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.

▪ 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 ionotropic glutamate receptors are ligand-gated ion channels that mediate the vast majority of excitatory neurotransmission in the brain. The cloning of cDNAs encoding glutamate receptor subunits, which occurred mainly between 1989 and 1992 ([Hollmann and Heinemann, 1994][1]), stimulated this

/pdf/the-glutamate-receptor-ion-channels-560mntczdb.pdf

The glutamate receptor ion channels

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

Patch-clamp measurements of Ca2+ currents and membrane capacitance were performed on slices of mouse adrenal glands, using the perforated-patch configuration of the patch-clamp technique. These recording conditions are much closer to the in vivo situation than those used so far in most electrophysiological studies in adrenal chromaffin cells (isolated cells maintained in culture and whole-cell configuration). We observed profound discrepancies in the quantities of Ca2+channel subtypes (P-, Q-, N-, and L-type Ca2+channels) described for isolated mouse chromaffin cells maintained in culture. Differences with respect to previous studies may be attributable not only to culture conditions, but also to the patch-clamp configuration used. Our experiments revealed the presence of a Ca2+ channel subtype never before described in chromaffin cells, a toxin and dihydropyridine-resistant Ca2+ channel with fast inactivation kinetics, similar to the R-type Ca2+ channel described in neurons. This channel contributes 22% to the total Ca2+ current and controls 55% of the rapid secretory response evoked by short depolarizing pulses. Our results indicate that R-type Ca2+ channels are in close proximity with the exocytotic machinery to rapidly regulate the secretory process.

https://www.jneurosci.org/content/jneuro/20/22/8323.full.pdf

R-Type Ca2+ channels are coupled to the rapid component of secretion in mouse adrenal slice chromaffin cells.

The calcium concentration, [Ca]i, in single rat peritoneal mast cells was measured by means of the new Ca indicator dye fura-2. Upon stimulation with antigen or compound 48/80, [Ca]i rose for seconds to values greater than 5 microM. These Ca transients did not depend on the presence of extracellular Ca, and they sometimes occurred spontaneously, especially in the presence of exogenous phosphatidylserine. Calcium transients did not necessarily lead to degranulation. Degranulation usually occurred during periods of somewhat elevated [Ca] (0.5-1 microM) following transients but was sometimes observed at [Ca]i less than or equal to 250 nM. We found no evidence that an antigen-induced Ca influx is required for degranulation.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1986.tb04176.x

Fast calcium transients in rat peritoneal mast cells are not sufficient to trigger exocytosis.

We have studied the effects of cAMP on synaptic transmission at the calyx of Held and found that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analog of cAMP) potentiated excitatory postsynaptic currents (EPSCs). Direct sampling of miniature EPSCs (mEPSCs) and nonstationary fluctuation analysis showed that mEPSCs were not modulated by cAMP, suggesting that the locus of modulation is presynaptic. Deconvolution was used to examine effects of cAMP on quantal-release rates. By using this method, it was shown recently that release probabilities of readily releasable vesicles are heterogeneous. Here, we show that cAMP selectively increases the number of vesicles with higher release probabilities, whereas a slow component of the EPSC, representing vesicles that fuse more slowly, is unchanged. cAMP increases the apparent Ca2+ sensitivity for secretion, but this increase does not reflect an increase in release probability necessarily but rather an increase in the number of highly sensitive vesicles.

https://www.pnas.org/content/pnas/98/1/331.full.pdf

Preferential potentiation of fast-releasing synaptic vesicles by cAMP at the calyx of Held

It has been known since the work of Katz and collaborators in the early 1950s that an increase in intracellular Ca ++ concentration ((Ca ++ )) is the immediate trigger for neurotransmitter release (1). Later work has shown that, next to Ca ++ , many other signaling pathways, particularly via cyclic AMP, modulate the release of both neurotransmitters and hormones (2-5). However, regulated secretion is a multistep process and the signaling mecha- nisms involved act at many stages. Biochemical and traditional electrophysiological techniques very often can- not distinguish whether a change in secretion is caused by regulation of ion channels, vesicle trafficking, or the exocytic process itself. My laboratory has made an effort to dissect the stimulus secretion pathway by developing assays in chromaffin cells (for catecholamine release) and at a glutamatergic central nervous synapse (the calyx of Held, a component of the auditory pathway), which permit the study of secretion in single cells under voltage clamp conditions (6, 7). This enables us to clearly distinguish between consequences of changes in electrical signaling, from those regarding the process of vesicle recruitment or the process of exocytosis.

/pdf/a-comparison-between-exocytic-control-mechanisms-in-adrenal-32llx4xib5.pdf

A comparison between exocytic control mechanisms in adrenal chromaffin cells and a glutamatergic synapse

https://www.cell.com/biophysj/pdf/S0006-3495(01)75848-1.pdf

Erwin Neher

Papers

R-Type Ca2+ channels are coupled to the rapid component of secretion in mouse adrenal slice chromaffin cells.

Fast calcium transients in rat peritoneal mast cells are not sufficient to trigger exocytosis.

Preferential potentiation of fast-releasing synaptic vesicles by cAMP at the calyx of Held

A comparison between exocytic control mechanisms in adrenal chromaffin cells and a glutamatergic synapse

Estimating synaptic parameters from mean variance, and covariance in trains of synaptic responses.