GLYCINE UPTAKE IN RAT CENTRAL NERVOUS SYSTEM SLICES AND HOMOGENATES: EVIDENCE FOR DIFFERENT UPTAKE SYSTEMS IN SPINAL CORD AND CEREBRAL CORTEX

Abstract— Evidence is presented that glycine is taken up by two different transport systems in rat CNS tissue slices; one system has relatively low affinity for glycine (K_m = 300 μ m ) and predominates in cerebral cortex, cerebellum and mid-brain, the other has a higher affinity for glycine (K_m = 40 μ m ) and is detectable only in spinal cord, medulla and pons. The low affinity transport system appears to be shared by other small neutral amino acids, whereas the high affinity system is very specific for glycine. Both transport systems were shown to be present in particles in homogenates of CNS tissue by incubation with glycine in vitro , and subcellular fractionation studies suggested that synaptosomes were partly responsible for such uptake. Various substances were tested as inhibitors of the high affinity uptake system for glycine in spinal cord slices; the most potent inhibitors were p -chloro-mercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine, imipramine, desipramine, hydrazinoacetic acid and haloperidol. No competitive inhibitors of the high affinity glycine uptake were found. It is suggested that the high affinity transport system is associated with inhibitory synapses where glycine is a transmitter.     

POSTNATAL CHANGES IN THE HIGH AFFINITY UPTAKE OF GLYCINE and GABA IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— High affinity uptake systems for GABA into slices of cerebral cortex and for glycine into slices of spinal cord have been demonstrated in rats of 1 and 10 days postnatal age and compared with the systems in tissue slices from adult rats. For both systems there was an increase in the maximal rate of uptake of the substrate with development. For glycine uptake there was no significant change in apparent K_m during development, whereas there was a four-fold increase in the apparent K_m for GABA uptake. There were some changes with development in the apparent substrate specificity of the two systems suggesting increased specificity with maturation. Bicuculline and strychnine, antagonists of the postsynaptic inhibitory actions of GABA and glycine, produced convulsions in 1-, 2- and 10-day-old rats following intraperitoneal injection of doses somewhat lower than those required to convulse adult rats. These findings are consistent with other evidence that glycine and GABA are functioning as inhibitory transmitters at least as soon as 1 day after birth.     

PYRIDOXINE DEFICIENCY AND DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM IN THE RAT

Pyridoxine (vitamin B₆) deficiency was produced in rats during the period of development of the central nervous system. The levels of pyridoxal phosphate and y-amino-butyric acid in whole brains of these rats were determined, together with the activities of glutamate decarboxylase (EC 4.1.1.15) and γ-aminobutyrate aminotransferase (EC 2.6.1.19). The lowered contents of pyridoxal phosphate and γ-aminobutyrate in the brains confirmed the existence of pyridoxine deficiency. The activity of the glutamate decarboxylase holo-enzyme was decreased, whereas the activity of the apoenzyme was increased. However, there appeared to be no difference in the activity of γ-aminobutyrate aminotransferase. Concomitantly, some electrophysiological parameters, such as EEG and auditory evoked potentials, were analysed. The EEG of pyridoxine-deficient animals showed spike activity, presumably indicative of the existence of seizures in many of the deficient rats. Evoked potentials presented abnormalities in their latency, wave form and response to repetitive stimuli, but the extent to which they were affected depended upon the intensity of the deficiency.     

COMPETITIVE INHIBITION OF GABA UPTAKE IN RAT BRAIN SLICES BY SOME GABA ANALOGUES OF RESTRICTED CONFORMATION

Abstract— trans -4-aminocrotonic acid, dl - cis -aminocyclohexane-ltarboxylic acid and 4-aminotetrolic acid were found to be competitive inhibitors of GABA uptake in rat brain slices. These inhibitors are analogues of extended conformations of GABA, which indicates that these conformations are important in the initial binding of this inhibitory transmitter to its transport carrier.     

