Ca2+ is a highly versatile intracellular signal that operates over a wide temporal range to regulate many different cellular processes. An extensive Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics. Rapid highly localized Ca2+ spikes regulate fast responses, whereas slower responses are controlled by repetitive global Ca2+ transients or intracellular Ca2+ waves. Ca2+ has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease.

/pdf/calcium-signalling-dynamics-homeostasis-and-remodelling-2oni038yeg.pdf

Calcium signalling: dynamics, homeostasis and remodelling

▪ Abstract Synaptic transmission is a dynamic process. Postsynaptic responses wax and wane as presynaptic activity evolves. This prominent characteristic of chemical synaptic transmission is a crucial determinant of the response properties of synapses and, in turn, of the stimulus properties selected by neural networks and of the patterns of activity generated by those networks. This review focuses on synaptic changes that result from prior activity in the synapse under study, and is restricted to short-term effects that last for at most a few minutes. Forms of synaptic enhancement, such as facilitation, augmentation, and post-tetanic potentiation, are usually attributed to effects of a residual elevation in presynaptic [Ca2+]i, acting on one or more molecular targets that appear to be distinct from the secretory trigger responsible for fast exocytosis and phasic release of transmitter to single action potentials. We discuss the evidence for this hypothesis, and the origins of the different kinetic phases...

/pdf/short-term-synaptic-plasticity-5d4dx79319.pdf

Short-Term Synaptic Plasticity

In cultures of dissociated rat hippocampal neurons, persistent potentiation and depression of glutamatergic synapses were induced by correlated spiking of presynaptic and postsynaptic neurons. The relative timing between the presynaptic and postsynaptic spiking determined the direction and the extent of synaptic changes. Repetitive postsynaptic spiking within a time window of 20 msec after presynaptic activation resulted in long-term potentiation (LTP), whereas postsynaptic spiking within a window of 20 msec before the repetitive presynaptic activation led to long-term depression (LTD). Significant LTP occurred only at synapses with relatively low initial strength, whereas the extent of LTD did not show obvious dependence on the initial synaptic strength. Both LTP and LTD depended on the activation of NMDA receptors and were absent in cases in which the postsynaptic neurons were GABAergic in nature. Blockade of L-type calcium channels with nimodipine abolished the induction of LTD and reduced the extent of LTP. These results underscore the importance of precise spike timing, synaptic strength, and postsynaptic cell type in the activity-induced modification of central synapses and suggest that Hebb’s rule may need to incorporate a quantitative consideration of spike timing that reflects the narrow and asymmetric window for the induction of synaptic modification.

https://www.jneurosci.org/content/jneuro/18/24/10464.full.pdf

Synaptic Modifications in Cultured Hippocampal Neurons: Dependence on Spike Timing, Synaptic Strength, and Postsynaptic Cell Type

The responses of vertebrate neurones to glutamate involve at least three receptor types. One of these, the NMDA receptor (so called because of its specific activation by N-methyl-D-aspartate), induces responses presenting a peculiar voltage sensitivity. Above resting potential, the current induced by a given dose of glutamate (or NMDA) increases when the cell is depolarized. This is contrary to what is observed at classical excitatory synapses, and recalls the properties of 'regenerative' systems like the Na+ conductance of the action potential. Indeed, recent studies of L-glutamate, L-aspartate and NMDA-induced currents have indicated that the current-voltage (I-V) relationship can show a region of 'negative conductance' and that the application of these agonists can lead to a regenerative depolarization. Furthermore, the NMDA response is greatly potentiated by reducing the extracellular Mg2+ concentration [( Mg2+]o) below the physiological level (approximately 1 mM). By analysing the responses of mouse central neurones to glutamate using the patch-clamp technique, we have now found a link between voltage sensitivity and Mg2+ sensitivity. In Mg2+-free solutions, L-glutamate, L-aspartate and NMDA open cation channels, the properties of which are voltage independent. In the presence of Mg2+, the single-channel currents measured at resting potential are chopped in bursts and the probability of opening of the channels is reduced. Both effects increase steeply with hyperpolarization, thereby accounting for the negative slope of the I-V relationship of the glutamate response. Thus, the voltage dependence of the NMDA receptor-linked conductance appears to be a consequence of the voltage dependence of the Mg2+ block and its interpretation does not require the implication of an intramembrane voltage-dependent 'gate'.

Magnesium gates glutamate-activated channels in mouse central neurones

Interneurons of the hippocampus

1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.

/pdf/improved-patch-clamp-techniques-for-high-resolution-current-tmno572o60.pdf

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or alteration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and Cl currents, lasts for approximately 5 min. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above approximately 1 microM ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].

/pdf/muscarinic-activation-of-ionic-currents-measured-by-a-new-xaerj4ehzt.pdf

Muscarinic activation of ionic currents measured by a new whole-cell recording method

1. Inward currents in chromaffin cells were studied with the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The intracellular solution contained 120 mM-Cs+ and 20 mM-tetraethylammonium (TEA+). Na+ currents were studied after blockade of Ca2+ channels with 1 mM-Co2+ applied externally. Ca2+ currents were recorded after eliminating Na+ currents with tetrodotoxin (TTX). The current recordings were obtained in cell-attached, outside-out and whole-cell recording configurations (Hamill et al. 1981).

2. Single channel measurements gave an elementary current amplitude of 1 pA at -10 mV for Na+ channels. This amplitude increased with hyperpolarization between -10 and -40 mV, but did not vary significantly between -40 and -70 mV.

3. The mean Na+ channel open time was 1 ms at -30 mV. This open time decreased both with depolarization and hyperpolarization. Its value was close to the time constant of inactivation, τh, above -20 mV.

