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.

Expression of interleukin-6 receptor alpha in normal and injured rat sciatic nerve.

Interleukin-6 (IL-6) is a pleiotropic cytokine synthesized by many different cells after appropriate stimulation. IL-6 binds first to the interleukin-6 receptor alpha (IL6-Ralpha) and then this complex binds to the signal-transducing gp130 receptor, forming a functional hexameric receptor complex. We observed by Western blot analysis with anti-IL6-Ralpha two bands of approximately 80 kDa and approximately 110 kDa in the rat sciatic nerve, cerebral cortex, spleen, pancreas and liver, corresponding to the mature glycosylated form and possibly to the dimer of the non-glycosylated precursor protein. By immunohistochemistry, high levels of IL6-Ralpha expression are observed in non-myelinating Schwann cells. In myelinating Schwann cells IL6-Ralpha is present as discrete dots in the perinuclear region, in distinct membrane domains of the Schwann cell sheath and at the nodes of Ranvier, suggesting that IL6-Ralpha is clustered both on the axonal side of the node and within the Schwann cells. After sciatic nerve crush injury IL6-Ralpha is upregulated in denervated Schwann cells between the myelin ovoids during the period of Schwann cell proliferation. The expression of IL6-Ralpha continues during the period of remyelination, suggesting that IL6-Ralpha might be involved in both Schwann cell proliferation and remyelination of the rat sciatic nerve.

Circadian modulation of the ryanodine receptor type 2 in the SCN of rodents.

We examined the temporal modulation of intracellular calcium release channels in the suprachiasmatic nucleus (SCN). We found a circadian rhythm in [3H]ryanodine binding that was specific to the SCN. The peak in the rhythm occurred at CT 7 and was due to an increase in Bmax, which correlated well with immunoblots showing an increase in RyR-2 expression in the SCN. Double immunohistochemical studies showed that RyR-2 was expressed exclusively in neurons. Ryanodine and caffeine applied around CT 7-9 advanced the clock phase in a hamster brain slice preparation. No rhythm of IP3R was seen in any of the brain areas studied. Our results indicate that RyR-2 exhibits an endogenous rhythm, which influences the intracellular calcium dynamics and thus modulates SCN activity.

P0 is constitutively expressed in the rat neural crest and embryonic nerves and is negatively and positively regulated by axons to generate non-myelin-forming and myelin-forming Schwann cells, respectively.

We show that in the rat, the major gene of PNS myelin, P0, is expressed long before myelination in the neural crest, Schwann cell precursors, and embryonic Schwann cells irrespective of whether they will myelinate or not. This myelin-independent P0 expression is constitutive and likely to serve as a specific marker for the Schwann cell lineage. The much higher P0 expression accompanying myelination is therefore not new gene expression but strong up-regulation of preexisting basal levels. We provide new evidence that the up-regulation to myelination-related levels depends on positive extrinsic signals and therefore does not represent a constitutive phenotype. P0 mRNA is not detectable in mature non-myelin-forming Schwann cells of the sympathetic trunk, but is detectable after transection, indicating that there is a P0-inhibitory signal associated with mature unmyelinated axons. Thus, the regulation of the P0 gene is complex, encompassing extrinsically signaled amplification superimposed on a highly lineage-specific and constitutive basal expression.

Loss of DNA loop supercoiling and organization in cells infected by herpes simplex virus type 1.

In cells infected by herpesviruses, a sequence of nuclear changes during interphase, as well as chromosomal aberrations during mitosis, are commonly observed. These changes suggest the progressive modification of host-cell chromatin. Previous studies have shown that the early chromatin modifications in cells infected by herpes simplex virus type 1 (HSV1) are not due to extensive breakdown of host-cell DNA or disruption of the nucleosomal structure. We have previously shown that infection by HSV1 induces single-stranded breaks in the host-cell DNA early in the course of infection, and that such breaks lead to modifications in the higher-order structure of host-cell chromatin. Here we report that virus-induced DNA breaks produce permanent long-term effects on the state of supercoiling and organization of the nuclear DNA loops, comparable to the DNA loop disorganization produced by high (and irreparable) doses of ultraviolet radiation.

Expression of type 1 inositol 1,4,5-trisphosphate receptor during axogenesis and synaptic contact in the central and peripheral nervous system of developing rat.

Release of intracellular Ca2+ is triggered by the second messenger inositol 1,4,5-trisphosphate, which binds to the inositol 1,4,5-trisphosphate receptor and gates the opening of an intrinsic calcium channel in the endoplasmic reticulum. In order to understand the importance of this mechanism in development, we have examined the distribution of the type 1 inositol 1,4,5-trisphosphate receptor during development, in some areas of the rat brain and spinal cord and in peripheral neurons, using in situ hybridization and immunohistochemistry. In brain, we find that type 1 inositol 1,4,5-trisphosphate receptor is expressed in neurons from very early in development; low levels of expression are first detected after the neurons have migrated to their final positions, when they start to differentiate and begin axonal growth. Increasing levels of expression are observed later in development, during the time of synaptogenesis and dendritic contact. Glial cells do not express type 1 inositol 1,4,5-trisphosphate receptor, except for a transient period of expression, probably by oligodendrocytes, in developing fibre tracts during the onset of myelination. In contrast with the brain, both grey and white matter of the spinal cord express type 1 inositol 1,4,5-trisphosphate receptor throughout development, and it remains present in the adult spinal cord. We also show, for the first time, that type 1 inositol 1,4,5-trisphosphate receptor is expressed in the peripheral nervous system. Strong labelling was observed in the dorsal root ganglia and during development this expression seems to coincide with the onset of axogenesis. These results suggest that type 1 inositol 1,4,5-trisphosphate may be involved in the regulatory mechanism controlling Ca2+ levels in neurons during the periods of cell differentiation, axogenesis and synaptogenesis.

