The role of glial glutamate transporters in maintaining the independent operation of juvenile mouse cerebellar parallel fibre synapses - PubMed (original) (raw)
The role of glial glutamate transporters in maintaining the independent operation of juvenile mouse cerebellar parallel fibre synapses
Paikan Marcaggi et al. J Physiol. 2003.
Abstract
There is controversy over the extent to which glutamate released at one synapse can escape from the synaptic cleft and affect receptors at other synapses nearby, thereby compromising the synapse-specificity of information transmission. Here we show that the glial glutamate transporters GLAST and GLT-1 limit the activation of Purkinje cell AMPA receptors produced by glutamate diffusion between parallel fibre synapses in the cerebellar cortex of juvenile mice. For a single stimulus to the cerebellar molecular layer of wild-type mice, increasing the number of activated parallel fibres prolonged the parallel fibre EPSC, demonstrating an interaction between different synapses. Knocking out GLAST, or blocking GLT-1 in the absence of GLAST, prolonged the EPSC when many parallel fibres were stimulated but not when few were stimulated. When spatially separated parallel fibres were activated by granular layer stimulation, the EPSC prolongation produced by stimulating more fibres or reducing glutamate transport was greatly reduced. Thus, GLAST and GLT-1 curtail the EPSC produced by a single stimulus only when many nearby fibres are simultaneously activated. However when trains of stimuli were applied, even to a small number of parallel fibres, knocking out GLAST or blocking GLT-1 in the absence of GLAST greatly prolonged and enhanced the AMPA receptor-mediated current. These results show that glial cell glutamate transporters allow neighbouring synapses to operate more independently, and control the postsynaptic response to high frequency bursts of action potentials.
Figures
Figure 2. Comparison of stimulus-response data and single bouton properties in wild-type and knock-out cells
A, mean EPSC amplitude as a function of stimulation intensity in 20 wild-type (filled circles) and 15 knock-out (open circles) cells: stimuli were adjusted to produce EPSC amplitudes near 160 pA, then doubled to produce the larger responses. B, paired-pulse facilitation ratio (PPF, for stimuli separated by 5 ms) in cells from 20 wild-type and 15 GLAST knock-out cells (mean EPSC amplitudes were 162 ± 17 and 157 ± 16 pA in wild-type and knock-out cells, respectively). C, ratio of the variance to the mean of the peak of the EPSC in 29 wild-type and 43 GLAST knock-out cells (mean EPSC amplitudes were 671 ± 21 and 653 ± 18 pA in wild-type and knock-out cells, respectively). D, plots of variance versus mean of EPSC amplitudes (normalised to the values in 3 mM [Ca2+]o), with release probability altered by raising or lowering [Ca2+]o in exchange for [Mg2+]o. Experiments for 4 cells from 15-day-old rats (filled squares, shown for comparison with mouse data) and 7 cells in 17-18-day-old GLAST knock-out mice (open circles) employed [Ca2+]o = 2, 2.5, 3 and 3.5 mM, while the experiments for 2 cells in 20-day-old wild-type mice used [Ca2+]o = 2, 3 and 4 mM. Best-fit parabolae shown are fits to eqn (5) and gave the release probabilities quoted in the text.
