Showing papers in "Journal of Neuroscience Research in 1981"
TL;DR: It is reported that the vimentin‐GFA transition in rat brain occurs during the 2nd‐3rd week, ie, at the time of rapid myelination when dividing glioblasts in white matter differentiate into oligodendroglia and interfascicular astrocytes.
Abstract: Extraction with Triton, a nonionic detergent, is a common procedure to prepare intermediate filament enriched fractions from cells maintained in culture. The Triton-Insoluble fraction is called a cytoskeletal preparation. Using this procedure, we previously demonstrated that vimentin and glial fibrillary acidic protein (GFA) are major cytoskeletal proteins of neuroglia, in newborn and adult rat brain, respectively. In the present communication we report that the vimentin-GFA transition in rat brain occurs during the 2nd-3rd week, ie, at the time of rapid myelination when dividing glioblasts in white matter differentiate into oligodendroglia and interfascicular astrocytes.
266 citations
TL;DR: It was demonstrated that the nerve‐fiber regeneration ceased after a few weeks if there was no distal nerve inserted into the tube, and the importance of optimizing the interaction between local factors and regenerating nerve fibers for reestablishment of functionally valuable motor units is discussed.
Abstract: Regeneration of severed peripheral nerves is unfortunately often incomplete, due to loss of nerve fibers and neuroma formation. A new approach is presented with the intention of improving the conditions for nerve repair. In the first of the two stages, a pseudosynovial tube is formed around a silicone rubber rod, surrounded by a stainless steel spiral, which was placed in the backs of rats. This tube, in the second stage, is used as a free "tube graft" to bridge gaps of about 10-12 mm lengths in the severed sciatic nerve. The tube was kept open by the metal spiral. Regenerating nerve fibers with their sprouts grew into the initially open space in the tube. A new nerve trunk was formed, comprised of closely packed myelinated and unmyelinated axons, organized into fascicles. Demonstration by electron microscopy and by EMG recording of reinnervation of foot muscles supported successful long-term results. The fascicles were delimited by perineurial and epineurial sheaths and, furthermore, showed signs of maturation. It was also demonstrated that the nerve-fiber regeneration ceased after a few weeks if there was no distal nerve inserted into the tube. The importance of optimizing the interaction between local factors and regenerating nerve fibers for reestablishment of functionally valuable motor units is discussed.
113 citations
TL;DR: Mouse brain astrocytes from primary cultures were found to contain both α1 and α2 adrenergic receptors, which were 2–3 times higher in mouse cerebral cortex than in glial cells.
Abstract: Mouse brain astrocytes from primary cultures were found to contain both alpha 1 and alpha 2 adrenergic receptors. 3H WB 4101 labeled one category of binding site (KD = 1.5 +/- 0.39 nM, Bmax = 64 +/- 7.9 fmoles/mg protein) with typical alpha 1 adrenergic specificity (WB 4101 greater than prazosin greater than yohimbine). The density of alpha 1 adrenergic receptors was 2-3 times higher in mouse cerebral cortex than in glial cells. Like rat brain [U'Pritchard et al, 1979; Rouot et al, 1980], mouse glial cells were found to contain two categories of 3H clonidine binding sites: high affinity sites, which were identical to the high but not to the low affinity sites found in rat brain, since 1) they displayed the same affinity for 3H clonidine (KD = 1.2 +/- 0.13 nM, n = 4) and the same typical alpha 2 adrenergic specificity (yohimbine greater than WB 4101 greater than prazosin); 2) the dissociation rate constant for clonidine binding was equal to 0.06 min-1, a value close to that found previously for the high affinity 3H clonidine binding sites in rat brain (0.05 min-1); and 3) divalent cations augmented and guanyl nucleotides reduced 3H clonidine binding as in rat brain. Na+ decreased 3H clonidine binding in a complex manner. The number of high affinity sites in glial cells (52 +/- 9.4 fmoles/mg protein, n = 4) was half the number found in mouse cerebral cortex (98 fmoles/mg protein). Low affinity 3H clonidine binding sites (KD = 81 +/- 18 nM, Bmax = 96 +/- 5.8 fmoles/mg protein, n = 3) were not fully characterized. In conclusion, glial cells contained the same alpha adrenergic receptors as those described in brain, but their physiological function is not yet known.
68 citations
TL;DR: The results suggest that a correlation may exist between certain KA‐induced lesions of the retina and putative glutamoreceptive neurons and may be useful in elucidating the differences between specific, Glu‐related toxicity and nonspecific toxicity of KA.
