caenorhabditis elegans.
The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.
1173216
UNC-13 is required for synaptic vesicle fusion in C. elegans
We analyzed the synaptic physiology of unc-13 mutants in the nematode C. elegans. Mutants of unc-13 had normal nervous system architecture, and the densities of synapses and postsynaptic receptors were normal at the neuromuscular junction. However, the number of synaptic vesicles at neuromuscular junctions was two- to threefold greater in unc-13 mutants than in wild-type animals. Most importantly, evoked release at both GABAergic and cholinergic synapses was almost absent in unc-13 null alleles, as determined by whole-cell, voltage-clamp techniques. Although mutant synapses had morphologically docked vesicles, these vesicles were not competent for release as assayed by spontaneous release in calcium-free solution or by the application of hyperosmotic saline. These experiments support models in which UNC-13 mediates either fusion of vesicles during exocytosis or priming of vesicles for fusion.
9089749
Synaptic vesicle endocytosis.
Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization.
41604904
Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.
Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.
4348719
Essential functions of synapsins I and II in synaptic vesicle regulation
SYNAPTIC vesicles are coated by synapsins, phosphoproteins that account for 9% of the vesicle protein1-3. To analyse the functions of these proteins, we have studied knockout mice lacking either synapsin I, synapsin II, or both. Mice lacking synapsins are viable and fertile with no gross anatomical abnormalities, but experience seizures with a frequency proportional to the number of mutant alleles. Synapsin-II and double knockouts, but not synapsin-I knockouts, exhibit decreased post-tetanic potentiation and severe synaptic depression upon repetitive stimulation. Intrinsic synaptic-vesicle membrane proteins, but not peripheral membrane proteins or other synaptic proteins, are slightly decreased in individual knockouts and more severely reduced in double knockouts, as is the number of synaptic vesicles. Thus synapsins are not required for neurite outgrowth, synaptogenesis or the basic mechanics of synaptic vesicle traffic, but are essential for accelerating this traffic during repetitive stimulation. The phenotype of the synapsin knockouts could be explained either by deficient recruitment of synaptic vesicles to the active zone, or by impaired maturation of vesicles at the active zone, both of which could lead to a secondary destabilization of synaptic vesicles.
6469759
Synapsin I deficiency results in the structural change in the presynaptic terminals in the murine nervous system
Synapsin I is one of the major synaptic vesicle-associated proteins. Previous experiments implicated its crucial role in synaptogenesis and transmitter release. To better define the role of synapsin I in vivo, we used gene targeting to disrupt the murine synapsin I gene. Mutant mice lacking synapsin I appeared to develop normally and did not have gross anatomical abnormalities. However, when we examined the presynaptic structure of the hippocampal CA3 field in detail, we found that the sizes of mossy fiber giant terminals were significantly smaller, the number of synaptic vesicles became reduced, and the presynaptic structures altered, although the mossy fiber long-term potentiation remained intact. These results suggest significant contribution of synapsin I to the formation and maintenance of the presynaptic structure.
36151274
Molecular evolution of the synapsin gene family.
Synapsins, a family of synaptic vesicle proteins, play a crucial role in the regulation of neurotransmission and synaptogenesis. They have been identified in a variety of invertebrate and vertebrate species, including human, rat (Rattus norvegicus), cow (Bos taurus), longfin squid (Loligo pealei), and fruit fly (Drosophila melanogaster). Here, synapsins were cloned from three additional species: frog (Xenopus laevis), lamprey (Lampetra fluviatilis), and nematode (Caenorhabditis elegans). Synapsin protein sequences from all these species were then used to explore the molecular phylogeny of these important neuronal phosphoproteins. The ancestral condition of a single synapsin gene probably gave rise to the vertebrate synapsin gene family comprised of at least three synapsin genes (I, II, and III) in higher vertebrates. Synapsins possess multiple domains, which have evolved at different rates throughout evolution. In invertebrate synapsins, the most conserved domains are C and E. During the evolution of vertebrates, at least two gene duplication events are hypothesized to have given rise to the synapsin gene family. This was accompanied by the emergence of an additional conserved domain, termed A. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:360-377, 1999.
