REGIONAL DISTRIBUTION OF MONOAMINES AND THEIR METABOLITES IN THE HUMAN BRAIN

Abstract— Noradrenaline (NA), dopamine (DA). 5-hydroxytryptamine (5-HT), 4-hydroxy, 3-methoxy-phenylethylene glycol (MHPG), homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolylacetic acid (5-HIAA) were measured in twenty areas of post-mortem brain from ten psychiatrically and neurologically normal patients. There was a marked difference, which did not appear to be related to sex, medication, cause of death or time between death and dissection, in amine and metabolite concentrations between brains. In the cortex, 5-HT, MHPG, HVA. DOPAC and S-HIAA were approximately even in their distribution; NA and DA could not be detected. In sub-cortical areas there were clear differences in the distribution of the three amines accompanied by less marked differences in the distribution of their respective metabolites.     

ENZYMES ASSOCIATED WITH THE METABOLISM OF CATECHOLAMINES, ACETYLCHOLINE AND GABA IN HUMAN CONTROLS AND PATIENTS WITH PARKINSON''S DISEASE AND HUNTINGTON''S CHOREA

—Tyrosine hydroxylase (TH), dopa decarboxylase (DDC), glutamic acid decarboxylase (GAD), choline acetyltransferase (CAT), and acetylcholinesterase (AChE) were measured in 18–55 areas of brain from humans post mortem. Individuals meeting sudden and unexpected death (22), patients dying in hospital with non–neurological illness (6), Parkinson's disease (12), Huntington's chorea (8), terminal coma (6) or head injury (2) were included in the series. The absolute values obtained compared favourably with some previous human studies where high values for these enzymes were obtained, as well as with monkey and baboon data. The regional distributions of the enzymes were also comparable to those previously reported in human and animal studies. A number of important points with regard to human tissue seemed to emerge from the study. The mode of death was not a factor in enzyme levels in non–neurological and non-coma cases. Post mortem delay did not seem to be a major factor either even though a substantial decline in GAD, TH and DDC could be demonstrated in rats left several hours between sacrifice and removal of the brain for assay. Age had a highly significant effect in certain areas of brain. The decline typically followed a curvilinear pattern (activity = A/age + B with the sharpest drops being in the younger age groups). DDC seemed to be the enzyme most severely affected by age but all the enzymes showed declines in certain brain areas, while in other areas there was no significant decline. All the enzymes were very depressed by coma from illness except AChE. TH and DDC in the brain stem were, however, not affected in the head injury cases. The Parkinsonian cases showed a sharply decreased TH activity in the substantia nigra, caudate and putamen. There were decreases in GAD in the globus pallidus (GP) and substantia nigra with marginal decreases in the neostriatum. CAT levels in the extrapyramidal nuclei were normal. In Huntington's chorea there was a substantial decrease in GAD in all the extrapyramidal structures. There was a patchy loss of CAT in the neostriatum and locus coeruleus.     

REGIONAL DISTRIBUTION OF HOMOCARNOSINE, HOMOCARNOSINE-CARNOSINE SYNTHETASE AND HOMOCARNOSINASE IN HUMAN BRAIN

Homocarnosine (HCarn) content varied over a 6-fold range in different regions of autopsied human brain, being highest in the dentate nucleus and the inferior olive, and lowest in the caudate nucleus and mesolimbic system. HCarn content was similar in biopsied and autopsied frontal cortex. Very little if any carnosine (Carn) was present in human brain, except for the olfactory bulb, where Carn may have comprised 20% of the imidazole dipeptides present. Only HCarn was present in human CSF. HCarn-Carn synthetase enzyme activity in biopsy specimens of human frontal and temporal cortex was approx 10 times greater than has been reported for rat cerebral cortex. The enzyme synthesized Carn 3–5 times as rapidly as HCarn, when β-alanine (β-Ala) or GABA substrate concentrations were 10 MM. The synthetase was found to have an apparent K_m of 1.8 mM for β-Ala, and 8.8 mM for GABA. HCarn-Carn synthetase activity decreases rapidly after brain death, and was not detectable in autopsied brain specimens frozen more than 6 h after patients’deaths. Homocarnosinase activity was determined in brain, using L-[γaminobutyryl-1-¹⁴C]HCarn as substrate, and measuring radioactive GABA produced by hydrolysis of HCarn at pH 7.2 in the presence of Co²⁺ ions. Homocarnosinase activity was similar in biopsied and autopsied human cerebral cortex, and appeared to be stable for at least 10 h after death in unfrozen brain. Differences in the regional distribution of HCarn-Carn synthetase and homocarnosinase activities, as well as regional differences in GABA content in human brain, do not readily account for regional differences in HCarn content, nor do they suggest a physiological role for HCarn.     

