Long-term potentiation at cerebellar parallel fiber-Purkinje cell synapses requires presynaptic and postsynaptic signaling cascades

Abstract

Long-term depression (LTD) and long-term potentiation (LTP) at cerebellar parallel fiber-Purkinje cell (PF-PC) synapses play critical roles in motor learning. The 1 Hz stimulation at PF-PC synapses induces a postsynaptically expressed LTP that requires a postsynaptic Ca(2+) transient, phosphatases, and nitric oxide (NO). However, the mechanism underlying 1 Hz PF-LTP remains unclear because none of the known events is related to each other. Here, we demonstrated that 1 Hz PF-LTP requires postsynaptic cytosolic phospholipase A2 α (cPLA2α)/arachidonic acid (AA) signaling and presynaptic endocannabinoid receptors. Using patch-clamp recording in cerebellar slices, we found that 1 Hz PF-LTP was abolished in cPLA2α-knock-out mice. This deficit was effectively rescued by the conjunction of 1 Hz PF stimulation and the local application of AA. 2-Arachidonoylglycerol and the retrograde activation of cannabinoid receptor 1 (CB1R) were also involved in 1 Hz LTP because it was blocked by the hydrolysis of 2-AG or by inhibiting CB1Rs. The amount of NO released was detected using an NO electrode in cultured granule cells and PF terminals. Our results showed that the activation of CB1Rs at PF terminals activated NO synthetase and promoted NO production. The 1 Hz PF-stimuli evoked limited NO, but 100 Hz PF stimulation generated a large amount. Therefore, 1 Hz PF-LTP, distinct from classical postsynaptically expressed plasticity, requires concurrent presynaptic and postsynaptic activity. In addition, NO of sufficient amplitude decides between the weakening and strengthening of PF-PC synapses.

Keywords: Purkinje cell; arachidonic acid; cPLA2α; endocannabinoids; long-term potentiation; parallel fiber.

Figure 1.

The 1 Hz PF-LTP is deficient in cPLA₂α KO mice. A, PF-LTP was induced by PF stimulation at 1 Hz for 5 min. Example EPSCs before (baseline) and after (t = 38 min) stimulation are shown. Both EPSC1 and EPSC2 were potentiated after stimulation. B, Time course of percentage change in EPSC1 amplitude (n = 20). Each data point indicates the average of three successive EPSCs evoked at 0.05 Hz. C, Time course of the PPF ratio (EPSC2/EPSC1) from a subset of the cells shown in B. D, Example EPSCs from one KO cell before (baseline) and after (t = 38 min) stimulation. EPSC amplitude was unaltered by the stimulation. E, Time course of percentage change of EPSC1 amplitude (n = 14). Each data point indicates the average of three EPSCs. F, Time course of the PPF ratio from cells shown in E. G, Time course of percentage change of EPSC1 amplitude when WT cells were locally perfused with 100 μm AACOCF3 before and during stimulation (n = 11), indicating that AACOCF3 blocked the LTP induction (n = 11). H, Time course of the PPF ratio of EPSCs from cells shown in G.

Figure 2.

AA is involved in PF-LTP. A, Example PF-EPSCs from WT cells stimulated for 2.5 min at 1 Hz (stim) or locally perfused with 2 μm AA overlapping the 1 Hz stimulation (stim+AA). Black and gray traces represent EPSCs before (baseline) and after (t = 38 min) the stimulation, respectively. EPSCs were potentiated in the stim+AA group but not in the stim group. B, Time course of percentage changes of EPSC amplitudes in “stim” and “stim+AA” groups. Each data point indicates the average of three EPSCs. C, Time course of the PPF ratio from cells shown in B. D, KO cells were locally perfused with 2 μm AA overlapping the 1 Hz stimulation for 5 min. Example PF-EPSCs before (baseline) and after (t = 38 min) stimulation are shown. E, Time course of percentage change of EPSC amplitude. Each data point indicates the average of three EPSCs. F, Time course of PPF ratio from cells shown in E. G, Time course of percentage change of PF-EPSC amplitude when KO cells were locally perfused with 2 μm AA overlapping 1 Hz stimulation for 5 min. These cells were internally perfused with 10 mm BAPTA. The mean EPSC amplitude at 38 min was 100 ± 4% of baseline (n = 9). p > 0.05 compared with baseline. H, Time course of PPF ratio of EPSCs from cells shown in G. I, Example EPSCs at 0 and 45 min when WT cells were perfused with 2 μm AA. J, Time courses of percentage change of EPSC amplitude (filled circles) and PPF ratio (open circles). Black bar represents the presence of AA.

