DOI: 10.5061/dryad.zs7h44jk0
title: Associative plasticity of granule cell inputs to cerebellar Purkinje cells

description: This data set contains a collection of electrophysiological whole cell
 patch clamp recordings of synaptic currents in cerebellar Purkinje cells
 and analysis of the pooled data for each type of experiment performed. The
 data were acquired using an EPC9 amplifier (HEKA, Germany) and PatchMaster
 software, and analyses performed in Igor Pro (Wavemetrics). Synaptic
 currents were recorded in response to the stimulation of granule cell
 axons in acute cerebellar slices, targeting both parallel fibers and
 ascending axons, to investigate the effects of synchronous activation of
 synaptic inputs to Purkinje cells. Granule cells of the cerebellum make up
 to 175 000 excitatory synapses on a single Purkinje cell, encoding the
 wide variety of information from the mossy fibre inputs into the
 cerebellar cortex. The granule cell axon is made of an ascending portion
 and a long parallel fibre extending at right angles, an architecture
 suggesting that synapses formed by the two segments of the axon could
 encode different information. There are controversial indications that
 ascending axon (AA) and parallel fibre (PF) synapse properties and
 modalities of plasticity are different. We tested the hypothesis that AA
 and PF synapses encode different information, and that association of
 these distinct inputs to Purkinje cells might be relevant to the circuit
 and trigger plasticity, similarly to the coincident activation of PF and
 climbing fibre inputs. Here, by recording synaptic currents in Purkinje
 cells from either proximal or distal granule cells (mostly AA and PF
 synapses respectively), we describe a new form of associative plasticity
 between these two distinct granule cell inputs. We show for the first time
 that synchronous AA and PF repetitive train stimulation, with inhibition
 intact, triggers long term potentiation (LTP) at AA synapses specifically.
 Furthermore, the timing of presentation of the two inputs controls the
 outcome of plasticity and induction requires NMDAR and mGluR1 activation.
 The long length of the PFs allows us to preferentially activate the two
 inputs independently, and despite a lack of morphological reconstruction
 of the connections, these observations reinforce the suggestion that AA
 and PF synapses have different coding capabilities and plasticity that is
 associative, enabling effective association of information transmitted via
 granule cells.
description: <em><strong>Slice
 preparation:</strong></em><strong>
 </strong>Experiments were performed on horizontal slices 300 µm
 thick cut from the cerebellum of 19-25 day-old Sprague-Dawley male or
 female rats. Briefly, rats were killed by decapitation under general
 anaesthesia following inhalation of the volatile anaesthetic isoflurane at
 a concentration of 3-4% in accordance with the Directive 2010/63/UE, and
 the cerebellum was quickly removed. After removal of the brainstem, the
 tissue was glued to the stage of a vibratome (Leica VT1200S, Germany).
 Slices were cut at a temperature of 34° C and subsequently kept in a
 vessel bubbled with 95% O<sub>2</sub> / 5%
 CO<sub>2</sub> at this temperature. Slice preparation and
 recordings were made in a bicarbonate buffered solution containing in mM:
 115 NaCl, 2.5 KCl, 1.3
 NaH<sub>2</sub>PO<sub>4</sub>, 26
 NaHCO<sub>3 </sub>, 2mM CaCl<sub>2</sub>, 1mM
 MgSO<sub>4</sub>, 0.1mM Ascorbic Acid, and 25
 glucose. <em><strong>Patch-clamp recording
 of synaptic currents:</strong></em> Whole-cell patch-clamp
 recordings were made from PCs, identified by their size and location at
 the edge of the molecular and GC layers, with an EPC10 amplifier (HEKA,
 Germany) and PatchMaster acquisition software. Bath temperature was kept
 at 30-32° C The internal solution contained in mM: 135 KGluconate, 10
 K<sub>2</sub> Creatine Phosphate, 10 HEPES, 0.01 EGTA, 2.5
 MgCl<sub>2</sub>, 2 ATPNa<sub>2</sub> and 0.4
 GTPNa, pH adjusted to 7.3 with KOH and osmolarity to 295 mOsm/kg. When
 filled with the internal solution, recording pipettes had a resistance
 between 3 and 4 MΩ. Membrane currents were recorded at a pipette potential
 of -60 mV (not corrected for junction potential of approximately -12 mV
 pipette-bath). Series resistance was 80-90 % compensated. During
 experiments, the preparation was visualised on an upright microscope
 (Olympus BFX51; 60x 0.9 NA water dipping objective) and the bath was
 continuously perfused at a rate of 5 ml/min (5 bath volumes per min) with
 solution equilibrated with 95% O<sub>2</sub> / 5%
 CO<sub>2</sub> to maintain pH and solution
 recirculated. Plasticity of GC to PC synapses was
 studied in horizontal cerebellar slices as these slices better preserve
 PFs running in the plane of cut. At this age, GC synapses are well
 established (Ichikawa et al., 2016). AAs and PFs were stimulated with
 patch pipettes slightly larger than those used for recordings filled with
 a Hepes-buffered external solution and positioned either in the molecular
 (lower two thirds) or the GC layer, as discussed in the result section.