4. Ensemble fluctuation analysis of Na+ currents gave results consistent with those of single channel measurements. Noise power spectra obtained between -35 mV and 0 mV could be fitted with a single Lorentzian. A range of Na+ channel densities of 1·5-10 channels per μm2 was calculated.

5. Cell-attached single Ca2+ channel recordings were obtained in isotonic BaCl2 solution. The single channel amplitude was 0·9 pA at -5 mV, and it became smaller for positive potential values.

6. At -5 mV, single Ba2+ currents appeared as bursts of 1·9 ms mean duration containing on the average 0·6 short gaps. The burst duration was larger at positive potentials.

7. Ensemble fluctuation analysis of Ca2+ channels was performed on whole-cell recordings in external solutions containing isotonic BaCl2 or external Ca2+ (Cao) concentrations of 1 and 5 mM. The unit amplitude calculated in the former case was similar to that obtained in single channel measurements.

8. Noise power spectra of Ca2+ or Ba2+ currents could be fitted by the sum of two, but not one, Lorentzian components.

9. Tail currents could be fitted by the sum of two exponential components. The corresponding time constants had values close to those obtained with noise analysis.

10. The rising phase of Ca2+ and Ba2+ currents was sigmoid. It could be fitted by the sum of three exponentials. The time constant of the largest amplitude component, τ1, was similar to the time constants of the slow component observed in noise and tail experiments. This time constant also corresponded to the burst duration obtained in single channel measurements.

11. The value of τ1 was larger in 5 mM-Cao and in isotonic Ba2+ than in 5 mM-Bao. Thus, the kinetic properties of Ca2+ channels depend on the nature and concentration of the permeating ion.

12. A simple kinetic scheme is proposed to model the activation pathway of Ca2+ channels.

13. Currents in 1 mM-Cao and 5 mM-Cao showed clear reversals around +53 mV and +64 mV respectively. The outward currents observed above these potentials are most probably due to Cs+ ions flowing through Ca2+ channels.

14. The instantaneous current—voltage relation was obtained from tail current data in the range -70 to +100 mV in 5 mM-Cao. The resulting curve displayed an inflexion point around the reversal potential.

15. Very little inactivation of Ca2+ currents was observed. However, a slow current decline was observed in some cells above +10 mV.

16. Conditioning prepulses to positive potentials had potentiating or depressing effects on Ca2+ currents depending on whether the test pulse lay below or above the maximal current potential. The potentiating effect may be linked to the slowest component of the current rise observed below +10 mV. The depressing effect may be related to the slow decline obtained above +10 mV.

17. Analysis of ensemble variance and of tail current amplitudes suggested that the opening probability of Ca2+ channels was at least 0·9 above +40 mV.

18. A slow rundown of Ca2+ currents was observed in whole-cell recordings. The speed of the rundown was dependent on intracellular Ca2+ concentration. The rundown was apparently due to a progressive elimination of the channels available for activation.

19. The density of Ca2+ channels (before rundown) was estimated at 5-15/μm2.

20. In cell-attached experiments, inward current channels were often seen to follow action potentials. These events did not appear to be the usual Na+ and Ca2+ currents. They were probably due to cation influx of either Na+ or Ba2+, depending on the pipette solution, through Ca2+-dependent channels. Voltage-independent single channel activity observed in whole-cell and outside-out recordings may be due to the same channels.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1982.sp014394

Sodium and calcium channels in bovine chromaffin cells

1. Bovine chromaffin cells were enzymatically isolated and kept in short term tissue culture. Their electrical properties were studied using recent advances of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). 2. When a patch pipette was sealed tightly to a chromaffin cell ('cell-attached configuration') current wave forms due to intracellular action potentials could be observed. The frequency of the wave forms was altered by changing the pipette potential. When acetylcholine was present in the pipette solution, acetylcholine-induced single channel currents were evident in the patch recording. Action potential wave forms were then often seen to follow acetycholine-induced single channel currents. 3. In the cell-attached configuration, large single channel current events did not resemble square pulses but showed exponential relaxations with time constants of the order of 50 ms. 4. After rupture of the patch of membrane, the pipette--cell seal remained stable ('whole-cell recording', Hamill et al. 1981). Chromaffin cells were found to have a resting potential of -50 to -80 mV, and an input resistance around 5 G omega. The high cell resistance accounts for the relaxing currents evident in the cell-attached configuration. 5. In the best cases, the effective time constant of the voltage clamp in the whole-cell recording mode was 15 microseconds. Exchange of small ions such as Na+ ions between pipette and cell interior solutions was then complete within 15 s. 6. Acetylcholine-induced currents were obtained at various acetylcholine concentrations. Single acetylcholine-induced channels had a slope conductance of 44 pS between -100 and -55 mV, and a mean duration of 27 ms at -80 mV (at room temperature).

A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine.

Although patch pipettes were originally developed for the recording of single channels, they can be of great advantage for more conventional recordings from whole cells, particularly when the cells under study are small. As described by Hamill et al. (1981), a membrane patch can be broken by a short pulse of suction after a gigaseal has been formed. Very often, this manipulation does not damage the pipette-membrane seal, so that a conductive pathway is established between the pipette and the cell interior, which is well insulated from the bath. It is our experience that this way of penetrating a cell inflicts much less damage on the cell than the standard microelectrode impalement.

Alain Marty

Papers

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

Muscarinic activation of ionic currents measured by a new whole-cell recording method

Sodium and calcium channels in bovine chromaffin cells

A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine.

Tight-Seal Whole-Cell Recording