Phylogenetic preservation of alpha3 Na+,K+-ATPase distribution in vertebrate peripheral nervous systems

The alpha(3) isoform of Na(+),K(+)-ATPase is uniquely expressed in afferent and efferent neurons innervating muscle spindles in the peripheral nervous system (PNS) of adult rats, but the distribution pattern of this isoform in other species has not been investigated. We compared expression of alpha(3) Na(+),K(+)-ATPase in lumbar dorsal root ganglia (DRG), spinal roots, and skeletal muscle samples of amphibian (frog), reptilian (turtle), avian (pigeon and chicken), and mammalian (mouse and human) species. In all species studied, the alpha(3) Na(+),K(+)-ATPase isoform was nonuniformly expressed in peripheral ganglia and nerves. In spinal ganglia, only 5-20% of neurons expressed this isoform, and, in avian and mammalian species, these alpha(3) Na(+),K(+)-ATPase-expressing neurons belonged to a subpopulation of large DRG neurons. In ventral root fibers of pigeons, mice, and humans, the alpha(3) Na(+),K(+)-ATPase was abundantly expressed predominantly in small myelinated axons. In skeletal muscle samples from turtles, pigeons, mice, and humans, alpha(3) Na(+),K(+)-ATPase was detected in intramuscular myelinated axons and in profiles of nerve terminals associated with the equatorial and polar regions of muscle spindle intrafusal fibers. These results show that the expression profiles for alpha(3) Na(+),K(+)-ATPase in the peripheral nervous system of a wide variety of vertebrate species are similar to the profile of rats and suggest that stretch receptor-associated expression of alpha(3) Na(+),K(+)-ATPase is preserved through vertebrate evolution.     

Target-determined expression of alpha3 isoform of the Na+,K+-ATPase in the somatic nervous system of rat

Factors that determine the differential expression of isoforms of Na(+),K(+)-ATPase in the nervous system of vertebrates are not understood. To address this question we studied the expression of alpha(3) Na(+),K(+)-ATPase in the L5 dorsal root ganglia (DRG) of developing rat, the normal adult rat, and the adult rat after peripheral axotomy. During development, the first alpha(3) Na(+),K(+)-ATPase-positive DRG neurons appear by embryonic day 21. At birth, the L5 DRG have a full complement (14 +/- 2%) of these neurons. By 15 days after sciatic nerve transection in adult rat, the number of alpha(3) Na(+),K(+)-ATPase-positive DRG neurons and small myelinated L5 ventral root axons decreases to about 35% of control counts. These results combined with data from the literature suggest that the expression of alpha(3) Na(+),K(+)-ATPase by rat somatic neurons is determined by target-muscle spindle-derived factors.     

Regulation of Na+ channel distribution in the nervous system

An important aspect of Na+ channel regulation is their distribution on neuronal membranes within the nervous system. The complexity of this process is brought by the molecular diversity of Na+ channels and differential regulation of their distribution. In addition, Na+ channel localization is a highly dynamic process depending on the status of the cell in vitro, and (patho)physiological condition of the organism in vivo. Nonetheless, the pharmacological manipulation of Na+ channel distribution should be possible and will hopefully bring safer and more-potent medicines in the future.     

Development of the electrosensory nervous system in Eigenmannia (Gymnotiformes): I. The peripheral nervous system

The nerves of the anterior lateral line system in embryonic and larval stages of the weakly electric gymnotiform fish Eigenmannia were visualized by injection of the fluorescent marker DiI into the primordium of the anterior (ALLN) and posterior (PLLN) lateral line nerves. Examination of developmental series reveals that the nerve fibers that innervate the electrosensory and mechanosensory components of the anterior lateral line system are present before the first mechanoreceptors and electroreceptors have differentiated. This suggests that nerve fibers might induce the formation of lateral line receptors. Whereas the innervation of the mechanoreceptive system is already established at an early stage, the afferent innervation of electroreceptors continues to arborize in the periphery, presumably by following pioneer axon pathways. The earliest recognizable stage of the anterior lateral line nerve ganglion (ALLNG) is evident 2 days after spawning. The ganglion shows two germinal cell masses that develop into the supraorbital-infraorbital and the hyomandibular placodes. The supraorbital-infraorbital placode forms the dorsal part of the ALLNG; the hyomandibular placode forms the ventral part of the ALLNG. Counts of ALLNG cells in embryonic, larval, and adult stages of Eigenmannia show that, at each stage examined, the number of ganglion cells is always significantly larger than the number of mechanoreceptors and electroreceptor units in the periphery. During development, the distribution of ALLNG cell diameters shifts from a unimodal distribution in juveniles to a bimodal distribution in adults, peaking at 8 microns and 18 microns. These results suggest that tuberous electroreceptive organs, which are innervated by the large ALLNG cells, may not be functional prior to day 18. Our results further suggest that the number of ALLNG cells correlates with the rate of induction of lateral line receptors in the periphery.     

