DAS MEDIKAMENTÖSE PARKINSONOID BEI DER RATTE ALS MODELL FÜR BIOCHEMISCHE UNTERSUCHUNGEN DES PARKINSONSYNDROMS BRAIN AMINOACIDS IN PHENOTHIAZIN-INDUCED PARKINSONISM DRUG-INDUCED PARKINSONISM IN THE RAT—A MODEL FOR BIOCHEMICAL INVESTIGATIONS OF THE PARKINSON-SYNDROME

Abstract The combined application of Majeptil² and Randolectil³ provokes an extra-pyramidal syndrome in rats. In four investigated brain areas (‘cortex’, ‘striatum’,‘thalamus’and cerebellum) the concentrations of the‘inhibitor’amino acids—glycine and γ-amino-butyric acid—were found to be increased, although a reduction of the amount of glutamic acid was observed.     

GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

EFFECTS OF POTASSIUM ON INDICATOR SPACES AND FLUXES IN SLICES OF BRAIN CORTEX FROM ADULT AND NEW-BORN RATS

—Inulin, sucrose and chloride spaces were measured in slices of brain cortex from adult and from new-born rats incubated in‘balanced', potassium-rich and sodium-rich media. The efflux of the radioactive markers was followed in the two first media and the following results were obtained: (1) In brain slices from new-born rats inulin and sucrose spaces were of identical magnitude (35 per cent). The space magnitude was essentially unaffected by excess potassium. The chloride space was somewhat larger than the inulin (sucrose) space, and the difference increased continuously but relatively slightly with the external potassium concentration. By far the largest amount (i.e. about 90 per cent) of the efflux of radioactive inulin, sucrose and chloride occurred from a rapidly exchanging compartment during incubation in both ‘balanced’ and potassium-rich media. (2) In brain slices from adult rats the inulin space (35 per cent) was significantly smaller than that of sucrose (50 per cent) and of chloride (65 per cent); it seemed to represent the extracellular space relatively well although 10 per cent of the efflux occurred from a slowly exchanging (probably intracellular) compartment. High concentrations of potassium led to a reduction of the inulin space which was probably a result of the concomitant intracellular swelling. The hyperosmolarity per se did not affect the space magnitude, but an increase of the sodium concentration exerted a competitive inhibition of potassium effects on the inulin space. Of the sucrose efflux, 20 per cent occurred from a slowly exchanging compartment in both ‘balanced’ and potassium-rich media, and 30 per cent of the chloride exchanged with this compartment when the tissue was incubated in a ‘balanced’ medium. An increase of the external potassium concentration caused a drastic increase of the chloride space and a reduction of the slowly exhanging fraction of chloride efflux to less than 10 per cent.     

Elevation of cyclic GMP levels in central nervous system by excitatory and inhibitory amino acids

—Guanosine 3′,5’cyclic monophosphate (cyclic GMP) levels in incubated slices of mouse cerebellum are increased 10-fold by glutamate and two-to three-fold by glycine or γ-aminobutyric acid (GABA). Glutamate also produces a 10-fold increase in adenosine 3′,5’cyclic monophosphate (cyclic AMP) in the same tissue. However, GABA decreases cyclic AMP levels 30-40 per cent, and glycine produces only a transient 50 per cent accumulation of this cyclic nucleotide. Theophylline slightly augments the accumulation of cyclic GMP produced by all three amino acids but markedly attenuates the accumulation of cyclic AMP produced by glutamate. In the absence of Ca²⁺, none of the three amino acids has any effect on cyclic GMP levels, and glutamate produces only a 50 per cent rise in cyclic AMP levels. The decrease of cyclic AMP levels produced by GABA is not affected by theophylline or by the absence of Ca²⁺. These data suggest an involvement of both cyclic GMP and cyclic AMP in the neurochemical actions of glutamate, GABA and glycine.     