39549475
Organogenesis in C. elegans: Positioning of neurons and muscles in the egg-laying system
One of the final stages in the development of egg-laying behavior in the nematode C. elegans is the organization of 8 motor neurons (2 HSN and 6 VC cells) and 8 muscles into a motor system to control the opening of the vulva. Using mutations that disrupt the development of specific components of the egg-laying system and laser microsurgery to ablate selected precursor cells, we have determined that the guidance of the egg-laying neurons and muscles, and in particular the VC neurons and vulval muscles, into the vulval region is dependent on interactions with surrounding epithelial and gonadal tissue and appears to be independent of neuron-neuron and neuron-muscle interactions. The development of the egg-laying system can be described as a series of cell interactions in which certain cells arise through induction and subsequently provide inductive cues themselves.
33876301
Localization of FMRF amide‐like peptides in Caenorhabditis elegans
The neuropeptide FMRFamide (Phe‐Met‐Arg‐Phe‐NH2) is a member of a large family of related peptides that have been found throughout the animal kingdom. By using an antiserum specific for the Arg‐Phe‐NH2 moiety, we have found that about 10% of the neurons in the nematode Caenorhabditis elegans are immunoreactive. Most of these neurons, which include sensory, motor, and interneurons, were identified on the basis of their number, position, and projection pattern and by analysis of characterized mutants. Neurons that were immunoreactive in hermaphrodite animals were generally also found in males, but each sex had, in addition, sex‐specific immunoreactive cells. Staining of hermaphrodite animals from different larval stages suggests that the onset of FMRFamide‐like expression is differentially regulated among the cells. We have found a possible neuromodulatory role for the related peptide FLRFamide (Phe‐Leu‐Arg‐Phe‐NH2). In an egg‐laying assay, FLRFamide by itself was not active but could potentiate a serotonin effect. The FMRFamide‐like immunoreactivity was also used as a marker to examine the differentiation of cells that normally undergo programmed cell death. Cells that are destined to die in the Pn.a lineages appear to differentiate and adopt the fate of lineally equivalent cells before cell death.
6550037
Neuronal Control of Locomotion in C. elegans Is Modified by a Dominant Mutation in the GLR-1 Ionotropic Glutamate Receptor
How simple neuronal circuits control behavior is not well understood at the molecular or genetic level. In Caenorhabditis elegans, foraging behavior consists of long, forward movements interrupted by brief reversals. To determine how this pattern is generated and regulated, we have developed novel perturbation techniques that allow us to depolarize selected neurons in vivo using the dominant glutamate receptor mutation identified in the Lurcher mouse. Transgenic worms that expressed a mutated C. elegans glutamate receptor in interneurons that control locomotion displayed a remarkable and unexpected change in their behavior-they rapidly alternated between forward and backward coordinated movement. Our findings suggest that the gating of movement reversals is controlled in a partially distributed fashion by a small subset of interneurons and that this gating is modified by sensory input.
4380457
CaMKII regulates the density of central glutamatergic synapses in vivo
Synaptic connections undergo a dynamic process of stabilization or elimination during development, and this process is thought to be critical in memory and learning and in establishing the specificity of synaptic connections. The type II calcium- and calmodulin-dependent protein kinase (CaMKII) has been proposed to be pivotal in regulating synaptic strength and in maturation of synapses during development. Here we describe how CaMKII regulates the formation of central glutamatergic synapses in Caenorhabditis elegans. During larval development, the density of ventral nerve cord synapses containing the GLR-1 glutamate receptor is held constant despite marked changes in neurite length. The coupling of synapse number to neurite length requires both CaMKII and voltage-gated calcium channels. CaMKII regulates GLR-1 by at least two distinct mechanisms: regulating transport of GLR-1 from cell bodies to neurites; and regulating the addition or maintenance of GLR-1 to postsynaptic elements.