Figure 3.

PF-LTP requires 2-AG production and CB1R activation. A, Example PF-EPSCs from WT cells that were stimulated for 5 min at 1 Hz. Cells were filled with either MAGL or FAAH. Black and gray traces represent EPSCs before (baseline) and after (t = 38 min) stimulation, respectively. EPSCs were potentiated in the FAAH group but not in the MAGL group. B, Time course of percentage change of EPSC amplitude in the MAGL and FAAH groups. Each data point indicates the average of three consecutive EPSCs. Black bar above represents the presence of MAGL or FAAH in the pipette. C, Time course of PPF ratio from cells shown in B. D, Example EPSCs derived from a WT cell before (baseline) and after (t = 38 min) stimulation with bath application of AM251. Cells were stimulated for 5 min at 1 Hz. E, Time course of percentage change of EPSC amplitude. AM251 was locally applied before and during stimulation. Each data point indicates the average of three consecutive EPSCs. The mean EPSC amplitude at 38 min was 98 ± 6% of baseline (n = 12). p > 0.05 compared with baseline. F, Time course of PPF ratio from EPSCs shown in E.

Figure 4.

NO is required for PF-LTP. A, LTP induction in WT cells perfused with l-NAME or oxymyoglobin (myoglobin) in the bath. Both groups were stimulated for 5 min at 1 Hz. Black and gray traces represent EPSCs before (baseline) and after (t = 38 min) stimulation, respectively. B, Time course of percentage change of EPSCs in l-NAME and oxymyoglobin groups. Each data point indicates the average of three consecutive EPSCs. Black bars represent the duration of application of l-NAME and myoglobin. C, Time course of PPF ratio from cells shown in B. D, The 1 Hz LTP was blocked by the inclusion of oxymyoglobin inside PCs. Representative traces show EPSCs before (baseline) and after (t = 38 min) stimulation. E, Time course of percentage change of EPSCs with internal oxymyoglobin (n = 10). The mean EPSC amplitude at 38 min was 100 ± 5% of baseline (n = 13). p > 0.05 compared with baseline. Each data point indicates the average of three consecutive EPSCs. The bar above represents the inclusion of oxymyoglobin in PCs (internal myoglobin). F, Time course of PPF ratio from cells shown in E. G, Example traces show EPSCs before (baseline) and after (t = 38 min) stimulation. The 1 Hz LTP was normal when 5 μm arginine was included in the recording pipettes. H, Time course of percentage change of EPSCs with internal arginine (n = 9). The mean EPSC amplitude at 38 min was 134 ± 3% of baseline (n = 13). p < 0.01 compared with baseline. Each data point indicates the average of three consecutive EPSCs. Bar indicates the duration of arginine included in PCs (internal arginine). I, Time course of PPF ratio from cells shown in H.

Figure 5.

CB1R activation produces NO in granule cells and at PF terminals. A, DIC image of cultured cerebellar granule cells (DIV7). Scale bar, 10 μm. B, ELISA detection of NOS in cultured granule cells. The NOS activity was calculated as the concentration of nitrite. The percentage changes of NOS activity were 166 ± 18% and 193 ± 20% of the control (Ctrl) when cells were exposed to WIN55 (WIN) for 5 min and 10 min, respectively. C, Schematic illustration showing how NO efflux was detected using the L-shaped NO electrode in cultured granule cells. Chemicals were locally applied onto cells as shown. D, Example traces of NO concentration recorded in the control (Ctrl), 5 μm WIN55 (WIN), and 5 μm WIN55 + 5 μm AM251 (WIN+AM251) groups. Gray arrows indicate the peaks of NO responses in the WIN and WIN+AM251 groups. Right bar graphs represent the averaged peak values in the three groups. E, DIC image illustrating the placement of the PF stimulation pipette, patch pipette, and sharp NO electrode in a cerebellar slice. The clamped cell in the center is a PC. Scale bar, 20 μm. F, Examples of NO trace recorded with 1 Hz stimulation (stim) and 1 Hz stimulation + 5 μm AM251 (stim+AM251). Black arrows indicate the peak of each response. Right bar graphs represent the averaged peak values. *p < 0.05. **p < 0.01.

