Figure 1.
Presynaptic Ca2+ elevation in response to bath application of NMDA in a voltage clamped basket cell.
The cell was voltage clamped at a holding potential of −60 mV in normal extracellular [Mg2+] (1 mM). It was perfused with the Ca2+ dye OGB-5N and part of its axon was imaged using 2-photon Ca2+ imaging. NMDA was added to the bath perfusion during the time indicated by bars (0.5 µM TTX throughout), while imaging a basket terminal belonging to the recorded cell. A: Fluorescence images from 2-photon laser scanning Ca2+ imaging at rest (left) and during the peak of the NMDA-induced current (right). B: Superposition of transmitted light and fluorescence images, showing that the fluorescent structure illustrated in A is a basket cell terminal in contact with a Purkinje cell soma. C: Time course of the NMDA-induced current and of the calcium-dependent fluorescent changes in 2 ROIs of the terminal as indicated by boxes in A, with matching colors in traces and in boxes.
Figure 2.
Wide field uncaging of MNI-glutamate reveals presynaptic NMDARs.
A: MNI-glutamate was uncaged in a wide area of the recording chamber by a UV-flash through the microscope objective (see Methods) in 0.1 mM [Mg2+] and in the presence of both TTX (0.5 µM) and NBQX (5 µM). Fluorescence levels are shown before (a, left) and after (a, right) the flash. b: Reconstruction of the MLI (somatodendritic compartment in black and axon in blue). c: Ca2+ transients recorded before TTX application in response to 4 propagated action potentials in an axonal region (blue, region 5 in a) and in a dendritic region (black, region indicated «den» in a). d: MNI-glutamate uncaging in the presence of TTX and NBQX evoked a NMDAR-mediated current (left bottom trace) and Ca2+ transients in dendrites (left upper trace) as well as in axonal areas 4 and 5. B: Another experiment following the same experimental paradigm. a: Cell morphology. b: Responses to 4 propagated action potentials obtained before TTX application in axonal area 1 and in a dendritic location. c: Global uncaging of MNI-glutamate in TTX elicited Ca2+ transients both in the axonal and in the dendritic compartment (upper blue and black traces respectively), together with an inward current (bottom).
Figure 3.
Activation of presynaptic ionotropic glutamate receptors using local glutamate uncaging.
A: Responses obtained in TTX in the absence of NBQX. a: Fluorescence image of a MLI filled with 40 µM Alexa 488 via the patch pipette. b: Current responses to 0.3 ms-long laser flashes that locally delivered glutamate from its photolabile precursor MNI-glutamate (1 mM) in the soma (1, black trace) and at various axonal locations (blue). c: The size of the actual uncaging spot was measured by drawing a line accross its image in fluorescence and fitting the projection of the spot on the line by a Gaussian curve (AU: fluorescence arbitrary units; full width at half maximum = 1.38 µm). B: A similar experiment performed in the presence of NBQX (5 µM). a: Fluorescence image. b: Current responses to 1 ms-long laser pulses at various locations indicated by numbers on the cell reconstruction. c: Spatial resolution of axonal uncaging using MNI-glutamate in the presence of NBQX. To construct this curve the relative location of the laser and of the preparation was moved in the direction orthogonal to the neurite, in 4 separate experiments. The data were fit to an exponential decay with a space constant of 1.40 µm. C: Average amplitude (left), 20–80% risetime (middle) and half-decay time (right) of the currents evoked in dendrites (den) and axons (ax) by local uncaging of MNI-glutamate in TTX, in the absence (control) or in the presence (NBQX) of NBQX (5 µM). Error bars indicate ± sem; associated numbers indicate numbers of current traces contributing to the mean. All these experiments were performed in low extracellular [Mg2+] (0.1 mM).
Figure 4.
Features of NMDAR-mediated axonal responses using local uncaging.
