Grant D. Nicol

Ceramide, a putative second messenger for nerve growth factor, modulates the TTX‐resistant Na+ current and delayed rectifier K+ current in rat sensory neurons

Because nerve growth factor (NGF) is elevated during inflammation and is known to activate the sphingomyelin signalling pathway, we examined whether NGF and its putative second messenger, ceramide, could modulate the excitability of capsaicin‐sensitive adult and embryonic sensory neurons. Using the whole‐cell patch‐clamp recording technique, exposure of isolated sensory neurons to either 100 ng ml−1 NGF or 1 μM N‐acetyl sphingosine (C2‐ceramide) produced a 3‐ to 4‐fold increase in the number of action potentials (APs) evoked by a ramp of depolarizing current in a time‐dependent manner. Intracellular perfusion with bacterial sphingomyelinase (SMase) also increased the number of APs suggesting that the release of native ceramide enhanced neuronal excitability. Glutathione, an inhibitor of neutral SMase, completely blocked the NGF‐induced augmentation of AP firing, whereas dithiothreitol, an inhibitor of acidic SMase, was without effect. In the presence of glutathione and NGF, exogenous ceramide still enhanced the number of evoked APs, indicating that the sensitizing action of ceramide was downstream of NGF. To investigate the mechanisms of action for NGF and ceramide, isolated membrane currents were examined. Both NGF and ceramide facilitated the peak amplitude of the TTX‐resistant sodium current (TTX‐R INa) by approximately 1.5‐fold and shifted the activation to more hyperpolarized voltages. In addition, NGF and ceramide suppressed an outward potassium current (IK) by ≈35 %. Ceramide reduced IK in a concentration‐dependent manner. Isolation of the NGF‐ and ceramide‐sensitive currents indicates that they were delayed rectifier types of IK. The inflammatory prostaglandin, PGE2, produced an additional suppression of IK after exposure to ceramide (≈35 %), suggesting that these agents might act on different targets. Thus, our findings indicate that the pro‐inflammatory agent, NGF, can rapidly enhance the excitability of sensory neurons. This NGF‐induced sensitization is probably mediated by activation of the sphingomyelin signalling pathway to liberate ceramide(s), wherein ceramide appears to be the second messenger involved in modulating neuronal excitability.

The cAMP transduction cascade mediates the PGE2‐induced inhibition of potassium currents in rat sensory neurones

1 The role of the cyclic AMP (cAMP) transduction cascade in mediating the prostaglandin E2 (PGE2)‐induced decrease in potassium current (IK) was investigated in isolated embryonic rat sensory neurones using the whole‐cell patch‐clamp recording technique. 2 Exposure to 100 μM chlorophenylthio‐adenosine cyclic 3′,5′‐monophosphate (cpt‐cAMP) or 1 μM PGE2 caused a slow suppression of the whole‐cell IK by 34 and 36 %, respectively (measured after 20 min), without a shift in the voltage dependence of activation for this current. Neither of these agents altered the shape of the voltage‐dependent inactivation curve indicating that the suppression of IK did not result from alterations in the inactivation properties. 3 To determine whether the PGE2‐mediated suppression of IK depended on activation of the cAMP pathway, cells were exposed to this prostanoid in the presence of the protein kinase A (PKA) inhibitor, PKI. The PGE2‐induced suppression of IK was prevented by PKI. In the absence of PGE2, PKI had no significant effect on the magnitude of IK. 4 Results obtained from protocols using different conditioning prepulse voltages indicated that the extent of cpt‐cAMP‐ and PGE2‐mediated suppression of IK was independent of the prepulse voltage. The subtraction of control and treated currents revealed that the cpt‐cAMP‐ and PGE2‐sensitive currents exhibited little time‐dependent inactivation. Taken together, these results suggest that the modulated currents may be delayed rectifier‐like IK. 5 Exposure to the inhibitors of IK, tetraethylammonium (TEA) or 4‐aminopyridine (4‐AP), reduced the control current elicited by a voltage step to +60 mV by 40‐50 %. In the presence of 10 mM TEA, treatment with cpt‐cAMP did not result in any further inhibition of IK. In contrast, cpt‐cAMP reduced IK by an additional 25‐30 % in the presence of 1 mM 4‐AP. This effect was independent of the conditioning prepulse voltage. 6 These results establish that PGE2 inhibits an outward IK in sensory neurones via activation of PKA and are consistent with the idea that the PGE2‐mediated sensitization of sensory neurones results, in part, from an inhibition of delayed rectifier‐like IK.

