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.     

Effects of dorsal root section and occlusion of dorsal spinal artery on the neurotransmitter candidates in rat spinal cord

In order to obtain further evidence of putative neurotransmitters in primary sensory neurons and interneurons in the dorsal spinal cord, we have studied the effects of unilateral section of dorsal roots and unilateral occlusion of the dorsal spinal artery on cholinergic enzyme activity and on selected amino acid levels in the spinal cord. One week after sectioning dorsal roots from caudal cervical (C₇) to cranial thoracic (T₂) levels, the specific activity of choline acetyltransferase (ChAT) was significantly decreased and acetylcholinesterase (AChE) showed a tendency to decrease in the dorsal quadrant on the operated side of the spinal cord. Dorsal root sectioning had little effect on the levels of free glutamic acid or other amino acids in the dorsal spinal cord. These results suggest that primary sensory neurons may include some cholinergic axons, and that levels of putative amino acid transmitters are not regulated by materials supplied by axonal transport from the dorsal root ganglia. By contrast, one week following unilateral occlusion of the dorsal spinal artery, the activities of ChAT and AChE were unchanged in the operated quadrant of the spinal cord, while decreases of Asp, Glu, and GABA, and an increase in Tau were detected. These findings are consistent with the proposals that such amino acids, but not ACh, may function as neurotransmitter candidates in interneurons of the dorsal spinal cord.Abbreviation used ACh acetylcholine - AChE acetylcholinesterase - Asp aspartic acid - ChAT choline acetyltransferase - GABA -aminobutyric acid - Glu glutamic acid - Gly glycine - SP substance P - Tau taurine     

Glutamic acid as a synpatic transmitter candidate in the dorsal sensory neuron: reconsiderations.

Two primary reasons which are emerging to suggest that glutamate is not a dorsal root transmitter are: 1) that free glutamate levels in the dorsal root vs. the ventral root are not sufficiently different to warrant a transmitter function in the dorsal root, and 2) the spinal cord glutamate levels do not significantly change (per g tissue) after dorsal root input section. Recent analyses suggest, however, that there is a highly significant depression of glutamate in the dorsal root when related to total free amino acid concentration changes after root injury. This is not seen in the peripheral nerve. Thus the dorsal vs. ventral root free glutamate concentration difference is highly significant metabolically. The failure to see a decrease in spinal cord gray glutamate levels after dorsal root section would appear to be explained by the fact that the spinal cord satellite cells and neurons have a higher free glutamate concentration than the entering dorsal roots along with a considerable perikaryal free amino acid pool for protein synthesis. This will mask any changes due to dorsal root section.Comparisons of excess free glutamate and substance P (the two leading dorsal root transmitter candidates) in the dorsal root compared to the ventral root have shown that there is a much larger excess of free glutamate in the dorsal root. This is true, even when considering the excitatory potency differences of these two substances. Thus, a very large free glutamate excess in the dorsal root is present with a relatively small concentration difference compared to the ventral root (where a transmitter role is not entertained). This fact could be of considerable metabolic significance in the regulation of transmitter levels of glutamate. The data available, therefore, are supportive of a possible glutamate transmitter role in a population of dorsal root fibers.     

QUANTITATIVE HISTOCHEMISTRY OF γ-AMINOBUTYRIC ACID IN CAT SPINAL CORD WITH SPECIAL REFERENCE TO PRESYNAPTIC INHIBITION

Abstract— A method is described for quantifying the GABA distribution in cat spinal cord at 200–500 μn resolution. Isolated spinal cord (L5–S1) was frozen and sectioned at about 150 μm thickness. The frozen tissue section was cut into 200 or 500 μm square blocks. The GABA content of each square tissue block was determined by enzymic micromethods and GABA distribution was mapped quantitatively. Average GABA concentrations were: 0·4 mmol/l. in white matter, 1·2 mmol/l. in ventral horn and 1·7 mmol/l. in dorsal horn. The highest concentrations of GABA (2–3 mmol/l.) were found in the dorsolateral part of dorsal horn. In order to destroy the interneurons of dorsal horn, the blood vessels supplying the dorsal horn of the lumbar enlargement were unilaterally cauterized. Seven to 30 days after operation, both the size of dorsal root potential and the GABA level in the dorsal horn were markedly reduced on the cauterized side. These results suggest that GABA is highly concentrated in the interneurons of dorsal horn and functions as a transmitter of presynaptic inhibition.     

