Absence of Parallel Fibre to Purkinje Cell LTD During Eyeblink Conditioning

Long-term depression (LTD) of parallel fibre/Purkinje cell synapses has been the favoured explanation for cerebellar motor learning such as classical eyeblink conditioning. Previous evidence against this interpretation has been contested. Here we wanted to test whether a classical conditioning protocol causes LTD. We applied a conditioning protocol, using a train of electrical pulses to the parallel fibres as the conditional stimulus. In order to rule out indirect effects caused by antidromic granule cell activation or output from Purkinje cells that might produce changes in Purkinje cell responsiveness, we focused the analysis on the first pulse in the conditional stimulus, that is, before any indirect effects would have time to occur. Purkinje cells learned to respond with a firing pause to the conditional stimulus. Yet, there was no depression of parallel fibre excitation after training.


Materials and Methods
All experiments were approved by the local Animal Experimentation Ethics Committee of Malmö-Lund, and all experiments were performed in accordance with relevant guidelines and regulations. Twelve male 1 year old ferrets were decerebrated and prepared for stimulation and recording in the cerebellum under anesthesia as previously described 8,17 . The experimental setup is shown in Fig. 1A. All recordings were made in a known blink-controlling area 14 of cortical lobule HVI and each Purkinje cell was identified as controlling the eyelid by short-latency complex spikes in response to electrical periocular stimulation (Fig. 1B). When the CS consists of forelimb or mossy fiber stimulation these Purkinje cells consistently develop typical conditioned responses 14 as shown in Fig. 1C. Here, the CS was a 100 Hz train of electrical stimuli (2-20 μA, 0.1 ms) applied directly to the parallel fibers. The location of parallel fiber stimulating electrodes was confirmed by eliciting simple spikes (Fig. 1D). The US consisted of two five-pulse 500 Hz trains of stimuli (30-400 μA, 0.1 ms) separated by 10 ms applied to ipsilateral climbing fibers. Parallel fibers and climbing fibers were stimulated with platinum tungsten electrodes (pulled and ground tips, 25 μm core diameter).
Two training protocols were used, one with a 300 ms CS, where the US was delivered 150 ms after CS onset and one with a 800 ms CS where the US was delivered 200 ms after CS onset. Using a CS that continues beyond the US serves two purposes. First, in standard protocols where the CS and US co-terminate the end of a Purkinje cell response might merely reflect the termination of the CS as opposed to reflecting learning of the interval between the CS and the US. Second, since each stimulus pulse excites the same population of parallel fibres it is of interest to assess the probability of spike elicitation beyond the CS-US interval where LTD but not conditioned responding would be expected (for references see 7 ). The intertrial interval was 15 +/− 1 s (randomized). For further details concerning recording techniques and analysis, see 8,17 .

Results
Twelve Purkinje cells were trained with a CS that consisted of a 100 Hz train of stimulus pulses to the pfs followed by a US consisting of direct climbing fibre stimulation as described previously 8 and as illustrated in Fig. 1A. To enable analysis of spike probability both before and after the expected US, this is the subset of cells in ref. 8 where the CS continued beyond the US. In 7/12 cells the CS lasted 300 ms and the US was delivered at 150 ms. In 5/12 cells the CS lasted 800 ms and the US was delivered at 200 ms. In every case, this training protocol resulted in a typical Purkinje cell CR, that is a strong suppression of simple spike firing that reaches a maximum just before the expected US onset and ending shortly after the US, even though the CS continued for an additional 150 or 600 ms ( Fig. 2A).
From the CS train, we selected three individual stimuli and determined the probability of a spike response 1-4 ms after each stimulus pulse over 10-20 trials. For comparison, we selected the first stimulus pulse ('early'), the pulse delivered 100 ms before the US ('middle') and the pulse delivered 150 ms after the US ('late').
Although there was some variability between cells, the main results were clear (Fig. 2B-D). The average probability of spike responses of the Purkinje cell to the 'early' and 'late' pf stimuli were virtually identical before

Discussion
The result show that, although the Purkinje cells exhibited clear conditioned pause responses, the conditioning protocol did not cause any measurable depression of the pf/Pc synapses.
It might be suggested that the reason that there was no LTD of the pf/Pc synapses activated by the first pf pulse, was that this set of synapses was activated too far ahead of the climbing fibre US, whereas later pf input, generated by the indirect loops, would be closer in time to the US. This suggestion is contradicted by the LTD literature, according to which the 150 ms CS-US interval employed in our experiments is well within the range for significant LTD 7 . Furthermore, as the CS is a direct train of repetitive stimuli to the same parallel fibers, the set of pf/ Pc synapses activated by the first stimulus pulse would also be activated by all the subsequent pulses, and most of them would therefore be closer in time to the US. A substantial proportion of the synapses should therefore have undergone LTD and some degree of reduced spiking probability should have occurred. The fact that hundreds of CS-US presentations did not cause any measurable decrease at all in the probability of a spike after the first CS pulse, is strong evidence that no LTD occurred.
LTD can remove excitation but it cannot by itself suppress Purkinje cell firing below its resting level. It can only do so in combination with inhibitory input 7,8,13 . The present finding is consistent with recent reports suggesting that the Purkinje cell CR is generated by an intrinsic mechanism that does not involve the inhibitory interneurons 8,12,13 so LTD is not needed.
Although we agree with most investigators that LTD probably has an important role in other forms of motor learning, the present results clearly support the conclusion, that a classical conditioning protocol does not cause any significant LTD and that, therefore, the learning of the Purkinje cell CR in classical conditioning is not due to LTD but involves a different kind of learning mechanisml 7,8,13 .