Experimental and modeling studies of motor network excitability of neonatal rat spinal cord in vitro

The present study has investigated certain mechanisms that control network excitability in the neonatal rat spinal cord in vitro, by means of intracellular recordings from single motoneurons, extracellular recordings from ventral roots, whole-cell clamp recordings from interneurons and modeling of electrical behavior of single motoneurons. Sulphonylurea drugs have been used to investigate the role of ATP sensitive K+ channels (KATP) in controlling the excitability of central neurons (Crepel et al., 1993; Mironov et al., 1998). In certain brain areas, which possess intrinsic electrical rhythmicity like the brainstem respiratory centers and associated nuclei, KATP channels pace the frequency of bursting and the duration of single bursts (Pierrefiche et al., 1996; Sharifullina et al., 2005). Because this mechanism relies on cyclic intracellular consumption and neosynthesis of ATP, it represents a powerful process to link neuronal electrical discharges to metabolic activity. In the spinal cord, inherent rhythmicity can be readily observed in locomotor networks which express a stable pattern of regular electrical discharges (Kiehn & Butt, 2003). Since we have previously shown that the ATP-dependent Na+-K+ pump is a major controller of spinal network bursting (Rozzo et al., 2002), we wondered whether periodic changes in intracellular ATP might control the activity of KATP conductances and thus limit neuronal excitability. One simple functional test for this possibility was to apply a KATP channel blocker like glibenclamide (Bryan et al., 2004) and to monitor resultant changes in network responses. This approach soon led us to unexpected results, which raised the issue of a novel mechanism to control spinal network excitability. Because sulphanylurea drugs are also efficient blockers of cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein involved in Cl- transport, it seemed interesting to explore the hypothesis that the neonatal rat spinal cord could express this proteins whose function might be expected to regulate Cl- dependent transmission operated by GABA and glycine. Application of glibenclamide (or tolbutamide), or the CFTR inhibitors (DPC or thiazolidinone CFTRinh-172) led to membrane potential hyperpolarization, input resistance increase and larger spike overshoot. These data were accompanied by a negative shift in the reversal potential of the GABA and glycine mediated effects. RT-PCR analysis and Western blotting showed CFTR gene function and protein expression in support of a functional role of CFTR to control Cl- homeostasis and neuronal excitability. Indeed, glibenclamide affected generation of spinal reflexes and rhythmic bursting. The present data allow us to formulate a new basis for the role of GABA and glycine as inhibitory neurotransmitters at an early postnatal stage of development. The effects of CFTR channel blockers were further examined by means of modeling the electrical behavior of motoneurons. To this end a 3D structure of motoneurons was simulated using a stochastic algorithm the design which was based on known morphological parameters. Discrete surface distribution of voltage-sensitive ion channels and passive membrane changes enabled simulation of experimental findings. The model suggested a somatic localization of CFTR channels providing hypothetical possibility of colocalization with GABA/glycine receptors and thus regulation of their functionality by Cl- homeostasis regulation.