Localization of aquaporin 1 water channel in the Schmidt-Lanterman incisures and the paranodal regions of the rat sciatic nerve.

Aquaporin 1 (AQP1) is a member of a family of small, integral membrane water-transporting proteins, which facilitate water movement across cell membranes in response to osmotic gradients. Several papers have studied the expression and function of the AQPs in the central nervous system. However, little is known about the AQPs in the peripheral nervous system (PNS). In the PNS, AQP1, AQP2 and AQP4 have been reported in both peripheral neurons and glial cells. In this work we studied the expression and localization of AQP1 in the rat sciatic nerve. We found that from the four AQPs we studied (AQP1, AQP2, AQP4 and AQP9) only AQP1 is expressed in the nerve by reverse transcription polymerase chain reaction (RT-PCR). AQP1 is also observed at the protein level by Western blot analysis. We also studied the localization of AQP1 in the sciatic nerve by immunohistochemistry. The results show that AQP1 is present in both myelinating and non-myelinating Schwann cells. In myelin internodes AQP1 is enriched in the Schmidt-Lanterman incisures and in some internodes it is also present in the abaxonal membrane. At the nodes of Ranvier, AQP1 co-localizes with actin in the paranodal regions of the nerve. Therefore, AQP1 might play an important role in myelin homeostasis maintaining the thermodynamic equilibrium across the plasma membrane in myelinated axons during electrical activity. Also the expression of AQP1 in non-myelinating Schwann cells supports the involvement of AQP1 in pain perception.

F-actin distribution at nodes of Ranvier and Schmidt-Lanterman incisures in mammalian sciatic nerves.

Very little is known about the function of the F-actin cytoskeleton in the regeneration and pathology of peripheral nerve fibers. The actin cytoskeleton has been associated with maintenance of tissue structure, transmission of traction and contraction forces, and an involvement in cell motility. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and intracellular transport therein. In this work, we analyze the distribution of F-actin at Schmidt-Lanterman Incisures (SLI) and nodes of Ranvier (NR) domains in normal, regenerating and pathologic Trembler J (TrJ/+) sciatic nerve fibers, of rats and mice. F-actin was quantified and it was found increased in TrJ/+, both in SLI and NR. However, SLI and NR of regenerating rat sciatic nerve did not show significant differences in F-actin, as compared with normal nerves. Cytochalasin-D and Latrunculin-A were used to disrupt the F-actin network in normal and regenerating rat sciatic nerve fibers. Both drugs disrupt F-actin, but in different ways. Cytochalasin-D did not disrupt Schwann cell (SC) F-actin at the NR. Latrunculin-A did not disrupt F-actin at the boundary region between SC and axon at the NR domain. We surmise that the rearrangement of F-actin in neurological disorders, as presented here, is an important feature of TrJ/+ pathology as a Charcot-Marie-Tooth (CMT) model.

Expression of IP3 receptor isoforms at the nodes of Ranvier in rat sciatic nerve.

Inositol 1,4,5-trisphosphate receptors (IP3R) are modulated by the second messenger IP3, which induces intracellular calcium release. Using immunohistochemical techniques, we show that the three isoforms are expressed in sciatic nerve. IP3R1 and IP3R2 are mainly present in the nucleus of Schwann cells. IP3R1 is also expressed in Schmidt-Lanterman incisures. IP3R3 is primarily localized at very high levels in nonmyelinating Schwann cells. Interestingly, the three isoforms are expressed at the nodes of Ranvier. IP3R1 is clustered at the node of Ranvier, in a distribution that is similar to the Nav1.6 sodium channels in the sciatic nerve. IP3R3 is present in the paranodal regions of the nodes. IP3R2 is concentrated in the vicinity of the node, and the outer Schwann cell cytoplasm similar to the Kv1.5 potassium channel.

Immunolocalization of the Na(+)-K(+)-2Cl(-) cotransporter in peripheral nervous tissue of vertebrates.

Efflux of Cl(-) through GABA(A)-gated anion channels depolarizes the cell bodies and intraspinal terminals of sensory neurons, and contributes to the generation of presynaptic inhibition in the spinal cord. Active accumulation of Cl(-) inside sensory neurons occurs through an Na(+)-K(+)-2Cl(-) cotransport system that generates and maintains the electrochemical gradient for this outward Cl(-) current. We studied the immunolocalization of the Na(+)-K(+)-2Cl(-) cotransporter protein using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the molecule. Western blots of frog, rat and cat dorsal root ganglion membranes revealed a single band of cotransporter immunoreactivity at approximately 160kDa, consistent with the molecular mass of the glycosylated protein. Deglycosylation with N-glycosidase F reduced the molecular mass to approximately 135kDa, in agreement with the size of the core polypeptide. Indirect immunofluorescence revealed strong cotransporter immunoreactivity in all types of dorsal root ganglion cell bodies in frog, rat and cat. The subcellular distribution of cotransporter immunoreactivity was different amongst species. Membrane labeling was more apparent in frog and rat dorsal root ganglion cell bodies than in cat. In contrast, cytoplasmic labeling was intense in cat and weak in frog, being intermediate in the rat. Cotransporter immunoreactivity also occurred in satellite cells, particularly in rat and cat dorsal root ganglia. The membrane region and axoplasm of sensory fibers were heavily labeled in cat and rat and less in frog. Three-dimensional reconstruction of confocal optical sections and dual immunolocalization with S-100 protein showed that the cotransporter immunoreactivity was prominently expressed in the nodal and paranodal regions of the Schwann cells. Ultrastructural immunolocalization confirmed the presence of immunoreactivity on the membranes of the axon and the Schwann cell in both the nodal region and the paranode. Treatment with sodium dodecylsulfate and beta-mercaptoethanol also uncovered intense cotransporter immunoreactivity in Schmidt-Lanterman incisures at the light microscopic level. The localization of the Na(+)-K(+)-2Cl(-) cotransporter protein is consistent with its function as a Cl(-)-accumulating mechanism in sensory neurons. Its distinctive presence in Schwann cells suggests that it could also be involved in K(+) uptake from the extracellular space, particularly in the paranodal region of myelinated axons, thereby regulating the extracellular ionic environment and the excitability of axons.