Abstract
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.
Highlights
It has been established that neurons in the lamina X of the spinal cord play an important role in somatosensory integration (Ness and Gebhart, 1987), visceral nociception (Lantéri-Minet et al, 1993, 1995; Cervero and Laird, 2004; Eijkelkamp et al, 2007), autonomic regulation (Deuchars and Lall, 2015) and modulation of motoneuron outputs (Bertrand and Cazalets, 2011)
We observed a small number of probidium iodide (PI)-positive cells in the lamina X located close to the surface of the tissue, whereas no PI-positive particles were detected in the deeper layers (>20 μm below the surface, Figure 3A)
The functional properties of lamina X neurons in ex-vivo spinal cord preparations were assessed by electrophysiological recordings made from individual cells
Summary
It has been established that neurons in the lamina X of the spinal cord play an important role in somatosensory integration (Ness and Gebhart, 1987), visceral nociception (Lantéri-Minet et al, 1993, 1995; Cervero and Laird, 2004; Eijkelkamp et al, 2007), autonomic regulation (Deuchars and Lall, 2015) and modulation of motoneuron outputs (Bertrand and Cazalets, 2011). The resolved cell visualization by this technique in ex-vivo spinal cord preparation was numerously confirmed in further studies of sensory input integration in the superficial dorsal horn (Szucs et al, 2013; Luz et al, 2015; Fernandes et al, 2016) Given all these benefits for the lamina X research, we used a combination of LED oblique illumination with ex vivo spinal cord preparation implementing a longitudinal hemisection at the sagittal midline (Wilson et al, 2007, 2010; Hinckley et al, 2010; Meyer et al, 2010; Bui et al, 2013; García-Ramírez et al, 2014) for direct access to the area around the central canal
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