Transmembrane AMPA receptor regulatory proteins and AMPA receptor function in the cerebellum.

Heterogeneity among AMPA receptor (AMPAR) subtypes is thought to be one of the key postsynaptic factors giving rise to diversity in excitatory synaptic signaling in the CNS. Recently, compelling evidence has emerged that ancillary AMPAR subunits-the so-called transmembrane AMPA receptor regulatory proteins (TARPs)-also play a vital role in influencing the variety of postsynaptic signaling. This TARP family of molecules controls both trafficking and functional properties of AMPARs at most, if not all, excitatory central synapses. Furthermore, individual TARPs differ in their effects on the biophysical and pharmacological properties of AMPARs. The critical importance of TARPs in synaptic transmission was first revealed in experiments on cerebellar granule cells from stargazer mice. These lack the prototypic TARP stargazin, present in granule cells from wild-type animals, and consequently lack synaptic transmission at the mossy fibre-to-granule cell synapse. Subsequent work has identified many other members of the stargazin family which act as functional TARPs. It has also provided valuable information about specific TARPs present in many central neurons. Because much of the initial work on TARPs was carried out on stargazer granule cells, the important functional properties of TARPs present throughout the cerebellum have received particular attention. Here we discuss some of these recent findings in relation to the main TARPs and the AMPAR subunits identified in cerebellar neurons and glia.

CNQX increases GABA-mediated synaptic transmission in the cerebellum by an AMPA/kainate receptor-independent mechanism.

GABA(A) receptor-mediated inhibitory synaptic transmission within the CNS is often studied in the presence of glutamate receptor antagonists. However, for nearly a decade it has been known that, in the hippocampus, one of the most commonly used alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), can increase the frequency of spontaneous GABA(A) receptor-mediated postsynaptic currents (sIPSCs). In the present study we examined the effect of CNQX and related compounds on GABA-mediated synaptic transmission in the cerebellum. At various stages of development, low concentrations of CNQX increased the frequency of sIPSCs recorded from granule cells. This effect was independent of the blocking action of CNQX on ionotropic glutamate receptors, as it was not observed with the broad-spectrum glutamate receptor antagonist kynurenate. No increase in sIPSC frequency was observed with the NMDA receptor antagonists D-AP5 or 7-ClK, the selective AMPA receptor antagonists GYKI 52466 or GYKI 53655, or the kainate receptor antagonist NS-102. In contrast, two other quinoxaline derivatives, NBQX and DNQX, were capable of increasing sIPSC frequency. These results demonstrate that the novel excitatory action of CNQX, unrelated to blockade of ionotropic glutamate receptors, is not restricted to the hippocampus and can be observed with structurally related compounds.

Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance.

Many neurons receive a continuous, or 'tonic', synaptic input, which increases their membrane conductance, and so modifies the spatial and temporal integration of excitatory signals. In cerebellar granule cells, although the frequency of inhibitory synaptic currents is relatively low, the spillover of synaptically released GABA (gamma-aminobutyric acid) gives rise to a persistent conductance mediated by the GABA A receptor that also modifies the excitability of granule cells. Here we show that this tonic conductance is absent in granule cells that lack the alpha6 and delta-subunits of the GABAA receptor. The response of these granule cells to excitatory synaptic input remains unaltered, owing to an increase in a 'leak' conductance, which is present at rest, with properties characteristic of the two-pore-domain K+ channel TASK-1 (refs 9,10,11,12). Our results highlight the importance of tonic inhibition mediated by GABAA receptors, loss of which triggers a form of homeostatic plasticity leading to a change in the magnitude of a voltage-independent K + conductance that maintains normal neuronal behaviour.

Slow deactivation kinetics of NMDA receptors containing NR1 and NR2D subunits in rat cerebellar Purkinje cells.

