Molecular and functional remodeling of electrogenic membrane of hypothalamic neurons in response to changes in their input.

Neurons respond to stimuli by integrating generator and synaptic potentials and generating action potentials. However, whether the underlying electrogenic machinery within neurons itself changes, in response to alterations in input, is not known. To determine whether there are changes in Na+ channel expression and function within neurons in response to altered input, we exposed magnocellular neurosecretory cells (MNCs) in the rat supraoptic nucleus to different osmotic milieus by salt-loading and studied Na+ channel mRNA and protein, and Na+ currents, in these cells. In situ hybridization demonstrated significantly increased mRNA levels for alpha-II, Na6, beta1 and beta2 Na+ channel subunits, and immunohistochemistry/immunoblotting showed increased Na+ channel protein after salt-loading. Using patch-clamp recordings to examine the deployment of functional Na+ channels in the membranes of MNCs, we observed an increase in the amplitude of the transient Na+ current after salt-loading and an even greater increase in amplitude and density of the persistent Na+ current evoked at subthreshold potentials by slow ramp depolarizations. These results demonstrate that MNCs respond to salt-loading by selectively synthesizing additional, functional Na+ channel subtypes whose deployment in the membrane changes its electrogenic properties. Thus, neurons may respond to changes in their input not only by producing different patterns of electrical activity, but also by remodeling the electrogenic machinery that underlies this activity.

Orphan nuclear receptor ROR alpha gene: isoform-specific spatiotemporal expression during postnatal development of brain.

We analyzed expression of mouse orphan nuclear receptor ROR alpha during postnatal development of rodent brain. Using a riboprobe corresponding to the 3'-end of mROR alpha cDNA a peak of ROR alpha expression was observed at postnatal 16 day (P16) in the Purkinje cells of cerebellum, neurons of the thalamus and the olfactory bulb. The hippocampus was also shown to express ROR alpha with an earlier peak at P7. Expression in cell types other than the Purkinje cells appeared transient. On the other hand, when a probe to the 5'-end of mROR alpha cDNA was used, we observed patterns of ROR alpha expression that are different from those observed with the 3'-probe. No specific transcripts of ROR alpha were detected with the 5'-probe in the Purkinje cells until P16. Additionally, the relative level of the hybridization signals with the 5'-probe and the 3'-probe were different among the various brain regions. Together with the previous findings that ROR alpha comprises at least four isoforms which differ from one another in their N-terminal regions, these observations suggest that the spatiotemporal expression of ROR alpha is under isoform-specific regulation. The timing of its expression suggests that ROR alpha may be involved in regulation of postnatal maturation of specific classes of neurons.

Manipulation of the delayed rectifier Kv1.5 potassium channel in glial cells by antisense oligodeoxynucleotides.

Glial cells have been shown to express several biophysically and pharmacology distinct potassium channel types. However, the molecular identity of most glial K+ channels is unknown. We have developed an antibody specific for the Shaker type potassium channel Kv1.5 protein, and demonstrate by immunohistochemistry the presence of this channel in glial cells of adult rat hippocampal and cerebellar slices, as well as in cultured spinal cord astrocytes. Immunoreactivity was particularly intense in the endfoot processes of astrocytes surrounding the microvasculature of the hippocampus. The specific contribution of this channel protein to the delayed rectifying K+ current of spinal cord astrocytes was determined by incubating these cells with antisense oligodeoxynucleotides complementary to the mRNA coding for Kv1.5 protein. Such treatment reduced delayed rectifier current density and shifted the potassium current steadystate inactivation, without altering current activation, cell capacitance, or cell resting potential. The tetraethylammonium acetate (TEA) sensitivity of astrocytic delayed rectifier current was enhanced following antisense oligodeoxynucleotide treatment, suggesting that Kv1.5 channel protein may provide a significant component of the TEA-insensitive current in this preparation. Our results suggest that Kv1.5 is widely expressed in glial cells of brain and spinal cord and that delayed rectifying K+ currents in astrocytes are largely mediated by Kv1.5 channel protein.

An orphan nuclear receptor, mROR alpha, and its spatial expression in adult mouse brain.

