Comparative analysis of sodium-dependent L-glutamate transport of synaptosomal and astroglial membrane vesicles from mouse cortex.

Uptake of [3H]L-glutamate into membrane vesicles prepared from either mouse cortical astrocyte cultures or synaptosomes was found to be an electrogenic sodium- and potassium-dependent transport process with saturable uptake kinetics. Pharmacological differences were revealed by using a variety of substrate analogues. L-trans-PDC inhibited the synaptosomal glutamate transport 2-4-fold stronger than the astroglial uptake. The substrate analogues DL-threo-beta-hydroxy-aspartate, DL-aspartate-beta-hydroxamate, L-aspartate and D-aspartate inhibited glutamate transport of astroglial and neuronal membrane vesicles in a distinctive manner, whereas D-glutamate, quisqualate and dihydrokainate had no effect in either case. Immunoblotting and immunocytochemical labeling with antibodies against the rat brain glutamate transporter revealed the selective reaction of a band at about 75 kDa mol. wt. and a specific pattern of astrocyte immunostaining.

Neuronal and glial gamma-aminobutyric acid+ transporters are distinct proteins.

In the central nervous system, two subtypes of sodium- and chloride-coupled GABA transporter exist. One is sensitive to ACHC, the other to beta-alanine. They are thought to be of neuronal and glial origin, respectively. GABA transport in membrane vesicles derived from astroglial cells was found to be sodium- and chloride-dependent, electrogenic and much more sensitive to beta-alanine than to ACHC. Immunoblotting with antibodies directed against a variety of sequences of the ACHC-sensitive transporter indicated that none of these epitopes was shared by the glial transporter.

Small conductance calcium-activated potassium channels of trout CNS: molecular structure, developmental expression, and partial biophysical characterization.

Two small conductance calcium-activated potassium channels that exhibited amino acid similarity with mammalian SK2 and SK3, respectively, were PCR cloned from the CNS of trout and sequenced. Upon heterologous expression in Sf21 insect cells trout SK2 (termed tSK2) produced a calcium-dependent, voltage-insensitive, and non-inactivating current with a single unit conductance of about 11 pS. This current was half maximally activated by 0.76 microM Ca(2+) and blocked by picomolar concentrations of apamin but not by TEA. Transcripts of both SK-related channels possessed a widespread distribution in the mature brain tissue of trout and outside the nervous system were detectable in muscle tissue as well as in liver. As revealed by RT-PCR analysis transcripts encoding tSK2 and tSK3 channels were early detectable during brain development (stage 30, shortly after hatching).

Molecular cloning and functional characterization of Shaw-related potassium channels of trout CNS.

Two Shaw-related potassium channels expressed in the CNS of trout were PCR cloned and sequenced: Traw1 was identified as a fish homologue to mammalian K(v)3.1, while Traw2 could not be exactly classified. Upon heterologous expression Traw1 exhibited a high threshold (-20 mV) non-inactivating delayed rectifier current that was efficiently blocked by submicromolar concentrations of TEA, 4-AP and quinine but not by alpha-DTX or apamin. The amplitude of the Traw1 induced current was reduced by the phorbol ester TPA, the effect being prevented by the proteinkinase inhibitor H7. Transcripts of both Shaw- related channels possess a widespread distribution in the mature brain tissue and outside the nervous system are detectable in muscle but not in liver. During brain development Traw1 mRNA was initially identified at stage 31 (shortly after hatching) while transcripts encoding Traw2 were detectable already at stage 28 (1 week before hatching).

Appearance of Myelin proteins during vertebrate evolution.

Myelin, defined as an arrangement of spirally fused unit membranes, is an acquisition of vertebrates and first appeared during evolution in Gnathostomata. In all species studied PNS and CNS myelins contain the myelin-associated glycoprotein (MAG) and the myelin basic protein (MBP). Throughout phylogeny PNS myelin is characterized by the major P(0) glycoprotein which is called IP in fishes. The PNS myelin proteins did not evolve further except for the addition of P(2) protein from reptiles onward. In Elasmobranchii and Chondrostei, PNS and CNS myelin proteins are similar. CNS myelin of actinopterygian fishes possesses a 36,000 Da protein (36K) in addition to P(0)-like IP glycoproteins. In tetrapod CNS myelin, P(0) is replaced by the proteolipid protein (PLP) and the Wolfgram protein (WP). Of particular interest in a transitional phylogenetic sense are the lungfish Protopterus, carrying glycosylated PLP (g-PLP) but no P(0), 36K or WP, and the bichir Polypterus, showing simultaneous presence of P(0), 36K and PLP. These results indicate that myelin proteins could be valuable molecular markers in establishing vertebrate phylogenetic relationships and in reconstructing the fish-tetrapod transition.