 The baseline amplitude of both AA and PF pathways were sampled with a pair
 of suprathreshold pulses at 50 ms interval, delivered every 10 s (Fig 1A
 top panel, stimulation was biphasic, 100-180 µs duration, 5 to 15V). The
 AA and PF test stimuli were separated by 0.5 s. Stimulation strength was
 adjusted between 5 and 15V to stimulate AA- and PF-EPSCs reliably. PF
 stimulation was usually very efficient as fibres are densely packed in the
 molecular layer and PF-EPSC amplitude typically increased smoothly with
 stimulation intensity. Although we originally worked with large amplitude
 PF-EPSCs (up to about 1nA), we aimed at amplitudes of a few hundred pA,
 closer to the amplitude of stimulated AA-EPSCs, by adjusting stimulation
 intensity. Granule cell somas and axons are more sparse in the granule
 cell layer and stimulation intensity was adjusted to activate EPSCs a few
 hundred pA when possible, but increasing stimulation intensity did not
 necessarily recruit larger EPSCs. Care was taken to avoid stimulating the
 local Purkinje cell axon and climbing fibre. Once a stable input was
 obtained, stimulation intensity was raised by one Volt to increase
 reliability. In most of the experiments AAs were tested first to avoid
 possible interference from mGluR1s activation and the release of
 endocannabinoid by PFs stimulation. No antagonist of the inhibitory inputs
 was applied. Evoked responses consisted of excitatory postsynaptic
 currents (EPSCs) that were often quickly followed by inhibitory
 postsynaptic currents (IPSCs) (mixed EPSC/IPSC, see supplementary material
 and supplementary fig.3). The low Cl<sup>-</sup> concentration
 in the intracellular solution ensured the reversal potential for
 Cl<sup>-</sup> was close to the value determined for PCs (-85
 mV, (Chavas & Marty, 2003)), and IPSCs were outward at the
 recording potential of -60 mV. After recording a baseline period of at
 least 10 minutes, we applied a stimulation protocol aimed at inducing
 plasticity. The recording configuration was switched to Current clamp and
 the potential set and maintained near -65 mV. The protocol applied (see
 Fig. 1A bottom panel) consisted in the synchronous stimulation of both
 inputs by a train of 15 pulses at 100 Hz repeated every 3 s a total of 100
 times. Following the induction protocol, the recording configuration was
 returned to Voltage clamp and test of alternate AA- and PF-EPSC amplitude
 resumed. In the result section the number of cells sometimes differ for
 the two pathways for a given set of experiments, because one of the inputs
 was sometimes lost during the 30 minutes following induction. Because the
 protocol was properly applied, the data for the remaining input were
 included in the results.
