Caged calcium in Aplysia pacemaker neurons. Characterization of calcium-activated potassium and nonspecific cation currents.

We have studied calcium-activated potassium current, IK(Ca), and calcium-activated nonspecific cation current, INS(Ca), in Aplysia bursting pacemaker neurons, using photolysis of a calcium chelator (nitr-5 or nitr-7) to release caged calcium intracellularly. A computer model of nitr photolysis, multiple buffer equilibration, and active calcium extrusion was developed to predict volume-average and front-surface calcium concentration transients. Changes in arsenazo III absorbance were used to measure calcium concentration changes caused by nitr photolysis in microcuvettes. Our model predicted the calcium increments caused by successive flashes, and their dependence on calcium loading, nitr concentration, and light intensity. Flashes also triggered the predicted calcium concentration jumps in neurons filled with nitr-arsenazo III mixtures. In physiological experiments, calcium-activated currents were recorded under voltage clamp in response to flashes of different intensity. Both IK(Ca) and INS(Ca) depended linearly without saturation upon calcium concentration jumps of 0.1-20 microM. Peak membrane currents in neurons exposed to repeated flashes first increased and then declined much like the arsenazo III absorbance changes in vitro, which also indicates a first-order calcium activation. Each flash-evoked current rose rapidly to a peak and decayed to half in 3-12 s. Our model mimicked this behavior when it included diffusion of calcium and nitr perpendicular to the surface of the neuron facing the flashlamp. Na/Ca exchange extruding about 1 pmol of calcium per square centimeter per second per micromolar free calcium appeared to speed the decline of calcium-activated membrane currents. Over a range of different membrane potentials, IK(Ca) and INS(Ca) decayed at similar rates, indicating similar calcium stoichiometries independent of voltage. IK(Ca), but not INS(Ca), relaxes exponentially to a different level when the voltage is suddenly changed. We have estimated voltage-dependent rate constants for a one-step first-order reaction scheme of the activation of IK(Ca) by calcium. After a depolarizing pulse, INS(Ca) decays at a rate that is well predicted by a model of diffusion of calcium away from the inner membrane surface after it has entered the cell, with active extrusion by surface pumps and uptake into organelles. IK(Ca) decays somewhat faster than INS(Ca) after a depolarization, because of its voltage-dependent relaxation combined with the decay of submembrane calcium. The interplay of these two currents accounts for the calcium-dependent outward-inward tail current sequence after a depolarization, and the corresponding afterpotentials after a burs

Dual mechanism for presynaptic modulation by axonal metabotropic glutamate receptor at the mouse mossy fibre-CA3 synapse

To investigate mechanisms responsible for the presynaptic inhibitory action mediated by the axonal group II metabotropic glutamate receptor (mGluR) at the mossy fibre-CA3 synapse, we used a quantitative fluorescence measurement of presynaptic Ca2+ in mouse hippocampal slices.Bath application of the group II mGluR-specific agonist (2S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV, 1 μM) reversibly suppressed the presynaptic Ca2+ influx (to 55·2 ± 4·6 % of control, n= 5) as well as field EPSPs recorded simultaneously (to 3·1 ± 2·0 %). Presynaptic fibre volley was not affected by 1 μM DCG-IV.A quantitative analysis of the inhibition of presynaptic Ca2+ influx and field EPSP suggested that DCG-IV suppressed the field EPSP to a greater extent than would be expected if the suppression were solely due to a decrease in the presynaptic Ca2+ influx.DCG-IV at 1 μM suppressed the mean frequency (to 73·8 ± 3·9 % of control, n= 11), but not the mean amplitude (to 97·0 ± 3·5 %), of miniature EPSCs recorded from CA3 neurones using the whole-cell patch-clamp technique.These results suggest that group II mGluR-mediated suppression is due both to a reduction of presynaptic Ca2+ influx and downregulation of the subsequent exocytotic machinery

Kainate receptor-mediated presynaptic inhibition at the mouse hippocampal mossy fibre synapse

