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S G Cull-Candy

Publications and source records attributed to S G Cull-Candy.

At least 37 records · Page 2Linked to original sources
PubMed1994

A comparison of non-NMDA receptor channels in type-2 astrocytes and granule cells from rat cerebellum.

1. Patch-clamp recording methods have been used to compare the pharmacological properties and single-channel characteristics of non-NMDA receptor channels in cerebellar type-2 astrocytes and granule cells. 2. In type-2 astrocytes whole-cell concentration-response curves for glutamate, quisqualate, AMPA and kainate gave EC50 values of 5.8, 3.8, 7.6 and 160 microM and Hill slopes of 1.65, 1.18, 1.64 and 1.65, respectively, resembling estimates for granule cell receptors. 3. The non-NMDA receptor antagonists CNQX and diCl-HQC (see Methods) inhibited whole-cell kainate currents in both cell types. The IC50 for CNQX antagonism of the kainate response was 536 nM in type-2 astrocytes, and 500 nM in granule cells. The IC50 for diCl-HQC was 3.5 microM in astrocytes and 3.7 microM in granule cells. 4. CNQX acted as a competitive antagonist of whole-cell kainate responses in type-2 astrocytes and granule cells giving Schild plots with a slope near 1. The equilibrium constant, K, for CNQX binding was 524 nM in astrocytes and 489 nM in granule cells. 5. Quisqualate and AMPA responses showed rapid desensitization in type-2 astrocytes with a ratio of steady-state to peak response of 0.09. Concanavalin A reduced this desensitization. 6. Non-NMDA channels in type-2 astrocytes and granule cells showed a low permeability to Ca2+ ions with a reversal potential, for kainate-activated whole-cell currents in isotonic Ca2+, of approximately -25 mV for astrocytes and -45 mV for granule cells. 7. Outside-out patches from type-2 astrocytes exhibited a range of single-channel conductances that were superficially similar to the glutamate-activated conductances in granule cells. However, the type-2 astrocytes were devoid of NMDA receptors, hence all of these conductances originated from non-NMDA channels. Their slope conductances were approximately 11, 21, 32, 42 and 52 pS. Amplitudes were verified with mean low-variance plots and single-channel current-voltage curves, which were linear. 8. There was also evidence of lower conductance kainate-activated channels in astrocyte patches. From noise analysis their estimated mean conductance was 1.9 pS, as described for the 'low-conductance' type kainate responses in cerebellar neurones. 9. Apparent open times, shut times and burst lengths of AMPA-activated (3-10 microM) channels were examined in patches from type-2 astrocytes, and kinetic properties of the 40 and 50 pS levels were compared with the lower levels. 10. Our results indicate some marked pharmacological similarities between non-NMDA receptor channels in type-2 astrocytes and granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy fiber-granule cell synapse.

We have analyzed the variance associated with the decay of the non-NMDA receptor component of synaptic currents, recorded from mossy fiber-granule cell synapses in cerebellar slices, to obtain a conductance estimate for the synaptic channel. Current fluctuations arising from the random channel gating properties were separated from those arising from the fluctuations in the population of channels by subtracting the mean excitatory postsynaptic current (EPSC) waveform scaled to the EPSC peak amplitude. A weighted mean single-channel conductance of approximately 20 pS was determined from the relationship between the mean current and the variance around the mean during the decay of evoked and spontaneous synaptic currents. This result suggests that high conductance non-NMDA channels, such as the 10-30 pS glutamate receptor channel previously characterized in granule cells, carry the majority of the fast component of the EPSC at this synapse. In addition, our data are consistent with the activation of surprisingly few (approximately 10) non-NMDA channels by a single packet of transmitter.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

Evidence for more than one type of non-NMDA receptor in outside-out patches from cerebellar granule cells of the rat.