We have examined the deactivation kinetics of native N-methyl-D-aspartate receptors (NMDARs) containing NR1 and NR2D subunits by patch-clamp recording from Purkinje cells in cerebellar slices from young rats. NMDAR-mediated whole-cell currents were elicited in response to bath application of 20 microM NMDA and 50 microM glycine. The NMDAR-mediated currents were small, with an average whole-cell conductance of approximately 750 pS. Following the rapid application of brief pulses (1-10 ms) of 1 mM glutamate to outside-out membrane patches, we observed a low-conductance type of single-channel activity which lasted up to 30 s after the removal of agonist. Analysis of individual channel openings revealed asymmetry of transitions between the main- and subconductance states - a characteristic of NR1/NR2D-containing NMDARs. The averaged macroscopic current exhibited a decay time course which was well described by a single exponential function with a time constant of approximately 3 s. We conclude that native NR1/NR2D-containing NMDARs, like their recombinant counterparts, display very slow deactivation kinetics. This feature should provide a means for identification of these receptors at synapses, and indicates that they do not contribute to the synaptic NMDAR currents so far described.

Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype.

Activity-dependent change in the efficacy of transmission is a basic feature of many excitatory synapses in the central nervous system. The best understood postsynaptic modification involves a change in responsiveness of AMPAR (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor)-mediated currents following activation of NMDA (N-methyl-D-aspartate) receptors or Ca2+-permeable AMPARs. This process is thought to involve alteration in the number and phosphorylation state of postsynaptic AMPARs. Here we describe a new form of synaptic plasticity--a rapid and lasting change in the subunit composition and Ca2+ permeability of AMPARs at cerebellar stellate cell synapses following synaptic activity. AMPARs lacking the edited GluR2 subunit not only exhibit high Ca2+ permeability but also are blocked by intracellular polyamines. These properties have allowed us to follow directly the involvement of GluR2 subunits in synaptic transmission. Repetitive synaptic activation of Ca2+-permeable AMPARs causes a rapid reduction in Ca2+ permeability and a change in the amplitude of excitatory postsynaptic currents, owing to the incorporation of GluR2-containing AMPARs. Our experiments show that activity-induced Ca2+ influx through GluR2-lacking AMPARs controls the targeting of GluR2-containing AMPARs, implying the presence of a self-regulating mechanism.

Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors in Golgi cells of the rat cerebellum.

1. To investigate the properties of N-methyl-D-aspartate receptors (NMDARs) in cerebellar Golgi cells, patch-clamp recordings were made in cerebellar slices from postnatal day 14 (P14) rats. To verify cell identity, cells were filled with Neurobiotin and examined using confocal microscopy. 2. The NR2B subunit-selective NMDAR antagonist ifenprodil (10 microM) reduced whole-cell NMDA-evoked currents by approximately 80 %. The NMDA-evoked currents were unaffected by the Zn2+ chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN; 1 microM) suggesting the absence of NMDARs containing NR2A subunits. 3. Outside-out patches from Golgi cells exhibited a population of 'high-conductance' 50 pS NMDAR openings. These were inhibited by ifenprodil, with an IC50 of 19 nM. 4. Patches from these cells also contained 'low-conductance' NMDAR channels, with features characteristic of NR2D subunit-containing receptors. These exhibited a main conductance of 39 pS, with a sub-conductance level of 19 pS, with clear asymmetry of transitions between the two levels. As expected of NR2D-containing receptors, these events were not affected by ifenprodil. 5. The NMDAR-mediated component of EPSCs, evoked by parallel fibre stimulation or occurring spontaneously, was not affected by 1 microM TPEN. However, it was reduced (by approximately 60 %) in the presence of 10 microM ifenprodil, to leave a residual NMDAR-mediated current that exhibited fast decay kinetics. This is, therefore, unlikely to have arisen from receptors composed of NR1/NR2D subunits. 6. We conclude that in cerebellar Golgi cells, the high- and low-conductance NMDAR channels arise from NR2B- and NR2D-containing receptors, respectively. We found no evidence for NR2A-containing receptors in these cells. While NR2B-containing receptors are present in both the synaptic and extrasynaptic membrane, our results indicate that NR1/NR2D receptors do not contribute to the EPSC and appear to be restricted to the extrasynaptic membrane.