18956893
NSF Binding to GluR2 Regulates Synaptic Transmission
Here, we show that N-ethylmaleimide-sensitive fusion protein (NSF) interacts directly and selectively with the intracellular C-terminal domain of the GluR2 subunit of AMPA receptors. The interaction requires all three domains of NSF but occurs between residues Lys-844 and Gln-853 of rat GluR2, with Asn-851 playing a critical role. Loading of decapeptides corresponding to the NSF-binding domain of GluR2 into rat hippocampal CA1 pyramidal neurons results in a marked, progressive decrement of AMPA receptor-mediated synaptic transmission. This reduction in synaptic transmission was also observed when an anti-NSF monoclonal antibody (mAb) was loaded into CA1 neurons. These results demonstrate a previously unsuspected direct interaction in the postsynaptic neuron between two major proteins involved in synaptic transmission and suggest a rapid NSF-dependent modulation of AMPA receptor function.
34052780
The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs.
In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.
163413
Interaction of the N-Ethylmaleimide–Sensitive Factor with AMPA Receptors
Glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system (CNS) and play important roles in synaptic plasticity and neuronal development. Recently, protein-protein interactions with the C-terminal domain of glutamate receptor subunits have been shown to be involved in the modulation of receptor function and clustering at excitatory synapses. In this paper, we have found that the N-ethylmaleimide-sensitive factor (NSF), a protein involved in membrane fusion events, specifically interacts with the C terminus of the GluR2 and GluR4c subunits of AMPA receptors in vitro and in vivo. Moreover, intracellular perfusion of neurons with a synthetic peptide that competes with the interaction of NSF and AMPA receptor subunits rapidly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), suggesting that NSF regulates AMPA receptor function.
27350829
Postsynaptic membrane fusion and long-term potentiation.
The possibility that membrane fusion events in the postsynaptic cell may be required for the change in synaptic strength resulting from long-term potentiation (LTP) was examined. Introducing substances into the postsynaptic cell that block membrane fusion at a number of different steps reduced LTP. Introducing SNAP, a protein that promotes membrane fusion, into cells enhanced synaptic transmission, and this enhancement was significantly less when generated in synapses that expressed LTP. Thus, postsynaptic fusion events, which could be involved either in retrograde signaling or in regulating postsynaptic receptor function or both, contribute to LTP.
6437168
Postsynaptic expression of tetanus toxin light chain blocks synaptogenesis in Drosophila
During the development of the nervous system embryonic neurons are incorporated into neural networks that underlie behaviour. For example, during embryogenesis in Drosophila, motor neurons in every body segment are wired into the circuitry that drives the simple peristaltic locomotion of the larva. Very little is known about the way in which the necessary central synapses are formed in such a network or how their properties are controlled. One possibility is that presynaptic and postsynaptic elements form relatively independently of each other. Alternatively, there might be an interaction between presynaptic and postsynaptic neurons that allows for adjustment and plasticity in the embryonic network. Here we have addressed this issue by analysing the role of synaptic transmission in the formation of synaptic inputs onto identified motorneurons as the locomotor circuitry is assembled in the Drosophila embryo. We targeted the expression of tetanus toxin light chain (TeTxLC) to single identified neurons using the GAL4 system. TeTxLC prevents the evoked release of neurotransmitter by enzymatically cleaving the synaptic-vesicle-associated protein neuronal-Synaptobrevin (n-Syb) [1]. Unexpectedly, we found that the cells that expressed TeTxLC, which were themselves incapable of evoked release, showed a dramatic reduction in synaptic input. We detected this reduction both electrophysiologically and ultrastructurally.
4325229
Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans
SYNAPTOTAGMIN, an integral membrane protein of the synaptic vesicle1,2, binds calcium and interacts with proteins of the plasma membrane4–6. These observations suggest several possible functions for synaptotagmin in synaptic vesicle dynamics: it could facilitate exocytosis by promoting calcium-dependent fusion3, inhibit exocytosis by preventing fusion7, or facilitate endocytosis of synaptic vesicles from the plasma membrane by acting as a receptor for the endocytotic proteins of the clathrin AP2 complex8. Here we show that synaptic vesicles are depleted at synaptic terminals in synaptotagmin mutants of the nematode Caenorhabditis elegans. This depletion is not caused by a defect in transport or by increased synaptic vesicle release, but rather by a defect in retrieval of synaptic vesicles from the plasma membrane. Thus we propose that, as well as being involved in exocytosis, synaptotagmin functions in vesicular recycling.