A: Responses to MNI-glutamate uncaging (arrow; AMPA receptors blocked with 5 µM NBQX; 0.5 µM TTX) at −40 and −70 mV are compared in the same axonal location in low external [Mg2+] (100 µM, upper traces) and in 2 mM Mg2+ (lower traces). B: Separate recording in 200 µM Mg2+, showing weakly voltage-dependent responses. C: Summary results. Numbers next to error bars (± sem) indicate the numbers of cells contributing to the means. D: Addition of AP-V (50 µM; blue) abolishes the uncaging response in the presence of NBQX (5 µM). E: Uncaging experiments using the novel cage MNI-NMDA. Left: Fluorescence view of a MLI filled with Alexa 488 through the patch pipette. Middle: Current traces obtained with focused 1 ms-long laser pulses delivered in the presence of MNI-NMDA (1 mM) at various locations indicated by numbers. Right: Reconstruction of the MLI with somatodendritic compartment in black and axon in blue.
Figure 5.
Axonal Ca2+ signals elicited by local activation of pre-NMDARs.
A: Representative experiment. Top: Fluorescence image (left) and reconstruction (right) of a MLI filled with Alexa 488 (20 µM) and OGB-1 (50 µM). Bottom: Local Ca2+ transients and associated somatic currents obtained in response to axonal (left) and dendritic (right) glutamate release (1 ms laser pulses; 0.9 mM MNI-glutamate; 5 µM NBQX; 0.5 µM TTX). B: Summary data. Left: In responsive axonal spots, the amplitude of Ca2+ transients are correlated to the corresponding somatic current (correlation coefficient R = 0.74). Right: Ca2+ transients elicited in TTX by local glutamate uncaging (Uncaging) had peak amplitudes similar to those obtained in the same axonal spots before TTX application using 4 propagated action potentials (Action potentials). Experiments were carried out in normal extracellular [Mg2+] (1 mM).
Figure 6.
NMDAR activation increases the mIPSC frequency in MLIs.
A: mIPSCs recorded from a representative MLI under control conditions (0.5 µM TTX, left) and in the presence of 30 µM NMDA (right). A modest inward current shift (control level indicated by continuous line on the right) represents activation of somatodendritic NMDARs. Note the marked mIPSC frequency increase. B, C: Similar experiments were performed in the continuous presence of Cd2+ (100 µM; B) or ifenprodil (10 µM, Ifen, C). D: Summary data. The percentage of increase of mIPSC frequency obtained with NMDA is plotted in various conditions: no drug (NMDA), MK801 (50 µM), Cd (Cd2+, 100 µM), Mibe (mibefradil, 10 µM), Da (dantrolene, 10 µM), Cd+Da (Cd2+ and dantrolene), L-NNA ((L)N-nitroarginine, 10 µM), Ifen (ifenprodil, 10 µM) and Zn (Zn2+, 300 nM). E: The presence of dantrolene (10 µM) does not significantly affect the frequency or the amplitude of mIPSCs. All experiments were carried out in normal extracellular [Mg2+] (1 mM).
Figure 7.
NMDA application increases premini frequency in voltage-clamped MLIs.
A: Spontaneous synaptic currents in the presence of TTX (preminis are identified as events having a peak amplitude of <30 pA; there is 1 such event in the upper trace, plus one somatodendritic miniature) and after further bath application of 20 µM NMDA (lower trace: 5 preminis plus 2 minis). The soma is voltage clamped at −60 mV. 2 of the miniature events, one in control and one in NMDA, have their peaks clipped off. B: Plots of 20–80% risetime as a function of peak amplitude in control (2 min duration) and in NMDA (also 2 min), from the same experiment as in A. Conventional miniature currents (minis) appear as a cluster of events with peak amplitudes >30 pA and risetimes <0.7 ms, and presynaptic miniature currents (preminis) appear as a non overlapping cluster of events with peak amplitudes <30 pA and risetimes >0.5 ms. Note that the frequencies of both preminis and minis increase in response to NMDA application. C: Peak amplitude histograms from the data in B (gray: control; open bars: NMDA). D: Dots: summary of 8 experiments (5 with 20 µM NMDA, and 3 with 50 µM NMDA), showing ratios of both premini and mini frequencies in NMDA over control periods. Only 1/8 experiment fails to show an increase in the premini frequency in response to NMDA. Open squares: summary of 4 experiments performed in the presence of NBQX (20 µM), Cd2+ (100 µM) and NMDA (50 µM). Experiments have been carried out in normal extracellular [Mg2+] (1 mM).