Our previous studies found that nerve growth factor (NGF), via ceramide, enhanced the number of action potentials (APs) evoked by a ramp of depolarizing current in capsaicin-sensitive sensory neurons. Ceramide can be metabolized by ceramidase to sphingosine (Sph), and Sph to sphingosine 1-phosphate (S1P) by sphingosine kinase. It is well established that each of these products of sphingomyelin metabolism can act as intracellular signalling molecules. This raises the question as to whether the enhanced excitability produced by NGF was mediated directly by ceramide or required additional metabolism to Sph and/or S1P. Sph applied externally did not affect the neuronal excitability, whereas internally perfused Sph augmented the number of APs evoked by the depolarizing ramp. Furthermore, internally perfused S1P enhanced the number of evoked APs. This sensitizing action of NGF, ceramide and internally perfused Sph was abolished by dimethylsphingosine (DMS), an inhibitor of sphingosine kinase. In contrast, internally perfused S1P enhanced the number of evoked APs in the presence of DMS. These observations support the idea that the metabolism of ceramide/Sph to S1P is critical for the sphingolipid-induced modulation of excitability. Both internally perfused Sph and S1P inhibited the outward K + current by 25-35% for the step to +60 mV. The Sph-and S1P-sensitive currents had very similar current-voltage relations, suggesting that they were likely to be the same. In addition, the Sph-induced suppression of the K + current was blocked by pretreatment with DMS. These findings demonstrate that intracellular S1P derived from ceramide acts as an internal second messenger to regulate membrane excitability; however, the effector system whereby S1P modulates excitability remains undetermined.

Neurotrophin-mediated signalling cascades can be initiated by activation of either the p75 neurotrophin receptor (p75 NTR ) or the more selective tyrosine kinase receptors. Previously, we demonstrated that nerve growth factor (NGF) increased the excitability of sensory neurons through activation of p75 NTR to liberate sphingosine 1-phosphate. If neurotrophins can modulate the excitability of small diameter sensory neurons through activation of p75 NTR , then brain-derived neurotrophic factor (BDNF) should produce the same sensitizing action as did NGF. In this report, we show that focally applied BDNF increases the number of action potentials (APs) evoked by a ramp of depolarizing current by reducing the rheobase without altering the firing threshold. This increased excitability results, in part, from the capacity of BDNF to enhance a tetrodotoxin-resistant sodium current (TTX-R I Na ) and to suppress a delayed rectifier-like potassium current (I K ). The idea that BDNF acts via p75 NTR is supported by the following observations. The sensitizing action of BDNF is prevented by pretreatment with a blocking antibody to p75NTR or an inhibitor of sphingosine kinase (dimethylsphingosine), but not by inhibitors of tyrosine kinase receptors (K252a or AG879). Furthermore, using single-cell RT-PCR, neurons that were sensitized by BDNF expressed the mRNA for p75 NTR but not TrkB. These results demonstrate that neurotrophins can modulate the excitability of small diameter capsaicin-sensitive sensory neurons through the activation of p75 NTR and its downstream sphingomyelin signalling cascade. Neurotrophins released upon activation of a variety of immuno-competent cells may be important mediators that give rise to the enhanced neuronal sensitivity associated with the inflammatory response.