THE REGIONAL DISTRIBUTION OF THE POLYAMINES SPERMIDINE AND SPERMINE IN BRAIN

Abstract— The distribution in brain of the polyamines spermidine and spermine is described in the rat, dog, sheep, rabbit and in man. The distribution pattern was about the same in all the species, spermidine concentration being highest in areas rich in white matter. The concentration of spermine was lower than that of spermidine and showed less variation from area to area. Rat brain polyamine content was the same in rats killed by decapitation as in those killed by rapid freezing in liquid nitrogen and was also unchanged up to 48 h after the death of the animal.     

THE REGIONAL DISTRIBUTION OF SIALOGLYCOPROTEINS, GANGLIOSIDES AND SIALIDASE IN BOVINE BRAIN

Abstract— Sialoglycoproteins and gangliosides were characterized in various bovine brain regions by determining the amount of sialic acid. Expressed per g dry weight, the gangliosidic sialic acid ranged from 11·20 to 1·93 μmol and the glycoprotein sialic acid from 8·93 to 1·84 μmol in grey and white matter respectively (values not corrected for incomplete release and breakdown during hydrolysis). Both the sialoglycoproteins and the gangliosides occur in highest concentration in areas predominating in neuronal cell bodies (cerebral grey, cerebellar grey, caudate nucleus). The lowest concentrations are found in those areas, consisting largely of myelinated fibre tracts and glial cells (pons, medulla, corpus callosum, cerebral white). Relative to the gangliosides the sialoglycoproteins are somewhat more concentrated in white matter.
The sialidase activity was investigated with endogenous substrate as well as with additional gangliosides or sialoglycopeptides. In all conditions the activity was much greater in grey matter than in white matter. The regional sialidase distribution more or less parallels the distribution of sialic acid in the various regions. At high substrate level the sialoglycopeptides inhibit the sialidase activity. There are indications that gangliosides are a far better substrate for brain sialidase than glycoproteins or glycopeptides. The possible significance of this phenomenon is discussed.     

THE REGIONAL AND SUBCELLULAR DISTRIBUTION OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Catechol-O-methyl transferase (COMT) activities determined in different regions of rat brain showed small variations. Highest activities were found in the hypothalamus and corpora quadrigemina, and lowest activities in the hippocampus and corpus striatum. The regional distribution of COMT was thus at variance with the distribution of DOPA decar- boxylase in this study and with the distribution of catecholamines and tyrosine hydroxylase reported in the literature. Determinations of the subcellular distribution of COMT in rat forebrain showed that 50 per cent of the activity was recovered in the high speed supernatant fluid and about 33 per cent in the crude mitochondrial fraction. Further separation of the latter by discontinuous sucrose gradients showed that the particulate COMT was found in the synaptosomal fraction in an occluded form. Full enzyme activity was only obtained after treatment with a detergent or after resuspension in water. After hypo-osmotic rupture of the crude mitochondrial fraction, COMT was recovered in the cytoplasmic fraction. The subcellular distribution of COMT was very similar to the ones of lactate dehydrogenase and DOPA decarboxylase. The proportions of soluble COMT obtained from homogenates of various regions of the brain differed from that of choline acetyl transferase and DOPA decarboxylase but were similar to that of lactate dehydrogenase. In conclusion, COMT is a cytoplasmic enzyme almost evenly distributed in the CNS. Its distribution does not resemble the distributions of the catecholamines or of the enzymes participating in the synthesis of catecholamines.     

PROPERTIES AND REGIONAL DISTRIBUTION OF TRYPTOPHAN HYDROXYLASE IN THE CHICKEN BRAIN

Tryptophan hydroxylase in young chicken brain had a pH optimum of 7.5–8, depending on the buffer used. It had apparent K_m values for tryptophan and tetrahydrobiopterin of 49 μM and 32 μM respectively. The enzyme in chicken brain, but not rat brain, was cold-shock labile but was stable for up to 4 days at — 20°C. Lability was observed both in tissues and homogenates of these tissues subjected to cold shock, but the extent of loss of activity varied between brain regions. Supernatant fractions did not lose activity after cold shock. The highest level of tryptophan hydroxylase was found in the rostral region of the chicken brainstem. High levels were also found in the caudal region of the brainstem, the midbrain, thalamus, caudate and cerebral cortex. The cerebellum and optic chiasma contained only traces of activity.     

REGIONAL DISTRIBUTION OF AMINO ACIDS IN HUMAN BRAIN OBTAINED AT AUTOPSY

Abstract— Contents (μmol/g wet wt.) of 35 free amino acids and related compounds were measured in 12 different regions of each of five human brains. Specimens were obtained at autopsy from patients who died suddenly without previous brain disease. These data may serve for later comparison with contents of amino compounds in similar regions of the brains of patients dying with various neurological or psychiatric disorders.
There were marked and consistent differences in the regional distribution of the following eight compounds: γ-aminobutyric acid, homocarnosine, glutamic acid, aspartic acid, taurine, cystathionine, glycerophosphoethanolamine, and phosphoethanolamine. These differences suggest that some of these compounds may have special physiological roles, including the possible mediation of synaptic transmission.
Human brain contains two previously unreported compounds, the mixed disulphide of cysteine and glutathione and α-(γ-aminobutyryl)-lysine. The latter dipeptide occurs in much higher concentrations in human brain than in the brains of lower mammals.     

