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Impact of parallel fiber to Purkinje cell long-term depression is unmasked in absence of inhibitory input

Simultaneous impairment of Purkinje cell inhibitory input and parallel fiber LTD severely affects cerebellar learning.


INTRODUCTION
During eyeblink conditioning, subjects typically hear a short beep or see a light [conditional stimulus (CS)] followed several hundred milliseconds later by an air puff on the eye [unconditional stimulus (US)]. As a result of repeated CS-US pairings, subjects will eventually learn to close their eye in response to the CS, which is called the conditioned response (CR). The CR is not simply a static reflex, but instead an acquired, precisely timed eyelid movement, the kinetic profile of which depends on the temporal interval between the CS and the US (Fig. 1A) [for review, see (1,2)]. During Pavlovian eyeblink conditioning, memory formation takes place in Purkinje cells (PCs) of defined areas of the cerebellar cortex (3)(4)(5)(6)(7)(8). These PCs receive inputs from the mossy fiber-parallel fiber (PF) system, which conveys sensory CS signals and input from single climbing fibers (CFs), which transmit the instructive US signal (Fig. 1B). During the conditioning process, these PCs acquire a well-timed suppression of their simple spike firing in response to the CS (6,9,10), thereby temporarily disinhibiting the cerebellar nuclei, which drive the overt eyeblink CR.
How can PCs time their simple spike suppression with such millisecond precision? Most models on eyeblink conditioning assume a critical role for long-term depression (LTD) of the PF-PC synapse (11,12). However, PCs show high firing frequencies that are intrinsically generated, even when the PF input is silenced (13)(14)(15), and animal models lacking PF-PC LTD can have completely normal eyeblink conditioning (16,17). A second contribution, which, in principle, may actively suppress the intrinsic PC simple spike activity, could be provided by molecular layer interneurons (MLIs), since they provide the PC with a powerful feed-forward inhibition (FFI) (5,18). However, removal of MLI-PC FFI induces compensation at the PF-PC synapse (19) and only mildly impairs eyeblink conditioning (6,20).
Here, we asked this question: To what extent does a concurrent disruption of PF-PC LTD and MLI-PC FFI affect Pavlovian eyeblink conditioning? To this end, we developed a mouse line in which we aimed to block both synaptic processes simultaneously (Fig. 1C). We generated GluR27-L7-2 mice by crossing GluR27 knock-in (KI) mice with L7-2 knockout (KO) mice. GluR27 KI mice have impaired PF-PC LTD, because they lack the last seven amino acids at the intracellular C-terminal tail, which hampers the interaction of GluR2 with PICK1 (protein interacting with C kinase 1) and GRIP1/2 (glutamate receptor interacting protein 1 and 2), and thereby the internalization of the AMPA receptor (21). L7-2 KO mice have impaired MLI-PC FFI, as they have a PC-specific ablation of the 2 subunit of -aminobutyric acid type A (GABA A ) receptors (Fig. 1C), (6,19).

