Repetitive applications of NMDA promote the facilitatory effect on mEPSC frequency.

<p>A. Current traces showing mEPSCs before (left) and after (right) a second application of NMDA. B (same cell as in A). Histogram representing the number of mEPSCs as a function of recording time (bin 10 s). During the short horizontal bars NMDA is applied at 10⁻⁵ M (bars 1 and 2) and at 5×10⁻⁶ M. (bar 3). C. Cumulative plots of inter-event intervals in control conditions (black curve) in the presence of NMDA after the first application (1, red curves) and second application (2, blue curve).</p

The effect of NMDA is specific for mEPSC.

<p>A. Histogram represents the number of mEPSCs as the function of the recording time (bin 10 s), NMDA is applied as indicated by the horizontal bar. Current traces represent average of events selected in control conditions between 0 and 50 sec (right), and at the peak of the increase of frequency induced by NMDA between 180 and 220 sec. Note that the two average currents display the same kinetic properties that fit with mEPSC (the trace on the left is more noisy because the average is constructed with few events). B. Left: mEPSCs recorded in control conditions (top trace) and after NMDA (bottom trace). Traces on the right (same cell), current trace in the presence of NBQX (top trace, mEPSCs are blocked) and after co-application of NMDA (bottom trace, no further effects of NMDA are detected).</p

The effect of NMDA on mEPSC frequency is seen in juvenile but not adult mice.

<p>A. Histogram representing the number of mEPSCs recorded in a Purkinje cell from a slice prepared from a P40 mouse as a function of recording time (bin 10 s). During the horizontal bars NMDA is applied at 5×10⁻⁴ Note the absence of NMDA effect on mEPSC frequency. B. The effect of NMDA on mEPSC mean frequency recorded in Purkinje cell from acute slices prepared with juvenile (left, 7 cells) and adult mice (right, 5 cells). A representation with box plots indicating the mean values (thick line) and median values (thin line) has been adopted because of the non-normal distribution of frequency values. The bottom and the top of the box represent the 25^th and 75 th percentile respectively.</p

Summary of NMDA effects on mEPSCs recorded in Purkinje cells recorded from slice cultures.

<p>Graph on the left represents % Purkinje cells in which an NMDA-mediated increase in mEPSC frequency is observed as a function of Purkinje cell age. Graphs in middle and on the right represent respectively the mEPSC mean frequency and mean amplitude before and after the first application NMDA recorded in Purkinje cells from 12–22 day old slice cultures. A representation with box plots indicating the mean values (thick line) and median values (thin line) has been adopted because of the non-normal distribution of mean frequency and mean amplitude values. The bottom and the top of the box represent the 25^th and 75 th percentile respectively. The ends of the whiskers represent the 5^th/95^th percentile.</p

NMDA binds a NMDA receptor and increases mEPSC frequency.

<p>A. The NMDA effect on mEPSC frequency is blocked in the presence of external Mg²⁺. Histogram on the left represents the number of mEPSCs as a function of recording time (bin 10 s), NMDA is applied as indicated by the two short horizontal bars, in the absence or presence of external Mg²⁺ (as indicated by the two horizontal long bars). Right: cumulative plot of the inter-event intervals when NMDA is applied in the absence (red curve) or presence (black curve) of Mg²⁺. B and C. The facilitatory effet of NMDA on mEPSC frequency is blocked by two NMDA receptor antagonists, MK801 and AP5 respectively. Left, histograms represent the number of mEPSCs as a function of recording time (bin 10 s). NMDA and NMDA receptor antagonists are applied as indicated by the horizontal bars. Right, cumulative plots of inter-event intervals in the presence of NMDA alone (red curves), or in the presence of receptor antagonists (black curves). Note that in the presence of Mg²⁺, MK801 and AP5, the distributions of inter-event intervals are shifted towards longer intervals.</p

NMDA increases the frequency of mEPSCs in Purkinje cells recorded in acute slices prepared from P15–16 mice.

