FMRFamide modulation of secretory machinery underlying presynaptic inhibition of synaptic transmission requires a pertussis toxin- sensitive G-protein

Abstract

The neuropeptide FMRFamide modulates synaptic transmission between identified neurons of the pond snail Helisoma trivolvis. FMRFamide causes a presynaptic inhibition of transmitter release by actions on ion channels and secretory machinery (Man-Son-Hing et al., 1989). The actions of FMRFamide on secretory machinery were studied using giant synapses that form between somata in culture. Using the calcium cage DM-nitrophen, synchronized, calcium-clamped release of neurotransmitter was promoted by UV photolysis. A series of UV flashes (15 msec duration) repeatedly promoted the transient synchronized release of neurotransmitter. Addition of FMRFamide reduced the magnitude of these flash-evoked inhibitory postsynaptic currents. Under conditions of synchronized transmitter release, FMRFamide modulates the secretory responsiveness to internal calcium. The release of neurotransmitter at somasoma synapses was determined to be quantal in nature. To test for the involvement of G-proteins in mediating the effects of FMRFamide on secretory machinery, the modulation of the frequency of miniature inhibitory postsynaptic currents (MIPSCs) was examined. Addition of FMRFamide reduced the frequency of MIPSCs without affecting intracellular free calcium measured with fura-2. Injection of a nonhydrolyzable analog of GTP, GTP gamma S, mimicked the effect of FMRFamide and reduced MIPSC frequency. Preinjection of the presynaptic soma with the A-protomer of pertussis toxin (PTX) prevented FMRFamide from reducing MIPSC frequency. Thus, a PTX-sensitive G-protein mediates the action of FMRFamide on secretory machinery. Similarly, preinjection of the presynaptic soma with PTX prevented FMRFamide from reducing the magnitude of action potential-evoked IPSC. Dose-response curves for the actions of FMRFamide on secretory machinery and calcium current were constructed and demonstrated that secretory machinery can be modulated at concentrations of FMRFamide (less than or equal to 10(-7) M) that do not affect calcium current magnitude. At a concentration of 10(-7) M FMRFamide, action potential-evoked synaptic transmission was reduced. Thus, synaptic transmission can be regulated by the modulation of secretory machinery, without a requirement for the modulation of ion channels.

Rab effector domain peptides stimulate the release of neurotransmitter from cell cultured synapses

“…MIPSC frequency was recorded for a control period of 3 min and all values were normalized to the mean MIPSC frequency during the control period. Microinjection of GTPyS in these experiments caused a small reduction in the frequency of spontaneous MIPSCs, as previously reported [8] (Fig. 1A) from a control value of 1.0 to 0.69 + 0.23 (n = 9) within 6 min after injection.…”

“…Identified neurons B5 and VD4 were isolated from the buccal and visceral ganglia, respectively, according to previously published techniques [8,16,17]. In brief, the central ganglia were removed from the animal, incubated for 15 min at room temperature in 0.2% trypsin (Sigma type IlI) in defined medium (DM; 50% Liebowitz-15 (L-15-Gibco) with additional Helisoma salts; [16]).…”

“…Previous studies have probed the role of GTP-binding proteins in synaptic transmission through the microinjection of guanine nucleotide analogs [8][9][10]. Presynaptic microinjection of non-selective G protein activators, such as the non-hydrolyzable analog of GTP, GTPyS, resulted in reduced action potential-evoked synaptic transmission.…”

GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

“…G-protein regulation might also be a unifying principle for other proposed cell biologic roles of GAP-43, such as the control of neurotransmitter release (31) and synaptic plasticity (4,47,48) of mature neurons. The activity of heterotrimeric G proteins is known to contribute to the regulation of neurotransmitter release (49), and pertussis toxin can block the generation of long-term potentiation in hippocampal slices (50).…”

Modulatory actions of the neuropeptide FMRFamide on synaptic transmission in Helisoma neurons

“…Since the effects of FMRFamide on the HVA calcium current and on the secretory machinery (Haydon et al, 1990) appear to be mediated by a PTX-sensitive G protein, I tested whether a PTX-sensitive G protein is also involved in the modulation of action potentialevoked synaptic transmission. Presynaptic somata were injected with the A protomer of PTX 4 hours prior to recording.…”