INTRODUCTION: The area around the central canal (or lamina X) is the most enigmatic lamina of the spinal cord. It is established that somatosensory integration, visceral nociception, autonomic regulation and modulation of motoneuron output are mediated by lamina X neurons, nevertheless their electrophysiological properties and functional connectivity are largely unknown. AIM(S): Electrophysiological investigations of lamina X neurons are hampered primarily by technical challenges in the preparation procedures, so we aimed to develop a reliable technique for functional studies of the neurons around the central canal. METHOD(S): Our approach relies on the method of oblique LED illumination for cell visualization in thick blocks of tissue, developed by Prof. Safronov and colleagues. RESULTS: We have developed simple, fast (5–10 min) and reliable lamina X preparation technique, that preserves whole spinal cord architecture including dorsal and ventral roots. In combination with oblique LED illumination, allowing cell visualization in thick blocks of tissue, our preparation enables visually guided patch clamp of lamina X neurons coupled with simultaneous stimulation of either afferent or efferent fibers with suction electrode. Our preparation also permits the usage of fluorescent approaches which might additionally boost the relevance of lamina X studies. First, injections of retrograde dye Fluorogold (or its analogues) into the peritoneum or lateral thalamus selectively label sympathetic preganglionic or projection neurons respectively, giving the possibility to work with these specific neuronal populations. Second, calcium transients might be recorded after loading the cells with either cell-impermeable (through the patch pipette) or cell-permeable (AM ester) Fura 2 calcium dye. CONCLUSIONS: We introduce a new methodology that combines electrophysiological and fluorescent approaches for the research of lamina X neuron functioning in physiological and pathological conditions. FINANCIAL SUPPORT: Supported by IBRO.
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