2.5. Simultaneous electrophysiology and two‐photon calcium imaging of lamina I neurons

Spinal cord lamina I neurons were recorded from in current‐clamp configuration, with simultaneous two‐photon calcium imaging as previously described (Harding et al., ²⁰²⁰). Briefly, slices were placed under a Zeiss 710 NLO system equipped with an AxioExaminer Z1 (Zeiss, Germany), and neurons were visualized under infrared‐differential interference contrast optics. Patch‐clamp recordings were made with recording pipettes of 7–10 MΩ, pulled by a Sutter P97 puller (Sutter Instruments, Navato, CA, USA). The external recording solution (artificial CSF) and perfusion speed was as above. The internal current‐clamp patch pipette solution consisted of (mM) 112 K‐gluconate, 8 KCl, 10 HEPES, 4 Mg‐ATP, 0.3 Na₂‐ATP, 10 phosphocreatine, 0.3 EGTA, 0.04 Alexa Fluor‐594, 0.11 Oregon Green Bapta‐1 (pH 7.3, 290 mOsm). Neurons were allowed to dialyze for 20–30 min before imaging to allow the fluorescent dye concentration to equilibrate. All recordings were performed between 24°C and 28°C. Current‐clamp recordings were performed as above. Bridge balance and pipette capacitance compensation were performed in all neurons. In all experiments, neurons were held between −70 and −80 mV (typically 0 to −20 pA holding current injection). Single action potentials were evoked with a 5 ms current injection of between 150 and 400 pA. Each recording was 15 s in duration, with the experimental current injection at 2 s into recording. A minimum of 15 s was given between the end of one recording and the beginning of the next to allow the neuron to return to baseline.

Two‐photon dual excitation of OGB‐1 (110 μM in recording pipette) and AF‐594 (40 μM in recording pipette) was achieved using a Coherent Chameleon Ultra Ti:Sapphire laser tuned to 800 nm (Coherent, USA). OGB‐1 and AF‐594 fluorescence were split based on emission spectra using NDD filter cubes (500–550 nm, 565–610 nm; Carl Zeiss Microscopy, Germany) and sent into NDD detectors (Carl Zeiss Microscopy, Germany). Two‐photon images were obtained using a 20× water‐immersion objective lens (Carl Zeiss Microscopy, Germany) and the Zen 2009 acquisition program (Carl Zeiss Microscopy, Germany). Laser power was kept between 0.3% and 0.7%, and gain was restrained to 650–850 for all calcium imaging experiments. Fluorescence data were acquired using line scan acquisition (1024 pixels × 1 pixel line scan, 2× averaging) at a rate of 133 Hz and saved as LSM files from Zen 2009 (Carl Zeiss Microscopy, Germany). Calcium imaging data were analysed as previously described, utilizing a custom‐made, semi‐automated MATLAB toolbox (Mathworks, USA) for analysis, entitled CIAT (Calcium Imaging Analysis Toolbox) (Harding et al., ²⁰²⁰). Use of the semi‐automated analysis toolbox, Calcium Imaging Analysis Toolbox, which automatically calculates peak response from averaged calcium imaging trials, was designed to remove experimental bias. For data in Figure 2, drug wash‐in experiments were randomized such that Z944 and DMSO vehicle trials were completed in alternating order where possible. Statistical analysis within this figure was performed on groups with an n > 5, of unequal size due to one neuron being removed for photobleaching.

Activity‐induced calcium transients in lamina I neurons are reduced by Z944. (a) Flattened projection of a two‐photon z‐stack of a lamina I neuron filled with Alexa Fluor‐594 via a patch pipette. White arrows represent line scan trajectory during calcium imaging. Scale bar represents 20 μm. (b) Left: Single action potentials induced by current injection (5 ms, 200 pA) from a membrane potential of −70 mV induce calcium transients in the somata and dendrites of lamina I neurons (black). Right: Recordings remain stable after vehicle administration (green, right). Traces are an average of eight neurons for both somatic and dendritic compartments. (c) Wash‐in of Z944 (2 μM) greatly reduces calcium transients in both the somata and dendrites (dark blue) and decreases action potential afterdepolarization (insets). Traces are an average of seven neurons for both somatic and dendritic compartments. For b and c, action potential traces are representative examples from a single neuron, calcium transients are average responses, ±SEM. For b and c, electrophysiology scale bar y axis = 30 mV, x axis = 2 s. Inset scale bar y axis = 30 mV, x axis = 100 ms. Calcium imaging scale bar y axis = ΔG/R 0.04, x axis = 2 s. (d) Quantification of the percent remaining peak calcium transient, as displayed in b and c after the administration of Z944 or vehicle. Comparisons performed with Student's unpaired t‐tests (P = .05 for soma, P <.05 for dendrite, n = 8 neurons for vehicle and 7 neurons for Z944 for both compartments). (e) Quantification of the percent remaining action potential area under the curve after administration of Z944 or vehicle (P = .05, Student's unpaired t‐test, n = 7 neurons for Z944 and vehicle conditions). *P < .05 for all t‐tests. All error bars represent SEM