Matthew J. Craner

Altered Sodium Channel Expression in Second-Order Spinal Sensory Neurons Contributes to Pain after Peripheral Nerve Injury

Peripheral nerve injury is known to upregulate the rapidly repriming Nav1.3 sodium channel within first-order spinal sensory neurons. In this study, we hypothesized that (1) after peripheral nerve injury, second-order dorsal horn neurons abnormally express Nav1.3, which (2) contributes to the responsiveness of these dorsal horn neurons and to pain-related behaviors. To test these hypotheses, adult rats underwent chronic constriction injury (CCI) of the sciatic nerve. Ten days after CCI, allodynia and hyperalgesia were evident. In situ hybridization, quantitative reverse transcription-PCR, and immunocytochemical analysis revealed upregulation of Nav1.3 in dorsal horn nociceptive neurons but not in astrocytes or microglia, and unit recordings demonstrated hyperresponsiveness of dorsal horn sensory neurons. Intrathecal antisense oligodeoxynucleotides targeting Nav1.3 decreased the expression of Nav1.3 mRNA and protein, reduced the hyperresponsiveness of dorsal horn neurons, and attenuated pain-related behaviors after CCI, all of which returned after cessation of antisense delivery. These results demonstrate for the first time that sodium channel expression is altered within higher-order spinal sensory neurons after peripheral nerve injury and suggest a link between misexpression of the Nav1.3 sodium channel and central mechanisms that contribute to neuropathic pain after peripheral nerve injury.

Changes of sodium channel expression in experimental painful diabetic neuropathy.

Although pain is experienced by many patients with diabetic neuropathy, the pathophysiology of painful diabetic neuropathy is not understood. Substantial evidence indicates that dysregulated sodium channel gene transcription contributes to hyperexcitability of dorsal root ganglion neurons, which may produce neuropathic pain after axonal transection. In this study, we examined sodium channel mRNA and protein expression in dorsal root ganglion neurons in rats with streptozotocin‐induced diabetes and tactile allodynia, using in situ hybridization and immunocytochemistry for sodium channels Nav1.1, Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9. Our results show that, in rats with experimental diabetes, there is a significant upregulation of mRNA for the Nav1.3, Nav1.6, and Nav1.9 sodium channels and a downregulation of Nav1.8 mRNA 1 and 8 weeks after onset of allodynia. Channel protein levels display parallel changes. Our results demonstrate dysregulated expression of the genes for sodium channels Nav1.3, Nav1.6, Nav1.8, and Nav1.9 in dorsal root ganglion neurons in experimental diabetes and suggest that misexpression of sodium channels contributes to neuropathic pain associated with diabetic neuropathy.

Fibroblast growth factor homologous factor 2B: association with Nav1.6 and selective colocalization at nodes of Ranvier of dorsal root axons.

Voltage-gated sodium channels interact with cytosolic proteins that regulate channel trafficking and/or modulate the biophysical properties of the channels. Nav1.6 is heavily expressed at the nodes of Ranvier along adult CNS and PNS axons and along unmyelinated fibers in the PNS. In an initial yeast two-hybrid screen using the C terminus of Nav1.6 as a bait, we identified FHF2B, a member of the FGF homologous factor (FHF) subfamily, as an interacting partner of Nav1.6. Members of the FHF subfamily share ∼70% sequence identity, and individual members demonstrate a cell- and tissue-specific expression pattern. FHF2 is abundantly expressed in the hippocampus and DRG neurons and colocalizes with Nav1.6 at mature nodes of Ranvier in myelinated sensory fibers in the dorsal root of the sciatic nerve. However, retinal ganglion cells and spinal ventral horn motor neurons show very low levels of FHF2 expression, and their axons exhibit no nodal FHF2 staining within the optic nerve and ventral root, respectively. Thus, FHF2 is selectively localized at nodes of dorsal root sensory but not ventral root motor axons. The coexpression of FHF2B and Nav1.6 in the DRG-derived cell line ND7/23 significantly increases the peak current amplitude and causes a 4 mV depolarizing shift of voltage-dependent inactivation of the channel. The preferential expression of FHF2B in sensory neurons may provide a basis for physiological differences in sodium currents that have been reported at the nodes of Ranvier in sensory versus motor axons.

Contactin regulates the current density and axonal expression of tetrodotoxin-resistant but not tetrodotoxin-sensitive sodium channels in DRG neurons

Contactin, a glycosyl‐phosphatidylinositol (GPI)‐anchored predominantly neuronal cell surface glycoprotein, associates with sodium channels Nav1.2, Nav1.3 and Nav1.9, and enhances the density of these channels on the plasma membrane in mammalian expression systems. However, a detailed functional analysis of these interactions and of untested putative interactions with other sodium channel isoforms in mammalian neuronal cells has not been carried out. We examined the expression and function of sodium channels in small‐diameter dorsal root ganglion (DRG) neurons from contactin‐deficient (CNTN–/–) mice, compared to CNTN+/+ litter mates. Nav1.9 is preferentially expressed in isolectin B4 (IB4)‐positive neurons and thus we used this marker to subdivide small‐diameter DRG neurons. Using whole‐cell patch‐clamp recording, we observed a greater than two‐fold reduction of tetrodotoxin‐resistant (TTX‐R) Nav1.8 and Nav1.9 current densities in IB4+ DRG neurons cultured from CNTN–/– vs. CNTN+/+ mice. Current densities for TTX‐sensitive (TTX‐S) sodium channels were unaffected. Contactins effect was selective for IB4+ neurons as current densities for both TTX‐R and TTX‐S channels were not significantly different in IB4– DRG neurons from the two genotypes. Consistent with these results, we have demonstrated a reduction in Nav1.8 and Nav1.9 immunostaining on peripherin‐positive unmyelinated axons in sciatic nerves from CNTN–/– mice but detected no changes in the expression for the two major TTX‐S channels Nav1.6 and Nav1.7. These data provide evidence of a role for contactin in selectively regulating the cell surface expression and current densities of TTX‐R but not TTX‐S Na+ channel isoforms in nociceptive DRG neurons; this regulation could modulate the membrane properties and excitability of these neurons.

