We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the a-conotoxin-and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channd (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N-or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K+-induced Ca'5 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FIX and sFVX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.Ca2' influx through voltage-dependent Ca2' channels (VDCCs) is the trigger for the release of neurotransmitters from the nerve terminals (1, 2). Three major types ofVDCC named T, L, and N were described in neuronal cells (3). The high-threshold L and N VDCCs are sensitive to the blocking effect of o-conotoxin (c-CgTX), and only the L type is affected by Ca2`channel antagonists of the 1,4-dihydropyridine (DHP) class. An intermediate-threshold VDCC channel called the P channel was identified in the; Purkinje cells of mammalian cerebellum and found to be insensitive to DHP and w-CgTX, but very sensitive to a low molecular weight fraction of the venom of the funnel-web spider Agelenopsis aperta (4). This funnel-web spider toxin (FTX) was also effective in blocking Ca2+ conductance and synaptic transmission at the squid giant synapse (4). Evoked release of neurotransmitter was shown to be dependent on Ca2+ influx through the N-type VDCC in sympathetic neurons by the inhibitory effect of w-CgTX and the lack of effect of DHP (5). By contrast, substance P release from dorsal root ganglia neurons (6, 7) and catecholamine release from chromaffin cells (8) are strongly inhibited by DHP, consistent with a major participation of L-type channels. However, mammalian motor nerve terminals are normally insensitive to either w-CgTX or DHP (9-11). Furthermore, in brain synaptosomes, K+-evoked Ca2' uptake and transmitter release are only partially sensitive to c-CgTX and DHP (12, 13). Thus the identity of the VDCC involved in transmitter release in the majority of the synapses at the mammalian central and peripheral nervous system has not been defined. The experiments presented here were designed to study the effect of FTX on transmitter release and Ca2+ influx at the mammalian neuromuscular junction and on Ca2+ uptake by cerebral cortex synaptosomes in order to determine whether a particular type of VDCC ...

The receptive field (RF) of retinal ganglion cells (RGCs) consists of an excitatory central region, the RF center, and an inhibitory peripheral region, the RF surround. It is still unknown in detail which inhibitory interneurons (horizontal or amacrine cells) and which inhibitory circuits (presynaptic or postsynaptic) generate the RF surround.To study surround inhibition, light-evoked whole-cell currents were recorded from RGCs of the isolated, intact rabbit retina. The RFs were stimulated with light or dark spots of increasing diameters and with annular light stimuli.Direct inhibitory currents could be isolated by voltage clamping ganglion cells close to the Na ϩ /K ϩ reversal potential. They mostly represent an input from GABAergic amacrine cells that contribute to the inhibitory surround of ganglion cells. This direct inhibitory input and its physiological function were also investigated by recording light-evoked action potentials of RGCs in the current-clamp mode and by changing the intracellular Cl Ϫ concentration. The excitatory input of the ganglion cells could be isolated by voltage clamping ganglion cells at the Cl Ϫ reversal potential. Large light spots and annular light stimuli caused a strong attenuation of the excitatory input. Both GABA A receptors and GABA C receptors contributed to this inhibition, and picrotoxinin was able to completely block it.Together, these results show that the RF surround of retinal ganglion cells is mediated by a combination of direct inhibitory synapses and presynaptic surround inhibition.

Combined electrophysiological and imaging techniques were used to study calcium currents (I Ca ) and their sites of origin at rod bipolar cells in rat retinal slices. We report here for the first time the successful whole-cell patch-clamp recording from presynaptic boutons that were compared with somatic recordings. TTX-resistant inward currents were elicited in response to depolarization. The kinetic and pharmacological properties of I Ca were very similar for recordings obtained from the soma and the presynaptic terminals. I Ca activated maximally between Ϫ30 and Ϫ20 mV was enhanced by Bay K 8644 and was blocked by isradipine and nifedipine. Peak amplitude and time to peak were Ϫ31.3 Ϯ 1.2 pA and 3.2 Ϯ 0.2 msec with somatic recordings (n ϭ 54), whereas the corresponding values were Ϫ31.6 Ϯ 6.1 pA and 3.2 Ϯ 0.7 msec in recordings obtained directly from terminals (n ϭ 6). I Ca showed little inactivation during sustained depolarizations. No T-type I Ca was observed with depolarizations from Ϫ90 mV. Concomitant with Ca 2ϩ entry, depolarization induced the appearance of transient outward currents that resembled IPSCs and were blocked by GABA and glycine receptor antagonists, suggesting that they arise from activation of amacrine feedback synapses. Upon depolarization, intracellular Ca 2ϩ ([Ca 2ϩ ] i ) rises were restricted to the presynaptic terminals with no somatic or axonal changes and were linearly dependent on pulse duration when using a low-affinity Ca 2ϩ indicator. In cone bipolar cells, I Ca inactivated markedly, and [Ca 2ϩ ] i rises occurred in the axon, as well as in the presynaptic terminals.

Bipolar cells in the vertebrate retina have been characterized as nonspiking interneurons. Using patch-clamp recordings from goldfish retinal slices, we find, however, that the morphologically well-defined Mb1 bipolar cell is capable of generating spikes. Surprisingly, in dark-adapted retina, spikes were reliably evoked by light flashes and had a long (1-2 s) refractory period. In light-adapted retina, most Mb1 cells did not spike. However, an L-type Ca2+ channel agonist could induce periodic spiking in these cells. Spikes were determined to be Ca2+ action potentials triggered at the axon terminal and were abolished by 2-amino-4-phosphonobutyric acid (APB), an agonist that mimics glutamate. Signaling via spikes in a specific class of bipolar cells may serve to accelerate and amplify small photo-receptor signals, thereby securing the synaptic transmission of dim and rapidly changing visual input.