TETANUS TOXIN AND AMINO ACID LEVELS IN CAT SPINAL CORD

Abstract— —The levels of the depressant amino acids found in appreciable amounts in cord extracts—α-alanine, cystathionine, GABA, glycine and serine—were not significantly influenced by tetanus toxin. This supports the view that the antagonism of spinal inhibition by the toxin is the result of an interference with transmitter release rather than a reduction in the amount of transmitter available for release.
The marked increase in aspartic acid levels found in the spinal cord after treatment with tetanus toxin may reflect the association of aspartic acid with the increased activity of spinal excitatory interneurones or the involvement of aspartic acid as a glycine precursor in spinal tissue.     

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.     

Spinal subarachnoid perfusion in the rat: glycine transport from spinal fluid

Abstract— A method was developed for perfusion of the spinal subarachnoid space in the rat. Bidirectional steady-state fluxes of [¹⁴C]glycine between spinal fluid and plasma were measured. [¹⁴C]glycine clearance from spinal fluid was 5-fold greater than its clearance from plasma. Glycine was transported out of spinal fluid by a saturable process, and the rate of transport was unaffected by the other depressant amino acids, GABA, β-alanine, and taurine. Perfused [¹⁴C]glycine and [³H]GABA distributed in an intracellular compartment in spinal cord. The preparation should be useful for study of the release of these inhibitory amino acids from the intact spinal cord.     

UPTAKE, RELEASE AND HOMO- AND HETERO-EXCHANGE DIFFUSIONS OF INHIBITORY AMINO ACIDS IN GUINEA-PIG CEREBELLAR SLICES

Abstract— GABA, taurine and β-alanine are taken up by guinea-pig cerebellar slices by both the high-and low-affinity uptake processes, whereas glycine is taken up only by the low-affinity process. A considerable amount of labelled GABA loaded in the slice is released by unlabelled external GABA and a minute amount is released by external β-alanine, glycine and taurine. External glycine and β-alanine releases labelled glycine loaded in the slice. Labelled taurine loaded is effectively released by external taurine and β-alanine, while labelled β-alanine loaded is released only by external β-alanine.
It is suggested that hetero-exchanges which are one-directional in some cases also take place between the amino acids in addition to homo-exchanges. Therefore, high-affinity uptake processes observed with GABA and taurine could be the result of the homo-exchange diffusions, while that of β-alanine could be due to either the homo-exchange or the hetero-exchange diffusions or both.
K⁺'-evoked releases of GABA and to a lesser extent, taurine are partially dependent upon the presence of Ca⁺ in the superfusion media, whereas that of glycine and probably that of β-alanine, are not, K⁺ -evoked releases of labelled GABA and taurine are larger when loaded by their high-affinity uptake systems than by their low-affinity uptake processes. The reverse is the case with labelled glycine and β-alanine. These results do not rule out the possibility that taurine might act as a neurotransmitter in the cerebellum.     

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.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.     

HIGH AFFINITY UPTAKE OF TRANSMITTERS: STUDIES ON THE UPTAKE OF l-ASPARTATE, GABA, l-GLUTAMATE AND GLYCINE IN CAT SPINAL CORD

Abstract— The uptake of l -aspartate, l -glutamate and glycine each appeared to be mediated by two kinetically distinct systems with apparent K_m's of the order of 10 ('high affinity') and 100 μM ('low affinity') in slices of cat spinal cord, whereas the uptake of GABA appeared to be mediated by a single system of high affinity. The high affinity uptake of these amino acids in slices of spinal grey matter was approximately 5 times faster than that in slices of spinal white matter. The high affinity uptake systems in the cord slices survived homogenisation of the tissue under conditions known to preserve nerve terminals. Subcellular fractionation studies indicated that osmotically-sensitive particles of equilibrium density equivalent to that of 1.0 m -sucrose were at least in part responsible for the uptake of these amino acids. Inhibition studies indicated that three structurally specific systems of high affinity transported these amino acids:

• 1 specific for glycine—not inhibited by GABA or any of the other depressant amino acids found in cat spinal cord;
• 2 specific for GABA—not inhibited by glycine, taurine, l -aspartate or l -glutamate and (3) specific for l -aspartate and l -glutamate—not inhibited by glycine or GABA but strongly inhibited by various acidic amino acids such as l -cysteate and l -cysteine sulphinate.

