Animals
All transgenic mice (Trpm4(tm1.1Mfre) 48 and wild type C57BL/6 were housed and bred at the University Medical Center Hamburg-Eppendorf (UKE) animal facility. Both female and male mice were used as indicated. All mice were housed with a 12-hour light/dark cycle and had water and food ad libitum. All procedures performed in mice were in compliance with German law and according to the guidelines of Directive 2010/63/EU. Protocols were approved by the local ethics committee (Behörde für Justiz und Verbraucherschutz (BJV), Lebensmittelsicherheit und Veterinärwesen). The ARRIVE guidelines for reporting the experiments were followed. No a priori determination of sample size was performed.
Mouse organotypic hippocampal slice cultures
Hippocampal slice cultures were prepared from P4-P7 male wild type C57BL/6 mice following a published protocol 49. Pups were anesthetized with 80% CO2/20% O2 and decapitated. Hippocampi were dissected in cold slice culture dissection medium containing (in mM): 248 sucrose, 26 NaHCO3, 10 glucose, 4 KCl, 5 MgCl2, 1 CaCl2, 2 kynurenic acid and 0.001% phenol red. The solution was saturated with 95% O2, 5% CO2, pH 7.4, 310-320 mOsm kg-1. Tissue was cut into 400 µM thick sections on a tissue chopper and cultured on porous membranes (Millipore PICMORG50) at 37° C in 5% CO2. No antibiotics were added to the slice culture medium which was partially exchanged (60-70%) twice per week and contained (for 500 ml): 394 ml Minimal Essential Medium (Sigma M7278), 100 ml heat inactivated donor horse serum (H1138 Sigma), 1 mM L-glutamine (Gibco 25030-024), 0.01 mg ml-1 insulin (Sigma I6634), 1.45 ml 5M NaCl (S5150 Sigma), 2 mM MgSO4 (Fluka 63126), 1.44 mM CaCl2 (Fluka 21114), 0.00125% ascorbic acid (Fluka 11140), 13 mM D-glucose (Fluka 49152).
Plasmid construction and expression
DNA encoding GCaMP6f and tdimer2 were each subcloned into a neuron-specific expression vector (pCI) under the control of the human synapsin-1 promoter using the HindIII and NotI restriction sites for GCaMP6f and the Acc65I and EcoRI restriction sites for tdimer2. To express GCaMP6f and tdimer2 in CA1 pyramidal neurons, gold particles (1.6 μm, 2.75 μg DNA per mg gold) were coated with an 8:3 ratio of expression vectors encoding GCaMP6f and tdimer2 (respectively). At DIV 7-9, slice cultures were ballistically transfected using a Helios gene gun (Bio-Rad). Experiments were conducted between DIV 14-21.
Two-photon microscopy
The two-photon imaging setup was custom built based on an Olympus BX51WI microscope. An Olympus LUMPlan W-IR2 60x 0.9 NA objective was used and image acquisition was controlled by the open-source software package ScanImage 3.7 50, which we modified to allow user-defined arbitrary line scans. A pulsed Ti:Sapphire laser (Chameleon, Coherent) was used to excite GCaMP6f and tdimer2 at 980 nm. Emitted photons were collected through the objective and oil-immersion condenser (1.4 NA, Olympus) with two pairs of photomultiplier tubes (H7422P-40, Hamamatsu). 560 DXCR dichroic mirrors and 525/50 and 607/70 emission filters (Chroma) were used to separate green and red fluorescence. Excitation light was blocked by short-pass filters (ET700SP-2P, Chroma).