 <em><strong>Analysis of evoked EPSCs:
 </strong></em>For the analysis of synaptic currents, raw
 current traces were exported to Igor Pro (Wavemetrics) and peak excitatory
 current amplitudes were measured as the minimum of the synaptic response
 (mixed EPSC/IPSC) over a time window overlapping the peak and spanning a
 few sampling points of the average EPSC. Since the plasticity protocol
 might affect EPSCs and IPSCs differently, and might therefore affect our
 estimate of the peak EPSC and its long term changes, we conducted a set of
 experiments to measure the IPSCs and EPSCs separately and confirmed that
 measuring the minimum of the mixed EPSC/IPSC gives a good estimate of EPSC
 amplitude and its long term changes (see supplementary materials and
 supplementary figure 3).
 <em><strong>Statistical
 significance</strong></em> was tested with non-parametric
 methods for most of the data sets. These do not require assumptions about
 the nature of the distribution of the variables (as parametric tests do);
 we used either the Wilcoxon signed rank test (non-parametric, for paired
 samples) or the Wilcoxon Mann Whitney test (non-parametric, for unpaired
 samples). The T-test was used for control data only, where N was big
 enough to show a normal distribution of the variables (see Supplementary
 figure 2). Tests were conducted using Igor Pro (Wavemetrics). All values
 given are mean ± SEM.
description: # Associative plasticity of granule cell inputs to cerebellar Purkinje
 cells
 [https://doi.org/10.5061/dryad.zs7h44jk0](https://doi.org/10.5061/dryad.zs7h44jk0) ## Description of the data and file structure We investigated the effect of repetitive synchronous stimulation of the ascending axon (AA) and parallel fibre (PF) synapses formed by granule cells onto Purkinje cells of the cerebellum to test whether co-activation of the inputs could trigger plasticity. ### Files and variables #### File: Archiv\_ContiAuger\_Elifepaper.zip **Description:** Files are organised around the figures of the paper and type of experiment performed. The Analysis_generalinfo file (pdf) describes the format of the raw data files and analysis files, and name convention used within the analysis files. Each type of experiment is associated with: \- a Data file (pdf) with a brief description of the experiment and a list of the recordings to be found in the raw data folder, with specific information when necessary. The data files of figures 2 and 3 describe variations of an experiment and corresponding files; \- a folder containing the raw data files (PatchMaster); PatchMaster data files dating from 2018 are made of 4 files (extensions .dat/.pul/.amp/.pgf). When opening the .dat file, PatchMaster will open the other extensions automatically. Later PatchMaster files are made of a single .dat file. PatchMaster files can be opened with PatchMaster running in Demo mode (freely available). \- an Igor Pro analysis file (pxp), with amplitude data averaged over 1 min bins and normalised to the control period for each individual recording, and average time course of the experiment for the different amplitude parameters, as presented in the corresponding figure. The files can be opened with IgorPro running in Demo mode. **Figure1_and_SupplFig1SupplFig2** folder contains folders: **- Control:** This folder contains the individual data for experiments where both the ascending axon (AA) and parallel fibre (PF) pathways were stimulated synchronously during the induction protocol. **- No Stim:** This file contains the individual data for experiments where no stimulation was applied to the synaptic pathways during the induction protocol. **- AA only:** This folder contains the individual data for experiments where only the AA pathway was stimulated during the induction protocol. **- PF only:** This folder contains the individual data for experiments where only the PF pathway was stimulated during the induction protocol. which all contain: \- a Raw data folder \- an Analysis file \- a Data file **Figure2** folder contains: \- Data file; **- AAdelay_+150ms**: This folder contains the individual data for experiments where, during induction, AA stimulation was delayed by 150 ms after the start of PF stimulation (Raw Data folder), and an analysis file; **- AAdelay_-150ms**: This folder contains the individual data for experiments where, during induction, AA stimulation was started 150 ms before the start of PF stimulation (Raw Data folder), and an analysis file; \- Analysis file for individual data points and averages for these and other experiments as in Fig 2C. **Figure3** folder contains: \- Data file for APV and CPCCOEt recordings; **- APV recordings** folder which contain a Raw Data folder and an analysis file; **- CPCCOEt recordings** folder with a Raw Data folder and an analysis file. **Figure4** and **Figure5** folders contain: \- a Raw data folder; \- an Analysis file; \- a Data file.