Characterization of nuclear neurokinin 3 receptor expression in rat brain.

Ligand-induced translocation of the G-protein-coupled receptor, neurokinin 3 (NK3-R), to the nucleus of hypothalamic neurons was reported using antibodies (ABs) raised against the C-terminal region of NK3-R. The current work was undertaken to substantiate the ability of NK3-R to enter the nucleus and identify which portion of the NK3-R molecule enters the nucleus. ABs directed at epitopes in the N-terminal and second extracellular loop of the rat NK3-R molecule were used to evaluate western blots of whole tissue homogenates and nuclear fractions from multiple brain areas. Specificity of the protein bands recognized by these ABs was demonstrated using Chinese hamster ovary (CHO) cells transfected with rat or human NK3-R. Both ABs prominently recognized a diffuse protein band of ∼56-65 kDa (56 kDa=predicted size) and distinct ∼70-kDa and 95-kDa proteins in homogenates of multiple brain areas. The ∼95-kDa protein recognized by the extracellular loop AB was enriched in nuclear fractions. Recognition of these proteins by ABs directed at different regions of the NK3-R supports their identification as NK3-R. The size differences reflect variable glycosylation and possibly linkage to different cytosolic and nuclear proteins. Recognition of protein bands by both ABs in nuclear fractions is consistent with the full-length NK3-R entering the nucleus. Hypotension increased the density of the ∼95-kDa band in nuclear fractions from the supraoptic nucleus indicating activity-induced nuclear translocation. Since NK3-R is widely distributed in the CNS, the presence of NK3-R in nuclei from multiple brain regions suggests that it may broadly influence CNS gene expression in a ligand-dependent manner.

Pulpitis increases the proportion of atypical nodes of Ranvier in human dental pulp axons without a change in Nav1.6 sodium channel expression.

Studies show a change in sodium channel (NaCh) expression after inflammatory lesions, and this change is implicated in the generation of pain states. We are using the extracted human tooth to study NaCh expression and here examine the expression of the major NaCh isoform located at nodes of Ranvier, Na(v)1.6, in normal and painful samples. Pulpal sections were double-labeled with human-specific Na(v)1.6 antibody and caspr antibody (paranodal protein to identify nodes). Confocal microscopy was used to obtain a z-series of optically-sectioned images of axon bundles surrounded by inflammatory cells in painful samples and of similar regions within the coronal pulp of normal samples. Nodes contained within these images were classified as typical or atypical as based on caspr staining relationships, and NIH ImageJ software was used to quantify the size and immunofluorescence staining intensity of Na(v)1.6 accumulations at these nodal sites. Results show no significant difference in the size or immunofluorescence staining intensity of Na(v)1.6 nodal accumulations located at either typical or atypical nodal sites (heminodes and split nodes) within axons in normal samples when compared to painful samples (n=9/each group). In contrast, there was a highly significant decrease in the proportion of typical nodal sites and an increase in atypical nodal sites in painful samples when compared to normal samples. The unchanged expression of Na(v)1.6 contrasts to our previous finding that showed an increased expression of Na(v)1.7 at both typical and atypical nodal sites within painful samples. Together, these findings suggest there is not a simple replacement of one isoform with another, but rather an increased co-expression of multiple isoforms at both intact and remodeling/demyelinating (atypical) nodal sites within the painful dental pulp. The resultant heterogeneous population of isoforms may produce unique axonal excitability properties that could contribute to spontaneous pain sensations that are common in toothache.

Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Nav1.7 sodium channels in primary afferents.

Induction of peripheral inflammation increases the expression of the Nav1.7 sodium channel in sensory neurons, potentially increasing their excitability. Peripheral inflammation also produces hyperalgesia in humans and an increase in nociceptive responsiveness in animals. To test the relationship between these two phenomena we applied a recombinant herpes simplex-based vector to the hindpaw skin of mice, which encoded both green fluorescent protein (GFP) as well as an antisense sequence to the Nav1.7 gene. The hindpaw was subsequently injected with complete Freund's adjuvant to induce robust inflammation. Application of the vector, but not a control vector encoding only GFP, prevented an increase in Nav1.7 expression in GFP-positive neurons and prevented development of hyperalgesia in both C and Adelta thermonociceptive tests. These results provide clear evidence of the involvement of an increased expression of the Nav1.7 channel in nociceptive neurons in the development of inflammatory hyperalgesia.

Nr-CAM and neurofascin interactions regulate ankyrin G and sodium channel clustering at the node of Ranvier.