1. Application of non-NMDA (non-N-methyl-D-aspartate) receptor agonists onto outside-out patches of cerebellar granule cells gave two characteristic types of response (in different patches) which we have referred to as 'high conductance' and 'low conductance' responses. At a qualitative level these patches could be readily distinguished by the size of the noise increase accompanying their membrane currents. 2. In high conductance patches both AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate gave discrete single-channel conductances (10-30 pS), while in low conductance patches, AMPA produced small discrete events (6-10 pS), and kainate opened channels with conductances too small to be directly resolved. All patches examined contained NMDA receptor channels with characteristic 50 and 40 pS conductance levels. 3. Despite the marked differences in single-channel conductances, kainate dose-response curves constructed for high and low conductance patches had similar EC50 values of approximately 150 microM. 4. Spectral analysis of low conductance kainate responses gave an estimated channel conductance of approximately 1.5 pS. In these same low conductance patches AMPA produced discrete openings with two conductance levels; their mean conductances (and relative proportions) were 6 (87%) and 10 pS (13%). 5. In high conductance patches, glutamate (10-30 microM), AMPA (3-10 microM), and kainate (10-30 microM), each activated non-NMDA channels with three multiple conductance levels. The amplitudes of these conductance levels (approximately 10, 20 and 30 pS) were similar for each of the agonists, and their relative proportions (i.e. areas in the amplitude histograms) were constant for all three agonists. In addition, the relative proportion of levels was constant between patches, and all three levels were invariably present. These observations are all consistent with the idea that the three multiple conductances originate from a single receptor channel, activated by AMPA, kainate and glutamate. 6. Non-NMDA single-channel current-voltage (I-V) plots showed outward rectification in high conductance patches. For all three multiple conductance levels the ratio of outward to inward single-channel slope conductance was 1.8 +/- 0.1 and this rectification remained present in symmetrical Na+ solutions. 7. In high conductance patches, the events produced by a rapid application of 20-50 microM glutamate were compared with those activated during steady-state application.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Rapid-time-course miniature and evoked excitatory currents at cerebellar synapses in situ.

Neurotransmission from mossy fibre terminals onto cerebellar granule cells is almost certainly mediated by L-glutamate. By taking advantage of the small soma size, limited number of processes and short dendrite length of granule cells, we have obtained high-resolution recordings of spontaneous miniature excitatory postsynaptic currents (m.e.p.s.cs) and evoked currents in thin cerebellar slices. Miniature currents have a similar time-course and pharmacology to evoked currents and consist of an exceptionally fast non-NMDA (N-methyl-D-aspartate) component (measured rise-time, 200 microseconds; estimated pre-filtered rise-time less than 100 microseconds; decay time constant, tau = 1.0 ms), followed by 50 pS NMDA channel openings that are directly resolvable. We could find no evidence for the recent proposal that miniature currents in granule cells are mediated solely by NMDA channels with a novel time course. The non-NMDA receptor component of m.e.p.s.cs has a skewed amplitude distribution, which suggests potential complications for quantal analysis. The difference in time course between the m.e.p.s.cs reported here and other synaptic currents in the brain could reflect differences in synaptic function or electrotonic filtering; the relative contribution of these possibilities has yet to be established.

Animals↗
PubMed1992

On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat.