Abstract: Short-term (15 minutes) in vitro exposure to kainic acid (KA), a rigid structural analog of L-glutamic acid (Glu), caused two morphologically distinct neuronal lesions in retinas of several species. In rabbit retina, one type of lesion was characterized by rapid swelling after exposure to low concentrations of KA (10(-4)M). This lesion was observed in elements of both plexiform layers and, more specifically, in cell bodies and neurites of horizontal cells that contact cones. A few cell bodies from the amacrine cell layer showed some limited swelling. The swelling was completely blocked when sodium was removed from the incubation medium. The second type of lesion was generally seen after longer exposures of after exposure to higher concentrations of KA and was evidenced by degeneration of neurons in the amacrine and ganglion cell layers. One exception was noted in that a few cells from the ganglion cell layer degenerated even under low exposure conditions. The second type of lesion was not blocked by removal of sodium ions. Photoreceptor cells appeared resistant to all effects of KA. The results suggest that a correlation may exist between certain KA-induced lesions of the retina and putative glutamoreceptive neurons. At the same time, the two types of retinal lesions produced by KA are morphologically and chemically differentiable and may be useful in elucidating the differences between specific, Glu-related toxicity and nonspecific toxicity of KA.
65 citations
TL;DR: There are multiple parallel but separate mechanisms of temporal regulation controlling myelinogenic gene expression, and that, among them, those factors that control MBP accumulation are more sensitive to disruption by the rigors of dissociated culture.
Abstract: The developmental regulation of sulfogalactosylceramide (sulfatide) synthesis, 2', 3'-cyclic nucleotide-3'-phosphohydrolase activity (CNP), and myelin basic protein (MBP) accumulation, three markers characteristic of myelinogenesis, were observed in dispersed cultures of fetal rat brain in spite of the absence of the formation of compact, multilamellar myelin. Sulfatide synthetic rate and CNP activity began to increase by 8 days in vitro (DIV) (approximately comparable to days post-natal) and reached their maxima by 20 DIV. MBP began to accumulate after 14 DEV and reached a maximum by 38 DIV. Thus, the temporal regulation of the onset of expression of these parameters, which is coordinate in vivo, has been dissociated into two sequential periods in vitro. Similarly, the regulatory mechanisms controlling the subsequent decline of the net expression of these three parameters were dissociated. Whereas, the increase in the net CNP activity ceased on schedule, the decline of sulfatide synthetic rate was delayed 20 days, and the accumulation of MBP underwent a net loss. These data suggest that there are multiple parallel but separate mechanisms of temporal regulation controlling myelinogenic gene expression, and that, among them, those factors that control MBP accumulation are more sensitive to disruption by the rigors of dissociated culture.
64 citations
TL;DR: A clonal cell line, IMR‐32, derived from a human neuroblastoma was found to contain a population of cells that respond to NGF by exhibiting morphological differentiation, and the effect of NGF on these cells is compared with that of other agents known to induce differentiation of IMR-32, including glioma‐conditioned media.
Abstract: The nerve growth factor protein (NGF) stimulates neurite outgrowth form embryonic sensory ganglia and sympathetic ganglia at all stages of development. In addition, NGF is required for the maintenance of the differentiated state in adult sympathetic ganglia. A clonal cell line, IMR-32, derived from a human neuroblastoma was found to contain a population of cells that respond to NGF by exhibiting morphological differentiation. The effect of NGF on these cells is compared with that of other agents known to induce differentiation of IMR-32, including glioma-conditioned media.
61 citations
TL;DR: Assays in the mutants at early ages were rendered feasible by the use of marker genes, which has allowed the diagnosis of the mutation at birth, 12 days before the expression of their typical tremor phenotype.
Abstract: Myelin basic protein (MBP) was quantified using a RIA technique in the spinal cord, cerebellum, diencephalon plus brainstem region and cerebral hemispheres of two dysmyelinating murine mutants, quaking (qk) and jimpy (jp) mice. Comparison was made with normal control values. The whole life-span has been investigated: ie, ages ranging from 0 to 26 days for the jp, O to one year for the qk, and prenatal stage to three years for the control animals. Assays in the mutants at early ages were rendered feasible by the use of marker genes, which has allowed the diagnosis of the mutation at birth, 12 days before the expression of their typical tremor phenotype. Special care was given to the period of early myelinogenesis in order to clarify the dysynchrony between the various parts of the central nervous system. In normal mice, MBP was already detected in the brain of 19-day-old embryos. During development, rapid accumulation of MBP first occurred in the spinal cord then in the diencephalon, the brainstem, the cerebellum, and finally in the cerebral hemispheres. In the 25-day-old jimpy mutant, levels of MBP were found dramatically decreased, never exceeding 6% of the normal controls in any of the areas investigated. The situation for the quaking mouse was quite different. This mutant could be investigated up to one year old. At that age, a high discrepancy was observed between the values found in the brain and in the spinal cord (respectively, 10% and 35%) compared to normal controls. In both mutants, not only were the levels of MBP decreased, but also its appearance during development was delayed. Nevertheless, in both mutants the caudo-rostral timing of myelination as assayed by MBP levels was maintained. Furthermore, the later myelination occurred, the stronger weas the deficit in MBP. Interestingly, in the quaking mutant, the specific plasticity of the spinal cord was exemplified by its ability to reduce constantly, even at an advanced age, its initial deficit of MBP.