AMINO ACID AND SUBSTANCE P CONTENTS IN SPINAL CORD OF CATS WITH EXPERIMENTAL HIND-LIMB RIGIDITY PRODUCED BY OCCLUSION OF SPINAL CORD BLOOD SUPPLY

Abstract— Experimental hind-limb rigidity of spinal origin was produced in cats by temporary occlusion of thoracic aorta and internal mammary arteries. In the lumbar segments (L6- S1) of these rigid cats, the monosynaptic reflex recorded from ventral roots was enhanced whereas the polysynaptic reflexes as well as the dorsal root reflexes were almost abolished. On morphological examination of the lumbar spinal cord, the number of interneurons was greatly reduced, whereas the small sized cells, presumably glial cells, were increased by about two times. Ventral horn motoneurons were also reduced. The lumbar spinal cords of the rigid cats were analysed for amino acid and substance P contents. Four major amino acids, aspartate, glutamate, glycine and GABA, were definitely reduced in both grey and white matter except that the glutamate level in the dorsal white was within the normal range. Content and distribution pattern of substance P were not altered in the lumbar cord of the rigid cats. These results are consistent with the notions that GABA occurs in the dorsal horn interneurons subserving primary afferent depolarisation, and that substance P is concentrated in primary afferent fibre terminals. The implications of the decrease of aspartate, glutamate and glycine in the spinal cord of rigid cats are discussed.     

DISTRIBUTION OF AMINO ACIDS AND OF EXO- AND ENDOPEPTIDASES ALONG VERTEBRATE AND INVERTEBRATE NERVES

Abstract— The proximo-distal gradients for representative peptidases, peptidylpeptide hydrolases, and amino acids were measured in segments of peripheral nerve from invertebrates and vertebrates and in the lobster brain and ventral cord.
Crustacean nerve was characterized by a large pool of free amino acids totaling 100–200 μmoles/g wet wt. In lobster nerve, the principal free amino acid was aspartic acid which comprised 55 per cent of the free pool, whereas in the rat sciatic nerve it comprised only 5 per cent. The principal free amino acid in rat sciatic nerve was taurine (32 per cent of the pool) and in lobster brain glycine comprised 30 per cent of the pool. No consistent patterns emerged for the gradients along the nerves for amino acids and hydrolytic enzymes. In the leg nerve of the lobster, concentrations of aspartic acid and arginine were higher in the proximal region, and concentrations of proline and alanine were higher in the distal region. Concentrations of most amino acids were higher in the proximal regions of crab nerve, of lobster brain and ventral cord, and of rat sciatic nerve.
Rat sciatic nerve exhibited a pronounced proximo-distal increase in activity of aminopeptidase (Leu-Gly-Gly). In lobster leg nerve, activity of neutral proteinase was higher in the proximal segment, whereas activity of acid proteinase was higher in the distal segment. The best examples of proximo-distal gradients were found in lobster brain and ventral cord; activities of endopeptidases, arylamidases (Leu- and Arg-βNA), and aminopeptidase were higher in the supra-esophageal ganglia or cephalothorax segments than in the distal regions.     

FAST AXONAL TRANSPORT IN VITRO IN THE SCIATIC SYSTEM OF THE FROG

Abstract— An in vitro system from the frog has been used to study fast axonal protein transport. The preparation, which was incubated in a specially made chamber, consisted of the gastrocnemius muscle, the sciatic nerve, the dorsal ganglia and part of the spinal cord. The parts were separated from each other by silicone grease barriers, which made it possible to follow the migration of labelled proteins from the spinal cord and ganglia, along the sciatic nerve, towards the muscle. About 80 per cent of transported proteins in the sciatic nerve originated from the dorsal spinal ganglia and moved antidromically at a rate of 60–90 mm per day at 18°C. The rapidly transported proteins were 90 per cent particulate and mainly associated with structures sedimenting in the microsomal fraction.
The effects of cyclohexirnide showed that the synthesis of rapidly moving proteins and their transport were separate processes. A low concentration of colchicine inhibited the transport when it was present in the medium surrounding the ganglia, but had no effect even at a higher concentration, when it was added to the nerve compartment. The presence of vinblastine at a low concentration in either of the two compartments completely arrested the protein transport. Likewise N-ethylmaleimide or p-chloromercuribenzene sulphonic acid in the nerve medium effectively blocked the fast transport. Results from experiments performed to test the possibility of disto-proximal flow and of transfer of proteins from the muscle to the nerve are discussed.     

