5404 Journal of Physiology (1996), 493.1, pp.167-173 167 Deactivation and desensitization of non-NMDA receptors in patches and the time course of EPSCs in rat cerebellar granule cells

R. Angus Silver, David Colquhoun, Stuart G. Cull-Candy and Brian Edmonds Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK

1. Spontaneous and evoked non-NMDA receptor-mediated EPSCs were recorded from cerebellar granule cells in slices at -24 and -34 '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 r = 0 6 ms at 24 °C) was substantially faster than desensitization ('r = 4 ms). Both processes were fitted by single exponential functions. 3. The decay of the fast component of the spontaneous EPSC (TEPSCfast = 09 ms at 23 °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 (rEPscslow = 8 ms at 23°C).

The time course of excitatory postsynaptic currents (EPSCs) the mossy fibre to granule cell EPSC (Silver, Traynelis & is determined by the time course of the concentration of Cull-Candy, 1992). We have investigated the mechanisms transmitter in the synaptic cleft and the kinetic properties that shape non-NMDA component EPSCs in granule cells by of the postsynaptic receptors. If the concentration of comparing the time- and temperature-dependent properties transmitter in the cleft declines slowly, relative to the EPSC, of EPSCs with those of deactivation and desensitization in both the time course of transmitter decay and the rate of isolated patches from the soma of cultured granule cells. A receptor desensitization would be expected to shape the preliminary report has been published in this journal EPSC (Trussell, Zhang & Raman, 1993; Barbour, Keller, (Silver, Colquhoun, Cull-Candy & Edmonds, 1994). Llano & Marty, 1994). In contrast, if the decay of transmitter is relatively fast, the EPSC decay would be determined predominantly by the rate of deactivation (defined here as METHODS the rate at which current declines after a jump to zero Recordings agonist concentration) (Magleby & Stevens, 1972; Katz & As previously described, 10- to 14-day-old Sprague-Dawley rats Miledi, 1973; Colquhoun, Jonas & Sakmann, 1992). Recent were decapitated and their brains rapidly removed and placed in studies on glutamatergic suggest that mechanisms ice-cold saline containing (mM): 125 NaCl, 2-5 KCl, 1 CaCl2, the of the 5 MgCl2, 125 NaH2PO4, 26 NaHCO3 and 15 glucose (pH 7-3 when underlying decay non-N-methyl-D-aspartate bubbled with 95 % 02-5 % CO2) (see Traynelis, Silver & Cull-Candy, (non-NMDA) component may vary at different synapses. 1993). Synaptic recordings were made from granule cells in 150 #sm Deactivation alone may account for the time course of EPSCs thick cerebellar slices. The effect of asynchrony of transmitter at some central synapses (Colquhoun et al. 1992; Hestrin, release on EPSC shape was minimized by examining spontaneous 1992), while at others, receptor desensitization may be currents and by selecting evoked currents that had a monotonic important in shaping the synaptic current (Trussell et al. rising phase (Traynelis et al. 1993). EPSCs were evoked at 1993). 0-5-1P0 Hz by stimulating (5-20 V; duration, 200 ,us) mossy fibre inputs with a patch electrode in the surrounding tissue. Spontaneous Cerebellar granule cells are electrically compact, allowing EPSCs were collected in the interstimulus period. Recordings were high resolution recordings of the non-NMDA component of made with an external solution containing (mM): 125 NaCl,

This manuscript was accepted as a Short Paper for rapid publication. 16f8 R. A. Silver and others J. Phy8iol.493.1