The presynaptic action of kainate (KA) receptor activation at the mossy fibre-CA3 synapse was examined using fluorescence measurement of presynaptic Ca2+ influx as well as electrophysiological recordings in mouse hippocampal slices.Bath application of a low concentration (0·2 μM) of KA reversibly increased the amplitude of presynaptic volley evoked by stimulation of mossy fibres to 146 ± 6 % of control (n = 6), whereas it reduced the field excitatory postsynaptic potential (EPSPs) to 30 ± 4 %.The potentiating effect of KA on the presynaptic volleys was also observed in Ca2+-free solution, and was partly antagonized by (2S,4R)-4-methylglutamic acid (SYM 2081, 1 μM), which selectively desensitizes KA receptors.The antidromic population spike of dentate granule cells evoked by stimulation of mossy fibres was increased by application of 0·2 μM KA to 160 ± 10 % of control (n = 6). Whole-cell current-clamp recordings revealed that the stimulus threshold for generating antidromic spikes recorded from a single granule cell was lowered by KA application.Application of KA (0·2 μM) suppressed presynaptic Ca2+ influx to 78 ± 4 % of control (n = 6), whereas the amplitude of the presynaptic volley was increased.KA at 0·2 μM reversibly suppressed excitatory postsynaptic currents (EPSCs) evoked by mossy fibre simulation to 38 ± 9 % of control (n = 5).These results suggest that KA receptor activation enhances the excitability of mossy fibres, probably via axonal depolarization, and reduces action potential-induced Ca2+ influx, thereby inhibiting mossy fibre EPSCs presynaptically. This novel presynaptic inhibitory action of KA at the mossy fibre-CA3 synapse may regulate the excitability of highly interconnected CA3 networks

Photolysis-induced suppression of inhibition in rat hippocampal CA1 pyramidal neurons

Whole cell patch clamp recording, Ca2+ measurement with ratiometric fluorescent dyes and photolysis of caged Ca2+ were combined to investigate the depolarization- and photolysis-induced suppression of inhibition (DSI and PSI) in rat hippocampal CA1 pyramidal cells.A 5-s depolarization from −70 mV to 0 mV or a 6-s photolysis of nitrophenyl-EGTA (NPE) in cell bodies could each depress the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) and the amplitude of evoked IPSCs while elevating intracellular Ca2+ concentration ([Ca2+]i).Within a cell the elevation of [Ca2+]i induced by depolarization was inversely related to that induced by photolysis, suggesting that higher [NPE] is more effective in releasing caged Ca2+ but also increases buffer capacity to reduce [Ca2+]i rises caused by Ca2+ influx through voltage-dependent Ca2+ channels.Both DSI and PSI were linearly related to [Ca2+]i, with a 50 % reduction in transmission occurring at about 3.6–3.9 μM.[Ca2+]i recovered more quickly than DSI, indicating that the duration of DSI is not set simply by the duration of [Ca2+]i elevation, but rather entails other rate-limiting processes.We conclude that DSI is activated by micromolar [Ca2+]i acting far from sites of Ca2+ entry through channels in the plasma membrane

Presynaptic target of Ca2+ action on neuropeptide and acetylcholine release in Aplysia californica

When buccal neuron B2 of Aplysia californica is co-cultured with sensory neurons (SNs), slow peptidergic synapses are formed. When B2 is co-cultured with neurons B3 or B6, fast cholinergic synapses are formed.Patch pipettes were used to voltage clamp pre- and postsynaptic neurons and to load the caged Ca2+ chelator o-nitrophenyl EGTA (NPE) and the Ca2+ indicator BTC into presynaptic neurons. The relationships between presynaptic [Ca2+]i and postsynaptic responses were compared between peptidergic and cholinergic synapses formed by cell B2.Using variable intensity flashes, Ca2+ stoichiometries of peptide and acetylcholine (ACh) release were approximately 2 and 3, respectively. The difference did not reach statistical significance.ACh quanta summate linearly postsynaptically. We also found a linear dose-response curve for peptide action, indicating a linear relationship between submaximal peptide concentration and response of the SN.The minimum intracellular calcium concentrations ([Ca2+]i) for triggering peptidergic and cholinergic transmission were estimated to be about 5 and 10 μm, respectively.By comparing normal postsynaptic responses to those evoked by photolysis of NPE, we estimate [Ca2+]i at the release trigger site elicited by a single action potential (AP) to be at least 10 μm for peptidergic synapses and probably higher for cholinergic synapses.Cholinergic release is brief (half-width ≈200 ms), even in response to a prolonged rise in [Ca2+]i, while some peptidergic release appears to persist for as long as [Ca2+]i remains elevated (for up to 10 s). This may reflect differences in sizes of reserve pools, or in replenishment rates of immediately releasable pools of vesicles.Electron microscopy revealed that most synaptic contacts had at least one morphologically docked dense core vesicle that presumably contained peptide; these were often located within conventional active zones.Both cholinergic and peptidergic vesicles are docked within active zones, but cholinergic vesicles may be located closer to Ca2+ channels than are peptidergic vesicles