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.

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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

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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

Calcium buffers in flash-light.

Around 1970 the fundamental signal mechanisms for communication between cells of the nervous system were known. Hodgkin and Huxley (1952) had provided the basis for understanding the nerve action potential. The concept of chemical transmission at synapses had received its experimental verification by detailed studies on excitatory and inhibitory postsynaptic potentials (see Katz, 1966, for a concise description of the electrical signals in nerve and muscle). The question of the molecular mechanisms underlying these signals was still open, however. Hodgkin and Huxley (1952) used the concept of voltage-operated gates for a formal description of conductance changes, and by 1970 the terms Na-channel and K-channel were used frequently (see review by Hille, 1970), although no direct evidence for the existence of channels was available from biological preparations. This was different for the case of artificial membranes. Miiller and Rudin (1963) introduced \"black-lipid membranes\" as experimental model systems, which in many respects resemble the bimolecular lipid membrane of living cells. These membranes are rather good insulators. However, when they are doped with certain antibiotics or proteins they become electrically conductive. Bean et al. (1969) and Hladky and Haydon (1970) showed that some of these dopants induce discrete, steplike changes in conductance when they are added in trace amounts. All the evidence suggested that the conductance changes observed represent the insertion of single pore-like structures into the membranes.

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Nobel lecture. Ion channels for communication between and within cells.

The analysis of transients in intracellular Ca2+ concentration ([Ca2+]) has a long and – unfortunately – not so bright history. Although it was recognized early on that Ca2+-indicator dyes act as Ca2+ buffers and distort the dynamical aspects of [Ca2+] transients (Timmerman & Ashley, 1986), this was largely neglected in the relevant literature. Researchers got away with ignoring such distortions, because of a peculiar property of Ca2+ buffers with respect to rapid [Ca2+] transients induced by action potentials: buffers reduce the amplitudes of such transients, but at the same time lengthen them by about the same factor. Therefore, the area under transients (or the integral over the deviation in [Ca2+] throughout the transient) is unchanged by buffers. Consequently, any Ca2+-sensing effector process is unaffected by their presence, provided that it is linear in [Ca2+] (Neher, 1998). However, many processes in biology are non-linear, which means that quite often the details of the [Ca2+] signal are relevant. The paper by Aponte et al. (2008) in this issue of The Journal of Physiology is an excellent example of a growing list of studies, in which investigators have made serious and successful attempts to measure unperturbed Ca2+ signals. Not surprisingly, it turned out that the signals reveal detail and allow conclusions about roles of [Ca2+] in synaptic transmission that go far beyond those seen with normal (i.e. distorted) Ca2+ imaging.

Early work on several types of neurons and chromaffin cells was restricted to measurement of [Ca2+] by fura-2, a high-affinity Ca2+ buffer. When such a dye is infused into cells via patch pipettes the endogenous properties of cells are overridden by the buffering action of the dye, but they can be estimated by extrapolation of a concentration series to zero buffer concentration (Neher & Augustine, 1992; Helmchen et al. 1997; Lee et al. 2000). Using this approach, the buffering ratio (i.e. the incremental ratio of bound Ca2+ to free Ca2+) of the cytoplasm of several cell types was found to be 40 or somewhat larger (see Neher, 1995 for review of early work). Fura-2 at 100 μm, which is often considered to be a low concentration of indicator dye, has a buffering ratio of approximately 330 at low [Ca2+]. Therefore, the buffering by the indicator dye dominates the Ca2+ dynamics; in fact, a Ca2+ transient following an action potential is attenuated by the dye by almost a factor of 10. More recently, low-affinity indicator dyes have become available, allowing researchers to better match the affinity of the dye to the amplitude of the expected signal (The Handbook, Invitrogen; http://probes.invitrogen.com/handbook/). Thus, complications by saturation of the dye are avoided and buffering effects of the indicator dye can be minimized.

In their current study (this issue) Aponte et al. first used fura-2 to carefully estimate the endogenous buffering ratio (κs) in apical dendrites of fast spiking hippocampal basket cells. They found a relatively large value of 202, meaning that upon the entry of 203 Ca2+ ions, 202 will be bound to endogenous Ca2+-binding sites and 1 will remain free. By extrapolation to zero fura-2 concentration they found the [Ca2+] increment during an action potential (AP) to be 43 nm and the unperturbed decay time constant of that signal to be 360 ms. The high value of κs made it relatively easy to also measure these properties directly, since the low-affinity dye fura-FF at 100 μm concentration has a Ca2+-binding ratio of only 24. Analysing averages from 10 single AP responses, Aponte et al. arrived at a peak excursion value of 39 nm for a single AP, which appropriately is about 10% lower than the value obtained by extrapolation. The [Ca2+] increment increased linearly with the number of action potentials for short high-frequency trains, while during intense stimulation [Ca2+] signalling was shifted into a non-linear range. The relatively long duration of the transient was implied to set the time window for coincidence detection during LTP.

In contrast, a recent study on the calyx of Held (a giant, calyx-like nerve terminal in the brainstem) showed large transients and quite non-linear behaviour, even at the single-spike level (Muller et al. 2007). The calyx has a low intrinsic buffering ratio of 40, AP-induced Ca2+ transients of 400 nm, and a biphasic decay of the Ca2+ transient, which is governed both by Ca2+-removal mechanisms and by slow binding of Ca2+ to the Ca2+-binding protein parvalbumin. This decay correlates with a rapid decay of paired-pulse facilitation (see also Atluri & Regehr, 1996; Caillard et al. 2000). However, these properties can be observed only when extreme care is taken not to override the intrinsic buffers by the dye and not to ‘washout’ endogenous mobile buffers by diffusional loss through the patch pipette.

Both recent studies, discussed here, stress the importance of a subtler handling of Ca2+ signals. Only when care is taken not to overwhelm the cell by Ca2+ buffering of indicators can one reveal important dynamic aspects, which govern the kinetics of paired-pulse synaptic facilitation or the fine-tuning of LTP induction.

Details of Ca2+ dynamics matter.

Noise analysis was performed on the membrane currents produced by the ion channel former gramicidin A in black lipid bilayer membranes. The average channel lifetime and the unit channel conductance can be determined from the autocorrelation function. The values agree with the independently obtained data from measurements of single channels. The dependence of this function on the channel density reveals information on the process of channel formation. The kinetic information is the same as that obtained by voltage clamp measurements.

The properties of ionic channels measured by noise analysis in thin lipid membranes.

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 (pfusion) 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 pfusion of SVs with a mature release machinery but reflect both pfusion 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.

Erwin Neher

Papers

Calcium buffers in flash-light.

Nobel lecture. Ion channels for communication between and within cells.

Details of Ca2+ dynamics matter.

The properties of ionic channels measured by noise analysis in thin lipid membranes.

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