Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion

The membrane protein syntaxin participates in several protein–protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.

Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes

Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-μs time resolution, respectively, for studying neurotransmitter receptors in giant (≈20-μm diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.

A neurotransmitter transporter encoded by the Drosophila inebriated gene

Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this paper, we describe the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In situ hybridization of ine transcripts to developing embryos revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na+/Cl−-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function. We conclude that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron.

Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels

Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein–protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.

Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. We have previously shown that metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. We identified mRNA for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. We found that this mRNA associates with translational complexes in synaptoneurosomes within 1–2 min after mGluR1 stimulation of this preparation, and we observed increased expression of FMRP after mGluR1 stimulation. In addition, we found that FMRP is associated with polyribosomal complexes in these fractions. In vivo, we observed FMRP immunoreactivity in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. We suggest that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections.

Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human Huntington disease gene

Loss of neurotransmitter receptors, especially glutamate and dopamine receptors, is one of the pathologic hallmarks of brains of patients with Huntington disease (HD). Transgenic mice that express exon 1 of an abnormal human HD gene (line R6/2) develop neurologic symptoms at 9–11 weeks of age through an unknown mechanism. Analysis of glutamate receptors (GluRs) in symptomatic 12-week-old R6/2 mice revealed decreases compared with age-matched littermate controls in the type 1 metabotropic GluR (mGluR1), mGluR2, mGluR3, but not the mGluR5 subtype of G protein-linked mGluR, as determined by [3H]glutamate receptor binding, protein immunoblotting, and in situ hybridization. Ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors were also decreased, while N-methyl-d-aspartic acid receptors were not different compared with controls. Other neurotransmitter receptors known to be affected in HD were also decreased in R6/2 mice, including dopamine and muscarinic cholinergic, but not γ-aminobutyric acid receptors. D1-like and D2-like dopamine receptor binding was drastically reduced to one-third of control in the brains of 8- and 12-week-old R6/2 mice. In situ hybridization indicated that mGluR and D1 dopamine receptor mRNA were altered as early as 4 weeks of age, long prior to the onset of clinical symptoms. Thus, altered expression of neurotransmitter receptors precedes clinical symptoms in R6/2 mice and may contribute to subsequent pathology.

SNAP-25 and synaptotagmin involvement in the final Ca(2+)-dependent triggering of neurotransmitter exocytosis.

In neurons, depolarization induces Ca2+ influx leading to fusion of synaptic vesicles docked at the active zone for neurotransmitter release. While a number of proteins have now been identified and postulated to participate in the assembly and subsequent disengagement of a vesicle docking complex for fusion, the mechanism that ultimately triggers neuroexocytosis remains elusive. Using a cell-free, lysed synaptosomal membrane preparation, we show that Ca2+ alone is sufficient to trigger secretion of glutamate and furthermore that Ca(2+)-signaled exocytosis is effectively blocked by antibodies and peptides to SNAP-25, a key constituent of the vesicle docking complex. In addition, Ca2+ inhibits the ability of synaptotagmin, a synaptic vesicle protein proposed as a calcium sensor and triggering device, to associate with this docking complex. These results support a model in which Ca(2+)-dependent triggering of neurotransmission at central synapses acts after ATP-dependent potentiation of the docking-fusion complex for membrane fusion.

Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release.

Synaptophysin (syp I) is a synaptic vesicle membrane protein that constitutes approximately 7% of the total vesicle protein. Multiple lines of evidence implicate syp I in a number of nerve terminal functions. To test these, we have disrupted the murine Syp I gene. Mutant mice lacking syp I were viable and fertile. No changes in the structure and protein composition of the mutant brains were observed except for a decrease in synaptobrevin/VAMP II. Synaptic transmission was normal with no detectable changes in synaptic plasticity or the probability of release. Our data demonstrate that one of the major synaptic vesicle membrane proteins is not essential for synaptic transmission, suggesting that its function is either redundant or that it has a more subtle function not apparent in the assays used.

Reduced sympathetic innervation after alteration of target cell neurotransmitter phenotype in transgenic mice.