POSTNATAL CHANGES IN THE POTASSIUM-STIMULATED, CALCIUM-DEPENDENT RELEASE OF RADIOACTIVE GABA AND GLYCINE FROM SLICES OF RAT CENTRAL NERVOUS TISSUE

—Using a simple apparatus designed to perfuse nervous tissue mini-slices retained on glass fibre filter discs, slices of adult (13 week) rat cerebral cortex and spinal cord were shown to release radioactive GABA and glycine, but not 2-amino-isobutyric acid, in response to increased potassium ion concentration of the perfusing medium. A major portion of this potassium-stimulated release was dependent upon the presence of calcium ions in the perfusing medium. Slices of cerebral cortex and spinal cord from rats of 1 day and 10 days postnatal age showed potassium-stimulated, calcium-dependent release of radioactive GABA and glycine respectively. These findings are consistent with other evidence that GABA and glycine are functioning as inhibitory transmitters in rats at least as soon as 1 day after birth.     

POSTNATAL ALTERATIONS IN EFFECTS OF POTASSIUM ON UPTAKE AND RELEASE OF GLUTAMATE AND GABA IN RAT BRAIN CORTEX SLICES

The uptake and release of glutamate and of GABA, as well as the effect of high potassium concentrations (35 or 80 mM) hereupon, were studied by aid of ¹⁴C-labelled amino acids in brain cortex slices from rats of different ages between birth and adulthood. Both the extent of the uptake (i.e. the tissue/medium ratio of ¹⁴C at, or close to, equilibrium) and the rate of uptake (i.e. the tissue/ medium ratio of ¹⁴C after short (5 min) incubation periods) increased with age. Differences were, however, found between glutamate and GABA, and the extent of the GABA uptake had a distinct maximum during the second postnatal week. At all ages, high concentrations of potassium caused a decrease in the rate of GABA uptake but were without effect on the rate with which glutamate was taken up. The release of the two amino acids occurred with approximately the same half-time (50 min) in slices from animals of at least 14 days of age. Before that time the release of glutamate was somewhat faster, whereas that of GABA was much slower, especially during the first postnatal week (half-time 90 min). The ontogenetic alterations in the effect of excess potassium were complex and varied both between the two potassium concentrations used and between the two amino acids. The results are thus compatible with the existence of different transport systems for the two amino acids, They also suggest that glutamate may exert other functions in addition to its role as a putative transmitter.     

EFFECTS OF 5,6-DIHYDROXYTRYPTAMINE ON MONOAMINERGIC NEURONES IN THE CENTRAL NERVOUS SYSTEM OF THE RAT

—The injection of 50 μg of 5,6-dihydroxytryptamine (5,6-HT) into a lateral ventricle of the rat depleted the spinal cord and various regions of the brain of indoleamines (presumably 5-HT) and 5-hydroxyindole acetic acid. The concentrations of 5-HT were measured by two different methods: the formation of a fluorescent derivative with o-phthalaldehyde, and the native fluorescence in hydrochloric acid. When the results of both methods were compared on the pons and medulla 4 days after injecting 5,6-HT, the loss in indoleamine appeared to be greater when o-phthalaldehyde was used. This suggests that the two methods may be measuring different compounds. According to both methods, the loss of 5-HT persisted for several days after the injection of 5,6-HT, but by 2 months 5-HT concentrations (measured only by the native fluorescence procedure), had recovered to near-normal values. The depletion of 5-HT was most pronounced in regions adjacent to the ventricular system and in the spinal cord. Initially, caudate and septum were more affected on the side of the injection, and later showed some permanent atrophy. The injection of up to 50 μg of 5,6-HT did not lead to any significant loss of noradrenaline or dopamine from the brain, or to any reduction in the activity of the enzyme tyrosine hydroxylase. The drug was a potent inhibitor of the uptake of [³H]5-HT by brain slices, but was less effective in inhibiting catecholamine uptake systems. These observations suggest a preferential action on tryptaminergic neurones. Larger doses of 5,6-HT caused a loss of catecholamines and tyrosine hydroxylase from the brain, and were severely toxic.     