Urokinase-type plasminogen activator expression by neurons and oligodendrocytes during process outgrowth in developing rat brain.

The expression of tissue- and urokinase-type plasminogen activators has been studied in developing cerebellum, hippocampus, cerebral cortex, olfactory bulb and olfactory mucosa of the rat by in situ hybridization. All identifiable neurons express urokinase mRNA from an early stage in their development, and this expression appears to coincide with the onset of axogenesis. For cerebellar granule cells, both axonal growth and urokinase expression are initiated before they migrate from the external granule layer; for the majority of neocortical neurons, however, both processes are commenced after the cells have migrated to the cortical plate. Neurons continue to express this protease in the adult. The large projection neurons exhibit the highest levels of message, the smaller interneurons having much lower levels except for hippocampal granule cells, which have notably high levels of expression. Glial cells generally do not express urokinase message, except for transient expression by oligodendrocytes in developing fibre tracts during the period of myelination. Thus for both neurons and oligodendrocytes, the onset of urokinase-type plasminogen activator expression coincides with their initiation of major process outgrowth, although neurons maintain this expression in the adult, possibly to retain a degree of synaptic plasticity. In contrast, although high levels of message for the related protease, tissue plasminogen activator, are found in the embryonic floor plate, in postnatal brain it is abundantly expressed only by ventricular ependymal cells and by cells in connective tissue surrounding the olfactory nerve.

The expression of tissue and urokinase-type plasminogen activators in neural development suggests different modes of proteolytic involvement in neuronal growth.

Tissue and urokinase-type plasminogen activators are serine proteases with highly restricted specificity, their best characterised role being to release the broad specificity protease plasmin from inactive plasminogen. It has frequently been suggested that these, and similar proteases, are involved in axonal growth and tissue remodelling associated with neural development. To help define what this role might be, we have studied the expression of the plasminogen activators in developing rat nervous tissue. Urokinase-type plasminogen activator mRNA is strongly expressed by many classes of neurons in peripheral and central nervous system. We have analysed its appearance in spinal cord and sensory ganglia, and found the mRNA is detectable by in situ hybridisation very early in neuronal development (by embryonic day 12.5), at a stage compatible with it playing a role in axonal or dendritic growth. Tissue plasminogen activator mRNA, on the other hand, is expressed only by cells of the floor plate in the developing nervous system, from embryonic day 10.5 and thereafter. Immunohistochemical and enzymatic analysis showed that active tissue plasminogen activator is produced by, and retained within, the floor plate. A mechanism is suggested by which high levels of tissue plasminogen activator produced by the stationary cells of the floor plate could influence the direction of growth of commissural axons as they pass through this midline structure.

Selective modification of the squid axon Na currents by Centruroides noxius toxin II-10.

We have studied the effects of the purified toxin II-10, from the venom of the scorpion Centruroides noxius Hoffmann, on the Na and K currents of voltage clamped squid giant axons. Extracellular applications of 10 microM of toxin II-10 produced a selective depression of peak Na currents, with no significant effects on the time course of K currents. On pharmacologically separated Na currents, low concentrations of toxin II-10 (0.28-1 microM) caused a reversible decrease in inward and outward peak INa, with little effect on either the maintained level of the currents or their turning-off. At high concentrations (greater than 3 microM), toxin II-10 drastically reduced the peak conductance and increased both the level of the maintained conductance, and the time course of its turning-off. It is suggested that, when applied extracellularly on squid axons, toxin II-10 primarily reduces the peak Na conductance by modifying the activation of fast-inactivating Na channels. At high concentrations (10 microM), the toxin also modifies the rate constants of the transition from the inactivated to the second open state of the channel (Chandler and Meves, 1970) thus producing an increased level of the maintained Na conductance. It is also very likely, however, that peak conductance and maintained conductance reflect two separate populations of Na channels on which toxin II-10 has a differentiated action. Under these conditions, toxin II-10 would be the first reported toxin which can pharmacologically separate the two types of channels.

Preliminary spectroscopic characterization of six toxins from Latin American scorpions.

This paper reports on spectroscopic studies of six toxins from the Latin American scorpions Centruroides noxius Hoffmann, Centruroides elegans Thorell and Tityus serrulatus Lutz and Mello. The isolation and purification of five of these toxins was described previously. The preparation of toxin II.9.2.2 from the venom of C. noxius is first described here. Circular dichroism and nuclear magnetic resonance spectra indicate similarities and differences between these scorpion toxins and previously characterized snake toxins. While there is evidence that the toxins from scorpions and snakes both contain extended beta-sheet secondary structures, the spectral properties of the scorpion toxins are overall of a different type from those of snake toxins. Among the six scorpion toxins those from T. serrulatus have spectral properties markedly different from those of the Centruroides species. Furthermore, thermal denaturation and amide proton exchange measurements showed that the globular structures of the Tityus toxins were markedly less stable and less rigid than those of the Centruroides toxins.