Monitoring Glutamate Release during LTP with Glial Transporter Currents  Christian Lüscher, Robert C Malenka, Roger A Nicoll  Neuron  Volume 21, Issue 2, Pages 435-441 (August 1998) DOI: 10.1016/S0896-6273(00)80552-8

Figure 1 Whole-Cell Recording of Synaptically Evoked Glutamate Transporter Currents in Hippocampal Glial Cells (A) Visualization of a glial cell (arrow) in stratum radiatum under DIC, with whole-cell recording pipette in place. Notice the dense web of dendrites from adjacent pyramidal neurons (double arrow) and the absence of visible processes of the glial cell. (B) Biocytin-filled glial cell. The cell shows a stellate morphology typical for astrocytes. Scale bars, 10 μm. (C) Voltage-clamp recordings of synaptically evoked responses in hippocampal glial cells. Response showing an initial outward component (reminiscent of an inverted field potential) followed by a large inward current lasting 5–10 s (1). After bath application of kyn (1.5 mM), the fiber component is still visible, followed by a fast inward current and a slow component fast forward current of lesser amplitude (2). This slow component is isolated after additional wash-in of trans-PDC and DHK (300 μM each) (3). The subtraction of this slow component reveals the synaptically evoked glutamate transporter current (2−3). (D) Superimposition of (1), (2), and (3) gives the temporal relationship (for discussion, see text). Scale bar, 5 pA/20 ms. (E) Recordings from glial cells in the stratum lucidum of the CA3 region of the hippocampus in the presence of kyn (1.5 mM). (E1) Even with a short train of stimuli, which is known to facilitate glutamate release from mossy fibers, only a slow component similar to (C3) could be recorded. (E2) This slow transient is not affected by PDC and DHK applied together (300 μM each) (baseline, 1; transporter blockers, 2). Neuron 1998 21, 435-441DOI: (10.1016/S0896-6273(00)80552-8)

Figure 2 Pharmacology of Synaptically Evoked Glutamate Transporter Currents (A) The glutamate transporter blocker trans-PDC (300 μM) blocked the evoked glial current (in presence of kyn) by 39.2% ± 6.2%, and DHK (300 μM) reduced the current by 50.4% ± 4.0%. Applied together, the current was abolished (95.4% ± 2.6%). (B) Modulating transmitter release by adenosine (Adeno) (10 μM) and its antagonist CPT (50 μM in the presence of 5 μM adenosine) decreases the response by 62.5% ± 7.9% or increases it by 55.5% ± 6.9%, respectively. L-AP4, a group 3 mGluR agonist and selective blocker of Schaffer collateral synapses onto interneurons, leaves the evoked glial transporter current unaltered (99.9% ± 2.6% of control), indicating that this group of synapses does not contribute in a significant manner to the response measured. Increasing stimulus strength (33%) (High stim) increases the size of the response. Note that the changes in glial transporter amplitude are of the same magnitude for all manipulations as the changes observed in the extracellular field response after the washout of kyn (n = 5 for each agonist, n = 4 for High stim, p > 0.2 for all manipulations, t test). Neuron 1998 21, 435-441DOI: (10.1016/S0896-6273(00)80552-8)

Figure 3 Correlation of PPF Ratio in Glial Recordings and Field Recordings (A) Overlay of synaptically evoked glutamate transporter currents at various interpulse intervals. Scale bar, 10 pA/100 ms. (B) Extracellular field recordings obtained in the same slice at identical interpulse intervals after washing out kyn. Scale bar, 0.1 mV/100 ms. (C) The graph shows all individual points from all experiments (one symbol per experiment, two points are off scale n = 7), as well as the regression line y = 1.028x −1.631, r = 0.87 and the 95% confidence interval for the slope and intercept. The arrow pointing to the x-axis indicates the cut-off point for detecting with 95% confidence an increase in the mean glial response. Neuron 1998 21, 435-441DOI: (10.1016/S0896-6273(00)80552-8)

