Synaptic Vesicle Docking Research Articles

Rac GTPase Plays an Essential Role in Exocytosis by Controlling the Fusion Competence of Release Sites

The role of small GTPases of the Rho family in synaptic functions has been addressed by analyzing the effects of lethal toxin (LT) from Clostridium sordellii strain IP82 (LT82) on neurotransmitter release at evoked identified synapses in the buccal ganglion of Aplysia. LT82 is a large monoglucosyltranferase that uses UDP-glucose as cofactor and glucosylates Rac (a small GTPase related to Rho), and Ras, Ral, and Rap (three GTPases of the Ras family). Intraneuronal application of LT (50 nm) rapidly inhibits evoked acetylcholine (ACh) release as monitored electrophysiologically. Injection of the catalytic domain of the toxin similarly blocked ACh release, but not when key amino acids needed for glucosylation were mutated. Intraneuronal application of competitive nucleotide sugars that differentially prevent glucosylation of Rac- and Ras-related GTPases, and the use of a toxin variant that affects a different spectrum of small GTPases, established that glucosylation of Rac is responsible for the reduction in ACh release. To determine the quantal release parameters affected by Rac glucosylation, we developed a nonstationary analysis of the fluctuations in postsynaptic response amplitudes that was performed before and after the toxin had acted or during toxin action. The results indicate that neither the quantal size nor the average probability for release were affected by lethal toxin action. ACh release blockage by LT82 was only caused by a reduction in the number of functional release sites. This reveals that after docking of synaptic vesicles, vesicular Rac stimulates a membrane effector (or effectors) essential for the fusion competence of the exocytotic sites.

PolyOne sees revenues improve from the fourth quarter

We investigated the distribution of N-type voltage-dependent calcium channels that mediate Ca2+ entry initiating transmitter release in the rat vestibular sensory epithelium. We used confocal microscopy to assess the in vitro labeling by fluorescent specific ligand binding, ω-conotoxin-GVIA and also the immunolabeling of presynaptic soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor (SNARE) proteins, syntaxin, 25,000 mol. wt synaptosome-associated protein and synaptotagmin: components of the neurotransmitter exocytosis machinery. We found that there was a close anatomical association between the voltage-gated calcium channels, the synaptic vesicle and synaptic membrane-associated proteins on the afferent nerve calyces and probably afferent boutons, which are postsynaptic compartments.Our data suggest that these peripheral afferent endings possess the presynaptic Ca2+ channels and the components of the presynaptic SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. They provide additional evidence for a secretory function and efferent role of these endings in hair cell neurotransmission.

Substrates regulate gamma-aminobutyric acid transporters in a syntaxin 1A-dependent manner.

Several ion channels and pumps are regulated by syntaxin 1A, a component of the synaptic vesicle docking and fusion apparatus. One such regulated protein is the rat brain gamma-aminobutyric acid (GABA) transporter GAT1. The N-terminal cytoplasmic domain of GAT1 directly interacts with syntaxin 1A; this interaction induces a decrease in the rate at which GABA and associated ions are transported. GAT1 function also is regulated by transporter substrates, raising the possibility that substrates mediate at least some of their effects by regulating the interaction between GAT1 and syntaxin 1A. In oocytes expressing GAT1 and syntaxin 1A, superfusion of transporter substrates increases the GAT1 transport rate. The substrate-induced rate change (i) is prevented by coapplication of GAT1 antagonists, (ii) does not occur in oocytes expressing GAT1 alone, and (iii) does not occur in oocytes expressing interaction-deficient syntaxin 1A mutants. In oocytes, and in hippocampal neurons that endogenously express both GAT1 and syntaxin 1A, substrate application results in a decrease in the fraction of syntaxin 1A that is bound to GAT1 on a time-scale comparable to the substrate-induced change in transport rates. These data suggest that substrate translocation regulates GAT1-syntaxin 1A interactions and provide a mechanism by which GABA transport can be increased during times of rising synaptic GABA concentrations.

Association between a polymorphism in the pseudoautosomal X-linked gene SYBL1 and bipolar affective disorder.

