Functional properties of lamina X neurons in the spinal cord remain unknown despite
the established role of this area for somatosensory integration, visceral nociception,
autonomic regulation and motoneuron output modulation. Investigations of neuronal
functioning in the lamina X have been hampered by technical challenges. Here we
introduce an ex-vivo spinal cord preparation with both dorsal and ventral roots still
attached for functional studies of the lamina X neurons and their connectivity using an
oblique LED illumination for resolved visualization of lamina X neurons in a thick tissue.
With the elaborated approach, we demonstrate electrophysiological characteristics
of lamina X neurons by their membrane properties, firing pattern discharge and
fiber innervation (either afferent or efferent). The tissue preparation has been also
probed using Ca2+ imaging with fluorescent Ca2+ dyes (membrane-impermeable or
-permeable) to demonstrate the depolarization-induced changes in intracellular calcium
concentration in lamina X neurons. Finally, we performed visualization of subpopulations
of lamina X neurons stained by retrograde labeling with aminostilbamidine dye to
identify sympathetic preganglionic and projection neurons in the lamina X. Thus, the
elaborated approach provides a reliable tool for investigation of functional properties
and connectivity in specific neuronal subpopulations, boosting research of lamina X of
the spinal cord