25 KCl, 2 CaCl2, 1P25 NaH2PO4, 26 NaHCO3 and 15 glucose Non-NMDA currents from patches were filtered at 3 kHz, and (pH 7-3 when bubbled with 95% 02-5% CO2). MgCl2 (1 mM) was sampled on-line at 100 kHz or recorded onto tape. added in some experiments. D-Amino-5-phosphonopentanoic acid Fitting and (20 uM), 20/uM 7-chlorokynurenic acid and 15 uM bicuculline analysis methiodide were added to the perfusate to block NMDA and Non-NMDA EPSCs recorded in granule cells were averaged by y-aminobutyric acid type A receptors. Patch pipettes (5-15 M.Q) aligning the onset of the current and the decay was fitted with the were made from thick-walled borosilicate glass (Clark sum of two exponentials. Electrical models of granule cells (Silver et Electromedical) and coated with Sylgard (Dow Corning). The al. 1992), and the fact that single NMDA channel openings can be pipette solution consisted of (mM): 110 CsF, 30 CsCl, 4 NaCl, 0 5 resolved in the synaptic current, indicate that the voltage at CaC12, 10 Hepes and 5 EGTA (pH 7 3). EPSCs were recorded using synapses located on the dendrites is well clamped. The capacitance, an L/M-EPC-7 (List) or an Axopatch 200A (Axon Instruments) input resistance and series resistance were determined for each cell amplifier and recorded on tape. Recordings were filtered at 4-8 kHz from the capacitative current response to a -10 mV voltage step. (-3 dB) and digitized from tape at 50-100 kHz. The mean values were: 3-2 + 0'3 pF, 6 + 2 GQl and 23 + 3 MQl (n = 12), respectively, at 24 °C; and 3'0 + 0 3 pF, 6 + 2 GQ and Non-NMDA receptors were not detectable in patches taken from 21 + 3 M.Q (n = 11), respectively, at 34 'C. Least-squares fits were the soma of granule cells in slices. Outside-out patches were made by fixing the asymptote of the exponential function to the therefore made from cultured granule cells prepared from 7-day-old pre-event baseline. Since the relative amplitude of fitted rats and used 3-5 days after plating (Cull-Candy, Howe & Ogden, exponentials depends on where t = 0 is taken to be, and in the case 1988). Application of glutamate (0 3-5 0 mM) was achieved by of EPSCs this is somewhat arbitrary, we have defined t = 0 as the rapid movement (using a piezotranslator, model P-244.40; Physik peak of the EPSC. Instrumente, Waldbronn, Germany) of the interface of solutions flowing from either side of a theta glass partition. Junction Temperature coefficient (Q1o) values were calculated from current responses to the shortest (200 ,us) jumps were usually (TT/1T2)lO/AT, where TTl and 1T2 are the mean time constants of within 90% of the steady-state value. The inflowing bath perfusate decay at lower and higher temperatures, respectively, and ATis the (1-2 ml min-') was heated with a Peltier device and the temperature difference. Errors for the numerator (e.g. TTlO0/AT) and temperature was monitored in the bath. Immersion of about 2 cm denominator were calculated using a Taylor series approximation. of the thin theta glass application tool, and the relatively low flow An approximate standard deviation for the Qlo was then obtained rates used (0-1-0-2 ml min-'), ensured that the perfusate in the using Fieller's theorem. P values were calculated with Student's t rapid application tool and bath were close to thermal equilibrium. and randomization tests.

A C Spontaneous EPSCs at 260C Evoked EPSCs at 25 00

rEPSCsIow -47 ms TEPSCsiow -103 ms

10 pA 50 TEPSCfast = 07 ms TEPSCfast 1-4 ms pA 5 ms 5 ms B D Evoked EPSCs at 350°C

7EPSCsIow = 4O0 ms 7EPSCslow - 7 9 ms

5 pA 100 pA 0 7 ms EPSCfast =5 5 ms EPSCfast = 5 ms

Figure 1. The non-NMDA component of EPSCs in cerebellar granule cells decays biphasically A, the average of 102 spontaneous EPSCs recorded from a granule cell at room temperature. The superimposed curve shows a least-squares fit of the sum of two exponentials to the EPSC decay (I(t) = -23'2exp(-t/0 69) - 3-5exp(-t/4 7), where I is current and t is time). B, the average of 233 EPSCs recorded at 34 °C. The superimposed curve is the least-squares fit to the EPSC (I(t) =-12-2exp(-t/0 49)-1-91 exp(-t/4 0)). C, the average of 63 evoked EPSCs at 25 °C with superimposed fit (I(t) = -46-5exp(-t/1-4) - 671exp(-t/10-3)). D, the average of 76 evoked EPSCs at 35 °C with superimposed fit (I(t) = -162-9exp(-t/0 67) - 14-2exp(-t/7 9)). All EPSCs were recorded at -70 mV. Part of the stimulus artifact in C and D was omitted for clarity. J Physiol.493.1 Time course of EPSCs and non-NMDA receptor currents 169