The high affinity uptake of these amino acids was not inhibited by any of the known antagonists of the postsynaptic actions of these amino acids—strychnine (glycine), bicuculline and benzyl penicillin (GABA), methioninesulphoximine and l -glutamate diethyl ester (l -aspartate and l -glutamate). p-Chloromercuriphenylsulphonate strongly inhibited the high affinity uptake of glycine and GABA but was much less effective as an inhibitor of l -aspartate/l -glutamate high affinity uptake. This is in good agreement with microelectrophoretic studies in which this mercurial was found to potentiate depression of neuronal firing induced by glycine and GABA much more readily than excitation induced by l -aspartate or l -glutamate. These findings suggest the importance of high affinity transport processes in the removal of amino acids from the synaptic environment.     

Identification and Function of Glycine Receptors in Cultured Cerebellar Granule Cells

Abstract: Poly(A)⁺ mRNA was isolated from cultured mouse cerebellar granule cells and injected into Xenopus oocytes. This led to the expression of receptors that evoked large membrane currents in response to glycine. Current-responses were also obtained after application of β-alanine and taurine, but these were very low relative to that of glycine (maximal β-alanine and taurine responses were 8 and 3% of that of glycine, respectively). The role of glycine receptors on K⁺-evoked transmitter release in cultured cerebellar granule cells was also assayed. Release of preloaded d -[³H]aspartate evoked by 40 m M K⁺ was dose dependently inhibited by glycine, and the concentration producing half-maximal inhibition was 50 μ M. Taurine, β-alanine, and the specific GABA_A receptor agonist isoguvacine also inhibited K⁺-evoked release, and the maximal inhibition was similar for all agonists (˜40%). The EC₅₀ value was 200 μ M for taurine, 70 μ M for β-alanine, and 4 μ M for isoguvacine. Bicuculline (150 μ M ) antagonized the inhibitory effect of isoguvacine (150 μ M ) but not that of glycine (1 m M ). In contrast, strychnine (20 μ M ) antagonized the inhibitory effect of glycine (1 m M ) but not that of isoguvacine (150 μ M ). The pharmacology of the responses to β-alanine and taurine showed that these agonists activate both glycine and GABA_A receptors. The results indicate that cultured cerebellar granule cells translate the gene for the glycine receptor and that activation of glycine receptors produces neuronal inhibition.     

Glutamate release from cerebellar granule cells differentiating in culture: Modulation of the K+-stimulated release by inhibitory amino acids

The properties of l-[³H]glutamate release with an emphasis on the modulation by inhibitory amino acids of the potassium-induced release were studied with cerebellar granule cells from 7-day-old rats cultured for 7 or 14 days. Spontaneous glutamate release from cells grown for 7 days was fast, being slightly enchanced in Na⁺-free medium. l-Glutamate, kainate and quisqualate stimulated the release whereas N-methyl-d-aspartate and taurine were without any effect. The potassium-evoked glutamate release was Ca²⁺-dependent and potentiated by l-glutamate and quisqualate. Stimulated release was strongly depressed by glutamatediethylester. This inhibition was antagonized by GABA but not by taurine. GABA and its structural analogues taurine, hypotaurine, β-alanine and glycine were all equally effective in depressing stimulated glutamate release. The inhibition by GABA could be blocked by GABA antagonist. Both K⁺-evoked release and the kainate-induced release of glutamate were significantly greater in 14-day-old than in 7-day-old cultures, but the other properties of release were similar. The demonstration of calcium-dependent and potassium-stimulated glutamate release from cerebellar granule cells is consonant with the proposed neurotransmitter role of glutamate in these cells. The release could be modulated by both glutamatergic substances and inhibitory amino acids, the effect of GABA probably being mediated by GABAergic receptors.     