In the past decade, several chromosomal regions have been analyzed for linkage with bipolar affective disorder (BPAD). There have been conflicting results regarding the involvement of X-chromosomal regions in harboring susceptibility genes for BPAD. Recently, a new candidate gene (SYBL1) for BPAD has been described on Xq28. SYBL1, which maps to the Xq pseudoautosomal region (PAR), encodes a member of the synaptobrevin family of proteins involved in synaptic vesicle docking, exocytosis, and membrane transport. A subsequent case-control association study, including 110 US-American patients with BPAD and 119 unrelated controls, investigated a potential etiological role of a novel polymorphism (G-->C transversion) in a regulatory region of the SYBL1 gene. In this analysis, the C allele showed a statistical trend to be more frequent in males with BPAD than in respective controls (P=0.06). This finding prompted us to verify whether a similar effect was also present in a larger German sample of 164 unrelated patients with BPAD (148 patients with BP I disorder, 16 patients with BP II disorder) and 267 controls. We observed a significantly increased frequency of genotypes homozygous for the C allele in females with BPAD in comparison with controls (P=0.017). Thus, our data strengthen the role of the SYBL1 gene as a candidate gene for BPAD.

Neuromuscular Transmission and Muscle Contractility in SNAP-25-deficient Coloboma Mice

Synaptosomal associated protein of 25 kDa (SNAP-25) is a cytoplasmic protein that participates in the docking and fusion of synaptic vesicles with the nerve terminal in preparation for neurotransmitter release. SNAP-25 is also a substrate for three of the seven serotypes of botulinum neurotoxin (BoNT). Intoxication by BoNT/A, /C1 or /E results in weakness and paralysis of skeletal muscle due to cleavage of SNAP-25 (and syntaxin 1a in the case /C1) at discrete serotype-specific sites. To elucidate the role of SNAP-25 in muscle function in more detail, contractility and neuromuscular transmission were studied in a mutant mouse model termed coloboma. The coloboma mutation results from a contiguous deletion of 1–2 centiMorgans on chromosome 2, which includes the entire SNAP-25 locus and three other identified genes. Homozygotes do not survive beyond gestation day 6; heterozygotes (C m/+) have a normal life-span but express reduced levels of SNAP-25 mRNA and protein in the brain. The consequences of the C m/+ mutation on twitch and tetanic tension, quantal release of neurotransmitter and spinal motoneuron expression of SNAP-25 were examined in the present study. Contrary to expectations, C m/+ mice exhibited no alteration in twitch tension and generated normal tetanic tension even at the highest frequency examined (800 Hz). Microelectrode recordings revealed that MEPP amplitude and frequency were both within control limits. The ventral spinal cord of C m/+ mice showed no deficiency in SNAP-25 content and immunohistochemical examination of nerve terminals in C m/+ mice disclosed that SNAP-25 levels and distribution were similar to those of control mice. It is concluded that spinal motor neurons up-regulate SNAP-25 to preserve vital neuromuscular function.

Neurotransmitter release through the V0 sector of V-ATPase.

Neurotransmitter release through the V0 sector of V-ATPase.

Mutations in the second C2 domain of synaptotagmin disrupt synaptic transmission at Drosophila neuromuscular junctions

The synaptic vesicle protein, synaptotagmin, has been hypothesized to mediate several functions in neurotransmitter release including calcium sensing, vesicle recycling, and synaptic vesicle docking. These hypotheses are based on evidence from in vitro binding assays, peptide and antibody injection experiments, and genetic knockout studies. Synaptotagmin contains two domains that are homologous to the calcium ion (Ca2+)-binding C2 domain of protein kinase C. The two C2 domains of synaptotagmin have broadly differing ligand-binding properties. We have focused on the second C2 domain (C2B) of synaptotagmin I, in particular, on a series of conserved lysine residues on β-strand 4 of C2B. This polylysine motif binds clathrin-adapter protein AP-2, neuronal calcium channels, and inositol high polyphosphates. It also mediates Ca2+-dependent oligomerization. To investigate the importance of these lysine residues in synaptic transmission, we have introduced synaptotagmin I (syt) transgenes harboring specific polylysine motif mutations into flies otherwise lacking the synaptotagmin I protein (sytnull). Electrophysiological analyses of these mutants revealed that evoked transmitter release is decreased by ≈36% and that spontaneous release is increased approximately twofold relative to sytnull flies that express a wild type syt transgene. Synaptotagmin expression in both the mutant and the wild type transgenic lines was equivalent, as measured by semiquantitative Western blot analysis. Thus, the alteration in synaptic transmission was due to the mutation and not to the level of synaptotagmin expression. We conclude that synaptotagmin interactions mediated by the C2B polylysine motif are required to attain full synaptotagmin function in vivo. J. Comp. Neurol. 436:4–16, 2001. © 2001 Wiley-Liss, Inc.