Diffusion calculations change. The values of the resistive and capacitative elements of the There is insufficient knowledge of the properties of release, cleft circuit were taken from granule cell recordings at 35 'C. The time- geometry, receptor density and binding kinetics to allow realistic dependent conductance change was determined by approximating calculation of the expected time course of transmitter the mean waveform of the evoked synaptic current with the sum of concentration. However, a rough estimate can be obtained if the three exponential functions. The differential equation describing cleft is treated as a cylinder of radius a in an infinite volume. With the equivalent circuit of the granule cell was solved numerically in an instantaneous line source at the centre, the fraction of material MathCad (MathSoft, Inc., Cambridge, MA, USA) using the remaining within the cylinder (q) at time t is: Runge-Kutta method. The shape of the simulated EPSP was similar to EPSPs recorded from granule cells in the presence of q(t) = 1 - exp(-a2/4Dt), NMDA receptor antagonists (D'Angelo, De Filippi, Rossi & where D is the diffusion constant. If the cylinder has an initially Taglietti, 1995). uniform concentration, then:

rr q(t) = 2J(J2(ua)/u)exp(-Dtu2) du, RESULTS As Fig. 1A shows, the decay of spontaneous EPSCs recorded where J, is a Bessel function of the first kind and order 1 (Crank, at 21-26 'C (mean 23 °C) had two components. In six cells 1986). For a = 0 5 /sm and D = 7-65 x 10-6 cm2 s-' (for glutamine the major component made up 89 + 1 % of the peak in free solution at 35 °C) these two functions give similar results. amplitude (mean + S.E.M.) and decayed rapidly (TEPSCfast = Modelling the granule cell EPSP 0 93 + 0-13 ms). The low-amplitude component was The electrical properties of granule cells were modelled to investigate relatively slow (rEPSCslow = 8-1 + 2-5 ms). Figure 1B what contribution the two components of the EPSC make to the illustrates that these components were also present at EPSP waveform. The shape of the EPSP was calculated from the higher temperatures (30-34 °C, mean 32 °C). TEPSCfast was passive properties of the granule cell using a single compartment 0-56 + 0 04 ms (amplitude 89 + 2 %, n = 6) and TEPSCslow model (Silver et al. 1992) with a time-dependent conductance was 6 0 + 1 0 ms (n = 6). The time to peak of spontaneous

A B Deactivation at 240C -ii Command step -i

Junction current

X = 0-66 ms 20 pA 10 ms Non-NMDA current C Deactivation at 330C -iL

4 pA 2 pA r = 0-23 ms 1 ms 1 ms

Figure 2. Rapid application of glutamate and the time course of non-NMDA receptor deactivation in granule cell patches A, a 200 /as duration command step used to drive a piezoelectric translator is shown (upper trace) with the solution exchange time course illustrated by the junction current shift (middle trace), after the patch was ruptured and the control solution diluted to 50 %. The decay of the junction current could be fitted with a single exponential (r = 90 ,us; not shown). Average of 28 non-NMDA receptor current responses to a 200 ,us application of 5 mm glutamate onto a granule cell patch at 24 °C (lower trace). The current decay resulting from channel deactivation could be fitted by a single exponential function (r = 0 55 ms) as shown by the superimposed curve. B, a 1 ms application of 5 mm glutamate (upper trace) is shown with the average of 59 non-NMDA currents at 24 °C (lower trace). Deactivation was fitted by a single exponential function with a time constant of 0 66 ms as shown by the superimposed curve. C, a deactivating current (average of 58 events) recorded at 33 °C, resulting from a 0 5 ms application of 5 mm glutamate (upper trace) was fitted by a single exponential function (T = 023 ms). All patches were held at -70 mV. 170 R. A. Silver and others J Physiol. 493.1

