Neuronal culture preparation
All procedures were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals Norway (FOTS 20135149/20157494/20170001). Wistar Hannover GLAST rat pups (n = 328), embryonic day 18 (E18) to postnatal day 10 (P10), were used for neuronal culture preparation. Briefly, following anaesthesia and decapitation, the brains were rapidly transferred into ice cold EBSS (Gibco, #24010043) solution containing 0.5% glucose (Sigma, #G8769) and 10 mM HEPES (Gibco, #15630056). Under a dissection microscope, the meninges and medulla oblongata were carefully removed, and the cerebellum was separated from the pons and the midbrain. Depending on the culture, either only the cerebellum or the cerebellum including pones was transferred to a 15 mL tube containing 20 U/mL papain (Worthington, #LK003178) dissolved in preparation solution and warmed up to 36 °C. The tube containing the cerebellar tissue was placed into the incubator for 15 minutes at 36 °C with occasionally swirling to digest the tissue. The papain solution was carefully removed with a fire-polished Pasteur, and the digestion reaction was stopped by addition of stop media (advanced DMEM/F12 solution (Gibco, #12634010) containing 0.5% glucose (Sigma, #G8769) and 10% foetal bovine serum (FBS, Gibco, #10500064) pre-warmed 36 °C. After 5 minutes of deactivation, the stop media was removed and 250 µL growth media containing 10% FBS per cerebellum was added. The tissue/media suspension was pipetted up and down with a fire polished Pasteur pipette at least 100 times until cells were separated.
Support cell layer
To prepare the support cell layer, 375.000 cells/mL from cerebellum including pones were seeded on cover slides from Neuvitro (#GG-12-1.5-PDL, 24 well, 500 µL/well; #GG-18-1.5-PDL, 12 well, 1 mL/well; #GG-25-1.5-laminin, 6 well, 2 mL/well) that were pre-coated with poly-D-lysine (PDL) alone or additionally with laminin. Cultures were maintained in 6-, 12-, or 24-well plates in growth media consisting of 45% advanced DMEM/F12 solution (Gibco, # 126340010), 45% NBM solution (Miltenyibiotec, #130-093-570), 1.5% B-27 serum-free supplement (Gibco, #17504044), 1.5% NB-21 serum-free supplement (Miltenyibiotec, #130-093-566), 1% sodium pyruvate (Invitrogen, #11360088), 1% heat-inactivated FBS (Invitrogen, #10500064), 2% Glutamax (Gibco, #35050038), 5 mg/mL D-glucose and 10 mM HEPES (Invitrogen, #15630056) at 36°C. Half of the culture medium was replaced every 7 days.
PN layer
E18- and P0-derived PN cultures were prepared by seeding 500.000 cells/mL from cerebellum without pones onto the support cell layer of different in vitro ages. P10-derived PN culture was prepared by seeding 750.000 cells/mL from the vermis of the cerebellum onto the support cell layer of different in vitro ages. The growth media was supplemented with insulin (Invitrogen, #12585014; 1:250, stock 4 mg/mL), progesterone (Sigma, #P8783, 1:2000, stock 80 mM), insulin-like growth factor 1 (IGF1; Promokine, #E-60840, 1:40000, stock 1 µg/µL) and Protein kinase C inhibitor K252a (Alomone, # K-150; IC50 25 nM). In long-term cultures that were maintained for more than 28 days in vitro the IGF1 and progesterone concentration were reduced to 10 ng/mL and 20 µM, respectively. K252a was supplemented for 21 days before the washout process started, its optimal concentration was experimental evaluated for each tested culture type. Half of the culture medium was replaced every 3.5 days for 6-well plates and every 2 days for 12- and 24-well plates. All experiments testing the yield of PNs from tissue from rats of different ages, different in vitro ages of the support cell layers, and K252a concentration were performed randomly, containing 3 to 6 probes per experimental setting and 5 independently repeats for each group and condition.
