Glass capillaries

Cells were transfected in vivo through in utero electroporation, as described previously (Inoue et al., 2012 (link)). Briefly, plasmids carrying monomeric red fluorescent protein (mRFP) downstream of a CAG promoter (Addgene, MA, USA) were prepared using the EndoFree Plasmid Kit (Qiagen, Hilden, Germany). Pregnant mice and rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) at E14.5 and E15.5, respectively, and their uterine horns were exposed. Plasmid DNA was dissolved in phosphate buffered saline (PBS) at a final concentration of 0.5 μg/μl with Fast Green (final concentration 0.05% [v/v]). Plasmids were injected into the lateral ventricle using a glass micropipette and a controlled pipette system (IM-30, Narishige, Tokyo, Japan). The micropipettes were generated from glass capillaries (outer diameter 1.0 mm; Harvard Apparatus, South Natick, MA, USA) that were pulled using a P-97 micropipette puller (Sutter Instrument Co., Novato, CA, USA). Electric pulses were produced by an electroporator (CUY21EDIT; NepaGene, Ichikawa, Japan) and delivered by a round plate forceps-type electrode with a 5-mm diameter (CUY650P5; NepaGene). Electric pulses (43 V, 50 ms) were applied five times at intervals of 950 ms. The uterine horns were then returned to the abdomen.

Glass capillaries [1.2 mm O.D. × 0.94 mm I.D., Harvard Apparatus (BF‐120‐94‐10)] were pulled into micropipettes using a micropipette puller (P‐97 Flaming Brown, Sutter instruments, Novato, CA). See User Manual and guidelines therein.

Glass capillaries (Harvard Apparatus (Holliston, MA, USA) Glass capillaries with 780-μm inner diameter) are pulled using a needle puller and micro forged to forge a holding pipette and an injection needle. The resulting injection needles are filled with mRNA solution diluted to 1 μg/μL in injection buffer (5 mM Tris-HCl pH = 7.4, 0.1 mM EDTA). The filled needle is positioned on a micromanipulator (Narishige MMO-4) and connected to a positive pressure pump (Eppendorf FemtoJet 4i). Embryos are placed in FHM drops covered with mineral oil under Leica (Wetzlar, Germany) TL Led microscope. Two-cell stage embryos were injected while holding with holding pipette connected to a Micropump CellTram Oil.

Before proceeding with the neuronal recordings, mice were allowed several days to get used to the electrophysiology setup, which was located in a dedicated room and contained a similar eyeblink apparatus within a light-isolated Faraday cage. During this period, trained mice received daily training sessions consisting of 100 trials for a maximum of 4 days to ensure at least 75% CRs. Neurons were recorded with glass capillaries (Ø = 2 mm, Harvard Apparatus) that were heated and pulled to obtain a 2- to 5-μm tip and filled with a 2M NaCl solution. The electrode was lowered into lobule simplex (Heiney et al., 2014 (link), Van Der Giessen et al., 2008 (link)) using a one-axis hydraulic manipulator (MMO-220A, Narishige). The obtained electrical signal was pre-amplified and digitized at a sampling frequency of 25 kHz using a TDT System 3 electrophysiology workstation. When a recording was stable, the animal was subjected to blocks of paired trials. Purkinje cells were identified by the presence of complex spikes. When we encountered cells that were located no further than 100 μm from Purkinje cells, had relatively low firing rates, and did not show complex spikes, we would record them if they seemed to modulate within the ISI. Offline analysis was used to verify these putative MLIs (see below).

Glass capillaries (Harvard Apparatus, 30-0053) were pulled and fire polished such that the end of the pipette tip was melted and sealed to a ~60μm rounded, bulbous end. Pipettes were attached to a motorized micromanipulator (Sutter Instrument, MP-225) used to maneuver the pipette in X, Y, and Z directions. Prior to mechanical touch of single cells, cells were brought into focus and the glass pipette was visualized using light microscopy in order to closely approach a single cell in a cell monolayer with the pipette. Then, 200 time series images with 2 second frame rate were collected using simultaneous DIC and Fluo-4 imaging. The pipette was then brought to the point of cell contact, as confirmed using visualization of pipette focus in DIC and initiation of Fluo-4 fluorescence. The pipette was then retracted for the remainder of the time series.

Baculovirus-mediated transduction of axolotl tissues was performed as described elsewhere^{25 (link)}. Briefly, Baculovirus was pseudotyped with vsv-ged gene, inserted into the rescue vector under the baculovirus polyhedrin promoter. Genes of interest (Tig1^wt-T2a-EGFP and Tig1^P155A-T2a-EGFP) were cloned into the baculovirus rescue vector under the CMV promoter using standard restriction enzyme methods. Pseudotyped baculoviruses carrying the gene of interest were generated by co-transfection of the rescue vector together with replication-incompetent baculovirus DNA into a modified Spodoptera frugiperda cell line (expresSF+, ProteinSciences Corporation). Upon culture expansion, recombinant baculoviruses were collected, concentrated and purified. Baculovirus titre was assessed by end-point dilution assay in SF-9 Easy Titer cells, a modified Spodoptera frugiperda cell line expressing a reporter egfp gene under the control of the baculovirus polyhedrin promoter.
Expression of the genes of interest in axolotl blastemas was achieved by injecting the corresponding baculovirus (2 µl each; Bv titre: c. 1 × 10¹²) into the upper arm blastemas as indicated. Injections were performed using a glass capillary needle pulled on a micropipette puller (Sutter Instrument Co. model P-97). Glass capillaries (1.2 mm OD, 0.9 mm ID borosilicate glass) were acquired from Harvard Apparatus.

A combined whole-cell/perforated patch dual voltage-clamp method was used to measure gap junctional conductance between cell pairs^{17 (link),73 (link)}. Coverslips with cells expressing Cx43, or Cx50, were transferred to a recording chamber on an inverted microscope with a fluorescence imaging system. Cells were perfused with a solution containing (in mM) NaCl, 150; KCl, 10; CaCl₂, 2; HEPES, 5 (pH 7.4); glucose, 5; CsCl, 2; and BaCl₂, 2. Patch clamp electrodes were filled with a solution containing (in mM) K⁺ aspartate^-, 120; NaCl, 10; MgATP, 3; HEPES, 5 (pH 7.2); EGTA, 10. For electrodes used in a perforated patch configuration, the solution was supplemented with 30–50 μM β-escin^{74 (link)}. Patch clamp electrodes were pulled from glass capillaries (Harvard Apparatus, Holliston, MA) with a horizontal puller (DMZ-Universal, Zeitz-Instrumente, Martinsried, Germany). The measured resistance of the electrodes was 2–5 MΩ. Gap junctional conductance was measured throughout each experiment, beginning when both cells in the pair had been successfully patched.