EPSCs was 0 37 + 0 03 ms at 23°C and 0-28 + 0-02 ms fast compared with deactivation at 24 'C. At 320C the (n = 6) at 32 0C. deactivation is much faster, but even in the worst case The decay time course of evoked EPSCs had well-separated (T = 0-22 ms), model calculations suggest that the time taken fast and slow components that were slower than those of to change the concentration should cause an error of less spontaneous EPSCs (Fig. 1 C). The evoked EPSCs had a than 20%. The time constant we observe for deactivation 24 mean peak conductance of 520 pS at 25 00, which is about at 0C is similar to the mean burst length of non-NMDA 3-fold larger than the miniature (quantal) conductance channels in granule cells measured under steady-state (Silver et al. 1992). Evoked decays were fitted with conditions (0-62 ms; Wyllie, Traynelis & Cull-Candy, 1993). EPSC In contrast to the rapid deactivation, the current the sum of two exponential components: a TEPSCfat of decay 1P5+ 0-1 ms (90 + 1 % of amplitude) and a of during sustained applications of glutamate (desensitization) TEPSCslow was slower. Typical of are shown 14 + 1 ms (n = 5) at 250C. At elevated temperatures examples desensitization in Fig. 3A and B. exponential to decay (35 0C; Fig. 1D) the mean decay time constants were: Single fits the phase of averaged currents elicited by a 30 ms application of TEPSCfast, 0 77 + 008 ms (93 + I % of amplitude); and TEPSCslow, 9 + 2 ms (n = 5). The time to peak for evoked glutamate yielded a mean time constant of 4-1 + 0 3 ms events was 0X81 + 0-08 ms at 250C and 0 43 + 0-02 ms at (n=26) at 240C and 2-13+0-05ms (n=5) at 320C. A 350C. lower concentration of glutamate (0 3 mM) produced much smaller currents with desensitization time courses (r = To investigate the mechanisms that contribute to the EPSC 4*5 + 0X8 ms at 240C, n = 6) that were similar (P = 0 64) decay, the time course of non-NMDA receptor-mediated to those found with higher concentrations of glutamate. currents was studied in outside-out patches from cultured granule cells. Non-NMDA currents were activated with rapid The mean single channel conductance of channels in patches applications of glutamate (5 mM). Figure 2A (top two traces) was estimated using non-stationary fluctuation analysis shows the command and application profile (liquid junction (Sigworth, 1980). The non-NMDA channel conductance current) for a 200 concentration jump. The macroscopic varied widely from patch to patch (4-44 pS) and the mean ,us = current decay conductance (29 + 2 pS, n 28) was higher than the value following glutamate removal (deactivation) found was similar for applications ranging from 0-2 to 1 ms (see for synaptic channels that underlie spontaneous Methods), and was well fitted by a single exponential function EPSCs (Traynelis et al. 1993). However, the deactivation and desensitization kinetics were homogeneous, showing no at both 240C (mean T = 0 61 + 0 05 ms; n = 18, Fig. 2A and apparent dependence on the estimated conductance (cor- B) and 320C (mean T = 0-35 + 0-05ms; n =5, Fig. 2C). The time course of the concentration change (Fig. 2A) was relation coefficients were -0 004 and -0 009, respectively).

A Desensitization at 25 °C -J

10 pA T = 3-63 ms 5 ms Figure 3. The time course of non-NMDA receptor desensitization in granule cell patches A, a 30 ms application of 5 mm glutamate (upper trace) is shown with the corresponding average of 40 desensitizing non-NMDA B currents at 25 °C (lower trace). The decay of the desensitizing Desensitization at 33 OC current was fitted by a single exponential function (T = 3-63 ms) as shown by the superimposed curve. B, a desensitizing current (average of 108 events) resulting from a 30 ms application of 5 mm glutamate at 33 °C (upper trace). The current was fitted by a single exponential function (r = 2 14 ms). All currents were measured at -70 mV.