THE DISTRIBUTION AND AXONAL TRANSPORT OF FREE AMINO ACIDS AND RELATED COMPOUNDS IN THE DORSAL SENSORY NEURON OF THE RAT, AS DETERMINED BY THE DANSYL REACTION

A method is reported for the quantitative analysis of free amino acids and related substances by the dansyl reaction, and the technique has been used for the analysis of tissues of the dorsal sensory neuron of the rat. A comparison of dorsal and ventral roots revealed no major qualitative differences, but glutamate and the very much less abundant amino acids, threonine and arginine occurred in significantly higher concentrations in dorsal roots as compared with ventral roots. After an 8 h period of dorsal root ligation in vivo, an apparently selective accumulation of alanine, glutamate, glycine and tyrosine occurred. These findings are compatible with the postulated transmitter role of glutamate at the terminals of primary afferent fibres and may indicate its subsequent transport towards the cord after synthesis in the ganglion. After the injection of [¹⁴C]glucose into the dorsal root ganglion, no rapid transport of any radiolabelled material along the axon could be detected. This finding is discussed in relation to current knowledge of the metabolic pathways involved in the synthesis of glutamate and related amino acids.     

Induction of locomotion in spinal tadpoles by excitatory amino acids and their agonists

Bath application of the excitatory amino acids L-aspartate and/or L-glutamate or their agonists N-methyl-D,L-aspartate and/or kainate elicited swimming movements in spinal tadpoles. Swimming cycles induced by the amino acids were in the frequency range of natural movements, and could be evoked after sectioning all dorsal roots in the exposed spinal segments. Locomotion was only elicited by L-aspartate or L-glutamate at low concentrations when the bath medium was rapidly circulated over the exposed surface of the spinal cord, and was of much shorter duration than the agonist-induced movements. These results indicate some differences between the actions of L-aspartate and L-glutamate and their agonists on the tadpole spinal cord.     

The action of quinolinate in the rat spinal cord in vitro

The responses of dorsal horn neurones to the excitatory amino acids quisqualate, kainate, N-methyl-D-aspartate (NMDA), and quinolinate have been examined in an in vitro preparation of the rat spinal cord. The antagonism of these responses by iontophoretically applied D-(-)-2-amino-5-phosphonovalerate (DAPV), kynurenate, and acridinate was tested, and the results were compared with data obtained from the spinal cord in vivo. The pattern of antagonism was similar in both preparations, although the potencies of agonists and antagonists were found to be significantly greater in vitro. The antagonism of amino acid induced firing of neurones was also recorded during the application of DAPV and kynurenate in the bathing medium. Dose-response curves and IC50 values were determined for these antagonists against all four agonists. The responses to quinolinate were antagonized differently from those to NMDA, quisqualate, or kainate, suggesting that quinolinate does not act specifically through the NMDA receptor as it does in other regions, nor does it appear to act via two or more of the three archetypal amino acid receptors. These findings suggest that a fourth amino acid receptor responsible for quinolinate's action in the spinal cord may exist.     

Specific effects of alpha-D,L-aminoadipic acid on synaptic transmission in frog spinal cord

The sucrose gap technique was employed to investigate both synaptic and amino acid evoked responses from motoneurones or primary afferents of frog spinal cord. alpha-D,L-Aminoadipic acid (alpha-D,L-AAD) selectively antagonized responses to acidic amino acids, especially aspartate. The drug was most effective in antagonizing the polysynaptic components of synaptic potentials evoked by dorsal root or lateral column stimulation but had little effect on their monosynaptic components. The ventral root dorsal root potential which is thought to be mediated by a pathway that does not involve acidic amino acids was insensitive to alpha-D,L-AAD. These data, which were confirmed by intracellular recording from motoneurones, provided further evidence for the role of acidic amino acids in polysynaptic pathways in frog spinal cord.     

