Biomedical subjects
Publications and source records attributed to F A Edwards.
Over the past two years, ATP has clearly been shown to act as a co-transmitter with GABA, glycine and probably glutamate in the central nervous system. Our understanding of the ATP-gated P2X receptors is progressing rapidly, and the pharmacology, stoichiometry and subunit combinations of heteropolymeric P2X channels has been substantially elucidated.
1. The effects of the naturally occurring neurosteroid tetrahydrodeoxycorticosterone (THDOC) on GABAA receptor-mediated miniature, spontaneous and evoked IPSCs was tested using patch-clamp techniques in slices of hippocampus and cerebellum from rats at two developmental stages ( approximately 10 and approximately 20 days postnatal). The cells studied were hippocampal granule cells and cerebellar Purkinje and granule cells. 2. Most miniature GABAergic currents (mIPSCs) decayed with two exponentials and neurosteroids caused a approximately 4-fold increase in the decay time constant of the second exponential at the highest concentration used (2 microM). Similar effects were seen at high concentrations of THDOC (1-2 microM) in all cell groups tested. No effects were seen on amplitude or rise time of mIPSCs. 3. The effects of THDOC (1 microM) were shown to be stereoselective and rapidly reversible, indicating that the neurosteroid binds to the GABAA receptor, rather than acting genomically. 4. At concentrations of THDOC likely to occur physiologically (50-100 nM), the decay time of IPSCs was also enhanced (25-50 %) in all cerebellar cell groups tested. In contrast, at 100 nM THDOC, seven of 11 hippocampal granule cells were sensitive from the 10 day group but the 20 day hippocampal granule cells showed no significant enhancement in the presence of these lower concentrations of THDOC. 5. The differences in sensitivity of hippocampal and cerebellar cells to THDOC are compared to data reported in the literature on regional development of expression of different receptor subunits in the brain and it is suggested that the progressive relative insensitivity of the 20 day hippocampal cells may depend on increasing expression of the delta subunit of the GABAA receptor and possibly an increase in the alpha4 subunit.
1. 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. 2. 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. 3. 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. 4. 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). 5. 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. 6. 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.
Explore the source record for details and available documents.
1. ATP and glutamatergic synaptic currents were compared in slices of rat medial habenula nucleus using whole-cell patch-clamp techniques. 2. In most cells low voltage stimulation resulted in glutamatergic responses and not purinergic responses. In five cells where ATP currents could be stimulated with low voltages, wash out of glutamate antagonists did not reveal evoked glutamate currents. Spontaneous glutamate currents confirmed washout of antagonist. 3. Modulation of release probability of glutamate and ATP, assessed by changes in failure rate of synaptic currents, was compared under conditions of different stimulation frequencies and in the presence of adenosine agonists and antagonists. 4. ATP release, but not glutamate release, was shown to be modulated by increased stimulation frequency which resulted in inhibition of ATP release via A2-like adenosine receptors. A1 receptors caused inhibition of both ATP and glutamate release. 5. Endogenous adenosine inhibited glutamate release via A1 receptors but only inhibited ATP release via A2-like receptors. 6. Attempts to inhibit the degradation of ATP to adenosine did not alter the frequency dependence of the failure rate. 7. We conclude, from the direct demonstration and from the differences in pharmacology and frequency dependence of the modulation of release, that ATP and glutamate responses are due to release from separate neurones.