Structure, heterologous expression, and adhesive properties of the P(0)-like myelin glycoprotein IP1 of trout CNS.

The IP1 protein of trout CNS myelin as well as an IP1/P(0) chimeric protein were stably expressed in CHO cells. Successful targeting of the recombinant proteins to the membrane surface was verified by immunofluorescence staining. Full-length expression of IP1 could be confirmed by Western blot analysis of proteins extracted from stably transfected CHO-cells. The adhesive properties of IP1 were studied by an in vitro aggregation assay in which microscopic examination was combined with electronic particle counting. While IP1 conveyed only a weak increase in cell aggregation of transfected CHO cells, the IP1/P0 chimera was much more effective. In the presence of specific antibodies, cell aggregation was strongly reduced. The adhesive properties of P(0)-like proteins are discussed considering recent crystallographic data on the atomic structure of the extracellular domain of mammalian P(0).

Biochemical characterization of the central nervous system myelin proteins of the rainbow trout, Salmo gairdneri.

Central nervous system myelin isolated from the rainbow trout (Salmo gairdneri) displays a very low median density on zonal gradient centrifugation, banding at approximately 0.32 M sucrose. Its proteins consist of a 36 K (36,000 mol.wt.) component, two Concanavalin A-reactive intermediate proteins IP1 (23,000 mol.wt.) and IP2 (26,200 mol.wt.), and two basic proteins BP1 and BP2, of which the latter co-migrates with rat SBP while BP1 is of slightly smaller size. The trout myelin proteins electrofocus at pH positions similar to those of their mammalian counterparts. Immunoblotting shows that antibodies against rat PNS myelin P0 glycoprotein are bound by IP1 and IP2, but not by 36K. None of the trout myelin proteins react with anti-rat CNS myelin proteolipid protein (PLP) antiserum. The basic proteins BP1 and BP2 bind strongly to antibodies directed against human myelin basic protein. In vivo injection of tritiated fucose or palmitate leads to radiolabeling of IP1 and IP2. Under autolytic in situ conditions the appearance of a glycosylated 20,000 mol.wt. component (IP0) is noted, with parallel reduction of both IP1 and IP2, indicating sequence homologies between IP1 and IP2. The 36K protein is not affected by autolysis.

In vitro differentiation of trout oligodendrocytes: evidence for an A2B5-positive origin.

The molecular differentiation of oligodendrocytes derived from larval trout brain was studied in dissociated cell cultures using a range of cell type and stage specific antibodies. By double-labeling immunostaining using A2B5 antibodies in conjunction with antibodies against the myelin glycoproteins IP1 and IP2 evidence was obtained that oligodendrocytes of trout in vitro originate from A2B5+ precursor cells, which in terms of morphology closely resemble 0-2A progenitors of the mammalian CNS. Most surprisingly these cells did not differentiate in vitro beyond the level of IP2 expression, which signifies the initial step of oligodendroglial development in vivo. Hence it appears that in trout oligodendrocytes the initiation of the developmental program is intrinsically regulated, whereas further maturation of the cells requires appropriate environmental stimulation.

Developmental expression of myelin proteins by oligodendrocytes in the CNS of trout.

Using immunohistochemical techniques, the pattern of cytoplasmic staining and the temporal order of expression of 5 major myelin components of oligodendrocytes were studied in the developing central nervous system of trout. The two myelin glycoproteins, IP1 and IP2, in the cytoplasm of glial cells showed a granular pattern of immunostaining, whereas the 36K protein was homogeneously distributed. Analysis of freshly dissociated cells during early stages of myelinogenesis revealed a constant chronological sequence of expression of myelin proteins by the oligodendrocytes: glycoprotein IP2 was the first protein to appear during glial development together with the galactocerebroside GalC at stage 28 followed by the 36K at stage 30 and finally IP1 at stage 32. The deposition of myelin proteins into the nascent myelin sheath occurred in the same chronological order as their expression by oligodendrocytes. Moreover myelin basic protein, which was not detectable in glial cells, on tissue sections was found to appear in parallel with IP2.

Ingrowth of optic nerve fibers and onset of myelin ensheathment in the optic tectum of the trout (Salmo gairdneri).

