Abstract.(1) A preparation is described which allows patch clamp recordings to be made on mammalian central nervous system (CNS) neurones in situ. (2) A vibrating tissue slicer was used to cut thin slices in which individual neurones could be identified visually. Localized cleaning of cell somata with physiological saline freed the cell membrane, allowing the formation of a high resistance seal between the membrane and the patch pipette. (3) The various configurations of the patch clamp technique were used to demonstrate recording of membrane potential, whole cell currents and single channel currents from neurones and isolated patches. (4) The patch clamp technique was used to record from neurones filled with fluorescent dyes. Staining was achieved by filling cells during recording or by previous retrograde labelling. (5) Thin slice cleaning and patch clamp techniques were shown to be applicable to the spinal cord and almost any brain region and to various species. These techniques are also applicable to animals of a wide variety of postnatal ages, from newborn to adult.

SUMMARY1. Synaptically connected neurones were identified in the granule cell layer of slices of 17-to 21-day-old rat hippocampus. Whole-cell current recording using the patch-clamp technique revealed synaptic currents ranging from less than 10 to 200 pA in symmetrical Cl-conditions, at a holding potential of -50 mV. These currents were blocked by 2 ftM-bicuculline, indicating that they result from the activation of postsynaptic y-aminobutyric acid receptor (GABAA-receptor) channels.2. Addition of tetrodotoxin (TTX, 1 1uM) resulted in the loss of most currents of more than 40 pA in amplitude. Currents which disappeared after TTX treatment were assumed to be the result of spontaneous presynaptic action potentials. The currents seen in the absence of TTX are referred to as spontaneously occurring inhibitory postsynaptic currents (IPSCs); those remaining in the presence of TTX were defined as miniature IPSCs. 3. Similar currents were observed when recording in the whole-cell configuration while extracellular stimulation was applied to a nearby neurone. These currents were also completely blocked by 2 /tM-bicuculline and by 0-5 JLm-TTX They were thus defined as stimulus-evoked IPSCs.4. The half rise time of both miniature and stimulus-evoked IPSCs was fast (<1Ims). The time course of decay of both miniature IPSCs and stimulus-evoked IPSCs could be well fitted with the sum of two exponentials. At a membrane potential of -50 mV, the mean decay time constants of the two components were 2-0+0-38 and 54-4+ 18 ms (mean+ S.D.) for miniature IPSCs (six cells) and 2-2+ 1-3 and 66 + 20 ms (three cells) for stimulus-evoked IPSCs.5. Stimulus-evoked IPSCs varied in amplitude from less than ten to hundreds of picoamperes. In eight of eleven cells histograms of IPSC amplitudes showed several clear peaks which, when fitted with the sum of Gaussian curves, were found to be equidistant. This is consistent with the view that stimulus-evoked IPSC amplitudes vary in a quantal fashion. The quantal size varied between 7 and 20 pA, at a membrane potential of -50 mV. 7. In one cell where the recording was stable for more than an hour, changing the membrane voltage from -50 to -120 mV increased the quantal size by a factor of 241, close to that expected if the current-voltage relation of IPSC peak amplitudes were linear.8. The peak of miniature IPSC amplitude histograms measured in the presence of 1 /sM-TTX was comparable with the quantal size of stimulus-evoked IPSCs.However, in all cells, a tail of larger amplitude miniature IPSCs was observed. The amplitudes in the tail in six of twelve cases were quantally distributed. 9. Single-channel currents activated by GABA, applied locally to outside-out patches isolated from the soma membrane of granule cells, indicated that GABAAreceptor channels had two conductance levels of 14 and 23 pS. Thus for IPSCs in hippocampal granule cells, one quantal current represents the simultaneous opening of less than thirty GABAA-receptor channels.10. The small number of postsynaptic channels mediating a quanta...