T = 2 14 ms 5 pA

5 ms J Physiol. 493.1 Time course of EPSCs and non-NMDA receptor currents 171

DISCUSSION EPSC reflects deactivation and removal of transmitter, the The decay of the fast component of spontaneous EPSCs is time to peak of spontaneous EPSCs will provide an substantially faster than the time course of desensitization, approximate upper limit for the period over which the suggesting that desensitization does not shape the initial transmitter concentration rises at the receptors. EPSC decay. It was observed that TEPSCfast is marginally Possible mechanisms underlying the decay of the small slower than the deactivation time constant, whereas they slow component of the EPSC include a slowly decaying would be expected to be similar if deactivation were the sole phase of transmitter concentration in the cleft, receptor factor that controlled the rate. This suggests that both desensitization or the presence of a population of slowly deactivation and the rate of transmitter removal influence deactivating postsynaptic receptors not present in the the rapid initial decay of the EPSC, though the deactivation somatic membrane (i.e. not present in the isolated patches). rate is likely to be the predominant factor. Consistent with To estimate the shape of the transmitter concentration this interpretation, the temperature dependence of TEPSCfast profile we have used simple diffusion calculations (Crank, (Qlo0 1P7 + 0 3) is similar to that for the time course of 1986; Fig. 4A). These admittedly oversimplified calculations deactivation of non-NMDA receptors in patches (Qlo, predict a decline in transmitter concentration that is rather 2O0 + 0G4). Although these results imply that desensitization slow at longer times (Fig. 4A; see also Holmes, 1995; Wahl, does not shape this component of the EPSC decay, they do Pouzat & Stratford, 1996). Persistence of a low concentration not preclude the possibility that desensitization occurs of glutamate in the cleft could therefore contribute to the during the synaptic current (Trussell et al. 1993; Barbour et shape of the slow component of the EPSC (Fig. 4A; see also al. 1994). If, as our data suggest, the initial decline of the Mennerick & Zorumski, 1995), either as a result of a slower

A 1 0- 1 00

c 08- 0+@ 0-4- D0-10

0-6 -

0 0~~~~~~~~~00~~D ON~ 0.4- D/2

0 Z 0-2-

0 1 2 3 4 5 Time (ms) B

10 mV

20 ms I Fast

Figure 4. Predicted time course of transmitter in the cleft and the effect of the slow component of the EPSC on the shape of the EPSP A, the time course of transmitter predicted from a simple diffusion model (see Methods). The two continuous curves show the predicted transmitter decay within a disc (radius, 0.5 uin) for two different values of the diffusion constant (D = 7 65 x 10-6 cm2 s-' and D/2). Both curves had a 'biphasic' shape (see text). The time course of the late phase of the transmitter decay is shown more clearly on a logarithmic scale in the inset. B, the slow component of the synaptic current contributes to the waveform of the synaptic potential. The trace marked 'Fast + slow' shows the simulated synaptic potential for a time- dependent conductance change similar to that underlying evoked EPSCs at 35 °C (G(t) = 2274exp(-t/0-765) + 98exp(-t/8-845) - 2372exp(-t/0-26), t in ms). The trace marked 'Fast' shows the simulated synaptic potential when only the fast component of conductance change was present (G(t) = 2274exp(-t/0 765) - 2274exp(-t/0 26)). Charge transfer during the slow component of the EPSC increases the peak and duration of the EPSP. 172 R. A. Silver and others J Physiol. 493.1 rate of desensitization of receptors activated nearly Overall, our results suggest that at the mossy fibre to simultaneously or as a result of delayed activation of distant granule cell the glutamate concentration in the cleft receptors. rises rapidly and has a fast decaying phase. Both simple diffusion calculations and detailed simulations of central Evoked EPSCs had a time to peak and a decay time course synaptic transmission (Holmes, 1995; Wahl et al. 1996) that were slower than those found for spontaneous and a concentration waveform that low-amplitude evoked events. When the probability of predict decays 'biphasically' with fast and slow phases, not as a single exponential, as transmitter release is high, transmitter released from has been assumed Jahr & neighbouring sites can overlap in the granule cell synaptic previously (Clements, Lester, Tong, Westbrook, 1992). The persistent component of transmitter cleft (Silver, Cull-Candy & Takahashi, 1996; see also Tong & could account for the time course and amplitude of the small Jahr, 1994). Transmitter overlap may slow the decay of slow EPSC tail. The fast component of the EPSC comprises transmitter concentration at the postsynaptic receptors 90% of the of the but (although this will depend on the spatial distribution of the amplitude synaptic current, the component carries a substantial fraction receptors relative to the release sites), and contribute to the smaller slow into the of the slower time course of the evoked EPSCs. The decay of the (-40 %) of the charge cell. Modelling granule cell slow of the current makes a fast component of the evoked EPSCs is therefore likely to be suggests that the component to both the and of the determined by a combination of the time course of contribution amplitude duration and transmitter decay, asynchrony of transmitter release and excitatory postsynaptic potential (Fig. 4B), may therefore be an factor in the non-NMDA receptor deactivation. Although evoked EPSCs important determining summation of fibre in the cerebellum. were slower than spontaneous EPSCs they had a time course temporal mossy inputs that was still too fast to be accounted for by desensitization alone.