Decreased levels of free d-aspartic acid in the forebrain of serine racemase (Srr) knock-out mice

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor is synthesized from l-serine by serine racemase (SRR). A previous study of Srr knockout (Srr-KO) mice showed that levels of d-serine in forebrain regions, such as frontal cortex, hippocampus, and striatum, but not cerebellum, of mutant mice are significantly lower than those of wild-type (WT) mice, suggesting that SRR is responsible for d-serine production in the forebrain. In this study, we attempted to determine whether SRR affects the level of other amino acids in brain tissue. We found that tissue levels of d-aspartic acid in the forebrains (frontal cortex, hippocampus and striatum) of Srr-KO mice were significantly lower than in WT mice, whereas levels of d-aspartic acid in the cerebellum were not altered. Levels of d-alanine, l-alanine, l-aspartic acid, taurine, asparagine, arginine, threonine, γ-amino butyric acid (GABA) and methionine, remained the same in frontal cortex, hippocampus, striatum and cerebellum of WT and mutant mice. Furthermore, no differences in d-aspartate oxidase (DDO) activity were detected in the forebrains of WT and Srr-KO mice. These results suggest that SRR and/or d-serine may be involved in the production of d-aspartic acid in mouse forebrains, although further detailed studies will be necessary to confirm this finding.     

Effect of acetyl-l-carnitine on extracellular amino acid levels in vivo in rat brain regions

Acetyl-l-carnitine (ALCAR) was found to have beneficial effects in senile patients. In recent years many of its effects on the nervous system have been examined, but its mechanism(s) of action remains to be elucidated. We previously reported that it causes release of dopamine in the striatum. In the present paper we report that ALCAR, when administered at intracerebral sites via microdialysis, stimulates the release of amino acids in a concentration-dependent and regionally heterogeneous manner. The effect was strong in the striatum and cerebellum, less so in the frontal cortex, and weak in the thalamus. Seven amino acids were measured: the increase in the level of aspartate, glutamate, and taurine was substantial, and the increase in the level of glycine, serine, threonine, alanine, and glutamine in the microdialysate was minor. The stimulatory effect of ALCAR on the release of amino acids in the striatum was inhibited by the muscarinic antagonist atropine, but was not inhibited by the nicotinic antagonist mecamylamine. The effect of ALCAR on the levels of most of the amino acids tested was independent of the presence of Ca²⁺ in the perfused. These results indicate that ALCAR, when administered intracerebrally at fairly high concentrations, can affect the level and the release not only of such neurotransmitters as acetylcholine and dopamine, but also of amino acids. The mechanism of action of ALCAR on the release of cerebral amino acids may involve the participation of muscarinic receptors or may be mediated through the release of dopamine, but the lack of Ca²⁺ dependence indicates a release from the cytoplasmic amino acid pool, possibly through the effect of ALCAR on cell membrane permeability.     

Experimental alcoholism in rats: protein synthesis in subcellular fractions from cerebellum, cerebral cortex and liver after long term treatment

Abstract— The incorporation in vivo of [³H]leucine into protein from subcellular fractions was determined in rats chronically ingesting 15 per cent ethanol for 8 months. Mitochondrial, microsomal and cell sap fractions from cerebellum, cortex cerebri and liver were investigated. The results showed a minor over-all depression of protein synthesis in cerebellum and cortex cerebri and a slight stimulation of the incorporation of leucine into protein from liver subcellular fractions. If the animals were abstinent 24 h before injection of the isotope, the incorporation of labelled amino acids into protein was markedly increased in cerebellum and cerebral cortex but not in liver.     