Mutations in the second C2 domain of synaptotagmin disrupt synaptic transmission at Drosophila neuromuscular junctions

The synaptic vesicle protein, synaptotagmin, has been hypothesized to mediate several functions in neurotransmitter release including calcium sensing, vesicle recycling, and synaptic vesicle docking. These hypotheses are based on evidence from in vitro binding assays, peptide and antibody injection experiments, and genetic knockout studies. Synaptotagmin contains two domains that are homologous to the calcium ion (Ca(2+))-binding C2 domain of protein kinase C. The two C2 domains of synaptotagmin have broadly differing ligand-binding properties. We have focused on the second C2 domain (C2B) of synaptotagmin I, in particular, on a series of conserved lysine residues on beta-strand 4 of C2B. This polylysine motif binds clathrin-adapter protein AP-2, neuronal calcium channels, and inositol high polyphosphates. It also mediates Ca(2+)-dependent oligomerization. To investigate the importance of these lysine residues in synaptic transmission, we have introduced synaptotagmin I (syt) transgenes harboring specific polylysine motif mutations into flies otherwise lacking the synaptotagmin I protein (syt(null)). Electrophysiological analyses of these mutants revealed that evoked transmitter release is decreased by approximately 36% and that spontaneous release is increased approximately twofold relative to syt(null) flies that express a wild type syt transgene. Synaptotagmin expression in both the mutant and the wild type transgenic lines was equivalent, as measured by semiquantitative Western blot analysis. Thus, the alteration in synaptic transmission was due to the mutation and not to the level of synaptotagmin expression. We conclude that synaptotagmin interactions mediated by the C2 B polylysine motif are required to attain full synaptotagmin function in vivo.

SNARE Proteins Are Critical for Regulated Exocytosis of ECP from Human Eosinophils

The SNARE hypothesis, describing a protein assembly–disassembly pathway, was recently proposed for the sequential steps of synaptic vesicle docking, activation, and fusion. To determine if SNARE proteins are involved in regulated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fractionation, and immunocytochemistry documented that vesicle-associated membrane protein-2 (VAMP-2), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR products amplified from a domain conserved among VAMP isoforms revealed identity only to VAMP-2 but not to VAMP-1 or cellubrevin. Functional experiments revealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophils, significantly inhibited both IgE receptor- and phorbol ester-mediated exocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabilized eosinophils. Thus, these results strongly suggest a critical role of SNAREs in regulated exocytosis in eosinophils.

Assembling the Presynaptic Active Zone: A Characterization of an Active Zone Precursor Vesicle

The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an ∼80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.

Vesicle-associated membrane protein isoforms in the tiger salamander retina.

Vesicle associated membrane protein (VAMP; also known as synaptobrevin) is a key component of the core complex needed for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Recent work indicates that the precise complement of presynaptic proteins associated with transmitter release and their isoforms vary among synapses, presumably conferring specific functional release properties. The retina contains two types of vesicular synapses with distinct morphologic, functional, and biochemical characteristics: ribbon and conventional synapses. Although the precise complement of presynaptic proteins is known to differ between conventional and ribbon synapses and among conventional synapses, the distribution of VAMP isoforms among retinal synapses has not been determined. The expression and localization of VAMP isoforms in the salamander retina, a major model system for studies of retinal circuitry, was examined by using immunocytochemical and immunoblotting methods. Both methods indicated that at least two VAMP isoforms were expressed in salamander retina. One isoform, recognized by an immunoglobulin M antibody that recognizes both mammalian VAMP-1 and VAMP-2, was associated with photoreceptor and bipolar cell terminals as well as many conventional synapses, and probably corresponds to mammalian VAMP-2. A different VAMP isoform associated with a subset of amacrine cells, was recognized only by antibodies directed against the N-terminus of mammalian VAMP-2. An antiserum directed against the N-terminus of mammalian VAMP-1 did not specifically recognize any salamander VAMPs in either immunocytochemical or immunoblotting experiments. Heterogeneous distribution of VAMP isoforms among conventional retinal synapses was confirmed by double labeling for synapsin I, a marker for conventional synapses. These studies indicate that VAMP isoforms are expressed heterogeneously among retinal synapses but cannot account for the differences in transmitter release characteristics at ribbon and conventional synapses. These results also corroborate previous studies in Xenopus indicating that the N-terminus of nonmammalian VAMP isoforms differs from their mammalian counterparts.

