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New approach for studying lamina X neurons of the spinal cord

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Krotov V., Tokhtamysh A., Dromaretskii A., Sheremet E., Belan P., Voitenko N.
Acta Neurobiologiae Experimentalis
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2017
|
tom 77
|
nr Suppl.1
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.
2

Subpopulation of c-fiber-activated lamina I projection neurons with unique input-output characteristics for the nmdar-dependent nociceptive coding

84%
Agashkov K., Krotov V., Krasniakova M., Shevchuk D., Liashenko A., Safronov B., Voitenko N., Belan P.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Spinal lamina I projection neurons (PNs) are key elements of the pain processing system, which relay peripheral input to supraspinal structures generating sensation of pain. The population of PNs is small (~5% of lamina I neurons) but very heterogeneous according to their intrinsic and synaptic properties. AIM(S): Investigate mechanisms of acute nociception encoding and evaluate input-output characteristics of lamina I PNs. METHOD(S): Whole-cell recordings from lamina I PNs retrogradely-labeled via the lateral PB area in an intact spinal cord preparation with attached dorsal roots. RESULTS: We identified a specific group of PNs (16%) with unique properties. In these PNs (SB-PNs), a stimulation of nociceptive afferents evoked gradual strength‑dependent amplification of afferent input expressed as an increase in the number of generated APs. Upon a root stimulation at C‑fiber‑intensity, the SB‑PN group generated more than 80% of spikes of the entire population of PNs, thus, being the major group of PNs codifying acute pain sensation. We have also identified several mechanisms of this nociceptive input amplification. First, SB‑PNs are intensively innervated by the high‑threshold A delta‑ and C‑afferents providing robust and reliable spike generation. Second, the nociceptive input was amplified by intrinsic bursting capabilities of SB PNs. Third, the afferent input was prolonged (to 0.-1.5 s) and potentiated (to -45 mV to -20 mV) by the NMDAR-dependent synaptic component forming intrinsic plateau potentials generated by SB‑PNs. The afferent stimulation increased, for several seconds, spontaneous excitatory drive to SB-PNs that became suprathreshold and evoked series of spikes. CONCLUSIONS: We have described a new type of lamina I PNs efficiently transmitting the main part of primary nociceptive input to supraspinal structures playing an important role in acute pain generation. A complex interplay between synaptic, intrinsic and network activities underlies unique nociceptive encoding features of this group of PNs. FINANCIAL SUPPORT: Supported by IBRO.
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