Single-channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes.

Many neurons express a multiplicity of GABAA receptor subunit isoforms. Despite having only a single source of inhibitory input, the cerebellar granule cell displays, at various stages of development, more than 10 different GABAA subunit types. This subunit diversity would be expected to result in significant receptor heterogeneity, yet the functional consequences of such heterogeneity remain poorly understood. Here we have used single-channel properties to characterize GABAA receptor types in the synaptic and extrasynaptic membrane of granule cells. In the presence of high concentrations of GABA, which induced receptor desensitization, extrasynaptic receptors in outside-out patches from the soma entered long-lived closed states interrupted by infrequent clusters of openings. Each cluster of openings, which is assumed to result from the repeated activation of a single channel, was to one of three main conductance states (28, 17, or 12 pS), the relative frequency of which differed between patches. Such behavior indicates the presence of at least three different receptor types. This heterogeneity was not replicated by individual recombinant receptors (alpha1beta2gamma2S or alpha1beta3gamma2S), which gave rise to clusters of a single type only. By contrast, the conductance of synaptic receptors, determined by fluctuation analysis of the synaptic current or direct resolution of channel events, was remarkably uniform and similar to the highest conductance value seen in extrasynaptic patches. These results suggest that granule cells express multiple GABAA receptor types, but only those with a high conductance, most likely containing a gamma subunit, are activated at the synapse.

NMDA receptor diversity in the cerebellum: identification of subunits contributing to functional receptors.

Recent studies of N-methyl-D-aspartate (NMDA) receptors have led to the suggestion that there are two distinct classes of native NMDA receptors, identifiable from their single-channel conductance properties. 'High-conductance' openings arise from NR2A- or NR2B-containing receptors, and 'low-conductance' openings arise from NR2C- or NR2D-containing receptors. In addition, the low-conductance channels show reduced sensitivity to block by Mg2+. The readily identified cell types and simple architecture of the cerebellum make it an ideal model system in which to determine the contribution of specific subunits to functional NMDA receptors. Furthermore, mRNA for all of these four NR2 subunits are represented in this brain region. We have examined NMDA channels in Purkinje cells, deep cerebellar nuclei (DCN) neurons and Golgi cells. First we find that NR2D-containing NMDA receptors give rise to low-conductance openings in cell-attached recordings from Purkinje cells. The characteristic conductance of these events cannot, therefore, be ascribed to patch excision. Second, patches from some DCN neurons exhibit mixed populations of high- and low-conductance openings. Third, Golgi cells also exhibit a mixed population of high- and low-conductance NMDA receptor openings. The features of these low-conductance openings are consistent with the presence of NR2D-containing NMDA receptors, as suggested by in situ hybridization data. On the other hand the existence of high-conductance channels, with properties typical of NR2B-containing receptors, was not expected. Our results provide new evidence about the subunit composition of NMDA receptors in identified cerebellar cells, and suggest that examination of single-channel properties is a potentially powerful approach for determining the possible subunit composition of native NMDA receptors.

Locus of frequency-dependent depression identified with multiple-probability fluctuation analysis at rat climbing fibre-Purkinje cell synapses.

