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

Many physiological processes are controlled by changes in membrane permeability. Inflow of sodium or calcium ions into a living cell can, for instance, trigger a nerve action potential, elicit muscle contraction, or lead to hormone release. These permeability changes occur in so-called ionic channels which open and close in response to chemical and electrical stimuli. The ion flow through individual channels can be recorded as an electrical signal. The typical signal is a rectangular current pulse of 1 to 2 pA (1-2 × 10−12A) amplitude and of 1 to 10 msec duration. Three examples of channels and their unitary currents are discussed: 1) The gramicidin channel, a model channel of known structure, which can be incorporated into artificial membranes; 2) the acetylcholine- activated channel, which is the best-characterized channel in biological membranes; and 3) the electrically activated Na channel, which is responsible for the nerve action potential.

Ion flow through individual membrane pores and its control by membrane voltage and by chemical reactions

In vitro calcium binding kinetics of calcium chelators as revealed by temperature-jump relaxation

Secretion via exocytosis is a process common to excitable as well as non-excitable cells. The notion that this process is entirely determined by a rise in [Ca]i is no longer tenable in view of recent reports demonstrating secretion at basal or even reduced levels of [Ca]i. It appears appropriate to distinguish between electrically excitable and electrically non-excitable cells. In the former, a rise in [Ca]i is the triggering event for secretion, whereas in the latter, second messengers seem to induce secretion while [Ca]i acts as a modulator of the rate of secretion. Conversely, second messengers may modulate Ca-induced secretion in excitable cells.

[The importance of calcium for secretion in excitable and non-excitable cells].

Abstract Glutamatergic synapses display variable strength and diverse short-term plasticity (STP), even for a given type of connection. Using non-negative tensor factorization (NTF) and conventional state modelling, we demonstrate that a kinetic scheme consisting of two sequential and reversible steps of release-machinery assembly and a final step of synaptic vesicle (SV) fusion reproduces STP and its diversity among synapses. Analyzing transmission at calyx of Held synapses reveals that differences in synaptic strength and STP are not primarily caused by variable fusion probability ( p fusion ) but determined by the fraction of docked synaptic vesicles equipped with a mature release machinery. Our simulations show, that traditional quantal analysis methods do not necessarily report p fusion of SVs with a mature release machinery but reflect both p fusion and the distribution between mature and immature priming states at rest. Thus, the approach holds promise for a better mechanistic dissection of the roles of presynaptic proteins in the sequence of SV docking, two-step priming and fusion and suggests a mechanism for activity-induced redistribution of synaptic efficacy. 

A sequential two-step priming scheme reproduces diversity in synaptic strength and short-term plasticity

Whole-cell membrane capacitance measure- ments are frequently used to monitor neuronal and nonneu- ronal secretory activity. However, unless individual fusion events can be resolved, the type of the fusing vesicles cannot be identified in these experiments. Here we apply statistical analysis of trial-to-trial variations between depolarization- induced capacitance increases of mouse adrenal chromaffin cells and obtain estimates for the capacitance contribution of individual exocytic vesicles between 0.6 and 2 fF. For com- parison, measurements of membrane capacitance were com- bined with amperometric recordings of catecholamine release during intracellular perfusion of chromaffin cells with high (Ca 21 ). Crosscorrelation of both signals yielded a mean capacitance contribution of individual catecholaminergic ves- icles of 1.3 fF. We suggest that depolarization-induced capac- itance increases in mouse adrenal chromaffin cells mainly represent fusion of chromaffin granules.

Estimation of mean exocytic vesicle capacitance in mouse adrenal chromaffin cells (exocytosisysynaptic-like microvesiclesyamperometryymembrane capacitanceynoise analysis)

Erwin Neher

Papers

Ion flow through individual membrane pores and its control by membrane voltage and by chemical reactions

In vitro calcium binding kinetics of calcium chelators as revealed by temperature-jump relaxation

[The importance of calcium for secretion in excitable and non-excitable cells].

A sequential two-step priming scheme reproduces diversity in synaptic strength and short-term plasticity

Estimation of mean exocytic vesicle capacitance in mouse adrenal chromaffin cells (exocytosisysynaptic-like microvesiclesyamperometryymembrane capacitanceynoise analysis)