Voltage-dependent sodium (Na(+)) channels are highly concentrated at nodes of Ranvier in myelinated axons and play a key role in promoting rapid and efficient conduction of action potentials by saltatory conduction. The molecular mechanisms that direct their localization to the node are not well understood but are believed to involve contact-dependent signals from myelinating Schwann cells and interactions of Na(+) channels with the cytoskeletal protein, ankyrin G. Two cell adhesion molecules (CAMs) expressed at the axon surface, Nr-CAM and neurofascin, are also linked to ankyrin G and accumulate at early stages of node formation, suggesting that they mediate contact-dependent Schwann cell signals to initiate node development. To examine the potential role of Nr-CAM in this process, we treated myelinating cocultures of DRG (dorsal root ganglion) neurons and Schwann cells with an Nr-CAM-Fc (Nr-Fc) fusion protein. Nr-Fc had no effect on initial axon-Schwann cell interactions, including Schwann cell proliferation, or on the extent of myelination, but it strikingly and specifically inhibited Na(+) channel and ankyrin G accumulation at the node. Nr-Fc bound directly to neurons and clustered and coprecipitated neurofascin expressed on axons. These results provide the first evidence that neurofascin plays a major role in the formation of nodes, possibly via interactions with Nr-CAM.

Contactin associates with Na+ channels and increases their functional expression.

Contactin (also known as F3, F11) is a surface glycoprotein that has significant homology with the beta2 subunit of voltage-gated Na(+) channels. Contactin and Na(+) channels can be reciprocally coimmunoprecipitated from brain homogenates, indicating association within a complex. Cells cotransfected with Na(+) channel Na(v)1.2alpha and beta1 subunits and contactin have threefold to fourfold higher peak Na(+) currents than cells with Na(v)1.2alpha alone, Na(v)1.2/beta1, Na(v)1.2/contactin, or Na(v)1.2/beta1/beta2. These cells also have a correspondingly higher saxitoxin binding, suggesting an increased Na(+) channel surface membrane density. Coimmunoprecipitation of different subunits from cell lines shows that contactin interacts specifically with the beta1 subunit. In the PNS, immunocytochemical studies show a transient colocalization of contactin and Na(+) channels at new nodes of Ranvier forming during remyelination. In the CNS, there is a particularly high level of colocalization of Na(+) channels and contactin at nodes both during development and in the adult. Contactin may thus significantly influence the functional expression and distribution of Na(+) channels in neurons.

Molecular organization of the nodal region is not altered in spontaneously diabetic BB-Wistar rats.

We examined the organization of the molecular components of the nodal region in spontaneously diabetic BB-Wistar rats. Frozen sections and teased fibers from the sciatic nerves were immunostained for nodal (voltage-gated Na(+) channels, ankyrin(G), and ezrin), paranodal (contactin, Caspr, and neurofascin 155 kDa), and juxtaparanodal (Caspr2, the Shaker-type K(+) channels Kv1.1 and Kv1.2, and their associated subunit Kvbeta2) proteins. All of these proteins were properly localized in myelinated fibers from rats that had been diabetic for 15-44 days, compared to age-matched, nondiabetic animals. These results demonstrate that the axonal membrane is not reorganized, so nodal reorganization is not likely to be the cause of nerve conduction slowing in this animal model of acute diabetes.

Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon.

Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.

Differential control of clustering of the sodium channels Na(v)1.2 and Na(v)1.6 at developing CNS nodes of Ranvier.

Na(v)1.6 is the main sodium channel isoform at adult nodes of Ranvier. Here, we show that Na(v)1.2 and its beta2 subunit, but not Na(v)1.6 or beta1, are clustered in developing central nervous system nodes and that clustering of Na(v)1.2 and Na(v)1.6 is differentially controlled. Oligodendrocyte-conditioned medium is sufficient to induce clustering of Na(v)1.2 alpha and beta2 subunits along central nervous system axons in vitro. This clustering is regulated by electrical activity and requires an intact actin cytoskeleton and synthesis of a non-sodium channel protein. Neither soluble- or contact-mediated glial signals induce clustering of Na(v)1.6 or beta1 in a nonmyelinating culture system. These data reveal that the sequential clustering of Na(v)1.2 and Na(v)1.6 channels is differentially controlled and suggest that myelination induces Na(v)1.6 clustering.

Progesterone treatment abolishes exogenously expressed ionic currents in Xenopus oocytes.

