Neurotransmitter release into the synapse is stimulated by calcium influx through ion channels that are closely associated with the transmitter release sites. This link may involve the membrane protein syntaxin, which is known to be associated with the release sites and to bind to the calcium channels. There is evidence that presynaptic calcium channels are downregulated by second messenger pathways involving G proteins. Here we use the patch-clamp technique to test whether calcium current is regulated by G proteins in a vertebrate presynaptic nerve terminal, and whether this regulation is affected by the linkage to syntaxin. The calcium current in the nerve terminal showed typical G-protein-mediated changes in amplitude and activation kinetics which were reversed by a preceding depolarization. These effects of the G protein were virtually eliminated if syntaxin was first cleaved with botulinum toxin C1. Our findings indicate that this sensitivity of the current to modulation by G proteins requires the association of the presynaptic calcium channel with elements of the transmitter release site, which may ensure that channels tethered at release sites are preferentially regulated by the G-protein second messenger pathway.

Although correlated neural activity is a hallmark of many regions of the developing nervous system, the neural events underlying its propagation remain largely unknown. In the developing vertebrate retina, waves of spontaneous, correlated neural activity sweep across the ganglion cell layer. Here, we demonstrate that L-type Ca(2+) channel agonists induce large, frequent, rapidly propagating waves of neural activity in the developing retina. In contrast to retinal waves that have been described previously, these L-type Ca(2+) channel agonist-potentiated waves propagate independent of fast synaptic transmission. Bath application of nicotinic acetylcholine, AMPA, NMDA, glycine, and GABA(A) receptor antagonists does not alter the velocity, frequency, or size of the potentiated waves. Additionally, these antagonists do not alter the frequency or magnitude of spontaneous depolarizations that are recorded in individual retinal ganglion cells. Like normal retinal waves, however, the area over which the potentiated waves propagate is reduced dramatically by 18alpha-glycyrrhetinic acid, a blocker of gap junctions. Additionally, like normal retinal waves, L-type Ca(2+) channel agonist-potentiated waves are abolished by adenosine deaminase, which degrades extracellular adenosine, and by aminophylline, a general adenosine receptor antagonist, indicating that they are dependent on adenosine-mediated signaling. Our study indicates that although the precise spatiotemporal properties of retinal waves are shaped by local synaptic inputs, activity may be propagated through the developing mammalian retina by nonsynaptic pathways.

The inhibition of presynaptic calcium channels via G-protein-dependent second messenger pathways is a key mechanism of transmitter release modulation. We used the calyx-type nerve terminal of the chick ciliary ganglion to examine which G-proteins are involved in the voltage-sensitive inhibition of presynaptic N-type calcium channels. Adenosine caused a prominent inhibition of the calcium current that was totally blocked by pretreatment with pertussis toxin (PTX), consistent with an exclusive involvement of Go/Giin the G-protein pathway. Immunocytochemistry was used to localize these G-protein types to the nerve terminal and its transmitter release face. We used two approaches to test for modulation by other G-protein types. First, we treated the terminals with ligands for a variety of G-protein-linked neurotransmitter receptor types that have been associated with different G-protein families. Although small inhibitory effects were observed, these could all be eliminated by PTX, indicating that in this terminal the Gifamily is the sole transmitter-induced G-protein inhibitory pathway. Second, we examined the kinetics of calcium channel inhibition by uncaging the nonselective and irreversible G-protein activator GTPγS, bypassing the receptors. A large fraction of the rapid GTPγS-induced inhibition persisted, consistent with a Go/Gi-independent pathway. Immunocytochemistry identified Gq, G11, G12, and G13as potential PTX-insensitive second messengers at this terminal. Thus, our results suggest that whereas neurotransmitter-mediated calcium channel inhibition is mainly, and possibly exclusively, via Go/Gi, other rapid PTX-insensitive G-protein pathways exist that may involve novel, and perhaps transmitter-independent, activating mechanisms.