A real-time quantitative PCR comparative study between rat optic and sciatic nerves: determination of neuregulin-1 mRNA levels.

Injured axons from peripheral nervous system (PNS) possess the ability to regenerate. In contrast, regeneration of injured axons does not occur in the central nervous system (CNS) or occurs to a limited extent. Previous works have shown that rat sciatic nerve conditioned medium (CM) produced PC12 cells neuronal-like differentiation and neurite outgrowth. In the present work, we compared the expression of neuregulin-1s (NRG-1s) from rat sciatic and optic nerves as members of the PNS and CNS, respectively. Sciatic nerve CM showed a higher neurotrophic activity on PC12 cells than rat optic nerve CM. RT-PCR analysis verified the presence of all three types of NRG-1 mRNAs and their receptors in both types of nerves. Real-time quantitative PCR (QPCR) assays showed that the relative expression levels of all three types of NRG-1 mRNAs were higher in optic nerves than in sciatic nerves. Eleven-day cultured optic nerves showed an increased in NDF and SMDF when compared to freshly isolated optic nerves, whereas GGF decreased. However, 11-day-cultured sciatic nerves only showed an increase in SMDF mRNA. Western blots corroborated the differences in NRG-1 expression profile for both types of nerves and their CMs. Incubation of both CMs with the anti-pan-NRG-1 antibody showed that the neurotrophic activity of the optic nerve CM increased, whereas the sciatic nerve CM remained unchanged. These results indicated that different NRG-1 levels are expressed upon nerve degeneration and the balance between those levels and other neurotrophic factors could have an important role on nerve regeneration.

Ionic currents in PC12 cells differentiated into neuron-like cells by a cultured-sciatic nerve conditioned medium.

The present work deals with the identification of the ionic currents found in PC12 cells differentiated into neuron-like cells by a 9-11-day cultured-sciatic nerve conditioned medium (CM). PC12 whole-cell currents were measured after chronic exposure to CM. The results obtained in these CM-treated cells reveal that the functional expression of Ca(2+) currents is increased, that Na+ currents are not affected, and that a transient K+ current and a K+ delayed rectifier (K+ dr) current are increased. The combination of nifedipine and omega-conotoxin GVIA (omega-CgTX) does not block completely the increased functional expression of the Ca(2+) current. The remaining current is blocked by omega-agatoxin TK indicating that P/Q-type channels are additionally contributing to the increase in Ca(2+) current. NGF-treated PC12 cells, used as positive controls, confirm that NGF increases the expression of voltage-dependent Na+ currents and of Ca(2+) currents. In addition, we found that NGF also increases a K+ dr-type current in these cells. The results obtained with the CM might be due to a molecule or a mixture of molecules released into the medium by the 9-11-day cultured sciatic nerves.

Neuregulin found in cultured-sciatic nerve conditioned medium causes neuronal differentiation of PC12 cells.

The present work deals with the search and identification of the molecule or combination of molecules, present in a medium conditioned by cultured rat-sciatic nerves (CM), able to cause neuronal differentiation of PC12 cells. The molecular mass range of the active fraction, as well as the thermostability and heparin affinity of the active component found in previous work, all characteristics shared with neuregulin (NRG) family members, led us to search for a NRG protein in the CM. Nerves were previously cultured for 8 days and the CM collected every 24 h, the following 3 days. The CM was concentrated (30,000 NMWL) and fractionated by quaternary ammonium chromatography and Cibacron blue affinity chromatography. The most active fraction B1.2 was further characterized by heparin affinity chromatography, size exclusion HPLC, Western blotting and immunoprecipitation. Results reveal abundance of NRG mRNA in the cultured nerves, presence of a 54 kDa NRG protein in the CM that increases along fractionation, and progressive diminution of fraction B1.2 differentiation activity on PC12 cells by gradual removal of the NRG protein by immunoprecipitation. The abundance of Schwann cells and the lack of axons in the cultured nerves suggest Schwann cells as the main NRG source, to which fibroblasts and perineurial cells might contribute.

Neuronal differentiation of PC12 and chick embryo ganglion cells induced by a sciatic nerve conditioned medium: characterization of the neurotrophic activity.