RESULTS
We first established in vitro that GluR27-L7-2 mice lack both PF-PC LTD and MLI-PC FFI. For PF-PC LTD induction, we used 100-Hz PF stimulation in conjunction with a single CF stimulation ( Fig. 1D) (22) at an interstimulus interval of 110 ms, which is an interval that closely mimics those used during eyeblink conditioning (23,24). We were unable to induce postsynaptic PF-PC LTD in GluR27-L7-2 mice, just like in the GluR27 mice [ Fig. 1E and table S1; see also (17)], whereas LTD could be readily induced in controls and L7-2 mice [ Fig. 1E and table S1; see also (19)]. We found that our LTD protocol briefly induced an initial LTD-like pattern (~2 min after LTD induction) in GluR27-L7-2 mice (Fig. 1D), which is in line with Piochon et al. (22); they show the same phenomenon using this protocol in patDp/+ mice with similar PF-PC LTD impairments. Most probably, this is due to the highfrequency (100 Hz) PF stimulation. There were no signs of presynaptic neurotransmitter release abnormalities at the PF-PC synapse in GluR27-L7-2 mice, in that paired-pulse facilitation was unaffected following PF stimulation at intervals varying from 50 to 200 ms ( Fig. 1F and table S1). To evaluate the inhibitory MLI to PC input, we measured the spontaneous inhibitory postsynaptic currents (sIPSCs) of PCs. We found that these sIPSCs were virtually absent in GluR27-L7-2 mice ( Fig. 1G and table S1), similar to what has been reported for L7-2 mice (19). Thus, GluR27-L7-2 mice show both impaired PF-PC LTD and MLI-PC FFI.
Next, we evaluated performance in eyeblink conditioning. The GluR27-L7-2, GluR27, L7-2, and control mice all showed a significant increase in CR percentage and fraction eyelid closure [FEC, ranging from 0 (fully open) to 1 (fully closed)] over the course of 10 acquisition sessions (Fig. 2, A and B, and table S2). However, compared to the other three groups, GluR27-L7-2 mice showed severely affected acquisition, in terms of both CR percentage and FEC (Fig. 2, A and B, and table S2). Nevertheless, CR timing, that is, latency to CR onset and CR peak time, was comparable to control mice: CRs initially had no preferred peak time in the CS-US interval (session 1), but after 10 days of training, it peaked around the onset of the expected US (session 10) (Fig. 2C). The smaller FEC but normal CR timing was even more obvious in the CS-only trials presented in the probe session (session 11) ( Fig. 3 and table S2).
In line with previous findings (17), GluR27 mice, lacking only PF-PC LTD, showed normal acquisition of eyelid CRs over the course of 10 days of training. Neither the percentage, nor FEC, nor timing of eyeblink CRs showed any abnormalities compared to control litters (Figs. 2 and 3 and table S2). The L7-2 mice, which lack MLI-PC inhibition, had a virtually normal acquisition in terms of CR percentage ( Fig. 2A and table S2), but their CRs did not reach the amplitude values of control mice at the end of the training (Figs. 2, B and C, and 3, A and C, and table S2). Although the average CR timing in L7-2 mice was not significantly different from that in controls (Figs. 2C and 3, A, B, D, and E, and table S2), we found a significantly higher coefficient of variation (CV) for the latency to CR peak, even after correcting for CR amplitude ( Fig. 3H and table S2). These data indicate that the timing of the CR peak in L7-2 mice is more jittered compared to that in the other three groups, which is mainly due to a right shift of CR peak times (Fig. 3, A and B).
The level of eyeblink conditioning in the GluR27-L7-2 mice was much more affected than what could be expected on the basis of the individual phenotypes observed in the LTD-deficient GluR27 mice or the FFI-deficient L7-2 mice. These data are in line with our hypothesis that the two mechanisms may both contribute to the simple spike suppression required for eyeblink conditioning (6) and that each may compensate for the other's absence to largely preserve conditioned behavior (25). Alternatively, the exceptionally strong behavioral phenotype in the GluR27-L7-2 mice could potentially result from general deficits in PC complex spike and simple spike firing levels (26), although they appear relatively normal in the GluR27 and L7-2 mice (17,19). We therefore recorded PC activity in the anterior lobe of awake GluR27-L7-2 mice, which should have the characteristic firing patterns of zebrin-negative modules, includ-ing that of the eyeblink microzone (5, 6, 14) (Fig. 4A). The complex spike activity of GluR27-L7-2 mice showed an average firing frequency of 1.13 Hz [95% confidence interval (CI), 0.91 to 1.34], and the simple spike pause following the complex spike was, on average, 15.55 ms (95% CI, 14.16 to 16.94), both frequencies being indistinguishable from those of GluR27 mice, L7-2 mice, or controls (Fig. 4B  and table S3). Likewise, their simple spike firing frequencies, which were relatively high at 105.07 Hz (95% CI, 94.55 to 115.59), as expected for the anterior lobe (14), were not significantly different from those of the other mutant lines or controls (Fig. 4C and table S3). Moreover, the regularity [that is, coefficient of variation for adjacent intervals (CV2)] of simple spike activity of PCs in the anterior lobe of GluR27-L7-2 mice was not significantly different from that in control mice ( Fig. 4D and table S3). showing the distribution of CR onset (dark filled bars) and CR peak time (light filled bars) relative to CS and US onset in paired CS-US trials for sessions 1, 4, 7, and 10. Green dashed lines indicate CS onset, and red dashed lines denote US onset; light green and light red fill indicate CS and US duration, respectively. In all groups, there is a clear development in CR onset and peak time: There are no clearly preferred times in the CS-US interval at the start of training (session 1), but toward the end of training (sessions 7 and 10), CR onset values are centered around 100 to 125 ms after CS onset, and CR peak times are located around the onset of the expected US. In all panels, we refer to "CR" if only trials are included with an FEC > 0.1 and to "FEC" if all trials are included. For complete statistics, we refer to table S2; *P < 0.05.