Fully grown oocytes of Xenopus laevis undergo resumption of the meiotic cycle when treated with the steroid hormone progesterone. Previous studies have shown that meiotic maturation results in profound downregulation of specific endogenous membrane proteins in oocytes. To determine whether the maturation impacts the functional properties of exogenously expressed membrane proteins, we used cut-open recordings from Xenopus oocytes expressing several types of Na(+) and K(+) channels. Treatment of oocytes with progesterone resulted in a downregulation of heterologously expressed Na(+) and K(+) channels without a change in the kinetics of the currents. The time course of progesterone-induced ion channel inhibition was concentration dependent. Complete elimination of Na(+) currents temporally coincided with development of germinal vesicle breakdown, while elimination of K(+) currents was delayed by approximately 2 h. Coexpression of human beta(1)-subunit with rat skeletal muscle alpha-subunit in Xenopus oocytes did not prevent progesterone-induced downregulation of Na(+) channels. Addition of 8-bromo-cAMP to oocytes or injection of heparin before progesterone treatment prevented the loss of expressed currents. Pharmacological studies suggest that the inhibitory effects of progesterone on expressed Na(+) and K(+) channels occur downstream of the activation of cdc2 kinase. The loss of channels is correlated with a reduction in Na(+) channel immunofluorescence, pointing to a disappearance of the ion channel-forming proteins from the surface membrane.

Modeling human congenital disorder of glycosylation type IIa in the mouse: conservation of asparagine-linked glycan-dependent functions in mammalian physiology and insights into disease pathogenesis.

The congenital disorders of glycosylation (CDGs) are recent additions to the repertoire of inherited human genetic diseases. Frequency of CDGs is unknown since most cases are believed to be misdiagnosed or unrecognized. With few patients identified and heterogeneity in disease signs noted, studies of animal models may provide increased understanding of pathogenic mechanisms. However, features of mammalian glycan biosynthesis and species-specific variations in glycan repertoires have cast doubt on whether animal models of human genetic defects in protein glycosylation will reproduce pathogenic events and disease signs. We have introduced a mutation into the mouse germline that recapitulates the glycan biosynthetic defect responsible for human CDG type IIa (CDG-IIa). Mice lacking the Mgat2 gene were deficient in GlcNAcT-II glycosyltransferase activity and complex N-glycans, resulting in severe gastrointestinal, hematologic, and osteogenic abnormalities. With use of a lectin-based diagnostic screen for CDG-IIa, we found that all Mgat2-null mice died in early postnatal development. However, crossing the Mgat2 mutation into a distinct genetic background resulted in a low frequency of survivors. Mice deficient in complex N-glycans exhibited most CDG-IIa disease signs; however, some signs were unique to the aged mouse or are prognostic in human CDG-IIa. Unexpectedly, analyses of N-glycan structures in Mgat2-null mice revealed a novel oligosaccharide branch on the "bisecting" N-acetylglucosamine. These genetic, biochemical, and physiologic studies indicate conserved functions for N-glycan branches produced in the Golgi apparatus among two mammalian species and suggest possible therapeutic approaches to GlcNAcT-II deficiency. Our findings indicate that human genetic disease due to aberrant protein glycosylation can be modeled in the mouse to gain insights into N-glycan-dependent physiology and the pathogenesis of CDG-IIa.

Fibroblast growth factor receptor 3 induces gene expression primarily through Ras-independent signal transduction pathways.

Fibroblast growth factor receptors (FGFR) are widely expressed in many tissues and cell types, and the temporal expression of these receptors and their ligands play important roles in the control of development. There are four FGFR family members, FGFR-1-4, and understanding the ability of these receptors to transduce signals is central to understanding how they function in controlling differentiation and development. We have utilized signal transduction by FGF-1 in PC12 cells to compare the ability of FGFR-1 and FGFR-3 to elicit the neuronal phenotype. In PC12 cells FGFR-1 is much more potent in the induction of neurite outgrowth than FGFR-3. This correlated with the ability of FGFR-1 to induce robust and sustained activation of the Ras-dependent mitogen-activated protein kinase pathways. In contrast, FGFR-3 could not induce strong sustained Ras-dependent signals. In this study, we analyzed the ability of FGFR-3 to induce the expression of sodium channels, peripherin, and Thy-1 in PC12 cells because all three of these proteins are known to be induced via Ras-independent pathways. We determined that FGFR-3 was capable of inducing several Ras-independent gene expression pathways important to the neuronal phenotype to a level equivalent of that induced by FGFR-1. Thus, FGFR-3 elicits phenotypic changes primarily though activation of Ras-independent pathways in the absence of robust Ras-dependent signals.

Patient perceptions of the specialty of emergency medicine.