59 citations
TL;DR: The evolution of the pattern of the major gangliosides showed a phase from 1 to 20 DIC which was characterized during the first half of this period by a high percentage of tri‐ and tetra‐sialoganglioside, thereby confirming the presence of well differentiated neurons, and the yin yang hypothesis was only partially confirmed.
Abstract: Cerebral hemispheres of 14- to 15-day-old mouse embryos were dissociated mechanically into single cells and cultured in polylysine-coated plastic flasks. The succession of changes and the identification of cell populations have been studied by transmission electron microscopy (TEM). Four distinct cell types can be clearly defined. Neuroblasts, oligodendroblasts, and astroblasts were used to denote embryonic states of neurons, oligodendrocytes, and astrocytes. Cell populations were: (1) neuroblasts; (2) astroblasts, either growing on the surface of the flasks and forming a basal layer of epithelioid cells or scattered at the surface of the bed layer; (3) oligodendroblasts, characterized by a high nucleocytoplasmic ratio and resting on top of the bed layer; (4) macrophages. Incorporation of [3H]thymidine into cells at different days in culture (DIC) delineated at least three periods of DNA synthesis (from 1 to 6, from 10 to 20, and from 25 to 60 DIC). High cell RNA/DNA and protein/DNA ratios indicated an increase in the cytoplasmic mass. Guanylate cyclase (EC 4.6.1.2; GC) and adenylate cyclase (EC 4.6.1.1; AC) activities and the ratio AC/GC also agreed with the existence of different periods in culture, but the yin yang hypothesis (postulated by Goldberg et al, 1975) was only partially confirmed. Concerning membrane constituents, the evolution of the pattern of the major gangliosides showed a phase from 1 to 20 DIC which was characterized during the first half of this period by a high percentage of tri- and tetra-sialogangliosides, thereby confirming the presence of well differentiated neurons. Choline acetyltransferase (EC 2.3.1.6; ChAc), acetylcholinesterase (EC 3.1.1.7; AChE), and [3H]muscimol binding (indicative of GABA-receptors) supported this conclusion. Another phase from 20 to 60 DIC showed a relatively higher content of GM3 and GD3 of the disialogangliosides GD1a and GD1b. These data favor a glial origin of the cells, also demonstrated by TEM and by immunohistochemistry with anti-glial fibrillary acidic protein (anti-GFAP) and anti-carbonic anhydrase isoenzyme II (anti-CAII; EC 4.2.1.1) sera.
Ultrastructural, biochemical, and immunohistochemical data are in agreement with the existence of successive and different periods of cell proliferation and differentiation which have been defined as: (1) mainly neuronal from 1 to 12 DIC; (2) astroglial and oligodendroglial between 10 and 20 DIC, and (3) possibly microglial from 25 to 40 DIC. This surface-adhering primary culture system should significantly enhance efforts to examine the roles played by hormones or other effectors in the regulation of myelination.
45 citations
TL;DR: The histochemical method of Häusler was employed to demonstrate carbonic anhydrase (CA, carbonate hydrolase, 4.2.1.1.) in tissue sections and the CA reaction was inhibited in the presence of 5 mM acetazolamide.
Abstract: The histochemical method of Hausler was employed to demonstrate carbonic anhydrase (CA, carbonate hydrolase, 4.2.1.1.) in tissue sections. The CA reaction was inhibited in the presence of 5 mM acetazolamide. In the frog and fish retinas the CA activity was positive in the Muller fiber, the laminated segment of the cones and the myelin of the axons, and was negative in the laminated segment of the rods, the photoreceptor ellipsoid and myoid, the horizontal, bipolar, amacrine and ganglion cells, and the pigment epithelium. In the spinal cord of cat and fish the CA activity was positive in the myelin of the axons, the perineuronal oligodendroglia and the protoplasmic astrocytes, and was negative in the cell body, dendrite, and axon of the neurons and in the fibrous astrocytes. In the dorsal root ganglion of the cat CA reaction was positive in the satellite cells and the myelin, and was negative in the neuronal cell body and its processes. The capillaries in central nervous tissue show no CA reaction. The erythrocyte is CA positive while the mitochondria are CA negative.
45 citations
TL;DR: Results provide further evidence that acetylcholinesterase activity is specifically associated with cortical cholinergic afferents and support the existence of cortical intrinsic Cholinergic neurons.
