The sodium channel Nav1.5a is the predominant isoform expressed in adult mouse dorsal root ganglia and exhibits distinct inactivation properties from the full-length Nav1.5 channel

Nav1.5 is the principal voltage-gated sodium channel expressed in heart, and is also expressed at lower abundance in embryonic dorsal root ganglia (DRG) with little or no expression reported postnatally. We report here the expression of Nav1.5 mRNA isoforms in adult mouse and rat DRG. The major isoform of mouse DRG is Nav1.5a, which encodes a protein with an IDII/III cytoplasmic loop reduced by 53 amino acids. Western blot analysis of adult mouse DRG membrane proteins confirmed the expression of Nav1.5 protein. The Na+ current produced by the Nav1.5a isoform has a voltage-dependent inactivation significantly shifted to more negative potentials (by approximately 5 mV) compared to the full-length Nav1.5 when expressed in the DRG neuroblastoma cell line ND7/23. These results imply that the alternatively spliced exon 18 of Nav1.5 plays a role in channel inactivation and that Nav1.5a is likely to make a significant contribution to adult DRG neuronal function.     

Neurogenesis in postnatal mouse dorsal root ganglia.

Neurogenesis continues in various regions of the central nervous system (CNS) throughout life. As the mitogen basic fibroblast growth factor (bFGF) can proliferate neuronal precursors of CNS neurons in culture, and is also upregulated within adult dorsal root ganglia following axotomy, it is possible that the postnatal dorsal root ganglia contain bFGF-responsive neuronal precursors. We undertook cell culture of postnatal mouse dorsal root ganglia to demonstrate neurogenesis. Basic FGF induced a cellular proliferative response in dorsal root ganglia cell culture. After 2 weeks in serum-free medium containing bFGF, neurons were rarely observed. However, following removal of bFGF and addition of trophic factors, many cells were observed that morphologically resembled dorsal root ganglia neurons, stained for neuronal markers, and generated action potentials. Furthermore, bromodeoxyuridine, used as a marker of cytogenesis, was detected in neurofilament-160(+) and/or microtubule-associated protein-2(+) cells that morphologically resembled neurons. In addition to bFGF, epidermal growth factor, nerve growth factor, and sonic hedgehog were also capable of generating spherical cell clusters that contained cells that stained for neuronal markers following the addition of trophic factors. These results suggest that early postnatal dorsal root ganglia contain neural precursors that appear to proliferate in response to various factors and can then be induced to differentiate into neurons. In conclusion, the existence of neural precursors and the possibility of neurogenesis in postnatal dorsal root ganglia may provide a greater range of plasticity available to somatosensory systems during growth or following injury, perhaps to replace ineffectual or dying neurons.     

Matrix metalloproteinase-2 is involved in myelination of dorsal root ganglia neurons

Matrix metalloproteinases (MMPs) comprise a large family of endopeptidases that are capable of degrading all extracellular matrix components. There is increasing evidence that MMPs are not only involved in tissue destruction but may also exert beneficial effects during axonal regeneration and nerve remyelination. Here, we provide evidence that MMP-2 (gelatinase A) is associated with the physiological process of myelination in the peripheral nervous system (PNS). In a myelinating co-culture model of Schwann cells and dorsal root ganglia neurons, MMP-2 expression correlated with the degree of myelination as determined by immunocytochemistry, zymography, and immunosorbent assay. Modulation of MMP-2 activity by chemical inhibitors led to incomplete and aberrant myelin formation. In vivo MMP-2 expression was detected in the cerebrospinal fluid (CSF) of patients with Guillain-Barré syndrome as well as in CSF and sural nerve biopsies of patients with chronic inflammatory demyelinating polyneuropathy. Our findings suggest an important, previously unrecognized role for MMP-2 during myelination in the PNS. Endogenous or exogenous modulation of MMP-2 activity may be a relevant target to enhance regeneration in demyelinating diseases of the PNS.     

Rat dorsal root ganglia express distinctive forms of the alpha2 calcium channel subunit

Calcium channel alpha2 delta subunit is a glycosylated structural subunit consistent of the alpha2 subunit and the delta peptide. Previous studies have indicated distinctive alpha2 subunit expression in rat spinal cord and dorsal root ganglia (DRG). This study examined whether differential glycosylation underlies the molecular basis of distinct alpha2 delta subunits. The migration patterns of deglycosylated alpha2 subunits from rat spinal cord, DRG, brain and skeletal muscle were compared in Western blots. The data reported indicate that there are two forms of the alpha2 subunit in DRG that are different from the alpha2 subunit in other tissues examined, at least at the glycosylation level. Thus, post-translational modification may be important in tissue specific and functional expression of the alpha2 delta subunit.     

Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis

Purpose:   Central nervous system plasticity is essential for normal function, but can also reinforce abnormal network behavior, leading to epilepsy and other disorders. The role of altered ion channel expression in abnormal plasticity has not been thoroughly investigated. Nav1.6 is the most abundantly expressed sodium channel in the nervous system. Because of its distribution in the cell body and axon initial segment, Nav1.6 is crucial for action potential generation. The goal of the present study was to investigate the possible role of changes in Nav1.6 expression in abnormal, activity-dependent plasticity of hippocampal circuits.
Methods:   We studied kindling, a form of abnormal activity-dependent facilitation. We investigated: (1) sodium channel protein expression by immunocytochemistry and sodium channel messenger RNA (mRNA) by in situ hybridization, (2) sodium current by patch clamp recordings, and (3) rate of kindling by analysis of seizure behavior. The initiation, development, and expression of kindling in wild-type mice were compared to Nav1.6 +/− med ^tg mice, which have reduced expression of Nav1.6.
Results:   We found that kindling was associated with increased expression of Nav1.6 protein and mRNA, which occurred selectively in hippocampal CA3 neurons. Hippocampal CA3 neurons also showed increased persistent sodium current in kindled animals compared to sham-kindled controls. Conversely, Nav1.6 +/− med ^tg mice resisted the initiation and development of kindling.
Discussion:   These findings suggest an important mechanism for enhanced excitability, in which Nav1.6 may participate in a self-reinforcing cycle of activity-dependent facilitation in the hippocampus. This mechanism could contribute to both normal hippocampal function and to epilepsy and other common nervous system disorders.     

Developmental regulation of carbonic anhydrase expression in mouse dorsal root ganglia

The development of proprioceptive neurons in mammalian dorsal root ganglia (DRG) remains poorly documented since few specific markers for these neurons are known. Recent studies suggest that carbonic anhydrase (CA) is a specific marker of this functionally defined neuronal population. The present study was designed to investigate the development of CA staining in sensory neurons. We investigated CA reactivity in mouse lumbar DRGs from embryonic day 13 (E13) to postnatal day 100 (P100) using a modified cytoenzymatic Hansson method. Neuronal CA reactivity was first detected during the perinatal stage (1–3% of DRG neurons) and increased progressively from P0 to P60 when it reached a plateau (about 30–33% of DRG neurons). Statistical morphometric analysis was used to define whether CA staining identifies the same population(s) during development. The results demonstrated that, whatever the stage of development, reactive neuronal cells are included in the well-defined large type A population. The possibility that neuronal CA expression is a reliable marker of the ‘functional activity’ of the proprioceptive neurons in mammals is discussed. The late developmental expression of the enzyme (after target innervation) raises the possibility of a regulation of the CA phenotype by neuron-target interactions.     

Abnormalities expressed in long-term cultures of dorsal root ganglia from the dystrophic mouse

We have compared the development in long-term tissue culture of dorsal root ganglia taken from normal and dystrophic mice. Cultures were prepared from late fetal (15–20 days) or neonatal mice of either the C57BL/6 dy^2j/dy^2j dystrophic(dy) or C57BL/6J +/+ (control) strain and maintained until fully myelinated (5 weeks or more). Analysis by light and electron microscopy indicated tha the substantial ensheathment failure present in certain dy nerve roots in vivo is not expressed in cultures; myelination and Schwann cell numbers are comparable to control cultures. On the other hand, many of the subtle abnormalities more recently described in distal parts of peripheral nerves of dystrophic mice are expressed in the dy cultures. These include: (a) discontinuity in the basal lamina surrounding both myelin-forming and non-myelinating Schwann cells; (b) elongated nodes of Ranvier occurring along otherwise well myelinated nerve fibres; (c) relatively short myelin internodes that are increased in thickness as well as irregularities of internodes length along a nerve fiber; (d) Schwann cell nuclei substantially displaced from the central point of myelin internodes; and (e) occasional regions of incomplete ensheathment of unmyelinated nerve fibers. In discussing these observations, we present arguments that the dy nerve lesion may be explained by the presence of an abnormality in the extracellular matrix of the peripheral nerve tissues of the dy mouse.     

