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.     

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.     

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.     

DECARBOXYLATION OF EXOGENOUS l-5-HYDROXYTRYPTOPHAN AFTER DESTRUCTION OF THE CEREBRAL RAPHE SYSTEM

Abstract— l -5-Hydroxytryptophan ( l -5-HTP) was administered intravenously to rats (12 mg/kg) after inhibition of the peripheral aromatic l -amino acid decarboxylase with l -α-hydrazino-α-methyl dopa (MK 486). The accumulation of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid in the cerebral cortex was measured 1, 2 and 4 h after injection of 5-HTP with automated assay techniques. Besides controls two groups of rats were studied: rats after inhibition of tryptophan-5-hydroxylase with p -chlorophenylalanine (pcpa) and subjects with a chronic lesion in the area of the raphe nucleus. The net accumulation of both measured 5-hydroxyindoles was diminished in rat cerebral cortex after degeneration of 5-HT containing nerve endings, compared with control animals and pcpa-treated rats. These results indicate that the formation of 5-HT in the cerebral cortex from exogenous l -5-HTP, after inhibition of the peripheral aromatic amino acid decarboxylase, occurs predominantly in 5-HT containing nerve endings possibly by a specific 5-HTP-decarboxylating enzyme.     

EFFECT OF THE ADMINISTRATION OF L-5-HYDROXYTRYPTOPHAN AND A MONOAMINE OXIDASE INHIBITOR ON GLUCOSE METABOLISM IN RAT BRAIN

The effects of the presence of large amounts of 5-HT and of its precursor 5-HTP in brain on cerebral utilization of glucose were studied. [U-¹⁴C]Glucose was injected to fed rats that had previously been treated with L-5-HTP, L-5-HTP and an inhibitor—N-[β-(2-chlorophenoxy)-ethyl]-cyclopropylamine hydrochloride (Lilly-51641)-of MAO, or Lilly-51641 alone. Such treatment increased the concentrations of 5-HTP and 5-HT in the brain. After treatment with 5-HTP and Lilly-51641, and to a lesser extent with Lilly-51641 alone, the concentration of glucose in plasma was increased. However, the uptake of glucose by the brain did not appear to be proportionately increased, and this suggested an impairment in this mechanism. After the administration of Lilly-51641 alone and more especially of Lilly-51641 plus 5-HTP, the concentration of glucose in the brain was increased. This increase was thought to be due to an impairment of glucose utilization, because the flux of ¹⁴C from glucose to amino acids in the brain was reduced. The concentrations of most major amino acids in the brain were not greatly affected by these treatments. GABA and alanine concentrations in the brain were modestly increased after treatment with 5-HTP alone or in combination with Lilly-51641. The present results suggest that the metabolism of glucose to amino acids in the brain is altered when the concentration of 5-HTP, or more especially that of 5-HT, in the brain is increased.     

MYELIN IN THE CENTRAL NERVOUS SYSTEM AS OBSERVED IN EXPERIMENTALLY INDUCED EDEMA IN THE RAT

The compact arrangement of cells in the normal white matter of the brain makes an analysis of cellular architecture difficult. To overcome this difficulty, cerebral edema was induced in rats by means of the unilateral intracerebral implantation of silver nitrate. Within 48 hr, the brains were fixed by perfusion with glutaraldehyde followed by immersion in Dalton's chrome-osmium. Sections of the callosal radiations were studied in the electron microscope. The untreated hemisphere appeared entirely unaltered, whereas in the edematous hemisphere the edema fluid separated individual cell processes and small groups of them. The myelin sheaths and their relationships to the axons appeared essentially unaltered. In this material, analysis of cellular architecture was relatively easy, and the widely held theory of spiral wrapping could be confirmed. In addition, several other aspects of the myelin and myelin-forming cell relationships became apparent in the edematous tissue. Most of these were later confirmed by extensive and careful study of the nonedematous tissue. These included the presence of occasional isolated cytoplasmic areas in myelin and the presence of two complete sheaths around a single axon. Other observations, such as the appearance of mitochondria and dense bodies within the outer loop and the separation of myelin lamellae, are apparently limited to the edematous tissue.     

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.     

ARYLSULPHATASE A (EC 3.1.6.1) ACTIVITY IN RAT CENTRAL NERVOUS SYSTEM DURING EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS

Abstract— The activity of arylsulphatase A (EC 3.1.6.1) was measured in several brain sub cellular fractions during experimental allergic encephalomyelitis in rats. In the acute stage there was a slight but significant increase in the brain homogenate in comparison to that from control animals. The alteration appeared to be localized in a subcellular fraction con stituted mainly by myelin fragments. The arylsulphatase A activity returned to the control level in the recovery stage.     

HISTOCHEMICAL DEMONSTRATION OF ACID RIBONUCLEASE IN THE CENTRAL NERVOUS SYSTEM

Abstract— The staining pattern of acid ribonuclease activity in the nervous tissues of rats has been studied with different fixatives and incubation media. Certain modifications in the method adopted (V orbrodt , 1961) produced a definite improvement in the localization as well as distinct subcellular distribution at the pHs studied. The modifications introduced consisted of preincubation of tissue sections with prostatic acid phosphatase, omission of acid phosphatase from the standard incubation medium and its addition at a later stage of incubation. This avoids much of the diffused cytoplasmic and filamentous background staining at pH 5.2–5.8, eliminates the irregular nuclear staining at pH 4.5 and replaces the strong nuclear positivity at pH 5.2 by a fine granular dispersion.
A study of the enzyme activity of subcellular fractions at the different pHs used was performed to confirm the histochemical localization.     

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.