Differential functional expression of cation-Cl- cotransporter mRNAs (KCC1, KCC2, and NKCC1) in rat trigeminal nervous system

GABA is the main inhibitory neurotransmitter in the adult brain, which causes Cl- influx into the cell via GABAA receptors. The direction of Cl- inflow is dependent on the Cl- gradient across the membrane. Cation-Cl- cotransporters have been considered to play pivotal roles in controlling intracellular Cl- concentration ([Cl-]i) of neurons; hence, they modulate the GABAergic function. To elucidate how these cotransporters are distributed in the trigeminal nuclei, we investigated the expressions of K+-Cl- cotransporters (KCC1 and KCC2) and Na+-K+-2Cl- cotransporter (NKCC1) mRNAs by using in situ hybridization histochemistry. KCC2 mRNA was expressed in the motor trigeminal nucleus (Mo5), the principal trigeminal nucleus (Pr5), and the spinal trigeminal nucleus (Sp5), but not in the trigeminal ganglion (TG) and the mesencephalic trigeminal nucleus (Me5). On the other hand, KCC1 and NKCC1 mRNAs were expressed in all the trigeminal nuclei. The resting [Cl-]i of Me5 neurons was significantly higher than that of Mo5 neurons. Thus, in primary sensory neurons such as the TG and the Me5, [Cl-]i would be higher than those in the other trigeminal nuclei because of the lack of KCC2 mRNA expression. Since Me5 neurons, but not Mo5 neurons, responded to GABA by depolarization, GABA would have differential physiological functions among trigeminal nuclei and TG.     

Immunohistochemical differential distribution of S-100 alpha and S-100 beta in the peripheral nervous system of the rat

The localization of the alpha subunit of the S-100 protein (S-100 alpha) and beta subunit (S-100 beta) was studied in the peripheral nervous system of the rat. In peripheral nerves, S-100 alpha and S-100 beta were found in the cytoplasm of Schwann cells. Axons were positively stained in part by S-100 alpha and almost totally by S-100 beta. In the dorsal root ganglia, S-100 alpha was found in satellite cells and their processes and in some neurons. S-100 beta was found in more of the large neurons, but almost all of the small neurons were negative for S-100 beta. In the anterior horn cells, S-100 beta staining was stronger than that of S-100 alpha. In Schwann cells, both S-100 alpha and S-100 beta were present on the rough endoplasmic reticulum, free ribosomes, and nucleus, as seen by electron microscopy. The S-100 alpha and S-100 beta in axons were associated with microtubules and neurofilaments.     

Expression of the large myelin-associated glycoprotein isoform during the development in the mouse peripheral nervous system

The developmental maximum expression of the large myelin-associated glycoprotein isoform (L-MAG) protein prior to that of the small myelin-associated glycoprotein isoform (S-MAG) in both the central and peripheral nervous systems (CNS, PNS) in mice was shown by immunoblotting techniques using specific antibodies to the L-MAG protein and the S-MAG protein. Both the L-MAG protein and the S-MAG protein were expressed earlier in the PNS than in the CNS, which reflects earlier myelination in the PNS. The peak of the L-MAG protein concentration was 8 days in the sciatic nerve and 15 days in the brainstem. The concentration of the S-MAG protein in the sciatic nerve reached a peak at 15 days, whereas in the brainstem it increased rapidly between 15 and 20 days and gradually thereafter. Thus, the preceding maximum expression of the L-MAG during active myelination in the PNS demonstrated here as well as in the CNS strongly suggests an important role for L-MAG in myelin formation.     