INTRANEURAL DISTRIBUTION OF EXOGENOUS NOREPINEPHRINE IN THE CENTRAL NERVOUS SYSTEM OF THE RAT

Catecholaminergic neurons, which take up and retain exogenous norepinephrine labeled with tritium, were studied by means of high resolution radioautography, in the substantia nigra, the substantia grisea periventricularis, and the locus coeruleus of the rat. Under the conditions required for the radioautographic detection of exogenous norepinephrine-³H, it was established that (1) glutaraldehyde was the most suitable fixative for preserving the labeled amine in situ; (2) norepinephrine-³H itself, rather than metabolites, accounted for most of the reactions detected in catecholaminergic neurons. At various time intervals after an intraventricular injection of norepinephrine-³H, the tracer reached a concentration 15–100 times higher, and disappeared at a slower rate, in presynaptic axons (t½:4 hr) than in nerve cell bodies (t½:0.8–1.3 hr). After pretreatment with a monoamine oxidase inhibitor, the radioautographic reactions increased and persisted longer, especially in the preterminal axons. Within neurons, the labeled amine was ubiquitously distributed in the nerve cell body and concentrated in presynaptic axons and synaptic terminals of various morphological types. Although large granular vesicles were usually present in the labeled axonal bulbs, no structural characteristic could be specifically ascribed to catecholaminergic neurons. It is suggested that exogenous norepinephrine bound to macromolecular complexes is present in all parts of catecholaminergic neurons and mainly concentrated within presynaptic axons.     

GABA UPTAKE IN RAT BRAIN SLICES: INHIBITION BY GABA ANALOGUES AND BY VARIOUS DRUGS

Abstract— 2-Hydroxy-, 2-chloro-, 2- and Cmethyl-GABA are linear competitive inhibitors of GABA uptake in rat brain slices. These analogues are thus potential substrates for the GABA transport system and possible'false transmitters'. 2-Hydroxy-GABA is the most potent inhibitor of GABA uptake yet described. No specific inhibitor of GABA uptake was revealed amongst the drugs tested.     

FREE 3-METHOXY-4-HYDROXYPHENYLETHYLENEGLYCOL IN THE CENTRAL NERVOUS SYSTEM OF THE RAT: SEMI-AUTOMATED FLUOROMETRIC ASSAY, TURNOVER AND EFFECTS OF DRUGS

A semi-automated fluorometric assay technique for free 3-methoxy-4-hydroxyphenylethyl-eneglycol (MOPEG) in rat brain and spinal cord is described. The method is based on a simple manually-performed two-step purification procedure using column chromatography on Sephadex G 10 and DEAE-Sephadex A 25 (borate form) respectively. After isolation MOPEG is converted into a fluorophore in a continuous flow system using ethylene diamine condensation in the presence of an oxidant. The MOPEG assay is highly sensitive (detection limit 2 ng/sample) and linear, with an overall recovery of approx 75%. Specificity is demonstrated by testing a number of compounds and confirmed by gas chromatography-mass spectrometry analysis. Treatment with clozapine and haloperidol (both neuroleptics), reser-pine (impairing intraneuronal storage) or phenoxybenzamine (α-adrenoceptor blocking agent) increased the content of MOPEG both in brain and spinal cord. Cerebral levels of MOPEG were decreased after injection of a single dose of the tricyclic antidepressant desipramine and after chronic destruction of the locus coeruleus by electrolytic lesion or by the administration of the neurotoxic drug 6-hydroxy-dopamine. Animals killed by microwave irradiation did not show lower MOPEG contents in brain than decapitated animals. These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated.     

