Volume 96, Issue 1, Pages 73-80.e4 (September 2017) Slow AMPAR Synaptic Transmission Is Determined by Stargazin and Glutamate Transporters  Hsin-Wei Lu, Timothy S. Balmer, Gabriel E. Romero, Laurence O. Trussell  Neuron  Volume 96, Issue 1, Pages 73-80.e4 (September 2017) DOI: 10.1016/j.neuron.2017.08.043 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 AMPARs Mediate a Slow EPSC at Mossy Fiber-UBC Synapse (A) Top: UBC was filled with Alexa 488 to visualize the location of the brush. Bottom: recording diagram: electrode placed nearby the brush to stimulate a mossy fiber. (B) Left: AMPAR antagonist GYKI-53655 (red) blocked both fast and slow EPSCs evoked by 10 stimuli at 100 Hz. Black, control. Right: this stimulus regime increased spike rate during the slow EPSC. (C) Expanded view of (B). Left: fast EPSCs evoked during train stimulation (triangles) depress profoundly. Right: a slow EPSC occurred after train stimulation stops. (D) Short-term depression kinetics of the fast EPSCs can be fit by a two-exponential decay (red). The peak of slow EPSC occurred ∼150 ms after end of stimulation. (E) The slow EPSC begins after stimuli stop, regardless of number of stimuli (10×–40×). Bars indicate duration of 100-Hz stimulation applied to the same cell. (F) Summary of the normalized time to peak from last stimulus of the slow EPSC versus number of stimuli from nine cells. Each symbol represents a different cell. Linear regression fit (red) showed no significant correlation. (G) Application of 5% dextran solution slowed the rise and decay of slow EPSC. Traces are normalized to the peak slow EPSC. (H) Summary of dextran’s effect on decay time constant and rise time of the slow current. Error bars given as ±SEM. Neuron 2017 96, 73-80.e4DOI: (10.1016/j.neuron.2017.08.043) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Transporters Determine Glutamate Time Course (Ai and Aii) Examples of the effect of TBOA on the response to synaptic stimulation. Dashed line indicates holding current in control (0 μM TBOA). TBOA increased the inward holding current, measured immediately before stimulation train (circle). TBOA decreased the steady-state inward current, measured at the end of the train (square) relative to the holding current of the same trace (circle) (i). TBOA increased the time to peak and the decay of the slow EPSC that occurs at the end of the stimulation train (triangle). In 50 μM TBOA (ii), the steady-state current became outward relative to the standing glutamate current. Addition of 50 μM GYKI blocked both standing current and the outward current, indicating that they were due to tonic AMPAR activation and desensitization, respectively. (B) Same traces as in (A), except that they are overlaid with baselines subtracted to show that steady-state current became outward in 50 μM TBOA. (C) TBOA increased the time to peak of the slow EPSC that begins at the offset of the synaptic stimuli. Paired t tests: 0 μM versus 10 μM: p = 7E–5, n = 11; 10 μM versus 50 μM: p = 0.0006, n = 8; 0 μM versus 50 μM: p = 9E–5, n = 8. (D) TBOA increased the decay of the slow EPSC that begins at the offset of the synaptic stimuli (measured from the inward peak to 10% of the baseline. Paired t tests: 0 μM versus 10 μM: p = 0.009, n = 11; 0 μM versus 50 μM: p = 0.018, n = 8. (E) The inward holding current was increased by TBOA and blocked by GYKI. Paired t tests: 0 μM versus 10 μM, p = 0.0003, n = 11; 10 μM versus 50 μM, p = 0.006, n = 8. Illustrated holding current values are relative to the holding current in the previous condition, such that the 10 μM value is relative to the 0 μM, 50 μM is relative to 10 μM, and GYKI is relative to 50 μM TBOA. Paired t test: 50 μM versus GYKI, p = 0.012, n = 6. (F) Change in steady-state current, measured between the 9th and 10th synaptic stimulus, relative to holding current. A positive change in steady state indicates that the current went outward during the stimulus train, as shown in (A) and (B). Paired t tests: 10 μM to 50 μM, p = 0.005, n = 8; 0 μM to 50 μM, p = 0.005, n = 8. Addition of 50 μM GYKI blocked the steady-state current, indicating that the outward current was an AMPAR-mediated current. Paired t test including cells that had an outward steady-state current: 50 μM to GYKI, p = 0.017, n = 6. (G) The first EPSC in the train in (A). (H) The first EPSC in the train peak normalized to illustrate small change in decay rate. (I) TBOA reduces amplitude of the first EPSC in the train. Paired t tests: 10 μM versus 50 μM, p = 0.006, n = 8; 0 μM versus 50 μM, p = 0.008, n = 8. (J) TBOA increases the decay of the first EPSC in the train. Paired t test: 0 μM versus 50 μM: p = 0.037, n = 8. Error bars given as ±SEM. Neuron 2017 96, 73-80.e4DOI: (10.1016/j.neuron.2017.08.043) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 AMPARs Are Desensitized and Recover during Slow EPSC (A) Experimental configuration. UV-laser spot was targeted to the brush to uncage MNI-glutamate after synaptic stimulation. (B) Uncaging at different times (arrowheads) during slow EPSC. Gray trace shows the control uEPSC. Inset: expanded view showing diminished size of uncaging response (∗; black trace) delivered 15 ms after final synaptic stimulation. Blue trace delivered 85 ms later shows recovery even though baseline current is the same as in black trace. (C) Summary of recovery time course shown in (B). Each symbol represents a different cell. The recovery was fit by a single exponential (black line). (D) Steady-state current of the slow EPSC evoked just before the test uncaging pulse plotted against the proportional recovery of test pulse amplitude. Slow EPSC was evoked by synaptic stimulation as in (B). Each color represents a different cell (n = 6). The rising and then falling curves show that the same steady-state current corresponds to two different recovery states, indicating that they were produced by different glutamate levels. (E) Paired-pulse uncaging also showed a desensitized second uEPSC (triangle). Note the undershoot current (arrow) in the second uEPSC. (F) Recovery time course for the second uEPSC in paired uncaging paradigm. Each symbol represents a different cell. Recovery was fit with a single exponential (black line). (G) Steady-state current of the slow current evoked by an uncaging pulse just before a second test uncaging pulse as in (E), plotted against the proportional recovery of test pulse amplitude. Neuron 2017 96, 73-80.e4DOI: (10.1016/j.neuron.2017.08.043) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Stargazer UBCs Showed Reduced Slow EPSC and Monotonic Dose-Response Relation (A) Overlay of EPSCs from a WT (black) and a stg UBC (red). The peak of the fast EPSC is scaled to the same level and each trace represents the median slow/fast ratio of each group. Added filtering was applied to the slow EPSC. Inset: expanded view of slow EPSC. (B) Plot of slow versus fast EPSC amplitudes from 32 WT and 21 stg UBCs. The peak amplitudes of slow, but not fast, EPSC in stg UBCs were significantly smaller compared to WT. (C) A GFP-labeled dissociated UBC with intact dendritic brush. (D) AMPAR-mediated currents evoked by glutamate (gray bar) at the indicated concentrations applied to dissociated WT and stg UBCs. (E and F) Comparison of absolute (E) and normalized (F) amplitudes of steady-state AMPAR currents in WT and stg UBCs. Error bars given as ±SEM. Neuron 2017 96, 73-80.e4DOI: (10.1016/j.neuron.2017.08.043) Copyright © 2017 Elsevier Inc. Terms and Conditions