Physiology and pathophysiology of Na(+)/H(+) exchange isoform 1 in the central nervous system

Na(+)/H(+) exchangers (NHEs) conduct the electroneutral exchange of proton (H(+)) and sodium (Na(+)) ions across cellular membranes down their concentration gradients. To date, nine NHE family members have been cloned from mammals and share a common secondary structure. The ubiquitous exclusive plasma membrane NHE isoform 1 (NHE1) is a major membrane transport mechanism in regulation of intracellular pH (pH(i)) and volume. In addition to its role in regulation of ionic homeostasis, NHE1 can directly interact with other regulatory cellular signaling pathways, including modulation of the activity of mitogen-activated protein kinases (MAPKs) and Akt/protein kinase B (PKB). Thus, NHE1 is a multifaceted regulator of cell migration, proliferation, and cell death. NHE1 also plays pivotal roles under a number of pathophysiological conditions such as osmotic stress, acidosis, and mechanical stress. NHE1 is the most abundant NHE isoform in the rat central nervous system (CNS). This review discusses distribution and regulation of NHE1, and its physiological roles in the CNS. Moreover, it includes an extensive presentation of studies on activation of NHE1 under ischemic conditions in the CNS and its impact on Na(+) and Ca(2+) ionic homeostasis as well as on cell survival and damage.     

Postnatal histogenesis in the peripheral nervous system.

The issue of postnatal neurogenesis has gained great importance over the last few years and the recent amazing scientific advancements, changing our viewpoint on the long-lasting "no new neurons" dogma, have opened promising new perspectives on the treatment of the damaged nervous system. While most of the researchers have focused on the central nervous system, the peripheral nervous system has received little attention so far with respect to postnatal histogenesis. To attract scientific attention on this issue, the present article was written with the aim of reviewing the body of literature on postnatal histogenesis in the various districts of the peripheral nervous system, from the historical roots to the most recent reports.     

(Na+ + K+)-ATPase in subcellular fractions of rat skeletal muscle.

Sodium and potassium-stimulated, magnesium-dependent, ouabain-sensitive ATPase has been used as an enzymic marker for muscle membrane (sarcolemma). However, the precise localization of this enzyme is still unknown. The enzyme activity was determined in muscle homogenates, sarcolemma, mitochondria, myosin B, and fragmented sarcoplasmic reticulum fractions isolated from frozen rat skeletal muscle. NaI-extracted fragmented sarcoplasmic reticulum isolated from frozen muscle (NaI-extracted high-speed fraction) contained a much higher specific (Na⁺ + K⁺)-ATPase activity (102 nmoles P₁/mg/min) than did a similar fraction isolated from fresh skeletal muscle (34 nmoles P₁/mg/min). NaI-treated muscle homogenate, sarcolemma, myosin B, and mitochondria had lower (Na⁺ + K⁺)-ATPase specific activity than the NaI-extracted high-speed fraction. The fragmented sarcoplasmic reticulum prepared from fresh or frozen muscle had the same calcium accumulating capacity; this capacity was almost abolished in the fragmented sarcoplasmic reticulum of both fresh and frozen muscle treated with NaI. The methods described may be useful in the study of muscle from humans or experimental animals where the amount of tissue available is limited. The results reemphasize the possible hazard in determining precise subcellular localization of enzyme activities in frozen tissues, as is a necessarily routine procedure in histochemistry.     

Changes in fibronectin distribution in the developing peripheral nervous system of the chick

The relationship of peripheral nerves with fibronectin was examined at different stages of the chick embryo using double immunofluorescent staining. Neurons were stained with a monoclonal antibody (E/C8) against intermediate filaments in neuronal processes, and fibronectin was stained with polyclonal antibodies. Prior to axonal outgrowth, fibronectin was distributed in a meshwork throughout the mesenchyme. However, soon after the initiation of axonal outgrowth, fibronectin began to disappear along neuronal pathways. Thus, during the period of active axonal growth, all neural tissues were marked by the striking absence of fibronectin. Interestingly, fibronectin reappeared along peripheral pathways soon after projection patterns were established. The presence of fibronectin in the substrate on which axons grow suggests that fibronectin may provide a permissive substrate for axon extension. The disappearance of fibronectin upon axon arrival suggests that neurons may modify the substrate of their pathway during outgrowth.     

Differential distribution of the G protein gamma3 subunit in the developing zebrafish nervous system.

