Effect of ischaemia on protein synthesis in neuron and glia-enriched fractions from the rabbit spinal cord

The incorporation of 14C-leucine into the post-mitochondrial supernatant and neuron, glia and myelin-enriched fractions isolated from the rabbit spinal cord was studied after ischaemia and subsequent recirculation. In the cell-free system, incorporation decreased to 55% of the control value after 40 min ischaemia, but proteosynthesis returned to the pre-ischaemic value after 3 h recirculation and remained at this level during further recirculation. The incorporation of amino acids into proteins of neurons and neuroglia differed from the cell-free system and from each other. In the enriched neuronal fraction, protein synthesis fall after ischaemia and also during the first hours of recirculation, but during further recirculation it rose to 60% above the control value. In the enriched glial fraction, specific radioactivity of proteins rose abruptly immediately after ischaemia and by the fourth day there was sixfold increase as compared with control values. The results indicate that the ischaemia-induced decrease in protein synthesis is only transient and that a significant increase occurs in the surviving cell populations, especially the neuroglia. The functional changes caused by spinal cord ischaemia are irreversible, however.     

Influence of blood sera from dogs subjected to ischemia and recirculation on incorporation of14C-amino acids in vitro

Incomplete ischemia of the spinal cord was produced in dogs by 40 min occlusion of the abdominal aorta that was followed by 5–40 min of recirculation. Amino acid incorporation into ribosomes in vitro in the presence of venous blood sera was estimated. The most significant reduction in incorporation was produced by sera of the dogs following a short recirculation period (5–10 min). No significant changes were observed at the end of the ischemic period nor at longer periods of recirculation. The decrease in incorporation might be the consequence of inactivation or absence of a substance stimulating polypeptide synthesis in vitro, normally present in blood sera of intact dogs, that temporarily loses its activity during recirculation.     

Distribution of choline acetyltransferase and acetylcholinesterase in the nervous system of the dog

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity were determined in 23 selected parts of the dog CNS and 4 parts of the peripheral nervous system. Maximum ChAT activity was found in the caudate nucleus and the ventral roots of the spinal cord. High activity was also present in the thalamus, the pons, the cerebral cortex, the medulla oblongata, the ventral spinal horns and the sciatic nerve. The lowest activity was measured in the cerebellum, the dorsal cord roots and the spinal ganglia. Maximum AChE activity was found in the caudate nucleus and the cerebellum. Relatively high activity was also present in the thalamus, the pons, the medulla oblongata, the grey matter of the spinal cord and the spinal ganglia. The lowest AChE activity was measured in the ventral and dorsal spinal roots.     

Vasoactive intestinal polypeptide immunoreactivity in the spinal cord of the guinea pig

Summary The distribution of vasoactive intestinal polypeptide-immunoreactive (VIP-IR) neurons in the lower medulla oblongata and the spinal cord has been analyzed in guinea pigs. This study includes results obtained by colchicine treatment and transection experiments. In the spinal cord, numerous VIP-IR varicosities were observed in the substantia gelatinosa of the columna dorsalis; some were also found in the substantia intermedia and the columna anterior. The spinal VIP-IR nerve fibers were mainly of intraspinal origin and oriented segmentally. VIP-IR nuclei in the spinal cord extended dorsally into corresponding regions of the caudal medulla oblongata, namely from the substantia intermedia medialis and lateralis into the vagus-solitarius complex and from the nucleus spinalis lateralis into the area of the nucleus reticularis lateralis. Additional VIP-IR perikarya were observed in the pars caudalis of the nucleus spinalis nervi trigemini. The VIP-IR nuclei within the caudal medulla oblongata probably form a continuous system with those localized within the spinal cord. They may be involved functionally in the modulation of cardiovascular and respiratory regulation in the guinea pig.Supported by the DFG, Carvas SFB 90     

Impairment of protein biosynthesis in the spinal cord in ischemia and recirculation

Polysomic profiles and ultrastructure of granulated endoplasmic reticulum of the dog spinal cord as well as incorporation of labelled amino acids to cytosol proteins during ischemia and recirculation are studied. Ischemia does not evoke serious changes of the studied parameters while several minutes after ligature removal there appear pronounced changes in the ribosome distribution confirmed by electron-microscopic examination. Complete restoration of the observed parameters after the induced ischemia requires two days.     