DISTRIBUTION OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE TRANSAMINASE IN SEVERAL AREAS OF THE CENTRAL NERVOUS SYSTEM OF THE RAT

—The regional distributions of serine hydroxymethyltransferase (SHMT) and glycine transaminase (GT) have been determined in five areas of the CNS of the rat. The SHMT activity per mg protein varied in these areas in the following order: medulia-pons and spinal cord > cerebellum > midbrain > telencephalon. The GT activity per mg protein was essentially the same in the four brain areas, whereas, in the spinal cord it was lower. The activity of GT did not correlate with the glycine content (r=?0.45. P > 0.05). However, SHMT activity per mg protein was correlated with the glycine content in four regions (the telencephalon, midbrain, medulla-pons and spinal cord; r= 0.997, P < 0.05). When the activity of SHMT was expressed per relative number of mitochondria, the enzyme levels were correlated with the glycine content in all five areas (r= 0.952, P < 0.05). The distribution of SHMT was determined in the primary subcellular fractions of the CNS. The SHMT activity in these areas of the CNS appeared to be located predominately in paniculate structures, while only 1 to 4 per cent was found in the soluble fraction. The crude nuclear (P₁) and the crude mitochondrial (P₂) fractions contained 90–97 per cent of the activity. Subfractionation of P₂ pellets obtained from the telencephalon, medulla-pons and spinal cord indicated the SHMT activity was localized in both ‘free’ and occluded mitochondria.     

INHIBITION OF THE UPTAKE OF GABA AND RELATED AMINO ACIDS IN RAT BRAIN SLICES BY THE OPTICAL ISOMERS OF NIPECOTIC ACID

R(-)-Nipecotic acid was a more potent inhibitor than the S(+)-isomer of the uptake of GABA, (+)-nipecotic acid, and β-alanine in rat brain slices. (-)-Nipecotic acid was an order of magnitude more potent as an inhibitor of GABA uptake than as an inhibitor of β-alanine uptake, whereas the (+)-isomer was less selective. (–)-Nipecotic acid was a weak inhibitor of L-proline uptake and of rat brain acetylcholinesterase activity. Kinetic studies showed that both isomers of nipecotic acid were competitive inhibitors of GABA uptake when added at the same time as GABA, but non-competitive inhibitors when preincubated with the tissue for 15 min before addition of GABA. The apparent slope inhibition constants, which were not influenced by preincubation, indicated that (–)-nipecotic acid has an affinity for the carrier some 5 times higher than that for (+)-nipecotic acid. (–)-Nipecotic acid stimulated the release of preloaded radioactive GABA from rat brain slices. These observations indicate that (–)-nipecotic acid is a substrate-competitive inhibitor of GABA which combines with the GABA carrier and is taken up. (?)-Nipecotic acid and (+)-2,4-diaminobutyric acid, on the basis of their absolute structures and inhibition kinetics, are proposed to interact in a similar way with the GABA transport system.     

HIGH AFFINITY UPTAKE SYSTEMS FOR TAURINE IN TISSUE SLICES AND SYNAPTOSOMAL FRACTIONS PREPARED FROM VARIOUS REGIONS OF THE RAT CENTRAL NERVOUS SYSTEM. CORRECTION OF TRANSPORT DATA BY DIFFERENT EXPERIMENTAL PROCEDURES

Abstract— —The uptake of taurine into tissue slices of specific regions of the rat central nervous system (CNS) was compared with the uptake of taurine into synaptosomal fractions prepared from the corresponding regions. Two different techniques for performing control experiments were also compared: procedure I, correction for the uptake of taurine obtained from duplicate incubations but at 2°c and procedure II, correction of taurine uptake into extracellular or extrasynaptosomal space measured by inulin uptake experiments plus correction for diffusion (non-saturable) processes.
Kinetic analyses of the uptake data in tissue slices utilizing the procedure I correction technique indicate that six regions of the rat CNS (spinal cord, diencephalon, cortex, striatum, hippocampus, and midbrain) possess high affinity uptake systems (K_m values approx 60 μM or less). The K_m value for the cerebellum (105.4 ± 15.7 μM) is intermediate between a high and low affinity uptake system while the K_m value for the pons-medulla (210.0 12.4 μM) is considered to be low affinity. When procedure II techniques were utilized for correcting the uptake data all eight regions demonstrated high affinity uptake systems (11.8–73.2μM).
Synaptosomal fractions prepared from the spinal cord, pons-medulla, diencephalon, and midbrain demonstrate high affinity uptake systems (procedure I) for taurine (10.3–47.2 μM) while the hippocampus, cortex, striatum, and cerebellum have intermediate (but still high affinity) values (59.4–96.4 μM). High affinity uptake systems (8.2–79.8 μM) were obtained for all eight regions of the rat CNS when procedure II was utilized for correction of the data.     