Abstract: The distribution of presynaptic markers for cholinergic and GABAergic neurons in the various layers of the lateral neocortex was determined with a technique in which sections of increasing depth horizontal to the pial surface were obtained on a frozen-stage microtome and analyzed for the markers. The specific activities of choline acetyltransferase and acetylcholinesterase had an uneven distribution in the cortex with lowest levels of activity associated with lamina II and III, whereas glutamate decarboxylase activity was highest in lamina II, III, and IV. Fetally-induced hypoplasia of the cortex resulted in an enrichment in both choline acetyltransferase and acetylcholinesterase activity in all layers with no significant change in the activity of glutamate decarboxylase. Lesion of the nucleus basalis-cortical cholinergic pathway resulted in marked reductions in the activities of acetylcholinesterase and choline acetyltransferase in all cortical layers and an elimination of the uneven distribution of these markers within the cortex. These results provide further evidence that acetylcholinesterase activity is specifically associated with cortical cholinergic afferents and support the existence of cortical intrinsic cholinergic neurons.
41 citations
TL;DR: The presence of taurine, GABA, or glycine, at concentrations of 5–25 mM, effectively counteracts the light‐induced ROS disruption and reduces labeled bicarbonate and tritiated water levels to those observed in the dark incubated ROS.
Abstract: Isolated frog rod outer segments (ROS) incubated in a Krebs-bicarbonate medium, and illuminated for 2 h, show a profound alteration in their structure. This is characterized by distention of discs, vesiculation, and a marked swelling. The light-induced ROS disruption requires the presence of bicarbonate and sodium chloride. Replacement of bicarbonate by TRIS or HEPES protects ROS structure. Also, substitution of sodium chloride by sucrose or choline chloride maintains unaltered the ROS structure. Deletion of calcium, magnesium, or phosphate does not modify the effect produced by illumination. An increased accumulation of labeled bicarbonate and tritiated water is observed in illuminated ROS, as compared with controls in the dark. The presence of taurine, GABA, or glycine, at concentrations of 5-25 mM, effectively counteracts the light-induced ROS disruption. Taurine (25 mM) reduces labeled bicarbonate and tritiated water levels to those observed in the dark incubated ROS.
TL;DR: Norepinephrine elicited a smaller increase in cyclic AMP from 155‐day‐old mice than at earlier ages, and the response continued to decrease with age; at 270 days, equal accumulation, and at 365 days, lower accumulation of cyclicAMP was detected in pcd cerebella.
Abstract: The Purkinje cell degeneration mutant (pcd) is characterized by a complete loss of cerebellar Purkinje cells. Norepinephrine causes an accumulation of cyclic AMP in the cerebellum of pcd that is far greater than in normal mice. Experiments were conducted 1) to correlated changes in the cyclic-nucleotide response with a histologic examination of the cerebellum during neuronal loss and 2) to examine the role of cyclic AMP catabolism and adenosine receptor interactions in the phenomenon. The greatest elevation in cyclic AMP occurred between 30 and 128 days of age when a severe astrocytic response was demonstrated throughout the cerebellar cortex. Purkinje cells had degenerated by 45 days of age. Norepinephrine elicited a smaller increase in cyclic AMP from 155-day-old mice than at earlier ages, and the response continued to decrease with age; at 270 days, equal accumulation, and at 365 days. lower accumulation of cyclic AMP was detected in pcd cerebella. During this time, the Purkinje cell debris had been removed, the granule cell layer was depleted of granule cells, and the molecular layer was deprived of a large number of parallel fibers. However, although phagocytosis of neuronal debris was completed, large numbers of astrocytic processes were still seen in the neuropil. Biochemical experiments in vitro established that the exaggerated accumulation of cyclic AMP in the presence of norepinephrine was not due to lower catabolism of cyclic AMP, a synergistic interaction with adenosine, or a result of lower protein in the pcd cerebellum. The correlates of heightened norepinephrine-stimulated accumulation of cyclic AMP with neuronal loss and the glial cell reaction might indicate that cyclic nucleotides play a role in controlling some glial cell functions, ie, proliferation, migration, and phagocytosis.
TL;DR: The data indicate that generation of action potentials in these human neuroblastoma cells is to a large extent a function of morphological differentiation.
Abstract: The SK-N-SH cell line is a human neuroblastoma which when grown under standard culture conditions remains relatively undifferentiated. The undifferentiated SK-N-SH cells are relatively inexcitable: they show only partial active responses to injections of current pulses and lack the depolarizing component of the action potential generating mechanism [Kuramoto et al, 1977]. In this study we report on the membrane properties of two subclones of the SK-N-SH, the SK-N-SH-IN (referred to as IN) and the SK-N-SH-Sy5Y (referred to as 5Y) which exhibit extensive morphological differentiation when grown with 1mM dibutyryl cAMP. Fully differentiated IN and 5Y cells have higher resting membrane potentials, in the range of -50 to -80 mV, and higher input resistance and time constants than do the undifferentiated SK-N-SH parental cell line. After 1 week in culture the differentiated IN and 5Y cells exhibit spike potentials in response to injection of current to the cell body. The presence of the Na2+-dependent depolarization was verified directly by the use of tetrodotoxin (TTX) and indirectly in experiments where veratridine (0.1 mM) markedly enhanced the influx of 22Na+. Taken together, the data indicate that generation of action potentials in these human neuroblastoma cells is to a large extent a function of morphological differentiation.
