Zones of enhanced glutamate release from climbing fibers in the mammalian cerebellum - PubMed (original) (raw)

Zones of enhanced glutamate release from climbing fibers in the mammalian cerebellum

Martin Paukert et al. J Neurosci. 2010.

Abstract

Purkinje cells in the mammalian cerebellum are remarkably homogeneous in shape and orientation, yet they exhibit regional differences in gene expression. Purkinje cells that express high levels of zebrin II (aldolase C) and the glutamate transporter EAAT4 cluster in parasagittal zones that receive input from distinct groups of climbing fibers (CFs); however, the physiological properties of CFs that target these molecularly distinct Purkinje cells have not been determined. Here we report that CFs that innervate Purkinje cells in zebrin II-immunoreactive (Z(+)) zones release more glutamate per action potential than CFs in Z(-) zones. CF terminals in Z(+) zones had larger pools of release-ready vesicles, exhibited enhanced multivesicular release, and produced larger synaptic glutamate transients. As a result, CF-mediated EPSCs in Purkinje cells decayed more slowly in Z(+) zones, which triggered longer-duration complex spikes containing a greater number of spikelets. The differences in the duration of CF EPSCs between Z(+) and Z(-) zones persisted in EAAT4 knock-out mice, indicating that EAAT4 is not required for maintaining this aspect of CF function. These results indicate that the organization of the cerebellum into discrete longitudinal zones is defined not only by molecular phenotype of Purkinje cells within zones, but also by the physiological properties of CFs that project to these distinct regions. The enhanced release of glutamate from CFs in Z(+) zones may alter the threshold for synaptic plasticity and prolong inhibition of cerebellar output neurons in deep cerebellar nuclei.

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Figures

Figure 1.

Parasagittal zones of Purkinje cells in EAAT4-EGFP mice. A, B, EGFP fluorescence in coronal sections of cerebella from EAAT4-EGFP (A) or wild-type (B) mice. C–F, Sections were immunostained for EAAT4 (C, D) and zebrin II (E, F). The boxed areas in A, C, and E are shown at higher magnification in G–I. Arrows highlight a single Purkinje cell that expressed high levels of EGFP, EAAT4, and zebrin II. Scale bars: A–F, 500 μm; G–I, 50 μm.

Figure 2.

CFs that innervate Z+ Purkinje cells elicit prolonged EPSCs as a result of enhanced glutamate release. A, CF EPSCs from representative Z+ (gray trace) or Z− (black trace) Purkinje cells. Traces at right were normalized to the peak amplitude. Histograms show the peak amplitude and weighted decay of CF EPSCs from Z+ (n = 15) and Z− (n = 14) Purkinje cells (**p < 0.01). B, Representative average CF EPSCs recorded from Z+ (gray trace) and Z− (black trace) Purkinje cells in the presence of 200 μ

cyclothiazide. Responses have been normalized to the peak amplitude. C, Average CF EPSCs recorded in the presence or absence of 2 m

γ-DGG from a Z+ (gray trace) and a Z− (black trace) Purkinje cell.

Figure 3.

Purkinje cells in Z+ zones exhibit larger glutamate transporter currents. A, EAAT4-mediated transporter currents elicited through photolysis of MNI-

-aspartate recorded from Z+ (gray trace) or Z− (black trace) Purkinje cells (_V_m = −70 mV). Arrowheads indicate the onset of 1 ms UV exposure. B, CF-evoked transporter currents recorded from Z+ or Z− Purkinje cells (_V_m = −70 mV). Red traces represent average responses.

Figure 4.

CF synaptic vesicles in Z+ and Z− zones contain a similar amount of glutamate. A, CF-induced mEPSCs recorded in the presence of SrCl2 (_V_m = −70 mV). B, C, Average amplitude (Z+: 17,792 events, n = 10; Z−: 6705 events, n = 9) and decay time (tau decay; Z+: 5520 events, n = 10; Z−: 1731 events, n = 8) of CF-evoked mEPSCs from Z+ and Z− Purkinje cells (mean ± SEM). The inset shows averaged scaled CF mEPSCs from representative Z+ (gray) and Z− (black) Purkinje cells.

Figure 5.

CFs in Z+ zones exhibit enhanced multivesicular release. A, CF EPSCs elicited through paired stimulation in the presence or absence of 1 m

γ-DGG (red trace; ±γ-DGG) from representative Z+ (gray) and Z− (black) Purkinje cells. Responses are normalized to peak of EPSC1. The histogram shows percentage inhibition and weighted decay of EPSC1 for Z+ (n = 11) and Z− (n = 10) Purkinje cells (**p < 0.01; ***p < 0.001). B, CF EPSCs in 1 m

γ-DGG normalized to the first peak in the absence of γ-DGG. C, CF EPSCs recorded as described in A; however, red traces show responses recorded in 120 n

NBQX. The histogram at right shows the inhibition by NBQX and the weighted decay for the first CF EPSC in Z+ (n = 9) and Z− (n = 9) Purkinje cells (mean ± SEM; ***p < 0.001). D, CF EPSCs recorded in the presence or absence of NBQX in C normalized to the peak amplitude of the first response. In all panels, recordings were performed in ACSF containing 0.5 m

CaCl2 and 3.3 m

MgCl2.

Figure 6.

CFs in Z+ zones contain a larger pool of release-competent vesicles. A, Top, Overlay of 10 consecutive EPSCs from representative Z+ (gray traces) and Z− (black traces) Purkinje cells collected at 0.033 Hz in 4 m

extracellular Ca2+ in the presence of 10 m

γ-DGG (to relieve AMPA receptors from saturation). Recordings were made at a holding potential of −40 mV. Bottom, Same traces presented at an enlarged scale. B, Graph of variance versus mean EPSC amplitude calculated from at least 20 consecutive CF EPSCs. CF responses in Z+ zones (red symbols) exhibited larger amplitudes (p = 0.02) and higher variance (p < 0.02) than CF responses in Z− zones (blue symbols), indicating that CFs in Z+ zones contain a larger pool of release-competent vesicles. Squares and error bars represent mean ± SEM.

Figure 7.

CFs in Z+ zones exhibit higher vGluT2 immunoreactivity. A, Native EGFP fluorescence in a coronal section of cerebellum from an EAAT4-EGFP mouse containing Z+ and Z− zones. B, vGluT2 immunoreactivity for the region shown in A, pseudocolored with warmer colors indicating higher pixel intensities. Scale bar, 100 μm. C, Plot of all pixels in Z+ (top) and Z− (bottom) zones from the section shown in A that exceeded a threshold set at 95% of pixel intensities measured within Z+ zones.

Figure 8.

Increased dynamic range of CF-induced complex spikes in Z+ Purkinje cells. A, CF-evoked complex spikes in Purkinje cells contained one to three spikelets (26–33 consecutive trials shown). B, Histograms showing the number of Purkinje cells in Z+ and Z− zones that exhibited complex spikes containing one to three spikelets. C, CF EPSCs with or without TBOA (left) and complex spikes plus TBOA (right) recorded from two Z− Purkinje cells (5 of 34 consecutive responses showed 4 spikelets). D, Histogram showing the number of Z− Purkinje cells that exhibited complex spikes containing one to three spikelets in TBOA (10 μ

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Zones of enhanced glutamate release from climbing fibers in the mammalian cerebellum - PubMed (original) (raw)