Protein composition and synthesis in the adult mouse spinal cord

Propepties of spinal cord proteins were studied in adult mice subjected to unilateral crush or electrical stimulation of sciatic nerve. The protein composition of spinal tissue was determined using SDS-polyacrylamide gel electrophoresis coupled with subcellular fractionation. Comparisons of mouse spinal cord and brain revealed similarities in the types but differences in the concentrations of myelin associated proteins, nuclear histones and other proteins. Comparisons with sciatic nerve proteins demonstrated differences in types of proteins but similarities in the concentration of myelin proteins and nuclear histones. The short term (<2 hrs.) incorporation of radioactive amino acids into spinal cord proteins revealed heterogeneous rates of incorporation. Neither nerve crush six days prior to testing nor sciatic nerve stimulation had a significant effect on the protein composition or amino acid incorporation rates of spinal cord tissue. These observations suggest that known differences in spinal cord function following alterations in nerve input may be dependent upon different mechanisms than have been found in the brain.     

Effects of incomplete ischemia and subsequent recirculation on free palmitate, stearate, oleate and arachidonate levels in lumbar and cervical spinal cord of rabbit

The effect of severe incomplete ischemia, induced by abdominal aorta ligation for 40 minutes, and subsequent recirculation for one and four days on accumulation of free fatty acids was studies in the lumbar and cervical part of rabbit spinal cord. Changes in free fatty acid levels were determined separately in gracile fascicle (Fg), dorsal part (Dp, without Fg) and ventral part (Vp) of both spinal cord regions. In lumbar spinal cord increases in free fatty acid levels, especially that of arachidonate, were observed in Fg, Dp and Vp a the end of the ischemic period. During recirculation all values were similar to nonischemic controls. In cervical spinal cord a slight increase in free fatty acid levels was found in Fg after four days of recirculation, and in Dp arachidonate and stearate levels were most markedly elevated after one day of recirculation. No changes at any interval were found in Vp of cervical spinal cord. The present results indicate that the experimental insult induced typical ischemic injury to spinal cord tissue demonstrated by fatty acid liberation from membrane lipids. This injury may affect neurotransmission and other processes and free fatty acids themselves impair tissue metabolism (inhibition of oxidative phosphorylation, edema precipitation, synthesis of eicosanoids) and thus restrict the possibilities to enhance recovery in the recirculation period.     

Developmental study of the shortened spinal cord in the adult tiger puffer fish, Takifugu rubripes (Teleostei)

ABSTRACT The spinal cords of vertebrates are generally divided into the cord proper and the minute filum terminale. While the spinal cord extends the entire length of the vertebral canal in the adult tiger puffer, Takifugu rubripes, the cord proper is greatly reduced in length and almost all of the canal is occupied by the filum terminale, which is tape-like rather than thread-like. The dorsal and ventral roots of the spinal nerves extend, respectively, above and below the filum terminale; as a whole, these form a massive cauda equina. Supramedullary cells are found in the rostral half of the medulla oblongata caudal to the cerebellum. In 4-mm long tiger puffers, the spinal cord is cylindrical and supramedullary cells are found in the rostral half of the cord. In 7-mm puffers, the longitudinally arranged ventral roots appear ventrally in the middle portion of the spinal cord. In 15-mm puffers, the dorsal and ventral roots run longitudinally along the spinal cord and have noticeably increased in number. Supramedullary cells are located in the rostral 15% of the cord. In 21-mm puffers, the spinal cord in large part becomes dorsoventrally flattened. In 30-mm puffers, the spinal cord becomes much flatter, and supramedullary cells now are located mainly in the medulla oblongata. These observations indicate that formation of the shortened spinal cord proper is due to at least two developmental processes. First, the elongation of the spinal cord proper is remarkably less than that of the vertebral canal. Second, the bulk of the spinal cord proper is translocated to the cranial cavity, where it is transformed into part of the medulla oblongata.     

Immunohistochemistry and Biosynthesis of N-Acetylaspartylglutamate in Spinal Sensory Ganglia

N-Acetylaspartylglutamate (NAAG) is a nervous system-specific dipeptide which has been implicated in chemical neurotransmission. Antisera were prepared against NAAG in order to study its cellular distribution. When these antisera were applied to tissue sections of rat spinal sensory ganglia, NAAG-like immunoreactivity was detected within a subpopulation of relatively large neuronal cell bodies in cervical, lumbar, and thoracic ganglia. In order to confirm the presence of NAAG within these neurons, the dipeptide was extracted and purified from spinal ganglia using high-performance liquid chromatography and its composition confirmed by amino acid analysis. Further, the biosynthesis of NAAG was studied in vitro by following the incorporation of either [3H]glutamine or [3H]glutamate into the glutamate residue of the purified dipeptide. [3H]Aspartate was not incorporated efficiently into NAAG under these conditions, suggesting a precursor role for the large N-acetylaspartate pool. The incorporation of radiolabeled amino acids into newly synthesized NAAG by spinal sensory ganglia was not inhibited by incubation of the cells with anisomycin or cycloheximide at concentrations which significantly inhibited protein synthesis. These data suggest that NAAG is present in a subpopulation of primary afferent spinal neurons and that its biosynthesis is mediated by a dipeptide synthetase.     