TL;DR: Acute administration of CDP‐choline was able to correct the impairment caused by hypoxic treatment of lipid metabolism in some brain regions, ie, cerebral hemispheres, cerebellum, and brainstem.
Abstract: Acute administration of CDP-choline (i.p. 100 mg/Kg b.w.), 10 min before the intraventricular injection of labeled precursors, (2-3H) glycerol and (1-14C)-palmitate, was able to correct the impairment caused by hypoxic treatment of lipid metabolism in some brain regions, ie, cerebral hemispheres, cerebellum, and brainstem. After CDP-choline treatment, an increase of the specific radioactivity of total lipids and of phospholipids was observed in mitochondria purified from the three above-mentioned brain regions of the hypoxic animals, while no effect on the other subcellular fractions was found. CDP-Choline had a stimulating effect particularly on the incorporation of both precursors into mitochondrial PC, PE, and polyglycerophosphatides isolated form the three brain regions examined. The results obtained show that the action of CDP-choline in restoring lipid metabolism was more pronounced in brain mitochondria, which, among subcellular fractions, were the most affected by the hypoxic treatment.
TL;DR: The putative high molecular weight form of guinea pig NGF does not contain a subunit with arginine esteropeptidase activity, and although there is an abundance of this enzymatic activity in the homogenate, it does not appear to be associated with the fractions containing NGF.
Abstract: Nerve growth factor (NGF) has been purified from the guinea pig prostate using a modification of the Bocchini-Angeletti method for isolating 2.5S NGF from mouse submaxillary glands. As with the mouse preparation, guinea pig prostate NGF appears to migrate as a high molecular weight entity at physiological pH. Following dissociation, NGF, active in neurite proliferation assays and similar in size to mouse 2.5S NGF, can be isolated by chromatography on a column of carboxymethyl-cellulose at pH 4.8. Based on gel filtration, SDS-polyacrylamide gel analysis, and amino-terminal sequence studies, this material consists of two, noncovalently linked, identical polypeptide chains each with a molecular weight of about 13,000. The amino-terminal third of the polypeptide chain is at least 90% identical to the corresponding region of the murine molecule, confirming the homology of the guinea pig prostate protein to NGFs obtained from different tissues in other species. However, in contrast to the mouse preparation, the putative high molecular weight form of guinea pig NGF does not contain a subunit with arginine esteropeptidase activity. Although there is an abundance of this enzymatic activity in the homogenate, it does not appear to be associated with the fractions containing NGF. This apparent difference in the mouse and guinea pig material is of interest because the mouse γ subunit, possessing the arginine esteropeptidase activity, has been alleged to participate in the processing of a precursor of the β NGF polypeptide chain.
TL;DR: The brains of Swiss‐Webster adult mice contain an NGF‐like substance which promotes neurite outgrowth in PC‐12 cells, which probably is NGF itself, since the effect is blocked by specific NGF antiserum.
Abstract: Two pools of seven brains each from adult Swiss-Webster mice were homogenized, and supernatants were collected for bioassay. PC-12 cells were placed in a bioassay plate at time zero, at a concentration of 10(4) cells per well, and primed for 48 hours in a medium containing 50 ng/ml of mNGF. The PC-12 cell bioassay for neurite outgrowth was conducted after primed cells were exposed to an NGF-free medium for 24 hours. Suitable controls for serum toxicity and cell viability were established. The sensitivity of the bioassay approximates 100 pg NGF/ml. The results showed 80-100% neurite outgrowth in wells exposed to brain pool supernatant (BPS) alone, and control level outgrowth (3-8%) in wells containing BPS and specific anti-beta-NGF antibody. Therefore, the brains of Swiss-Webster adult mice contain an NGF-like substance which promotes neurite outgrowth in PC-12 cells. The substance probably is NGF itself, since the effect is blocked by specific NGF antiserum.
TL;DR: It appears that oligodendrocytes undergo a transient dedifferentiation during their recovery from the trauma of isolation, not unlike the central chromatolysis seen in neurons following axonal injury.
Abstract: The changes in the ultrastructure of oligodendrocytes with time in culture were examined by transmission electron microscopy. Oligodendrocytes were isolated from ovine white matter and maintained in Dulbecco's modified Eagle's medium supplemented with 20% horse serum. One day after being placed in plastic culture dishes, most cells aggregate into clusters that do not attach to the surface for several days. Cells within these clusters form junctional complexes resembling gap junctions, their nuclear chromatin becomes uniformly dispersed, they are rich in polyribosomes, and their cytoplasm is less osmiophilic than oligodendrocytes in ovine white matter or cells fixed immediately after isolation. At this time, the cells in culture fit the description of light oligodendrocytes. After several days in culture, the cells become attached to the plastic surface, chromatin once again becomes clumped beneath the nuclear membrane, rough endoplasmic reticulum accumulates within the cytoplasm and the cells extend long processes containing cytoplasmic organelles. From these observations, it appears that oligodendrocytes undergo a transient dedifferentiation during their recovery from the trauma of isolation, not unlike the central chromatolysis seen in neurons following axonal injury. After acclimatization to tissue culture conditions, isolated oligodendrocytes show many of the morphologic features seen in vivo and therefore offer a good model to study myelination.