1. EPSCs were recorded under whole-cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12- to 14-day-old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired-pulse depression and all-or-none appearance in response to a graded stimulus. 2. Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca2+] and [Mg2+]. Quantal parameters were estimated from plots of coefficient of variation (CV) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non-uniform release probability. This 'multiple-probability fluctuation' (MPF) analysis gave an estimate of 510 +/- 50 for the number of functional release sites (N) and a quantal size (q) of 0.5 +/- 0.03 nS (n = 6). 3. Control experiments, and simulations examining the effects of non-uniform release probability, indicate that MPF analysis provides a reliable estimate of quantal parameters. Direct measurement of quantal amplitudes in the presence of 5 mM Sr2+, which gave asynchronous release, yielded distributions with a mean quantal size of 0.55 +/- 0.01 nS and a CV of 0.37 +/- 0.01 (n = 4). Similar estimates of q were obtained in 2 mM Ca2+ when release probability was lowered with the calcium channel blocker Cd2+. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 microM) reduced both the evoked current and the quantal size (estimated with MPF analysis) to a similar degree, but did not affect the estimate of N. 4. We used MPF analysis to identify those quantal parameters that change during frequency-dependent depression at climbing fibre-Purkinje cell synaptic connections. At low stimulation frequencies, the mean release probability (pr) was unusually high (0.90 +/- 0.03 at 0.033 Hz, n = 5), but as the frequency of stimulation was increased, pr fell dramatically (0.02 +/- 0.01 at 10 Hz, n = 4) with no apparent change in either q or N. This indicates that the observed 50-fold depression in EPSC amplitude is presynaptic in origin. 5. Presynaptic frequency-dependent depression was investigated with double-pulse and multiple-pulse protocols. EPSC recovery, following simultaneous release at practically all sites, was slow, being well fitted by the sum of two exponential functions (time constants of 0.35 +/- 0.09 and 3.2 +/- 0.4 s, n = 5). EPSC recovery following sustained stimulation was even slower. We propose that presynaptic depression at CF synapses reflects a slow recovery of release probability following release of each quantum of transmitter. 6. The large number of functional release sites, relatively large quantal size, and unusual dynamics of transmitter release at the CF synapse appear specialized to ensure highly reliable olivocerebellar transmission at low frequencies but to limit transmission at higher frequencies.

Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.

Non-NMDA glutamate receptor subunits of the AMPA-preferring subfamily combine to form ion channels with heterogeneous functional properties. We have investigated the effects of RNA editing at the Q/R site, splice variation of the "flip/flop" cassette, and multimeric subunit assembly on the single-channel conductance and kinetic properties of the recombinant AMPA receptors formed from GluR2 and GluR4 expressed in HEK 293 cells. We found that AMPA receptor single-channel conductance was dependent on the Q/R site editing state of the subunits comprising the channel. Calcium-permeable (unedited) channels had resolvable single-channel events with main conductance states of 7-8 pS, whereas fully edited GluR2 channels had very low conductances of approximately 300 fS (estimated from noise analysis). Additionally, the flip splice variant of GluR4 conferred agonist-dependent conductance properties reminiscent of those found for a subset of AMPA receptors in cultured cerebellar granule cells. These results provide a description of the single-channel properties of certain recombinant AMPA receptors and suggest that the single-channel conductance may be determined by the expression of edited GluR2 subunits in neurons.

A direct comparison of the single-channel properties of synaptic and extrasynaptic NMDA receptors.

The assumption that synaptic and extrasynaptic glutamate receptors are similar underpins many studies that have sought to relate the behavior of channels in excised patches to the macroscopic properties of the EPSC. We have examined this issue for NMDA receptors in cerebellar granule cells, the small size of which allows the opening of individual synaptic NMDA channels to be resolved directly. We have used whole-cell patch-clamp recordings to determine the conductance and open time of NMDA channels activated during the EPSC and used cell-attached and outside-out recordings to examine NMDA receptors in somatic membrane. Conductance and open time of synaptic channels were indistinguishable from those of extrasynaptic channels in cell-attached patches. However, the channel conductance in outside-out patches was 20% lower than in cell-attached recordings. This change was partially reduced by dantrolene and phalloidin, suggesting that it may involve depolymerization of actin following Ca2+ release from intracellular stores. Our results demonstrate that synaptic and extrasynaptic NMDA receptors have similar microscopic properties. However, NMDA channel conductance is reduced following the formation of an outside-out patch.

Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors.

1. To investigate the origin and functional significance of a recently described tonic GABAA receptor-mediated conductance in cerebellar granule cells we have made recordings from cells in cerebellar slices from rats of different ages (postnatal days P4 to P28). 2. During development there was a dramatic change in the properties of GABA-mediated synaptic transmission. The contribution to GABAA receptor-mediated charge transfer from the tonic conductance (GGABA), relative to that resulting from discrete spontaneous postsynaptic currents (sPSCs), was increased from 5% at P7 to 99% at P21. GGABA was reduced by bicuculline, tetrodotoxin and by lowering extracellular Ca2+, and was initially present only in those cells which exhibited sPSCs. 3. At P7 sPSCs were depolarizing, occasionally triggering a single action potential. By P18 the GABA reversal potential was shifted close to the resting potential and GGABA produced a shunting inhibition. Removal of GGABA by bicuculline increased granule cell excitability in response to current injection. 4. This novel tonic inhibition is present despite the low number of Golgi cell synapses on individual granule cells and appears to result from 'overspill' of synaptically released GABA leading to activation of synaptic and extrasynaptic GABAA receptors.

Identification of a native low-conductance NMDA channel with reduced sensitivity to Mg2+ in rat central neurones.

1. We have identified a new type of NMDA channel in rat central neurones that express mRNA for the NR2D subunit. We have examined single NMDA channels in cerebellar Purkinje cells (which possess NR1 and 2D), deep cerebellar nuclei (NR1, 2A, 2B and 2D) and spinal cord dorsal horn neurones (NR1, 2B and 2D). 2. In Purkinje cells, NMDA opened channels with a main conductance of 37.9 +/- 1.1 pS and a subconductance of 17.8 +/- 0.7 pS, with frequent transitions between the two levels. 3. NMDA activated low-conductance ('38/18 pS') events (along with high-conductance--'50/40 pS'--openings) in some patches from deep cerebellar nuclei and dorsal horn neurones. Our evidence suggests that 38/18 pS and 50/40 pS events arose from distinct types of NMDA receptors. 4. The transitions for 38/18 pS events were asymmetrical: steps from 38 to 18 pS were more frequent (72.2%) than steps from 18 to 38 pS. This feature appeared common to the 38/18 pS events in all three cell types, suggesting similarity in the low-conductance channels. 5. The 38/18 pS channels in Purkinje cells exhibited characteristic NMDA receptor properties, including requirement for glycine, antagonism by D-2-amino-5-phosphonopentanoic acid (D-AP5) and 7-chlorokynurenic acid, and voltage-dependent block by extracellular Mg2+. 6. The mean open time for the 38 pS state (0.74 +/- 0.07 ms) was significantly briefer than that for the 18 pS state (1.27 +/- 0.18 ms). 7. Mg2+ block of low-conductance NMDA channels in Purkinje cells was less marked than block of 50/40 pS channels in cerebellar granule cells. 8. The time course of appearance of 38/18 pS NMDA channels matched the expression of mRNA for the NR2D subunit. Thus 38/18 pS events were present in > 70% of Purkinje cell patches in 0- to 8-day-old animals, and absent by postnatal day 12. 9. We propose that the 38/18 pS NMDA channels identified here (associated with the NR2D subunit), and the other low-conductance NMDA channel associated with the NR2C subunit, may together constitute a functionally distinct subclass of native NMDA receptors.

Non-NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites.

