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RNA interference appears as a technique of choice to identify gene candidate or to evaluate gene function. To date, a main problem is to achieve high transfection efficiencies on native cells such as adult neurons. In addition, transfection on organ or mass culture does not allow to approach the cellular diversity. Dorsal root ganglia are composed with several cell types to convey somato-sensory sensations. Single-cell electroporation is the most recent method of transfection that allows the introduction into cells, not only dyes or drugs, but also large molecules such plasmid DNA expression constructs. In the present study, the application of the RNA interference technique with the use of single-cell electroporation was evaluated in primary culture of adult sensory neurons. With the use of fluorescent dextran as a co-transfectant, we first determined the non-specific siRNA concentration leading to cell death. Efficacy of siRNA at the non-toxic concentration was demonstrated at the protein level by extinction of GFP fluorescence in actin-GFP neurons and by the inhibition of the intracellular Cl- concentration increase following activation of the membrane co-transporter Na+-K+-2Cl- in regenerating axotomized sensory neurons. Altogether, these data show that delivery of siRNAs by single-cell electroporation leads to the induction of functional RNA interference.

In addition to its inhibitory action, reports have shown that, in sensory neurons, GABA can be responsible for excitatory effects leading to painful behavior. The cellular mechanisms for these excitatory effects remain largely unknown. Although the high intracellular chloride concentration allows GABA(A) receptor activation to depolarize all adult sensory neurons, we show that GABA, acting through GABA(A) receptors, can generate, in vitro, action potential and intracellular Ca(2+) increase only in a subset of neurons expressing a prominent T-type Ca(2+) current. When recorded from Cav3.2(-/-) mice, T-type Ca(2+) current was totally abolished in this morphologically identified subset of neurons and GABA(A) receptors activation did not induce electrical activity nor intracellular Ca(2+) increase. In addition to gene inhibition, pharmacological analysis of Ca(2+) channel subunits shows the amplifying role of T-current in GABA(A) current-induced membrane depolarization and the involvement of both T-current and high voltage activated Ca(2+) current in GABA(A)-induced intracellular Ca(2+) increase. Altogether, these data establish that the Cav3.2/alpha1H, T-current is responsible for GABA-induced cell excitability and intracellular Ca(2+) increase. Our results reveal a positive cross-talk between T-channel and GABA(A) receptor in adult sensory neurons and indicate that Cav3.2/alpha1H, T-type Ca(2+) channel may be the molecular determinant for excitatory effects of GABA in peripheral somatosensory system.

Peripheral nerve section promotes regenerative, elongated neuritic growth of adult sensory neurons. Although the role of chloride homeostasis, through the regulation of ionotropic GABA receptors, in the growth status of immature neurons in the CNS begins to emerge, nothing is known of its role in the regenerative growth of injured adult neurons. To analyze the intracellular Cl- variation after a sciatic nerve section in vivo, gramicidin perforated-patch recordings were used to study muscimol-induced currents in mice dorsal root ganglion neurons isolated from control and axotomized neurons. We show that the reversal potential of muscimol-induced current, E(GABA-A), was shifted toward depolarized potentials in axotomized neurons. This was attributable to Cl- influx because removal of extracellular Cl- prevented this shift. Application of bumetanide, an inhibitor of NKCC1 cotransporter and E(GABA-A) recordings in sensory neurons from NKCC1-/- mice, identified NKCC1 as being responsible for the increase in intracellular Cl- in axotomized neurons. In addition, we demonstrate with a phospho-NKCC1 antibody that nerve injury induces an increase in the phosphorylated form of NKCC1 in dorsal root ganglia that could account for intracellular Cl- accumulation. Time-lapse recordings of the neuritic growth of axotomized neurons show a faster growth velocity compared with control. Bumetanide, the intrathecal injection of NKCC1 small interfering RNA, and the use of NKCC1-/- mice demonstrated that NKCC1 is involved in determining the velocity of elongated growth of axotomized neurons. Our results clearly show that NKCC1-induced increase in intracellular chloride concentration is a major event accompanying peripheral nerve regeneration.