DISCUSSION
Together, these data indicate that the strong phenotype in eyeblink conditioning in the GluR27-L7-2 mice cannot be explained by the abnormal baseline firing patterns of their PCs. Instead, the most parsimonious explanation is that the simple spike suppression required for eyeblink conditioning is facilitated by both PF-PC LTD and MLI-PC FFI and that ablating only one of these mechanisms will elicit processes that can at least partly compensate for the deficit induced. It has been shown that deleting GABA A receptors from PCs, as occurs in the L7-2 mice, will reduce the efficacy of the excitatory PF to PC synapse (19). These presumptively homeostatic mechanisms may well keep the excitatory-inhibitory inputs in balance and still permit simple spike suppression to be induced. This notion is further supported by the observation that combined PF and CF stimu-lation may lead to concurrent LTD of the PF-PC synapse and LTP of the PF-MLI synapse in vivo (27).
L7-2 mice have a stronger phenotype, in both their simple spike firing regularity (higher CV2) and CR peak time regularity (lower CV), than GluR27-L7-2 mice. It is unknown whether more regular simple spikes and more jittered CR peak times are causally related, but this finding does suggest that, in addition to simple spike rate coding, simple spike temporal coding contains important functional features for cerebellar learning (26). It is interesting that the added PF-PC LTD impairment in the GluR27-L7-2 mice seems to partially "rescue" simple spike and CR peak time regularity. In terms of simple spikes, the reduced attenuation of PF-PC input in this mutant could lead to a stronger displacement of simple spike timings due to stronger PF input, in absence of FFI. A plausible explanation for the lack of a CR timing phenotype in GluR27-L7-2 mice could be the very low CR expression and the fact that a real timing deficit is more prominently present in movements with bigger amplitude. It should be noted that, in all analyses on CR timing, we corrected for FEC, since it is known that CRs that peak too early or too late typically have an amplitude or FEC value of less than 0.25 ( fig. S2).
Whether induction of PF-PC LTD is completely blocked in the GluR27-L7-2 mice under physiological circumstances is unclear. Recently, it was shown that some PF-PC LTD can still be induced in GluR27 mice when the PF stimulus conditions are intensified; the CF stimulus is replaced by a strong somatic depolarization of the PC, and their stimulus interval is shortened (28). As pointed out by Hesslow et al. (23) and Titley and Hansel (24), many of the LTD-induction protocols have temporal characteristics that do not match those of eyeblink conditioning. Therefore, we have chosen to induce LTD with a protocol that closely mimics the temporal aspects of eyeblink conditioning, similar to what has been used previously by the Hansel group (22), using a more subtle PF stimulation followed by a single CF activation after a 110-ms delay. We find a significant behavioral phenotype that can be associated with an LTD blockage following the GluR27 genetic manipulation in conjunction with MLI blockage. Thus, even if LTD induction would only be partly impaired in the GluR27 mouse (depending on the induction protocol used), our behavioral data indicate that this difference in sensitivity for LTD induction is apparently still sufficient to allow for behavioral differences to occur.
Here, we used the PCP2-cre 2Mpin/J line, for which it has been reported that Cre may also be expressed by ±3% of the MLIs (29). Still, for our research on the GluR27-L7-2 mutants, this line appeared suitable since we intended to block MLI-PC transmission and since the GluR27-L7-2 mutants can be expected to have a 3% MLI Cre expression similar to that of the L7-2 mutants, which served as controls. Thus, this potential caveat cannot explain the severe learning phenotype in the triple mutants.
Although eyeblink conditioning in GluR27-L7-2 mice was severely affected, they do undeniably show some CRs at the end of the training. These eyeblink CRs are properly timed; that is, they peak around the onset of the expected US. Moreover, they do not show the higher CV for the latency to CR peak as found in the L7-2 mutants (Fig. 3H), which probably occurred because of their relatively low expression level. Our findings in the GluR27-L7-2 mice suggest additional neural mechanisms that contribute to PC simple spike suppression. It is unclear at this point whether PF-PC LTD and MLI-PC inhibition are indeed the only factors driving the PC simple spike suppression or only facilitate it by optimizing the PC's inputs that transmit the CS information. In line with recent findings supporting a more neurocentric instead of exclusively circuitsynaptocentric view on learning, such a mechanism might well involve a temporal memory that is partly formed inside the PC itself (20,30). Such a cell-intrinsic learning mechanism, however, may still depend on optimized synaptic inputs provided by PFs and MLIs. Our finding that only a concurrent disruption of PF-PC LTD and MLI-PC inhibition could severely impair eyeblink conditioning, but not completely block learning, rejects the idea that a single form of neural plasticity is essential and sufficient, and it supports the notion that synaptic and intrinsic plasticity synergistically contribute to form a temporal memory in the cerebellum (25).