Reversible effects of hypoxia on neurons in mouse dorsal root ganglia in vitro

Mouse dorsal root ganglia (DRG) were isolated and maintained in a tissue chamber. Membrane potential of ‘A-type’ neurons was recorded with intracellular electrodes. When the supply of oxygen was reduced, cells depolarized by a few mV and then maintained a stable membrane potential or partially repolarized. During depolarization the action potential was reduced in amplitude and the hyperpolarizing afterpotential was depressed. Reoxygenation within 15–88 min was followed by a brief period of hyperpolarization and then complete recovery. In about 60% of the cells, invasion of the cell soma by impulses triggered by dorsal root (DR) stimulation failed during hypoxia while action potentials could still be evoked by stimulation of the peripheral nerve and by direct intracellular stimuli. Conduction from DR into the peripheral nerve stump was unchanged indicating that the blockade of DR-evoked impulse conduction occurred at the bifurcation of the axon. Results with paired pulse stimulation indicated that impulses passing the axon bifurcation leave a long lasting ( 25 ms) post-spike subnormal period. In DRG cells treated with tetraethylammonium (TEA) the calcium-mediated ‘shoulder’ of the action potential was curtailed during oxygen withdrawal. In contrast to CNS neurons, DRG cells did not show early hypoxic hyperpolarization, nor the delayed hypoxic spreading depression-like depolarization. The findings support the suggestion that the reversible depression of synaptic potentials in the CNS during the early phase of hypoxia is caused by a combination of conduction failure at axon branch points and curtailment of voltage calcium currents of presynaptic terminals, both effects resulting in reduced transmitter output.     

Axonal dystrophy of dorsal root ganglion sensory neurons in a mouse model of Niemann-Pick disease type C

Niemann-Pick disease type C (NP-C) is a progressive and fatal neurological disorder characterized by intracellular accumulation of cholesterol and glycolipid. A Balb/c-npc1 mutant strain is a genetically authentic murine model of NP-C, and homozygous mice show progressive weight loss and tremor or ataxia until death at 12-14 weeks of age. Neuropathologically, this model is known to faithfully reproduce the cardinal histologic features of NP-C including neuronal storage, appearance of swollen axons (spheroids), and neuronal loss, although the cellular mechanisms of neural degeneration are largely unknown. To investigate the mode of neural degeneration of sensory neurons in NP-C, we studied the central processes of dorsal root ganglion (DRG) neurons at the level of the medullary dorsal column nuclei and the spinal dorsal horn with special attention to the ultrastructural changes of presynaptic axon terminals. The appearance of axonal spheroids in the dorsal column nuclei and the loss of axons in the spinal nerve roots were assessed quantitatively. We show that the gracile nuclei develop numerous axonal spheroids after only 3 weeks. At 6 and 9 weeks, dystrophic axons, which were separated from simple axonal spheroids by the ultrastructural presence of distinctive tubulo-vesicular elements, progressively increased in size and number. These neuropathological findings are identical to those of gracile axonal dystrophy (GAD) of the normal aging mouse. Presynaptic elements were exclusively involved in spheroid formation. The cuneate nuclei and the spinal dorsal horn revealed fewer axonal spheroids and only rare dystrophic changes. This was associated with a significant drop in the number of L4-5 dorsal root axons in NP-C mouse at 9 weeks of age compared with controls. These results support the existence of a length-dependent axonopathy in the central processes of DRG neurons and are consistent with the view that altered axonal transport, which is implicated in the pathogenesis of GAD in physiological aging, may be an underlying mechanism in neuronal degeneration in NP-C. Clinically, the premature development of GAD may be responsible for ataxia, one of the early manifestations of this disease.     

