Characterization of a calcium current in a vertebrate cholinergic presynaptic nerve terminal

Abstract

Calcium currents were recorded from a cholinergic presynaptic nerve terminal in the chick ciliary ganglion using the whole-cell voltage- clamp technique. The presynaptic element of this synapse is in the form of a calyx that envelops the postsynaptic ciliary neuron. A method was developed to isolate the ciliary neuron, expose the calyx, and apply patch-clamp electrodes under visual control. The presynaptic Ca current activated at +30 mV with a fast time constant of about 1.5 msec and deactivated at -80 mV with a time constant of about 0.5 msec, values that are consistent with a role in action-potential-dependent transmitter release. The calyx Ca current was blocked by 0.1 mM Cd or 2 microM omega-conotoxin and was resistant to voltage-dependent inactivation. The presynaptic Ca channel exhibits similarities to the N- type group but differs from these by the minimal voltage-dependent inactivation. This type of channel, designated CaN-PT (N-like, presynaptic terminal), may play a key role in transmitter release at many vertebrate fast-transmitting synapses.

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

“…Chick ciliary ganglia were dissociated enzymatically and plated on polylysine-coated coverslips, and calyx nerve terminals were identified under the light microscope by the attached axon and by staining with 4-Di-2-Asp (Figures 1B and 1C) as described (Stanley and Goping, 1991;Stanley, 1991). The location of each calyx was recorded by noting its position relative to the coordinates of an electron microscopy grid attached to the underside of the coverslip and by a sketch of the surrounding terrain (see Experimental Procedures).…”

“…We searched for calcium channels by tagging the channels with colloidal gold particles via co-conotoxin GVlA (o~CTx), a toxin that irreversibly blocks both transmission and virtually all the presynaptic calcium current (Stanley and Goping, 1991;Stanley and Atrakchi, 1990;Yawo and Momiyama, 1993) at this synapse. The test calyx terminals were incubated with biotinylated o~-CTx (b-co-CTx) followed by 30 nm avidin-gold particles.…”

“…Chick embryo ciliary ganglia (15-day-old) were dissociated as described (Stanley and Goping, 1991;Stanley, 1991) but were incubated with enzymes for 150 min to increase the yield of isolated calyces. The preparation was plated on a poly-L-lysine-coated coverslip and incubated at 37°C in minimal essential medium (MEM) to allow the dissociated cells to attach.…”

“…In contrast, the chick ciliary ganglion calyx synapse is extraordinary for its sheet-like presynaptic terminal that can completely envelope the postsynaptic ciliary neuron ( Figure 1A) (Hess, 1965;de Lorenzo, 1960;Landmesser and Pilar, 1972;Stanley, 1992). This nerve terminal has been used previously to record whole-terminal calcium currents (Stanley, 1989;Martin et al, 1989;Stanley and Goping, 1991;Yawo and Momiyama, 1993) and transmitter release site-associated single calcium channels (Stanley, 1991(Stanley, ,1993. The isolated calyx nerve terminal has a flat and extensive transmitter release face that is particularly favorable for direct examination with AFM.…”

A Nitric Oxide/Cyclic GMP‐Dependent Protein Kinase Pathway Alters Transmitter Release and Inhibition by Somatostatin at a Site Downstream of Calcium Entry

“…Direct recording from most presynaptic nerve terminals is difficult because of their small size (Stanley and Goping, 1991;Borst and Sakmann, 1996;Yazejian et al, 1997). Therefore, cell preparations where the cell body releases transmitter in a Ca2+-dependent manner are useful for studying the modulation of transmitter release.…”