Figure 7. EPSC prolongation produced by block of GLT-1 is larger when more fibres are stimulated
A, specimen parallel fibre EPSCs of different amplitude evoked by molecular layer stimulation in the same GLAST knock-out cell. B, EPSCs in the same cell and with the same stimulus strength as A, after application of 200 μM DHK. C and D, data in A and B normalised to have the same peak. E, weighted decay time constant for different stimulation intensities, in the absence and presence of DHK, measured as in _A_-D and normalised to the value (4.83 ± 0.44 ms) for an EPSC amplitude near 640 pA in control solution, in 7 knock-out cells. The amplitudes and time constants of the fast and slow exponential components of the EPSC decay (in the order _A_fast, τfast, _A_slow, τslow) were as follows. For low stimulation, in control: 118 ± 12 pA, 3.69 ± 0.19 ms, 3.05 ± 2.07 pA, 22.8 ± 8.1 ms (4 out of 7 cells showed a slow component); in DHK: 76.4 ± 11.0 pA, 3.07 ± 0.41 ms, 20.4 ± 8.9 pA, 25.1 ± 10.6 ms (5 out of 7 cells showed a slow component). For medium stimulation, in control: 539 ± 44 pA, 4.09 ± 0.47 ms, 66.5 ± 35.5 pA, 17.4 ± 6.1 ms (5 out of 7 cells showed a slow component); in DHK: 482 ± 44 pA, 4.37 ± 0.68 ms, 124 ± 35 pA, 36.3 ± 7.9 ms (all cells showed a slow component). For high stimulation, in control: 1060 ± 196 pA, 4.40 ± 0.67 ms, 451 ± 187 pA, 21.4 ± 10.6 ms (6 out of 7 cells showed a slow component); in DHK: 1102 ± 61 pA, 5.91 ± 0.62 ms, 338 ± 52 pA, 50.9 ± 8.7 ms (all cells showed a slow component).
Figure 10. The effect of GLAST knock out, and of blocking GLT-1 with DHK, on the EPSC generated by trains of stimuli to the parallel fibres
A, response of a Purkinje cell in a wild-type slice to 10 stimuli at 200 Hz in the absence and presence of 200 μM DHK. Stimulus intensity was adjusted to produce an initial EPSC of amplitude ≈170 pA (‘low stim’, mean value 168 ± 21 pA in 20 cells). B, as for A, but in a knock-out slice (mean initial EPSC amplitude 160 ± 21 pA in 15 cells). C, same cell as in A but at double the stimulation intensity (‘high stim’), producing an initial EPSC amplitude of ≈1100 pA (mean value 1068 ± 125 pA). D, same cell as in B, but at double the stimulation intensity (mean initial EPSC amplitude 1105 ± 111 pA). E, peak current amplitude evoked by the train, normalised to the amplitude of the first EPSC in the train in control conditions, for data as in _A_-D from 20 wild-type and 15 GLAST knock-out cells. F, time needed for the current evoked by the train to decay to half its amplitude at the end of the train. G, total charge transfer evoked by the train (integrated from the start of the train to 1 s after the train), normalised to the amplitude of the first EPSC in the train in control conditions.
Figure 1. Knocking out glial GLAST transporters prolongs parallel fibre synaptic currents
A, specimen EPSCs at −70 mV evoked in Purkinje cells by parallel fibre stimulation in the molecular layer, adjusted to produce the same EPSC amplitude in a wild-type cell (+/+) and a cell from a mouse with GLAST knocked out (−/−). Stimulus artefacts removed for clarity. Smooth lines through the EPSC decays are fits of eqn (1), with weighted time constants (τw, from eqn (2)) as shown. B, weighted decay time constant as a function of the series resistance remaining after ≈90 % compensation, _R_S, in 43 cells from wild-type mice. Almost-horizontal dotted line is a linear regression through the data. Vertical dotted line is the upper limit of 1MΩ which we set for acceptable values of _R_S. C and D, mean (±
s.e.m
.) values of time from the start of the stimulus to the EPSC peak (C), and τw (D), for EPSCs with amplitudes around 620 pA in 37 wild-type and 47 knock-out cells. The amplitudes and time constants of the fast and slow exponential components of the EPSC decay (in the order _A_fast, τfast, _A_slow, τslow) for the data in D were: in wild-type cells, 542 ± 16 pA, 4.06 ± 0.22 ms, 21.3 ± 12.1 pA, 31.1 ± 11.2 ms (13 out of 37 cells showed a slow component; for the other 24 cells _A_slow was set to 0 and no τslow value was included in the average); and in knock-out cells: 486 ± 14 pA, 4.34 ± 0.19 ms, 59.6 ± 12.0 pA, 19.7 ± 2.8 ms (35 out of 47 cells showed a slow component).