The presynaptic cytomatrix of brain synapses.

Synapses are principal sites for communication between neurons via chemical messengers called neurotransmitters. Neurotransmitters are released from presynaptic nerve terminals at the active zone, a restricted area of the cell membrane situated exactly opposite to the postsynaptic neurotransmitter reception apparatus. At the active zone neurotransmitter-containing synaptic vesicles (SVs) dock, fuse, release their content and are recycled in a strictly regulated manner. The cytoskeletal matrix at the active zone (CAZ) is thought to play an essential role in the organization of this SV cycle. Several multi-domain cytoskeleton-associated proteins, including RIM, Bassoon, Piccolo/Aczonin and Munc-13, have been identified, which are specifically localized at the active zone and thus are putative molecular components of the CAZ. This review will summarize our present knowledge about the structure and function of these CAZ-specific proteins. Moreover, we will review our present view of how the exocytotic and endocytic machineries at the site of neurotransmitter release are linked to and organized by the presynaptic cytoskeleton. Finally, we will summarize recent progress that has been made in understanding how active zones are assembled during nervous system development.

SNARE Proteins Are Critical for Regulated Exocytosis of ECP from Human Eosinophils

The SNARE hypothesis, describing a protein assembly–disassembly pathway, was recently proposed for the sequential steps of synaptic vesicle docking, activation and fusion. To determine if SNARE proteins are involved in regulated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fractionation, and immunocytochemistry documented that vesicle-associated membrane protein-2 (VAMP-2), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR products amplified from a domain conserved among VAMP isoforms revealed identity only to VAMP-2 but not to VAMP-1 or cellubrevin. Functional experiments revealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophils, significantly inhibited both IgE receptor- and phorbol ester-mediated exocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabilized eosinophils. Thus, these results strongly suggest a critical role of SNAREs in regulated exocytosis in eosinophils.

Role of the conserved WHXL motif in the C terminus of synaptotagmin in synaptic vesicle docking.

Synaptotagmin (Syt) I, an abundant synaptic vesicle protein, consists of one transmembrane region, two C2 domains, and a short C terminus. This protein is essential for both synaptic vesicle exocytosis and endocytosis via its C2 domains. Although the short C terminus is highly conserved among the Syt family and across species, little is known about the exact role of the conserved C terminus of Syt I. In this paper, we report a function of the Syt I C terminus in synaptic vesicle docking at the active zones. Presynaptic injection of a peptide corresponding to the C-terminal 21 amino acids of Syt I (named Syt-C) into the squid giant synapse blocked synaptic transmission without affecting the presynaptic action potential or the presynaptic Ca(2+) currents. The same procedure repeated with a mutant C-terminal peptide (Syt-CM) had no effect on synaptic transmission. Repetitive presynaptic stimulation with Syt-C produced a rapid decrease in the amplitude of the postsynaptic potentials as the synaptic block progressed, indicating that the peptide interferes with the docking step rather than the fusion step of synaptic vesicles. Electron microscopy of the synapses injected with the Syt-C peptide showed a marked decrease in the number of docked synaptic vesicles at the active zones, as compared with controls. These results indicate that Syt I is a multifunctional protein that is involved in at least three steps of synaptic vesicle cycle: docking, fusion, and reuptake of synaptic vesicles.