Calcium imaging
An organotypic hippocampal slice culture was transferred to the chamber at the two-photon microscope and bathed in oxygenated ACSF containing (in mM): 135 NaCl, 2.5 KCl, 4 CaCl2, 4 MgCl2, 10 Na-HEPES, 12.5 D-glucose, 1.25 NaH2PO4. To identify spines and dendrites responding to electrical stimulation of Schaffer collateral axons (two 0.2 ms paired pulses with an interpulse interval of 40 ms), frame scans (at 15.625 Hz) of oblique dendrites were acquired. After finding a responding spine, user-defined arbitrary line scans (500 Hz) that crossed the responding spines and dendrite were used to quantify the calcium response to stimulation, improving time resolution and signal to noise. Responses to stimulation were acquired at 0.0167 Hz. A stable baseline was acquired for at least twenty minutes. After baseline, 9-phenanthrol (30 μM) was washed into the chamber and responses were acquired for another twenty minutes. Finally, fresh ACSF containing no drugs was washed into the chamber and responses were acquired for a final twenty minutes. For analysis, regions of interest (ROI) were drawn over spines and dendrites in ImageJ and the relative percent change in GCaMP6f green fluorescence was calculated as 100 * (F-F0) / F0 where F is the fluorescence intensity and F0 is the average fluorescence intensity prior to stimulation. Peak amplitude and area under the curve (AUC) were extracted from within the response time-window after smoothing in Matlab with a moving average span of 5. Baseline, drug and washout responses over each twenty minute period were then averaged (Fig. 1).
Immunohistochemistry
Trpm4+/+ and Trpm4-/- mice were anaesthetized with an intraperitoneal injection of 10 mg ml−1 esketamine hydrochloride (Pfizer), 1.6 mg ml−1 xylazine hydrochloride (Bayer) in water (100 µl per 10 g of body weight). Mice were then perfused with 0.1 M phosphate buffer and fixed in 4% paraformaldehyde (PFA). For immunohistochemistry, 40 µm thick free floating sections were washed 3 x 5 min. in 1x PBS followed by 10 min. in 0.5% sodium borohydride (Sigma #S-9125) in 1x PBS. Slices were again washed 3 x 5 min. in 1x PBS. Slices were incubated in blocking solution for 1 hour (0.3% BSA Sigma #A-4503, 10% horse serum Invitrogen #16050-130, 0.3% Triton X-100). Slices were then incubated overnight at 4° C in Carrier + (0.2 % BSA, 1 % horse serum and 0.3% Triton X-100) with primary antibody against TRPM4 (Rabbit polyclonal, 1:200, Alomone ACC-044; rabbit polyclonal, 1:100, Abcam ab104572; goat polyclonal, 1:500, Santa-cruz SC-27540). TRPM4 rabbit anti-serum (targeting the N-terminus), a gift from Dr. J Marc Simard (University of Maryland School of Medicine), was reconstituted in water and used at a concentration of 1:200. The next day slices were washed 3 x 5 min. In 1x PBS. Secondary goat anti-rabbit antibodies coupled to Alexa-488 (Invitrogen Cat. # A-11008, 1:1000) in Carrier + were incubated with slices for 2-5 hours. Slices were then mounted and imaged with a confocal microscope (Leica) for Abcam and Dr. Simard’s antibodies with a 63x oil immersion objective [HCX PLA PO 63x NA 1.32] and Olympus Fluoview FV 1000 with a 60x oil immersion objective [UPLSAPO 60x NA 1.35] for Alomone and secondary only). Excitation/emission spectra and filters for Alexa-488 were selected using the automatic dye selection function of the confocal software. Acquisitions were taken with the same laser intensity and PMT voltages for samples acquired within the same batch of stainings. The antibodies from Alomone and Dr. Simard target the N-terminus and the Abcam antibody targets the C-terminus of TRPM4.
Experimental autoimmune encephalomyelitis (EAE) induction
Male and female Trpm4-/- and their wild-type (Trpm4+/+) littermates were used for EAE (Trpm4(tm1.1Mfre)48) induction at 3-5 months old. We immunized mice subcutaneously with 200 μg MOG35–55 (EP02030, peptides&elephants, Hennigsdorf, Germany) in complete Freund's adjuvant (BD) containing 2 mg ml−1 Mycobacterium tuberculosis (H37Ra, BD). We injected 200 ng pertussis toxin (EMD Millipore) intraperitoneal on the day of immunization and 48 h later. We scored the mice daily for clinical signs by the following system: 0, no clinical deficits; 1, tail weakness; 2, hind limb paresis; 3, partial hind limb paralysis; 3.5, full hind limb paralysis; 4, full hind limb paralysis and forelimb paresis; 5, premorbid or dead. Hippocampal slices were prepared at scores ≥ 3 or shortly after the score began to decrease 11-18 days after EAE induction (see Electrophysiology, below).