EFFECT OF NOREPINEPHRINE ON 32P INCORPORATION INTO INDIVIDUAL PHOSPHATIDES IN SLICES FROM DIFFERENT AREAS OF THE GUINEA PIG BRAIN1

Abstract—

• 1 Incorporation of ³²P into phosphatidic acid was significantly increased in the presence of an appropriate concentration of norepinephrine, either 0.1 mm , or 1 mm , or both, in slices of guinea pig cerebellar cortex, olfactory bulbs, cerebral cortex, hypothalamus and thalamus; it was not significantly affected at either concentration of norepinephrine in slices of corpus striatum and the posterior and anterior colliculi.
• 2 Incorporation of ³²P into phosphatidylinositol was significantly increased in the presence of norepinephrine in a concentration of either 0.1 mm , or 1 mm , or both, in three of the areas studied—the olfactory bulbs, cerebral cortex, and thalamus.
• 3 There was no significant effect of 0.1 mm -norepinephrine on ³²P incorporation into phosphatidylcholine or phosphatidylethanolamine plus phosphatidylserine in any of the areas studied. Norepinephrine (1 mm ) significantly inhibited ³²P incorporation into phosphatidylcholine in cerebral cortex, hypothalamus, corpus striatum and anterior colliculus; ³²P incorporation into phosphatidylethanolamine plus phosphatidylserine was inhibited by 1 mm -norepinephrine in cerebral cortex, hypothalamus, and posterior and anterior colliculi.
• 4 In slices of cerebellar cortex, 0.1 mm and 1 mm -norpinephrine did not significantly affect either the specific activity or the level of nucleotide ～ P in the tissue.
• 5 The physiological significance of changes in phosphatide metabolism in response to neurotransmitters in the central nervous system is discussed.

     

DEVELOPMENTAL CHANGES IN Na+, K+ AND ATP AND IN THE LEVELS AND TRANSPORT OF AMINO ACIDS IN INCUBATED SLICES OF RAT BRAIN

Abstract—

• 1 Upon incubation, slices of brain tissue took up fluid; the degree of swelling increased with increasing age. No sweiling occurred in slices from foetal brain. Since this swelling was associated with increases in the inulin space, the percentage of inulin space in slices at the end of incubation increased during brain development.
• 2 Most of the capacity for ion transport seemed to be absent from foetal brain. In vivo and in slices, Na⁺ was very high and K⁺ was very low in comparison to levels at other ages. There was a rapid change around birth, but no significant change at later ages. Upon incubation, Na⁺ levels increased in other slices, but not in slices of foetal brain.
• 3 Upon incubation of the slices, ATP levels were restored to levels close to those in the living brain; there were no significant alterations in available energy during development to explain changes in amino acid transport.
• 4 The composition of the free pool of cerebral amino acids in vivo changed with development, with some compounds (glutamic acid and related compounds) increasing, others (mostly‘essential’amino acids) decreasing, with age. These changes were not linear with time, and the level of a compound might exhibit several peaks during development.
• 5 The uptake (influx) of taurine, glutamate and glycine into brain slices increased rapidly during the foetal and early neonatal periods, reached a maximum between 2 and 3 weeks of postnatal age and then declined to adult levels. The levels of steady-state uptake with glycine also exhibited a maximal peak at 2-3 weeks of postnatal age. Steady-state uptake of taurine and glutamate reached adult levels by about 3 weeks of age.
• 6 The pattern of inhibition of amino acid transport by two specific amino acid analogues changed during development for some amino acids (GABA, glycine and glutamate), indicating an alteration in substrate specificity.
• 7 The results demonstrate complex changes in cerebral amino acid transport during development, with several maxima or minima and with changes in specificity for at least some compounds.

     