EFFECTS OF INGESTION OF A CARBOHYDRATE-FAT MEAL ON THE LEVELS AND SYNTHESIS OF 5-HYDROXYINDOLES IN VARIOUS REGIONS OF THE RAT CENTRAL NERVOUS SYSTEM

—The concentrations of tryptophan, serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in spinal cord and most brain regions increase 2 h after fasted rats begin to consume a carbohydrate-fat meal: indole levels rise in all portions of the brain studied, but the increase is not statistically significant in the hypothalamus and corpus striatum. The rate at which the brain synthesizes 5-hydroxy-indoles (as estimated in vivo by measuring 5-hydroxytryptophan accumulation following an injection of the decarboxylase inhibitor RO4-4602) is also accelerated in all of the regions in which the experimental diet elevates tryptophan, 5-HT and 5-HIAA levels. These observations indicate that the previously reported increase in brain 5-hydroxyindole levels following consumption of a protein-free meal reflects accelerated serotonin synthesis, and occurs within both the cell bodies and the terminals of serotonin-containing neurons. It is possible that diet-induced changes in neuronal serotonin levels influence the quantities of the neurotransmitter released into synapses, either spontaneously or in response to drugs.     

EFFECT OF ZUCLOMIPHENE ON CHOLESTEROL FORMATION IN THE DEVELOPING CENTRAL NERVOUS SYSTEM OF THE RAT

Abstract— The effect of zuclomiphene, a hypocholesterolemic agent, on developing rat CNS cholesterol biosynthesis was examined. Sterol content and composition was studied in relation to age in four regions of the CNS, cerebrum, brain stem, spinal cord and cerebellum. Sterol content of all four regions was slightly lower in drug-treated animals than in controls. Brain stem and spinal cord were more susceptible to the effects of zuclomiphene than were cerebrum and cerebellum. Drug treatment resulted in the accumulation of desmosterol and zymosterol (5 x -cholesta-8,24-dien-3β-ol) in all CNS regions. After 15 days of drug treatment, desmosterol constituted more than 50% of the total sterol in the four examined regions. Six to 9% of the total sterol was zymosterol.
Examination by electron microscopy indicated only minimal morphological changes. Occasionally, neuronal membranous cytoplasmic inclusion bodies were evident.     

THE FORMATION AND STRUCTURE OF MYELIN SHEATHS IN THE CENTRAL NERVOUS SYSTEM

The development and structure of myelin sheaths have been studied in the optic nerves of rats and of Xenopus laevis tadpoles. Both potassium permanganate- and osmium-fixed material was examined with the electron microscope. In the first stage of myelinogenesis the nerve fibre is surrounded by a cell process which envelops it and forms a mesaxon. The mesaxon then elongates into a loose spiral from which the cytoplasm is later excluded, so that compact myelin is formed. This process is similar to myelinogenesis in the peripheral nervous system, although in central fibres the cytoplasm on the outside of the myelin is confined in a tongue-like process to a fraction of the circumference, leaving the remainder of the sheath uncovered, so that contacts are possible between adjacent myelin sheaths. The structure of nodes in the central nervous system has been described and it is suggested that the oligodendrocytes may be the myelin-forming cells.