1. The mechanism of action of picrotoxin on GABA receptor channels in rat sympathetic neurones has been investigated with whole-cell clamp. In addition, the action of picrotoxin on single GABA channels has been examined in outside-out membrane patches from these cells. 2. Picrotoxin, at concentrations which dramatically reduced the amplitude of whole-cell GABA currents, did not alter the spectral time constants or single-channel conductance estimated by analysis of GABA-activated current noise. This was observed at potentials both negative and positive to the GABA reversal potential (i.e. for both inward and outward GABA currents). In control conditions, the slow and fast time constants from GABA noise were 40 +/- 14 ms and 2 +/- 0.4 ms, while the estimated single-channel conductance was 14 +/- 2 pS. In the presence of picrotoxin, the time constants and estimated single-channel conductance were 41 +/- 5 ms, 2.7 +/- 0.6 ms and 15 +/- 2.3 pS. 3. Picrotoxin did not alter the shape of the whole-cell GABA current-voltage relationship, indicating that the steady-state block was not voltage dependent. The lack of effect of picrotoxin on the GABA noise spectra and the lack of outward rectification makes it unlikely that picrotoxin acts by a simple voltage-dependent (or voltage-independent) channel blocking mechanism. In the presence of picrotoxin the reversal potential for GABA remained at approximately 0 mV in symmetrical chloride. 4. Distributions of total burst durations, obtained from single-channel records with low concentrations of GABA, were fitted with three or four exponential components. Picrotoxin had no consistent effect on the time constants of the total burst length distributions. It also did not alter the amplitude of the main conductance state. However, picrotoxin did reduce the frequency of channel openings. 5. The application of brief ionophoretic pulses of GABA, to cells under whole-cell voltage clamp, revealed that the rate of onset of block by picrotoxin was accelerated in the presence of GABA. In the absence of agonist, picrotoxin produced a more slowly equilibrating block. 6. Our data are consistent with a mechanism whereby picrotoxin binds preferentially to an agonist bound form of the receptor and stabilizes an agonist-bound shut state. This could, for example, mean that picrotoxin enhances the occurrence of a desensitized state or an allosterically blocked state.

Animals↗
PubMed1991

Excitatory amino acid receptor-channels in Purkinje cells in thin cerebellar slices.

Glutamate receptors of the N-methyl-D-aspartate (NMDA) and non-NMDA type serve different functions during excitatory synaptic transmission. Although many central neurons bear both types of receptor, the evidence concerning the sensitivity of cerebellar Purkinje cells to NMDA is contradictory. To investigate the receptor types present in Purkinje cells, we have used whole-cell and outside-out patch-clamp methods to record from cells in thin cerebellar slices from young rats. At a holding potential of -70 mV (in nominally Mg(2+)-free medium, with added glycine) NMDA caused a whole-cell current response which consisted of a dramatic increase in the frequency of synaptic currents. In the presence of tetrodotoxin (TTX) and the gamma-aminobutyric acidA (GABAA) receptor antagonist bicuculline, spontaneous synaptic currents and responses to NMDA were inhibited. In a proportion of cells a small polysynaptic response to NMDA persisted, which was further reduced by the non-NMDA receptor antagonist 6-cyano-2,3-dihydro-7-nitroquinoxalinedione (CNQX). The non-NMDA glutamate receptor agonists kainate (KA), quisqualate (QA) and s-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (s-AMPA), evoked large inward currents due to the direct activation of receptors in Purkinje cells. NMDA applied to excised membrane patches failed to evoke any single-channel currents, whereas s-AMPA and QA caused small inward currents accompanied by marked increases in current noise. Spectral analysis of the s-AMPA noise in patches gave an estimated mean channel conductance of approximately 4 pS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Currents through single glutamate receptor channels in outside-out patches from rat cerebellar granule cells.