METABOLIC COMPARTMENTATION IN THE BRAIN: EFFECTS OF A CENTRAL NERVOUS SYSTEM DEPRESSANT, 1-HYDROXY-3-AMINO-PYRROLIDONE-2

—The effect of 1-hydroxy-3-aminopyrrolidone-2(HA-966), a CNS depressant, was studied on the metabolism of [¹⁴C]glucose and [³H]acetate in the brain in mice. HA-966 had a marked effect on glucose metabolism. The conversion of glucose carbon into amino acids associated with the tricarboxylic acid cycle (‘cycle’) was severely reduced, while the concentration of brain glucose was approximately doubled. Relative to the specific radioactivity of glucose in the brain, the specific radioactivity of alanine was 60–70 per cent of the control, indicating a reduction in the rate of glycolysis, and those of the‘cycle’amino acids were also lowered. A reduction in‘cycle’flux of 30–35 per cent was estimated. It was established that the depressed glucose utilization flux was not due to either impaired uptake of glucose from blood to brain or to hypothermia. In contrast to [¹⁴C]glucose, there was no change in the labelling of the amino acid fraction from [³H]acetate, which is preferentially metabolized in the 'small’compartment believed to be associated with glia. Thus it seems that CNS depression caused by HA-966 resulted in a selective decrease in energy production in the‘large’metabolic compartment where glucose is oxidized preferentially and which is believed to be associated with neuronal structures. The results also suggested that communication between the metabolic compartments mediated via glutamine and GABA was reduced, since the labelling from [³H]acetate of glutamine was increased and that of GABA decreased by HA-966.     

FREE AMINO ACIDS AND RELATED COMPOUNDS IN FIVE REGIONS OF BIOPSIED CAT BRAIN

Abstract— Contents (μmol/g wet wt.) of 34 free amino acids and related compounds were measured in grey matter from three areas of cerebral cortex, from the cerebellum, and from the caudate nucleus in unanaesthetized cats with classical cerveau isolé preparations. Brain specimens were frozen in liquid nitrogen within 10 s of removal; thus, the values found were expected to approximate those which occur in living cat brain. Levels of most of the compounds measured were lower than those previously reported for the cat. In the case of GABA, alanine, and ethanolamine, the lower values found seemed attributable to the rapid freezing of brain tissue, and may more closely approximate levels occurring in living cat brain. On the other hand, the relatively low levels of aspartic and glutamic acids found may have resulted from use of the cerveau isolé preparation. Little difference in levels of amino compounds was found among the three cerebral cortical areas examined. However, there were significant differences in the contents of a number of amino acids between cerebral cortex and the cerebellum or caudate nucleus. These differences resembled those previously observed in autopsied human brain. The content of GABA was two-fold higher in biopsied cat cortex than in biopsied human cortex, whereas the content of cystathionine was only 10 per cent of that in human cortex. Homocarnosine and α-(γ-aminobutyryl)-lysine, two GABA-containing dipeptides found in relatively large amounts in human brain, were not detectable in cat brain. Living cat brain contained two amino acids not previously reported for this species:putreanine and ɛ- N -methyllysine.     

THE COMPARTMENTATION OF GLUTAMATE AND ITS METABOLITES IN FRACTIONS OF NEURON CELL BODIES AND NEUROPIL; STUDIED BY INTRAVENTRICULAR INJECTION OF [U-14C]GLUTAMATE

Abstract—

• 1 The in vivo metabolism of glutamate in rat neuron cell bodies and neuropil was studied after intraventricular injection of (U-¹⁴C)glutamic acid followed by separation of the tissue into neuronal and neuropil fractions.

• 2 The losses of amino acid and of radioactivity during the fractionation were equivalent. Recoveries were: glutamate, 32; glutamine, 15; aspartate, 25; GABA, 41; alanine, 30 per cent. In the washed cell fractions glutamine was 45 per cent and alanine 132 per cent higher in the neuronal fraction, glutamate was 62, GABA 77 and aspartate 95 per cent of neuropil levels. This contrasted with results obtained previously for in vitro incorporation. Calculation from these results indicated that 28 per cent of the original cell suspension was neuronal, 72 per cent neuropil. In the final cell preparations, 29 per cent of the neuron cell bodies and 26 per cent of the neuropil were recovered.

• 3 Specific activity of glutamate in the neuronal fraction 15 min after injection was higher than in the original suspension, but had declined to 30 per cent of its initial value by 2 h. In the neuropil, specific activity of glutamate was below that of the cell suspension at 15 min, but at later times rose above it by up to 40 per cent.