1. Excitatory postsynaptic currents (EPSCs) were recorded under whole-cell voltage clamp from granule cells in slices of rat cerebellum. EPSCs from individual mossy fibre inputs were identified by their all-or-none appearance in response to a graded stimulus. Excitatory synaptic transmission was investigated at room temperature (approximately 24 degrees C) and at near-physiological temperature (approximately 34 degrees C) by analysing current fluctuations in the peak and decay of the non-N-methyl-D-aspartate (non-NMDA) component of EPSCs. 2. In a subset of synapses the mean EPSC amplitude remained unchanged as the probability of transmitter release was substantially lowered by raising the extracellular [Mg2+] and lowering [Ca2+]. These synapses were considered to have only one functional release site. Single-site synapses had small EPSCs (139 +/- 16 pS, n = 5, at 24 degrees C) with a large coefficient of variation (c.v. = 0.23 +/- 0.02, n = 5) and an amplitude distribution that was well fitted by a Gaussian distribution in four out of five cases. The EPSC latency had a unimodal distribution and its standard deviation had a temperature dependence with a temperature coefficient (Q10; range, 24-35 degrees C) of 2.4 +/- 0.4 (n = 4). 3. Peak-scaled non-stationary fluctuation analysis of single-site EPSCs indicated that the mean conductance of the underlying non-NMDA channels was 12 +/- 2 pS (n = 4) at 35 degrees C. Upper and lower limits for mean channel open probability (Po), calculated from fluctuations in the EPSC peak amplitude, were 0.51 and 0.38, respectively. These estimates, together with the open probability of the channel when bound by transmitter, suggest that only about 50% of the non-NMDA channels were occupied following the release of a quantum of transmitter. 4. At some multi-site synapses EPSCs had a low c.v. (0.4 +/- 0.01, n = 5) at 34 degrees C and non-stationary fluctuation analysis gave a parabolic variance-mean current relationship. This suggests that practically all of the non-NMDA receptors were occupied by glutamate at the peak of EPSC. The channel open probability (Po = 0.84 +/- 0.03, n = 5) at these 'saturated' multi-site synapses will therefore equal the open probability of the channel when bound by transmitter (Po,max). 5. Non-stationary fluctuation analysis of EPSCs from 'saturating' multi-site synapses indicated that 170 +/- 40 postsynaptic non-NMDA channels were exposed to transmitter at the peak of the EPSC. The mean conductance of the synaptic channels was 10 +/- 2 pS (n = 5) at 34 degrees C. 6. At synapses with multiple release sites the EPSC decay time became faster when release probability was lowered (by reducing the external [Ca2+]/[Mg2+] ratio), indicating that the transmitter concentration profile depended on release probability. No such speeding of the EPSC decay was observed at single-site synapses. 7. Our results suggest that release of a packet of transmitter from a single release site does not saturate postsynaptic non-NMDA receptors at cerebellar mossy fibre-granule cell synapses. However, at multi-site synapses transmitter released from neighbouring sites can overlap, changing the transmitter concentration profile in the synaptic cleft. We conclude that the level of postsynaptic receptor occupancy can depend on the probability of transmitter release at individual multi-site synapses.

Functional correlation of NMDA receptor epsilon subunits expression with the properties of single-channel and synaptic currents in the developing cerebellum.

NMDA receptor (NMDAR) subunits epsilon 1-epsilon 4 are expressed differentially with respect to brain region and ontogenic period, but their functional roles still are unclear. We have compared an epsilon 1 subunit-ablated mutant mouse with the wild-type to characterize the effect of epsilon subunit expression on NMDAR-mediated single-channel currents and synaptic currents of granule cells in cerebellar slices. Single-channel and Western blot analyses indicated that the epsilon 2 subunit disappeared gradually during the first postnatal month in both wild-type and mutant mice. Concomitantly, the voltage-dependent Mg2+ block of NMDAR-mediated EPSCs (NMDA-EPSCs) was decreased. Throughout the developmental period studied, postnatal day 7-24 (P7-P24), the decay time course of NMDA-EPSCs in epsilon 1 mutant (-/-) mice was slower than in wild-type mice. We suggest that the expression of the epsilon 3 subunit late in development is responsible for a reduction in the sensitivity of NMDA-EPSCs to block by extracellular Mg2+ and that receptors containing the epsilon 1 subunit determine the fast kinetics of the NMDA-EPSCs.

