Voltage dependence of 5‐hydroxytryptamine release at a synapse between identified leech neurones in culture.

Abstract

SUMMARY1. The release of 5-hydroxytryptamine (5-HT) from presynaptic terminals has been studied by the voltage-clamp technique at synapses made by isolated Retzius and pressure (P) sensory neurones dissected from the leech C.N.s. and maintained in tissue culture. At these synapses facilitation, depression and modulation of release occur with action potentials and with voltage-clamp pulses.2. Depolarization of Retzius cells from a constant holding potential by steps of varying amplitude (5 ms in duration) caused graded release of 5-HT. The steep transfer function for release using these short test pulses resembled that seen at the giant synapse of the squid: synaptic potentials increased markedly with presynaptic depolarizations beyond -25 mV and decreased with large depolarizing pulses beyond +40 mV.3. When the steady holding potential of voltage-clamped Retzius cells was suddenly displaced to a new value within the range of -40 mV to -85 mV, there followed a slow but smaller change of the post-synaptic P-cell membrane potential in the same direction. After an initial delay of about 40 ms, the post-synaptic potential reached its new level with an exponential time course and a time constant of 0 7 s. Since Retzius and P cells are not electrically coupled, these effects can be accounted for by alterations in tonic release of transmitter.4. Changes of presynaptic holding potential to a more depolarized level resulted in an increase in voltage noise recorded in the P cell. Conversely, hyperpolarization from a depolarized level reduced noise. Noise analysis showed that these changes could be accounted for by quantal events with a mean amplitude of about 0-15 mV. This value is similar to that for spontaneous miniature potentials and quantal fluctuations observed at synapses between Retzius and P cells. I. D. DIETZEL, P. DRAPEA U AND J. G. NICHOLLS responses evoked by depolarizations to 0 mV with pulses of 5 ms duration were reduced in amplitude as the holding potential of the Retzius cell was increased from the resting value of -45 to -75 mV. For example, depolarization to 0 mV starting from -45 mV evoked synaptic potentials as much as ten times larger than those evoked by depolarizations to 0 mV starting from -75 mV. With step depolarizations or hyperpolarizations of the holding potential, the effect on release evoked by brief pulses to 0 mV built up over an exponential time course similar to that found for the effect of holding potential on tonic release (r = 0 7 s).6. Frequency-dependent facilitation of transmission occurred with paired stimuli applied to Retzius cells. The time constant of decay of facilitation was similar (approximately 100 ms) with impulses and under voltage-clamp conditions. The time course of the decay of facilitation was independent of the holding potential. It is concluded that two-shock facilitation and modulation of release by changes in the resting potential depend on different presynaptic mechanisms.

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

“…The effects of two neurotoxins, TeNT and BoNT/A, were studied. When Retzius cells are paired with P-cells, a stable serotonergic synapse with well defined physiological properties is established (Fuchs et al, 1982;Dietzel et al, 1986;Bruns et al, 1993;Bruns and Jahn, 1995). Furthermore, the large-sized Retzius cell forms its synaptic contact with the postsynaptic P-cell over a short distance (Liu and Nicholls, 1989), making this preparation well suited for microinjection of neurotoxins.…”

Requirement for Tyrosine Phosphatase during Serotonergic Neuromodulation by Protein Kinase C

“…P cells were isolated from Hirudo medicinalis and cultured as described previously (Dietzel et al, 1986). Because 5-H T activates both chloride and cation conductances in the P cell (Sanchez-Armass et al, 1991), the latter was isolated during intracellular recordings by blocking potassium channels with 10 mM diaminopyridine and by replacing external chloride with sulfate in the recording solution (C atarsi et al, 1995), which contained 130 mM Na 2 SO 4 , 4 mM K 2 SO 4 , 5 mM MgSO 4 , 10 mM 3,4-diaminopyridine, 10 mM glucose, and 10 mM H EPES, pH 7.4, adjusted to 330 mOsm.…”

Tyrosine phosphorylation during synapse formation between identified leech neurons.

“…Cell cultures R and P cells were isolated from the segmental ganglia (excluding reproductive ganglia) of the leech Hirudo medicinalis (Ricarimpex, Audenge, France) and cultured as described previously (Dietzel et al 1986). Briefly, after exposing the desheathed ganglia to collagenase (Type XI; Sigma), the cell bodies of R and P cells were removed by aspiration into a micropipette.…”

“…1982;Dietzel, Drapeau & Nicholls, 1986;Kuffler, Nicholls & Drapeau, 1987;Drapeau & SanchezArmass, 1988 Merz & Drapeau, 1991). The depolarizing, extrasynaptic response may be involved in neuromodulation by 5-HT (Kristan & Nusbaum, 1982;Belardetti, Brunelli, Demontis & Sonetti, 1984).…”