Intracellular Ca2+ 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 Ca2+ imaging method in adult rat spinal cord slices without complex genetic modifications and characterize primary afferent-evoked intracellular Ca2+ responses in spinal dorsal horn neurons.

L5 lumbar spinal cord slices from adult rats were stained with a Ca2+ indicator. The relationship between intracellular Ca2+ signals and electrophysiological responses induced by dorsal root stimulation was examined. Additionally, the effects of analgesics, anesthetics, and hyperalgesics on the Ca2+ responses were analyzed.

Monophasic intracellular Ca2+ 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 Ca2+ responses coincided with action potential generation, whereas the falling phase did not. Dorsal root stimulation-induced Ca2+ 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 Ca2⁺ responses.

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.