Differential regulation of evoked peptide release by voltage-sensitive calcium channels in rat sensory neurons

To determine whether the sensitizing action of prostaglandins on sensory neurons are due to modulation of voltage-sensitive calcium channels (VSCC) we examined the effects of inhibiting these channels on PGE2-induced enhancement of evoked peptide release from isolated dorsal root ganglion neurons. The inhibitory effects of the VSCC blockers on stimulated release were dependent upon the type of chemical agent used to evoke the release. Bradykinin-stimulated release of immunoreactive substance P (iSP) and calcitonin gene-related peptide (iCGRP) was attenuated by the N-type VSCC blocker, omega-conotoxin GVIA (100 nM), but was unaffected by blockade of L-type (1 microM nifedipine) or P-type (200 nM omega-agatoxin IVA) VSCC. In contrast, potassium-stimulated release of peptides was inhibited by nifedipine, but not by omega-conotoxin GVIA or omega-agatoxin IVA. None of the VSCC blockers tested attenuated capsaicin-stimulated release of iSP and iCGRP. The combination of 1 microM nifedipine and 100 nM omega-conotoxin GVIA reduced the whole cell calcium current 89% +/- 1.7%. Administration of 100 nM PGE2 potentiated bradykinin- and capsaicin-evoked peptide release by 2-3-fold. Neither nifedipine nor omega-conotoxin GVIA attenuated the PGE2-mediated potentiation of bradykinin-evoked release, and neither omega-conotoxin GVIA nor omega-agatoxin IVA blocked the potentiation of capsaicin-evoked release induced by PGE2. These results indicate that the sensitizing actions of PGE2 as measured by enhanced peptide release, are not mediated by L-, N-, or P-type VSCC.

NGF-mediated sensitization of the excitability of rat sensory neurons is prevented by a blocking antibody to the p75 neurotrophin receptor

Nerve growth factor (NGF) can play a causal role in the initiation of hyperalgesia. Recent work demonstrates that NGF can act directly on nociceptive sensory neurons to augment their sensitivity to a variety of stimuli. Based on the existing literature, it is not clear whether this sensitization is mediated by the high-affinity TrkA receptor or the low-affinity p75 neurotrophin receptor. We examined whether a blocking antibody to the p75 neurotrophin receptor can prevent the NGF-induced enhancement of excitability in capsaicin-sensitive small-diameter sensory neurons that have been isolated from the adult rat. In this report, pretreatment with the p75 blocking antibody completely prevents the NGF-induced increase in the number of action potentials evoked by a ramp of depolarizing current as well as the suppression of a delayed rectifier-type of potassium current(s) in these neurons. Although the sensitization by NGF was blocked, the antibody had no effect on the capacity of ceramide, a putative downstream signaling molecule, to either enhance the excitability or inhibit the potassium current. These results indicate that NGF can increase the excitability of nociceptive sensory neurons through activation of the p75 neurotrophin receptor and its consequent liberation of ceramide from neuronal sphingomyelins.

Intracellular sphingosine 1-phosphate mediates the increased excitability produced by nerve growth factor in rat sensory neurons.

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.

