Three-dimensional neuron-astrocyte construction on matrigel enhances establishment of functional voltage-gated sodium channels.

This study aimed to investigate and compare cell growth manners and functional differences of primary cortical neurons cultured on either poly-d-lysine (PDL) and or Matrigel, to delineate the role of extracellular matrix on providing resemblance to in vivo cellular interactions in nervous tissue. Primary cortical neurons, obtained from embryonic day 15 mice pups, seeded either on PDL- or Matrigel-coated culture ware were investigated by DIC/bright field and fluorescence/confocal microscopy for their morphology, 2D and 3D structure, and distribution patterns. Patch clamp, western blot, and RT-PCR studies were performed to investigate neuronal firing thresholds and sodium channel subtypes Nav1.2 and Nav1.6 expression. Cortical neurons cultured on PDL coating possessed a 2D structure composed of a few numbers of branched and tortuous neurites that contacted with each other in one to one manner, however, neurons on Matrigel coating showed a more complicated dimensional network that depicted tight, linear axonal bundles forming a 3D interacted neuron-astrocyte construction. This difference in growth patterns also showed a significant alteration in neuronal firing threshold which was recorded between 80 < Iinj > 120 pA on PDL and 2 < Iinj > 160 pA on Matrigel. Neurons grown up on Matrigel showed increased levels of sodium channel protein expression of Nav1.2 and Nav1.6 compared to neurons on PDL. These results have demonstrated that a 3D interacted neuron-astrocyte construction on Matrigel enhances the development of Nav1.2 and Nav1.6 in vitro and decreases neuronal firing threshold by 40 times compared to conventional PDL, resembling in vivo neuronal networks and hence would be a better in vitro model of adult neurons.

Nav1.2 is expressed in caudal ganglionic eminence-derived disinhibitory interneurons: Mutually exclusive distributions of Nav1.1 and Nav1.2.

Nav1.1 and Nav1.2 are the voltage-gated sodium channel pore-forming alpha I and II subunits, encoded by the genes SCN1A and SCN2A. Although mutations of both genes have similarly been described in patients with epilepsy, autism and/or intellectual disability, their expression sites in brain are largely distinct. Nav1.1 was shown to be expressed dominantly in parvalbumin (PV)-positive or somatostatin (SST)-positive inhibitory neurons and in a sparsely-distributed subpopulation of excitatory neurons. In contrast, Nav1.2 has been reported to be dominantly expressed in excitatory neurons. Here we show that Nav1.2 is also expressed in caudal ganglionic eminence (CGE)-derived inhibitory neurons, and expressions of Nav1.1 and Nav1.2 are mutually-exclusive in many of brain regions including neocortex, hippocampus, cerebellum, striatum and globus pallidus. In neocortex at postnatal day 15, in addition to the expression in excitatory neurons we show that Nav1.2 is expressed in reelin (RLN)-positive/SST-negative inhibitory neurons that are presumably single-bouquet cells because of their cortical layer I-limited distribution, and vasoactive intestinal peptide (VIP)-positive neurons that would be multipolar cell because of their layer I/II margin and layer VI distribution. Although Nav1.2 has previously been reported to be expressed in SST-positive cells, we here show that Nav1.2 is not expressed in either of PV-positive or SST-positive inhibitory neurons. PV-positive and SST-positive inhibitory neurons derive from medial ganglionic eminence (MGE) and innervate excitatory neurons, while VIP-positive and RLN-positive/SST-negative inhibitory neurons derive from CGE, innervate on inhibitory neurons and play disinhibitory roles in the neural network. Our results therefore indicate that, while Nav1.1 is expressed in MEG-derived inhibitory neurons, Nav1.2 is expressed in CGE-derived disinhibitory interneurons in addition to excitatory neurons. These findings should contribute to understanding of the pathology of neurodevelopmental diseases caused by SCN2A mutations.

Expression of sodium channel α subunits 1.1, 1.2 and 1.6 in rat hippocampus after kainic acid-induced epilepsy.

Voltage-gated Na(+) channels control neuronal excitability and are the primary target for the majority of anti-epileptic drugs. This study investigates the (sub)cellular expression patterns of three important brain-associated Na(+) channel α subunits: NaV1.1, NaV1.2 and NaV1.6 during epileptogenesis (induced by kainic acid) using time points that cover the period from induction to the chronic phase of epilepsy. NaV1.1 immunoreactivity was persistently reduced at 1 day, 3 weeks and 2 months after SE in CA1 and CA3. About 50% of the NaV1.1-positive interneurons was lost at one day after SE in all regions investigated. In the hilus a similar reduction in NeuN-positive neurons was found, while in the CA1 and CA3 region the loss in NeuN-positive neurons only reached 15% in the chronic phase of epilepsy. This implies a stronger shift in the balance between excitation and inhibition toward excitation in the CA1 and CA3 region than in the hilus. NaV1.2 immunoreactivity in the inner molecular layer of the dentate gyrus was lower than control at 1 day after SE. It increased at 3 weeks and 2 months after SE in the inner molecular layer and overlapped with sprouted mossy fibers. NaV1.6 immunoreactivity in the dendritic region of CA1 and CA3 was persistently reduced at all time-points during epileptogenesis. Some astrocytes expressed NaV1.1 and NaV1.6 at 3 weeks after SE. Expression data alone are not sufficient to explain changes in network stability, or infer causality in epileptogenesis. These results demonstrate that hippocampal sub-regional expression of NaV1.1, NaV1.2 and NaV1.6 Na(+) channel α subunits is altered during epileptogenesis in a time and location specific way. This implies that understanding epileptogenesis has to take into account several distinct and type-specific changes in sodium channel expression.

Block of a subset of sodium channels exacerbates experimental autoimmune encephalomyelitis.

Voltage-gated sodium channels (Navs) are involved in several aspects of the pathogenesis of multiple sclerosis (MS). Within acute MS plaques, they are expressed along demyelinated axons. Studies in experimental autoimmune encephalomyelitis (EAE) demonstrated a neuroprotective effect of non-specific Nav blockers. Further, block of specific Navs involved in MS is suggested to have an advantage over non-specific blockers. We investigated the effects of the synthetic Midi peptide in EAE, as it potently and specifically blocks Nav1.2, Nav1.4 and Nav1.6. Administration of this Midi peptide worsens the clinical disease pattern and Nav1.2 and Nav1.6 expression levels were elevated in brain but not in spinal cord of Midi-treated mice, implicating that Navs play a complex role in the pathogenesis of EAE.