Constant current isolated stimulator

Borosilicate recording electrodes were manufactured as previously described [19 (link)]. Internal solution consisted of (mM): K-gluconate (110), KCl (5), HEPES (50), EGTA (0.05), MgSO₄ (4), ATP (4), GTP (0.2), phosphocreatine (9), and pH to 7.4 with 1 M KOH. Whole-cell voltage clamp recordings were made from Bergmann glia (holding potential -80 mV) and Purkinje neuron (holding potential -70 mV) somata in the Purkinje cell layer. Currents were low pass filtered at 4–5 kHz and sampled at 25 kHz, using Spike2 software (CED, Cambridge, UK). Series resistances ranged from 5 to 15 MΩ and were compensated by >85% in Purkinje neuron recordings, but uncompensated in glial recordings.
Parallel fibres were stimulated with a patch electrode (~1–2 MΩ) filled with bath solution and positioned in the molecular layer, connected to an isolated constant current stimulator (6.5–90 μA, 80 μs; Digitimer, Welwyn Garden City, UK). Stimulus was delivered as a pair of pulses with a 100 ms interval at a frequency of either 0.033 Hz or 1 Hz.

Two-photon imaging and physiology data were acquired using National Instruments boards and SciScan (Scientifica) and WinWCP (University of Strathclyde) respectively. Electrical stimulation was via a tungsten bipolar electrode (WPI) and an isolated constant current stimulator (Digitimer). Optical stimulation was via wide field irradiance with 473 nm LED light (CoolLED) as described above. Data was analyzed using custom routines written in Python 3.6. Current step data was analyzed using routines based around the Neo and eFEL packages. For synaptic connectivity experiments, amplitudes of PSPs were taken as averages over a 1-ms time window around the peak. For connectivity analysis, a cell was considered connected if the light-induced response was greater than 6 times the standard deviation of baseline.
Two-photon image reconstruction and analysis was carried out using VIAS and NeuronStudio^{83 (link)}, before further analysis was carried out using custom scripts in Python 3.6 based on the NeuroML package from the Human Brain Project. The online Human Brain Project morphology viewer was used for visualizing reconstructed neurons.

Borosilicate recording electrodes were manufactured as previously described [6 (link)]. Internal solution consisted of (mM) K-gluconate (110), KCl (5), HEPES (50), EGTA (0.05), MgSO₄ (4), ATP (4), GTP (0.2), phosphocreatine (9), and pH to 7.4 with 1 M KOH. For 16 Hz tetanus and forskolin experiments the internal solution was supplemented with 10 mM BAPTA. Whole-cell voltage clamp recordings were made from Bergmann glia (holding potential −80 mV) and Purkinje neuron (holding potential −70 mV) somata in the Purkinje cell layer. Currents were low pass filtered at 4-5 kHz and sampled at 25 kHz, using Spike2 software (CED, Cambridge, UK). Series resistances ranged from 5 to 15 MΩ and were compensated by >85% in Purkinje neuron recordings but uncompensated in glial recordings.
Parallel fibres were stimulated with a patch electrode (~1-2 MΩ) filled with bath solution and positioned in the molecular layer, connected to an isolated constant current stimulator (5–40 μA, 80 μs; Digitimer, Welwyn Garden City, UK). Stimulus was delivered as a pair of pulses with a 100 ms interval at a frequency of 0.033 Hz. Tetanic stimulation was delivered at 16 Hz (single pulses) for a 15 s period.
Experiments were performed at room temperature.

Borosilicate recording electrodes were manufactured as previously described [15 (link)]. Internal solution consisted of (mM): K-gluconate (110), KCl (5), HEPES (50), EGTA (0.05), MgSO₄ (4), ATP (4), GTP (0.2), phosphocreatine (9), and pH to 7.4 with 1 M KOH, plus 1 mg/mL Lucifer yellow. Whole-cell voltage clamp recordings were made from Bergmann glia (holding potential -80 mV) somata in the Purkinje cell layer. Currents were low pass filtered at 4–5 kHz and sampled at 25 kHz, using Spike2 software (CED, Cambridge, UK). Series resistances ranged from 5 to 15 MΩ and were uncompensated.
Parallel fibres were stimulated with a patch electrode (~1–2 MΩ) filled with bath solution and positioned in the midpoint of the molecular layer, connected to an isolated constant current stimulator (100 μA, 80 μs; Digitimer, Welwyn Garden City, UK). Stimulus was delivered as a pair of pulses with a 100 ms interval at a frequency of either 0.033 Hz or 1 Hz; a stimulus protocol that has been shown to be the most effective mechanism for depression of ectopic release [15 (link)], due to the increased release probability for the facilitated second pulse.