Development of the quantal properties of evoked and spontaneous synaptic currents at a brain synapse.

In many studies of central synaptic transmission, the quantal properties of miniature synaptic events do not match those derived from synaptic events evoked by action potentials. Here we show that at mossy fiber-granule cell (MF-gc) synapses of mature cerebellum, evoked excitatory postsynaptic currents (EPSCs) are multiquantal, and their amplitudes vary in discrete steps, whereas miniature (m)EPSCs are monoquantal or multiquantal with quantal parameters identical to those of the EPSCs. In contrast, at immature MF-gc synapses, EPSCs are multiquantal, but their amplitudes do not vary in discrete steps, whereas most mEPSCs seem to be monoquantal with a broad and skewed amplitude distribution. The results demonstrate that quantal variance decreases during synaptic development. They also directly confirm the quantal hypothesis of neurotransmission at a mature brain synapse.

Development of action potential-dependent and independent spontaneous GABAA receptor-mediated currents in granule cells of postnatal rat cerebellum.

The postnatal development of spontaneous GABAergic transmission between cerebellar Golgi cells and granule cells was investigated with voltage-clamp recording from rat cerebellar slices, in symmetrical Cl- conditions. Between postnatal days 7 and 14 (P7-14), bicuculline- and TTX (tetrodotoxin)-sensitive spontaneous inhibitory postsynaptic currents (sIPSCs), occurred at high frequency in 56% of granule cells. Between P10 and P14, sIPSCs were superimposed on a tonic current of -12 +/- 1.8 pA at -70 mV, that was accompanied by noise with a variance of 17 +/- 3 pA2. Both the current and noise were inhibited by bicuculline. TTX blocked the bicuculline-sensitive current and noise by approximately 60%. Between P18 and P25, sIPSCs were less frequent; all cells showed tonic, bicuculline-sensitive currents, but these were partially inhibited by TTX (approximately 35%). Between P40 and P53, sIPSCs were rare; tonic, bicuculline-sensitive currents and noise were greater in amplitude, with mean values of -17 pA and 22 pA2 at -70 mV, they were present in all cells but they were not inhibited by TTX. Glycine receptor channels that were expressed in immature, but not adult cells, did not mediate spontaneous currents. Our results indicate that spontaneous transmission onto cerebellar granule cells in immature animals consists primarily of action potential-dependent, phasic release of vesicular GABA. This generates GABAA receptor-mediated sIPSCs. The effects of GABA transporter blockers suggest that it also produces the TTX-sensitive current-noise, as GABA spills out of synapses to activate extrasynaptic receptors or receptors in neighbouring synapses. In older animals, action potential-independent release of transmitter is predominant and results in tonic activation of GABAA receptors. This does not appear to be spontaneous vesicular release of GABA. Neither does it appear to be reversed uptake of GABA, although further work is required to rule out these possibilities.

Ca2+ entry following store depletion in SH-SY5Y neuroblastoma cells.

Ca2+ entry following Ca2+ store depletion was examined in the human neuroblastoma cell line, SH-SY5Y, by measuring the concentration of intracellular free Ca2+ ([Ca2+]i) with fura-2. Application of the muscarinic agonist oxotremorine-M (oxo-M) caused an increase in [Ca2+]i. This consisted of a peak, mediated by release of Ca2+ from internal stores followed by a sustained plateau, mediated by Ca2+ entry across the plasma membrane. The Ca2+ entry resulted from depletion of intracellular Ca2+ stores This pathway was further characterized in the presence of thapsigargin, an inhibitor of the Ca2+ ATPase involved in replenishing IP3-sensitive stores. Stores were first depleted with oxo-M and thapsigargin in the absence of extracellular Ca2+. After washout of oxo-M, subsequent exposure to Ca2+ evoked reproducible increases in [Ca2+]i. Application of oxo-M plus Ca2+ had little effect on the increases in [Ca2+]i, indicating that in SH-SY5Y cells, agonist-dependent pathways contribute little to Ca2+ entry following store depletion. Mn2+, Sr2+ and Ba2+ were permeable through this pathway. Mn2+ and Ba2+ also showed slight permeability in the absence of store depletion. Ca2+ entry following store depletion was blocked by La3+ (IC50 = 75 nM) and by SKF 96365. La3+ blocked Mn2+ entry through the pathway activated by store depletion but did not affect basal Mn2+ permeability. These results indicate that SH-SY5Y neuroblastoma cells have an agonist-independent Ca2+ entry pathway activated by store depletion.

Differential inhibition of Ca2+ channels in mature rat cerebellar Purkinje cells by sFTX-3.3 and FTX-3.3.