1. Single-channel currents evoked in outside-out membrane patches from rat cerebellar granule cells by glutamate, aspartate, N-methyl-D-aspartate (NMDA), kainate and quisqualate were studied. Each agonist produced openings to five discrete amplitude levels. At a membrane potential of -70 mV, these levels correspond to single-channel conductances of about 8, 17, 30, 40 and 50 pS. NMDA, aspartate and glutamate evoked mainly 50 pS openings and also substantial numbers of 40 pS events. Kainate evoked primarily 8 and 17 pS openings. 2. The relative proportion of openings to each conductance level showed no dependence on membrane potential. At membrane potentials negative to -100 mV, current-voltage plots for 30, 40 and 50 pS openings showed substantial inward rectification. 3. With NMDA, aspartate and glutamate, the most common type of direct transition was between the 50 pS open level and the shut level. Transitions between the 30, 40 and 50 pS levels were also relatively common. With few exceptions, 8 and 17 pS openings appeared to arise directly from, and return directly to, the shut level. The differences between granule cells and certain other central neurones, in the types of transitions associated with NMDA receptor channels, provide evidence for the existence of more than one type of NMDA receptor. 4. Four exponential components were identified consistently in the shut-time distributions that were obtained with NMDA, aspartate and glutamate. Mean time constants for the briefest two components were 30 to 65 microseconds and 0.65 to 1.00 ms. The mean duration of these 'gaps within bursts' differed for different agonists, but did not vary with membrane potential. 5. Two exponential components were distinguished in most open-time distributions for the 50 pS level (time constants, 0.9-1.2 and 3.2-3.9 ms at -100 mV), whereas open-time distributions for 30 and 40 pS events were described adequately by single exponentials with time constants below 1.0 ms. The duration of 50 pS openings decreased with hyperpolarization. 6. Mean open times for 8 and 17 pS events produced by NMDA, aspartate and glutamate were 0.3 to 0.7 ms. The longest such openings were observed with quisqualate. 7. Three exponential components were present in distributions of burst length, and of total open time per burst, that were obtained with NMDA, aspartate and glutamate. The slowest two burst-length components had mean time constants of 1.7-2.4 and 10.6-13.0 ms and originated from the kinetic behaviour of the 50 pS state.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Single channels activated by high concentrations of GABA in superior cervical ganglion neurones of the rat.

1. Single-channel currents evoked by high concentrations of GABA (10-2000 microM) have been analysed to investigate the characteristics of GABAA receptor channels in outside-out patches from rat sympathetic neurones. When high concentrations of GABA were applied to a patch, channel openings occurred in prolonged clusters (3.8 +/- 3.7 s (mean +/- S.D.) at 50 microM-GABA) consisting, on average, of 350 apparent openings per cluster. Individual clusters were separated by long silent intervals. 2. Channel openings were to many (often ill-defined) conductance states (range 7-36 pS), but the most frequently observed conductance level was approximately 30 pS, (29.6 +/- 0.34 pS). Only these clusters during which the channel was open to this main state conductance for at least 95% of the cluster open time were used in the analysis of probability of being open. Other less frequently observed conductance levels were 15-18 and 22-23 pS, while levels of 33-36 and 7-9 pS were occasionally, but reliably, observed. 3. Bursts within clusters were defined as a series of openings separated by closed intervals shorter than some critical value, tc. At 50 microM-GABA the mean burst length was 439 +/- 434 ms (+/- S.D., tc = 50 ms). 4. The probability of being open, po, during bursts within clusters has been analysed as a function of GABA concentration. As expected, increasing the concentration of GABA resulted in an overall increase in po. However, for a given agonist concentration there was a wide spread in po, far greater than that predicted for a population of identical and independent receptor channels (demonstrated by comparison with stimulated channel activity). 5. The wide range of po values at a particular concentration of GABA was not due to inappropriate selection of tc. At 50 microM-GABA the range of po values was similar for tc of 20-1000 ms, although the overall mean po became lower (0.64 rather than 0.81). 6. On the basis of simulated channel activity, it appears that most clusters which do not contain multiple openings, arise from the activity of one individual channel. Furthermore, there was no detectable tendency for gaps between bursts to be shorter in the middle of a cluster than at its ends. Therefore it is unlikely that variability in po arose from overlapping activity of two or more channels. 7. The values for po, mean open time, and mean shut time for bursts within the same cluster, and between different clusters, were compared by a randomization test.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Activation of glutamate receptors and glutamate uptake in identified macroglial cells in rat cerebellar cultures.