Figure 3. EPSC prolongation by GLAST knock out is larger when more parallel fibres are stimulated
A, EPSCs with different amplitudes (produced by molecular layer stimulation of different intensities) in a specimen wild-type (+/+) cell. B, as A, but for a cell from a GLAST knock-out mouse (−/-). C and D, data in A and B normalised to have the same peak, with values of weighted decay time constant indicated (subscript indicates amplitude of EPSC in pA). E, mean (±
s.e.m
.) weighted decay time constants measured at 3 EPSC amplitudes in each of 9 wild-type and 19 knock-out cells, and rescaled to have the values 1 and 1.18 for 620 pA EPSCs in wild-type and knock-out cells, respectively, to reproduce the relative τw values in the much larger sample of cells in Fig. 1_D_ (the actual ratio of τw values for the −/− and +/+ cells studied here was 1.15). The amplitudes and time constants of the two exponential components from which these data were calculated are included in the data in Fig. 4 legend. EPSC amplitudes could not be set exactly to the same three desired values (approximately 130, 620 and 1670 pA) in each cell; horizontal error bars show the scatter around the mean values. Dotted lines are linear regressions, the slopes of which differ significantly (P = 1.2 × 10−5). Horizontal lines indicate the +/+ data compared for the p values given. _F_-I, the observed variation of τw with EPSC amplitude is not due to changes in series resistance voltage errors and voltage non-uniformity. F, EPSC evoked in a wild-type cell with a strong stimulus in the absence and presence of GYKI 52466 (40 μM), and evoked by a weaker stimulus (grey trace) that produces an amplitude similar to that seen with the strong stimulus in GYKI 52466. G, the data in F normalised to the same peak and fitted with double exponential decays. H, the amplitude reduction produced by reducing the stimulus strength (grey) is similar (P = 0.73) to that produced by GYKI 52466 (black) in 11 wild-type cells. I, a significant fractional reduction of τw is produced by reducing the stimulus strength (grey, P = 0.004 comparing low and high stimulus strength) but not by applying GYKI 52466 (black, P = 0.88 comparing the absence and presence of GYKI) in the same 11 wild-type cells. The amplitudes and time constants of the fast and slow exponential components of the EPSC decay (in the order _A_fast, τfast, _A_slow, τslow) were, for reduced stimulus strength: 173 ± 20 pA, 2.92 ± 0.31 ms, 5.1 ± 2.9 pA, 7.46 ± 1.27 ms (3 out of 11 cells showed a slow component); and for GYKI 52466: 159 ± 17 pA, 3.14 ± 0.31 ms, 16.3 ± 7.7 pA, 25.1 ± 13.9 ms (7 out of 11 cells showed a slow component).
Figure 4. Knocking out GLAST selectively increases charge transfer by the slower decaying component of the EPSC at large EPSC amplitudes
A_-C, charge transfer by the faster exponential component of eqn (1) (left panel), and by the slower exponential component of eqn (1) (right panel), for EPSCs with amplitudes around 130 pA (A), 620 pA (B) and 1670 pA (C) (A and C, same cells as in Fig. 3_E; B, all cells from Figs 3_E_ and 1_D_). The amplitudes and time constants of the fast and slow exponential components of the EPSC decay (in the order _A_fast, τfast, A_slow, τslow) for the cells of Fig. 3_E were as follows. In wild-type cells: for 130 pA EPSCs 117 ± 8 pA, 3.45 ± 0.45 ms, 2.63 ± 1.82 pA, 45.9 ± 20.2 ms (3 out of 9 cells showed a slow component); for 620 pA EPSCs 542 ± 16 pA, 4.06 ± 0.22 ms, 21.3 ± 12.1 pA, 31.1 ± 11.2 ms (13 out of 37 cells showed a slow component); for 1670 pA EPSCs 1352 ± 41 pA, 4.06 ± 0.49 ms, 146 ± 71 pA, 28.8 ± 16.3 ms (6 out of 9 cells showed a slow component). In knock-out cells: for 130 pA EPSCs 114 ± 5 pA, 3.89 ± 0.26 ms, 3.37 ± 1.74 pA, 18.01 ± 5.98 ms (6 out of 19 cells showed a slow component); for 620 pA EPSCs 486 ± 14 pA, 4.34 ± 0.19 ms, 59.6 ± 12.0 pA, 19.2 ± 2.8 ms (35 out of 47 cells showed a slow component); for 1670 pA EPSCs 1125 ± 102 pA, 4.70 ± 0.48 ms, 374.1 ± 98.3 pA, 19.8 ± 4.5 ms (17 out of 19 cells showed a slow component).