RIBEYE, a Component of Synaptic Ribbons: A Protein's Journey through Evolution Provides Insight into Synaptic Ribbon Function

Photoreceptor cells utilize ribbon synapses to transmit sensory signals at high resolution. Ribbon synapses release neurotransmitters tonically, with a high release rate made possible by continuous docking of synaptic vesicles on presynaptic ribbons. We have partially purified synaptic ribbons from retina and identified a major protein component called RIBEYE. RIBEYE is composed of a unique A domain specific for ribbons, and a B domain identical with CtBP2, a transcriptional repressor that in turn is related to 2-hydroxyacid dehydrogenases. The A domain mediates assembly of RIBEYE into large structures, whereas the B domain binds NAD + with high affinity, similar to 2-hydroxyacid dehydrogenases. Our results define a unique component of synaptic ribbons and suggest that RIBEYE evolved in vertebrates under utilization of a preexisting protein to build a unique scaffold for a specialized synapse.

Persistence of botulinum neurotoxin A demonstrated by sequential administration of serotypes A and E in rat EDL muscle

Botulinum neurotoxin serotypes A (BoNT/A) and E (BoNT/E) inhibit neurotransmitter release from peripheral cholinergic nerve terminals by cleaving different sites on SNAP-25, a protein involved in synaptic vesicle docking and exocytosis. Since recovery from BoNT/A is protracted, but reversal of BoNT/E intoxication is relatively rapid, it was of interest to determine whether sequential exposure to BoNT/A and BoNT/E could provide insight into the factors responsible for persistence of BoNT action. Extensor digitorum longus (EDL) muscles from rats were injected locally with 5 mouse LD 50 units of BoNT/A or 20 mouse LD 50 units of BoNT/E; these doses were selected to produce total paralysis of EDL muscles within 48 hr. Additional groups of rats were injected sequentially with either BoNT/A followed 48 h later by BoNT/E or with BoNT/E followed 48 h later by BoNT/A. Muscle tensions were elicited in situ in response to supramaximal stimulation of the peroneal nerve to monitor recovery from BoNT intoxication. Tensions returned to 53% and 94% of control, respectively, 7 and 15 days after injection of BoNT/E. In contrast, tensions in muscles injected with BoNT/A returned to only 2% and 12% of control at these time points. Preparations injected sequentially with BoNT/A followed by BoNT/E or with BoNT/E followed by BoNT/A exhibited slow recovery times resembling those recorded in the presence of BoNT/A alone. Pronounced atrophy of the EDL muscle was observed in rats injected with BoNT/A or in those receiving serotype combinations in either sequence, whereas no loss of muscle mass was observed in animals treated with BoNT/E alone. Data suggesting that BoNT/E can enter BoNT/A-treated preparations was obtained by findings that 3,4-diaminopyridine, which readily reversed muscle paralysis after BoNT/A exposure, lost this ability within 1 h of BoNT/E addition. Evidence that BoNT/E was able to cleave SNAP-25 at its characteristic site during sequential neurotoxin exposure was demonstrated by western blot analysis of cultured primary cortical neurons. Since the sequential exposure studies indicate that recovery from BoNT intoxication is lengthened by exposure to serotype A, but not shortened by exposure to serotype E, the duration of BoNT/A intoxication appears to be determined predominantly by the intracellular stability of catalytically active BoNT/A light chain.

SNAP-25 Functional Domains in SNARE Core Complex Assembly and Glutamate Release of Cerebellar Granule Cells

Synaptosomal associated protein of 25 kDa (SNAP-25) is a member of the SNARE protein complex that has been implicated in synaptic vesicle docking and fusion. In this report, we have generated SNAP-25 mutants and assayed their functions in SNARE complex formation and glutamate release from cultured rat cerebellar granule cells. In vitro binding studies show that a deletion mutant lacking the C-terminal 181-206 amino acid sequence inhibits the formation of the SNARE core complex. Additional deletion of an N-terminal 1-31 amino acid sequence abolished this inhibitory activity. Adenovirus-mediated gene transfer is used to overexpress wild-type and mutant SNAP-25 in cerebellar granule cells. Neurons overexpressing the wild-type protein show slight reductions in glutamate release, ranging from 10 to 15% in both the developing and mature granule cells. A 30-35% inhibition is obtained with the C-terminal deletion mutant, and the inhibitory effect is abolished in the N- and C-terminal double deletion mutant. These results demonstrate that the SNARE core complex exists in a dynamic and reversible state, and the formation of the core complex is necessary for neurotransmitter release in neurons.