Subjects and generation of mice
For all experiments, we used male and female mice with a C57Bl/6 background (individually housed under 12-hour light/12-hour dark cycles with food ab libitum). For in vitro electrophysiology experiments, we used mice aged 5 to 20 weeks; for in vivo electrophysiology and eyeblink conditioning, mice were 15 to 25 weeks old. We made use of three different transgenic mouse lines. First, GluR27 KI mice lack the last seven amino acids at the intracellular C-terminal tail, thereby disrupting the interaction of GluR2 with PICK1 and GRIP1/2, in turn disrupting the internalization of AMPA receptors, which impairs PF-PC LTD (21,31). Second, L7-2 mice have a PC-specific (L7) ablation of the 2 subunit of the GABA A receptors, resulting in impaired FFI provided by MLIs (6,19). For the L7-2 mice, we first generated 2I77lox mice by flanking exon 4 of the Gabrg2 gene with loxP sites and changing the codon encoding F77 in exons 4 to I, which resulted in a neutral amino acid substitution. Next, homozygous 2I77lox were crossed with mice hemizygous for the PC-specific L7-cre transgene and heterozygous for 2I77lox (32,33). For experiments, L7-cre 2I77lox/2I77lox mice (L7-2) and 2I77lox/2I77lox mice (controls) were used. Third, GluR27-L7-2 mice, which lack both PF-PC LTD and MLI FFI, were generated by crossbreeding female mice hemizygous for L7-cre and homozygous for 2I77lox/ 2I77lox with male homozygous GluR27 KI mice (= F 0 ). Next, F 1 female mice hemizygous for L7-cre and heterozygous for both 2I77lox/2I77lox and GluR27 were crossed with F 1 male mice heterozygous for both 2I77lox/2I77lox and GluR27. This F 1 breeding generated F 2 mice, which were hemizygous for L7-cre and homozygous for both 2I77lox/2I77lox and GluR27, and F 2 mice homozygous for both 2I77lox/2I77lox GluR27. F 2 mice were used in experiments. F 2 mice homozygous for both 2I77lox/2I77lox and GluR27 were pooled with GluR27 KI mice. As controls, we used pooled data from littermate GluR27 s and 2I77lox/2I77lox mice. All mice were genotyped by performing polymerase chain reaction analyses of genomic DNA at 2 to 3 weeks postnatal and once again postmortem. All experiments were approved by the Dutch Ethical Committee for animal experiments and were in accordance with the Institutional Animal Care and Use Committee (Erasmus MC).

PF-PC transmission
For PF-PC transmission, we used various interstimulus intervals (50 to 200 ms). For CF stimulation, similar electrodes (filled with ACSF) were positioned near the patched PC soma in the surrounding granule layer.