1. Patch-clamp methods have been used to examine the action of excitatory amino acids on three types of glial cell in cultures of rat cerebellum, namely type-1-like astrocytes, type-2 astrocytes and oligodendrocytes. In addition we have examined glutamate sensitivity of the precursor cell (the O-2A progenitor) that gives rise to type-2 astrocytes and oligodendrocytes. 2. Glutamate (30 microM), quisqualate (3-100 microM), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA, 10-30 microM) and kainate (10-500 microM) were applied to cerebellar type-2 astrocytes examined under whole-cell voltage clamp. Each of these agonists induced inward currents in cells held at negative membrane potentials. The currents reversed direction near 0 mV holding potential. N-Methyl-D-aspartate (NMDA, 30-100 microM) or aspartate (30 microM) in the presence of glycine (1 microM) did not evoke any whole-cell current changes in type-2 astrocytes. 3. The distribution of glutamate receptors in type-2 astrocytes was mapped with single- or double-barrelled ionophoretic pipettes containing quisqualate or kainate. Application of these agonists (current pulses 100 ms, 50-100 nA) to cells held at -60 mV evoked inward currents of 20-120 pA in the cell soma and 10-80 pA in the processes. Responses could also be obtained at the extremities of processes (approximately 60 microns from the soma). 4. Quisqualate or kainate (at 30 microM) applied to O-2A progenitor cells from rat cerebellum or optic nerve induced whole-cell currents (quisqualate 20-30 pA; kainate 20-50 pA, holding potential, Vh = -60 mV) that reversed near 0 mV. In common with type-2 astrocytes, the progenitor cells did not respond to NMDA (30 microM). 5. Type-1-like astrocytes produced large inward currents to glutamate (30 microM). These currents remained inward-going at holding potentials as positive as +80 mV and were not accompanied by any apparent noise increase. This result can be explained by the presence of an electrogenic glutamate uptake carrier. In cells kept up to 4 days in vitro, quisqualate, kainate and NMDA each failed to produce any whole-cell current changes, indicating the absence of receptors in type-1-like astrocytes at this stage in culture. Furthermore the glutamate uptake currents in type-1-like astrocytes were inhibited when external Na+ was replaced by Li+, although Li+ was found to pass through the glutamate channel in type-2 astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Pharmacological properties and H+ sensitivity of excitatory amino acid receptor channels in rat cerebellar granule neurones.

1. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate receptor channels have been examined in rat cerebellar granule neurones with whole-cell and single-channel patch-clamp methods. The whole-cell peak and steady-state aspartate and NMDA currents were reversibly inhibited by extracellular protons; the IC50 (concentration producing half-maximal inhibition) for the full H+ inhibition curve for NMDA receptors corresponded to pH 7.3, near to physiological pH. (S)-AMPA and kainate whole-cell currents were inhibited by protons with IC50 values that corresponded to pH 6.3 and 5.7, respectively; these receptors were, however, insensitive to H+ concentrations that inhibited NMDA receptor responses. 2. Proton inhibition of the NMDA, AMPA and kainate receptor-mediated responses was voltage insensitive, and did not involve a shift in reversal potential. 3. The EC50 (concentration producing half-maximal effect) for aspartate calculated from the whole-cell dose-response curve was similar at pH 6.8 and 7.6 (mean 11.2 microM). Although the EC50 for glycine potentiation of the aspartate response was marginally increased from 273 nM at pH 7.6 to 373 nM at pH 6.8, H+ inhibition was not overcome by up to 1 mM-external glycine. Inhibiting concentrations of H+ appropriate for AMPA and kainate receptors did not markedly alter the EC50 values determined for (S)-AMPA (3.4 microM) and kainate (114 microM) at pH 7.2. 4. Treatment of neurones with N-ethylmaleimide, iodoacetic acid, dithiothretiol or diethyl pyrocarbonate did not influence proton inhibition of NMDA receptor responses. However, treatment with diethyl pyrocarbonate, which potentiated aspartate responses, appeared to reduce the effectiveness of Zn2+ inhibition of NMDA receptors. 5. Desensitization of whole-cell NMDA and (S)-AMPA currents was studied with ionophoretic application of agonist to the cell soma. Whole-cell aspartate currents desensitized rapidly, irrespective of the glycine concentration. Increased H+ concentrations did not detectably alter the ratio of peak/steady-state current, or the time constants describing the onset of, or recovery from, desensitization. The time constant describing desensitization of (S)-AMPA-induced whole-cell currents also appeared unchanged by inhibiting pH (6.2). 6. The amplitudes of aspartate- or NMDA-activated single-channel multiple conductance levels were unchanged by decreasing the pH to 6.8.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Conductance and kinetic properties of single nicotinic acetylcholine receptor channels in rat sympathetic neurones.