Synthetic funnel web spider toxin (sFTX-3.3) is a polyamine amide analogue of FTX, a toxin fraction isolated from the venom of the funnel web spider, Agelenopsis aperta, that blocks P-type Ca2+ channels. The structures of these polyamine containing compounds are not identical: sFTX-3.3 contains an amide carbonyl oxygen that is absent from the predicted structure of native FTX. Recently, a compound called FTX-3.3 was synthesized with the structure predicted for native FTX. We have compared the effects of polyamine amide sFTX-3.3 and polyamine FTX-3.3, on Ca2+ channel currents in the soma of mature rat cerebellar Purkinje neurons, in which the predominant Ca2+ channels are defined as P-type. Differential inhibition by sFTX-3.3 and FTX-3.3 revealed three populations of Ca2+ channels. One group, mediating approximately 66% of the current, was blocked by sFTX-3.3 with an IC50 (concentration producing half maximal inhibition) of 33 nM or by FTX-3.3 with an IC50 of 55 pM. A second population (5-25% of the total current) was inhibited by sFTX-3.3 with an IC50 of 33 nM, but was insensitive to FTX-3.3, while a third (10-30%) was blocked by FTX-3.3 with an IC50 of 125 nM and was resistant to sFTX-3.3. These channels also showed distinctive current-voltage relationships. Our results suggest that P-type Ca2+ channels in mature rat cerebellar Purkinje cells may be subdivided according to pharmacological and biophysical properties.

Allosteric interactions at L-type calcium channels between FPL 64176 and the enantiomers of the dihydropyridine Bay K 8644.

Functional interactions between the enantiomers of the dihydropyridine 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridi ne carboxylic acid methyl ester (Bay K 8644) and the benzoylpyrrole methyl 2,5-dimethyl-4-[2(phenylmethyl)benzoyl]-H-pyrrole-3-carboxylate (FPL 64176) were investigated on L-type Ca++ channels in guinea pig ileal longitudinal smooth muscle. The effects of these drugs, when applied individually, were as described in earlier studies. For instance, both (-)-(S)-Bay K 8644 and FPL 64176 caused concentration-dependent contraction, which is consistent with Ca++ channel activation, whereas (+)-(R)-Bay K 8644 gave concentration-dependent relaxation, which is consistent with Ca++ channel inhibition. The activities of the different drugs were dependent on the extracellular levels of KCI. When applied in combination, however, the responses evoked were not those predicted from the effects of the drugs applied individually. Contractions produced by FPL 64176 (25 nM to 1 microM) were abolished in the presence of 100 nM (-)-(S)-Bay K 8644 but were potentiated by 10 to 150 nM (+)-(R)-Bay K 8644 and inhibited by 1 microM (+)-(R)-Bay K 8644. Conversely, contractile responses to (-)-(S)-Bay K 8644 were abolished by 100 nM FPL 64176. In the presence of 1 microM FPL 64176, however, (-)-(S)-Bay K 8644 gave concentration-dependent relaxation of the muscle, which is consistent with Ca++ channel inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

P-type calcium channels in the somata and dendrites of adult cerebellar Purkinje cells.

The pharmacological and single-channel properties of Ca2+ channels were studied in the somata and dendrites of adult cerebellar Purkinje cells. The Ca2+ channels were exclusively of the high threshold type: low threshold Ca2+ channels were not found. These high threshold channels were not blocked by omega-conotoxin GVIA and were inhibited rather than activated by BAY K 8644. They were therefore pharmacologically distinct from high threshold N- and L-type channels. Funnel web spider toxin was an effective blocker. The channels opened to conductance levels of 9, 14, and 19 pS (in 110 mM Ba2+). These slope conductances were in the range of those reported for N- and L-type channels. Our results are in agreement with previous reports suggesting that Ca2+ channels in Purkinje cells can be classified as P-type channels according to their pharmacology. The results also suggest that distinctions among Ca2+ channel types based on the single-channel conductance are not definitive.

Differential expression by nerve growth factor of two types of Ca2+ channels in rat phaeochromocytoma cell lines.

