Wild-type Dorsal Root Ganglia Neurons Research Articles

Inactivation of vimentin in satellite glial cells affects dorsal root ganglion intermediate filament expression and neuronal axon growth in vitro

Peripheral nerve trauma and regeneration are complex events, and little is known concerning how occurrences in the distal stump affect the cell body's response to injury. Intermediate filament (IF) proteins underpin cellular architecture and take part in nerve cell proliferation, differentiation and axon regeneration, but their role in these processes is not yet fully understood. The present study aimed to investigate the regulation and interrelationship of major neural IFs in adult dorsal root ganglion (DRG) neurons and satellite glial cells (SGCs) following sciatic nerve injury. We demonstrated that the expression of neural IFs in DRG neurons and SGCs after axotomy depends on vimentin activity. In intact DRGs, synemin M and peripherin proteins are detected in small neurons while neurofilament L (NFL) and synemin L characterize large neurons. Both neuronal populations are surrounded by vimentin positive- and glial fibrillary acidic protein (GFAP)-negative SGCs. In response to axotomy, synemin M and peripherin were upregulated in large wild-type DRG neurons and, to a lesser extent, in vim−/− and synm−/− DRG neurons, suggesting the role for these IFs in axon regeneration. However, an increase in the number of NFL-positive small neurons was observed in vim−/− mice, accompanied by a decrease of peripherin-positive small neurons. These findings suggest that vimentin is required for injury-induced neuronal IF remodeling. We further show that vimentin is also indispensable for nerve injury-induced GFAP upregulation in perineuronal SGCs and that inactivation of vimentin and synemin appears to accelerate the rate of DRG neurite regeneration at early stages in vitro.

Constitutional mislocalization of Pten drives precocious maturation in oligodendrocytes and aberrant myelination in model of autism spectrum disorder

There is a strong genetic association between germline PTEN mutation and autism spectrum disorder (ASD), making Pten-mutant models exemplary for the study of ASD pathophysiology. We developed the Ptenm3m4 mouse, where Pten is largely restricted from the nucleus, which recapitulates patient-like, autism-related phenotypes: behavioral changes, macrocephaly, and white matter abnormalities. This study aimed to investigate the contribution of oligodendrocyte (OL) lineage differentiation and functional changes in myelination to the white matter phenotype. OL lineage differentiation and myelination in Ptenm3m4 mice was studied using immunohistochemical and electron microscopic analyses. We also used primary oligodendrocyte progenitor cells (OPCs) to determine the effect of the Ptenm3m4 mutation on OPC proliferation, migration and maturation. Finally, we assessed the myelinating competency of mutant OLs via co-culture with wildtype dorsal root ganglia (DRG) neurons. The in vivo analyses of Ptenm3m4/m3m4 murine brains showed deficits in proteolipid protein (Plp) trafficking in myelinating OLs. Despite the increased expression of myelin proteins in the brain, myelin deposition was observed to be abnormal, often occurring adjacent to, rather than around axons. Mutant primary OPCs showed enhanced proliferation and migration. Furthermore, mutant OPCs matured precociously, exhibiting aberrant myelination in vitro. Mutant OPCs, when co-cultured with wildtype DRG neurons, showed an inability to properly ensheath axons. Our findings provide evidence that the Ptenm3m4 mutation disrupts the differentiation and myelination programs of developing OLs. OL dysfunction in the Ptenm3m4 model explains the leukodystrophy phenotype, a feature commonly associated with autism, and highlights the growing importance of glial dysfunction in autism pathogenesis.

Metallothionein-I/II Promotes Axonal Regeneration in the Central Nervous System

The adult CNS does not spontaneously regenerate after injury, due in large part to myelin-associated inhibitors such as myelin-associated glycoprotein (MAG), Nogo-A, and oligodendrocyte-myelin glycoprotein. All three inhibitors can interact with either the Nogo receptor complex or paired immunoglobulin-like receptor B. A conditioning lesion of the sciatic nerve allows the central processes of dorsal root ganglion (DRG) neurons to spontaneously regenerate in vivo after a dorsal column lesion. After a conditioning lesion, DRG neurons are no longer inhibited by myelin, and this effect is cyclic AMP (cAMP)- and transcription-dependent. Using a microarray analysis, we identified several genes that are up-regulated both in adult DRGs after a conditioning lesion and in DRG neurons treated with cAMP analogues. One gene that was up-regulated under both conditions is metallothionein (MT)-I. We show here that treatment with two closely related isoforms of MT (MT-I/II) can overcome the inhibitory effects of both myelin and MAG for cortical, hippocampal, and DRG neurons. Intrathecal delivery of MT-I/II to adult DRGs also promotes neurite outgrowth in the presence of MAG. Adult DRGs from MT-I/II-deficient mice extend significantly shorter processes on MAG compared with wild-type DRG neurons, and regeneration of dorsal column axons does not occur after a conditioning lesion in MT-I/II-deficient mice. Furthermore, a single intravitreal injection of MT-I/II after optic nerve crush promotes axonal regeneration. Mechanistically, MT-I/II ability to overcome MAG-mediated inhibition is transcription-dependent, and MT-I/II can block the proteolytic activity of α-secretase and the activation of PKC and Rho in response to soluble MAG.

