Reversing cerebellar long-term depression

Abstract

The discovery of a postsynaptically expressed form of cerebellar parallel fiber-Purkinje cell long-term potentiation (LTP) raises the question whether this is the long-sought resetting mechanism for long-term depression (LTD). Extracellular monitoring of PC spikes enables stable prolonged recordings of parallel fiber-Purkinje cell synaptic efficacy. LTD, saturated by repeated induction protocols, can be reversed by a single round of postsynaptic LTP or nitric oxide (NO), enabling LTD to be reinduced. Conversely, after postsynaptic LTP has been saturated, one round of LTD permits fresh postsynaptic LTP. By contrast, after saturation of LTD, induction of presynaptic LTP or application of forskolin leaves LTD still saturated. Likewise, presynaptic LTP cannot be reversed by LTD. Therefore postsynaptic LTP mediated by NO without postsynaptic Ca2+ elevation, unlike presynaptic LTP mediated by cAMP, is a true counterbalance to LTD mediated by coincidence of NO plus postsynaptic Ca2+

Fig. 1.

Extracellular spike recordings from PCs and the data analysis process. (Upper) Twenty sweeps are superimposed. The stimulus artifact of PFs stimulation at 50 ms was not counted as an event. The two parallel lines are the upper and lower thresholds. (Inset) Expended time scale shows the details of PC spikes recordings. (Lower) Integrated number of spikes from 20 sweeps during 200 ms. The staircase-like pattern is due to the regularity of spiking as a response to PFs stimulation and was characteristic in many experiments.

Fig. 2.

Stability of extracellular recordings of PC activity. The ability to use this method of extracellular recordings over an extended period was tested by recording the spike probability in response to PF stimulation every 10–20 min over a period of 90 min. The recording showed small changes in number of spikes during 90-min experiments with P > 0.3, indicating that the differences between data sets are not significant (n = 13).

Fig. 3.

Modulation of synaptic efficacy between PF and PC. The extracellular spike probability recording reflects synaptic modulations of PCs. (A) PF stimulation before any training of PCs served as a baseline and was followed by LTD induction by using costimulation of PFs and CF at 1 Hz for 30 s, inducing a significant decrease in spike probability (P = 0.008). Within 2- to 3-Hz LTD inductions there was no more significant reduction in spike probability recordings. (B and C) The same protocol was applied to LTP induction, both 1- and 4-Hz LTP showed a significant increase in spike numbers (P = 0.0004) and were mostly saturated after the second induction. (D) A sample of the spike data recorded before and after 1-Hz LTP induction. In the lowermost graph is shown an integrated number of spikes over 100 ms. The stimulation pulse is at 50 ms.

Fig. 4.

Pharmacologically induced LTP occluded synaptically induced LTP. LTP was pharmacologically induced by application of either an NO donor (NaNONO 10 μM for 5 min) to induce NO-dependent LTP (A) (n = 7) or forskolin (50 μM for 5 min) to induce cAMP-dependent LTP (C) (n = 4). Data were recorded every 10 min after the end of drug application. The saturation of increased PF evoked spiking was slower than the synaptically induced LTP and took at least 1 h to saturate. (B and D) NO or forskolin induced LTP occludes 1- and 4-Hz LTP, respectively (n = 4). The number of spikes evoked by PF stimulation could not be further increased by 1- or 4-Hz induction after pharmacological induction of LTP by NO and forskolin, respectively. (B Insets) Ten sweeps before (Left Inset) and 1 h after (Right Inset) NaNONO application. P values between pretreated and after drug application are indicated in the bar graph.

Fig. 5.

Reversal of LTD by 1- but not 4-Hz LTP. The LTD induction protocol was delivered three times (LTD #1–3) to ensure saturation. Then either 1-(A) (n = 7) or 4-Hz LTP (B) (n = 5) protocols were applied. The next LTD protocol (LTD #4) showed that only 1-Hz (A) and not 4-Hz stimulation (B) reversed the saturation of LTD and allowed renewal again. Subsequent induction of 1-Hz LTP (B) allowed LTD reinduction (LTD #5).

Fig. 6.

Pharmacological reversal of LTD. LTD was reversed by NO-(A)(n = 4) but not cAMP-driven (B)(n = 2) LTP. Triple LTD inductions (LTD #1–3) ensured saturation. Thereafter, either NaNONO (A) (10 μM for 5 min) or forskolin (B) (50 μM for 5 min) were applied, and enhanced spike probability was measured 1 h later. The next attempt to depress the synapse again, LTD #4, showed that only NO (A) and not forskolin reversed the saturated LTD and allowed reinduction. A subsequent 1-Hz LTP protocol (B) but not a 4-Hz LTP protocol (A) allowed a fifth LTD protocol (LTD #5) to depress the synapse significantly.

Fig. 7.

Reversal of 1- but not 4-Hz LTP by LTD. (A) After saturation of 1-Hz LTP by three inductions with 10-min intervals (1 Hz #1–3), an LTD protocol reduced spike probability back to nearly the initial (PF stimulation) value (n = 5). The final 1-Hz LTP protocol (1 Hz #4) resulted in a new increase in spike probability (P < 0.0007), meaning that LTD #1 had desaturated 1-Hz LTP. (B) Saturation of 4-Hz LTP by three inductions (4 Hz #1–3) was not reversed by a subsequent LTD induction (LTD #2), because the fourth round of 4-Hz stimulation (4 Hz #4) failed to show any increase in spike probability (n = 3).