The early differentiation of the optic pathway of the trout was studied by means of autoradiography, silver impregnation and electron microscopy. Ingrowth of the optic nerve fibers into the optic tectum was consistently shown by tracer application and Golgi studies to occur at stage 28, about one week before hatching. Fibers being arranged in discrete bundles were rapidly growing through the longitudinal axis of tectum and at stage 33 reached its posterior end. Cross sections of these fiber bundles at different positions revealed myelin ensheatment to be initiated at the end of stage 34 at the anterior pole of the tectum. Since in the optic nerve of the trout the onset of myelination occurred even earlier (stage 33), it is assumed that this differentiation process follows a rostro-caudal gradient during development of the optic pathway.

Protein analysis of myelin isolated from the CNS of fish: developmental and species comparisons.

The protein composition of myelin isolated from the CNS of four different fish species (trout, goldfish), eel, Tilapia) was analysed by SDS-polyacrylamide gel electrophoresis and compared with that of pig and rat brain. Thereby the following features were found typical to the myelin of fish: (1) a basic protein of particular low molecular weight, (2) the entire absence of Wolfgram protein, (3) the appearance of an additional major component of medium molecular weight (around 36,000) and (4) a strong Con A-affinity exhibited by the intermediate proteins, being most clearly discernible in trout and eel myelin. During development of the trout brain, in particular, a myelin fraction could first be isolated from the brainstem at 14 days after hatching and the myelin yield steadily increased during the first year of life. In tectum and cerebellum main myelin accumulation was stated during the third and seventh month. Concomitantly marked changes in the relative distribution of major myelin proteins were observed: especially the IP1-protein showed a marked increase during the first half year, while the relative amount of the 36-K protein was gradually declining during the same time.

A morphological and biochemical study of myelinogenesis in fish brain.

Myelinogenesis in different brain parts of trout was studied comparatively by means of histochemistry and biochemistry. A more detailed approach, including ultrastructural analysis, to characterize this important differentiation process, particularly in the optic tectum, revealed a good correlation between the structural development of myelinated axons and the lipid composition of membranes. The molar ratio of cholesterol to phospholipids and galactolipids proved to be most suitable for monitoring myelination in fish CNS.

Cell cultures enriched in oligodendrocytes from the central nervous system of trout in terms of phenotypic expression exhibit parallels with cultured rat Schwann cells.

Oligodendrocytes were isolated from the white matter of young trout by Percoll density centrifugation of enzymatically dissociated tissue and cultured on poly-D-lysine-coated petri dishes. Using antisera recognizing myelin-specific compounds of fish CNS (36K, IP2) up to 72% of the isolated cells could be identified as oligodendrocytes with an average yield of 4 x 10(6) cells per gram of wet tissue. Taken in culture, the cells rapidly regenerated their processes and soon acquired a morphology closely resembling mammalian oligodendrocytes in vitro. On the other hand, in terms of phenotypic expression, interesting parallels were revealed with the known in vitro behavior of Schwann cells: Galactocerebroside, which in mammalian oligodendrocytes is persistently expressed over longer periods of time in vitro, rapidly disappeared from the surface of cultured trout oligodendrocytes. In contrast, the fish CNS myelin glycoprotein IP2, which like IP1 is immunologically related to the major myelin product of Schwann cells, P0, was continuously expressed over several weeks in culture. Two other myelin protein constituents, 36K and IP1, transiently declined in vitro, but later on fully reappeared in the glial cells of trout. The present cell culture system offers an experimental model for studying in vitro the factors underlying oligodendroglial regeneration and remyelination in the fish CNS.

Molecular cloning and tissue expression of a cDNA encoding IP1--a P0-like glycoprotein of trout CNS myelin.

A full-length cDNA encoding a major structural glycoprotein of trout CNS myelin (IP1) was cloned and sequenced. The deduced amino acid sequence exhibited a significant structural homology with the P0 proteins of rat PNS and shark CNS. Sequence conservation was strongest in the extracellular domain, and it included the position of the two cysteine residues required for stabilization of an immunoglobulin-like secondary structure as well as those of the single N-glycosylation acceptor site. The cytoplasmic domain was shorter by 38 amino acids than those of rat and shark P0 and except for a high proportion of basic amino acids did not show any appreciable sequence homology. A single mRNA species of 2 kb was identified by northern blotting, which was expressed in brain tissue but not in liver. By in situ hybridization a selective labeling of myelinating glial cells in the trout CNS and PNS was revealed. The developmental appearance of the IP1 transcript closely coincided with a period of active myelin deposition in most regions of the trout brain.

Changes in ion channel expression during in vitro differentiation of trout oligodendrocyte precursor cells.

