Graham A. R. Johnston

Amino acid transmitters in the mammalian central nervous system

2. Evidence for A m i n o Acids as T ransmi t t e r s . . . . . . . . . . . . . . . . . . . . 99 2.1. Synthesis and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 2.2. Synapt ic Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 2.3. Postsynapt ic Act ion . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 2.4. Postsynapt ic An tagon i s t s . . . . . . . . . . . . . . . . . . . . . . . . . 103 2.5. Inac t iva t ion and R e m o v a l . . . . . . . . . . . . . . . . . . . . . . . . . 104

THE STRUCTURAL SPECIFICITY OF THE HIGH AFFINITY UPTAKE OF l‐GLUTAMATE AND l‐ASPARTATE BY RAT BRAIN SLICES

Abstract— The high affinity uptake system for l‐glutamate and l‐aspartate in rat cerebral cortex may not be specific for these likely excitatory synaptic transmitters, as threo‐3‐hydroxy‐dl‐aspartate, l‐cysteinesulphinate, l‐cysteate and d‐aspartate strongly inhibit the observed high affinity uptake of l‐[3H]glutamate by rat brain slices in a manner consistent with linear competitive inhibition. These substances should therefore be considered as possible substrates for the transport system. Each of these four acidic amino acids excites central neurones in a manner similar to excitation induced by l‐glutamate, and as each might occur in brain tissue, their possible synaptic role should be investigated.

Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat.

Summary (1) A study has been made of the effects of bicuculline, a phthalide isoquinoline convulsant alkaloid, on the inhibition of spinal neurones by amino acids and the synaptic inhibition of spinal reflexes in cats anaesthetised with pentobarbitone sodium. (2) The inhibitory effects on spinal neurones of glycine, β-alanine, l -α-alanine, taurine and nor -adrenaline were not affected by electrophoretically administered bicuculline, whereas the alkaloid was a reversible antagonist of the actions of GABA, β-hydroxy-GABA, δ-aminovaleric acid, muscimol and imidazole acetic acid. (3) The classification of amino acids depressing the firing of spinal neurones into ‘glycine-like’ and ‘GABA-like’ is identical when either strychnine or bicuculline is used as an antagonist: strychnine blocks the action of glycine-like amino acids, bicuculline that of GABA-like amino acids. (4) Bicuculline administered intravenously in convulsant doses does not affect the strychnine-sensitive inhibition of spinal motoneurones, but reduces both the prolonged inhibition of these cells which is sensitive to picrotoxin and dorsal root potentials evoked by impulses in cutaneous and muscle afferents. (5) Since GABA has a hyperpolarizing inhibitory transmitter-like action on motoneurones, the proposal is made that this amino acid, released at inhibitory axodendritic or axo-axonic synapses, is responsible for the prolonged inhibition of spinal motoneurones by repetitive impulses in afferent fibres (‘presynaptic’ inhibition). (6) The relationship of primary afferent depolarization to this inhibition is discussed. (7) The effects of laudanosine, gelsemine and dendrobine on spinal neurones resemble that of strychnine.

A pharmacological study of the depression of spinal neurones by glycine and related amino acids.

SummaryAn analysis has been made in anaesthetised cats of the depression by glycine and related amino acids of the firing of spinal dorsal horn interneurones, Renshaw cells and cortical neurones. In general, electrophoretically administered glycine was a more potent depressant of interneurones than GABA. The reverse was true for cortical neurones, whereas these two amino acids were approximately equally effective upon Renshaw cells. Strychnine blocked the depressant action of α- and β-amino acids, but not that of γ- and higher ω-amino acids. Only convulsants having a strychnine-like effect on spinal post-synaptic inhibition blocked the action of glycine. The depression of spinal neurones produced by glycine or GABA was not affected by structural analogues of glycine and GABA that were not depressants, or by substances influencing amino acid transport systems. Some evidence was obtained for the enzymic inactivation of electrophoretically administered glycine in spinal tissue. The results are discussed in terms of the involvement of a glycine-like amino acid as a major spinal inhibitory transmitter.

The hyperpolarization of spinal motoneurones by glycine and related amino acids

SummaryElectrophoretically administered glycine, β-alanine and GABA hyperpolarize spinal motoneurones in cats anaesthetized with pentobarbitone. The reversal potential for these hyperpolarizations is similar to that of inhibitory postsynaptic potentials. Alterations in intracellular K+ and Cl− ion concentrations, and intracellular injection of a series of anions of different hydrated ion size, affect inhibitory and amino acid potentials in the same fashion. Hence it is probable that glycine, β-alanine and GABA produce an alteration in membrane permeability similar to that produced by spinal inhibitory synaptic transmitters. Strychnine reversibly blocks the action of inhibitory transmitters, glycine and β-alanine, but is without effect on the hyperpolarizing action of GABA.These results indicate that glycine may be a major spinal inhibitory transmitter, in which case strychnine affects spinal postsynaptic inhibition by limiting the action of glycine upon subsynaptic inhibitory receptors.