<p>A, B. Upper panels illustrate current traces recorded at −60 mV in the presence of TTX and Gabazine before (left) and after NMDA application (right). Lower panels show histograms representing the number of mEPSC as a function of recording time (bin 10 s). NMDA 10⁻⁴ M is applied as indicated by the horizontal bars. Note in A that the NMDA effect on mEPSC frequency is observed after the third application of NMDA, whereas in B the response is observed after a long-lasting (10 minute) application of NMDA.</p

NMDA increases the frequency of mEPSCs in Purkinje cells recorded in a young (P18) slice culture.

<p>Upper record: currents traces obtained at −40 mV in the presence of TTX and Gabazine before (left) and after NMDA application (right). Transient inward currents represent mEPSCs. Lower graphs: (same cell as in A) cumulative probability plots of inter-event intervals (left) under control conditions (black trace) and in the presence of NMDA (red trace); amplitude histogram distribution under control conditions (middle) and in the presence of NMDA (right). In both cases the amplitude distribution was fitted using a gaussian function with a peak value indicated on each graph. Note that in the presence of NMDA the distribution of inter-event intervals is shifted towards shorter intervals whereas the amplitude distribution is almost unchanged.</p

A postsynaptic response to NMDA in old Purkinje cells.

<p>A. NMDA-induced inward current recorded in a 45 day old Purkinje cell in the presence of the AMPA receptor antagonist NBQX. B. (left) represents the number of Purkinje cells responding to NMDA with an inward current as a function of their age. B (right) mean amplitude of the NMDA inward current (10⁻⁵ M, for 30 s). C, single NMDA channel recordings in outside-out patches, left: current traces illustrating the NMDA channels, middle: I/V plots obtained with two outside-out giving a conductance close to 45 pS, right: voltage dependent blockade of the NMDA channels.</p

ET depolarizes granule cells in cultured slices.

<p>A, Schematic representation of the recording configuration (Whole Cell). B–E, typical membrane potential changes recorded in granule cells (using the Current Clamp mode) adjusted at −60 mV, after application of 10⁻⁷ M ET but without (B, <i>n</i> = 15) or after pre-treatment for 10 min with (C, <i>n</i> = 6) Bicuculline (Bicu, 10⁻⁵ M), (D, <i>n</i> = 7) CNQX (10⁻⁵ M) or, (E, <i>n</i> = 8) a cocktail of Bicuculline (10⁻⁵ M), CNQX (10⁻⁵ M) and TTX (10⁻⁶ M). F, quantification of the delay and amplitude of the depolarization induced by ET. For the corresponding <i>n</i>, see above. All comparisons <i>vs</i> ET alone are <i>n.s.</i> G, Changes in membrane resistance of the granule cells before (white bar) and 5 min after 10⁻⁷ M ET (black bar) (<i>n</i> = 15, <i>p<</i>0.001). Same scale for all voltage traces.</p

ET stimulates excitatory and inhibitory synaptic transmission onto the Purkinje cells.

<p>A, right: spontaneous PSC detected in voltage-clamped Purkinje cells maintained at −60 mV, in absence (Cont) or 5 min after ET (10⁻⁷ M) application. The relative mean frequencies (Freq) and amplitudes (Ampl) of spontaneous EPSC (upper graph) or IPSC (lower graph), before (white bar) or 5–7 min after 10⁻⁷ M ET was added (black bar), <i>n</i> = 15 distinct experiments. B–D, same kind of measurements but after pre-treatment (B) with TTX (10⁻⁶ M for 10 min, <i>n</i> = 18, (C) with bicuculline (10⁻⁵ M for 5 min) to block the IPSC (<i>n</i> = 17), or (D) CNQX (10⁻⁵ M for 5 min) to block the EPSC (<i>n</i> = 18). The frequencies and amplitudes are presented as percent of control condition (i.e. without any treatment, white bars) or after pre-treatment (grey bars), and after subsequent application of ET (black bars). **: <i>p</i><0.01, *: <i>p</i><0.05, otherwise <i>n.s.</i> Same scale for all current traces.</p