The properties of central ATP-mediated synaptic currents were studied using whole-cell patch-clamp recording in rat medial habenula slices. Release was shown to be calcium dependent with a Hill coefficient of approximately 2. The voltage dependence of synaptic current amplitudes was approximately linear. Some reduction of the synaptic current amplitudes was observed at 10 mM extracellular calcium, suggesting calcium block/permeability of the channels. This was confirmed by observation of current-voltage reversal potentials in different calcium concentrations. We estimate that the channels underlying half the synapses showed a negligible calcium permeability. In the other four out of eight synapses the results suggest a very high calcium permeability with an estimated PCa/PCs of > 10. Thus, at -70 mV, in 1 mM calcium, more than 15% of the ATP-mediated synaptic current is estimated to be carried by calcium, but only at synapses with calcium-permeable channels. Net current through these synaptic channels is also controlled by the voltage dependence of synaptic current decay time constants (increasing e-fold for 158 mV depolarization) and by a strong dependence of transmitter release on the frequency of stimulation of the presynaptic neurone, with failure rates increasing 3-fold as stimulation rates were increased from 1 to 10 Hz.
In addition to the widespread excitatory transmission mediated by glutamate receptors, P2X receptors also mediate fast excitatory synaptic transmission in the brain. The receptors in the brain have some features which are different from the more extensively characterized peripheral P2X receptors, possibly suggesting a difference in the subunits making up the protein. Perhaps the most notable feature of the central receptors is a higher Ca2+ permeability than seen in other areas, with a linear current voltage relation. The potential danger to the postsynaptic cell of the high Ca2+ permeability of neuronal P2X receptors is discussed and various forms of inbuilt features of the synapse and receptors are outlined which would combine to protect the neurons from excessive Ca2+ influx and consequent danger of cell death. These features include a rapid breakdown of transmitter in the cleft, which not only removes the ATP from the cleft quickly, but also results in the production of adenosine. Evidence for the interaction of ATP-mediated transmission and stimulation of purinoceptors by this adenosine is presented from experiments using patch-clamp recording in brain slices. This demonstrates an elegant negative feedback mechanism by which a fast excitatory transmitter can be inactivated by breakdown to a product which acts as a slow inhibitory modulator, controlling release of the original transmitter.
Application of patch-clamp techniques to brain slices has resulted in an enormous increase in the resolution of synaptic currents in mammalian central neurones. This improved resolution has allowed direct observation of miniature and evoked synaptic currents, leading, in agreement with other findings, to the conclusion that the quantal size of synaptic currents in the brain is limited by the number of postsynaptic receptors. Possible explanations for the skewed miniature distribution, observed at all fast central synapses are discussed, with reference to anatomical observations. As a result, a model is proposed which is consistent with much of the apparently contradictory data on the induction and maintenance of long-term potentiation. In addition to the study of mechanisms of synaptic transmission, improved resolution provided by the patch-clamp technique has allowed resolution of synaptic currents, in the brain, mediated by ATP. The role of ATP as a central neurotransmitter is discussed in terms of the above findings.
One of the major controversies in neuroscience concerns whether the expression of long-term potentiation (LTP) is a pre- or postsynaptic phenomenon, with apparently contradictory data being the norm. The model that is outlined in this article combines anatomical and electrophysiological evidence to allow apparently contradictory data to be compatible. Development of LTP involves both influx of Ca2+ through NMDA receptors, and activation of another factor, perhaps the metabotropic glutamate receptor. These two processes might result, respectively, in the insertion of activation of additional postsynaptic receptors, and the growth of microfilaments that could split simple synapses into perforated synapses, consisting of multiple active zones. Whether the latter occurred, and at what rate, would be likely to depend on multiple factors, such as temperature, the metabolic state of the cell, buffering of Ca2+, and the concentration of factors such as nitric oxide. These subtle experimental variables would thus determine whether the dominant effect observed was pre- or postsynaptic.
Detailed knowledge of the anatomy of central synapses is essential to the interpretation of the vast quantity of electrophysiological findings that have been published in recent years. When their function is considered, it is not surprising that, in both anatomy and electrophysiology, fast central synapses show important differences to the neuromuscular junction. This review concentrates on the detailed anatomy of the common excitatory synapses that impinge on dendritic spines, but also refers to other glutamatergic and GABAergic synapses. This information is brought together with present knowledge of the electrophysiology of fast neurotransmission in the brain. Various types of evidence are outlined, explaining why it is now widely accepted that release of transmitter from a single vesicle virtually saturates the small number of receptors available on the postsynaptic membrane of central synapses. Finally, the anatomic literature suggests that a particular type of spine synapse, which electron microscopy reveals to have a perforated active zone, may represent a synapse with high efficacy. This suggestion is shown to be completely compatible with the electrophysiological data, and a model is presented that shows that all the apparently conflicting data in the field of long-term potentiation could be compatible. This stresses the need for cooperative collaboration between laboratories that have apparently conflicting findings.