• 4 Radioactivity was detected in aspartate and glutamine 15 min after injection and GABA by 60 min after injection. In the original cell suspension the specific activity of glutamine was higher than that of glutamate at all times (the Waelsch effect) but aspartate and GABA were lower than glutamate.

• 5 In the neuronal fraction the specific activity of glutamine was below that of glutamate at all times, indicating a precursor-product relationship. In the neuropil fraction, glutamine specific activity remained above glutamate for the first hour.

• 6 These results are discussed in relation to the interpretation of the Waelsch effect in terms of metabolic compartmentation.

     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

Contents of several amino acids in the cerebellum, brain stem and cerebrum of the 'staggerer', 'weaver' and 'nervous' neurologically mutant mice.

The content of glutamate, GABA, aspartate, glycine and alanine was determined in the cerebellum, brain stem and cerebrum of three different mutant mice which have been named ‘staggerer’, ‘weaver’ and ‘nervous’ on the basis of neurological symptoms. In the ‘staggerer’ and ‘weaver’ mutants there is an almost complete absence of granule cells in the cerebellar cortex while in the ‘nervous’ mutant there is a loss of Purkinje cells (and to a lesser extent a loss of granule cells) in the cerebellar cortex. In the cerebellum of the ‘weaver’ mutant, the content of glutamate was signficantly lower (P < 0.025) than control values (8.77 ± 0.76 vs 12.0 ± 1.3 μmol/g tissue wet wt) and the contents of GABA and glycine were significantly greater than normal levels. In the cerebellum of the ‘staggerer’ mutant, the content of glutamate was significantly lower (6.62 ± 0.70 μmol/g) and the contents of glycine and alanine significantly higher than control values. In the cerebrum and brain stem regions of the staggerer mutant, weaver mutant and the normals the contents of the five amino acids were the same. The contents of glycine and alanine in the cerebellum, GARA and glycine in the brain stem and GABA and alanine in the cerebrum of the nervous mutants were higher than control values. The data are discussed in terms of a possible role for glutamate functioning as an excitatory transmitter when released from the cerebellar granule cells.     

FREE AMINO ACIDS AND RELATED COMPOUNDS IN BIOPSIES OF HUMAN BRAIN

Abstract— Contents (μmol/g wet wt.) of 35 free amino acids and related compounds were measured in biopsies of human brain from ten patients. Brain specimens were frozen in liquid nitrogen within 10 sec of their removal at neurosurgery; thus, the values found should approximate those which occur in living brain.
Levels in free pools of biopsied cerebral cortex of most of the amino acids that are constituents of proteins were only 20-50 per cent of those found in autopsied cortex. The content of cystine and ethanolamine was much lower in biopsied than in autopsied cortex. Concentrations of GABA in biopsied cortex were only 20 per cent as high as those found in autopsied cortex, and levels of γ-aminobutyryl dipeptides were also significantly lower in biopsied cortex. Amounts of cystathionine in biopsied cortex varied markedly, but averaged much higher than in autopsied cortex; a single biopsy specimen of cerebellar grey matter had a cystathionine content 36-fold greater than the mean found in autopsied cerebellum.
Appreciable variability in contents among cortical biopsies was found for glycerophosphoethanolamine, phosphoethanolamine, ethanolamine, taurine, aspartic acid, glutamic acid, glutamine, and GABA, as well as for cystathionine. Whether this variability occurred between different subjects, or between different cortical areas, was not clear, although the former possibility was suggested by findings in multiple cortical biopsies from one patient.     