1. The unitary conductance of nicotinic acetylcholine (ACh) receptor channels in rat sympathetic neurones has been studied. Conductance estimates varied from 26-48 pS with a mean of 36.8 pS in 1 mM-Ca2+. The main conductance level varied from patch to patch and the presence (or absence) of additional conductance levels also varied. 2. The channels showed large open channel noise and experiments with 300 mM-NaCl in the patch pipette substantially increased the open channel noise. The appearance of detectable step-like transitions within this noise strongly suggested the existence of closely spaced discrete levels. 3. Removal of divalent cations from the external solution increased the unitary channel conductance. Altering the main permeant ion in divalent-free solutions gave the following conductance sequence: K+ (93 pS) greater than Cs+ (61 pS) greater than Na+ (51 pS) greater than Li+(23 pS). 4. Replacement of Na+ by Cs+ in the external solution considerably reduced the current evoked by ACh in whole-cell recordings and the channel-opening frequency in outside-out patches. 5. The kinetic properties of channels activated by ACh and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) were also studied. At low concentrations of ACh and DMPP the gap distributions were complex and best fitted by the sum of four exponential components. Individual activations (bursts) were interrupted by the two shortest closed periods the briefer of which had time constants of 36 microseconds for ACh and 67 microseconds for DMPP. 6. The distribution of burst lengths had two components for each agonist, each component making up about 50% of the total area under the distribution. For ACh, the time constant of the longer component (12.2 ms) was similar to the decay time constant of excitatory postsynaptic potentials (EPSCs) at similar temperature and potential. For DMPP the time constant of the longer component was 17.6 ms. 7. The relative number of brief gaps per long burst was much larger for ACh than for DMPP. Therefore the corrected mean open time for ACh (0.86 ms) was much shorter than that for DMPP (2.3 ms). 8. In terms of receptor mechanism, the values of the channel opening equilibrium constant (beta/alpha) estimated from these numbers (ACh, 23; DMPP, 25) suggest that both agonists are efficaceous. 9. DMPP is a potent blocker of the channel with an equilibrium dissociation constant (KB) of around 50 microM and blockage gaps of around 1 ms duration. ACh also blocks the channel but with a higher KB of around 470 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1990

Proton inhibition of N-methyl-D-aspartate receptors in cerebellar neurons.

Mammalian neurons contain at least three types of excitatory amino-acid receptors, selectively activated by N-methyl-D-aspartate (NMDA) or aspartate, (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate ((S)-AMPA) and kainate. An important aspect of NMDA receptors is their regulation by a variety of factors such as glycine, Mg2+ and Zn2+ that are present in vivo. We show here that NMDA receptor responses are selectively inhibited by protons, with a 50% inhibitory concentration (IC50) that is close to physiological pH, implying that NMDA receptors are not fully active under normal conditions. (S)-AMPA and kainate responses remain unchanged at similar pH levels. Proton inhibition is voltage-insensitive and does not result either from fast channel block, a change in channel conductance, or an increase in the 50% excitatory concentration (EC50) of aspartate/NMDA or glycine. Instead, protons seem to decrease markedly the opening frequency of 30-50 pS NMDA channels, and reduce the relative proportion of longer bursts. This feature of NMDA receptors could be relevant to neurotoxic activation of NMDA receptors during ischaemia, as well as to seizure generation, as extracellular proton changes occur during both of these pathological situations. Furthermore, these results may have implications for normal NMDA receptor function as transient changes in extracellular protons occur during synaptic transmission.

Animals↗
PubMed1990

Rectification of currents activated by nicotinic acetylcholine receptors in rat sympathetic ganglion neurones.