Tumor necrosis factor α and interleukin-1β stimulate the expression of cyclooxygenase II but do not alter prostaglandin E2 receptor mRNA levels in cultured dorsal root ganglia cells

&NA; Tumor necrosis factor α (TNFα) and interleukin 1β (IL‐1β) are pro‐inflammatory cytokines capable of altering the sensitivity of sensory neurons. Because sensitization elicited by IL‐1β and TNFα is blocked by inhibition of the inducible enzyme, cyclooxygenase‐II (COX‐2), we examined whether these cytokines could increase COX‐2 expression in dorsal root ganglion (DRG) cultures. Treatment of cell cultures with either IL‐1β or TNFα increases immunoreactive COX‐2, as measured by immunoblotting, in a time‐ and concentration‐dependent manner. A 24‐h pretreatment with 10 ng/ml IL‐1β or 50 ng/ml TNFα augmented COX‐2 expression 50‐ and 8‐fold over basal levels, respectively. Immunohistochemistry established the presence of COX‐2‐like immunoreactivity in both neuronal and non‐neuronal cells in culture. The addition of IL‐1 receptor antagonist blocked the induction of COX‐2 expression by IL‐1β, but did not alter TNFα‐stimulated increases in COX‐2, indicating that the mechanism of TNFα is not limited to increasing the expression of IL‐1β. The basal and TNFα‐induced expression of COX‐2 was not dependent on the presence of NGF in the growth media. IL‐1β and TNFα treatment for 24 h enhanced prostaglandin E2 (PGE2) production 2–4‐fold, which was blocked by pretreatment with the COX‐2 inhibitor, NS‐398. Exposing cultures to PGE2, IL‐1β, or TNFα for 24 h did not alter PGE2 receptor (EP) mRNA levels. These results indicate that TNFα and IL‐1β induce the functional expression of COX‐2 but not EP receptors in DRG cells in culture and suggest that cytokine‐induced sensitization of sensory neurons is secondary to prostaglandin production and not alterations in EP receptors.

Brain-derived neurotrophic factor enhances the excitability of rat sensory neurons through activation of the p75 neurotrophin receptor and the sphingomyelin pathway

Neurotrophin‐mediated signalling cascades can be initiated by activation of either the p75 neurotrophin receptor (p75NTR) or the more selective tyrosine kinase receptors. Previously, we demonstrated that nerve growth factor (NGF) increased the excitability of sensory neurons through activation of p75NTR to liberate sphingosine 1‐phosphate. If neurotrophins can modulate the excitability of small diameter sensory neurons through activation of p75NTR, 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 INa) and to suppress a delayed rectifier‐like potassium current (IK). The idea that BDNF acts via p75NTR 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 p75NTR but not TrkB. These results demonstrate that neurotrophins can modulate the excitability of small diameter capsaicin‐sensitive sensory neurons through the activation of p75NTR 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.

Prostaglandin E2 inhibits the potassium current in sensory neurons from hyperalgesic Kv1.1 knockout mice

Prostaglandin E(2) (PGE(2)) enhances the sensitivity of sensory neurons to various forms of noxious stimulation. This occurs, in part, by the suppression of a delayed rectifier-like potassium current in these neurons. However, the molecular identity of this current remains unclear. Recent studies demonstrated that a mutant mouse lacking a delayed rectifier potassium channel gene, Kv1.1, displayed lowered thresholds to thermal stimulation in behavioral assays of pain perception, i.e. the Kcna1-null mice were hyperalgesic. Here we examined whether PGE(2) can alter the sensitivity of Kcna1-null mice to noxious stimulation and examine the capability of PGE(2) to inhibit the potassium current in these knockout mice. Behavioral assays were used to assess the effect of PGE(2) on either thermal hyperalgesia or mechanical sensitivities. In addition, the whole-cell patch-clamp technique was used to study the effects of PGE(2) on the total potassium current recorded from isolated mouse sensory neurons. Even with a reduced threshold to thermal stimulation, PGE(2) could still sensitize the response of Kcna1-null mice to thermal and mechanical stimulation by amounts that were similar to that in wild type mice. The activation properties of the potassium current were similar for both the wild type and the Kcna1-null mice, whereas the inactivation properties were different in cells exhibiting large amounts of steady-state inactivation (>50%) measured at +20 mV. PGE(2) suppressed the total potassium current in both groups of mice by 40-50% without altering the voltage dependence of activation. In addition, PGE(2) produced similar amounts of suppression in both groups of mice when currents were examined with the steady-state inactivation protocol. Based on these results, it is unlikely that Kv1.1 is the molecular identity of the potassium channel(s) modulated by PGE(2) to sensitize nociceptive sensory neurons. Also, the enhanced thermal sensitivity as observed in the Kcna1-null mice might be due to more central neurons of the pain sensing pathway.