REGIONAL AND SUBCELLULAR DISTRIBUTION OF AMINOTRANSFERASES IN RAT BRAIN

Abstract— Aminotransferase activity was measured in various areas of the nervous system of the rat (cortical grey matter, midbrain, corpus callosum, spinal cord and sciatic nerve) and in subcellular fractions of rat brain (nuclei, mitochondria and cytosol). Activity was low or absent in the sciatic nerve relative to that in the other areas, with the exception of incubation of glutamate with oxaloacetate (25 per cent of the activity found in brain) and of asparagine with 2-oxoglutarate (65 per cent of the activity found in brain). The distribution of enzymic activity was not homogeneous; alanine-2-oxoglutarate aminotransferase was highest in cortical grey matter; leucine- and GABA-2-oxoglutarate aminotransferases were highest in midbrain. Incubation of phenylalanine or tyrosine with 2-oxoglutarate gave similar activities in grey matter and midbrain. Activity generally was higher in the grey matter than in corpus callosum or spinal cord. However, incubations of methionine with 2-oxoglutarate, or glutamine with glyoxylate, gave similar activities in the three areas studied from the brain, whereas incubations of glutamate with glyoxylate gave highest activity in the corpus callosum. Only incubations of asparagine with 2-oxoglutarate, and glutamate with glyoxylate, gave significant activity in the nuclear subcellular fraction. Aminotransferase activity of phenylalanine, tyrosine or GABA with 2-oxoglutarate, or ornithine or glutamine with glyoxylate, was localized to mitochondria. The remaining reactions studied (glutamate with oxaloacetate; leucine, alanine, methionine or asparagine with 2-oxoglutarate and glutamate with glyoxylate) demonstrated activity in both the mitochondrial fraction and the soluble supernatant fraction.     

EFFECT OF OXOTREMORINE ON THE APPARENT REGIONAL TURNOVER OF ACETYLCHOLINE IN MOUSE BRAIN

The effect of oxotremorine (1 mg kg^-1 i.p.) on the steady state concentration of acetylcholine (ACh) and choline (Ch) and the transformation of radioactive choline ([³H]Ch) was studied in different brain regions of the mouse following death by microwave irradiation of the head. Oxotremorine significantly increased the concentration of endogenous ACh in the cortex and hippocampus and of endogenous Ch in the cortex. Pretreatment with atropine (5 mg kg^-1 i.p.) prevented the increase in ACh. The biosynthesis of radioactive ACh ([³H]ACh) was decreased in all brain regions. Atropine (5 mg kg^-1) pretreatment counteracted this effect of oxotremorine (1 mg kg^-1), while methylatropine (5 mg kg^-1) had no effect except in the striatum. A calculation of the apparent turnover rate of ACh showed that oxotremorine (1 mg kg^-1) decreased the turnover in the cortex, hippocampus, midbrain. and striatum.     

DEVELOPMENTAL CHANGES OF ENZYMES ASSOCIATED WITH ENERGY METABOLISM AND THE SYNTHESIS OF SOME NEUROTRANSMITTERS IN DISCRETE AREAS OF HUMAN NEOCORTEX

Developmental changes in pyruvate kinase (PK), lactate dehydrogenase (LDH), α-glycerophosphate dehydrogenase (α-GPD), glucose-6-phosphate dehydrogenase (G6P), succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), choline acetyltransferase (CAT), glutamate decarboxylase (GAD), activities were measured in early autopsy material in the following areas of human neocortex: area 4 (motor cortex), area 17 (visual cortex), area 40 (gyrus supramarginalis, associative cortex). Changes with age were analysed from 8 fetal weeks to adult age. The important points emerging from this study are: 1. Enzymes associated with glycolytic pathways show a high activity in early fetal period, decline through to the end of the active phase of neurogenesis and then, rise continuously to the end of the first year of life. 2. G6P, an enzyme associated with the oxidative segment of the pentose phosphate pathway, shows a high activity at 8 fetal weeks and, gradually declines through to the end of the active phase of neurogenesis; it then either does not change significantly (motor cortex) or increases slightly. 3. Enzymes related to the tricarboxylic pathway have a low level of activity throughout the first half of gestation, and then rise markedly during the last fetal months and the first year after birth. SDH increase is of much higher magnitude (× 10) than that observed for glycolytic enzymes (×4). For the enzymes of oxidative metabolism, motor cortex is the most advanced area, while associative cortex matures more slowly. 4. CAT activity at 8 fetal weeks is high in visual cortex and declines to the fifth month. After that time, there is a continuous rise until the age of 11 years. Although the time pattern in reaching the adult value is different in motor and associative cortex, there exists a continuous increase from fetal onset to adult level in both areas. Developmental changes in GAD activity are very unusual. The development of activity lags behind that of CAT and commences after birth. After a steady rise in the first year of life, the activity decreases after this age.     

