Clustering autism: using neuroanatomical differences in 26 mouse models to gain insight into the heterogeneity.

Autism is a heritable disorder, with over 250 associated genes identified to date, yet no single gene accounts for >1-2% of cases. The clinical presentation, behavioural symptoms, imaging and histopathology findings are strikingly heterogeneous. A more complete understanding of autism can be obtained by examining multiple genetic or behavioural mouse models of autism using magnetic resonance imaging (MRI)-based neuroanatomical phenotyping. Twenty-six different mouse models were examined and the consistently found abnormal brain regions across models were parieto-temporal lobe, cerebellar cortex, frontal lobe, hypothalamus and striatum. These models separated into three distinct clusters, two of which can be linked to the under and over-connectivity found in autism. These clusters also identified previously unknown connections between Nrxn1α, En2 and Fmr1; Nlgn3, BTBR and Slc6A4; and also between X monosomy and Mecp2. With no single treatment for autism found, clustering autism using neuroanatomy and identifying these strong connections may prove to be a crucial step in predicting treatment response.

Phosphorylation and modulation of a kainate receptor (GluR6) by cAMP-dependent protein kinase.

Ligand-gated ion channels gated by glutamate constitute the major excitatory neurotransmitter system in the mammalian brain. The functional modulation of GluR6, a kainate-activated glutamate receptor, by adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) was examined with receptors expressed in human embryonic kidney cells. Kainate-evoked currents underwent a rapid desensitization that was blocked by lectins. Kainate currents were potentiated by intracellular perfusion of PKA, and this potentiation was blocked by co-application of an inhibitory peptide. Site-directed mutagenesis was used to identify the site or sites of phosphorylation on GluR6. Although mutagenesis of two serine residues, Ser684 and Ser666, was required for complete abolition of the PKA-induced potentiation, Ser684 may be the preferred site of phosphorylation in native GluR6 receptor complexes. These results indicate that glutamate receptor function can be directly modulated by protein phosphorylation and suggest that a dynamic regulation of excitatory receptors could be associated with some forms of learning and memory in the mammalian brain.

Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7.

To study the role of mGlu7 receptors (mGluR7), we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7(-/-)). After the serendipitous discovery of a sensory stimulus-evoked epileptic phenotype, we tested two convulsant drugs, pentylenetetrazole (PTZ) and bicuculline. In animals aged 12 weeks and older, subthreshold doses of these drugs induced seizures in mGluR7(-/-), but not in mGluR7(+/-), mice. PTZ-induced seizures were inhibited by three standard anticonvulsant drugs, but not by the group III selective mGluR agonist (R,S)-4-phosphonophenylglycine (PPG). Consistent with the lack of signs of epileptic activity in the absence of specific stimuli, mGluR7(-/-) mice showed no major changes in synaptic properties in two slice preparations. However, slightly increased excitability was evident in hippocampal slices. In addition, there was slower recovery from frequency facilitation in cortical slices, suggesting a role for mGluR7 as a frequency-dependent regulator in presynaptic terminals. Our findings suggest that mGluR7 receptors have a unique role in regulating neuronal excitability and that these receptors may be a novel target for the development of anticonvulsant drugs.

Altered glutamate and GABA release within thalamocortical circuitry in metabotropic glutamate receptor 4 knockout mice.