We have cloned cDNA encoding a mouse nuclear receptor mROR alpha which is a homolog of human retinoic acid receptor-related orphan receptor (hROR alpha). Cotransfection experiments revealed that mROR alpha activates transcription through a retinoic acid responsive element of the laminin B1 gene (lamRARE), but not through a RARE of RAR beta gene (beta RARE) or a synthetic palindromic thyroid hormone responsive element (TREpal). The most distal AGGTCA half-site among the three half-sites of lamRARE was sufficient for binding of mROR alpha and consequently for activation of transcription. Transactivation by mROR alpha was dependent on serum in culture medium after transfection, suggesting the presence of a possible ligand. Northern hybridization and in situ hybridization analyses revealed that mROR alpha is expressed in specific areas of the brain including thalamus and olfactory bulb as well as cerebellum where it is present at highest levels in Purkinje cells. In addition to regionally heterogeneous expression in brain, its expression was temporally regulated during differentiation of P19 cells into neural cells, but not into muscle cells. These observations suggest that mROR alpha plays important roles as a transcription factor not only in differentiation of neural cell lineages but also in the mature brain.

The beta 1 subunit mRNA of the rat brain Na+ channel is expressed in glial cells.

Although the molecular characteristics of glial Na+ channels are not well understood, recent studies have shown the presence of mRNA for rat brain Na+ channel alpha subunits in astrocytes and Schwann cells. In this study, we asked whether the mRNA for the rat brain Na+ channel beta 1 subunit is expressed in glial cells. We performed in situ hybridization using a complementary RNA probe for the coding regions of the rat brain Na+ channel beta 1 subunit mRNA and detected beta 1 subunit mRNA in cultured rat optic nerve astrocytes and sciatic nerve Schwann cells. The beta 1 subunit was amplified by reverse transcription-polymerase chain reaction in rat optic and sciatic nerves, which lack neuronal somata but contain astrocytes and Schwann cells, respectively. Doublet bands of the beta 1 subunit mRNA were amplified from both optic and sciatic nerves. Through the cloning and sequencing of these bands, we confirmed the amplification of a mRNA highly homologous to the previously cloned rat brain Na+ channel beta 1 subunit (beta 1.1) and a novel form of the beta 1 subunit mRNA (beta 1.2), which is closely homologous to beta 1.1 but contains an additional 86-nucleotide insert in 3' noncoding regions. Two beta 1 subunit mRNAs were also amplified from rat brain and skeletal muscle, but not from rat liver or kidney. These results indicate that rat brain Na+ channel beta 1 subunit mRNAs are expressed in glial cells as well as in neurons.

Demyelination in spinal cord injury and multiple sclerosis: what can we do to enhance functional recovery?

Demyelination in white matter tracts has been observed in experimental and human spinal cord injury. The pathophysiology of demyelinated axons depends, in part, on their ion channel organization. Myelinated axons display a complementary distribution of sodium channels (clustered in the nodal axon membrane) and fast potassium channels (in the internodal axon membrane). The low density of sodium channels in the internodal axon membrane will impede conduction after demyelination. Moreover, "unmasked" potassium channels will tend to clamp the axon membrane close to EK, interfering with conduction in demyelinated axons. Pharmacologic blockade of these potassium channels can increase the safety factor for conduction in demyelinated axons. Restoration of conduction in demyelinated axons, so that action potentials can traverse the zone without myelin, appears to underly clinical remissions in patients with multiple sclerosis and may occur in some patients with spinal cord injury. At a cellular level, conduction through demyelinated axon regions can be facilitated by several mechanisms, including remyelination, development of excitability in demyelinated regions (which requires an adequate density of sodium channels), and impedance matching. Astrocytes have been shown to establish a specific relationship with sodium channel-rich regions of the axon membrane, and may play a role in the deployment and/or maintenance of sodium channels within the demyelinated axon membrane. Calcium influx appears to play a critical role in the cascade of events leading to secondary injury after spinal cord trauma. Recent observations suggest the hypothesis that myelin damage in spinal cord trauma may be mediated, at least in part, by influx of calcium into an intracellular compartment. As the route of calcium entry is identified and characterized, it may be possible to design strategies that will limit secondary injury after CNS trauma. The deleterious effects of calcium in injured white matter accumulate gradually, which suggests the potential reversibility of dysfunction in spinal cord tracts if treated early after trauma.

Organization of ion channels in the myelinated nerve fiber.

The functional organization of the mammalian myelinated nerve fiber is complex and elegant. In contrast to nonmyelinated axons, whose membranes have a relatively uniform structure, the mammalian myelinated axon exhibits a high degree of regional specialization that extends to the location of voltage-dependent ion channels within the axon membrane. Sodium and potassium channels are segregated into complementary membrane domains, with a distribution reflecting that of the overlying Schwann or glial cells. This complexity of organization has important implications for physiology and pathophysiology, particularly with respect to the development of myelinated fibers.