Deactivation and desensitization of non-NMDA receptors in patches and the time course of EPSCs in rat cerebellar granule cells.

1. Spontaneous and evoked non-NMDA receptor-mediated EPSCs were recorded from cerebellar granule cells in slices at approximately 24 and approximately 34 degrees C. The EPSC decay was fitted with the sum of two exponential functions. 2. The time courses of non-NMDA receptor deactivation and desensitization were determined with fast concentration jumps of glutamate onto patches from cultured granule cells. Deactivation (decay time constant tau = 0.6 ms at 24 degrees C) was substantially faster than desensitization (tau = 4 ms). Both processes were fitted by single exponential functions. 3. The decay of the fast component of the spontaneous EPSC (tau EPSCfast = 0.9 ms at 23 degrees C) was marginally slower than deactivation but too fast to be determined by desensitization. Our results suggest that the decay of this component is set by both the rate of decline of transmitter concentration and channel deactivation. 4. A simple diffusion model predicts that the time course of transmitter in the cleft declines slowly during the later stages of its action. The slow phase of transmitter removal could account for the time course of the slow component of the spontaneous EPSC (tau EPSCslow = 8 ms at 23 degrees C).

Effect of RNA editing and subunit co-assembly single-channel properties of recombinant kainate receptors.

1. Patch-clamp methods have been used to examine single-channel properties of recombinant GluR5 and GluR6 kainate-preferring glutamate receptors which differ in a single amino acid residue as a result of RNA editing at the Q/R (glutamine/arginine) site. Subunits were expressed alone or in combination with the high-affinity kainate receptor subunit KA - 2 in transfected human embryonic kidney (HEK-293) cells. 2. In outside-out patches, unedited homomeric GluR6(Q) receptors exhibited directly resolved domoate-activated single-channel conductances of 8, 15 and 25 pS. Variance analysis of GluR6(Q) responses gave a mean conductance of 5.4 pS, while the edited isoform GluR6(R) had an unusually low channel conductance (225 fS). 3. Homomeric channels composed of GluR5(Q) subunits exhibited three conductance states of 5, 9 and 14 pS characterized by prolonged burst activations in the presence of domoate. In contrast, the GluR5(R) subunit, which has not previously been reported to form functional homomeric receptors, had an extremely low conductance (< 200 fS). 4. Heteromeric GluR6(Q)/KA-2 kainate receptors gave single-channel events indistinguishible from homomeric GluR6(Q) channels. Conversely, openings produced by GluR5(Q)KA-2 and GluR5(Q) receptors differed from each other in their kinetic properties. The primary effect of co-expression of KA-2 with GluR5(Q) was a dramatic shortening in channel burst length. 5. Spectral and variance analyses were used to estimate mean single-channel conductances of heteromeric edited receptor-channels; channel conductances were 950 fS for GluR5(R)KA-2 receptors and 700 fS for GluR6(R)/KA-2 receptors. Both receptor types had significantly higher conductances than the respective homomeric channels, GluR5(R) and GluR6(R). 6. We conclude that Q/R site editing dramatically reduces single-channel conductance. Furthermore, we find similarity between the kainate receptor-channels described in sensory neurones and the recombinant GluR5(Q) homomeric channel. Characterization of recombinant single-channel properties could therefore aid identification of the native kainate receptors.

Intracellular spermine confers rectification on rat calcium-permeable AMPA and kainate receptors.