Involvement of Tyr1472 Phosphorylation of NMDA Receptor NR2B Subunit in Postherpetic Neuralgia in Model Mice

BackgroundPostherpetic neuralgia is spontaneous pain and allodynia that persist long after the disappearance of the cutaneous lesions caused by herpes zoster. Inoculation of mice with herpes simplex virus-1 causes herpes zoster-like skin lesions and herpetic and postherpetic pain. Although NMDA receptors have been suggested to be involved in postherpetic pain as in other types of neuropathic pain, the neural mechanism remains unclear. NMDA receptor NR2B subunit is the most tyrosine-phosphorylated protein in the brain, and Tyr1472 is the major phosphorylation site of this subunit.ResultsTo elucidate the role of Tyr1472 phosphorylation of the NR2B subunit in herpetic and postherpetic allodynia, we inoculated herpes simplex virus-1 into the unilateral hind paw of knock-in mice with a mutation of Tyr1472 of the NR2B subunit to Phe (Y1472F-KI). On day 7 post-inoculation, acute herpetic allodynia was observed in more than 80% of the inoculated wild-type and Y1472F-KI mice. Y1472F-KI mice showed significantly reduced intensity and incidence of postherpetic allodynia on days 45–50 post-inoculation as compared with wild-type mice. The innervation in the skin at the postherpetic neuralgia phase was retained to a greater extent in the Y1472F-KI mice. The level of activating transcription factor-3 mRNA, a marker of axonal damage, increased much less in the dorsal root ganglia (DRGs) of Y1472F-KI mice than in those of wild-type mice; and the level of nerve growth factor mRNA significantly increased in wild-type mice, but not at all in Y1472F-KI mice on day 7 post-inoculation. Production of nerve growth factor was at the basal level in the skin of both groups of mice on day 50 post-inoculation. Nerve growth factor and glial cell-derived neurotrophic factor stimulated neurite outgrowth of cultured DRG neurons from Y1472F-KI mice, similarly or less so as they did the outgrowth of those from wild-type mice. Wild-type DRG neurons were more susceptible to glutamate neurotoxicity than Y1472F-KI ones.ConclusionsTaken together, the present data suggest that phosphorylation of the NR2B subunit at its Tyr1472 is involved in the development of postherpetic allodynia due to nerve damage and that the nerve damage at the acute herpetic phase is correlated with the incidence of postherpetic pain.

Differential clustering of Caspr by oligodendrocytes and Schwann cells

Formation of the paranodal axoglial junction (PNJ) requires the presence of three cell adhesion molecules: the 155-kDa isoform of neurofascin (NF155) on the glial membrane and a complex of Caspr and contactin found on the axolemma. Here we report that the clustering of Caspr along myelinated axons during development differs fundamentally between the central (CNS) and peripheral (PNS) nervous systems. In cultures of Schwann cells (SC) and dorsal root ganglion (DRG) neurons, membrane accumulation of Caspr was detected only after myelination. In contrast, in oligodendrocytes (OL)/DRG neurons cocultures, Caspr was clustered upon initial glial cell contact already before myelination had begun. Premyelination clustering of Caspr was detected in cultures of oligodendrocytes and retinal ganglion cells, motor neurons, and DRG neurons as well as in mixed cell cultures of rat forebrain and spinal cords. Cocultures of oligodendrocyte precursor cells isolated from contactin- or neurofascin-deficient mice with wild-type DRG neurons showed that clustering of Caspr at initial contact sites between OL processes and the axon requires glial expression of NF155 but not of contactin. These results demonstrate that the expression of membrane proteins along the axolemma is determined by the type of the contacting glial cells and is not an intrinsic characteristic of the axon.

Increased 5-HT3-mediated signalling in pelvic afferent neurons from mice deficient in P2X2 and/or P2X3 receptor subunits

Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT3 receptors, respectively. We have investigated the changes in P2X and 5-HT3-mediated signalling in pelvic afferent neurons in mice deficient in P2X2 and/or P2X3 subunits by whole-cell recording of L6–S2 dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X3−/− neurons, whereas sustained currents were absent in P2X2−/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X2/P2X3Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT3 receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X 2−/−, P2X3−/− and P2X2/P2X3Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X2/P2X3Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X2/P2X3Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X2/P2X3Dbl−/− preparations. These data suggest that P2X receptors containing the P2X2 and/or P2X3 subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X2 or P2X3 receptor genes. This effect is more pronounced when both subunits are absent.