Lentiviral gene editing
The full-length L7 promoter region (1005 bp) was custom cloned by SBI System Bioscience into construct pCDH-L7-MCS-copGFP (#CS970S-1), and viral particles with a yield of 2.24 x 109 ifus/mL were produced. Freshly prepared PNs of E18 or P0 cerebellum suspended in growth media containing no serum were incubated for 10 minutes at 37 °C with 1.22 x 106 viral particles/mL before seeding onto the supplement structure layer containing coverslip or live-cell imaging 35 mm µ-dish (Ibidi, #80136). Media was changed after 3 days, and transfection efficiency evaluated by live-cell imaging microscopy at 24 hours post transfection and then daily up to 21 days and weekly up to 169 days in culture, respectively. Lentiviral transfections of PNs in culture were performed 1 day after feeding at 15 days in vitro (DIV) and DIV29 by applying 2.5 x 106 viral particles/mL. The neuronal development of the GFP-expressing PNs was followed by obtaining 10 independent 3x3 tile scan using the Zyla camera configuration of 2048x2048 with the CFI Plan Apochromat Lambda dry objective 10x0.45 (pixel size 603 nm) or 20x0.75 (pixel size 301 nm) at the Andor Dragonfly microscope system (Oxford Instruments company). Experiments were repeated three times.
Immunohistochemical characterisation of cell types
To evaluate PN yield, the distribution ratios of other cell types of the cerebellum, and their synaptic interactions, cultures were washed with pre-warmed 0.1 M PBS (Gibco, #70013016) and fixed with 1.5-4% paraformaldehyde (PFA, pH 6-7.2; ThermoScientific, #28908) containing 0.5% sucrose for 15 minutes at 36 °C. Tris-based or citric acid-based heat-induced antigen retrieval (pH 9 and pH 6, respectively; 45 min, 85 °C) 13 was perform when necessary (see Table 1). Cultures were quenched with 1xPBS containing 50 mM NH4Cl (PBSN), permeabilised with 0.2% Triton X-100 (Sigma, #T9284) in PBSN (5 min, 36°C), rinsed with PBSN containing 0.5% cold water fish gelatine (Sigma, #G7041)(PBSNG, 3x15 min), and incubated with primary antibodies over-night at 4°C in PBSNG containing 10% Sea Block (ThermoScientific, #37527), 0.05% Triton X-100 and 100 μM glycine (Sigma, #G7126) to visualise the different cerebellar cell types, including Purkinje neurons and their synaptic interactions (Table 1). The coverslips were rinsed with PBSNG (3x20 min) and incubated with highly cross-absorbed donkey secondary antibodies conjugated to CFTM488/594/647-Dye (Biotium, #20014, #20115, #20046, #20015, #20152, #20047, #20074, #20075, #20169, #20170; 1:400) for 2 hours at 22°C in PBSNG containing 2.5% Sea Block. To remove unbound secondary antibody, coverslips were rinsed with PBSN (3x20 min), and briefly dipped into MilliQ water before mounted in hardening ProlongTM Glass Antifade Reagent (Invitrogen, #P36981) onto cover slides. After 2 days of hardening at 18-21°C in the dark, cover slides were stored at 4 °C until imaging.
PN counting and imaging
PNs were counted manually and blind by screening the coverslips using a Leitz Diaplan fluorescence microscope equipped with CoolLED pE-300white lamp. For dendritic tree branch analysis and determination of maturity and synaptic interaction, 10 Z-stack images per cover slide were collected in five independent and randomized experiments at 0.5-1 μm intervals with the Zyla camera configuration (2048x2048) using the the Andor Dragonfly microscope system equipped with a CFI Plan Apochromat Lambda S LWD 40x1.14 water objective (pixel size 151 nm), a 60x1.20 oil objective (pixel size 103 nm), or a CFI SR HP Apo TIRF 100x1.49 oil objective (pixel size 60 nm) to detect DAPI and CFTM488/594/647 dye emission. Stacks were superimposed with Fusion software (Oxford Instruments). 3D surface visualization of synapses was performed using Oxford Instruments analysis software IMARIS 9.3.1 and the filament tracer tool 14.