Expression analysis of Trpm4 in cortical neurons from healthy and EAE mice
Healthy and EAE mice in the chronic phase of the disease were sacrificed with CO2. Cortices were homogenized in EZ buffer (Sigma) and the 500 g pellet washed once. Nucleic pellets were resuspended in Nuclei Buffer (NB, 430 mM sucrose, 2 mM MgCl2, 25 mM KCl, 65 mM glycerophosphate, 5% glycerol, 1 mM EDTA, 1% BSA) and washed twice. Final pellets were resuspended in NB + 0.2U/µl Ribolock (Thermofisher) and passed through MACS filters. Nuclei were stained using anti-NeuN-AF647 (1:1000) and PI (1:2000) and NeuN positive nuclei were sorted into 5 ml tubes. Total RNA was extracted with RNeasy Mini Kit (Qiagen) and mRNA reverse transcribed to obtain cDNA for quantitative PCRs.
Electrophysiology
Female and Male Trpm4+/+ and Trpm4-/- mice were sacrificed between 2 and 6 months of age. Mice were briefly anesthetized with 80% CO2 /20% O2 prior to decapitation. The brain was dissected and immersed in ice-cold solution containing (in mM): 110 choline chloride, 25 NaHCO3, 25 D-glucose, 11.6 sodium L-ascorbate, 7 MgSO4, 1.25 NaH2PO4, 2.5 KCl, 0.5 CaCl2, continuously bubbled with 95% O2 and 5% CO2, pH 7.4. Coronal slices (300 μm thick) were cut using a vibratome and were allowed to recover at 34 °C for 30 minutes in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 26.2 NaHCO3, 11 D-glucose, 1 NaH2PO4, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2. Slices were then kept in the same solution at room temperature until used. After at least 1 hour at room-temperature, a slice was placed in the recording chamber and continuously perfused with oxygenated ACSF supplemented with D-Serine (30 μM) to occupy the second agonist binding site of NMDA receptors, bicuculline-methochloride (10 μM) to block GABAA receptors and SK-channels 37 and 0.1% DMSO (see below). Recordings were performed using either a Multiclamp 700B or an Axopatch 200B amplifier (Molecular Devices). Recordings were controlled and digitized using National Instruments A/D boards and Ephus software in the Matlab environment 51. Current-clamp recordings from CA1 pyramidal cells were performed at 23-25 °C (Figures 1-3) or at 30-32 C° (Figure 4). Patch pipettes with a tip resistance of 3-5 MΩ were filled with (in mM): 135 K-gluconate, 10 HEPES, 4 MgCl2, 4 Na2-ATP, 0.4 Na-GTP, 10 Na2-phosphocreatine, 3 L-ascorbic acid and 3 QX-314 chloride (to block voltage-gated sodium channels). For recordings done in healthy Trpm4+/+ and Trpm4-/- mice, the experimenter was not blind to genotype. Recordings made from EAE mice were performed blind to the genotype. To evoke excitatory postsynaptic potentials (EPSPs), a monopolar electrode was placed in the stratum radiatum and 0.2 ms pulses were delivered using an ISO-Flex stimulator (A.M.P.I.). The stimulation intensity was set to produce an EPSP of approximately 40 mV. After a stable baseline was achieved (at least five minutes), a TRPM4 antagonist, either 9-phenanthrol (30 μM) or glibenclamide (20 μM), was washed into the bath. Both 9-phenanthrol and glibenclamide were dissolved in DMSO and resulted in a final 0.1% DMSO total concentration in the ACSF. An equivalent concentration of DMSO was included in the drug-free ACSF. Trials were acquired at 0.08 - 0.1 Hz.
Analysis & Statistics
Calcium imaging experiments were analyzed using ImageJ and repeated-measures ANOVA was used for statistical analysis. Electrophysiology analysis was completed in Matlab. Individual trials were then averaged into one minute bins (Figs. 3 b-c, 4 b-c, 6 b-c, 7 b-c). For statistics, averages of one minute bins were made from the last five minutes of baseline and within 15-22 minutes after washing in the drug (Figs. 3 d-h, 4 d-h, 6 d-h, 7 d-h). Analysis of electrophysiological recordings from EAE animals was conducted blind. All statistics were performed using Graphpad Prism (version 8). For paired analyses, the Wilcoxon matched pairs signed rank test was used. For analysis of the role of genotype and clinical score, a two-way ANOVA was used for each parameter. p less than 0.05 was considered significant.