Dual Ca2+ modulation of glycinergic synaptic currents in rodent hypoglossal motoneurones

Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp recording and Ca²⁺ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca²⁺ (Ca_i) in glycinergic synapse modulation. Ca²⁺ influx through voltage-gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired-pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212-2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca²⁺-dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Ca_i buffering (20 m m BAPTA). Our findings demonstrate two independent mechanisms by which Ca²⁺ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR-mediated responses. This dual Ca²⁺-induced regulation might be important for feedback control of neurotransmission in a variety of glycinergic networks in mammalian nervous systems.     

Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals

Retinal bipolar cells convey light-evoked potentials from photoreceptors to ganglion cells and mediate the initial stages of visual signal processing. They do not fire Na⁺-dependent action potentials (APs) but the Mb1 class of goldfish bipolar cell exhibits Ca²⁺-dependent APs and regenerative potentials that originate in the axon terminal. I have examined the properties of Ca²⁺-dependent APs in isolated bipolar-cell terminals in goldfish retinal slices. All recorded terminals fired spontaneous or evoked APs at frequencies of up to 15 Hz. When an AP waveform was used as a voltage stimulus, exocytosis was evoked by single APs, maintained throughout AP trains and modulated by AP frequency. Furthermore, feedback inhibition of the Ca²⁺ current ( I _Ca) by released vesicular protons reduced depression of exocytosis during AP trains. In the absence of K⁺ current inhibition, step depolarizations and AP waveforms evoked a rapidly activated outward current that was dependent on Ca²⁺ influx ( I _K(Ca)). I therefore investigated whether proton-mediated feedback inhibition of I _Ca affected the activation of I _K(Ca). A transient inhibition of I _K(Ca) was observed that was dependent on exocytosis, blocked by high-pH extracellular buffer, of similar magnitude to inhibition of I _Ca but occurred with a delay of 2.7 ms. In addition, the amplitude of APs evoked under current clamp was inhibited by the action of vesicular protons released by the APs. Protons released via exocytosis may therefore be a significant modulator of Ca²⁺-dependent currents and regenerative potentials in bipolar-cell terminals.     

Fast erg K+ currents in rat embryonic serotonergic neurones

Ether-à-go-go -related gene (erg) channels form one subfamily of the ether-à-go-go (EAG) K⁺ channels and all three erg channels (erg1–3) are expressed in the brain. In the present study we characterize a fast erg current in neurones in primary culture derived from the median part of rat embryonic rhombencephala (E15–16). The relatively uniform erg current was regularly found in large multipolar serotonergic neurones, and occurred also in other less well characterized neurones. The erg current was blocked by the antiarrhythmic substance E-4031. Single-cell RT-PCR revealed the expression of erg1a, erg1b, erg2 and erg3 mRNA in different combinations in large multipolar neurones. These cells also contained neuronal tryptophan hydroxylase, a key enzyme for serotonin production. To characterize the molecular properties of the channels mediating the native erg current, we compared the voltage and time dependence of activation and deactivation of the neuronal erg current to erg1a, erg1b, erg2 and erg3 currents heterologously expressed in CHO cells. The biophysical properties of the neuronal erg current were well within the range displayed by the different heterologously expressed erg currents. Activation and deactivation kinetics of the neuronal erg current were fast and resembled those of erg3 currents. Our data suggest that the erg channels in rat embryonic rhombencephalon neurones are heteromultimers formed by different erg channel subunits.     

Role of anion-cation interactions on the pre-steady-state currents of the rat Na+-Cl−-dependent GABA cotransporter rGAT1

The effects of sodium and chloride on the properties of the sodium-dependent component of the 'pre-steady-state' currents of rGAT1, a GABA cotransporter of the Na⁺-Cl⁻-dependent family, were studied using heterologous oocyte expression and voltage clamp. Reductions in either extracellular sodium or chloride shifted the charge-voltage ( Q-V ) and time constant-voltage (τ- V ) characteristics of the process towards more negative potentials. The shift induced by sodium (TMA⁺, tetramethylammonium substitution) was stronger than that induced by chloride (acetate substitution), and the shift of τ was accompanied by a decrease in its maximum value. Increasing extracellular Ca²⁺ did not produce significant shifts in Q - V and τ- V curves. The negative shift of the Q - V curve upon chloride reduction and the decrease in the value of the relaxation time constant, τ, when either sodium or chloride were lowered, contrasted with the prediction of the Hill-Boltzmann interpretation of the process. Analysis of the unidirectional rate constants under different conditions revealed that both sodium and chloride shifted the outward rate more than the inward rate; furthermore, the shifts induced by sodium were larger than those induced by chloride. These observations are qualitatively compatible with the existence of a selective vestibule at the mouth of the transporters, acting similarly to a Donnan system.