METABOLISM OF PUTATIVE TRANSMITTERS IN INDIVIDUAL NEURONS OF APLYSIA CALIFORNICA

The activities of acetylcholinesterase (acetylcholine acetyl-hydrolase; EC 3.1.1.7) and catechol-O-methyl transferase (S-adenosylmethionine: catechol-O-methyl transferase; EC 2.1.1 .a) were measured in the various ganglia of the nervous system of Aplysia californica and in some of the individually identifiable neurons in these ganglia. All of the neurons studied had measurable levels of activity for both enzymes. Since different individual neurons exhibited approximately the same level of activity we concluded that neither of these enzymes could be used to classify neurons as ‘cholinergic’ vs. ‘aminergic’ or ‘cholinoceptive’ vs. ‘aminoceptive'. The ubiquitous distribution of either or both of these enzymes in different single neurons may be related to glial contamination.     

METABOLISM OF PUTATIVE TRANSMITTERS IN INDIVIDUAL NEURONS OF APLYSIA CALIFORNICA: AROMATIC AMINO ACID DECARBOXYLASE

Abstract— The activities of aromatic amino acid decarboxylase (EC 4.1.1.26) in various ganglia, nerve trunks, and individual identifiable neurons of Aplysia culifornica were measured. The distribution of the decarboxylase enzyme is ubiquitous throughout the central nervous system of the Aplysia . Every Aplysia neuron tested contained some decarboxylase activity. The presence of this particular synthetic enzyme in an Aplysia neuron, therefore, cannot be used to classify these neurons as 'aminergic'.     

THE TOXIC EFFECT OF SODIUM GLUTAMATE ON RAT RETINA: CHANGES IN PUTATIVE TRANSMITTERS AND THEIR CORRESPONDING ENZYMES

Abstract– Subcutaneous administration of high doses of sodium glutamate to rats during their first week after birth produced an almost total loss of choline acetyltransferase, a 90% reduction in glutamate decarboxylase and 70% reductions in acetylcholinesterase and DOPA decarboxylase activities in the adult retina. In addition there was a 70% decrease in GABA and 35-55% decrease in aspartate, glutamate, glycine, alanine and glutamine. No reduction in taurine was observed. The results support the view that the enzymes are mainly localized in the interneurons of retina and that taurine is present in the photoreceptor cells.
Glutamate treatment was also followed by a small reduction in choline acetyltransferase and glutamate decarboxylase of the superior colliculus and in choline acetyltransferase of hippocampus, whereas no changes could be detected in the lateral geniculate body of the adult rat. Unilateral enucleation performed on 1-day-old animals did not alter choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase and DOPA decarboxylase activities in the superior colliculus and in the lateral geniculate body of the adult rat.     

RELEASE OF RADIOACTIVE GLUTAMIC ACID FROM THIN SECTIONS OF GUINEA-PIG OLFACTORY CORTEX IN VITRO

Slices from the guinea-pig olfactory cortex were incubated in the medium containing [¹⁴C]glutamate and release of radioactive compounds was subsequently studied in the standard or high potassium media or during repetitive stimulation of the lateral olfactory tract (LOT) while electrical activity of the tissue was monitored. In 50 mm -potassium concentration, the pre- and postsynaptic potentials were completely suppressed and effluxes of total ¹⁴C and [¹⁴C]glutamate increased. No significant increase in [¹⁴C]glutamine was found. When Ca²+ concentration was reduced from 2·4 to 0·12 mm , the postsynaptic potential disappeared and release of [¹⁴C]glutamate in 50 mm -potassium decreased to about a third of that in 2·4 mm -Ca²⁺. Repetitive LOT stimulation enhanced release of total ¹⁴C in thinner slices but caused no significant increase in [¹⁴C]glutamate efflux. These findings were discussed in relation to the possibility that glutamate is a mediator between the LOT fibres and cortical neurons.     