Figure 5. Granular layer stimulation evokes briefer parallel fibre EPSCs than molecular layer stimulation in GLAST knock-out mice
A, specimen parallel fibre EPSCs of similar amplitude recorded in the same wild-type cell (+/+) when stimulating the granule cell axons either in the molecular layer (ML) or the granular layer (GL), with weighted decay time constants (τw) indicated. B, as for A but in a GLAST knock-out cell (−/-). C, ratio of τw for GL and ML stimulation in 6 wild-type and 12 knock-out cells. P values are for t tests comparing data with unity. Amplitudes of EPSCs were 653 ± 72 pA for GL and 660 ± 52 pA for ML stimulation in the +/+ cells, and 578 ± 68 pA for GL and 578 ± 70 pA for ML stimulation in the −/− cells (not significantly different: P = 0.94 for +/+ and 0.998 for −/−). D and E, charge transfer by the faster exponential component (D), and by the slower exponential component of eqn (1) (E), for ML and GL stimulation in the 12 knock-out cells. The amplitudes and time constants of the fast and slow exponential components of the EPSC decay (in the order _A_fast, τfast, A_slow, τslow) were: for ML stimulation, 467 ± 58 pA, 3.34 ± 0.41 ms, 47.5 ± 18.1 pA, 9.12 ± 1.90 ms (7 out of 12 cells showed a slow component); and for GL stimulation: 484 ± 55 pA, 2.99 ± 0.20 ms, 19.9 ± 14.1 pA, 14.5 ± 2.8 ms (4 out of 12 cells showed a slow component). F, τw as a function of EPSC amplitude for GL stimulation in 23 knock-out cells (includes some cells stimulated only in the GL layer). This experiment (unlike that of Fig. 3_E) required inclusion of data from different cells because the range of EPSC amplitudes which can be generated by altering the intensity of GL stimulation is much smaller than for ML stimulation.
Figure 6. Block of GLT-1 transporters prolongs parallel fibre EPSCs in the GLAST knock-out
A, parallel fibre EPSCs evoked by molecular layer (ML) stimulation in a wild-type (+/+) Purkinje cell in the absence (control) and presence of 200 μM dihydrokainate (DHK). B, as for A, but in a GLAST knock-out (−/-) cell. Inset shows time at the peak of the EPSCs (vertical dotted lines); smooth black curves through grey data are multi-exponential fits to define the peak better and the thicker grey trace is in DHK. C, as for B, but with stimulation in the granular layer (GL). _D_-F, mean values of EPSC amplitude (D), time from the start of the stimulus to the peak of the EPSC (E), and weighted decay time constant (F) in DHK relative to the value in the same cell in control solution, for 6 wild-type cells stimulated in the ML (control EPSC amplitude was 666 ± 33 pA), 18 knock-out cells stimulated in the ML (control EPSC amplitude was 608 ± 12 pA), and 4 knock-out cells stimulated in the GL (control EPSC amplitude was 555 ± 64 pA, not significantly different from ML stimulation, P = 0.47). P values are for t tests comparing data with unity. The amplitudes and time constants of the fast and slow exponential components of the EPSC decay (in the order _A_fast, τfast, _A_slow, τslow) were as follows. For ML stimulation in wild-type, in control: 597 ± 33 pA, 4.80 ± 0.15, 18.5 ± 15.8 pA, 23.0 ± 10.5 ms (2 out of 6 cells showed a slow component); in DHK: 588 ± 61 pA, 4.87 ± 0.21 ms, 17.3 ± 10.9 pA, 18.3 ± 3.2 ms (2 out of 6 cells showed a slow component). For ML stimulation in knock-out cells, in control: 502 ± 28 pA, 4.54 ± 0.38 ms, 71.0 ± 25.2 pA, 24.4 ± 5.3 ms (16 out of 18 cells showed a slow component); in DHK: 499 ± 21 pA, 5.63 ± 0.59 ms, 93.7 ± 15.7 pA, 43.0 ± 4.4 ms (all 18 cells showed a slow component). For GL stimulation in knock-out cells, in control: 441 ± 115 pA, 2.99 ± 0.48 ms, 46.7 ± 21.9 pA, 8.80 ± 0.74 ms (2 out of 4 cells showed a slow component); in DHK: 502 ± 66 pA, 3.34 ± 0.58 ms, 33.9 ± 22.4 pA, 36.9 ± 13.1 ms (3 out of 4 cells showed a slow component). G and H, charge transfer by the faster (G) and slower (H) exponential components of eqn (1), for ML stimulation in the 18 knock-out cells in the absence (control) and presence of DHK.