Effect of aging on 24-hour changes in dopamine and serotonin turnover and amino acid and somatostatin contents of rat corpus striatum

This study examined the 24-hour changes in a number of transmitters in the corpus striatum of young and middle-aged male Wistar rats. The contents of excitatory amino acids (glutamate, aspartate) and inhibitory amino acids (gamma-aminobutyric acid, GABA; taurine, glycine) and of somatostatin were measured in 2-month- and 18- to 20-month-old rats killed at six different time points along the 24-hour cycle. The striatal serotonin and dopamine turnover was also measured. Both young and middle-aged rats showed significant 24-hour variations in striatal glutamate and aspartate contents; only in young rats these variations fitted a cosine function, with acrophase during the first part of rest span. Mesor values of striatal excitatory amino acid contents were lowest in middle-aged rats. Significant 24-hour variations in striatal contents of GABA, taurine, and glycine occurred in young rats, while only striatal GABA exhibited 24-hour changes in middle- aged rats (acrophases during the first part of rest span). For every inhibitory transmitter, the mesor values in middle-aged rats were significantly lower than in young rats. The 24-hour variation of the striatal somatostatin content showed acrophase during the first part of rest span, mesor values and amplitude being lowest in middle-aged rats. Aging rats exhibited significantly higher mesor values of striatal serotonin turnover (34% increase) and lower mesor values of dopamine turnover (69% decrease) than their younger counterparts. Some of the circadian modifications of motor function seen in aging rats could be related to the striatal transmitter changes reported herein.     

THE INFLUENCE OF SODIUM, POTASSIUM AND LANTHANUM ON AMINO ACID RELEASE FROM SPINAL–MEDULLARY SYNAPTOSOMES

Abstract— The effects of electrical pulses and raised potassium on glycolysis and the release of the putative transmitter amino acids glutamate, aspartate, glycine and GABA from spinal medullary synaptosomes is studied and compared with effects on glutamine, alanine and serine. The actions of ouabain, p -hydroxymercuribenzoate ( p -HMB), lanthanum chloride and lowered sodium levels on the release of all the amino acids is examined. All agents caused a change in the pattern of release of the amino acids, mostly, producing an increase, but only lanthanum, p -HMB and lowered sodium prevented the stimulus-induced release.     

Physiological and chemical analysis of neurotransmitter candidates at a fast excitatory synapse in the jellyfish Cyanea capillata (Cnidaria, Scyphozoa)

Motor nerve net (MNN) neurons in the jellyfish Cyanea capillata communicate with one another by way of fast, bidirectional excitatory chemical synapses. As is the case with almost all identified chemical synapses in cnidarians, the identity of the neurotransmitter at these synapses is unclear. MNN neurons are large enough for stable intracellular recordings. This, together with the fact that they can be exposed, providing unlimited access to them and to their synapses, prompted a study of the action of a variety of neurotransmitter candidates, including those typically associated with fast synapses in higher animals. Only the amino acids taurine and β-alanine produced physiological responses consistent with those of the normal EPSP in these cells. Moreover, chemical analysis revealed that both taurine and β-alanine are present in the neurons and released by depolarization. These various findings strongly suggest that either or both of these amino acids, or a closely related compound is the neurotransmitter at the fast chemical synapses between MNN neurons.     

Changes in Amino Acids and Nitric Oxide Concentration in Cerebrospinal Fluid During Labor Pain

This study analyzes the relationship between amino acids and pain perception during active labor. Cerebrospinal fluid (CSF) levels of the excitatory amino acids (EAAs)—glutamate, aspartate and their amide forms, inhibitory amino acids (IAAs)—glycine, γ-amino butyric acid (GABA) and taurine and nitric oxide (NO) related compounds—arginine and citrulline (by-product of NO synthesis) were compared between pregnant women at term pregnancy with labor pain (n = 38) and without labor pain (Caesarian section; n = 30). The levels of aspartate, glycine, GABA and citrulline were significantly higher; whilst taurine was significantly lower in the labor pain group. These findings suggest that aspartate and NO are associated with labor pain. An inhibitory role for the IAA taurine and a pronociceptive role for glycine in labor pain are proposed.     