EFFECT OF STIMULATION OF EXCITATORY NERVE TRACT ON RELEASE OF GLUTAMIC ACID FROM OLFACTORY CORTEX SLICES IN VITRO

Abstract— Thin sections prepared from the olfactory cortex of the guinea pig were incubated in a medium containing [¹⁴C]glutamate, and release of radioactive compounds and electrical activity were subsequently examined in the presence of l -cysteate. The postsynaptic potential was almost completely suppressed in the medium containing l -cysteate, whereas the presynaptic potential was unaffected. Repetitive stimulation of the excitatory input of the lateral olfactory tract enhanced release of radioactive glutamate. The facilitatory effect of lateral olfactory tract stimulation increased with increase in stimulus frequency and was dependent on calcium. Release of radioactive gluiamine was not enhanced by lateral olfactory tract stimulation. Phenobarbitone sodium markedly depressed both the postsynaptic potential and the effect of lateral olfactory tract stimulation on glutamate release. These results indicate that stimulation to the lateral olfactory tract enhances liberation of glutamate from the tract nerve terminals.     

THE TOXIC EFFECT OF SODIUM GLUTAMATE AND DL-α-AMINOADIPIC ACID ON RAT RETINA: CHANGES IN HIGH AFFINITY UPTAKE OF PUTATIVE TRANSMITTERS

Subcutaneous administration of high doses of sodium glutamate to new born rats was used to destroy retinal interneurons and ganglion cells. Such treatment was accompanied by 90% reduction in the high affinity uptake of choline, 60–70% reductions in the uptakes of GABA, diamino-n-butyric acid and glycine and 30–40% reductions in the uptakes of asparatate and glutamate measured on retinal homogenates from 30-day-old rats. The high affinity uptakes of β-alanine and taurine were unchanged. Preincubation of retinal homogenates with 1 mM β-alanine or 100 μM diamino-n-butyric acid severely reduced the high affinity GABA uptake in control and experimental animals. In intact retinae, however, the glutamate treatment increased the high affinity uptake of β-alanine by 70%, whereas that of diamino-n-butyric acid was reduced by 40% and the high affinity uptakes of GABA and glutamate were unchanged. Four hours after injection of the gliotoxic compound DL-α-aminoadipic acid into the vitreous body of 30-day-old rats, the Müller cells could no longer be identified. This lesion was accompanied by 55% reduction in the high affinity uptake of β-alanine and 25% reduction in the uptakes of GABA and glutamate on intact retinae. The high affinity uptakes of diamino-n-butyric acid, choline and the enzyme activities of choline acetyltransferase and glutamate decarboxylase were unchanged under these conditions. After 24 h, however, the Müller cells could be recognized again, and the β-alanine uptake had normalized.     

EFFECT OF VINBLASTINE AND COLCHICINE ON UPTAKE AND RELEASE OF PUTATIVE TRANSMITTERS BY SYNAPTOSOMES AND ON BRAIN ACTOMYOSIN-LIKE PROTEIN

Abstract— The effects of several inhibitors, including vinblastine and colchicine, on the accumulation of a number of putative transmitters by a rat brain synaptosomal preparation and their subsequent release by excess K⁺ was examined. In addition, the effect of the alkaloids on the ATPase activity of the actomyosin-like protein, neurostenin, isolated from the synaptosomal preparation, was studied. The uptakes of radioactive glutamate, GABA, dopamine and norepinephrine were energy-dependent, as evidenced by their susceptibility to 0.01 mM carbonyl cyanide m-chlorophenylhydrazone (Cl-CCP), 01 mM ouabain and temperature. The active accumulations of GABA, dopamine and norepinephrine were also greatly inhibited by 1 mM6-hydroxydopamine (6-OHDA), 01 mM mersalyl, 0.05–0.25mM vinblastine and 0.1–1.0 mM colchicine. Vinblastine was approximately 10-fold more potent (K₁, ?0.1 mM) than colchicine as an inhibitor. The release of actively accumulated dopamine or norepinephrine by excess K⁺ (increasing the [K⁺] from 5 to 30 mM) was inhibited somewhat when vinblastine was present during the entire incubation period. If the synaptosomes were preloaded with the radioactive compounds prior to addition of vinblastine, there was no discernible effect on the relative amount of material released by excess K⁺. However, the addition of inhibitor under the latter conditions caused a leakage of radioactivity into the medium even without excess K⁺ being present. Glutamate accumulation was somewhat different from that of GABA, dopamine or norepinephrine. Although it required energy for uptake, 6-OHDA, mersalyl, vinblastine or colchicine were not inhibitory. Studies of the oxidative metabolism of glutamate and GABA by this synaptosomal preparation indicated that the mechanisms of inhibition by vinblastine was not attributable to a metabolic effect. Both vinblastine and colchicine inhibited the Mg²⁺-stimulated, but not the Ca²⁺-activated ATPase of neurostenin. This effect was probably attributable to an interaction of the vinblastine with the neurin moiety of this actomyosin-like protein. We suggest that the inhibitory phenomena exhibited by vinblastine and colchicine in this synaptosomal preparation arose from the effect of these alkaloids on the neurin associated with the synaptic membrane.     