Axonal damage and demyelination in long-term dorsal root ganglia cultures from a rat model of Charcot-Marie-Tooth type 1A disease

Clinical progression in hereditary and acquired demyelinating disorders of both the central and peripheral nervous system is mainly due to a time-dependent axonal impairment. We established 90-day dorsal root ganglia (DRG) cultures from a rat model of Charcot-Marie-Tooth type 1A (CMT1A) neuropathy to evaluate the structure of myelin and axons, and the expression of myelin-related proteins and cytoskeletal components, by morphological and molecular techniques. Both wild-type and CMT1A cultures were rich in myelinated fibres. Affected cultures showed dysmyelinated internodes and focal myelin swellings. Furthermore, uncompacted myelin and smaller axons with increased neurofilament (NF) density were found by electron microscopy, and Western blots showed higher levels of nonphosphorylated NF. Confocal microscopy demonstrated an abnormal distribution of the myelin-associated glycoprotein which, instead of being expressed at the noncompact myelin level, showed focal accumulation along the internodes while other myelin proteins were normally distributed. These findings suggest that CMT1A DRG cultures, similarly to the animal model and human disease, undergo axonal atrophy over a period of time. This model may be utilized to study the molecular changes underlying demyelination and secondary axonal impairment. As axonal damage may occur after just 3 months and tissue cultures represent a strictly controlled environment, this model may be ideal for testing neuroprotective therapies.     

Acetylcholinesterase in the development of chick dorsal root ganglia

Acetylcholinesterase is expressed in chick dorsal root ganglia neurons very early in development. Since the physiological role of the enzyme in these cells is still obscure, it appeared of interest to investigate its modifications in the course of development. The specific activity of acetylcholinesterase in chick dorsal root ganglia increases, during in ovo development, from day E5 to day E13; after day E13 there is a decrease. Conversely, when acetylcholinesterase activity was expressed on a per ganglion basis, a continuous increase in the level of the enzyme until day E20 was observed. Acetylcholinesterase is a polymorphic enzyme and its molecular forms have different cellular localizations. Two globular forms, a tetramer (G4) and a dimer (G2), are present in the ganglia, as in chick brain. G4 is the major form at day E5, where it represents about 85% of the activity. This form shows a progressive decrease since day E8, and at day E20 exhibits activity levels similar to those of G2. It is known that acetylcholinesterase-producing cells are also able to release the enzyme in the extracellular space. We determined the release of acetylcholinesterase by cultured dorsal root ganglia neurons at various developmental stages: acetylcholinesterase release is significantly increased at day E20, as compared to younger stages, and 90% of the enzyme released is G4.     

Ultrastructural effects of herpes simplex virus type 2 infection of rat dorsal root ganglia in culture.

The ultrastructural consequences of herpes simplex virus type 2 (strain R-2) infection of organotypic cultures of embryonic rat dorsal root ganglia were studied. The intial consequences (dilation of endoplasmic reticulum and/or Golgi apparatus and distortion of mitochondrial structure) occurred in the cytoplasm. These effects were followed by several nuclear changes including loss of nucleoplasm, and margination of chromatin. Extensive nuclear membrane proliferation was accompanied by the viral maturation process. Two previously unreported observations were made. First, productive virus replications occurred in glial cells, as well as in neurons. Mature, enveloped virus was produced by nuclear budding and envelopment in the cytoplasm in both cell types. Second, neurons were observed to participate in polykaryocyte formation with other neurons and with glial cells. These polykaryocytes were usually composed of only three or four cells. Neuronal-glial polykaryocytes were more prevalent than neuronal-neuronal polykaryocytes. In general, however, the ultrastructural changes in neurons and glial cells in culture were consistent with previously reported changes occurring in nervous tissue of experimental animals suffering from acute herpes simplex virus infections. Therefore, the utilization of this in vitro system to further study the pathogenesis of acute herpetic infections of sensory ganglia appears to be reasonable.     

Imunoreactivity of zinc transporter 7 (ZNT7) in mouse dorsal root ganglia

In the present study, we showed for the first time the localization of ZNT7 immunoreactivity in the mouse dorsal root ganglion (DRG) by means of immunohistochemistry and confocal laser scanning microscopy. Our results revealed that ZNT7 immunoreactivity was abundantly expressed in the nerve cells of the mouse DRG. Strong ZNT7 immunoreactivity was predominantly distributed in the perinuclear region of positive cells, while the nuclei were devoid of staining. Double immunofluorescence labeling of ZNT7 and TGN38 revealed a colocalization of the two antigens in the Golgi apparatus. In addition, the presence of labile zinc ions was detected with in vivo zinc selenium autometallography (AMG). AMG observations showed that the zinc staining pattern was also predominately located in the perinuclear Golgi area, like the ZNT7 immunostaining pattern in the DRG. These observations strongly suggest that ZNT7 may play an important role in facilitating zinc transport into the Golgi apparatus from the cytosol in the mouse DRG.