1. Two clones of rat phaeochromocytoma PC12 cells have been used to study the expression of Ca2+ channels and their possible involvement in neuronal differentiation. One clone differentiated morphologically when exposed to nerve growth factor (NGF) for 4 days (PC12 cells), while the other clone was insensitive to NGF, but differentiated morphologically in the presence of ouabain (0.1 mM) for 7 days (PC12-mutant cells). 2. Whole-cell Ba2+ currents through Ca2+ channels were measured in PC12 cells at a test potential (Et) of +10 mV, from two holding potentials (Eh) of -90 and -30 mV (I-90 and I-30). NGF-induced differentiation increased I-90 by 248% and I-30 by 133%. The cells that differentiate in the presence of ouabain had only small, if any, Ba2+ currents that did not appear to change during morphological differentiation or after the addition of NGF. 3. Barium currents in PC12 cells could be separated into two components by selective antagonists. The component of I-90 that could be inhibited by omega-conotoxin GVIA (omega-CgTX) in NGF-differentiated cells was 458 +/- 84 pA (mean +/- S.E.M.), compared with 79 +/- 44 pA in native cells. I-30 was reduced by 50 +/- 17 pA in NGF-treated cells and was virtually insensitive to the toxin in native cells. By contrast, the dihydropyridine (DHP) isradipine reduced I-30 in NGF-treated cells by 30 +/- 8 pA and in native cells by 20 +/- 3 pA. 4. Radioligand binding studies with 125I-omega-CgTX in PC12 cell membrane fragments and in PC12 cells showed a 2- to 3-fold increase in maximal binding capacity after NGF exposure, while mutant cells showed no such change in binding capacity after treatment with NGF or ouabain. Staurosporine inhibited the effect of NGF on 125I-omega-CgTX binding. [3H](+)-isradipine binding capacity was increased 1.8-fold by NGF in depolarized PC12 cells while no change was observed in mutant cells after NGF or ouabain. There was no interaction between omega-CgTX and DHP binding sites. 5. Both the electrophysiological and the binding data indicate a preferential expression of omega-CgTX-sensitive Ca2+ channels (N type) over isradipine-sensitive channels (L type) in PC12 cells treated with NGF. By contrast, ouabain-induced differentiation of a mutant PC12 cell line, that lacks functional NFG receptors, was not associated with the expression of Ca2+ channels.

Whole-cell current noise produced by excitatory and inhibitory amino acids in large cerebellar neurones of the rat.

1. Membrane noise and current changes produced by glutamate and related excitatory amino acids have been examined in cultured large cerebellar neurones (including Purkinje cells), with whole-cell patch-clamp methods. The sensitivity of these neurones to the inhibitory amino acids gamma-aminobutyric acid (GABA) and glycine has also been studied. 2. The neurones formed inhibitory synapses in culture, and displayed spontaneous synaptic currents. Reducing the pipette Cl- concentration (i.e. intracellular synaptic currents. Reducing the pipette Cl- concentration (i.e. intracellular concentration) caused a negative shift in their reversal potential, and the currents could be blocked with bicuculline (10 microM), suggesting that they were mediated by GABAA receptors. Spontaneous synaptic activity was also considerably reduced in the presence of 3 microM-tetrodotoxin. 3. Analysis of the increase in whole-cell current noise produced by the application of GABA (3 microM) gave noise spectra that were fitted by two Lorentzian components with slow and fast time constants of 23.6 and 1.9 ms at a membrane potential (Vm) of -110 mV. The mean single-channel conductance estimated from GABA noise was gamma noise = 12 pS. Glycine (10 microM) whole-cell current responses were Cl(-)-mediated and reversibly abolished by 1 microM-strychnine. 4. Bath application of excitatory amino acids gave whole-cell current changes accompanied by an increase in synaptic activity. Postsynaptic responses to the excitatory amino acids were more readily seen after the inhibitory synaptic currents had been abolished by bicuculline. Membrane current changes were obtained in response to the putative transmitters glutamate and aspartate, and the agonists NMDA (N-methyl-D-aspartate), ibotenate, quisqualate and kainate. Their reversal potential was approximately -5 mV. 5. A majority of noise spectra produced by the various glutamate receptor agonists were fitted by two Lorentzian components; the rest were fitted with a single Lorentzian component. The noise time constants were apparently not dependent on the type of glutamate agonist used to activate the receptor channels. Pooling data for all agonists gave a mean time constant for single-component spectra of tau noise = 4.8 +/- 0.3 ms; for two-component spectra the time constants were tau 1 = 22.7 +/- 1.8 ms and tau 2 = 2.2 +/- 0.12 ms (Vm = -110 to -50 mV). It is likely that the two components present in whole-cell noise spectra reflect complex kinetics of glutamate receptor channels. 6. The mean single-channel conductance was estimated from whole-cell noise for the various excitatory amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

On the multiple-conductance single channels activated by excitatory amino acids in large cerebellar neurones of the rat.