Preferential expression of IGF-I in small DRG neurons and down-regulation following injury

In this study, we examined the expression of insulin-like growth factor I (IGF-I) and its receptor (IGF-IR) in dorsal root ganglia (DRG) neurons in two rodent models of nerve injury: sciatic nerve axotomy and streptozotocin-induced (STZ) painful diabetic neuropathy. We demonstrate that IGF-I and its receptor are preferentially expressed in small (< 25 μm diameter) DRG neurons. There is a significant down-regulation in the expression of IGF-I and IGF-IR in the small DRG neurons of STZ rats by 59% and 71%, respectively. A parallel reduction in expression is shown in axotomized < 25 μm diameter DRG neurons for IGF-I (47%) but not for IGF-IR. The loss of IGF-I support to a population of predominantly nociceptive neurons may contribute to neuropathic pain observed in these models.

Abnormal Purkinje cell activity in vivo in experimental allergic encephalomyelitis

Cerebellar deficits in multiple sclerosis (MS) tend to persist and can produce significant disability. Although the pathophysiological basis for these deficits is not clear, it was recently reported that the expression of the sensory neuron-specific sodium channel Nav1.8 (which is not normally expressed within the cerebellum) is aberrantly upregulated within Purkinje cells in experimental allergic encephalomyelitis (EAE) and in human MS. The expression of Nav1.8 in cultured Purkinje cells has been shown to alter the activity pattern of these cells in vitro by decreasing the number of spikes per conglomerate action potential and by contributing to the production of sustained, pacemaker-like activity upon depolarization, suggesting the hypothesis that, in pathophysiological situations where Nav1.8 is upregulated within Purkinje cells, the pattern of activity in these cells will be altered. In the present study, we examined this hypothesis in vivo in mice with EAE. Our results demonstrate a reduction in the number of secondary spikes per complex spike and irregularity in the temporal organization of secondary spikes in Purkinje cells from mice with EAE in which Nav1.8 is upregulated. We also observed abnormal bursting activity in Purkinje cells from mice with EAE, which was not observed in control animals. These results demonstrate functional changes in Purkinje cells in vivo within their native cerebellar environment in EAE, a model of MS, and support the hypothesis that misexpression of Nav1.8 can contribute to cerebellar deficits in neuroinflammatory disorders by altering the pattern of electrical activity within the cerebellum.

Upregulation and colocalization of p75 and Nav1.8 in Purkinje neurons in experimental autoimmune encephalomyelitis.

Recent studies have indicated that, in addition to demyelination and axonal degeneration, a third factor, dysregulated ion channel expression, contributes to the pathophysiology of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). Consistent with this suggestion, upregulated expression of sodium channel Na(v)1.8 is observed in Purkinje neurons in EAE and MS, and biophysical studies indicate that aberrant expression of Na(v)1.8 produces abnormal Purkinje cell firing which may contribute to the development of cerebellar ataxia. However, the molecular mechanisms that contribute to the upregulation of Na(v)1.8 in Purkinje cells in EAE and MS have not yet been determined. Previous studies have shown that neurotrophic factors can modulate sodium channel expression and that elevated levels of NGF are present in EAE and MS. Using immunocytochemical methods, we examined the relationship between the upregulation of Na(v)1.8 and the expression of the NGF receptors p75 and TrkA in EAE. Here we demonstrate that upregulation of Na(v)1.8 is associated with expression of p75 and low levels of TrkA in the majority of Purkinje cells in EAE. These findings, together with previous studies demonstrating a modulatory role of NGF on sodium channel expression, suggest that NGF acting via p75 contributes to the upregulation of Na(v)1.8 in Purkinje cells in EAE.

Sodium channel expression in hypothalamic osmosensitive neurons in experimental diabetes

Vasopressin is synthesized by neurons in the supraoptic nucleus of the hypothalamus and its release is controlled by action potentials produced by specific subtypes of voltage-gated sodium channels expressed in these neurons. The hyperosmotic state associated with uncontrolled diabetes mellitus causes elevated levels of plasma vasopressin, which are thought to contribute to the pathologic changes of diabetic nephropathy. We demonstrate here that in the rodent streptozotocin model of diabetes there are increases in expression of mRNA and protein for two sodium channel &agr;-subunits and two &bgr;-subunits in the neurons of the supraoptic nucleus. Transient and persistent sodium currents show parallel increases in these diabetic neurons. In the setting of chronic uncontrolled diabetes, these changes in sodium channel expression in the supraoptic nucleus may be maladaptive, contributing to the development of secondary renal complications.