The study objective was to determine emergency department (ED) patients' perceptions of the specialty of emergency medicine. We surveyed a convenience sample of adult ED patients regarding their knowledge of the specialty of emergency medicine. Responses included: 22% believing that ED physicians have their own practice outside the ED; 26% of patients with primary care physicians expected to be seen by their primary care physician in the ED; 19% thought ED physicians care for patients after admission; 26% thought that ED physicians perform surgery, 62% perceived emergency medicine to be a specialty; 15% have heard of the American College of Emergency Physicians; 71% thought that ED physicians are board certified and 15% thought paramedics were ED physicians. Patients estimated ED physicians' mean annual mean salary to be $100,000 and 61% believe that ED physicians are hospital employees. In conclusion, the specialty of emergency medicine is not well understood by our patients.

Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses.

Voltage-gated sodium channels perform critical roles for electrical signaling in the nervous system by generating action potentials in axons and in dendrites. At least 10 genes encode sodium channels in mammals, but specific physiological roles that distinguish each of these isoforms are not known. One possibility is that each isoform is expressed in a restricted set of cell types or is targeted to a specific domain of a neuron or muscle cell. Using affinity-purified isoform-specific antibodies, we find that Na(v)1.6 is highly concentrated at nodes of Ranvier of both sensory and motor axons in the peripheral nervous system and at nodes in the central nervous system. The specificity of this antibody was also demonstrated with the Na(v)1.6-deficient mouse mutant strain med, whose nodes were negative for Na(v)1.6 immunostaining. Both the intensity of labeling and the failure of other isoform-specific antibodies to label nodes suggest that Na(v)1.6 is the predominant channel type in this structure. In the central nervous system, Na(v)1.6 is localized in unmyelinated axons in the retina and cerebellum and is strongly expressed in dendrites of cortical pyramidal cells and cerebellar Purkinje cells. Ultrastructural studies indicate that labeling in dendrites is both intracellular and on dendritic shaft membranes. Remarkably, Na(v)1.6 labeling was observed at both presynaptic and postsynaptic membranes in the cortex and cerebellum. Thus, a single sodium channel isoform is targeted to different neuronal domains and can influence both axonal conduction and synaptic responses.

A possible role for nerve growth factor in the augmentation of sodium channels in models of chronic pain.

Inflammation induces an upregulation of sodium channels in sensory neurons. This most likely occurs as a result of the retrograde transport of cytochemical mediators released during the inflammatory response. The purpose of this study was to determine the effect of the subcutaneous administration of one such mediator, nerve growth factor (NGF), on the production of sodium channels in neurons of the rat dorsal root ganglion. For this, hindpaw withdrawal from either a thermal or mechanical stimulus was measured in rats at selected intervals for up to 2 weeks following injections of NGF. Sodium channel augmentation was then examined in dorsal root ganglia using site-specific, anti-sodium channel antibodies. Both thermal and mechanical allodynia was observed between 3 and 12 h post-injection. The hyperalgesic response returned to baseline by approximately 24 h post-injection. Sodium channel labeling was found to increase dramatically in the small neurons of the associated dorsal root ganglia beginning at 23 h, reached maximum intensity by 1 week, and persisted for up to 3 months post-injection. Pre-blocking NGF with anti-NGF prevented the NGF-induced decrease in paw withdrawal latencies and significantly reduced the intensity of sodium channel labeling. The results indicate that NGF is an important mediator both in the development of acute hyperalgesia and in the stimulation of sodium channel production in dorsal root ganglia during inflammation.

Immunolocalization of sodium channel isoform NaCh6 in the nervous system.

Sodium channel 6 (NaCh6) is the alpha-subunit of a voltage-gated sodium channel expressed in the rat nervous system. The mRNA for this isoform has been shown to be expressed in both neuronal and glial cells by in situ hybridization. To examine localization of NaCh6 protein, polyclonal antibodies specific for NaCh6 were generated against peptides from two cytoplasmic domains and a fusion protein from an extracellular domain. Affinity-purified antibodies were used to localize NaCh6 in the brain, spinal cord, peripheral nervous system, and neuromuscular junction. There was widespread labeling of neurons in the brain and spinal cord. NaCh6 was present in both sensory and motor pathways. Radial glial cells in the cerebellum were intensely labeled for both GFAP and NaCh6. At the subcellular level, NaCh6 is found in axons, dendrites, and the cell body. Motor neurons and primary sensory neurons in dorsal root ganglia had strong cytoplasmic and axonal staining. Nodes of Ranvier in peripheral nerve and in the spinal cord were also intensely labeled. Motor neuron axons near the neuromuscular junction were labeled up to, but not including, terminal boutons. Dendrites of pyramidal cells in the cortex, hippocampus, and cerebellum were labeled. NaCh6 is the first NaCh subtype to be localized either at the node of Ranvier or to a dendrite. We conclude that NaCh6 is widely distributed in the central and peripheral nervous systems and is likely to be important for the electrical properties of the axon and dendrite.