GABA uptake in rat central nervous system: comparison of uptake in slices and homogenates and the effects of some inhibitors.

Abstract— Fifty‐two substances were tested as inhibitors of the uptake of [3H]GABA in slices of rat cerebral cortex. Among GABA analogues tested, only the 2‐fluoro, 3‐hydroxy and 2‐amino compounds had affinities for the uptake mechanism comparable to that of GABA. [3H]GABA uptake was also potently inhibited by p‐chloromercuriphenylsulphonate, N‐ethylmaleimide, chlorpromazine and haloperidol. No inhibitors were found to act in a competitive manner with respect to GABA. [3H]GABA uptake was also examined in homogenates of cerebral cortex and other regions of CNS. There was a rapid uptake of [3H]GABA into particles when homogenate samples were incubated with the labelled amino acid; this uptake had similar kinetic properties and inhibitor sensitivity to that observed in slices of intact tissue. Density gradient centrifugation experiments indicated that the particles responsible for the uptake of [3H]GABA in homogenates were probably synaptosomes. Uptake of [3H]GABA also occurred in slices and homogenates of rat spinal cord, and evidence was obtained by the simultaneous labelling of homogenates with [14C]glycine and [3H]GABA that these two amino acids were taken up by different nerve terminals in this region.

Antagonism between bicuculline and GABA in the cat brain

Abstract (1) An investigation was made of the effects of bicuculline on the inhibition by amino acids of neurones in the lateral vestibular nucleus, the ventrobasal thalamus, and the cerebellar, hippocampal and cerebral cortices of cats anaesthetized with pentobarbitone sodium. (2) In all regions, bicuculline reversibly antagonized the action of GABA without modifying that of glycine. In contrast strychnine suppressed the inhibitory effect of glycine but not that of GABA. (3) The inhibition by β-alanine of the firing of thalamic, cerebellar, hippocampal and cerebral cortical neurones was reduced by both bicuculline and strychnine; the inhibition of lateral vestibular neurones by β-alanine, like that of spinal neurones, was insensitive to bicuculline. (4) The classification of depressant amino acids on the basis of antagonism by bicuculline and strychnine is different when these substances are tested on spinal and supraspinal neurones. (5) Concentrations of bicuculline adequate to modify the sensitivity of Purkinje cells to GABA had little or no effect on the inhibition of these neurones by noradrenaline. (6) The relatively high degree of specificity of bicuculline as a GABA antagonist indicates that this alkaloid is of considerable value in determining whether or not a particular inhibitory pathway could operate by releasing GABA as an inhibitory transmitter.

The 'ABC' of GABA receptors: a brief review.

1. In the mammalian central nervous system, GABA is the main inhibitory neurotransmitter. GABA is a highly flexible molecule and, thus, can exist in many low‐energy conformations. Conformationally restricted analogues of GABA have been used to help identify three major GABA receptors, termed GABAA, GABAB and GABAC receptors.

The specificity of strychnine as a glycine antagonist in the mammalian spinal cord

SummaryAn investigation was made of the influence of strychnine on the depression of the firing of spinal interneurones and Renshaw cells by glycine, GABA, nor-adrenaline and 3-hydroxytyramine. Administered electrophoretically or intravenously, strychnine blocks the effect of glycine more readily than that of the other depressants. Such specific antagonism of glycine action by relatively low concentrations of strychnine may be competitive in nature, but technical difficulties precluded a full assessment of the type of antagonism. The effects of relatively high concentrations of strychnine on the action of the other depressants probably result from interference with membrane permeability changes. The findings are considered to support previous proposals that glycine is the transmitter at spinal strychnine-sensitive inhibitory synapses.

UPTAKE AND RELEASE OF D‐ AND L‐ASPARTATE BY RAT BRAIN SLICES

D‐Aspartate is accumulated by slices of adult rat cortex by a high affinity uptake which is abolished if the sodium ions in the incubation medium are replaced by choline. A small uptake of D‐aspartate takes place if the sodium ions are replaced by lithium ions. It appears likely that D‐aspartate shares the same transport system with L‐aspartate, and that the uptake of D‐aspartate is into the same osmotically‐sensitive particles as those which accumulate L‐aspartate. D‐Aspartate is released from cerebral cortex slices by raised potassium concentrations, provided calcium is present in the perfusing buffer.