Inhibition of phototrophically growing Chromatium vinosum D by glycine

Abstract The effect of glycine on phototrophically growing Chromatium vinosum D was investigated in the presence and absence of various amino acids. Inhibition of autotrophic cells was more pronounced than that of heterotrophic cells. Cells of growth-inhibited cultures were misshaped, showing vacuolation and partial protoplast formation. Cells exposed to glycine in the presence of equimolar amounts of either d - or l -alanine did not show any inhibition of growth. Neither serine, leucine, valine, isoleucine nor α-aminoisobutyric acid could counteract the inhibition of growth by glycine. The inhibitory effect of glycine on growth is thus likely to be caused by a competition between glycine and alanine during cell wall synthesis. The possible functions of glycine transport system(s) in the natural habitat of C. vinosum are discussed.     

Long-lasting effects of audiogenic seizures on neurotransmitter amino acids in Rb mice

The existence of long-lasting (15–18 h) alterations of neurotrasmitter amino acid levels following a single or repeated acoustic stimulations in audiogenic seizure-prone Rb1 and Rb2 mice and suizure-resistant Rb3 mice were investigated. The levels of glutamate, aspartate, glycine, taurine, and of some of their precursors: glutamine and serine were determined. Fourteen brain areas were examined. Alterations were found only in 6 brain areas (pons, olfactory bulbs, superior colliculus, inferior colliculus, olfactory tubercles and raphe). Most frequent occuring changes were observed in pons and olfactory tubercles. These changes concerned mainly the excitatory amino acids, glutamate, and aspartate. Alterations of taurine, glycine and serine were also recorded.Abbreviations GABA 4-aminobutyrate - Tau taurine - Gly glycine - Asp aspertate - Glu glutamate - Gln glutamine - Ser serine - OB olfactory bulbs - OT olfactory tubercles - Sr striatum - Se septum - Hy hypothalamus - Th thalamus - Hi hippocampus - A amygdala - SC superior colliculus - IC inferior colliculus - FC frontal cortex - C cerebellum - P pons medulla - Ra raphe - AA neurotransmitter amino acids - I inhibitory - E excitatory - SSL steady-state level Plesant memories of Lawrence Austin's sojourn in my group at Strasbourg gather upon me when I dedicate this article on this occasion for the contribution that Lawrence Austin has made for the cause of neurochemical researchers.     

GABA- and glycine-immunoreactive synapses in spinal cord of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Using the method of the double immune label combined with two antibodies, i.e., monoclonal antibodies to gamma-aminobutyric acid (GABA) and polyclonal antibodies to glycine, the distribution of gamma-aminobutyric acid- and glycine-immunoreactive synapses on motoneurons and primary afferent axons was studied in the frog Rana temporaria spinal cord. An analysis of all labeled boutons on the dendrites and soma of motoneurons showed the existence of three categories of immunoreactive synapses as follows: 7% were labeled for GABA, 23% were labeled for glycine, and approximately 70% were immunoreactive to both GABA and glycine. These results confirm the predominant role of glycine in the postsynaptic inhibition of motoneuronal activity. Three similar populations of synaptic boutons were also founded on primary afferent axons, including one GABA-immunoreactive (25%) and one glycine-immunoreactive (5%); the majority of the immunoreactive synapses had the colocalization of two inhibitory transmitters. As a rule, the higher proportion of axo-axonal synapses was organized in synaptic triads. The possible simultaneous roles of glycine as a transmitter of postsynaptic inhibition and as a transmitter that mediates the process of the autoreception of glutamate in the axo-axonal synapses on the primary afferent fibers are discussed.     

The metabolism in vivo of glycine and serine in eight areas of the rat central nervous system

Abstract— Results of studies designed to estimate the rates at which glycine is derived from various possible sources in discrete areas of the rat CNS are reported. These results suggest that glycine is derived predominantly by de novo synthesis, presumably via the established pathways leading from glucose through serine to glycine. The content of glycine ranged from a low of approximately 0-6 μmol/g in the cerebellum and telencephalon to a high of 5·5 μmol/g in the spinal cord grey matter; however, based on its estimated rate of synthesis from serine, there appeared to be no correlation between the content of glycine and its rate of synthesis in the various areas studied. The flux of glycine from blood into the CNS was slower (0·03-0·15 μmol/g/h depending on the CNS structure) than that of serine (0·15-0·23 μmol/g/h) and both amino acids entered various CNS areas at rates unrelated to their respective tissue contents. These data have been discussed with regard to the putative transmitter function of glycine in the spinal cord and brainstem.     