1. Whole-cell recordings were made from cerebellar granule cells cultured in high-K+ medium to induce expression of Ca(2+)-permeable AMPA receptors. Current-voltage (I-V) plots of agonist-evoked responses showed varying degrees of inward rectification, but became linear within 5-10 min. 2. Recombinant Ca(2+)-permeable kainate receptors, composed of GluR6(Q)/KA-2 subunits, exhibited rectifying whole-cell I-V plots that became linear in outside-out patches. 3. Loss of rectification in granule cells was prevented by including 100 microM spermine in the pipette; the degree of rectification was then correlated with Ca2+ permeability. 4. Spermine also prevented loss of rectification in patches containing GluR6(Q)/KA-2 receptors (IC50, 1.7 microM). 5. We suggest that spermine, or a similar cellular constituent, may act as a cytoplasmic factor conferring inward rectification on Ca(2+)-permeable non-NMDA receptors, and that 'washout' of this factor underlies the observed loss of rectification.

Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum.

1. Patch-clamp methods have been used to characterize GABA-and glycine-activated channels and spontaneous synaptic currents in granule cells in thin cerebellar slices from 7- to 20-day-old rats. 2. All granule cells responded to 10 microM GABA, while approximately 60% responded to 100 microM glycine. With repeated against application, whole-cell responses to GABA, but not those to glycine, declined over a period of minutes unless the pipette solution contained Mg-ATP. 3. Whole-cell concentration-response curves gave EC50 values at 45.2 and 99.6 microM and Hill slopes of 0.94 and 2.6 for GABA and glycine, respectively. At saturating concentrations, currents evoked by GABA were fivefold larger than those evoked by glycine. 4. Whole-cell current-voltage (I-V) relationships of GABA- and glycine-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with F- shifted the GABA reversal potential to a more negative value. 'Instantaneous' I-V relationships produced by ionophoretic application of GABA were linear, while 'steady-state' I-V relationships produced by ramp changes in potential showed outward rectification. For glycine, 'steady-state' I-V plots were linear. 5. Responses to GABA were blocked by the GABAA receptor antagonists bicuculline (15 microM), SR-95531 (10 microM) and picrotoxinin (100 microM) while responses to glycine were selectively blocked by strychnine (200 nM), indicating the presence of two separate receptor types. 6. In outside-out membrane patches, GABA opened channels with conductances of 16 and 28 pS. The proportion of openings to each of the conductances varied between patches, possibly indicating the activation of two distinct channel types. Glycine-activated single-channel currents had conductances of 32, 55 and 104 pS. Single-channel I-V relationships were linear. 7. Spontaneous synaptic currents with a rapid rise time and biexponential decay were present in more than half of the cells examined. These currents were eliminated by bicuculline (15 microM) or SR-95331 (10 microM) and were greatly reduced in frequency by tetrodotoxin (TTX; 300 nM), suggesting that they were mediated by GABA and arose from spontaneous activity in Golgi interneurones. In granule cells where this spontaneous synaptic activity was apparent, glycine and low concentrations of GABA increased the frequency of the synaptic currents.(ABSTRACT TRUNCATED AT 400 WORDS)

NMDA-receptor channel diversity in the developing cerebellum.

In the cerebellum, NMDA (N-methyl-D-aspartate) receptors play an important role in neuronal differentiation and excitatory synaptic transmission. During early cerebellar development, marked changes occur in the distribution of messenger RNAs encoding various NMDA-receptor subunits. To determine whether these changes result in the appearance of functionally distinct NMDA receptors, we have recorded single-channel currents in rat cerebellar granule cells during the period of their migration from the external germinal layer to the inner granular layer. Here we show that before synapse formation, pre-migratory and migrating granule cells express NMDA receptors possessing single-channel properties similar to those previously described for many central neurons. In contrast, mature post-migratory cells also express an atypical form of NMDA receptor that has a lower single-channel conductance and distinct kinetic behaviour. The properties of these 'low-conductance' channels correspond to those described for recombinant NMDA receptors formed by coexpression of NR1 and NR2C subunits. The NR2C subunit appears postnatally and is found predominantly in the adult cerebellum. Our data demonstrate developmental changes in NMDA-receptor properties at the single-channel level, and suggest that in the cerebellum the expression of a specific subunit protein results in a distinct form of native receptor.