Knockdown of the sphingosine-1-phosphate receptor S1PR1reduces pain behaviors induced by local inflammation of the rat sensory ganglion

Sphingosine 1-phosphate (S1P) is a key immune mediator regulating migration of immune cells to sites of inflammation. S1P actions are mediated by a family of five G protein-coupled receptors. Sensory neurons express many of these receptors, and in vitro S1P has excitatory effects on small-diameter sensory neurons, many mediated by the S1P receptor 1 (S1PR1). This study investigated the role of S1P in regulating the sensitivity of DRG neurons. We found that in vivo perfusion of the normal L5 DRG with S1P increased mechanical sensitivity. Microelectrode recordings in isolated whole ganglia showed that large- and medium-diameter cells, as well as small-diameter cells, increased firing in the presence of S1P. To further determine the role of S1PRs, we examined the effects of in vivo S1PR1 knockdown in the L4 and L5 sensory ganglia. Small interfering RNA directed against S1PR1 did not affect baseline mechanical sensitivity in normal animals, in which S1P levels are expected to be low. However, when the L5 ganglion was locally inflamed, a procedure that leads to rapid and sustained mechanical hypersensitivity, S1PR1 siRNA injected animals showed significantly less hypersensitivity than animals injected with scrambled siRNA. Reduced expression of S1PR1, but not S1PR2 or S1PR3, was confirmed with qPCR methods. The results indicate that the S1PR1 receptors in sensory ganglia cells may play an important role in regulating behavioral sensitivity during inflammation.

Synaptic and membrane properties of neurons in the dorsomedial hypothalamus

Studies in intact rats have shown that the dorsomedial hypothalamus (DMH) plays a key role in generating stress-induced physiologic changes, including activation of the hypothalamic-pituitary-adrenal axis through direct projections to paraventricular hypothalamic nucleus (PVN). However, little is known about the cellular properties of DMH neurons. We employed whole-cell patch-clamp recording techniques to characterize membrane properties and spontaneous post-synaptic currents (PSCs) in DMH neurons, including those projecting to PVN (identified by prior injection of DiI into PVN), in rat hypothalamic slices. DMH neurons (n=86 total) had uniform membrane properties. However, PVN-projecting neurons (n=32) had higher action potential (AP) thresholds, and fired fewer APs in response to current injection. Spontaneous PVN-projecting neurons (n=20) also fired APs at lower rates (4.8+/-0.6 Hz) than spontaneous neurons of unknown projection (n=38; 7.3+/-1.1 Hz). Spontaneous PSCs were observed in all neurons: One population expressed rapid decay characteristics (1.5-2.0 ms) and was blocked by non-NMDA ionotropic glutamate receptor antagonists NBQX or CNQX. Remaining PSCs reversed near E(Cl), were blocked by the GABA(A) receptor antagonists picrotoxin or bicuculline methiodide (BMI), and had longer decay time constants (4.5-6.0 ms) that were modulated by pentobarbital. Tetrodotoxin markedly reduced the frequency of PSCs sensitive to NBQX but not to BMI. Thus, DMH is made up of electrophysiologically similar neurons and PVN-projecting neurons are less excitable than neurons of unknown projection. Furthermore, as suggested by studies in intact rats, neurons in the DMH, including those projecting to the PVN, are regulated by tonic GABA(A) and non-NMDA glutamate receptor-mediated synaptic transmission.