The metabotropic glutamate receptor 4 is highly expressed presynaptically on thalamocortical neurons that are involved in the pathogenesis of generalized absence seizures. Mutant mice devoid of metabotropic glutamate receptor 4 are completely resistant to absence seizures induced by low doses of GABA type A receptor antagonists. The purpose of this study was to test the hypothesis that there is altered glutamate and GABA release within thalamocortical circuitry in mice devoid of metabotropic glutamate receptor 4. Extracellular GABA and glutamate release were determined in ventrobasal thalamus, the nucleus reticularis thalami and laminae I-III, and IV-VI of cerebral cortex (laminae I-III of cerebral cortex, and laminae IV-VI of cerebral cortex) using in vivo microdialysis techniques on awake, free moving mice. A significant increase of both basal and K(+)-evoked glutamate release was detected in the ventrobasal thalamus, the nucleus reticularis thalami and laminae IV-VI of cerebral cortex of mice devoid of metabotropic glutamate receptor 4 mice. There also was a significant increase in both basal and K(+)-evoked GABA release in the mice devoid of metabotropic glutamate receptor 4, but a significant decrease of GABA release in laminae IV-VI of cerebral cortex. However, there was no alteration of either GABA or glutamate release in laminae I-III of cerebral cortex, cortical laminae that are not involved in absence seizures. These data indicate that deletion of the metabotropic glutamate receptor 4 gene results in a selective perturbation of glutamate and GABA release within the thalamocortical circuitry involved in the pathogenesis of absence seizures.

Neurochemical changes in rat brain amines after short- and long-term inhibition of monoamine oxidase by a low dose of tranylcypromine.

The effects of short- and long-term administration of a low dose of tranylcypromine on brain and urine levels of several biogenic amines and on brain activity of monoamine oxidases (MAO) A and B were investigated. MAO-A and MAO-B were inhibited by greater than 85% on day 1, and this inhibition continued to increase over the course of the study (42 days). Levels of 5-hydroxytryptamine in brain continued to increase up to day 21 and did not decline from day 21 to day 42, and levels of tranylcypromine itself continued to increase up to day 42. Dopamine concentrations peaked at day 10 and were not significantly different from that value by day 42. Brain levels of tryptamine and beta-phenylethylamine showed dramatic elevations after the first dose of the drug and remained essentially unchanged from those high values throughout the course of the drug treatment. Brain and urine increases in tryptamine and beta-phenylethylamine showed similar patterns, whereas urinary 5-hydroxytryptamine excretion reached maximal levels earlier than did brain levels.

Developmental expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body of the rat.

A preembedding immunocytochemical method for light microscopy was used to study the postnatal development of expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body (MNTB) of the rat. Immunoreactivity for mGluR4a was localized in axonal endings wrapping the principal globular neurons in MNTB, known as calyces of Held. The percentage of calyces of Held immunoreactive for mGluR4a increased progressively from postnatal day 3 (PND3), showing the highest density of labeled calyces by PND9. From this postnatal age on, a gradual reduction in the number of mGluR4a-immunopositive calyces of Held was observed, reaching the lowest level of labeled profiles in adult tissue. The developmental expression of mGluR4a in calyces of Held correlates well with previous studies in young animals showing a modulation of synaptic neurotransmission by group III mGluRs in these giant excitatory synapses made on MNTB principal neurons. All these observations together suggest that the expression of mGluR4a mainly between PND7 and PND12 might be relevant to the maturation and modulation of synaptic transmission at the calyces of Held.

Immunocytochemical localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of the rat.

This study evaluates the localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of rats using preembedding immunocytochemical methods. At the light microscopic level, punctate labeling was evident in layers Ia and Ib of the piriform cortex, and immunolabeled fibers were present in layers II and III. Following bilateral destruction of the olfactory bulb, the density of labeled puncta in layer Ia decreased. These results suggest that the receptor is present on the terminals of the lateral olfactory tract (LOT). Electron microscopic evaluation of layers Ia and Ib revealed that mGluR4a was localized in synaptic terminals in layers Ia and Ib. The terminals had clear, round synaptic vesicles and terminated on asymmetric synapses on dendritic spines and shafts. There was also immunolabeling of some dendritic profiles in layers Ia and Ib that were postsynaptic to unlabeled presynaptic terminals. These observations suggest that mGluR4a is present on presynaptic terminals in the layers of the piriform cortex that receive LOT and associational synapses. This is the same area in which previous studies have revealed the presence of mGluR7 and mGluR8, suggesting that all three receptors may be colocalized.