TL;DR: The lipid metabolism in guinea pig brain after intermittent hypoxia, prolonged for 80 hrs, was markedly impaired and the labeling of the major phospholipids present – ie, phosphatidylcholine (PC) andosphatidylethanolamine (PE) – in the mitochondria of the three brain regions examined decreased after hypoxic treatment.
Abstract: The lipid metabolism in guinea pig brain after intermittent hypoxia, prolonged for 80 hrs, was markedly impaired. The in vivo incorporation of (2-3H) glycerol and (1-14C) palmitate into lipids of microsomes, mitochondria, myelin, and synaptosomes, purified form cerebral hemispheres, was significantly lower in the hypoxic animals than in the controls. The same effect was observed on the incorporation of labeled precursors into lipids of mitochondria purified from cerebellum and brainstem. In particular, the labeling of th major phospholipids present - ie, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) - in the mitochondria of the three brain regions examined decreased after hypoxic treatment.
TL;DR: Dissociated brain cell cultures are a valid model for the study of biochemical parameters related to the synthesis of brain lipids during development and suggest the existence of intrinsic factors regulating the biochemical differentiation.
Abstract: Several metabolic activities in dissociated cultures of newborn mouse brain were compared to the situation in vivo. The developmental activity pattern of cerebroside-sulfotransferase, cyclic nucleotide phosphohydrolase, and beta-hydroxy-beta-methyl glutaryl-coenzyme A-reductase and the synthesis and deposition of sulfatide and cholesterol in culture were estimated. The enzyme activity patterns in vivo and in culture are the same. Since the cultures show very little myelin formation, the parallel increase of enzyme activities necessary for myelination in vivo and in culture suggest the existence of intrinsic factors regulating the biochemical differentiation. In addition, the formation of the products, determined in culture, follows the patterns of the enzyme activities. Dissociated brain cell cultures are therefore a valid model for the study of biochemical parameters related to the synthesis of brain lipids during development.
TL;DR: The present observations suggest that the anticonvulsant effect of taurine might be mediated by 4‐AP‐calcium‐taurine interactions.
Abstract: The effect of intraperitoneally injected taurine against the convulsive activity induced by 4-aminopyridine (4-AP) was studied in 12- to 15-day-old mice. At a dose of 2.6 mg/kg, taurine increased the latency of clonic seizures from 7 to 20 minutes, reduced the incidence of tonic seizures from 92% to 30% and the postconvulsive mortality from 80% to 31%. The injection of EDTA prior to the administration of taurine prevented the protective effects of the amino acid. GABA and glycine at the same doses did not protect against 4-AP-induced seizures. 4-AP caused a small increase (19%) in 45Ca accumulation by mice brain synaptosomes incubated in a Krebs-HEPES medium containing low CaCl2 (0.1 mM) and also slightly potentiated the veratrine and potassium-induced increase in calcium accumulation. 4-AP at concentrations of 1-2 mM caused a marked increase (100%-500%) of 45 Ca accumulation by synaptosomes incubated in a Krebs-bicarbonate medium containing 2.5 mM CaCl2. This increase was completely antagonized by taurine but not by GABA of glycine. The present observations suggest that the anticonvulsant effect of taurine might be mediated by 4-AP-calcium-taurine interactions.
TL;DR: In the hypothalamus as well as in cerebral hemispheres, the specific activities of both enzymes was highest on the 13th fetal day and decline thereafter until the 20–22nd post‐natal day, with a plateau around birth.
Abstract: The ontogenesis of neuropeptide degrading enzymes was studied in the mouse brain, from the 10th gestational day up to adulthood. Two activities were followed: the pyroglutamate aminopeptidase and the post-proline cleaving enzyme, using either TRH or specific fluorogenic peptides as substrates. In the hypothalamus as well as in cerebral hemispheres, the specific activities of both enzymes was highest on the 13th fetal day and decline thereafter until the 20–22nd post-natal day, with a plateau around birth. In contrast, a classical peptidase, the leucyl-arylamidase increased only in fetal life, and reached the adult level before birth.
TL;DR: The cerebellum and cervical spinal cord, two locus coeruleus target sites, displayed different trends in tyrosine hydroxylase activity throughout development and aging.