离体运动神经元对腹外侧索刺激的突触反应特征

应用新片大鼠脊髓薄片运动神经元细胞内记录技术,对电刺激腹外侧索诱发的突触反应进行了电生理特性分析。结果在２８个测试的ＭＮ中,２２人有兴奋性突触后电位反应,其中２个跟随在抑制性突触反应这后,６个还对单或串刺激产生慢ＥＰＳＰ反应;ＶＬＦ－ＥＰＳＰ的潜伏期频数分布呈峰坡性偏态;同－ＭＮ的ＶＬＦ－ＥＰＳＰ与腹根ＥＰＳＰ间有典型的空间总和。     

Effect of Impact Trauma on Neurotransmitter and Nonneurotransmitter Amino Acids in Rat Spinal Cord

N-Methyl-D-aspartate (NMDA) administration exacerbates neurological dysfunction after traumatic spinal cord injury in rats, whereas NMDA antagonists improve outcome in this model. These observations suggest that release of excitatory amino acids contributes to secondary tissue damage after traumatic spinal cord injury. To further examine this hypothesis, concentrations of free amino acids were measured in spinal cord samples from anesthetized rats subjected to various degrees of impact trauma to the T9 spinal segment. Levels of excitatory and inhibitory neurotransmitter amino acids [gamma-aminobutyric acid (GABA), glutamate, aspartate, glycine, taurine] and levels of nonneurotransmitter amino acids (asparagine, glutamine, alanine, threonine, serine) were determined at 5 min, 4 h, and 24 h posttrauma. Uninjured surgical (laminectomy) control animals showed modest but significant declines in aspartate and glutamate levels, but not in other amino acids, at all time points. In injured animals, the excitatory amino acids glutamate and aspartate were significantly decreased by 5 min posttrauma, and remained depressed at 4 h and 24 h as compared with corresponding laminectomy controls. In contrast, the inhibitory amino acids, glycine, GABA, and taurine, were decreased at 5 min postinjury, had partially recovered at 4 h, and were almost fully recovered at 24 h. The nonneurotransmitter amino acids were unchanged at 5 min posttrauma and significantly increased at 4 h, with partial recovery at 24 h. At 4 h postinjury, severe trauma caused significantly greater decreases in aspartate and glutamate than did either mild or moderate injury. These findings are consistent with the postulated role of excitatory amino acids in CNS trauma.     

THE DISTRIBUTION OF GLYCINE, GABA, GLUTAMATE AND ASPARTATE IN RABBIT SPINAL CORD, CEREBELLUM AND HIPPOCAMPUS

The distribution of glycine, GABA, glutamate and aspartate was measured among about 60 subdivisions of rabbit spinal cord, and among the discrete layers of cerebellum, hippocampus and area dentata. A more detailed mapping for GABA was made within the tip of the dorsal horn of the spinal cord. Spinal ventral horn and dorsal root ganglion cell bodies were analyzed for the amino acids and for total lipid. The distribution of lipid and lipid-free dry weight per unit volume was also determined in spinal cord. Calculated on the basis of tissue water, glycine in the cord is highest in lateral and ventral white matter immediately adjacent to the ventral grey. The distribution of GABA is almost the inverse of that of glycine with highest level in the tip of dorsal horn. It is most highly concentrated in the central 75% of Rexed layers III and IV. Aspartate in the tip of ventral horn is 4-fold higher than in the tip of the dorsal horn and 3 times the average concentration in brain. Glutamate was much more evenly distributed and is relatively low in concentration with slightly higher levels in dorsal than in ventral grey matter. Large cell bodies in both ventral horn and dorsal root ganglion contained high levels of glycine. As reported by others, GABA was found to be high in cerebellar grey layers, area dentata, and regio inferior of hippocampus. Glycine was moderately high in cerebellar layers but moderate to low in hippocampus and area dentata.     

Postnatal changes in the dorsal root reflex in the isolated spinal cord of the hamster,Mesocricetus auratus

Spontaneous activity has been demonstrated in the lumbar dorsal roots of isolated spinal cord preparations taken from animals ranging in age from 2 to 65 days. Peaks of activity were recorded at 2 and 5 weeks of age, with mean firing frequencies of 33 Hz and 28 Hz respectively. The firing frequency in weeks 3 and 4 was lower (15 Hz) as was the frequency in cords taken from animals older than 6 weeks. The pattern of the spontaneous dorsal root activity changed during the first 5 weeks of life. In cords taken from animals less than 10 days old, the roots fired single action potentials, producing a single broad peak in Inter Spike Interval plots (ISI). Dorsal root recordings made from cords taken from animals in weeks 2 and 3 of life exhibited both single spikes and bursts of action potentials. By the end of the third week of life, individual spike activityhad declined and the bursts of action potentials characteristic of the adult pattern had become dominant, producing a bimodal ISI plot. Cross correlation analysis of dorsal root and dorsal horn activity in lumbar segments up to five segments apart, revealed an increasing degree of correlation developing over the first 4 weeks of postnatal life. Dorsal horn responses to dorsal root stimulation in cords taken from young animals were prolonged, lasting in excess of 250 msec. In the third week of life, the duration of the excitatory component of the response was reduced to approximately 50 msec by the development of an inhibitory phase.     

Demonstration of Sensory-Motor Innervation of Postmetamorphic Forelimb Regenerates of Triturus Alpestris (Urodela) on Cryotome Serial Sections Using Horseradish Peroxidase

A method has been developed to obtain horseradish peroxidase-treated serial sections containing spinal cord as well as bilateral ventral and dorsal roots, dorsal root ganglia and spinal nerves. Young postmetamorphic newts (Triturus alpestris) served as experimental animals. After cryotome cross sectioning the forelimb region of the trunk, slices 80 μm in thickness were mounted serially with up to 15 sections per slide. This facilitated subsequent staining manipulations and made partial loss of sections less likely.     

THE EFFECT OF HYPOCHOLESTEREMIC AGENTS ON MYELINOGENESIS

Abstract— Three drugs known to inhibit biosynthesis of cholesterol, Clofibrate, 20, 25-diazacholesterol and AY-9944 were administered by stomach intubation to suckling rats. At weaning the rats were killed and subcellular fractions, including myelin, were prepared from the brains and spinal cords and analysed for sterol content. Central nervous tissue fractions from Clofibrate-treated rats showed some decrease in total sterols, but the sterol species were qualitatively normal. AY-9944 given to rats caused high amounts of 7-dehydro-cholesterol to accumulate in all brain and spinal cord fractions with the highest amounts (32–38 percent of total sterols) in myelin. In diazasterol-treated rats desmosterol reached 48 per cent of the sterols of myelin. A group of rats was allowed to survive after the final drug intake (21 days) and their brain and spinal cord sterol content followed up to 60 days. At 30 days the proportion of dehydrocholesterol or desmosterol comprised over half the total myelin sterol. By 60 days of age the 7-dehydrocholesterol had almost completely disappeared from all fractions while substantial amounts of desmosterol were retained in myelin. Myelination was retarded by treatment with AY-9944 and 20, 25-diazasterol, possibly by the limited amount of sterols available. The metabolism of the abnormal myelin constituents in drug-treated animals is discussed in relation to the molecular structure of the myelin membrane.     

Demonstration of sensory-motor innervation of postmetamorphic forelimb regenerates of Triturus alpestris (Urodela) on cryotome serial sections using horseradish peroxidase

A method has been developed to obtain horseradish peroxidase-treated serial sections containing spinal cord as well as bilateral ventral and dorsal roots, dorsal root ganglia and spinal nerves. Young postmetamorphic newts (Triturus alpestris) served as experimental animals. After cryotome cross sectioning the forelimb region of the trunk, slices 80 microns in thickness were mounted serially with up to 15 sections per slide. This facilitated subsequent staining manipulations and made partial loss of sections less likely.     

POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2-OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10-day-old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1- and 10-day-old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2-oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10-day-old rats suggests that the activity of this enzyme reflects cell growth rate.     

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.