Axonal GABAA stabilises excitability in unmyelinated sensory axons secondary to NKCC1 activity

GABA depolarized sural nerve axons and increased the electrical excitability of C-fibres via GABAA R. Axonal excitability responses to GABA increased monotonically with the rate of action potential firing. Action potential activity in unmyelinated C-fibres is coupled to NKCC1 loading of axonal chloride. Activation of axonal GABAA R stabilised C-fibre excitability during prolonged low frequency (2.5 Hz) firing. NKCC1 maintains intra-axonal chloride to provide feed-forward stabilisation of C-fibre excitability and thus support sustained firing. ABSTRACT: GABAA R mediated depolarisation of DRG axonal projections in the spinal dorsal horn is implicated in pre-synaptic inhibition. Inhibition, in this case, is predicated on an elevated intra-axonal chloride concentration and a depolarizing GABA response. We report here that the peripheral axons of DRG neurons are also depolarised by GABA and this results in an increase in the electrical excitability of unmyelinated C-fibre axons. GABAA R agonists increased axonal excitability while GABA excitability responses were blocked by GABAA R antagonists and were absent in mice lacking the GABAA R β3 subunit selectively in DRG neurons (AdvillinCre or snsCre ). Under control conditions, excitability responses to GABA became larger at higher rates of electrical stimulation (0.5 - 2.5 Hz). However, during NKCC1 blockade electrical stimulation rate did not affect GABA response size, suggesting that NKCC1 regulation of axonal chloride is coupled to action potential firing. To examine this, activity-dependent conduction velocity slowing (ADS) was used to quantify C-fibre excitability loss during 2.5 Hz challenge. ADS was reduced by GABAA R agonists and exacerbated by either GABAA R antagonists, β3 deletion or NKCC1 blockade. This illustrates that activation of GABAA R stabilises C-fibre excitability during sustained firing. We posit that NKCC1 acts in a feed-forward manner to maintain an elevated intra-axonal chloride in C-fibres during ongoing firing. The resulting chloride gradient can be utilised by GABAA R to stabilise axonal excitability. The data imply that therapeutic strategies targeting axonal chloride regulation at peripheral loci of pain and itch may curtail aberrant firing in C-fibres.