FMRFamide (i.e. Phe-Met-Arg-Phe-NH2) application to the C2 neurone of Helix caused a depolarizing response which consisted of a large, rapidly developing, and rapidly desensitizing inward current, underlain by a smaller, slower inward current which did not desensitize. Both currents were carried through sodium-selective channels which were insensitive to D-tubocurarine, and the to the fast sodium channel blockers tetrodotoxin (TTX) and lignocaine. Only the faster, desensitizing current could be blocked by amiloride. FMRFamide also activated two types of unitary inward currents with slightly differing amplitudes in outside-out patches taken from the C2 neurone, both through sodium-selective ion channels. Only the smaller unitary currents readily desensitized and were susceptible to block by amiloride, and they also activated more rapidly. Unitary currents of both types were recorded in outside-out patches in the absence of freely diffusible intracellular mediators, and were also activated when guanosine 5'-O-(2-thiodiphosphate) (GDP [beta-S]) was included in the recording pipette solution. This supports a tight receptor/channel coupling for both responses, with no involvement of GTP-binding proteins. Further, the very fast rate of activation of the smaller channels, which generally carry the major part of the FMRFamide-induced current, strongly indicates that these channels are ligand gated.

Dopamine gates a fast excitatory response in Helix C2 neurones. Whole cell, and multiple unitary dopamine-gated currents showed variable decay rates and desensitization properties, suggesting the presence of more than one channel type. Manipulation of internal free [Ca2+] by various procedures (external zero Ca2+ or 1 mM Co2+, prolonged depolarization, A23187, or flufenamic acid), affected both the amplitude and decay time for the response, and also suggested the presence of separate fast and slowly decaying components. Responses were prolonged by intracellular fluoride a non specific phosphatase inhibitor, and attenuated and shortened by the protein kinase inhibitors H7 and staurosporine, and the calmodulin inhibitor W7. Phorbol ester potentiated and prolonged the response and this effect was reversibly antagonized by the specific protein kinase C inhibitor chelerythrine. Different dopamine-activated unitary currents were distinguished in outside-out patches by conductance (5, 8, 12 and 15pS), rate of recovery from desensitization, and pattern of openings. Discrimination of slow and fast components of the response was possible with apomorphine, ADTN, and caffeine. Paradoxically the dopamine antagonists chlorpromazine and spiperone, but not dopamine itself, stimulated sustained activity of 5pS unitary currents which did not desensitize in outside-out patches. Modulation of different channels underlying the fast dopamine response by protein kinase C, and possibly other mechanisms, provides a potent means of controlling excitatory dopaminergic synaptic transmission.

SUMMARYTwO different responses to serotonin have been observed. One response was a depolarization accomphnied by a decrease in membrane conductance. This response was enhanced at depolarized potentials and reduced at hyperpolarized potentials; the apparent conductance change was also reduced at hyperpolarized potentials indicating some voltage sensitivity of the response. The other response was a depolarization accompanied by an increased membrane conductance. The response was enhanced at hyperpolarized potentials and reversed to a hyperpolarization at -35 to -60 mV. The total number ofresponsive neurones was small (5%). This might be explained by a deficiency of serotonergic input to the recorded cells, since it was shown autoradiographically that very few neuroties in the cultures used exhibited a specific high-affinity uptake for the transmitter, and hence probably contained it.

Unitary potassium (K+) currents of several different conductances have been recorded from the growth cones of isolated Cl neurones from Helix aspersa. The isolated neurones were maintained in culture for up to 1 week. Similar unitary currents were recorded in the growth cones of other isolated Helix neurones. The activity of one type of unitary K+ current recorded from the growth cones of the Cl neurone and other neurones was very similar to that described for the S-channel of the perikarya of Aplysia sensory neurones. Another type of unitary K+ current showed fast flickering and reduced amplitude when the membrane was held at large positive potentials, which is suggestive of channel block by some agent. The conductances of the K+ channels in the growth cones of isolated Cl neurones were generally smaller than those recorded in this and in previous studies from the perikarya of Cl neurones in situ. However, unitary K+ currents recorded from the perikaryon of the Cl neurone, and from other identified neurones, in culture also had lower conductances than those recorded in situ. The mean resting potential of the isolated neurones was smaller than those from neurones in situ. This and other results suggested that reduced intracellular K+ concentration in the isolated neurones might be an important factor in deciding the conductance of the recorded channels.