Diltiazem

The protocol for imaging spontaneous Ca²⁺ fluxes in primary cultures of mouse midbrain neurons has been previously published (Bancroft & Srinivasan, 2020 (link)). Briefly, cultures were placed in a gas free recording buffer containing (mM): 154 NaCl, 5 KCl, 2 CaCl₂, 0.5 MgCl₂, 5 D-glucose, 10 HEPES, pH adjusted to 7.4 with NaOH (all purchased from Sigma-Aldrich). Imaging was performed using a confocal microscope (Fluoview 1200, Olympus) with a ×40, 0.8 NA water-immersion objective (Olympus). We used a 488-nm laser line to excite GCaMP6f and eGFP, and a 569-nm laser line for tdTomato and RCaMP. The imaging frame was clipped to allow for a sampling rate of 1 frame per second. For all experiments, spontaneous activity was imaged for 300 s, followed by peptide/drug application. Recording buffers were bath perfused using a peristaltic pump at a rate of 2 ml/min. For each field of view imaged, a corresponding z-stack of both GCaMP6f and TH-tdTomato expression was captured and co-localization was used to identify TH⁺ cells. Diltiazem was purchased from Tocris (Minneapolis, MN), Mibefradil and 4-aminopyridine (4-AP) were purchased from Sigma-Aldrich, and cyclopiazonic acid (CPA) was purchased from Abcam.

Diltiazem, verapamil, and 2-aminoethoxydiphenylborane (2-APB), were purchased from Tocris Biosciences. Lithium chloride was purchased from Sigma. Drugs were dissolved in sterile water or DMSO. Prior to drug studies, cells were distributed into multiple smaller plates, and treated in parallel, under identical conditions, allowing for within sample matching. To ensure even application, concentrated lithium (1000×) was added to growth media and distributed from a common drug solution into each culture plate. Additional drugs were handled in a similar manner, alone or with lithium. Vehicle controls for solvents were used when indicated.