PF-PC LTD induction
For PF-PC LTD induction, we recorded from lobules V and VI. Recordings were done in voltage clamp, except for the tetanus, which consisted of 100-Hz PF stimulation in eight pulses followed by a 110-ms delay of single CF activation at 1 Hz for 5 min. We evaluated the synaptic plasticity by the change in PF-EPSC (baseline at 0.05 Hz) relative to the mean value calculated during the 5-min-long baseline pre-tetanus (22). Data analysis Data analysis was performed using Clampfit software (Molecular Devices).

Eyeblink conditioning Surgery
Mice were anesthetized with an isoflurane/oxygen mixture (5% for induction, 1.5 to 2% for maintenance), and body temperature was kept constant at 37°C. Eyes were protected against drying using an eye lubricant (Duratears). After a local scalp injection of bupivacaine hydrochloride (2.5 mg/ml, Bupivacaine Actavis), we made a sagittal scalp incision of 2 to 3 cm length. Next, we carefully removed the exposed periosteum and roughened the surface of the skull using an etchant gel (Kerr). After this, a small messing block (10 mm × 4 mm × 3 mm) with one screw thread and two additional pinholes was placed on the skull using OptiBond primer and adhesive (Kerr) and Charisma (Heraeus Kulzer). The surgical placement of this so-called pedestal allowed for head fixation during the eyeblink conditioning experiments.

Behavioral training
All behavioral experiments were conducted using head-fixed mice that were placed on top of a cylindrical treadmill on which they were allowed to walk freely (Fig. 1A). The treadmill consisted of a foam roller (diameter, ±15 cm; width, ±12 cm; Exervo, TeraNova EVA) with a horizontal metal rod through the axis that was connected with a ball bearing construction to two solid vertical metal poles. A horizontal messing bar was fixated to the same vertical poles at 3 to 5 cm above the treadmill. Mice were head-fixed to this bar using one screw and two pins, thereby ensuring perfect head fixation [for further details, see (34)]. National Instruments (NI-PXI) equipment was used to control experimental parameters and to acquire the eyelid position signal. Eyelid movements were recorded with the MDMT, which makes use of an NVE giant magnetoresistance magnetometer, positioned above the left upper eyelid, that measures movements of a minuscule magnet (1.5 mm × 0.7 mm × 0.5 mm) that is placed on the left lower eyelid of the animal with superglue (cyanoacrylate). This way, MDMT allows high spatiotemporal detection of eyelid kinematics [for further details, see (35)]. The CS was a green LED light (CS duration, 280 ms; LED diameter, 5 mm) placed 10 cm in front of the mouse's head. Because we performed our experiments in almost complete darkness, this small LED light was a salient stimulus, which could be easily detected by both eyes. The US consisted of a weak air puff applied to the eye (30 psi; duration, 30 ms), which was controlled by an API MPPI-3 pressure injector, and delivered via a 27.5-gauge needle that was perpendicularly positioned at 0.5 to 1 cm from the center of the left cornea. The training consisted of 3 daily habituation sessions, 1 baseline measurement, 5 or 10 daily acquisition sessions, and 1 probe session immediately after the last training session. During the habituation sessions, mice were placed in the setup for 30 to 45 min, during which the air puff needle (for US delivery) and green LED (for CS delivery) were positioned properly but no stimuli were presented. On the day of acquisition session 1, each animal first received 20 CS-only trials as a baseline measure, to establish that the CS did not elicit any reflexive eyelid closure. During each daily acquisition session, the animals received either 100 or 200 paired CS-US trials, separated over 10 or 5 daily acquisition sessions, respectively. The interval between the onset of CS and that of US was set at 250 ms. Because of an inherent 14-ms delay in the delivery of the air puff, we triggered the air puff at 236 ms after CS onset so that it would hit the cornea exactly at 250 ms after CS onset. The intertrial interval was set according to the following constraints: At least 10 s had to elapse, the eyelid had to be open below a predetermined threshold of 50% of a full eyelid closure, and eyelid position had to be stable for at least 2 s for a trial to begin. Immediately after acquisition session 10, we started a probe session, during which we presented another 100 paired CS-US trials, but now intermingled with 30 CS-only trials. During all training sessions, the experimenter carefully inspected threshold and stability parameters and adjusted them if necessary. All experiments were performed at approximately the same time of day by the same experimenter.