Abstract: Tyrosine hydroxylase activity was measured in the region of locus coeruleus, cerebellum, cervical spinal cord, lumbar sympathetic ganglia, and iris throughout most of the life span of the chicken (8 days of incubation to 5 years) to compare developmental trends in tyrosine hydroxylase activity in noradrenergic cell bodies and in axon terminals in both the central and peripheral nervous system. Fluorescence histochemistry and retrograde transport of horseradish peroxidase were used to characterize further the coeruleo-cerebellar projections. Tyrosine hydroxylase activity was detected in the cerebellum as early as 8 days of incubation, which is the earliest stage so far reported. The greatest increase in total tyrosine hydroxylase activity in the region of the locus coeruleus and cerebellum occurred during the embryonic period. There was a more pronounced increase in the cerebellum than in the locus coeruleus region. This is in contrast to the cervical spinal cord where tyrosine hydroxylase activity increased at approximately the same rate during the embryonic and post-hatching periods. Moreover, the cerebellum and cervical spinal cord, two locus coeruleus target sites, displayed different trends in tyrosine hydroxylase activity throughout development and aging.
In both structures examined in the peripheral nervous system, the greatest increase in total tyrosine hydroxylase activity occurred during the post-hatching period, with a greater rise in the cell bodies of the lumbar sympathetic ganglia than in the noradrenergic terminals of the iris. In both the central and peripheral nervous system, total tyrosine hydroxylase activity continued to increase in noradrenergic terminals long after hatching reaching the highest levels at 7 months when the chicken is considered fully mature. During aging, 16 months to 5 years, there was a greater decrease in total tyrosine hydroxylase activity in the terminals of noradrenergic neurons than in the cell bodies in both the central and peripheral nervous system, a phenomenon that was more marked in the peripheral nervous system than in the brain.
TL;DR: It is suggested that p‐TA may act as a modulator of neurotransmission, particularly that mediated by DA in the central nervous system.
Abstract: Extracellular recording and iontophoresis techniques were used to study the interactions of the trace amine, p-tyramine (p-TA) with putative neurotransmitters on single neurones in the cerebral cortex and caudate nucleus of the rat p-TA, when applied with weak iontophoretic currents which did not result in any change in neuronal firing rate, caused a pronounced potentiation of depressant responses to iontophoretically applied dopamine (DA) Depressant responses of cortical neurones to noradrenaline were also markedly potentiated by weak background application of p-TA This potentiating action of p-TA was related to the amount of the trace amine applied and was apparently specific for catecholamines, since depressant responses to 5-hydroxytryptamine and gamma-aminobutyric acid were unaffected Excitatory responses to iontophoretically applied glutamate were also unaltered by weak application of p-TA Excitatory responses to iontophoretically applied glutamate were also unaltered by weak applications of p-TA Excitatory responses of most neurones to acetylcholine (ACh) were also unaffected by p-TA in the cortex and caudate nucleus However, responses to ACh of a small number of cells in both brain areas were reduced in size during weak applications of p-TA It is suggested that p-TA may act as a modulator of neurotransmission, particularly that mediated by DA in the central nervous system
TL;DR: The relationship between induction of glutamine synthetase activity by dexamethasone and binding of the steroid to cytosolic glucocorticoid receptors was examined in sensitive C6 and resistant C6H glial cell cultures.
Abstract: The relationship between induction of glutamine synthetase activity by dexamethasone and binding of the steroid to cytosolic glucocorticoid receptors was examined in sensitive C6 and resistant C6H glial cell cultures. Glutamine synthetase activity increased 3-4-fold when C6 cultures were exposed to 7.6 x 10(-6) M dexamethasone. This inductive response was reversible, dose-dependent (ED50 approximately 2 x 10(-8) M), required de novo protein and RNA synthesis, and was elicited only by glucocorticoid steroids. Progesterone, but not epicortisol, antagonized the dexamethasone-induced enzyme increase. In contrast, only a slight inductive effect was observed in dexamethasone-treated C6H cells. Competitive binding assays demonstrated that specific binding of [3H]-dexamethasone to cytosolic receptors was also dose-dependent. The ED50 was approximately 10(-8) M for both C6 and C6H cells. Scatchard analysis revealed that each C6 cell contained approximately 10,800 receptor sites and that the equilibrium dissociation constant (Kd) was 4.5 x 10(-9) M. Each C6H cell possessed approximately 12,200 sites, and the Kd was 6.7 x 10(-9) M. Unlabeled dexamethasone and cortisol (but not epicortisol) competed effectively with [3H]-dexamethasone for binding to cytosolic receptor sites and nuclear sites of both cell types. These results suggest that induction of glutamine synthetase activity in dexamethasone-treated C6 cells is a glucocorticoid-directed response. Since C6H cells are refractory in this regard but contain functional cytosolic receptors which interact with cell nuclei, the basis for their resistance appears to involve some step beyond these cellular processes.
TL;DR: The direct receptor effects of the ergot derivatives in vitro may contribute to understanding their in vivo effects on behavior and in predicting their therapeutic potential in neurological and neuroendocrine disorders.