ONTOGENESIS AND REGIONAL DISTRIBUTION OF HISTAMINE AND HISTAMINE-N-METHYLTRANSFERASE IN THE GUINEA PIG BRAIN1

The ontogenetic development and the regional distribution of histamine (HA) and histamine-N-methyltransferase (HMT, EC 2.1.1.8) in guinea pig brain and pituitary gland were studied. The samples were taken every fourth day beginning on the 28th foetal day. The HA concentration in the brains of the youngest foetuses was almost undetectable. A significant increase in HA concentrations occurred between days 40 and 44, which coincides with the period of rapid growth of nerve cell processes in this species. After this, a steep increase continued to the end of gestation in the hypothalamus, and to a lesser degree in the medulla-midbrain and in the forebrain. In all parts of the brain the adult HA levels were reached by the time of birth. The HMT activity increased 15-fold from the 28th foetal day to the adult and reached ca. 80% of the adult activity by the time of birth. The HMT activity developed earlier in the midbrain than in the forebrain or in the cerebellum, but after the birth the regional distribution of HMT was fairly even. In the pituitary gland the HA concentration and HMT activity increased hundredfold and tenfold, respectively. The developmental patterns of HA and HMT in the guinea pig brain give support to the concept that HA might be a neurotransmitter in the brain.     

PROPERTIES AND REGIONAL DISTRIBUTION OF HISTIDINE DECARBOXYLASE IN RAT BRAIN

—Properties of the histamine-forming enzyme in rat brain were studied, utilizing a sensitive fluorometric assay. The optimum pH was related to substrate concentration and found to be6·4 at 10^?2m -histidine; the apparent Km was about 4·10^?4m ; enzyme activity was inhibited by α-hydrazino -histidine and brocresine but was not affected by α-methyl DOPA or benzene. These different data suggest that the 'specific’histidine decarboxylase (EC 4.1.1.22)—and not the aromatic l -aminoacid decarboxylase—is involved. Determination of enzyme activity and histamine level in different areas of the rat brain revealed important regional differences, the two values being roughly parallel.     

REGIONAL LEVELS OF GABA IN THE BRAIN: RAPID SEMIAUTOMATED ASSAY AND PREVENTION OF POSTMORTEM INCREASE BY 3-MERCAPTO-PROPIONIC ACID

Abstract— A simple and rapid semiautomated assay for GABA in central nervous tissue is described. The method is based on a simple manual procedure of isolating GABA from tissue extracts on small CM Sepharose Cl 6B columns, followed by an automated fluorimetric detection (continuous flow system) with o -phthalaldehyde (OPA) and β-mercapto-ethanol (β-ME) at an alkaline pH. GABA is separated from other compounds that fluoresce in our detection system. By using low concentration of OPA and β-ME and allowing only a short reaction time with these reagents, the detection is specific towards GABA. The detection limit of the assay is 1 nmol.
A procedure is described for the prevention of postmortem GABA increase in rat and mouse brain by intravenous injection of 3-mercapto-propionic acid (1.2 nmol/kg) 2min before decapitating the animal. This treatment and microwave irradiation result in similar GABA levels in mouse brain and substantia nigra tissue from rat brain. We found a great conformity in regional GABA levels in the rat and the mouse brain.     

REGIONAL AND SUBCELLULAR DISTRIBUTION OF PROTEIN CARBOXYMETHYLASE IN BRAIN AND OTHER TISSUES

Protein carboxymethylase, an enzyme that transfcrs the methyl group of S-adenosyl-L-methionine to carboxyl groups of proteins and endogenous acceptor proteins were examined in nerve and endocrine tissues. The highest protein carboxymethylase activity was found in the brain, followed by the testis, pituitary and heart. On the other hand, the tissue with the highest level of endogenous substrate(s) was the pituitary. The nearly identical specific activity ratio for two different protein substrates in all tissues examined, suggests that one enzyme is responsible for carboxymethylase activity in different tissues. The subcellular distribution of the enzyme in brain showed a high concentration in the soluble fraction, presumably representative of the enzyme in the cytosol of cell bodies. Considerable enzyme activity was also found in brain synaptosomes which was increased by osmotic lysis. Protein carboxymethylase was shown to accumulate proximally to a ligation of the rat sciatic nerve. A possible physiological role for protein carboxymethylase in neuronal function is discussed.