Figure 8. Synaptic crosstalk occurs at physiological temperature
A, specimen parallel fibre EPSCs evoked by molecular layer stimulation in a wild-type Purkinje cell at 27 °C and 37 °C. _B_-D, temperature dependence of the time from the start of the stimulus to the peak of the EPSC (B), the EPSC weighted decay time constant (C), and the EPSC amplitude normalised to its value at 27 °C (D), in 10 wild-type Purkinje cells. Mean amplitude of EPSC at 27 °C was 2320 ± 290 pA, and temperature dependence of amplitude did not depend significantly on EPSC amplitude at 27 °C for amplitudes between 1300 and 3400 pA (not shown). E, EPSC weighted decay time constant as a function of EPSC amplitude at 35-37 °C, in 6 GLAST knock-out cells (−/-) in the absence and presence of DHK (200 μM). From D, the amplitudes of EPSC studied here (≈365 and ≈1215 pA) correspond to amplitudes of ≈257 and ≈856 pA at 27 °C. The amplitudes and time constants of the fast and slow exponential components of the EPSC decay (in the order _A_fast, τfast, _A_slow, τslow) were as follows. For low stimulation, in control: 269 ± 54 pA, 2.45 ± 0.44 ms, 51.8 ± 48.8 pA, 9.96 ± 3.90 ms (2 out of 6 cells showed a slow component); in DHK: 307 ± 54 pA, 2.98 ± 0.45 ms, 24.1 ± 8.4 pA, 14.5 ± 2.6 ms (5 out of 6 cells showed a slow component). For high stimulation, in control: 982 ± 165 pA, 2.90 ± 0.45 ms, 142 ± 118 pA, 23.6 ± 5.7 ms (5 out of 6 cells showed a slow component); in DHK: 908 ± 99 pA, 3.87 ± 0.50 ms, 132 ± 29 pA, 34.8 ± 3.5 ms (all cells showed a slow component). F, charge transfer by the faster and slower exponential components of eqn (1), for the data in E at an EPSC amplitude of ≈365 pA. G, as F, but for the data in E with an EPSC amplitude of ≈1215 pA. H and I, in wild-type cells at 36 °C τw varies with EPSC amplitude, and this is not due to changes in series resistance voltage errors and voltage non-uniformity. H, the fraction of EPSC amplitude remaining is not significantly different (P = 0.17) when stimulation strength is reduced (grey) from strong (producing an EPSC amplitude of ≈ 1560 pA) to weak (≈320 pA amplitude), or when GYKI 52466 (40 μM) is applied during strong stimulation (black), in 5 wild-type cells at 36 °C. I, a significant fractional reduction of τw is produced by reducing the stimulus strength (grey, P = 0.004 comparing low and high stimulus strength) but not by applying GYKI 52466 (black, P = 0.75 comparing the absence and presence of GYKI) in the same 5 wild-type cells. The amplitudes and time constants of the fast and slow exponential components of the EPSC decay (in the order _A_fast, τfast, _A_slow, τslow) were: for reduced stimulus strength, 293 ± 49 pA, 1.93 ± 0.08 ms, 1.69 ± 1.69 pA, 22.8 ms (1 out of 5 cells showed a slow component); and for GYKI 52466, 389 ± 36 pA, 2.26 ± 0.09 ms, 1.85 ± 0.84 pA, 29.0 ± 9.4 ms (3 out of 5 cells showed a slow component).