1. Single-channel currents evoked by excitatory amino acids have been examined in outside-out patches from large cerebellar neurones (including Purkinje cells) in tissue culture. L-Glutamate (3-10 microM), L-aspartate (3-10 microM), NMDA (N-methyl-D-aspartate, 10-50 microM), ibotenate (50 microM), quisqualate (3-50 microM), and kainate (3-50 microM) all produced single-channel currents with multiple amplitudes. 2. Single-channel currents recorded over a range of patch potentials had a mean interpolated reversal potential of -3.8 +/- 0.5 mV. The directly resolvable multiple conductance levels could be classified into five main groups, with mean values (averaged for all agonists) of: 47.9 +/- 0.7, 38.5 +/- 0.8, 27.8 +/- 1.4, 18.2 +/- 0.5 and 8.3 +/- 0.6 pS. 3. From the relative areas under current amplitude histograms it was estimated that the percentage of openings with conductances greater than 30 pS was about 83% with NMDA, 79% with glutamate and 78% with aspartate. In some patches, the majority of greater than 30 pS events evoked by these agonists were to the maximum conductance of 48 pS, whereas in other patches there were more 38 pS openings than 48 pS openings. Only 27% of quisqualate openings, and about 10% of kainate openings, were greater than 30 pS. 4. Of the small amplitude (less than 20 pS) events, 93% of quisqualate openings were to the 8 pS level whereas approximately 87% of less than 20 pS currents produced by NMDA, glutamate and aspartate were to the 18 pS level (the remainder being 8 pS). Direct transitions could occur between certain levels (including events above and below 30 pS) suggesting that these are sublevels of multiple-conductance channels. The most frequently occurring transitions were between the 48 and 38 pS levels, and the 38 and 18 pS levels. 5. Channel openings occurred in bursts, within which individual openings were separated either by brief closures (gaps), or by direct transitions between the multiple conductance levels. The briefest of these gaps (less than 200-400 microseconds) could represent a mixture of transitions to lower conductance levels as well as partially resolved complete shuttings. The mean duration of the longer gaps within bursts, thought to represent complete but partially resolved shuttings was 1.05 +/- 0.25 ms (pooled for all agonists). 6. Burst-length distributions could be fitted with the sum of three exponentials. The briefest component may have arisen from brief single openings. The two slower components probably reflect the existence of two kinetically distinct open states.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids.

L-GLUTAMATE and L-aspartate are thought to have a widespread function as synaptic transmitters in the mammalian central nervous system and there are at least three types of neuronal glutamate receptors, which can be activated by the selective agonists N-methyl-D-aspartate (NMDA), quisqualate and kainate. Recent experiments indicate that glutamate receptors also occur in astrocytes. We have used patch-clamp methods to determine whether one type of macroglial cell, the type-2 astrocyte, possesses glutamate receptors, as previously proposed from neurochemical studies. We find that glutamate and related amino acids can evoke whole-cell and single-channel currents in type-2 astrocytes from rat cerebellum. Although these cells are found mainly in white matter, where neurotransmission does not occur, their processes are closely associated with axons at nodes of Ranvier, suggesting that such receptors are involved in neuronal-glial signalling at the node. Our experiments show that glial cells possess quisqualate- and kainate-receptor channels but lack receptors for NMDA. Interestingly, these glutamate channels exhibit multiple conductance levels that are similar in amplitude to the neuronal glutamate channels.

Multiple-conductance channels activated by excitatory amino acids in cerebellar neurons.

In the mammalian central nervous system amino acids such as L-glutamate and L-aspartate are thought to act as fast synaptic transmitters. It has been suggested that at least three pharmacologically-distinguishable types of glutamate receptor occur in central neurons and that these are selectively activated by the glutamate analogues N-methyl-D-aspartate (NMDA), quisqualate and kainate. These three receptor types would be expected to open ion channels with different conductances. Hence if agonists produce similar channel conductances this would suggest they are acting on the same receptor. Another possibility is suggested by experiments on spinal neurons, where GABA (gamma-amino butyric acid) and glycine appear to open different sub-conductance levels of one class of channel while acting on different receptors. By analogy, several types of glutamate receptor could also be linked to a single type of channel with several sub-conductance states. We have examined these possibilities in cerebellar neurons by analysing the single-channel currents activated by L-glutamate, L-aspartate, NMDA, quisqualate and kainate in excised membrane patches. All of these agonists are capable of opening channels with at least five different conductance levels, the largest being about 45-50 pS. NMDA predominantly activated conductance levels above 30 pS while quisqualate and kainate mainly activated ones below 20 pS. The presence of clear transitions between levels favours the idea that the five main levels are all sub-states of the same type of channel.

Glutamate and aspartate activated channels and inhibitory synaptic currents in large cerebellar neurons grown in culture.

Patch-clamp methods were used to examine large (greater than 30 microns) cerebellar neurons of the rat, maintained in cell culture. Cells possessed voltage-activated transient inward Na+ currents which were sensitive to tetrodotoxin. Spontaneous synaptic currents, present in whole-cell recordings, were abolished by bicuculline and picrotoxin and were carried by Cl-. Cells produced inward currents in response to the transmitter candidates glutamate and aspartate and also to the glutamate agonists kainate, quisqualate, N-methyl-D-aspartate and ibotenate. Analysis of glutamate and aspartate-current noise has been used to derive characteristics of the excitatory channels. Single channel currents have also been observed directly in whole-cell and outside-out patches. Both glutamate and aspartate are able to activate channels which were blocked by Mg2+ and had a maximum conductance of 50 pS.