Dendritic tree branch analysis
The PN dendritic tree development was evaluated by analysing 10 group-dependent PNs per experiment in 10 independent experiments. The order and length of the dendritic arbours was determined using an open-source ImageJ and Fiji plugin Simple_Neurit_Tracer (Neuroanatomy) 15.
Micro-electrode array (MEA) recordings
Primary cultures of E18-derived PNs at a concentration of 500.000 cells/mL were plated onto PDL precoated 24 well format plates of the Multiwell-MEA-system (Multi Channel System-MCS, Reutlingen, Germany). Each well contains 12 PEDOT coated gold micro-electrodes (30 µm diameter, 300 µm space, 3 x 4 geometrical layout) on glass base to facilitate visual checking (Multi Channel System-MCS, #890850, 24W300/30G-288). The amplifier (data resolution: 24 bit; bandwidth: 0.1 Hz to 10 kHz, modifiable via software; default 1 Hz to 3.5 kHz; sampling frequency per channel: 50 kHz or lower, software controlled; input voltage range: ± 2500 mV), stimulator (current stimulation: max. ± 1 mA; voltage stimulation: max. ± 10 V; stimulation pattern: pulse or burst stimulation sites freely selectable) and heating element (regulation: ± 0.1 °C) were integrated into the Multiwell-MEA-headstage which is driven by the MCS-Interface Board 3.0 Multiboot. The Multiwell recording platform was covered by a mini incubator to provide 5% CO2 and balanced air. Electrophysiological signals were acquired at a sampling rate of 20kHz through the commercial software Multiwell-Screen. Plates were tested every second day from day 5 in vitro for spontaneous activity. Raw voltage traces were recorded for 120 seconds, saved, and analysed using offline MCS-Multiwell-Analyzer to calculate spike rate and burst activity, including network properties. Two experimental settings were tested: First, spontaneous spike activity was recorded in PN culture media (45% advanced DMEM/F12 solution, 45% NBM solution, 1.5% B-27 serum-free supplement, 1.5% NB-21 serum-free supplement, 1% sodium pyruvate, 1% heat-inactivated FBS, 2% Glutamax, 5 mg/mL D-glucose, 10 mM HEPES, 16 µg/mL insulin, 25 ng/mL IGF1, 40 µM progesterone, 5 nM K252a) for 63 days. Second, spontaneous spike activity was recorded for the first 28 days in PN culture media that was then exchanged to previously described organotypic brain slice culture media 2 (30% advanced DMEM/F12 solution, 20% MEM solution (Gibco, #41090028), 25% EBSS solution (Gibco, #24010043), 25% heat-inactivated horse serum (Sigma, #H1138), 2% GLUTAMAX, 5 mg/ml D-glucose and 2% B-27 serum-free supplement) for the remaining 45 days of the experiment.
Patient sera
Sera were obtained from two untreated patients with gynecological cancer and PCD who had Yo antibodies against CDR2 and CDR2L (anti-Yo1 and anti-Yo2) but lacked antibodies to P/Q-type voltage-gated calcium channel (VGCC) proteins 2. A pool of sera from 100 healthy age-matched donors (non-hCDR100p) was used as a control. Sera were not heat-inactivated before use. The sera were stored at the PND Biobank #133/2015 or the Biobank for diagnostic cancer marker #188.05 with approval of the regional ethics committee, Western-Norway.
In vitro PCD model
Twenty-eight days post-seeding, the culture medium was replaced with medium containing 4 μL/mL human serum positive for Yo antibodies. PN culture was collected 2 or 4 days after commencement of treatment to evaluate the antibody effects. Each independent experiment included treatment (anti-Yo1-and anti-Yo2) and positive (non-hCDR100p) control to account for variations in cell survival between culture preparations. All treatments were performed in triplicate and five PNs were analyzed per sample.