Voltage-gated ionic currents were studied in cultured trout oligodendrocyte precursor cells derived from larval trout brain with the whole-cell mode of the patch-clamp technique. These bipolar cells which carry the ganglioside epitope A2B5 on their surface differentiated in vitro into immature multipolar oligodendrocytes expressing the myelin glycoprotein IP2, which signifies the initial step of oligodendroglial development in fish CNS. Depolarization above -40 mV activated a fast transient sodium inward current that was eliminated by substituting Na+ for choline and blocked in the presence of 1 microM TTX. The kinetics and the voltage-dependence of inactivation (half-maximal inactivation at -68 mV) resembled those of sodium currents described in mammalian oligodendrocyte precursor cells and CNS neurons. The expression of Na+ channels was developmentally regulated, since high amplitudes were measured only in A2B5+ cells with a characteristic bipolar morphology of glial progenitors. Depolarizing voltage steps, additionally elicited outward potassium currents that were sensitive to external 4-AP. In a subpopulation of cells this outward current consisted of a sustained and a transient component. The amplitude of both components were dependent on the prepulse potential.

Immunohistochemical localization of myelin basic protein in the developing optic system of trout.

Immunofluorescent localization of basic protein (BP) in the CNS of trout was performed using a heterologous antiserum raised against human BP. Bright specific fluorescence was confined to the myelin sheath of axons in most regions of the brain, whereas neuronal pericarya and dendrites as well as glial cells were entirely negative. In the tectum immunofluorescence was characteristically distributed in two rows of horizontally aligned patches, most evidently reflecting the orderly array of myelinated fiber bundles in the stratum opticum and stratum album. During development of the retino-tectal pathway, the appearance of BP followed a distinct rostro-caudal gradient firstly reaching the anterior ventral border of the tectum by larval stage 34. Furthermore, on cross sections through the optic nerve a specifically organized pattern of myelin formation was revealed by immunohistochemistry, which strikingly coincided with the pattern of fiber outgrowth.

Myelin deposition in the optic tectum of trout as monitored by enzymatic and morphometric analyses.

The activity of arylsulfatase A and 2'3'-cyclic nucleotide 3'-phosphohydrolase was studied in the brain of trout in parallel to the structural differentiation of tissue from early larval stages into adulthood. Whereas in the optic tectum, phosphodiesterase activity could not be detected before the second month after hatching in brainstem, the enzyme had already reached 80% of adult level. In tectum it was from the fourth to the seventh month that this enzyme dramatically increased, thereby reaching about the adult level. The developmental profile of arylsulfatase A was profoundly different, since 1) considerable activity was found in tectum at early larval stages and 2) the activity showed a peak between two and six months and then dropped markedly. Morphometric analysis of the two myelinated layers of trout tectum support and extend the biochemical results leading to the conclusion that the phosphodiesterase activity reflects the prevailing degree of myelination, whereas the developmental profile of the sulfolipid-metabolizing enzyme indicates the rate of myelin accumulation.

Calcium binding sites of synaptosomes from insect nervous system as probed by trivalent terbium ions.

Terbium binding to synaptosomes from the central nervous system of the locust was studied by fluorescence spectroscopy and electron microscopy. The protein-sensitized fluorescence of terbium was used to characterize the calcium binding sites of synaptosomes. As judged by electron microscopy and x-ray analysis, terbium ions produced electron-dense patches in regular arrays on the outer surface of synaptosomal membranes and induced marked aggregation of synaptic vesicles in isolated terminals.

Characterization of antibodies against major fish CNS myelin proteins: immunoblot analysis and immunohistochemical localization of 36K and IP2 proteins in trout nerve tissue.

Antisera against the trout CNS myelin proteins 36K and IP2 were prepared in rabbits and characterized by immunoblot analysis and immunohistochemistry. The anti-36K antiserum exclusively stained its corresponding antigen from trout CNS myelin but failed to recognize any myelin polypeptide from either trout PNS or mammalian CNS and PNS. Antibodies against the IP2 glycoprotein specifically cross-reacted with related intermediate proteins (IP) of both CNS and PNS myelin from trout but only faintly labeled the PO protein of mouse peripheral nerve. Immunohistochemical localization of both antigens in the CNS of young trout was confined to the myelin sheath, except that anti-36K antiserum also stained oligodendrocytes. Nodes of Ranvier, neuronal cell bodies, and dendrites, as well as other glial elements, were negative. Specificity of the immunofluorescent reaction was established by crossed immunoadsorption experiments. Whereas on adjacent sections through trout brain both antigens exhibited a nearly identical distribution pattern, immunostaining in peripheral nerves was seen only with anti-IP2 antibodies.