The Posttranslational Arginylation of Proteins in Different Regions of the Rat Brain

The posttranslational incorporation of arginine into proteins catalyzed by arginyl-tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.     

GLUTAMATE AND RELATED AMINO ACIDS IN CAT SPINAL ROOTS, DORSAL ROOT GANGLIA AND PERIPHERAL NERVES

Of the free amino acids found in extracts of cat spinal roots, dorsal root ganglia and peripheral nerves, only glutamate was present in disproportionately high concentrations in those parts of the dorsal roots between ganglia and spinal cord. This distribution suggests that the high dorsal root levels of glutamate may result from synthesis in dorsal root ganglia and subsequent transport towards the spinal cord. Four excitant amino acids were detected in the extracts: aspartate, cysteate, cysteine sulphinate and glutamate. The unique regional distribution of glutamate is consistent with the proposed role of this amino acid as an excitatory transmitter at the terminals of primary afferent fibres.     

PROTEIN SYNTHESIS IN THE ISOLATED MAUTHNER NERVE FIBRE OF GOLDFISH

Abstract— Mauthner nerve fibres isolated from the spinal cord of goldfish were incubated, in the presence of radioactive amino acids for varying periods of time. It was found that the Mauthner fibre synthesizes proteins in the absence of cell nuclei. Amino acid incorporation showed sensitivity to puromycin and to acetoxycycloheximide but resistance to chloramphenicol. Only slight inhibition was caused by actinomycin-D. The contribution of the denuded axon to the total protein synthesis was about 30 per cent per unit length Mauthner fibre. The remaining activity was due to the myelin sheath compartment. Fractionation experiments showed that the incorporation in the sheath was due to components other than the myelin lamellae. The subcellular distribution of newly synthesized proteins in the isolated and incubated Mauthner fibre was compared to that found in the incubated spinal cord. The results strongly suggested the existence in the Mauthner fibre of a primary microsomal, rather than a mitochondrial, protein synthesizing system.     

Somatostatin-immunoreactive nerve cell bodies and fibers in the medulla oblongata et spinalis.

Complete serial sectioning of the medulla oblongata in monkey, cat, guinea pig, and japanese dancing mouse and incubation for somatostatin-immunoreaction was carried out. Numerous regions of the medulla oblongata such as the nucleus reticularis gigantocellularis, nucleus cuneatus et gracillis, nucleus raphe magnus, nucleus tractus solitarius, nucleus vestibularis, and parts of the oliva contain dense networks of somatostatin-immunoreactive nerve fibers. Cell bodies were seen in the nucleus reticularis medullae oblongatae. In the spinal cord the sections from each segment were analyzed, showing the highest concentrations of somatostatinergic fibers in the substantia gelantinosa of the columna dorsalis. Cell bodies were seen in the zona intermedia centralis, especially in the upper cervical segments. Many positive fibers were also seen in the entire zona intermedia and the columna ventralis. Especially prominent was the immunoreactivity in the zona intermediolateralis of the thoracic segments and the columna ventralis of the lower lumbar and sacral segments.     

Encephalomyelitis in mice experimentally infected with Akabane virus.

Lesions in the central nervous system of mice, induced by intracerebral injection of Akabane virus, were observed by the fluorescent antibody technique and histological method. Fluorescent antigens were recognized in the cytoplasm of nerve cells, but were not detected exactly in any other part. Fluoresced nerve cells were distributed almost all over the central nervous system, especially in medulla oblongata and spinal cord. The appearance of fluorescent antigens was followed by histological changes. So-called Nissl's acute severe degeneration was observed in nerve cells in the area where the fluorescent antigens were distributed. Spongy foci were seen in medulla oblongata and spinal cord. Virus was recovered from brain and spinal cord, but not from any other visceral organ or blood. Akabane virus showed an affinity to nerve cells and caused primary nonpurulent encephalomyelitis when inoculated intracerebrally to mice.     