OLFACTORY CILIA IN THE FROG

Olfactory epithelium from the frog was examined in the living state by light microscopy and in the fixed state by electron microscopy. Particular attention was paid to the layer of cilia and mucus which covers the surface of the epithelium. The olfactory cilia differed from typical cilia in that they (a) arose from bipolar neurons and had centrioles near their basal bodies, (b) were up to 200 microns in length, of which the greater part was a distal segment containing an atypical array of ciliary fibers, (c) were often immotile, (d) had their distal segments arranged in parallel rows near the surface of the mucus, and (e) had many vesicles along their shafts and had splits in the array of fibers in their distal segments. These specializations make the olfactory cilia similar to cilia found on other sensory cells and support the theory that they are the locus where electrical excitation in the olfactory organ is initiated by contact with odorous substances.     

CHOLINESTERASE IN DENERVATED END PLATES AND MUSCLE FIBRES

Parallel studies were made of cholinesterase activities and localizations in denervated rat and rabbit gastrocnemius muscle. Koelle's histochemical reaction was used for demonstrating the localization of cholinesterases. Enzyme activities in whole sliced muscle were measured by electrometric titration. The Cartesian ampulla-diver technique was used for cholinesterase activity determinations in end plate regions or in small pieces of the muscle fibre itself. No changes in the activity of cholinesterases (ChE) were found in the whole denervated muscle which would account for its chemical supersensitivity. The ChE distribution pattern was changed so that the end plate region became less active in the denervated muscle than in the normal one. The decrease in ChE activity in the end plates seems to be largely compensated for by an increase of this enzyme elsewhere in the muscle. A possible connection between the spatial spread of cholinesterase activity and the enlargement of the acetylcholine-sensitive surface is discussed.     

NORADRENALINE METABOLIZING ENZYMES IN NORMAL AND SYMPATHETICALLY DENERVATED VAS DEFERENS

—A surgical technique for sympathetically denervating the vas deferens has been evaluated biochemically. A slight fall in soluble muscle protein content and no significant change in DNA content of the operated vas deferens were found. This indicates that the surgical procedure causes only a slight degree of tissue damage and may be useful for investigating the cellular localization and properties of noradrenaline metabolizing enzymes. In three species examined (rat, guinea pig and rabbit), monoamine oxidase activity of the vas deferens fell by approximately 50 per cent after denervation. The time course of the fall in monoamine oxidase activity of rat vas deferens was parallel to that of the disappearance of noradrenaline suggesting that this proportion of the total enzyme activity had a neuronal localization. The remaining enzyme activity is presumably located extraneuronally. Significant falls in catechol-O-methyl transferase activity were found in rat and rabbit vas deferens after denervation but not in guinea pig. The rabbit and rat vas deferens had respectively approximately 60 and 30 per cent of the catechol-O-methyl transferase activity associated with the sympathetic nerves. A complete loss of DOPA decarboxylase and tyrosine hydroxylase activities occurred in rat vas deferens after denervation, suggesting that these noradrenaline synthesizing enzymes have an entirely neuronal localization.     