Novel structure having antagonist actions at both the glycine site of the N-methyl-D-aspartate receptor and neuronal voltage-sensitive sodium channels: biochemical, electrophysiological, and behavioral characterization.

A novel series of N-substituted 4-ureido-5,7-dichloro-quinolines were synthesized to contain pharmacophores directed at voltage-sensitive sodium channels (VSNaCs) and N-methyl-D-aspartate (NMDA) receptors. These compounds were shown to act in a use-dependent manner as antagonists of VSNaCs and to act as selective competitive antagonists at the strychnine-insensitive glycine recognition site of NMDA receptors. These agents had little or no effect on alpha-adrenergic receptors, other glutamate receptors, or sites other than the glycine site on the NMDA receptor, and did not block voltage-sensitive calcium channels in vitro. In vivo, the compounds were active in preventing or reducing the signs and symptoms of neurohyperexcitability and had anxiolytic properties. Unlike benzodiazepines, N-substituted 4-ureido-5, 7-dichloro-quinolines showed little interaction with the sedative effects of ethanol, but were effective in controlling ethanol withdrawal seizures. The combined actions of these compounds on VSNaCs and NMDA receptors also impart properties to these compounds that are important for preventing and reducing excitotoxic neurodegeneration, but these compounds lack the undesirable side effects of other agents used for these purposes.

Dependence of nodal sodium channel clustering on paranodal axoglial contact in the developing CNS.

Na(+) channel clustering at nodes of Ranvier in the developing rat optic nerve was analyzed to determine mechanisms of localization, including the possible requirement for glial contact in vivo. Immunofluorescence labeling for myelin-associated glycoprotein and for the protein Caspr, a component of axoglial junctions, indicated that oligodendrocytes were present, and paranodal structures formed, as early as postnatal day 7 (P7). However, the first Na(+) channel clusters were not seen until P9. Most of these were broad, and all were excluded from paranodal regions of axoglial contact. The number of detected Na(+) channel clusters increased rapidly from P12 to P22. During this same period, conduction velocity increased sharply, and Na(+) channel clusters became much more focal. To test further whether oligodendrocyte contact directly influences Na(+) channel distributions, nodes of Ranvier in the hypomyelinating mouse Shiverer were examined. This mutant has oligodendrocyte-ensheathed axons but lacks compact myelin and normal axoglial junctions. During development Na(+) channel clusters in Shiverer mice were reduced in numbers and were in aberrant locations. The subcellular location of Caspr was disrupted, and nerve conduction properties remained immature. These results indicate that in vivo, Na(+) channel clustering at nodes depends not only on the presence of oligodendrocytes but also on specific axoglial contact at paranodal junctions. In rats, ankyrin-3/G, a cytoskeletal protein implicated in Na(+) channel clustering, was detected before Na(+) channel immunoreactivity but extended into paranodes in non-nodal distributions. In Shiverer, ankyrin-3/G labeling was abnormal, suggesting that its localization also depends on axoglial contact.

Mice deficient for tenascin-R display alterations of the extracellular matrix and decreased axonal conduction velocities in the CNS.

Tenascin-R (TN-R), an extracellular matrix glycoprotein of the CNS, localizes to nodes of Ranvier and perineuronal nets and interacts in vitro with other extracellular matrix components and recognition molecules of the immunoglobulin superfamily. To characterize the functional roles of TN-R in vivo, we have generated mice deficient for TN-R by homologous recombination using embryonic stem cells. TN-R-deficient mice are viable and fertile. The anatomy of all major brain areas and the formation and structure of myelin appear normal. However, immunostaining for the chondroitin sulfate proteoglycan phosphacan, a high-affinity ligand for TN-R, is weak and diffuse in the mutant when compared with wild-type mice. Compound action potential recordings from optic nerves of mutant mice show a significant decrease in conduction velocity as compared with controls. However, at nodes of Ranvier there is no apparent change in expression and distribution of Na+ channels, which are thought to bind to TN-R via their beta2 subunit. The distribution of carbohydrate epitopes of perineuronal nets recognized by the lectin Wisteria floribunda or antibodies to the HNK-1 carbohydrate on somata and dendrites of cortical and hippocampal interneurons is abnormal. These observations indicate an essential role for TN-R in the formation of perineuronal nets and in normal conduction velocity of optic nerve.