AMINO ACID INCORPORATION INTO PROTEIN IN NEURONAL CELL BODY AND NEUROPIL FRACTIONS IN VITRO

Abstract—

• 1 The incorporation of radioactivity from [³H]lysine into acid-insoluble material in vitro in mixed cell suspensions and isolated neuronal and neuropil fractions has been followed.
• 2 In the mixed cell suspension, incorporation was linear in fresh preparations for up to 60 min. In cold stored preparations, incorporation began to fall off after 30 min. Incorporation, at 4-11 pmol/mg protein/h, was intermediate between that in the tissue slice and in a cell-free preparation. Addition of a mixture of non-labelled amino acids at 1 mM produced a 30-40 per cent inhibition of incorporation. Molar rates of incorporation of glutamate and tryptophan into the mixed cell fractions were respectively 73 and 1-4 times that of lysine.
• 3 Only 8 per cent of the incorporated radioactivity could be recovered in soluble as opposed to particulate material. After hydrolysis of the protein, followed by paper chromatography and autoradiography, radioactivity was detected only in the position corresponding to lysine.
• 4 Incorporation in the separated cell fractions was not markedly reduced by the centri-fugation procedure. Incorporation into the neuronal fraction was 2-2-6 times that into the neuropil fraction, depending on the amino acid used. Incorporation into both was reduced by some 40 per cent by addition of an amino acid mixture.
• 5 Comparison of in vivo and in vitro data suggest that the differences in rate of incorporation are characteristic of neurons and neuropil in situ.

     

The effects of desiccation and rehydration on the lone star tick

This study describes the effects of desiccation and rehydration on the water content, haemolymph volume (per cent), osmolarity, and concentrations of Na, K, Mg, and Ca in the haemolymph of the lone star tick, Amblyomma americanum.The water content percentages of ‘severely desiccated’, ‘moderately’ and ‘fully hydrated’ ticks were 46·0, 52·8, and 60·3 per cent respectively. The lowest and highest of these were near the minimum and maximum possible.The haemolymph volume (per cent) of ‘severely desiccated’ ticks was regulated near the level of ‘moderately hydrated’ ticks despite significant decreases in total body water content and increases in osmolarity and concentration of sodium. Conversely, the change from ‘severely desiccated’ to ‘moderately hydrated’ ticks can be viewed as causing an increase in total body water, decrease in blood osmolarity and sodium, but little change in haemolymph volume (per cent).Most of the water taken up by ‘moderately hydrated’ ticks (while becoming ‘fully hydrated’) was added to the haemolymph. At the same time, there was little change in the blood osmolarity or haemolymph concentration of sodium. Conversely, the change of ‘fully’ to ‘moderately hydrated’ ticks was marked by a substantial loss of haemolymph volume (per cent) but little change in osmolarity and concentration of sodium.The concentration of potassium was regulated over the full range of desiccating and hydrating conditions. The lone star tick appeared less able to regulate its haemolymph concentrations of Ca and Mg; both fluctuated at the same rate, but inversely as the haemolymph volume (per cent).It appears that a carefully controlled movement of solutes (Na the predominant cation) between haemolymph and non-haemolymph tissue is intimately linked with haemolymph volume regulation and movement of water into the haemolymph during hydration.     

Cerebral carbohydrate metabolism during acute hypoxia and recovery

Abstract— The levels of ATP, ADP, AMP and phosphocreatine, of four amino acids, and of 11 intermediates of carbohydrate metabolism in mouse brain were determined after: (1) various degrees of hypoxia; (2) hypoxia combined with anaesthesia; and (3) recovery from severe hypoxia. Glycogen decreased and lactate rose markedly in hypoxia, but levels of ATP and phosphocreatine were normal or near normal even when convulsions and respiratory collapse appeared imminent. During 30 s of complete ischaemia (decapitation) the decline in cerebral ATP and phosphocreatine and the increase in AMP was less in mice previously rendered hypoxic than in control mice. From the changes we calculated that the metabolic rate had decreased by 15 per cent or more during 30 min of hypoxia. Hypoxia was also associated with decreases of cerebral 6-phosphogluconate and aspartate, and increases in alanine, γ-aminobutyrate, α-ketoglutarate, malate, pyruvate, and the lactate :pyruvate ratio. Following recovery in air (10 min), increases were observed in glucose (200 per cent), glucose-6-phosphate, phosphocreatine and citrate, and there was a fall in fructose-1, 6-diphosphale. Similar measurements were made in samples from cerebral cortex, cerebellum, midbrain and medulla. Severe hypoxia produced significant increases in lactate and decreases in glycogen in all areas; γ-aminobutyrate levels increased in cerebral cortex and brain stem, but not in cerebellum. No significant changes occurred in ATP and only in cerebral cortex was there a significant fall in phosphocreatine. Phosphocreatine, ATP and glycogen were determined by quantitative histochemical methods in four areas of medulla oblongata, including the physiological respiratory centre of the ventromedial portion. After hypoxia, ATP was unchanged throughout and the changes (decreases) in phosphocreatine and glycogen were principally confined to dorsal medulla, notably the lateral zone. Thus there is no evidence that respiratory failure is caused by a ‘power’ failure in the respiratory centre. It is suggested that in extremis a protective mechanism may cause neurons to cease firing before high-energy phosphate stores have been exhausted.     