STUDIES ON INCREASED ACID HYDROLASE ACTIVITIES IN DENERVATED MUSCLE

Denervation of rat hemidiaphragm led to large increases in the activity of acid proteinase, cathepsin B1 and β-glucuronidase in the muscle. The increases were not confined to the endplate regions of the muscle. Mononuclear cells extracted from normal and denervated extensor digitorum longus and diaphragm muscles contained only a small proportion of the acid proteinase and cathepsin B1 activities in the muscle. Actinomycin D, but not methotrexate, prevented the increase in acid proteinase and cathepsin B1 normally resulting from denervation. Brij/58 increased acid proteinase activity to a smaller extent in the denervated muscle than in the control. The length of the distal nerve stump remaining after denervation influenced the enzyme increases, but acid proteinase and cathepsin B1 appeared to be affected differently. The significance of the post-denervation increase in hydrolase activities is still unknown.     

ALANINE METABOLISM IN RAT CORTEX IN VITRO

Abstract— (1) The metabolism of [U-¹⁴C]alanine was followed in slices of rat cerebral cortex and its interaction with glucose, pyruvate and the metabolic inhibitors fluoracetate and malonate was studied.
(2) Alanine did not stimulate respiration above endogenous levels or affect the rate of oxygen uptake with glucose or pyruvate as cosubstrate. Radioactivity found in CO₂ from labelled alanine was only 6 per cent of that from labelled pyruvate. Lactate was not formed from alanine.
(3) After 2 h incubation with [U-¹⁴C]alanine the specific activities of glutamate, glutamine and GABA were 20–30 per cent that of alanine. All these specific activities except glutamate were lowered by addition of glucose, but with pyruvate as cosubstrate the specific activity of glutamate was increased by 87 per cent above the level with alanine alone.
(4) The effect of alanine as cosubstrate with [U-¹⁴C]pyruvate was to reduce the specific activity of GABA and of glutamine, but not glutamate or lactate; thus there was not an equal dilution of all the metabolites of pyruvate.
(5) Fluoracetate diminished respiration and the production of CO₂ from [U-¹⁴C]-alanine only slightly; the addition of malonate as well practically abolished both. Fluoracetate lowered incorporation from alanine into all the amino acids, and radioactivity could not be detected in glutamine at all; addition of malonate lowered the specific activity of glutamate to 25 per cent but increased that into aspartate, GABA and glutamine.
(6) The interpretation of these data in terms of known pathways of alanine metabolism is discussed.     

DEVELOPMENTAL CHANGES IN GLYCOLYSIS IN RAT CEREBRAL CORTEX

Abstract— The ATP concentration in infant rat cerebral cortex slices which were incubated aerobically with glucose (5 m m ) as substrate was much higher than in those from the adult. The higher ATP concentration in slices from young rat was also obtained when they were incubated aerobically with pyruvate (10 m m ), dl -lactate (20 m m ) and dl -3-nydroxybutyrate (20 m m ) However, when the slices were incubated anaerobically with glucose, the ATP concentration was very low. Thus, the formation of ATP in the slices from the young rat was thought to be mainly due to their oxidative metabolism, as in those from the adult. The amounts of glycolytic key enzymes in rat cerebral cortex (hexokinase. phosphofructokinase and pyruvate kinase) increased with age. Glycolysis was actually shown to be less active in the cerebral slices from young rats than from the adult. In addition it is known that the tricarboxylic acid cycle enzymes in rat cerebrum also increase with age. Consequently, the activity with respect to ATP formation must be lower in the cerebral cortex slices from young rats than from the adult. The fact that ATP was nevertheless higher in the slices from young rats may be explained by a lower rate of degradation. Developmental increases in the amounts of Na⁺-K⁺-ATPase and Mg²⁺ -ATPase in rat cerebral cortex were greater than those of the glycolytic key enzymes. These are discussed in relation to the observation that the rate of aerobic glycolysis in slices from cerebral cortex of young rats was not increased by d -glutamate (5 m m ) and high potassium (50 m m ).