Abstract: Bromocriptine, lergotrile, lisuride, metergoline, and the Sandoz ergot derivatives 25-397, 29-712, and 29-717 have been tested for their ability to inhibit the synaptic receptor binding of spiroperidol, 5-hydroxytryptamine (5-HT), d-lysergic acid diethylamide (LSD), quinuclidinyl benzilate (QNB), WB.4101, and gamma-aminobutyric acid (GABA). Only GABA binding was not affected, and QNB binding was decreased only by lergotrile and metergoline at high concentrations. The most potent inhibitors of the other ligands were bromocriptine and lisuride for spiroperidol (1-2 nMM), metergoline for 5-HT (29 nM), lisuride for LSD (15 nM), and lergotrile for WB.4101 (17 nM). The direct receptor effects of the ergot derivatives in vitro may contribute to understanding their in vivo effects on behavior and in predicting their therapeutic potential in neurological and neuroendocrine disorders.
TL;DR: Cultured oligodendrocytes showed selectivity in their synthesis of gangliosides, which was expressed in the type ofganglioside synthesized as well as in the change of incorporation over time in culture.
Abstract: Gangliosides are enriched in the nervous system compared to other tissues. The synthesis of gangliosides by monolayer cultures of isolated oligodendrocytes has not previously been investigated. Cells were labeled with (3H) galactose at preselected times and gangliosides isolated by phase partition, purified, and identified by chromatography. Cultured oligodendrocytes showed selectivity in their synthesis of gangliosides, which was expressed in the type of ganglioside synthesized as well as in the change of incorporation over time in culture. For the first ten days, there was very little incorporation of (3H) galactose in gangliosides, but this was followed by a stimulation of uptake for GM3, GM1/GD3, and GD1 gangliosides, reaching a maximum after approximately 25-30 days in vitro. There was little incorporation into GM2 or trisialogangliosides throughout the life of the cultures. Since oligodendrocytes synthesize extensive membranes during this period, one may speculate that the de novo-synthesized gangliosides are used for membranes.
TL;DR: It appears that GABA receptor development continues after eye opening in rabbits (day 9–10) and that receptor maturation is delayed by at least two to three days with respect to published reports of GABA uptake and evoked release.
Abstract: Bovine and rabbit retina synaptosomal fractions bind the potent and specific GABA agonist, [3H]-muscimol with high affinity and limited capacity. The high degree of pharmacological specificity and the subcellular distribution of these binding sites are similar to those reported for [3H]-GABA binding sites. These observations suggest that these sites represent the recognition sites for GABA receptors. The specific binding of [3H]-muscimol in retinal homogenates from different aged rabbits reveal a distinct developmental profile with a fivefold to six fold increase in binding between days 5 and 13. Thus, it appears that GABA receptor development continues after eye opening in rabbits (day 9-10) and that receptor maturation is delayed by at least two to three days with respect to published of GABA uptake and evoked release.
TL;DR: Na+‐independent taurine binding with properties corresponding to a postsynaptic interaction could not be detected in any of the regions studied and the possibility of Na+‐dependent t aurine binding, representing binding to uptake sites or to post Synaptic receptors for GABA and glycine, is discussed.
Abstract: [3H]-taurine binding to membranes from different regions from rat brain was studied. Binding to membranes from cerebral cortex and its subcellular fractions, hypothalamus, olfactory bulb and cerebellum was measured. Binding to membranes from dorsal root ganglion was also determined. Na+-dependent taurine binding was consistently observed in all the membranes except those from dorsal root ganglion. A KD = 4.06 muM was obtained for binding to membranes from cerebral cortex. Na+-dependent taurine binding was displaced by 20 muM strychnine or bicuculline. Na+-dependent taurine binding with properties corresponding to a postsynaptic interaction could not be detected in any of the regions studied. The possibility of Na+-dependent taurine binding, representing binding to uptake sites or to postsynaptic receptors for GABA and glycine, is discussed.
TL;DR: Met 5‐enkephalin blocks the active transport of GABA across plasma membranes of rabbit Deiters' neurons prepared by microdissection, being reversed by the antagonist Naloxone.
Abstract: Met 5-enkephalin blocks the active transport of GABA across plasma membranes of rabbit Deiters' neurons prepared by microdissection. Such an effect is receptor-mediated, being reversed by the antagonist Naloxone.
This result shows that Deiters' neurons bear enkephalin receptors, and suggests a hypothesis on the mechanism of enkephalin-mediated neuronal inhibition.
TL;DR: Dihydropteridine reductase was purified approximately 1,700‐fold from human outdated blood platelets and two forms of the enzyme, A and B, were resolved.
Abstract: Dihydropteridine reductase was purified approximately 1,700-fold from human outdated blood platelets. Two forms of the enzyme, A and B, were resolved. They have the same Km values for 2-amino-6,7,-dimethyl-4-hydroxydihydropteridine (46 microM vs 49 microM), but the A form has a Km for NADH that is two times higher than that of the B form (20 microM vs 9 microM).