ATP receptors continue to be found in an ever wider range of tissues in the brain and the periphery. Much of the recent research on these receptors focuses on features that would distinguish ATP-mediated excitatory synaptic transmission from that mediated through glutamate or acetylcholine receptors. These features include kinetics of the synaptic currents, voltage-independent calcium permeability of the underlying channels, interactions with adenosine, and various direct modulators of the ATP-gated ion channel.
ATP receptor-mediated responses in peripheral and central neurons have many characteristics which suggest that ATP may act as a fast neurotransmitter. While the receptors underlying these responses have properties which are similar to other ligand-gated ion channels which mediate fast neurotransmission, the nature of their calcium permeability and the rapid breakdown of ATP to adenosine may confer unique properties on ATP mediated synaptic transmission. The evidence that ATP acts as a fast neurotransmitter is reviewed and the properties of ATP and its receptor channels are discussed in terms of synaptic transmission.
Until now, the only well documented, fast excitatory neurotransmitter in the brain has been glutamate. Although there is evidence for adenosine 5'-triphosphate (ATP) acting as a transmitter in the peripheral nervous system, suggestions for such a role in the central nervous system have so far not been supported by any direct evidence. Here we report the recording of evoked and miniature synaptic currents in the rat medial habenula. The fast rise time of the currents showed that they were mediated by a ligand-activated ion channel rather than a second messenger system, thus limiting the known transmitter candidates. Evidence was found for the presence on the cells of glutamate, gamma-aminobutyric acid, acetylcholine and ATP receptors, but not for 5-hydroxytryptamine (5HT3) or glycine receptors. The evoked currents were unaffected by blockers of glutamate, gamma-aminobutyric acid or acetylcholine receptors but were blocked by the ATP receptor-blocker, suramin and the desensitizing ATP receptor-agonist alpha,beta-methylene-ATP. Our evidence identifies for the first time synaptic currents in the brain, mediated directly by ATP receptors.
Explore the source record for details and available documents.
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)
Thin slices (100-200 microns) from cardiac ventricles of newborn (1-14 days old) rats were used for patch-clamp recordings. High resistance seals (greater than 10 GOhms) could be obtained on this preparation without enzymatic treatment of tissue. Resting potential for cardiac fibres, measured in whole-cell configuration, ranged between -30 and -65 mV. Averaged sodium currents and inward rectifying potassium single-channel currents, recorded in cell-attached mode, displayed similar features to those previously reported for isolated rat ventricular cells. Application of patch-clamp techniques to cardiac slices allows single-channel recordings without complicated procedures of cell isolation. Moreover, possible alteration of channel properties caused by proteolytic enzymes can be avoided. This technique provides a new tool for studying electrophysiological properties of heart cells in situ.
1. 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 microM-bicuculline, indicating that they result from the activation of postsynaptic gamma-aminobutyric acid receptor (GABAA-receptor) channels. 2. Addition of tetrodotoxin (TTX, 1 microM) 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 microM-bicuculline and by 0.5 microM-TTX. They were thus defined as stimulus-evoked IPSCs. 4. The half rise time of both miniature and stimulus-evoked IPSCs was fast (less than 1 ms). 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. 6. Decreasing the Ca2+ and increasing the Mg2+ concentration in the extracellular solution decreased the number of peaks in the IPSC amplitude histogram but did not affect the size of the quantal event.(ABSTRACT TRUNCATED AT 400 WORDS)