Ectopic release of glutamate contributes to spillover at parallel fibre synapses in the cerebellum - PubMed (original) (raw)

Ectopic release of glutamate contributes to spillover at parallel fibre synapses in the cerebellum

Saju Balakrishnan et al. J Physiol. 2014.

Abstract

In the rat cerebellar molecular layer, spillover of glutamate between parallel fibre synapses can lead to activation of perisynaptic receptors that mediate short- and long-term plasticity. This effect is greatest when clusters of fibres are stimulated at high frequencies, suggesting that glutamate clearance mechanisms must be overwhelmed before spillover can occur. However, parallel fibres can also release transmitter directly into the extracellular space, from 'ectopic' release sites. Ectopic transmission activates AMPA receptors on the Bergmann glial cell processes that envelop parallel fibre synapses, but the possible contribution of this extrasynaptic release to intersynaptic communication has not been explored. We exploited long-term depression of ectopic transmission, and selective pharmacology, to investigate the impact of these release sites on the time course of Purkinje neuron excitatory postsynaptic currents (EPSCs). Depletion of ectopic release pools by activity-dependent long-term depression decreased EPSC decay time, revealing a 'late' current that is present when fibres are stimulated at low frequencies. This effect was enhanced when glutamate transporters were inhibited, and reduced when extracellular diffusion was impeded. Blockade of N-type Ca(2+) channels inhibited ectopic transmission to Bergmann glia and decreased EPSC decay time. Similarly, perfusion of the Ca(2+) chelator EGTA-AM into the slice progressively eliminated ectopic transmission to glia and decreased EPSC decay time with closely similar time courses. Collectively, this evidence suggests that ectopically released glutamate contributes to spillover transmission, and that ectopic release therefore degrades the spatial precision of synapses that fire infrequently, and may make them more prone to exhibit plasticity.

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Figures

Figure 1

A, representative whole-cell recordings of glial ESCs generated by paired pulse stimulation (10 ms interval) of parallel fibres in transverse cerebellar slices at 0.033 Hz (first panel). Raising stimulation frequency to 0.2 Hz (second panel) and 1 Hz (third panel) for 10 min leads to a progressive depression of ESCs. After returning baseline frequency to 0.033 Hz for 10 min (fourth panel), the persistent depression is evident in comparison with initial recordings (grey trace). B, representative recordings of EPSCs from Purkinje neurons under the same stimulation conditions as in A. Note persistent decrease in decay time. C, aggregate data of EPSC amplitude (Amp) and decay time (Dec) from Purkinje neurons (n = 17) and ESC amplitude in Bergmann glia (BGC; n = 5). Data are mean ± SEM normalized to initial values at 0.033 Hz. *P < 0.0001 (single sample t test). D, plot of change in decay time after recovery against initial ESPC amplitude for all Purkinje neurons (n = 17) with linear regression (dashed line). E, subtraction of ESCs (left panel) and EPSCs (right panel) recorded after recovery from initial recordings at 0.033 Hz shows the current sensitive to depression. Grey traces are from all recorded cells, black traces are mean currents.

Figure 2

A, first panel: representative EPSCs recorded from Purkinje neurons before (grey trace) and after (black trace) addition of 200 μ

TBOA for 10 min, during stimulation at 0.033 Hz. Second panel: raising baseline frequency to 0.2 Hz for 10 min after TBOA treatment decreased both ESPC amplitude and decay time (black trace). Third panel: mean ± SEM aggregate data from n = 7 cells for amplitude (Amp) and decay time (Dec) at 0.033 Hz (grey columns) and 0.2 Hz (black columns) after addition of TBOA, normalized to values before addition of TBOA. *P = 0.009 and 0.001 (paired t test) for change after 0.2 Hz stimulation in mean amplitude and decay time, respectively. B, representative EPSCs recorded from Purkinje neurons before (grey trace) and after (black trace) bath incubation with 2.5% dextran for 5 min. Second panel shows traces normalized to amplitude. Third panel shows mean ± SEM amplitude (Amp) and decay time (Dec) for six cells, normalized to values before addition of dextran. *P = 0.043 and 0.023 (single sample t test) for change in mean amplitude and decay time after dextran, respectively. C, representative EPSCs recorded from Purkinje neurons in slices incubated at 34°C stimulated at 0.033 Hz (grey trace) and 0.2 Hz for 10 min (black trace). Second panel shows traces normalized to amplitude. Third panel shows mean ± SEM amplitude (Amp) and decay time (Dec) for eight cells at 0.2 Hz, normalized to values at 0.033 Hz. *P = 0.034 (single sample t test).

Figure 3

A, representative traces of glial ESCs (BGC, first panel) and Purkinje neuron EPSCs (PN, second panel) before (grey trace) and 10 min after (black trace) addition of 3 μ

of the N-type Ca2+ channel blocker ω-conotoxin GVIA to the bath. B, concentration–response relationship for glial ESC amplitude (open circles; IC50 = 0.25 μ

) and Purkinje neurons EPSC decay time (filled circles; IC50 = 0.28 μ

) against conotoxin concentration. Lines are fits to the Hill equation with slope 1.

Figure 4

A, paired whole cell recordings from anatomically adjacent Bergmann glia (BGC, first panel) and Purkinje neurons (PN, second panel) before (grey traces) and 30 min after (black traces) incubation with 20 μ

EGTA-AM. Stimulus artefacts are blanked for clarity. B, time course of EGTA-AM effect on glial ESC amplitude (filled squares) and Purkinje neuron EPSC amplitude (filled circles) and decay time (open circles) after addition to bath at t = 0. Data are mean ± SEM from four pairs of cells.

Figure 5

A, representative recordings of glial ESCs (BGC, first panel) and Purkinje neuron EPSCs (PN, second panel) before (grey trace) and 30 min after (black trace) addition of the GluA2-deficient AMPAR pore blocker NASP (50 μ

). B, time course of NASP's effect on glial ESCs (squares), Purkinje neuron EPSC amplitude (filled circles) and EPSC decay time (open circles). Data are mean ± SEM from six cells.

Figure 6

A, diagram of electrode positioning for stimulation of closely associated parallel fibres in the molecular layer (ML, left panel), and more distributed parallel fibres from stimulating somata in the granular layer (GL, right panel), during recording from a single Purkinje neuron (grey). B, representative recordings of EPSCs from a single Purkinje neuron when stimulated in the molecular layer (ML, first panel) or the granular layer (GL, second panel). The third panel shows an overlay of the traces (molecular layer in grey). C, representative recordings of EPSCs from a Purkinje neuron stimulated in the granular layer at 0.033 Hz (first panel) and 0.2 Hz (second panel) for 10 min. The third panel shows an overlay of the traces (0.033 Hz in grey). D, mean ± SEM of n = 9 cells for amplitude (left panel) and decay time (right panel) when stimulating in the molecular layer (grey, ML) or granular layer (black, GL). *P = 0.028 (paired t test). E, mean ± SEM of n = 6 cells for amplitude (left panel) and decay time (right panel) when stimulating in the granular layer at 0.033 Hz (grey) and 0.2 Hz (black). No statistically significant differences were observed.

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Ectopic release of glutamate contributes to spillover at parallel fibre synapses in the cerebellum - PubMed (original) (raw)