1. The inward rectification of the whole-cell current evoked by acetylcholine (ACh) and other nicotinic agonists in rat sympathetic ganglion neurones has been studied using patch-clamp recording techniques. The selective nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (20 microM) induced an average peak current of -367 pA at -50 mV but no detectable outward current at +50 mV. Similar observations were made with ACh and carbachol. 2. The current-voltage relation of the whole-cell response induced by DMPP was linear in the negative voltage range; however, there was no detectable outward current in the voltage range 0 to about +70 mV. Above +70 mV an outward current became clearly detectable. Rapid depolarizing jumps in the holding potential failed to reveal any rapidly decaying outward current. 3. The rectification was not alleviated by changing the main permeant cation, by removal of divalent cations from the intracellular or extracellular solutions or by altering the pH buffer in the extracellular solution from HEPES to Tris. 4. Intracellular magnesium ions can block the channel. This effect increases with depolarization, but dissociation outwards (i.e. permeation by Mg2+) appears to relieve the block at more extreme positive potentials. This effect alone, or in combination with the voltage dependence of the burst length, is unlikely to be able to account for the whole-cell rectification in intact cells, much less that seen in cells perfused with Mg2(+)-free intracellular medium. 5. When the reversal potential was shifted to approximately -50 mV (by the use of impermeant cations) nicotinic agonists produced small outward currents in the membrane potential range -20 to +10 mV while shifting it to about +40 mV produced small inward currents in the potential range 0 to +20 mV. The rectification therefore appears to be independent of the direction of current flow and is maximum at a potential positive to 0 mV. 6. At positive potentials the receptors desensitized much less than at negative potentials in the continued presence of agonist. Thus, exposure of the cells to a steady application of 30 microM-ACh produced no detectable response if the cell was at a positive potential, but when the cell was stepped to a negative potential in the continued presence of ACh (at a time when much of the ACh current would be expected to have desensitized), ACh induced a large inward current. The onset of the ACh current had a time constant of 10 ms. It then decayed with a time constant of 790 ms as desensitization developed.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1989

Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids.

L-GLUTAMATE and L-aspartate are thought to have a widespread function as synaptic transmitters in the mammalian central nervous system and there are at least three types of neuronal glutamate receptors, which can be activated by the selective agonists N-methyl-D-aspartate (NMDA), quisqualate and kainate. Recent experiments indicate that glutamate receptors also occur in astrocytes. We have used patch-clamp methods to determine whether one type of macroglial cell, the type-2 astrocyte, possesses glutamate receptors, as previously proposed from neurochemical studies. We find that glutamate and related amino acids can evoke whole-cell and single-channel currents in type-2 astrocytes from rat cerebellum. Although these cells are found mainly in white matter, where neurotransmission does not occur, their processes are closely associated with axons at nodes of Ranvier, suggesting that such receptors are involved in neuronal-glial signalling at the node. Our experiments show that glial cells possess quisqualate- and kainate-receptor channels but lack receptors for NMDA. Interestingly, these glutamate channels exhibit multiple conductance levels that are similar in amplitude to the neuronal glutamate channels.

Animals↗
PubMed1989

Voltage-activated membrane currents in rat cerebellar granule neurones.

