Associative plasticity of granule cell inputs to cerebellar Purkinje cells
Data files
Dec 10, 2024 version files 1.62 GB
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Archiv_ContiAuger_Elifepaper.zip
1.62 GB
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README.md
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Abstract
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.
README: Associative plasticity of granule cell inputs to cerebellar Purkinje cells
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.
Methods
Slice preparation: 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% O2 / 5% CO2 at this temperature. Slice preparation and recordings were made in a bicarbonate buffered solution containing in mM: 115 NaCl, 2.5 KCl, 1.3 NaH2PO4, 26 NaHCO3 , 2mM CaCl2, 1mM MgSO4, 0.1mM Ascorbic Acid, and 25 glucose.
Patch-clamp recording of synaptic currents: 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 K2 Creatine Phosphate, 10 HEPES, 0.01 EGTA, 2.5 MgCl2, 2 ATPNa2 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% O2 / 5% CO2 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- concentration in the intracellular solution ensured the reversal potential for Cl- 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.
Analysis of evoked EPSCs: 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).
Statistical significance 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.