Ca2+ ion permeability and single-channel properties of the metabotropic slow EPSC of rat Purkinje neurons

Abstract

The slow EPSC (sEPSC) of cerebellar parallel fiber --> Purkinje neuron synapses is mediated by metabotropic glutamate receptor type 1 (mGluR1) activation of nonselective cation channels. Here, the channel properties were studied with uniform calibrated photorelease of L-glutamate with ionotropic receptors blocked, allowing isolation of postsynaptic processes, or with parallel fiber stimulation or mGluR1 agonist application. Evoked current and fluorescence from Ca(2+) indicators were recorded. Noise analysis of the mGluR1 current gave a single-channel conductance of 0.6 pS and showed low open probability at maximal mGluR1 activation. Similar small single-channel conductances were obtained with the mGluR1 agonist (S)-dihydroxyphenylglycine, with parallel fiber or climbing fiber stimulation. The mGluR1 current fluctuations were unaffected by potassium channel blockers. Photoreleased L-glutamate triggered a Ca(2+) concentration increase in the distal dendrites with a time course similar to that of the mGluR1 current. The proximal dendritic and somatic Ca(2+) changes were delayed with respect to the current. Ca(2+) channel blockers and the phospholipase Cdelta inhibitor 1-[6-[((17delta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione, which inhibits mGluR1-activated intracellular Ca(2+) release, did not prevent the dendritic Ca(2+) concentration increase. Polyamine naphthylacetyl spermine and cationic adamantanes that block the pore of the channel were used to vary the mGluR1 current over a wide range in each cell but still at maximal mGluR1 activation. The Ca(2+) influx was inhibited in parallel with the current. The results show that the mGluR1-activated current and the sEPSC are attributable to small-conductance, low-open probability Ca(2+)-permeable cation channels that will mediate spine-specific Ca(2+) influx during the parallel fiber sEPSP.

Figure 1.

sEPSC evoked at low PF stimulation intensity, 20 d PN in a transverse slice, stimulation in the granule cell layer, 32°C. A, Current-clamp recording showing a single action potential evoked in PN by 25 μA 100 μsec stimulation at 1 Hz in the granule cell layer. No receptor antagonists are present. Inset, Action potential on a faster time scale. B, Same PN in voltage clamp, –67 mV, with 20 μm NBQX added to the bathing solution. Trains of stimuli at the same intensity as in A evoke an sEPSC with 10 pulses at 25 Hz (top) or 50 Hz (bottom) delivered every 10 sec.

Figure 2.

Contribution of voltage-gated Ca²⁺ channels to the rise of the mGluR1-evoked potential. Membrane potential change (A) and current at –75 mV (B) evoked by photolytic release of 70 μm l-glutamate from NI-caged glutamate in a 20 d PN before and after addition of 50 μm CdCl₂ are shown. NBQX (100 μm), AP-5 (50 μm), bicuculline (10 μm), and TTX (1 μm) are present.

Figure 3.

Intracellular [Ca²⁺] increase associated with the sEPSP. A, Fluorescence image from a PN loaded with 500 μm fluo-4. The external solution contained NBQX (100 μm), AP-5 (50 μm), bicuculline (10 μm), TTX (1 μm), and AGA4A (1 μm). B, Membrane potential change (top trace) and fractional change in calcium fluorescence (ΔF/F; bottom trace) in the square region (distal dendrite) depicted in A evoked by photolytic release of 70 μm l-glutamate from NI-caged glutamate 20 min after the addition of 2.5 μm U-73122. C, Mean and SEM (5 cells) of the peak membrane potential change (left histogram) and the peak calcium fluorescence change (in the distal dendrite; right) in control conditions or 20 min after addition of 2.5 μm U-73122. D, Mean and SEM (9 cells) of the delay (in seconds) between the peak of the membrane potential and the peak of the fractional change in calcium fluorescence in the distal dendrite (>100 μm from the soma), in the proximal dendrite, and in the soma. U-73122 is absent. E, Mean and SEM (6 cells) of the peak membrane potential change (left) and of the peak fluorescence change ΔF/F in the distal dendrite (right) in control conditions or 20 min after addition of 100 μm NA-spermine.

Figure 4.

