The expression of cerebellar LTD in culture is not associated with changes in AMPA-receptor kinetics, agonist affinity, or unitary conductance

Abstract

Cerebellar long-term synaptic depression (LTD) is a model system of neuronal information storage that is expressed postsynaptically as a functional down-regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. What properties of postsynaptic AMPA receptors are changed? Several lines of evidence argue against changes in AMPA-receptor kinetics. Neither LTD evoked in cultured granule-cell Purkinje cell (PC) pairs nor an LTD-like phenomenon evoked by phorbol ester application was associated with alterations in evoked AMPA receptor-mediated excitatory post-synaptic current (EPSC) or mEPSC kinetics. LTD produced by pairing glutamate pulses with depolarization was not altered by prior application of the desensitization-reducing compound cyclothiazide. Finally, rapid application of glutamate to lifted PCs revealed no significant alterations in AMPA-receptor kinetic properties after LTD induction. When this system was used to apply varying concentrations of glutamate, no alteration in AMPA-receptor glutamate affinity was seen after LTD induction. Finally, peak-scaled nonstationary fluctuation analysis was applied to estimate AMPA-receptor unitary conductance before and after LTD induction in a cultured cell pair, and this analysis too revealed no significant change. These results suggest that cerebellar LTD may be expressed solely as a reduction in the number of functional AMPA receptors in the postsynaptic density [Wang, Y.-T. & Linden, D. J. (2000) Neuron 25, 635-664].

Figure 1

LTD in cultured PCs is not associated with changes in the shape of either granule cell-evoked or miniature AMPA-EPSCs. (A) Evoked AMPA-EPSCs from a representative granule cell–PC pair (bathed in 0.1 mM D-AP5). Each trace is the average of 25 consecutive responses recorded starting 7 min pre- and 15 min postLTD. (Bars = 10 msec, 10 pA.) (B) Normalized kinetic measures from populations of cells that received either LTD induced by pairing granule-cell stimulation with depolarization (measured with granule cell-evoked EPSCs, n = 6) or an LTD-like phenomenon induced by bath application of phorbol-12, 13-diacetate (PDA; 0.2 μM at t = 0–15 min; measured with mEPSCs recorded in TTX, n = 5) or an LTD-like phenomenon induced by bath application of 200 μM glutamate plus 20 mM KCl (at t = 0–10 min; also measured with mEPSCs, n = 5)

Figure 2

Manipulations designed to alter AMPA-receptor desensitization have no effect on the expression of cerebellar LTD. (A) Cyclothiazide, a blocker of AMPA-receptor desensitization, does not affect subsequent LTD. Application of cyclothiazide starting at t = 0 min (0.1 mM, indicated by thin horizontal line above scale) produces a large potentiation of glutamate-evoked AMPA currents. After reduction in the duration of the iontophoretic test pulse to match baseline current amplitude (indicated by the downward arrow near t = 10 min), LTD was induced by glutamate depolarization conjunction (heavy horizontal bar at t = 15 min). (Inset) Single glutamate-evoked inward currents recorded in a representative PC at the times indicated. (Bars = 1 sec, 50 pA.) (B) The complementary experiment to that shown in A. Prior LTD induction (followed by software renormalization, indicated by upward arrow) does not affect the response to subsequent application of 0.1 mM cyclothiazide. (C) Alternating test pulses of glutamate and kainate (which is a weakly desensitizing AMPA-receptor agonist) were delivered to the same site on cultured PC dendrites before and after induction of LTD by glutamate/depolarization conjunction (heavy horizontal bar at t = 0 min). (Inset) Single glutamate- and kainate-evoked inward currents recorded in a representative PC at the times indicated. (Bars = 1 sec, 30 pA.)

Figure 3

Rapid delivery of glutamate with a fast-flow perfusion system fails to reveal changes in AMPA-receptor kinetics in lifted, cultured PCs. (A) Responses to 1.5-msec pulses of 1 mM glutamate (indicated by heavy vertical bars) in a representative PC were measured by using perforated-patch recording. Each trace is the mean of five consecutive responses. (Bars = 20 msec, 300 pA.) (B) Normalized kinetic measures from populations of cells that received LTD induction by glutamate/depolarization conjunction reveal no significant difference in AMPA currents evoked pre- or postLTD. A population of 10 PCs was divided into two groups of 5, which received paired-test pulses at intervals of either 50 or 150 msec. The 10–90% rise time and 50% decay time measurements are from pooled data, whereas the paired-pulse depression (PPD) measurement is shown separately for each group.

Figure 4

A dose-response function shows no change in AMPA-receptor glutamate affinity after induction of LTD. (A) Representative single responses to doses of glutamate in a lifted, cultured PC measured −20–0 min before and 10–30 min after induction of LTD by glutamate/depolarization conjunction with perforated-patch recording. Cyclothiazide (0.1 mM) was present throughout the experiment to block AMPA-receptor desensitization. (Bars = 25 msec, 300 pA.) (B) A dose-response function relating mean glutamate-evoked current amplitude to glutamate concentration in seven PCs, before and after LTD induction. Current amplitudes have been normalized to the maximum response in each group (pre- vs. postLTD).

Figure 5

Peak-scaled nonstationary fluctuation analysis fails to show changes in γ after induction of LTD in cultured granule cell–PC pairs. (A) LTD was induced by granule cell-depolarization conjunction (at t = 0 min, indicated by the heavy horizontal bar). (Inset) Averages of 40 granule cell-evoked EPSCs recorded in a representative PC at the times indicated. The light horizontal lines show the periods in which evoked EPSCs were collected for subsequent peak-scaled nonstationary fluctuation analysis. (Bars = 10 msec, 10 pA.) (B) Peak-scaled nonstationary fluctuation analysis: a current–variance relationship for a representative granule cell–PC pair. The variance in the fluctuation of the decays of individual EPSCs recorded either before or after LTD induction is plotted as a function of the mean EPSC-decay amplitude. The dashed line indicates the background variance; the solid lines are parabolic least-squares fits for each group of points. (C) Estimates of unitary conductance (γ) based upon parabolic fits for 10 cells in which LTD was induced and 6 cells exposed to a submaximal dose (0.1 μM) of the AMPA-receptor antagonist NBQX. Horizontal lines connect the pre- and postdata points for individual cells.