Figure 6.

NMDAR-mediated NO release from SCs. A, Schematic illustration showing the stimulation protocols at PF-SC synapses and the localization of postsynaptic AMPARs and NMDARs in SCs. PF stimulation (100 Hz or 1 Hz) was applied at PF-SC synapses as high- and low-frequency stimuli, respectively. AMPARs are located in the center, whereas NMDARs are extrasynaptic. B, Representative recordings from one SC in response to 5 (top) and 60 (bottom) PF stimuli given at 1 Hz. Cells were clamped at 40 mV and perfused with GYKI53655 to block AMPAR responses. Time scales: top, 1 s; bottom, 11 s. C, Representative recordings from one SC in response to 5 PF stimuli given at 100 Hz. Cells were clamped at 40 mV and perfused with GYKI53655 to block AMPAR-mediated responses. High-frequency stimulation induced an outward current, which was blocked by the application of 50 μm d-AP5 (bottom). D, DIC image illustrating the recording configuration for NO release from one SC. Glass pipettes for PF stimulation and whole-cell recording in the SC as well as an NO electrode were placed on the slices as shown in the illustration. ML, Molecular layer; GC/PC, granule cells/Purkinje cells. Scale bar, 20 μm. E, Examples of NO recording from one SC evoked by 1 Hz and 100 Hz PF stimulation. Slices were perfused with Mg²⁺-free aCSF containing GYKI53655. The responses for 1 Hz and 100 Hz PF stimulation are shown in the top and bottom as labeled. Time scales: top, 1 s; bottom, 400 ms. The black arrow indicates the peak of NO release. The gray trace in the bottom represents that 50 μm d-AP5 blocked PF stimulation-induced NO release. F, Examples of NO recording when PFs were given the induction for PF-LTD (PF-LTD stim). Slices were perfused with Mg²⁺-free aCSF containing GYKI53655. The shadowed background shows the duration of PF-LTD stimulation. The black and gray traces represent the NO responses with PF-LTD stimulation and PF-LTD stimulation plus d-AP5 (stim+AP5), respectively, showing that d-AP5 blocked most of the NO release. The black arrows indicate the peak of NO release in each condition. G, Averaged peak values of NO efflux under different stimulation conditions. Five pulses at 1 Hz (5 stim at 1 Hz): 0, n = 12. Single pulse at 100 Hz (5 stim at 100 Hz): 4.9 ± 1.3 nm, n = 9. Single pulse at 100 Hz with d-AP5 (5 stim at 100 Hz+AP5): 0.3 ± 0.2 nm, n = 6. PF-LTD stimulation (PF-LTD stim): 17.4 ± 1.5 nm, n = 6. PF-LTD stimulation with d-AP5 (PF-LTD stim+d-AP5): 2.3 ± 0.5 nm, n = 4. For comparison, the released NO in response to 5 min PF stimulation at 1 Hz shown in Figure 5F is replotted (5 min at 1 Hz, gray bar). *p < 0.05. **p < 0.01. H, Example traces of PF-EPSCs before (baseline) and after (t = 38 min) 1 Hz stimulation when slices were perfused with d-AP5. I, Time course of percentage change of EPSC amplitude in response to 5 min stimulation at 1 Hz (stim; n = 10) and the overlapping application of 50 μm d-AP5 with 1 Hz stimulation (stim+AP5). Each data point indicates the average of three successive EPSCs evoked at 0.05 Hz. J, PPF ratios from a subset of the cells shown in I.

Figure 7.

A schematic illustration shows the major signaling molecules involved in the induction of 1 Hz PF-LTP (red lines) and PF-LTD (black lines). Red and black flash signs indicate the stimulations for LTP and LTD, respectively. The stimulation for LTD simultaneously activates postsynaptic AMPARs and mGluRs on PCs and NMDARs on interneurons. Dotted lines indicate the unclear cascades. Bright brown circles represent shared molecules (NO, Ca²⁺, cPLA₂α, and CB1R) in LTP and LTD.