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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

THE ALTERATION OF THE REGENERATIVE ACTIVITY AND THE CELL CYCLE OF PARTIALLY HEPATECTOMIZED RAT LIVER FOLLOWING ADMINISTRATION OF d-GALACTOSAMINE

A single injection of d-galactosamine given to rats at different times after partial hepatectomy (PH) changes the pattern of regenerative proliferation. When administered during the pre-replicative phase of regeneration, the onset of DNA synthesis and the increase in labelling index after injection of ³H-thymidine are delayed by about 12 hr. The injection of d-galactosamine at 24 hr after PH inhibits the drop in DNA synthesis occurring normally during the following 12 hr period. This was detected by a high labelling index and by an increased specific activity of DNA. The findings indicate a lengthening of the S phase, while G₂ and M remain normal. Two modes of action of d-galactosamine on the cell cycle are discussed.     

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.     

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.