The interpretation of our results is based on the assumption BARBOUR, B., KELLER, B. U., LLANO, I. & MARTY, A. (1994). that the kinetic behaviour of non-NMDA channels in Prolonged presence of glutamate during excitatory synaptic culture and in slices is the same. Our conclusions would be transmission to cerebellar Purkinje cells. 12, 1331-1343. complicated if the non-NMDA receptor subtypes present at CLEMENTS, J. D., LESTER, R. A., TONG, G., JAHR, C. E. & synapses in slices were different from those present at the WESTBROOK, G. L. (1992). The time course of glutamate in the soma of cultured granule cells. In situ hybridization studies synaptic cleft. Science 258, 1498-1501. indicate that mRNA for both the flip and the flop isoforms COLQUHOUN, D., JONAS, P. & SAKMANN, B. (1992). Action of brief of the glutamate receptor subunit GluR-D (GluR-4) are pulses of glutamate on AMPA/kainate receptors in patches from present in the cerebellar granule cells of rats at around P12 different neurones of rat hippocampal slices. Journal of Physiology 458, 261-287. (postnatal day 12; Mosbacher, Schoepfer, Monyer, Burnashev, CRANK, J. (1986). The Mathematics of Diffusion, 2nd edn, pp. 29-31. Seeburg & Ruppersburg, 1994). The time course of Oxford University Press, Oxford. desensitization (T 4 ms) of non-NMDA receptors in CULL-CANDY, S. G., HOWE, J. R. & OGDEN, D. C. (1988). Noise and patches from cultured granule cells is similar to that for the single channels activated by excitatory amino acids in rat cerebellar flip isoform of receptors containing the GluR-D subunit granule neurones. Journal of Physiology 400, 189-222. expressed in Xenopus oocytes (Mosbacher et al. 1994). D'ANGELO, E., DE FILIPPI, G., Rossi, P. & TAGLIETTI, V. (1995). However, both homomeric and heteromeric recombinant Synaptic excitation of individual rat cerebellar granule cells in situ: non-NMDA receptors containing the flop isoform of the evidence for the role of NMDA receptors. Journal of Physiology GluR-D subunit desensitize fast (Tr 1 ms) relative to 484, 397-413. receptors containing the flip isoform. Thus, the rapidly HESTRIN, S. (1992). Activation and desensitization of glutamate decaying component of the EPSC of animals around this activated channels mediating fast excitatory synaptic currents in age could be due to desensitization if flop receptors are the visual cortex. Neuron 9, 991-999. present at the synapse (Mosbacher et al. 1994). We think, HOLMES, W. R. (1995). Modeling the effect of glutamate diffusion and uptake on NMDA and Non-NMDA receptor saturation. Biophysical however, that this explanation is unlikely to account for our Journal 69, 1734-1747. results because the decay time course of large evoked EPSCs KATZ, B. & MILEDI, R. (1973). The binding of acetylcholine to becomes faster as presynaptic release probability is reduced receptors and its removal from the synaptic cleft. Journal of (Silver et al. 1996). Any simple desensitization mechanism Physiology 231, 549-574. would be expected to yield the opposite result since a MAGLEBY, K. L. & STEVENS, C. F. (1972). A quantitative description decrease in transmitter concentration in the synaptic cleft of end-plate currents. Journal of Physiology 223, 173-197. (see above) would be expected to slow desensitization, not MENNERICK, S. & ZORUMSKI, C. F. (1995). Presynaptic influence on speed it up. Furthermore, the fact that the slow component the time course of fast excitatory synaptic currents in cultured of the EPSC is also faster for small, spontaneous EPSCs hippocampal cells. Journal of 15, 3178-3192. than for multi-quantal evoked EPSCs would be consistent MOSBACHER, J., SCHOEPFER, R., MONYER, H., BURNASHEV, N., with the idea that transmitter diffusion rather than receptor SEEBURG, P. H. & RUPPERSBERG, J. P. (1994). A molecular desensitization is predominant in shaping the EPSC decay determinant for submillisecond desensitization in glutamate at later times. receptors. Science 266, 1059-1062. J Physiol. 493.1 Time course of EPSCs and non-NMDA receptor currents 173

