Visualization of primary afferent-evoked excitation of spinal dorsal horn neurons using an intracellular Ca imaging technique in adult rat spinal cord slices.

Purpose: Intracellular Ca imaging is a valuable tool for studying neuronal activity; however, its application in the spinal cord of mature animals remains underdeveloped. This study aimed to establish an intracellular Ca imaging method in adult rat spinal cord slices without complex genetic modifications and characterize primary afferent-evoked intracellular Ca responses in spinal dorsal horn neurons.

Methods: L5 lumbar spinal cord slices from adult rats were stained with a Ca indicator. The relationship between intracellular Ca signals and electrophysiological responses induced by dorsal root stimulation was examined. Additionally, the effects of analgesics, anesthetics, and hyperalgesics on the Ca responses were analyzed.

Results: Monophasic intracellular Ca responses were observed with A-fiber intensity stimulation, while biphasic responses were noted with C-fiber intensity stimulation. These responses were not photobleached after repeated measurements (n = 12). The rising phase of Ca responses coincided with action potential generation, whereas the falling phase did not. Dorsal root stimulation-induced Ca responses were significantly suppressed by morphine (10 μM, 43.9 ± 4.9% of control, n = 8) but not by remimazolam (10 μM, 98.0 ± 2.0% of control, n = 8). Conversely, bicuculline (40 μM, 288.4 ± 48.4% of control, n = 10) and high concentrations of tranexamic acid (3, 10 mM, 132.6 ± 19.9%, 152.6 ± 25.3%, respectively, n = 8) significantly enhanced Ca⁺ responses.

Conclusion: This is a simple and effective approach to examining the effects of drugs that target the spinal cord and investigating nociceptive transmission and modulation mechanisms in the spinal dorsal horn.