The present work deals with the finding and characterization of a neurotrophic factor present in serum-free Dulbecco's modified Eagle's medium in which rat sciatic nerves previously cultured for 9 days were maintained for 24 h. This sciatic nerve conditioned medium (SNCM) produced neuronal differentiation and neurite outgrowth on PC12 cells, as well as survival and differentiation of eight-day old chick embryo dorsal root ganglion (E8-DRG) and ciliary ganglion (E8-CG) neurons. SNCM activity was decreased by dilution, heating and trypsin treatment; it was not inhibited by anti-NGF and anti-bFGF antibodies; and it was not mimicked by CNTF, laminin and fibronectin. By utilizing its neurite-promoting activity on PC12 cells, experiments oriented to purify the factor were carried out. Ultrafiltration, heparin-affinity chromatography and size-exclusion high pressure liquid chromatography (HPLC) were employed. The ability of SNCM to induce PC12 cell, E8-DRG and E8-CG neuronal differentiation, the heparin affinity of the active SNCM protein, and the size-exclusion HPLC elution characteristics of the active protein suggest that the active component of the SNCM is, in all probability, a novel sciatic nerve neurotrophic factor (SNTF).

Order-disorder phenomena in myelinated nerve sheaths: V. Effects of temperature on rat sciatic and optic nerves, and structural differences between the two types of nerve.

We describe in this work X-ray scattering and electron microscope studies of rat sciatic and optic nerves as a function of temperature. The scattering experiments were analyzed as described in the previous papers of this series: a variety of parameters were determined, some of which characterize the lattice disorder, others the structure of the motif. The main results are the following. All the parameters determined by the X-ray scattering study vary with temperature and the temperature-dependence is specific for the type of nerve (sciatic or optic). Most of the disorder-related parameters display a minimum or a maximum in the vicinity of physiological temperature (38 degrees C in rat); this observation, strongly supported by the electron microscope study, shows that the degree of organization of myelin is highest near physiological temperature. The structure of the motif, as revealed by the electron density profile, is fairly different in the two types of nerves (in contrast with the assumption made by previous workers); the structure also varies with temperature and the temperature-induced alterations are nerve-type specific. In the two types of nerve the thickness of the lipid bilayer varies with temperature as expected for a lipid-containing system with hydrocarbon chains in the disordered conformation. In sciatic nerve the thickness of the (thinner) cytoplasmic polar layer, which is also the layer most affected by lattice disorder in this type of nerve, decreases dramatically with increasing temperature. In optic nerve, in which lattice disorder predominantly affects the extracellular layer, the thickness of both the cytoplasmic and the extracellular layer is barely affected by temperature.

Expression of sodium channels with different saxitoxin affinity during rat forebrain development.

This work characterizes the development of the saxitoxin (STX)-sensitive Na+ channels from rat whole forebrain between embryonic day 15 (E15) and postnatal day 90 (P90), both with binding studies and with single channel studies. The Na+ channel total mRNA and the individual mRNAs encoding Na+ channels I, II and III were also determined. The total STX binding rose about 40-fold from E15 to reach a plateau at P30 and its temporal course correlated with the expression of Na+ channel total mRNA. Low affinity and high-affinity STX binding sites, predominant in embryonic and postnatal forebrains, respectively, were found. The single channel studies of batrachotoxin-modified channels also revealed two main populations. In E15 only low-affinity channels (KD = 32.7 nM; 200 mM NaCl) and in P30 only high affinity ones (KD = 1.6 nM) were present. At P0 channels with intermediate affinity (KD range 3-34 nM) were observed. The increase in affinity was due to a gradual increase in the STX association rate.

Order-disorder phenomena in myelinated nerve sheaths. IV. The disordering effects of high levels of local anaesthetics on rat sciatic and optic nerves.

Sequences of 15 minute X-ray scattering spectra were recorded with rat sciatic and optic nerves, superfused with tetracaine-containing Ringer solutions. The spectra were analysed using the algorithm advocated in this series of papers. The main results, as a function of the time of exposure to tetracaine, were: the mean value of the repeat distance increases; its variance decreases; the average number of membrane pairs per coherent domain decreases; the fraction of isolated membrane pairs increases. Eventually, the spectra were observed to give way to the continuous intensity curve of a single, isolated membrane pair. At all stages of the experiment the continuous intensity curves were found to differ from one type of nerve to the other, and to be invariant, for each type of nerve, with respect to the tetracaine treatment. The X-ray scattering study clearly identified the nature of the structural differences between the two types of myelin sheaths: in that of native sciatic nerves, packing disorder preferentially affects the cytoplasmic space of the membrane pair, and tetracaine disrupts the packing in that space; in the myelin of optic nerves it is the external space that is preferentially affected by packing disorder and disrupted by tetracaine. The time-course of the structure parameters showed that, at any stage of the experiment, tetracaine acts preferentially on the more highly disordered regions of the structure and totally disrupts them. These results corroborate earlier conclusions reported in the previous papers of this series. An electron microscope study was also performed on tetracaine-treated nerves: the results, in close agreement with those of the X-ray scattering study, neatly confirm the conclusions given above. In a more general way, the remarkable agreement between the results of the analysis of the X-ray scattering spectra and the electron microscope observations strongly supports the validity of the physical model used in this series of papers and the correctness of the mathematical treatment that we advocate. Finally, the relations between this work and the work of others are discussed. It must be stressed that the present work bears on the toxic rather than on the anaesthetic effects of tetracaine.