EFFECT OF METHIONINE AND METHIONINE SULPHOXIMINE ON RAT BRAIN S-ADENOSYL METHIONINE LEVELS

Rat brain SAM levels were markedly increased after methionine administration, whereas the convulsant, L-methionine-dl-sulphoximine (MSO), produced a 35 per cent decrease in whole brain content of S-adenosyl-L-methionine (SAM). When methionine was given in combination with MSO, SAM levels were not decreased. Studies on the regional distribution of SAM revealed only a small variation between regions (from 24 nmol/g in midbrain to 49-5 nmol/g in striatum). SAM levels were reduced by about 50 per cent in the cerebellum, striatum, cortex and hippocampus 3 and 6 h after MSO. It is proposed that abberant cerebral methylation processes may be involved in the genesis of the MSO seizure.     

Amino acid neurotransmitter alterations in three sublines of Rb mice differing by their susceptibility to audiogenic seizures

The levels of inhibitory amino acids (Tau, Gly), or excitatory amino acids (Glu, Asp) and Gln, precursor of GABA, have been determined, under resting conditions, in 17 brain areas of 3 sublines of inbred Rb mice displaying different responses to an acoustic stimulus. Rb1 mice were clonictonic seizure-prone, Rb2 mice were clonic seizure-prone and Rb3 mice were seizure resistant. Profile of distribution in the brain of each one of these amino acids differed. Maximum to minimum level ratio was higher for Tau (3.8) than for Glu or Asp or Gln (2). The level of Gly was similar in 13 out of the 17 areas examined. Multiple inter-subline differences were recorded for each amino acid. These differences have been analyzed considering the seizure susceptibility or severity of the three Rb sublines. Common lower levels (approximately –20%: Rb1/Rb3, Rb2/Rb3) of Gln in Temporal Cortex may be implicated in seizure susceptibility. Seirure severity (Rb1/Rb2) seems to correlate, in some areas, with additional lower amounts of GABA already reported and, to a lower extent, of Asp (–19% in striatum, inferior colliculus and cerebellum), of Tau and Gly; a tendency for a rise in Gln content was observed in certain others (10–20% in olfactory bulb, thalamus, hypothalamus, substantia nigra, and frontal, temporal and occipital cortex). The data and correlations recorded provide guidelines for further investigations for synaptosomal and metabolic alterations in the three sublines of the same strain of Rb mice.Abbreviations used GABA 4-aminobutyrate - Tau taurine - Gly glycine - Asp aspartate - Glu glutamate - Gln glutamine - GEPR genetically epilepsy-prone rat - OB olfactory bulbs - OT olfactory tubercles - Sr striatum - Se septum - Hy hypothalamus - Hi hippocampus - Th thalamus - A amygdala - SC superior colliculus - IC interior colliculus - SN substantia nigra - FCx frontal cortex - TCx temporal cortex - OCx occipital cortex - C cerebellum - P pons - Ra raphe