SIGWORTH, F. J. (1980). The variance of sodium current fluctuations at the node of Ranvier. Journal of Physiology 307, 97-129. SILVER, R. A., COLQUHOUN, D., CULL-CANDY, S. G. & EDMONDS, B. (1994). Mechanisms underlying decay of the fast component of EPSCs in rat cerebellar granule cells. Journal of Physiology 476.P, 67P. SILVER, R. A., CULL-CANDY, S. G. & TAKAHASHI, T. (1996). Non- NMDA glutamate receptor occupancy and open probability at a rat cerebellar synapse with single and multiple release sites. Journal of Physiology (in the Press). SILVER, R. A., TRAYNELIS, S. F. & CULL-CANDY, S. G. (1992). Rapid- time-course miniature and evoked excitatory currents at cerebellar synapses in situ. Nature 355, 163-166. TONG, G. & JAHR, C. E. (1994). Multivesicular release from excitatory synapses of cultured hippocampal . Neuron 12, 51-59. TRAYNELIS, S. F., SILVER, R. A. & CULL-CANDY, S. G. (1993). Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy fiber-granule cell synapse. Neuron 11, 279-289. TRUSSELL, L. O., ZHANG, S. & RAMAN, I. M. (1993). Desensitization of AMPA receptors upon multiquantal neurotransmitter release. Neuron 10, 1185-1196. WAHL, L. M., POUZAT, C. & STRATFORD, K. J. (1996). Monte Carlo simulation of fast excitatory synaptic transmission at a hippo- campal synapse. Journal of Neurophysiology 75, 597-608. WYLLIE, D. J. A, TRAYNELIS, S. F. & CULL-CANDY, S. G. (1993). Evidence for more than one type of non-NMDA receptor in outside- out patches from cerebellar granule cells of the rat. Journal of Physiology 463, 193-226.

Acknowledgements We thank the , the Howard Hughes Medical Institute (International Research Scholars Award to S.G.C.-C.) and the MRC for support, Juliann Thomas for cell culture, David Attwell, Philippe Behe, Beverley Clark, Mark Farrant, Dirk Feldmeyer and Alasdair Gibb for comments on the manuscript, Dirk Feldmeyer for help with some experiments and Stephen Traynelis for software. Author's present address B. Edmonds: Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA. Author's email address R. A. Silver: [email protected]

Received 23 January 1996; accepted 19 March 1996.