Data analysis
Individual eyeblink traces were analyzed automatically with custom computer software (LabVIEW or MATLAB). First, 2000-ms eyeblink traces were imported and filtered in forward and reverse directions with a low-pass Butterworth filter using a cutoff frequency at 50 Hz. Second, trials with significant activity in the 500-ms pre-CS period (>7 * interquartile range) were regarded as invalid for further analysis. Third, trials were normalized by aligning the 500-ms pre-CS baselines and calibrating the signal so that the size of a full blink was 1 (FEC). Fourth, in valid normalized trials, all eyelid movements larger than 0.1 and with a latency to CR onset between 50 and 250 ms and a latency to CR peak of 100 to 250 ms (both relative to CS onset) were considered as CRs. For CS-only trials in the probe session, we used the exact same criteria except that the latency to CR peak time was set at 100 to 500 ms after CS onset. Additionally, we determined for each individual trial the following parameters: (i) maximum eyelid closure (= FEC) in the CS-US interval, (ii) CR amplitude, (iii) latency to CR onset, (iv) latency to CR peak, (v) latency to UR onset, and (vi) latency to first UR peak. For (i), we used all valid trials; for (ii) to (iv), we only used the trials in which a CR was present; for (v) and (vi), we used US-only trials in the baseline session. Fifth, on the basis of this trial-by-trial analysis, we calculated for each session per mouse the percentage of eyeblink CRs and the mean median, SD, and CV of all other outcome measures. Sixth, statistical effects of session and genotype on all outcome measures were established in R version 1.1.442 using linear mixed-effect models (LMMs). All our outcome measures except for CR peak time included group, session number (categorical), and their interaction as fixed effects. The random effect included mouse. (R syntax: lme (measure) ~ group + session + session:group), data = all_data, random = list (mouse = pdDiag ( form = ~ poly (session, 2))), method = "REML", na.action = na.exclude)). For CR peak time, we used a modified model, adding FEC as a fixed effect, since it is known that smaller CRs at the beginning of training have worse timing compared to CRs at the end of training (Fig. 2C and  fig. S2). (R syntax: lme (CRpeaktime) ~ group + session + session:group + FEC), data = all_data, random = list (mouse = pdDiag ( form = ~ poly (session,2))), method = "REML", na.action = na.exclude)). Data were considered statistically significant if P < 0.05, and we used false discovery rate correction for multiple comparisons.

In vivo PC recordings Surgery
Mice were surgically prepared following the same procedure as described for eyeblink conditioning. Additionally, a craniotomy (diameter, 2 to 3 mm) was performed on the occipital bone to perform extracellular PC recordings from both the anterior and posterior cerebellum. A small rim of Charisma was made around the craniotomy, and anti-inflammatory (dexamethasone, 4 mg/ml) solution was applied inside, after which the chamber was closed with a very low viscosity silicone elastomer sealant (Kwik-Cast, World Precision Instruments). After surgery, mice had 5 days to recover. Recordings Extracellular PC recordings were conducted using head-fixed mice that were placed on top of a cylindrical treadmill system as described above. Mice were first habituated for 3 days to the treadmill. PCs were recorded from vermal lobules I to V and lobules VI to X using glass micropipettes (OD, 2.0 mm; ID, 1.16 mm; tip, 2 to 5 m, impedance, 2 to 5 milliohms; Harvard Apparatus). Single-unit PCs were recorded for 2 to 5 min. Single-unit recording was identified online by the presence of a short simple spike pause (20 to 50 ms) after each complex spike. To verify recording locations, we made small alcian blue injections after recording sessions.

Analysis
In vivo recordings were analyzed offline using custom scripts in MATLAB (Mathworks) and SPSS 24. Average simple spike and complex spike firing were calculated. Simple spike firing regularity was investigated using the CV2 value, which was calculated as 2 * | ISI n+1 − ISI n | / (ISI n+1 − ISI n ) .