1. Voltage and current recordings were made from visually identified non-pyramidal neurones in slices of layer IV of rat primary visual cortex using the whole-cell configuration of the patch clamp technique. These neurones are characterized by a high input resistance (0.5-2 G omega) and a non-adaptive behaviour of action potential frequency following depolarizing current injection, which suggests that they are stellate cells. 2. Excitatory postsynaptic currents (EPSCs) were recorded from these neurones during focal stimulation of neighbouring cells by a second patch pipette, the tip of which was placed on the soma of the stimulated cell. The response amplitude as a function of stimulus strength showed a sharp increase at a critical stimulus strength suggesting that stimulus-evoked currents represent unitary EPSCs. 3. In most cases the latencies of stimulus-evoked EPSCs were unimodally distributed with means in the range of 2.1-3.6 ms. In some experiments two peaks were seen in the distribution of latencies. The EPSC rise times, measured as the time from 20 to 80% peak amplitude, fell into a distribution ranging from 0.1 to 0.8 ms with a peak at 0.2 ms. The EPSC decay time course at -70 mV membrane potential was fitted by a single exponential with a time constant of 2.39 +/- 0.99 ms (mean +/- S.D.). The rise and decay times were independent of EPSC peak amplitudes. 4. The peak amplitude of successive unitary EPSCs, elicited by a constant stimulus, fluctuated at random. At a holding potential of -70 mV the peak amplitudes varied between 5 and 90 pA. In two out of ten cells the histogram of peak amplitudes could be well fitted by the sum of several equidistant Gaussians with a peak distance of around 10 pA. This suggests that the quantal conductance change underlying the peak current fluctuations is of the order of 100 pS. 5. At membrane potentials more positive than -70 mV the decay of stimulus-evoked EPSCs showed two components with very different time courses. In standard extracellular solution the current-voltage (I-V) relation for the fast component was almost linear whereas the slow component showed a J-shaped I-V relation with a region of negative slope conductance between -30 and -70 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously investigated P2X receptor‐mediated synaptic currents in medial habenula neurones and shown that they can be calcium permeable. We now investigate the receptor properties of glutamate, the other, more abundant excitatory transmitter, to determine its receptor subtypes and their relative calcium permeability. This may have implications for the physiological role of the P2X receptors which mediate synaptic currents. Using fast application of ATP, L‐glutamate or kainate to nucleated patches, glutamate receptors were determined to be of the AMPA subtype but no functional P2X receptors were detected. The deactivation and desensitization rates of the AMPA channel were determined to have time constants of 1·77 ± 0·21 ms (n= 10) and 4·01 ± 0·85 ms (n= 9) at ‐60 mV, respectively. AMPA receptors recovered from desensitization with two exponential components with time constants of 21·08 ± 2·95 and 233·60 ± 51·1 ms (n= 3). None of the deactivation or desensitization properties of the GluR channels depended on membrane potential. The current‐voltage relationship under different ionic conditions revealed that the GluR channel was equally permeable to Cs+ and Na+ but relatively impermeable to Ca2+ (PCa/PCs= 0·13, n= 6). For both synaptic currents and somatic currents activated by fast application of L‐glutamate to nucleated patches, decay time constants were similar at ±60 mV in the presence of Mg2+ ions. Thus GluR channels appear to be of the AMPA subtype and not the NMDA subtype. Thus, under the conditions of this study, neurones of the medial habenula lack functional NMDA receptors and possess AMPA receptors that have low permeability to Ca2+. We conclude that the P2X receptor‐mediated synaptic currents are the only calcium‐permeable fast‐transmitter gated currents in these neurones which may be important for their physiological function.

SUMMARYProcedures are described for recording postsynaptic currents from neurones in slices of rat brain using patch clamp techniques. The method involves cutting brain slices (120-300 ,m thick) with a vibrating microtome followed by localization of cell somata, which can be clearly seen with Nomarski differential interference contrast optics in the light microscope. Tissue covering the identified cell is then removed mechanically and standard patch clamp techniques are applied. Using these methods, spontaneously occurring and stimulus-evoked inhibitory postsynaptic currents (IPSCs) were recorded from neurones in rat hippocampus at greatly improved resolution. In the presence of tetrodotoxin, to block presynaptic action potentials, spontaneous IPSCs seldom exceeded 25 pA. Evoked IPSCs elicited by constant electrical stimulation of a presynaptic neurone were larger and fluctuated in their amplitudes. Singlechannel currents, activated by the putative inhibitory transmitter y-aminobutyric acid (GABA), had a size of about 1 pA. The number of postsynaptic channels activated by a packet of inhibitory transmitter is probably not more than thirty, nearly two orders of magnitude smaller than previously reported estimates for CNS synapses. This might reflect matching of synaptic efficacy to the high input resistance of hippocampal neurones and could be a requirement for fine tuning of inhibition.

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