Pharmacological profiles of the metabotropic glutamate receptor ligands.

Metabotropic glutamate receptors (mGluRs) are a family of G-protein coupled receptors that are expressed in the central and peripheral nervous systems. The purpose of this study was to compare the ligand binding selectivity profiles of the mGluR agonist [(3)H]L-AP4 and the novel radiolabeled phenylglycine antagonist [(3)H]CPPG at all eight rat mGluR subtypes expressed in transfected human embryonic kidney cells. At a concentration of 30 nM [(3)H]L-AP4, no specific binding was detected in membranes expressing the group I receptors mGluR1a or mGluR5a, or in membranes expressing the group II mGluRs, mGluR2 and mGluR3. Among the group III mGluRs, specific [(3)H]L-AP4 binding was detected in cells expressing mGluR4a and mGluR8a but not in cells expressing mGluR6 or mGluR7a. The binding of [(3)H]CPPG showed an exceptional pattern of selectivity amongst the mGluR subtypes; at a concentration of 20 nM [(3)H]CPPG, a high level of specific binding was seen in membranes containing mGluR8a but not in any of the other mGluR subtypes. The affinity constant (K(D)) calculated for [(3)H]CPPG binding to mGluR8a was 183 nM. In competition experiments, the phosphono-substituted phenylglycine congeners including MPPG, (RS)-PPG, and unlabeled CPPG were the most potent inhibitors of [(3)H]CPPG binding while non-phosphonated compounds such as L-glutamate and MCPG were substantially less potent. These results demonstrate that [(3)H]L-AP4 and [(3)H]CPPG can be used as probes to selectively label group III mGluRs and that CPPG and related phenylglycine derivatives are useful for studying differences in the ligand recognition sites of highly homologous mGluRs.

Cloning and characterization of a metabotropic glutamate receptor, mGluR4b.

An alternative spliced variant of metabotropic glutamate receptor subtype mGluR4a, termed mGluR4b was isolated from a rat cDNA library. Subtype mGluR4b was identical to the previously described mGluR4a, except for the last 64 amino acids in the C-terminal region in which were replaced by 135 new amino acids in mGluR4b. Recombinant baculoviruses coding for mGluR4a and mGluR4b were expressed in Spodoptera frugiperda, Sf-9, insect cells and characterized pharmacologically by measuring [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding and second messenger formation. [3H]-L-AP4 binding to membranes prepared from Sf-9 cells expressing mGluR4a and mGluR4b revealed respective affinities (Kd) of 480 and 360 nM and maximal binding densities (Bmax) of 4.2 and 0.8 pmol/mg protein. The ligand selectivity of mGluR4a and mGluR4b was similar: L-AP4 > L-serine-O-phosphate > L-glutamate > L-2-amino 2-methyl-4-phosphonobutyrate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate > or = quisqualate. A decrease in the affinity of [3H]-L-AP4 was observed in the presence of 0.1 mM guanosine 5'-O-(3-thio)trisphosphate-gamma-S, indicating that mGluR4a and mGluR4b were functionally coupled to G-proteins in Sf-9 cells. Agonists of mGluR4 caused a minor decrease in forskolin-induced cAMP formation in Sf-9 cells expressing either mGluR4a or mGluR4b, suggesting that both receptors are coupled to adenylate cyclase in an inhibitory manner. Thus, mGluR4a and mGluR4b share a common signal transduction pathway and pharmacology when expressed in Sf-9 insect cells.

Purification of proteolipid protein and production of specific antiserum.

Proteolipid protein, the major protein in CNS myelin, was purified using several chromatographic steps. No detergents were used. Chromatography in organic solvents on lipophilic Sephadex (LH-60) for delipidation, on ion exchange chromatography for protein separation, and again on lipophilic Sephadex (LH-20) for desalting, produced homogeneous preparations of proteolipid protein. Rabbit antibodies to proteolipid protein were produced after 2 injections of a total of 500 micrograms of protein. By immunotransfer and absorption experiments, it was found that the antiserum bound to proteolipid protein and a closely related protein, DM-20; it did not bind to basic protein or other myelin proteins.

Characterization of two alternatively spliced forms of a metabotropic glutamate receptor in the central nervous system of the rat.

Amplification of complementary DNA by the polymerase chain reaction and anti-peptide antibodies were used to characterize the expression of two alternatively spliced forms of a metabotropic glutamate receptor (mGluR1 alpha and mGluR1 beta) in the central nervous system of the rat. Polymerase chain reaction analysis showed that mGluR1 alpha was the predominate of the two forms in the cerebellum, diencephalon, mesencephalon, olfactory bulb and brainstem, while mGluR1 beta was the major form present in the hippocampus. Approximately equal amounts of the two receptors were expressed in the cerebral cortex, septum and striatum. Immunochemical analyses of the two receptors were conducted in the rat cerebellum and hippocampus. An mGluR1 alpha-specific antibody labelled a protein with a relative molecular weight of 146,000 on immunoblots of the hippocampus and cerebellum. Immunoblot analysis of the developmental expression of mGluR1 alpha in the hippocampus and cerebellum demonstrated that in both structures, the levels of mGluR1 alpha were at or near their maximum levels in the adult brain. In contrast, two mGluR1 beta-specific antibodies failed to detect mGluR1 beta on immunoblots of brain tissue, thus precluding an immunocytochemical analysis of this receptor. Although low levels of a higher-molecular weight protein, possibly a dimeric form of mGluR1 beta were seen with one of the mGluR1 beta-specific antibodies, we hypothesize that some of the mGluR1 beta present in brain tissue may undergo proteolytic cleavage of the carboxy terminus. Immunocytochemical analysis of mGluR1 alpha showed that very high levels of this receptor were expressed in Purkinje cell bodies and dendrites. In the granule cell layer, some Golgi neurons were immunostained. The granule cells were not labelled. In the hippocampus, mGluR1 alpha immunoreactivity was present in interneurons of the stratum oriens and the dentate hilar region. Double-labelling studies demonstrated that these interneurons were also immunopositive for the neuropeptide somatostatin. The presence of mGluR1 alpha in cells of the hippocampus that are associated with the release of somatostatin, suggest that this receptor could play a role in regulating hippocampal excitability in both normal and epileptic tissues.

Localization of AMPA receptors in the hippocampus and cerebellum of the rat using an anti-receptor monoclonal antibody.

The primary amino acid sequences of the kainate binding proteins from the amphibian and avian central nervous systems are homologous with the functional alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors that have been cloned from rat brain. In this study, we have analysed the anatomical and subcellular distribution of the alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors in the rat hippocampus and cerebellum, using a monoclonal antibody that was raised against a kainate binding protein purified from frog brain. Immunoblots of rat hippocampus and cerebellum, and membranes from COS cells transfected with rat brain alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor cDNAs (GluR1, GluR2, or GluR3) showed a major immunoreactive band migrating at a relative molecular weight of 107,000. In the cerebellum, an additional immunoreactive protein of approximately 128,000 mol. wt was also seen on immunoblots probed with the antibody. The distribution of this protein is apparently restricted to the cerebellum since the 128,000 mol. wt band was not present in other brain areas examined. The identity of the 128,000 mol. wt cerebellar protein is not known. Immunocytochemical analyses of the hippocampus demonstrated that alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptor subunits are present in the cell bodies and dendrites of pyramidal cells. The granule cells were also immunostained. All of the pyramidal cell subfields were heavily labeled. In the pyramidal cell bodies, a high level of immunoreactivity was observed throughout the cytoplasm. In the cerebellum, the Purkinje cell bodies and dendrites also displayed very high levels of immunoreactivity. In addition to the Purkinje neurons, the Bergmann glia and some Golgi neurons were clearly immunostained. Subcellular fractionation and lesioning experiments using the excitotoxin domoic acid indicated that the alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor subunits were associated with postsynaptic membranes. Direct visualization of the immunoreactivity using electron microscopy confirmed the postsynaptic localization of the staining in the dendritic areas in both the hippocampus and the cerebellum. Thus, unlike the kainate binding proteins, which are found primarily extrasynaptically in the frog and on glial cells in the chicken cerebellum, the GluR1, GluR2, and GluR3 receptor subunits are localized to the postsynaptic membrane in the dendrites of neurons in the rat central nervous system.

Isolation, localization, and cloning of a kainic acid binding protein from frog brain.

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.

Constraints on proper folding of the amino terminal domains of group III metabotropic glutamate receptors.

The glutamate binding site of the G-protein coupled metabotropic glutamate receptors (mGluRs) is contained within the large extracellular amino terminal domain (ATD) of the receptor. In this study, we examined the ligand binding properties and cellular dispositions of the membrane-bound mGluR4 and mGluR8 subtypes of mGluRs, and a series of truncated versions of these receptors. Truncation of the ATDs of mGluR4 and mGluR8 40 amino acids upstream of the first transmembrane domain produced soluble proteins that were secreted into the cell culture media of transfected human embryonic kidney cells. The soluble receptors retained ligand binding capabilities. Additional constructs of the ATDs of mGluR4 and mGluR8 were assessed for their ability to bind the agonist [(3)H]L-AP4 and for secretion from cells. A shorter mGluR4 construct truncated 98 amino acids upstream from the first transmembrane domain failed to bind [(3)H]L-AP4, while the analogous mGluR8 construct displayed a low level of binding. Unlike the full-length receptors, which were expressed on the cell surface, or the soluble constructs which were secreted, the shorter constructs were primarily associated with intracellular membranes. These observations suggest that the cysteine-rich region may be important for efficient secretion, but not absolutely obligatory for ligand binding. Surprisingly, longer constructs encoding the entire ATDs of mGluR4 and mGluR8 failed to bind ligand and were localized intracellularly. Together, these findings demonstrate that there are strict limitations on the proper folding of truncated versions of the ATDs of mGluR4 and mGluR8. Specifically, all of the leucine-isoleucine-valine binding protein homology region, and part of the cysteine-rich region is required for optimal secretion in a soluble form that retains ligand binding activity.

Localization of the human mGluR4 gene within an epilepsy susceptibility locus(1).

The family of metabotropic glutamate receptors (mGluRs) consists of eight homologous G-protein coupled receptors. Several of the mGluRs, including the mGluR4 receptor subtype, are localized presynaptically; activation of this receptor induces an inhibition of neurotransmitter release from nerve terminals. Disruption of the mGluR4 gene in mice results in impaired motor and spatial learning, and alterations in seizure susceptibility. In this study, we have determined the structure of the human mGluR4 gene, as well as its chromosomal localization. A comparison of the gene structure of mGluR4 with the highly homologous mGluR6 receptor subtype reveals that both of the genes contain ten exons with similar exon/intron boundaries. A refined localization of mGluR4 was carried out by constructing a bacterial artificial chromosome clone contig of the region surrounding the gene. Thirteen sequence tagged sites (STSs) were identified within this contig. The gene was localized to chromosome 6 band p21.3 by fluorescence in situ hybridization (FISH). The mapping of the mGluR4 gene indicates that it is approximately 1 megabases centromeric of the major histocompatibility complex and 5 megabase from the GABA(B)R1 gene. The mGluR4 gene also falls within a susceptibility locus for juvenile myoclonic epilepsy suggesting a potential link to this form of epilepsy.

Cloning of a G protein-activated inwardly rectifying potassium channel from human cerebellum.