Abnormal GABAAReceptor-Mediated Currents in Dorsal Root Ganglion Neurons Isolated from Na–K–2Cl Cotransporter Null Mice

We have recently disrupted Slc12a2, the gene encoding the secretory Na-K-2Cl cotransporter in mice (NKCC1) (Delpire et al., 1999). Gramicidin perforated-patch and whole-cell recordings were performed to study GABA-induced currents in dorsal root ganglion (DRG) neurons isolated from wild-type and homozygote NKCC1 knock-out mice. In wild-type DRG neurons, strong GABA-evoked inward current was observed at the resting membrane potential, suggesting active accumulation of Cl(-) in these cells. This GABA-induced current was blocked by picrotoxin, a GABA(A) receptor blocker. The strong Cl(-) accumulation that gives rise to depolarizing GABA responses is caused by Na-K-2Cl cotransport because reduction of external Cl(-) or application of bumetanide induced a decrease in [Cl(-)](i), whereas an increase in external K(+) caused an apparent [Cl(-)](i) accumulation. In contrast to control neurons, little or no net current was observed at the resting membrane potential in homozygote NKCC1 mutant DRG neurons. E(GABA) was significantly more negative, demonstrating the absence of Cl(-) accumulation in these cells. Application of bumetanide induced a positive shift of E(GABA), suggesting the presence of an outward Cl(-) transport mechanism. In agreement with an absence of GABA depolarization in DRG neurons, behavioral analysis revealed significant alterations in locomotion and pain perception in the knock-out mouse. Our results clearly demonstrate that the Na-K-2Cl cotransporter is responsible for [Cl(-)](i) accumulation in DRG neurons and that via regulation of intracellular Cl(-), the Na-K-2Cl cotransporter participates in the modulation of GABA neurotransmission and sensory perception.

Sodium channel expression in NGF-overexpressing transgenic mice.

Dorsal root ganglion (DRG) neurons depend on nerve growth factor (NGF) for survival during development, and for the maintenance of phenotypic expression of neuropeptides in the adult. NGF also plays a role in the regulation of expression of functional sodium channels in both PC12 cells and DRG neurons. Transgenic mice that overexpress NGF under the keratin promoter (hyper-NGF mice) show increased levels of NGF in the skin from embryonic day 11 through adulthood, hypertrophy of the peripheral nervous system and mechanical hyperalgesia. We show here that mRNA levels for specific sodium channel isotypes are greater in small (< 30 microm diameter) DRG neurons from hyper-NGF mice compared to wild-type mice. Hybridization signals for sodium channel subunits alphaII and beta2 displayed the most substantial enhancement in hyper-NGF mice, compared to wild-type mice DRG, and mRNA levels for alphaI, NaG, Na6, SNS/PN3, NaN, and beta1 were also greater in hyper-NGF DRG. In contrast, the levels of alphaII and PN1 mRNAs were similar in neurons from hyper-NGF and wild-type DRG. Whole-cell patch-clamp studies showed no significant differences in the peak sodium current densities in hyper-NGF vs. wild-type DRG neurons. These data demonstrate that DRG neurons in wild-type mice have a heterogeneous pattern of sodium channel expression, which is similar to that previously described in rat, and suggest that transcripts of some, but not all, sodium channel mRNAs can be modulated by long-term overexpression of NGF.

Influence of leukemia inhibitory factor on galanin/GMAP and neuropeptide Y expression in mouse primary sensory neurons after axotomy.

The effect of unilateral transection of the sciatic nerve on expression of immunoreactive galanin (GAL), galanin-message-associated peptide (GMAP) and neuropeptide tyrosine (NPY) in dorsal root ganglia (DRGs) was studied in wild-type mice and in leukemia inhibitory factor (LIF)-deficient mice. In normal and contralateral DRGs small numbers of weakly fluorescent GAL- and GMAP-positive neuronal cell bodies and numerous positive fibers were observed. No NPY-positive cell bodies but a few fibers surrounding blood vessels were seen. In LIF deficient mice hardly any GAL- or GMAP-positive neurons or fibers were seen, nor was NPY-like immunoreactivity present in cell bodies. After axotomy there was a dramatic upregulation of all three peptides in wild-type DRG neurons, whereby 50-60% of the neuron profiles, encompassing both small and large profiles, were GAL- and GMAP-immunoreactive (IR). About one third of all neuron profiles, mainly large ones, were NPY-positive. In LIF-deficient mice this upregulation was much less pronounced. Thus GAL- and GMAP-IR neuron profiles were reduced by 65-70% compared with the wild-type mice. The number of NPY-positive neuron profiles was reduced to half but this difference was not significant. There was also an ipsilateral decrease in fluorescence intensity for all three peptide immunoreactivities in the LIF-deficient mice as compared with wild-type mice after axotomy. There was no apparent difference in size between, respectively, GAL- and GMAP-positive profiles when comparing LIF-deficient and wild-type mice before or after axotomy. There were, however, no small NPY-IR profiles in the LIF-deficient group. The present results suggests that LIF is important for the dramatic upregulation of GAL and GMAP seen after axotomy. It may also be important for the normal expression of galanin in mouse DRGs, since wild-type mice seemed to have somewhat more positive cell bodies and more fluorescent fibers. LIF seems to be less important for the control of NPY synthesis, but may be involved in NPY regulation in small-sized neurons.