G proteins play an essential role in the transduction and propagation of extracellular signals across the plasma membrane. It was once thought that the G protein alpha subunit was the sole regulator of intracellular molecules. The G protein betagamma complex is now recognized as participating in many signaling events. While screening a zebrafish cDNA library to identify members of the protein 4.1 superfamily (Kelly, G.M., Reversade, B., Biochem. Cell Biol. 75 (1997), 623), we fortuitously identified a clone that encodes a zebrafish G protein gamma subunit. The 666 nucleotides of the zebrafish G protein gamma subunit cDNA encodes a polypeptide of 75 amino acids with high degree of homology to human, bovine, rat and mouse gamma subunits. BLAST search analysis of GenBank revealed that the zebrafish gamma subunit is 93% identical and 97% similar to the mammalian gamma3 subunit. The gamma3 gene was mapped to the zebrafish linkage group 21, approximately 10.76 cRays from bf, a gene with sequence homology to the human properdin factor gene. RT-PCR and in situ hybridization analyses first detected gamma3 mRNA during late somitogenesis, where it was expressed preferentially in the Vth cranial nerve, the forebrain and in ventrolateral regions of the mid- and hindbrain including the spinal cord. The ability of the zebrafish gamma3 subunit to form a signaling heterodimeric complex with a beta subunit was tested using a human beta2 subunit. The gamma3 formed a heterodimer with beta2 and the complex was capable of binding calmodulin in a calcium-dependent manner. Overexpression of the beta2gamma3 complex in zebrafish embryos lead to the loss of dorsoanterior structures and heart defects, possibly owing to an up-regulation of mitogen-activated protein kinase activity and/or decline in protein kinase A signaling. Together, these data imply that a betagamma heterodimer plays a role in signal transduction events involving G protein coupled receptors and that these events occur in specific regions in the nervous system of the developing zebrafish.     

Neurofilament distribution and organization in the myelinated axons of the peripheral nervous system

The nature of neurofilament organization within the axonal cytoskeleton has been the subject of controversy for many years. Previous reports have suggested that neurofilaments are randomly distributed in the radial dimension of the myelinated axon. Randomness of distribution implies that there is no interaction between neurofilaments, while order in distribution suggest the presence of forces between neurofilaments. To address the issue of randomness vs. order, we evaluated neurofilament distribution by two different statistical approaches—nearest-neighbor distance and the Poisson tile-counting method. Neurofilament nearest-neighbor distances in a myelinated axon differ from nearest-neighbor distances of a set of random points with similar density (40.6 ± 7.0nm vs.30.7 ± 12.9nm, P < 0.0001). The Poisson tile-counting method also indicated that neurofilament distribution is different from a random distribution, under conditions of appropriate tile size and masking of other organelles. To further characterize the distribution of neurofilaments, we compared the relationship between nearest-neighbor distance and density for three sets of data: evenly spaced points, randomly distributed points and measured neurofilament coordinates. Neurofilaments do not conform to either evenly spaced or random distribution models. Instead, neurofilament distribution falls into an intermediate position between evenly spaced and random distributions. This study also demonstrates that the nearest-neighbor distance method of assessing neurofilament distribution offers several technical and theoretical advantages to the Poisson tile-counting method.     

Inhibition of purified (Na,K)-ATPase activity from porcine cerebral cortex by NO generating drugs

We tested the effects of several nitric oxide (NO) generating compounds on the activity of sodium-potassium adenosine 5′-triphosphatase [(Na⁺,K⁺)-ATPase] purified from porcine cerebral cortex. Sodium nitroprusside (SNP),S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1) and (dl)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR 3) inhibited the (Na⁺,K⁺)-ATPase activity dose dependently. Superoxide dismutase, a NO scavenger, and sulfhydryl (SH) compounds, reduced-form glutathione (rGSH) and dithiothreitol (DTT), prevented the inhibitory action of SNAP, SIN-1 and NOR 3 but not of SNP, when applied simultaneously with NO generating compounds, and this enzyme inhibition could be reactivated by the incubation with these SH compounds but not with SOD. The inhibitory action by SNP was magnified by simultaneous application of DTT. These results suggest that NO generating compounds, SNAP, SIN-1 and NOR 3 but not SNP, may release NO or NO-derived products and may inhibit (Na⁺,K⁺)-ATPase activity by interacting with a SH group at the active site of the enzyme.