Quantitative estimation of Ca²⁺ influx during the mGluR1-activated current. A, Fluorescence image from a PN loaded with 1 mm bis-fura-2 recorded with the intensified CCD camera. B, mGluR1 current at –75 mV (top trace) evoked by release of 112 μm l-glutamate and corresponding fluorescence changes, (F_i – F)/F_i (bottom traces a–c), in the regions (a–c) indicated in A. The timing of the flash is indicated by the arrow. (F_i – F)/F_i traces were corrected for the loss of fluorescence attributable to the byproduct release (see Materials and Methods). C, Fluorescence from a PN loaded with 1 mm bis-fura-2 (top) and region of PMT recording in the dendrites (bottom). D, Time course of mGluR1 currents (top traces 1–3) and of the corresponding (F_i – F)/F_i (bottom traces 1–3) recorded with the PMT in the region indicated in C. Recordings were done 0 (1), 9 (2), and 21 (3) min after the addition of 250 μm IEM1460 to the external solution. The arrow indicates the timing of the flash releasing 112 μm l-glutamate. The control solution contained 0.5 mm glutathione, and no correction was made to fluorescence data. E, Plot of the maximum rate of change of total Ca²⁺ D_Ca = (F_i – F)/F_i against the peak mGluR1 current for the PN shown in C. Filled circles are data from D. F, Plot of the maximum rate of change of (F_i – F)/F_i against the peak mGluR1 current for six PNs in which progressive block of the mGluR1 current and of the bis-fura-2 fluorescence signal was obtained either with 250 μm IEM1460 (n=5) or with 300 μm rimantidine (n = 1). Both currents and (F_i – F)/F_i slopes were normalized to the control in each cell. Each PN is represented by a different symbol. G, Maximum rate of change of (F_i – F)/F_i plotted against the peak mGluR1 current for three cells without U-73122. Normalized data; mGluR1 current was progressively blocked with 250 μm IEM1460.

Figure 5.

Fluctuation analysis of the mGluR1 current, Purkinje neuron, 21 d, voltage clamp, –75 mV, 32°C. One hundred micromolar NBQX, 1 μm TTX, and 1 μm AGA4A were present in the external solution. A, Current activated by 56 μm l-glutamate released at the time indicated by the arrow. Low-gain DC 2 kHz and high-gain bandpass records (2–100 Hz, –3 dB, 8-pole Butterworth) are shown. B, Variance calculated in 0.5 sec segments of the bandpass record. C, Variance in 0.5 sec segments plotted against mean current. Linear regression constrained to the baseline variance and current gives an initial slope of –74 fA.

Figure 6.

Fluctuation analysis of the mGluR1 current with K channels and I_H blocked, Purkinje neuron, voltage clamp, –75 mV, 32°C. A, Current activated by 56 μm l-glutamate released at the time indicated by the arrow. A low-gain DC 2 kHz and high-gain bandpass records (2–100 Hz, –3 dB, 8-pole Butterworth) are shown. B, Plot of the variance against mean current in 0.5 sec data segments. Initial slope, –38 fA. C, Same PN as in A and B. Activation of mGluR1 receptors by bath perfusion of 100 μm DHPG during the time indicated by the bar. Low-gain DC and high-gain bandpass records (1–100 Hz, –3 dB) are shown. D, Plot of variance against mean current evoked by DHPG, 1 sec data segments; initial slope, –42 fA.

Figure 7.

Fluctuation analysis of sEPSC evoked by PF stimulation. sEPSC was evoked by 10 pulses at 100 Hz, 60 V, and 100 μsec in the molecular layer of a transverse slice from a 16 d rat cerebellum, –70 mV, 25°C. The external solution contained 10 μm NBQX. The internal solution contained K gluconate with 0.5 mm EGTA and 1 mm N-(2,6-Dimethylphenylcarbamoylmethyl)triethylammonium bromide. A, Mean current DC 2 kHz and high-gain bandpass 5–100 Hz (–3 dB, 8-pole Butterworth) records. B, Plot of variance in 200 msec segments of data against the mean current. The fitted regression line constrained to baseline has a slope of –31 fA.

Figure 8.

Low open probability of maximally activated mGluR1 current. A, Plot of variance against mean current for a 20 d PN activated by 112 μm l-glutamate. The external solution contained 100 μm NBQX, 1 μm TTX, 1 μm AGA4A, 20 μm ZD7288, 10 μm paxilline, and 1 μm apamin. Data are shown without correction (open symbols) and after correction (filled symbols) for the effects of depolarization in the distal dendrites. Dendritic cable and pipette–soma series resistances are 17 and 6 MΩ, respectively. The fitted line has a slope of –31 fA. B, Plot of variance against mean current for a 21 d PN activated by 100 μm DHPG. Solutions are as in A. Data are shown without correction (open symbols) and after correction (filled symbols) for the effects of depolarization in the distal dendrites. Dendritic cable and pipette–soma series resistances are 11.6 and 7.4 MΩ, respectively. The fitted line has a slope of –45 fA.