Figure 9. The effect of blocking glutamate transporters with a combination of TBOA, DHK and GLAST knock out
A, parallel fibre EPSCs evoked by molecular layer (ML) stimulation in a wild-type (+/+) Purkinje cell in the absence (control) and presence of 200 μM DHK + 200 μM TBOA. Inset shows time at peak of the EPSCs (vertical dotted lines); smooth black curves through grey data are multi-exponential fits to define the peak better and the thicker trace is in DHK + TBOA. B, As for A, but in a GLAST knock-out (−/-) cell. C, as for B, but with stimulation in the granular layer (GL). _D_-F, mean values of EPSC amplitude (D), time from the start of the stimulus to the peak of the EPSC (E), and weighted decay time constant (F) in DHK + TBOA relative to the value in the same cell in control solution, for 6 wild-type cells stimulated in the ML (control EPSC amplitude was 666 ± 33 pA), 6 knock-out cells stimulated in the ML (control EPSC amplitude was 615 ± 46 pA, P = 0.39 compared with wild-type), and 6 knock-out cells stimulated in the GL (control EPSC amplitude was 483 ± 50 pA, P = 0.08 compared with ML stimulation). P values are for t tests comparing data with unity. G and H, charge transfer by the faster (G) and slower (H) exponential components of eqn (1), in the absence and presence of DHK + TBOA, for the cells of _D_-F (black bars: ML +/+; white bars: ML −/−; shaded bars: GL −/−). P values are for paired t tests comparing data with and without DHK + TBOA. The amplitudes and time constants of the fast and slow exponential components of the EPSC decay (in the order _A_fast, τfast, _A_slow, τslow) were as follows. For ML stimulation in wild-type, in control: 597 ± 33 pA, 4.80 ± 0.15 ms, 18.6 ± 15.8 pA, 23.0 ± 10.5 ms (2 out of 6 cells showed a slow component); in DHK + TBOA: 450 ± 34 pA, 8.92 ± 1.57 ms, 121 ± 26 pA, 42.6 ± 5.6 ms (all cells showed a slow component). For ML stimulation in knock-out, in control: 511 ± 26 pA, 5.20 ± 0.62 ms, 30.3 ± 14.1 pA, 33.6 ± 12.2 ms (all cells showed a slow component); in DHK + TBOA: 377 ± 61 pA, 6.53 ± 1.14 ms, 97.8 ± 11.9 pA, 79.9 ± 21.0 ms (all cells showed a slow component). For GL stimulation in knock-out, in control: 389 ± 77 pA, 2.91 ± 0.32 ms, 34.3 ± 21.3 pA, 17.3 ± 8.4 ms (3 out of 6 cells showed a slow component); in DHK + TBOA: 383 ± 37 pA, 3.43 ± 0.31 ms, 28.4 ± 3.6 pA, 87.6 ± 9.8 ms (all cells showed a slow component).
References
- Arnth-Jensen N, Jabaudon D, Scanziani M. Cooperation between independent hippocampal synapses is controlled by glutamate uptake. Nat Neurosci. 2002;5:325–331. -PubMed
- Asztély F, Erdemli G, Kullmann DM. Extrasynaptic glutamate spillover in the hippocampus: dependence on temperature and the role of active glutamate uptake. Neuron. 1997;18:281–293. -PubMed
- Auger C, Attwell D. Fast removal of synaptic glutamate by postsynaptic transporters. Neuron. 2000;28:547–558. -PubMed
- Barbour B. Synaptic currents evoked in Purkinje cells by stimulating individual granule cells. Neuron. 1993;11:759–769. -PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases