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.     

Distribution of some enzymes associated with the metabolism of glutamate, aspartate, gamma-aminobutyrate and glutamine in cat spinal cord

The activities of glutamine synthetase, glutaminase, glutamate decarboxylase, GABA aminotransferase, glutamate dehydrogenase, and aspartate aminotransferase were measured in four areas of the cat spinal cord and in dorsal and ventral roots. Five of the six enzymes showed identical distribution patterns; i.e. the activities in the dorsal and ventral gray matter were equal and those of dorsal and ventral white matter were equal. No statistical differences in the mean enzyme activities in the dorsal and ventral roots were found. Glutamate decarboxylase was the only enzyme which had a different pattern. The enzyme activity in dorsal gray was twice that of ventral gray; the same pattern as the GABA concentration in both these areas. The glutamine synthetase activities in the cord areas and roots correlated with the glutamine distribution reported earlier. Thus, the distribution of glutamine (not a transmitter) and GABA (questionable transmitter) in gray matter are dictated by their synthesizing enzymes, whereas the distribution of glutamate and aspartate (likely transmitter suspects) cannot be explained on the basis of enzyme activities. Therefore, the enzyme activities may be related to the amino acid levels primarily in metabolic compartments, whereas the excess of certain amino acids in specific areas of the cord and roots may be related to functional compartments accumulated for use in synaptic transmission.     

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.     

Hypothalamic substance P as a candidate for transmitter of primary afferent neurons.

A peptide that exerts a depolarizing action on frog spinal motoneurons was found in the dorsal root of bovine spinal nerve. Pharmacological, chemical, and immunological properties of this motoneuron-depolarizing peptide were investigated and the results indicated that the peptide is identical with an undecapeptide, substance P, recently isolated from bovine hypothalamus by M.M.Chang and S.E.Leeman. The amount of hypothalamic substance P in bovine dorsal root determined by bioassay or radioimmunoassay was 24-130 pmole/g wet wt, whereas that in the ventral root was 9-27 times less. The effects of synthetic hypothalamic substance P on the isolated spinal cord of the frog and the newborn rat were studied. The peptide exerted a powerful depolarizing action on the motoneurons, its potency being about 200 times higher than that of L-glutamate. Distribution of substance P in the cat spinal cord was studied. The concentration of the peptide was highest in the dorsal part of dy lowered. When the dorsal root of the cat was ligated, substance P accumulated in a high concentration on the ganglion side of the ligature. These results, taken together, support the hypothesis that hypothalamic substance P is an excitatory transmitter of primary afferent neurons.     

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.     

Locomotion induced by spinal cord stimulation in the neonate rat in vitro.

The present studies employed the neonate rat brain stem-spinal cord preparation to determine whether electrical stimulation of the lumbosacral enlargement (LE) of the spinal cord itself can be used to elicit locomotion, and whether or not such stimulation persists in inducing locomotion following midthoracic spinal cord transection or hindlimb deafferentation. Results suggest that (1) stimulation of the dorsal columns or ventral funiculus of the LE is effective in inducing airstepping in the neonatal rat brain stem-spinal cord limb-attached preparation; (2) central disconnection by midthoracic spinal cord transection does not alter LE-stimulation-induced airstepping and may lead to an increase in stepping frequency if suprathreshold stimulation is used; and (3) dorsal root section also leads to an increase in the frequency of suprathreshold LE-stimulation-induced locomotion, but there is not further increase in frequency if a spinal cord transection is performed in addition to dorsal rhizotomy.     

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     

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.     

Cholinergic modulation of the synaptic transmission in the frog spinal cord

It was found during experiments on isolated frog spinal cord involving extracellular recording from the dorsal roots (sucrose bridging) and intracellular recording from motoneurons by microelectrodes that 10 mM of the M-cholinomimetic arecoline produces motoneuronal depolarization which is matched by depolarizing electronic ventral root potentials and a rise in motoneuronal input resistance. Arecoline changes synaptic transmission by increasing the amplitude of postsynaptic potentials during intracellular recording and that of motoneuronal reflex discharges in the ventral roots but reduces the duration of dorsal root potentials. In the presence of arecoline, L-glutamate-induced motoneuronal response increases. Facilitation of synaptic transmission produced by arecoline in the spinal cord is bound up with cholinergic M₂- activation, since it is suppressed by atropine but not by low concentrations of pirenzipine; it is also coupled with a reduction in adenylcyclase activity. When motoneuronal postsynaptic response has been suppressed, as in the case of surplus calcium or theophylline, arecoline produces an inhibitory effect on the amplitude of motoneuronal monosynaptic reflex discharges which is suppressed by pirenzipine at a concentration of 1×10^–7 M. This would indicate the presence at the primary afferent terminals of presynaptic cholinergic M₁ receptors which mediate its inhibition of impulses of transmitter release. This effect is independent of changes in cyclic nucleotide concentration.A. M. Gorkii Medical Institute, Donetsk. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 399–405, May–June, 1987.     

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.     

Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord

Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro-caudally, somatostatin content was about 50% higher in lumbar-sacral cord than in cervical or thoracic levels. The dorso-ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met-enkephalin and substance P were also measured after the above surgical manipulations. Met-enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the origin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.     

Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies.

In order to analyse the spinal tract formation at early stages of development in avian embryos, chick-quail spinal cord chimeras were prepared and species-specific monoclonal antibodies (MAb) were developed. MAbs CN, QN and CQN uniquely stained chick, quail, and both chick and quail nervous tissues, respectively. All three antibodies appeared to bind to the same membrane molecule, but to different epitopes. Cord reversal revealed the features of axonal growth of both cord interneurons and dorsal root ganglion cells. Quail cord interneurons grew along an originally ventral marginal layer in the quail cord transplanted in a reversed position, then turned toward the ventral side at the boundary between the graft and the host, and grew along the host chick ventral marginal layer. Central axons of dorsal root ganglia were restricted to the ventrolateral region of the cord which originally formed the dorsal funiculus. These results suggest that cord interneurons and dorsal root ganglion cells actively select to grow along specific regions of the cord and that spinal tract formation appears to be determined by cord cells, and not by sclerotome cells.     

The actions of serotonin on frog primary afferent terminals and cell bodies

The actions of serotonin (5-HT) were studied in the isolated frog spinal cord and dorsal root ganglion preparations. In the spinal cord, 5-HT increased the spontaneous activity recorded from dorsal roots, facilitated evoked spinal reflexes and produced fast and slow primary afferent depolarization (PAD). A direct action of 5-HT on primary afferent terminals is likely since 5-HT induced PAD remained in the presence of 1 microM tetrodotoxin and 2 mM Mn2+. The direct action of 5-HT on primary afferent terminals was blocked by methysergide and attenuated by concentrations of Mn2+ in excess of that required to block transmitter release. Cell bodies of the dorsal root ganglion were also depolarized by 5-HT. A slow hyperpolarization occasionally followed the initial depolarization. The depolarizing action of 5-HT in the dorsal root ganglion was also attenuated by treatment with Mn2+. It is concluded that 5-HT acts directly on frog primary afferents and that this influence may involve a calcium sensitive process. The dorsal root ganglion response to 5-HT appears to be a suitable model of the afferent terminal response.     

THE EFFECT OF CHRONIC DORSAL ROOT SECTION ON THE CONCENTRATION OF FREE AMINO ACIDS IN THE RABBIT SPINAL CORD

Abstract— Amino acids may be involved in primary afferent excitatory neurotransmission in the spinal cord. To test this possibility the effect of chronic dorsal root section on amino acid levels of the rabbit spinal cord has been investigated. Dorsal roots L6-S2 were sectioned under anaesthesia. Control animals were subjected to similar surgical procedures but the dorsal roots were left intact. Electromyogram recordings taken 6 days after surgery confirmed the absence of sensory input to the lower lumbosacral cord of dorsal root sectioned animals although motor function was retained. In contrast to this control animals exhibited normal reflex activity. The spinal cord was removed from each animal and extracted in trichloracetic acid for subsequent analysts of amino acids on an autoanalyser. Sections of cord were retained for histological determination of neuronal degeneration. Comparison of amino acid levels in dorsal root sectioned and control animals revealed that the only excitatory amino acid to be significantly reduced by dorsal root section wasaspartic acid (–50 percent X although glutamic acid was also reduced (– 30 per cent). Two inhibitory amino acids, cystathionine and GABA, were also significantly depleted (– 50 and - 35 per cent). The possible involvement of these amino acids in spinal cord neurotransmission is discussed.     

谷氨酸转运抑制剂对器官型培养脊髓片的影响

观察谷氨酸转运体抑制剂苏一羟天冬氨酸(Threo-hydroxyaspartate,THA)对器官型培养的脊髓片的影响,探讨谷氨酸在运动神经元损伤中的作用。取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,在培养液中加入不同浓度THA(50μmol／L、100μmol／L、5001μmol／L),用神经元的特异性免疫组化染色剂SMI-32,非磷酸化神经丝标记物,对脊髓腹角α运动神经元进行鉴定,用单克隆抗钙网膜蛋白(calretinin)抗体对背角中间神经元进行记数,测定培养液中乳酸脱氢酶(LDH)的含量,并与对照组比较。结果显示对照组α运动神经元数目恒定,THA可以引起剂量依赖性的培养液中LDH含量增高和α运动神经元数目减少,而脊髓背角的中间神经元损伤相对较轻,其中THA100μmol／L组在体外培养4周后出现类似于肌萎缩侧索硬化(ALS)的病理改变：α运动神经元数目较对照组明显减少,而脊髓背角的中间神经元数目无显著变化。细胞外谷氨酸增高主要对运动神经元造成损伤,脊髓运动神经元较感觉神经元对谷氨酸的兴奋毒作用更加敏感。     

Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat

Immunohistochemical and radioimmunoassay studies revealed that both CGRP- and SP-like immunoreactivity in the caudal spinal trigeminal nucleus and tract, the substantia gelatinosa and the dorsal cervical spinal cord as well as in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglion is markedly depleted by capsaicin which is known to cause degeneration of a certain number of primary sensory neurons. Higher brain areas and the ventral spinal cord were not affected by capsaicin treatment. Furthermore CGRP and substance P-like immunoreactivity were shown to be colocalized in the above areas and to coexist in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglia. It is suggested that CGRP, like substance P, may have a neuromodulatory role on nociception and peripheral cardiovascular reflexes.     

Dissymmetry of the origins of the roots of the spinal cord in the dog

The spinal cord in 25 non-inbred dogs has been studied macro-microscopically. The dissymmetry in the arrangement level in the right and left root bases on the dorsal surface of the spinal cord is much greater than on the ventral surface. The same as in the human being, the dissymmetry is the greatest in the thoracic part (as compared to other spinal parts). On the ventral surface of the spinal cord both along the anterior and posterior margin of the root bases, there is a right-sided dissymmetry (with cranial shift); on the dorsal surface it is present only at the roots along the posterior margin. The dissymmetry of the dog spinal cord is quantitatively estimated along its whole extension.     

Dorsal spinal cord inhibits oligodendrocyte development

Oligodendrocytes are the myelinating cells of the mammalian central nervous system. In the mouse spinal cord, oligodendrocytes are generated from strictly restricted regions of the ventral ventricular zone. To investigate how they originate from these specific regions, we used an explant culture system of the E12 mouse cervical spinal cord and hindbrain. In this culture system O4(+) cells were first detected along the ventral midline of the explant and were subsequently expanded to the dorsal region similar to in vivo. When we cultured the ventral and dorsal spinal cords separately, a robust increase in the number of O4(+) cells was observed in the ventral fragment. The number of both progenitor cells and mature cells also increased in the ventral fragment. This phenomenon suggests the presence of inhibitory factor for oligodendrocyte development from dorsal spinal cord. BMP4, a strong candidate for this factor that is secreted from the dorsal spinal cord, did not affect oligodendrocyte development. Previous studies demonstrated that signals from the notochord and ventral spinal cord, such as sonic hedgehog and neuregulin, promote the ventral region-specific development of oligodendrocytes. Our present study demonstrates that the dorsal spinal cord negatively regulates oligodendrocyte development.     

Ionotropic Glutamate Receptors in Spinal Nociceptive Processing

Glutamate is the predominant excitatory transmitter used by primary afferent synapses and intrinsic neurons in the spinal cord dorsal horn. Accordingly, ionotropic glutamate receptors mediate basal spinal transmission of sensory, including nociceptive, information that is relayed to supraspinal centers. However, it has become gradually more evident that these receptors are also crucially involved in short- and long-term plasticity of spinal nociceptive transmission, and that such plasticity have an important role in the pain hypersensitivity that may result from tissue or nerve injury. This review will cover recent findings on pre- and postsynaptic regulation of synaptic function by ionotropic glutamate receptors in the dorsal horn and how such mechanisms contribute to acute and chronic pain.     

Neurochemical and Immunocytochemical Studies on the Distribution of N-Acetyl-Aspartylglutamate and N-Acetyl-Aspartate in Rat Spinal Cord and Some Peripheral Nervous Tissues

HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.