Figure 4 Induction of LTP in the Presence of CNQX (A) A diagram of the experimental setup (for explanation, see text). (B) Grouped data from a set of field experiments with brief application of CNQX and washout immediately after the tetanization (arrow). Under these conditions, we were able to obtain substantial LTP in S1 and complete recovery of the control path S2. Insets show representative traces from one experiment. Scale bar, 0.2 mV/20 ms, n = 5. (C) Control experiment in which the inducing stimulation protocol was applied under 100 μM D-APV. The two pathways wash out at the same rate. Note also that the envelope of extracellular field response during the tetanus is blocked (inset; scale bars, 0.2 mV/20 ms), compared with the envelope in (D). (D) Simultaneous field recording of two pathways with long application of CNQX. After CNQX (10 μM) had blocked the responses, a series of tetani were applied to one pathway (S1, see arrow). Note that the tetanized pathway recovers more rapidly following the removal of CNQX, but recovery is incomplete, owing to the long application of the drug. Neuron 1998 21, 435-441DOI: (10.1016/S0896-6273(00)80552-8)

Figure 5 PTP, but Not LTP, of Synaptically Evoked Glial Currents (A) PTP of glutamate transporter current evoked by tetanic stimulation (4 × 100 stimuli at 50/100 Hz, as indicated by arrows) given in the experiment shown in Figure 4D. Representative averaged traces before and immediately after tetanus and 3 min after induction are shown above the graph. The control path (open symbols) does not show any change, indicating the independence of the two inputs. Scale bar, 2 pA/20 ms. (B) Group data for seven cells showing a mean PTP of 207% ± 21% that decays within 2 min to baseline (100% ± 4%). Open symbols indicate control pathway. Neuron 1998 21, 435-441DOI: (10.1016/S0896-6273(00)80552-8)

Figure 6 Constant Transmitter Release with LTP, as Monitored with Whole-Cell Recording of Synaptically Evoked Glutamate Transporter Currents (A) Slope measurement of extracellular field potential recordings in CA1. (B) Simultaneous whole-cell voltage-clamp recording of a hippocampal glial cell in stratum radiatum of CA1. The experiment is started in the ionotropic glutamate receptor blocker kyn (1.5 mM) to isolate the synaptically evoked glutamate transporter current responses in the two pathways (S1 and S2). Kyn is then washed out and a baseline for the field is obtained. LTP is induced by four tetani (100 Hz, 1 s). Ten minutes later, kyn is reapplied and the glutamate transporter currents are again monitored. (C) To compensate for the slow rundown of the responses, the currents are expressed as a ratio between the two pathways (S1/ S2). This ratio did not change during LTP. (D) Representative traces of the extracellular field recording and glial cell responses corresponding to the time points indicated in (A) and (B). Scale bars for field recordings, 0.2 mV/10 ms; for glial recordings, 5 pA/20 ms. Neuron 1998 21, 435-441DOI: (10.1016/S0896-6273(00)80552-8)

Figure 7 Summary of Experiments Monitoring Glutamate Transporter Current during LTP (A) Group field potential data showing the average of six experiments. (B) The synaptically evoked glutamate transporter currents as expressed by the ratio S1/S2 remain unchanged (92% ± 7% of baseline value, p > 0.2). (C) Normalized PPF ratios of the glial response before and after the induction of LTP. Each experiment is represented by one symbol. The bar graphs represent mean and SEM n = 6. The ratio does not change, indicating that the sensitivity of the assay is maintained. Neuron 1998 21, 435-441DOI: (10.1016/S0896-6273(00)80552-8)

Figure 8 Summary Diagram of All Experiments Figure shows the correlation between field response and glial response for the manipulations described in the text. Manipulations known to affect release probability (adenosine, CPT, PPF, and PTP) and number of release sites (increased stimulation strength) fall onto the line of identity. In contrast, the two experiments in which LTP was induced (see Figures 4, 5, 6) show an unchanged glial response. Neuron 1998 21, 435-441DOI: (10.1016/S0896-6273(00)80552-8)