Based on sequence homology with the rat atrial G protein-coupled muscarinic potassium channel (GIRK1 or KGA1/KGB1), a human cDNA encoding a G protein-activated inwardly rectifying K+ channel (HGIRK1) was isolated. The cDNA encodes a protein of 501 amino acids and shares 99% identity to rat GIRK1 in its total amino acid sequence. Southern blot analysis of genomic DNA indicates a high degree of conservation among various species. In the human population a useful NlaIII restriction fragment length polymorphism was found in the coding sequence of HGIRK1. Co-expression of HGIRK1 and the 5-HT1A receptor in Xenopus oocytes resulted in opening of the channel upon treatment with serotonin. HGIRK1 currents showed strong inward rectification and could be blocked by extracellular Ba2+. Northern blot analysis shows that HGIRK1 expression in human is most abundant in the brain, while lower levels are round in kidney and heart.

Altered spatial learning and memory in mice lacking the mGluR4 subtype of metabotropic glutamate receptor.

The glutamate analog, L-2-amino-4-phosphonobutyric acid (L-AP4) is a selective agonist for several members of the metabotropic glutamate receptor (mGluR) family. Activation of presynaptic mGluRs by L-AP4 causes a suppression of synaptic transmission in the central nervous system. In this study, the role of 1 subtype of mGluR in the nervous system was investigated by analyzing mutant mice lacking the L-AP4-sensitive receptor, mGluR4. Experiments designed to probe hippocampal function showed no impairments in acquisition of spatial learning in the water maze task. However, in a spatial reversal learning task, the mutant mice exhibited significantly accelerated learning performance. Furthermore, in a probe trial administered 6 weeks posttraining, these mice showed impaired spatial accuracy. The results suggest that the mutant mice differed in their ability to learn and integrate new spatial information into previously formed memory traces and that their use of stored spatial information also was altered. Thus, the presynaptically expressed mGluR4 plays a role in the processing of spatial information.

Characterization of the oligosaccharide side chains on kainate binding proteins and AMPA receptors.

The amino acid sequences of the kainate binding proteins (KBPs) from frog and chicken brain are homologous with the carboxy terminal half of the rat brain AMPA receptors. In this study, we have characterized the oligosaccharide side chains present on the KBPs from chicken and frog brain, and the AMPA receptors (GluR1, GluR2, and GluR3) from rat brain. Deglycosylation of the asparagine-linked carbohydrates present on the chicken, frog, and rat receptor subunits with N-glycanase, resulted in decreases in the relative molecular weights (M(r)) of 3.4, 3.4, and 5.1 kDa respectively. Thus the percent of asparagine linked carbohydrate (based on M(r) values derived from SDS polyacrylamide gels) of the 49 kDa chicken, the 48 kDa frog, and the 107 kDa receptor rat subunits is 6.9, 7.1, and 4.8 percent respectively. No shifts in the M(r) were detected after treatment with neuraminidase indicating that sialic acid does not appear to be a major component of these receptors. Lectin binding studies demonstrated that both asparagine-linked and serine/threonine-linked oligosaccharides were present in the chicken, frog, and rat proteins. The data indicate that at least one of the asparagine linked oligosaccharide side chains appear to be of the complex or non-bisected hybrid type in all three species. The similarities in the glycosyl moieties of the chicken and frog kainate KBPs and the rat brain AMPA receptors suggests that the homology in the amino acid sequences between these proteins may extend to homology in their oligosaccharide sides chains as well.

Synaptotagmin-like expression in the motor nerve terminals of crayfish.

Synaptotagmin-like immunoreactivity was shown to be localized at crayfish neuromuscular junctions by whole mount immunocytochemistry. Synaptotagmin-like immunoreactivity was present in both phasic and tonic excitatory terminals and inhibitory nerve terminals. Immunoblots indicated that the antibody DSYT-2 raised against Drosophila synaptotagmin, labelled proteins at relative molecular weights of 87 kDa and 107 kDa in crayfish ganglion and neuromuscular preparations.