Segmental release of amino acid neurotransmitters from transcranial stimulation

The present study used microdialysis techniques in an intact rabbit model to measure the release of amino acids within the lumbar spinal cord in response to transcranial electrical stimulation. Dialysis samples from the extracellular space were obtained over a stimulation period of 90 minutes and were examined using high pressure liquid chromatography. Neuronal excitation was verified by recerding corticomotor evoked potentials (CMEPs) from the spinal cord. A significant increase in the release of glycine and taurine compared to sham animals was measured after 90 minutes of transcranial stimulation. Glutamate and aspartate release was not significantly elevated. GABA concentrations were consistently low. CMEP components repeatedly showed adequate activation of descending fiber pathways and segmental interneuron pools during dialysis sampling. Since glycine, and to a lesser extent taurine, have been shown to inhibit motor neuron activity and are closely associated with segmental interneuron pools, suprasegmental modulation of motor activity may be, in part, through these inhibitory amino acid neurotransmitters in the rabbit lumbar spinal cord.     

STUDIES OF THE UPTAKE AND RELEASE OF [3H]β-ALANINE BY FROG SPINAL SLICES

Abstract— [³H]β-Alanine was accumulated by frog spinal cord slices by two transport components with estimated K_m values of 31 M ('high-affinity') and 11 HIM ('low affinity') respectively. The high affinity uptake exhibited sodium ion and energy dependence, temperature sensitivity, had a very low V_max (10.4 nmol/g/min) compared to GABA and glycine, was competitively inhibited by GABA (K_t 2 M), and was significantly reduced by the presence of glycine and of taurine in the incubating medium.
When slices preloaded with [³H]β-alanine were superfused with medium containing depolarizing concentrations of potassium ions, there was a small, but consistent, increase in [³H]β-alanine efflux: 1.4 times prestimulation rates in 40 mM potassium. When the superfusate was altered by omission of calcium and addition of concentrations of magnesium (10 mm), manganese (1 mM), and cobalt (1 mM) ions sufficient to block reflex transmission in the isolated in vitro frog cord, the potassium-evoked release was not blocked. Release was decreased by lanthanum ions (1 mM). Release of [³H]GABA and [³H]glycine in parallel experiments was inhibited by magnesium, manganese, cobalt and lanthanum. Veratridine significantly increased the release of [³H]GABA and [³H]glycine but not of [³H]β-alanine.
These observations demonstrate the non-specificity of β-alanine uptake and the unconventional nature of the calcium-dependence of β-alanine release and therefore do not lend support to the hypothesis that β-alanine functions as a neurotransmitter in frog spinal cord.     

Transport of GABA from the perfused ventricular system of the cat

Abstract— The transport of GABA was studied in anaesthetized cats undergoing ventriculo-cisternal perfusion with radioactive GABA. Steady-state clearance of GABA from the CSF was greater than that of other amino acids previously studied, and was saturated at lower substrate concentrations, with an apparent K_t of 5·4 × 10^-5 M, after correcting for non-saturable transport. GABA clearance was inhibited by the inclusion of taurine or β-alanine in the perfusion fluid, but not by a number of the common neutral and acidic amino acids. Study of punch biopsies of brain tissue taken adjacent to the venticular system, at the completion of perfusions, showed accumulation of radioactive GABA in the tissue to values four times higher than those found in the perfusion fluid. Of the radioactivity which had been removed from the ventricular system, only 11 per cent remained in the brain at the completion of the perfusion. Excised cat choroid plexus showed a saturable uptake of GABA which was inhibited by inclusion of taurine, β-alanine, or β-guanidino propionic acid in the incubation medium.