Gene transfer into nervous tissue is a powerful tool for the analysis of gene function. By using a rat hippocampal slice culture preparation, we show here that Semliki Forest virus (

Synaptically released glutamate activates ionotropic and metabotropic receptors at central synapses. Metabotropic glutamate receptors (mGluRs) are thought to modulate membrane conductances through transduction cascades involving G proteins. Here we show, in CA3 pyramidal cells from rat hippocampus, that synaptic activation of type 1 mGluRs by mossy fiber stimulation evokes an excitatory postsynaptic response independent of G-protein function, while inhibiting an afterhyperpolarization current through a G-protein-coupled mechanism. Experiments using peptide activators and specific inhibitors identified a Src-family protein tyrosine kinase as a component of the G-protein-independent transduction pathway. These results represent the first functional evidence for a dual signaling mechanism associated with a heptahelical receptor such as mGluR1, in which intracellular transduction involves activation of either G proteins or tyrosine kinases.

A fast ATP‐mediated synaptic current was identified in CA3 pyramidal cells in organotypic hippocampal slice cultures. In the presence of inhibitors for ionotropic glutamate and GABA receptors, extracellular stimulation in the pyramidal cell layer evoked fast synaptic currents that reversed near 0 mV, reflecting an increase in a non‐selective cationic conductance. This response was mimicked by focal application of ATP. Antagonists of ionotropic P2X receptors reduced both synaptic and ATP‐induced currents. Using a pharmacological approach, the source of synaptically released ATP was determined. Synaptic ATP responses were insensitive to presynaptic blockade of GABAergic transmission between interneurons and CA3 pyramidal cells with the μ‐opioid receptor agonist D‐Ala2,MePhe4,Met(O)5‐ol‐enkephalin (FK33‐824), but were blocked by adenosine, which inhibits glutamate release from synaptic terminals in the hippocampus. However, selective inhibition of mossy fibre glutamatergic transmission with the metabotropic glutamate receptor group II agonist (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine (DCG IV) did not affect the response. This result points to the associational fibres as the source of the ATP‐mediated synaptic response. These results suggest that ATP, coreleased with glutamate, induces a synaptic response in CA3 pyramidal cells that is observed mainly under conditions of synchronous discharge from multiple presynaptic inputs.

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.Synaptic transmission is based on the rapid exocytosis of neurotransmitters from synaptic vesicles (1). Exocytosis occurs at active zones, where vesicles are docked for Ca 2ϩ -triggered fusion with the presynaptic plasma membrane (2). The exocytotic event is coupled to an endocytotic cascade that recycles fused synaptic vesicles for additional rounds of regulated fusion (3). Several proteins of the synaptic vesicle, plasma membrane, and presynaptic cytosol have been implicated in neurotransmitter release (4, 5). Syntaxin-1A, a neuronal homologue of a family of proteins involved in membrane fusion (6-8), plays a key role in exocytosis. First, this plasma membrane protein is a substrate of botulinum C1 (Bot-C1), a toxin that inhibits transmitter release by the proteolytic activity of its disulfide-linked light chain (9). Second, Drosophila lacking syntaxin suffer embryonic lethality and a loss of synaptic transmission (10, 11).In vitro, syntaxin 1A participates in a number of interactions that are believed to be essential for neurotransmitter release. Syntaxin-binding partners include Ca 2ϩ channels, complexin, and synaptotagmin, a putative Ca 2ϩ receptor for transmitter release, as well as mammalian homologues of the Unc13 and secϪ1 protein families (4,5,12). Syntaxin also binds SNAP-25 (synaptosomal associated protein of 25 kDa), a peripheral plasma membrane protein, and synaptobrevin, a major integral membrane protein of synaptic vesicles (13,14). Together these proteins form a stable ternary complex of 7S (15) that resists dissociation by SDS (14). This 7S complex has been proposed to mediate the docking and fusion of synaptic vesicles at the presynaptic plasma membrane, because it binds the fusion catalyzing proteins SNAP (soluble NSF attachment protein) and NSF (for N-ethylmaleimide-sensitive fusion protein) (15, 16). The resulting higher-order SNARE (SNAP receptor) complex of 20S dissociates upon hydrolysis of ATP by NSF, and this dissociation is thought to promote membrane fusion (15-17).To investigate how syntaxin's interactions with presynaptic proteins contribute to the trafficking events that underlie neurotransmitter release, we have perturbed syntaxin function in vivo by microinjecting Bot-C1 and recombinant fragments of synt...