Periaxonal ensheathment of lobster giant nerve fibres as revealed by freeze-fracture and lanthanum penetration.

Sheath structure and permeability have been studied in the nerve fibres of lobster (Panulirus argus) walking limbs, in particular the individually ensheathed larger giant fibres, 100-150 microns in diameter, of which there are five or six in a peripheral bundle. They are easily distinguished and can be separated from neighbouring fibre bundles in which smaller giant axons (65-80 microns diameter) and many axons of much smaller diameter (5-15 microns) are ensheathed together. Each of the larger giant axons is enveloped by a Schwann cell layer outside of which is a multilayered sheath consisting of one-cell thick belts of flattened cells and interleaved zones of collagen fibrils and extracellular matrix. The cells in each belt lack basal lamina and, after freeze-fracture, as well as in thin sections, exhibit intercellular gap junctions and incomplete, fascia type, tight junctions; their most striking aspect is an exceedingly large number of exo-endocytic profiles. Permeability to lanthanum chloride in the bathing medium studied before or during fixation both in intact nerves and in nerves with surgically breached (slit) epineurium showed penetration of lanthanum tracer between the cells around the giant fibres, but the electron-dense tracer was excluded from the Schwann cell layer and the periaxonal space unless the epineurium had been slit. The extent of lanthanum diffusion was evaluated by transmission electron microscopy of thin sections and confirmed by X-ray microanalysis (EDAX) of comparable selected areas in such sections. The results indicate structural similarities but distinct permeability differences between the multilayered sheath surrounding the lobster giant axons and the vertebrate nerve perineurium. Other ultrastructural details provided by the freeze-fracture replicas concern the distribution of intramembrane particles in the axolemma and the Schwann and sheath cell membranes.

[Electron microscopy of giant nerve fibers]. / Microscopia electrónica de fibras nerviosas gigantes.

The present paper is a brief review on the ultrastructural details of giant nerve fibers and their possible correlation to some physiological findings which have been reported in the squid giant axon. In both, thin sections and freeze-fracture replicas, the most striking feature is the exceeding amount of paired membranes populating the Schwann cell layer. These membranes represent permeable intercellular clefts connecting the axon surface to the endoneurial extracellular space, thus leaving the axolemma as the only continuous barrier between the axoplasm and the neuron exterior. Close apposition of the axon and Schwann cell at the level of structural complexes involving both cells plasma membranes are observed in sections and replicas. These zones could represent the morphologic expression of the functional coupling reported in the same preparation. Besides, the Schwann cell appears to be very active according to the amount of exo-endocytotic profiles seen in all its fracture faces. Finally, the endoneurial cells are different in the various giant fibers studied: in the squid they appear as spongy cells, whereas in the lobster they exhibit an extraordinary amount of exo-endocytosis mixed with some gap and a few incomplete tight junctions, and in the crayfish they present the same features as the adaxonal glia.

Microscopia electrónica de fibras nerviosas gigantes. / [Electron microscopy of giant nerve fibers]

The present paper is a brief review on the ultrastructural details of giant nerve fibers and their possible correlation to some physiological findings which have been reported in the squid giant axon. In both, thin sections and freeze-fracture replicas, the most striking feature is the exceeding amount of paired membranes populating the Schwann cell layer. These membranes represent permeable intercellular clefts connecting the axon surface to the endoneurial extracellular space, thus leaving the axolemma as the only continuous barrier between the axoplasm and the neuron exterior. Close apposition of the axon and Schwann cell at the level of structural complexes involving both cells plasma membranes are observed in sections and replicas. These zones could represent the morphologic expression of the functional coupling reported in the same preparation. Besides, the Schwann cell appears to be very active according to the amount of exo-endocytotic profiles seen in all its fracture faces. Finally, the endoneurial cells are different in the various giant fibers studied: in the squid they appear as spongy cells, whereas in the lobster they exhibit an extraordinary amount of exo-endocytosis mixed with some gap and a few incomplete tight junctions, and in the crayfish they present the same features as the adaxonal glia.