1. Voltage-activated currents have been recorded from cerebellar granule neurones in explant cultures from young rats (1-9 days old). Cells were examined with whole-cell patch-clamp methods. Depolarizing pulses from a pre-pulse potential of -100 mV evoked a rapidly activated transient inward current, and an outward current which decayed in two phases. The ionic dependence, kinetics and pharmacological properties of these currents have been studied. 2. Peak inward Na+ currents in cells from 7-day-old rats were in the range 350-450 pA. No evidence was found for the presence of calcium currents. Thus, inward current was unchanged in zero Ca2+, 1 mM-EGTA solution. No inward current was obtained in medium containing 10 mM-Ba2+ and tetrodotoxin (TTX). Supplementing the pipette (i.e. intracellular) solution with Mg-ATP did not reveal any Ca2+ current. 3. Depolarizing steps (from -100 mV) in TTX-containing solution gave an early transient outward current and a late outward current. The transient current resembled IA described in other cells, and reversed close to EK in both normal and elevated potassium concentrations, indicating that K+ is the predominant charge carrier. Depolarizing steps from -50 mV failed to give a transient outward current, and gave only a slowly rising current which resembled the late potassium current, IK. 4. Inactivation of the transient current was examined by applying test depolarizations from increasingly negative pre-pulse potentials (-50 to -120 mV): half-inactivation occurred at -72 mV. Transient outward currents decayed exponentially with time constants, tau, of 7.3-25.3 ms at 0 mV. The time course of removal of inactivation in cells held at -50 mV, and given increasingly long pre-pulses to -100 mV, was exponential with tau = 35 ms. 5. Both transient and late outward currents were reversibly abolished by addition to the bathing medium of 10 mM-Ba2+ or 1 mM-quinine. Outward K+ current was not dependent on external calcium. Tetraethylammonium (20 mM) selectively reduced the late outward current; the peak transient current was reduced by less than 20%. 4-Aminopyridine (2 mM) showed little selectivity between transient and late outward currents. 6. It is concluded that cerebellar granule cells from young rats possess voltage-activated inward Na+ current as well as two types of K+ current, IA and IK. In terms of neuronal functioning, the properties of the transient outward current may confer a role in regulating excitability and in repolarization, but a definitive statement will require knowledge of the cellular location and relative densities of channels in granule cells in vivo.

4-Aminopyridine↗
PubMed1989

Whole-cell current noise produced by excitatory and inhibitory amino acids in large cerebellar neurones of the rat.

1. Membrane noise and current changes produced by glutamate and related excitatory amino acids have been examined in cultured large cerebellar neurones (including Purkinje cells), with whole-cell patch-clamp methods. The sensitivity of these neurones to the inhibitory amino acids gamma-aminobutyric acid (GABA) and glycine has also been studied. 2. The neurones formed inhibitory synapses in culture, and displayed spontaneous synaptic currents. Reducing the pipette Cl- concentration (i.e. intracellular synaptic currents. Reducing the pipette Cl- concentration (i.e. intracellular concentration) caused a negative shift in their reversal potential, and the currents could be blocked with bicuculline (10 microM), suggesting that they were mediated by GABAA receptors. Spontaneous synaptic activity was also considerably reduced in the presence of 3 microM-tetrodotoxin. 3. Analysis of the increase in whole-cell current noise produced by the application of GABA (3 microM) gave noise spectra that were fitted by two Lorentzian components with slow and fast time constants of 23.6 and 1.9 ms at a membrane potential (Vm) of -110 mV. The mean single-channel conductance estimated from GABA noise was gamma noise = 12 pS. Glycine (10 microM) whole-cell current responses were Cl(-)-mediated and reversibly abolished by 1 microM-strychnine. 4. Bath application of excitatory amino acids gave whole-cell current changes accompanied by an increase in synaptic activity. Postsynaptic responses to the excitatory amino acids were more readily seen after the inhibitory synaptic currents had been abolished by bicuculline. Membrane current changes were obtained in response to the putative transmitters glutamate and aspartate, and the agonists NMDA (N-methyl-D-aspartate), ibotenate, quisqualate and kainate. Their reversal potential was approximately -5 mV. 5. A majority of noise spectra produced by the various glutamate receptor agonists were fitted by two Lorentzian components; the rest were fitted with a single Lorentzian component. The noise time constants were apparently not dependent on the type of glutamate agonist used to activate the receptor channels. Pooling data for all agonists gave a mean time constant for single-component spectra of tau noise = 4.8 +/- 0.3 ms; for two-component spectra the time constants were tau 1 = 22.7 +/- 1.8 ms and tau 2 = 2.2 +/- 0.12 ms (Vm = -110 to -50 mV). It is likely that the two components present in whole-cell noise spectra reflect complex kinetics of glutamate receptor channels. 6. The mean single-channel conductance was estimated from whole-cell noise for the various excitatory amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

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