心外膜应用腺苷时c—fos在脊髓延髓和丘脑中的表达

在１２只切断两侧缓冲神经和迷走神经的麻醉大鼠,观察了心外膜应用腺苷对脊髓,延髓和丘脑ｃ－ｆｏｓ原部基因表达的影响。结果显示：心外膜应用腺苷组大鼠,动脉血压和心率无明显变化;脊髓Ｔ３节段背角,延髓巨细胞旁外侧核以及丘脑的腹后外侧核,后核,中央外侧核和束旁核等部位Ｆｏｓ蛋白样免疫阳性反应神经元显著增加;而在溶剂对照组大鼠,仅见少数ＦＬＩ细胞。     

Peptide YY-like immunoreactivity in the central nervous system of the rat

The concentration of peptide YY (PYY)-like immunoreactivity in rat brain and spinal cord was determined by radioimmunoassay. The highest concentrations were found in the cervical spinal cord (18.1 +/- 1.3 ng/g, mean +/- S.E.M.) and in the medulla oblongata (16.3 +/- 1.5 ng/g). Lower amounts were found in the pons and in the hypothalamus. Chromatographic analysis of the PYY-like immunoreactivity from various regions of the brain revealed 95% of the immunoreactive material to be indistinguishable from synthetic porcine PYY. PYY-immunoreactive nerve cell bodies could be demonstrated by immunocytochemistry in the medulla oblongata of colchicine-treated rats, the largest group of cells being found in the midline area between and partly in the raphe pontis and obscurus nuclei. Another large group of immunoreactive cells was detected more laterally in the medial parts of the gigantocellular reticular nucleus. A few cells, finally, were seen in the dorsal parts of the medulla, including the nucleus of the solitary tract. Varicose nerve fibers displaying PYY immunoreactivity were observed in many parts of the hypothalamus, pons, medulla and spinal cord.     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

Histoenzmological mapping of ATPase and 5-nucleotidase in the spinal cord and medulla oblongata of hedgehog (Paraechinus micropus)

The contribution presented deals with the distribution of adenosine triphosphatase (ATP-A) and 5-nucleotidase (AMP-A) in the spinal cord and medulla oblongata of hedgehog. The highlights of this study are: (1) AMP-A activity is stronger in neuropil than in neurons, in all the areas of spinal cord and medulla oblongata. In the nerve cells the enzyme is localized at the peripheries of the neurons, whereas the cytoplasm and nuclei are completely free from enzymatic activity. Reaction in blood vessels is quite high both in gray and white matter. (2) ATP-A activity is seen mainly at the peripheries of the neurons. The neuropil activity varies from mild to intense. Reaction in blood vessels is quite strong in all the areas. (3) Fibrous bundles and tracts are negative for both the enzymes. (4) In general, the activity of ATP-A and AMP-A is strongest in cranial nerve nuclei, irrespective of their sensory or motor nature. The distribution of these enzymes has been correlated with the functions of various nuclei of spinal cord and medulla oblongata in hedgehog, and compared with other mammals.     

An orthograde labelling study of axons of the dorsal motor nucleus of the vagus to rat cardiac ganglia as demonstrated by autoradiography

Central organization of the cardiac vagus has not been clarified. Retrograde changes produced in medulla oblongata neurons after section of vagal branches has favored the dorsal motor nucleus of the vagus (DMNX). Current information concerning the origin, course, and termination of vagal preganglionic fibers within cardiac ganglia is conflicting. The explicit purpose of this study was to determine if vagal fibers originated specifically within the DMNX proper. Fibers within the cardiac ganglia were labelled with 3H-leucine following injection into the DMNX. 12 adult albino rats were studied. DMNX were injected with 25 microCi 3H-leucine reconstituted to microliter. Animals were sacrificed by transcardial perfusion following a 4-day survival period. Serial cross-sections of the caudal pons, medulla oblongata, and thoracic viscera were processed for autoradiography. DMNX possessed a heavy incorporation of the radiochemical. Label was observed within the axons of the vagi. Cardiac ganglia contained labelled vagal fibers in close proximity to the postganglionic somata. Cardiac ganglia containing labelled preganglionic vagal axons were located in the cardiac plexuses and in the epicardium. Results show a labelled vagal preganglionic input to cardiac ganglia from the DMNX.     

Changes in the protein composition of the synaptic structures of the brain of rats in tetanus intoxication]

As shown by the method of electrophoresis on polyacrylamide gel, the number of proteins with low electrophoretic mobility proved to be increased in the triton extract fractions of synaptic structures isolated from spinal cord of rats with local tetanus; no changes in the protein spectrum were revealed in the dodecyl-sulphate extract. In vitro tetanus toxin stimulated the lysin-H3 incorporation into the total proteins of synaptosomes of rat brain cortex.