Neurotransmitters play a variety of important roles during nervous system development. In the present study, we hypothesized that neurotransmitter phenotype of both projecting and target cells is an important factor for the final synaptic linkage and its specificity. To test this hypothesis, we used transgenic techniques to convert serotonin/melatonin-producing cells of the pineal gland into cells that also produce dopamine and investigated the innervation of the phenotypically altered target cells. This phenotypic alteration markedly reduced the noradrenergic innervation originating from the superior cervical ganglia. Although the mechanism by which the reduction occurs is presently unknown, quantitative enzyme-linked immunoassay showed the presence of the equivalent amounts of nerve growth factor (NGF) in the control and transgenic pineal glands, suggesting that it occurred in a NGF-independent manner. The results suggest that target neurotransmitter phenotype influences the formation of afferent connections during development.

Citrate modulates the regulation by Zn2+ of N-methyl-D-aspartate receptor-mediated channel current and neurotransmitter release.

The effect of the two metal-ion chelators EDTA and citrate on the action of N-methyl-D-aspartate (NMDA) receptors was investigated by use of cultured mouse cerebellar granule neurons and Xenopus oocytes, respectively, to monitor either NMDA-evoked transmitter release or membrane currents. Transmitter release from the glutamatergic neurons was determined by superfusion of the cells after preloading with the glutamate analogue D-[3H]aspartate. The oocytes were injected with mRNA isolated from mouse cerebellum and, after incubation to allow translation to occur, currents mediated by NMDA were recorded electrophysiologically by voltage clamp at a holding potential of -80 mV. It was found that citrate as well as EDTA could attenuate the inhibitory action of Zn2+ on NMDA receptor-mediated transmitter release from the neurons and membrane currents in the oocytes. These effects were specifically related to the NMDA receptor, since the NMDA receptor antagonist MK-801 abolished the action and no effects of Zn2+ and its chelators were observed when kainate was used to selectively activate non-NMDA receptors. Since it was additionally demonstrated that citrate (and EDTA) preferentially chelated Zn2+ rather than Ca2+, the present findings strongly suggest that endogenous citrate released specifically from astrocytes into the extracellular space in the brain may function as a modulator of NMDA receptor activity. This is yet another example of astrocytic influence on neuronal activity.

Long-range signaling within growing neurites mediated by neurotrophin-3

In addition to well established trophic functions, neurotrophins acutely affect neurotransmitter secretion from the presynaptic nerve terminal, influence synaptic development, and may serve as selective retrograde messengers that regulate synaptic efficacy. The crucial question related to the mechanisms of neurotrophin-mediated signaling is whether acute effects of neurotrophins are spatially restricted to the activated synapses. Here we have used a local perfusion technique for local delivery of neurotrophin-3 (NT-3) to various regions of developing Xenopus embryo neurons in culture. Within minutes after a focal exposure of a soma or a small (≈30 μm in length) axonal segment to NT-3, we observed an increase in the spontaneous neurotransmitter secretion from the presynaptic nerve terminals located ≈300–400 μm away from the site of NT-3 application. Secretory activity along the axonal shaft was not affected. Our findings suggest that the NT-3-mediated signal may rapidly travel through neuronal cytoplasm over unexpectedly long distances and modulate neurotransmitter release specifically at the presynaptic nerve terminals.

A third member of the synapsin gene family

Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuron-specific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12–13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.

Developmental emergence of different forms of neuromodulation in Aplysia sensory neurons

The capacity for neuromodulation and biophysical plasticity is a defining feature of most mature neuronal cell types. In several cases, modulation at the level of the individual neuron has been causally linked to changes in the functional output of a neuronal circuit and subsequent adaptive changes in the organism’s behavioral responses. Understanding how such capacity for neuromodulation develops therefore may provide insights into the mechanisms both of neuronal development and learning and memory. We have examined the development of multiple forms of neuromodulation triggered by a common neurotransmitter, serotonin, in the pleural sensory neurons of Aplysia californica. We have found that multiple signaling cascades within a single neuron develop sequentially, with some being expressed only very late in development. In addition, our data suggest a model in which, within a single neuromodulatory pathway, the elements of the signaling cascade are developmentally expressed in a “retrograde” manner with the ionic channel that is modulated appearing early in development, functional elements in the second messenger cascade appearing later, and finally, coupling of the second messenger cascade to the serotonin receptor appearing quite late. These studies provide the characterization of the development of neuromodulation at the level of an identified cell type and offer insights into the potential roles of neuromodulatory processes in development and adult plasticity.