Active Zones on Motor Nerve Terminals Contain α3β1 Integrin

Active zones are the sites along nerve terminals where synaptic vesicles dock and undergo calcium-dependent exocytosis during synaptic transmission. Here we show, by immunofluorescent staining with antibodies generated against Xenopus laevis integrins, that alpha3beta1 integrin is concentrated at the active zones of Xenopus motor nerve terminals. Because integrins can link extracellular matrix molecules to cytoskeletal elements and participate in the formation of signaling complexes, the localization of integrin at active zones suggests that it may play a role in the adhesion of the nerve terminals to the synaptic basal lamina, in the formation and maintenance of active zones, and in some of the events associated with calcium-dependent exocytosis of neurotransmitter. Our findings also indicate that the integrin composition of the terminal Schwann cells differs from that of the motor nerve terminals, and this may account at least in part for differences in their adhesiveness to the synaptic basal lamina.

Identification of DNA variants in the SNAP-25 gene and linkage study of these polymorphisms and attention-deficit hyperactivity disorder.

The gene for the synaptic vesicle docking fusion protein, synaptosomal-associated protein of 25 kDa (SNAP-25), has been implicated in the etiology of attention-deficit hyperactivity disorder (ADHD) based on the mouse mutant strain coloboma. This neutron-irradiation induced mouse strain is hemizygous for the deletion of the SNAP-25 gene and displays spontaneous hyperactivity that is responsive to dextroamphetamine. Because of these characteristics, this strain has been suggested to be a mouse model for ADHD. We identified using single stranded conformational polymorphism analysis (SSCP) four DNA sequence variants in the 3' untranslated region of the human SNAP-25 gene. We searched for polymorphisms in the 3' untranslated region because the intron/exon structure of this gene has not yet been determined. We tested for linkage of this gene and ADHD using two of the identified polymorphisms that change a restriction enzyme recognition site. We examined the transmission of the alleles of each of these polymorphisms and the haplotypes of both polymorphisms using the transmission disequilibrium test in a sample of 97 small nuclear families consisting of a proband with ADHD, their parents, and affected siblings. We observed biased transmission of the haplotypes of the alleles of these two polymorphisms. Our findings are suggestive of a role of this gene in ADHD.

SNAP-25 regulation during adrenal gland development: Comparison with differentiation markers and other SNAREs

Synaptosomal-associated protein of 25 kDa (SNAP-25) is one of a limited number of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) that play a major role in membrane docking of synaptic vesicles and secretory granules during regulated exocytosis. We have previously shown that SNAP-25 levels differ between noradrenergic and adrenergic chromaffin cell populations of the adult adrenal gland. We examine SNAP-25 expression by immunofluoresence in cells of the sympathoadrenal lineage in the rat during late embryonic and postnatal development. In parallel, tyrosine hydroxylase was used to identify sympathoadrenal cells, phenylethanolamine N-methyltransferase to distinguish adrenergic from noradrenergic chromaffin cells, and chromogranin A to define the presence of secretory granules. In addition, SNAP-25 protein and mRNA levels were followed in adrenal gland extracts by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Protein levels were compared with those of other molecules also implicated in organelle trafficking, including syntaxin 1 and vesicle-associated membrane protein (VAMP-2) and the nonneuronal analogues SNAP-23 and cellubrevin. This study provides evidence that SNAP-25 is expressed early during development in sympathoadrenal neurons and migrating cells. It is detected in intra-adrenal chromoblasts as soon as they enter the adrenal primordium. Its differential expression between catecholamine chromaffin cell phenotypes is already evident from the 17th embryonic day, future noradrenergic cells appearing to express higher levels than adrenergic cells. The granule maturation marker chromogranin A is expressed in chromaffin cells later than SNAP-25. Both SNAP-25 protein and mRNA increased rapidly in the adrenal gland in the perinatal period to peak during the first postnatal week, after which levels dropped dramatically to adult values. In contrast, levels of both syntaxin and SNAP-23 appeared to remain fairly constant throughout adrenal gland development. VAMP-2 expression increased gradually around birth to reach maximal levels during the first two postnatal weeks, and then decreased slightly. Cellubrevin levels also appeared to increase gradually until adult values were attained by the end of the second postnatal week. The threefold increase of SNAP-25 mRNA shortly after birth compared to the low adult levels suggests that during this period SNAP-25 is implicated in additional functions than regulated secretion, possibly associated with cellular growth or maturation.