Masaru Sorimachi

A Gas Chromatographic Method for the Determination of N‐Acetyl‐l‐Aspartic Acid, N‐Acetyl‐α‐ Aspartylglutamic Acid and β‐Citryl‐l‐Glutamic Acid and Their Distributions in the Brain and Other Organs of Various Species of Animals

Abstract: A simple and sensitive gas‐chromatographic method for the determination of N‐acetyl‐l‐aspartic acid (NA‐Asp), N‐acetyl‐α‐aspartylglutamic acid (NA‐Asp‐Glu) and β‐citryl‐l‐glutamic acid (β‐CG) was developed. The organ, regional and phylogenetic distributions of these compounds were studied. NA‐Asp and NA‐Asp‐Glu were highly concentrated in nervous tissue, and less than 1% of the amounts in the nervous tissues were found in nonnervous organs. These two compounds showed a reciprocal relationship in their regional distribution in mature brains, but such a relationship was not evident or was even reversed in immature brains. The two compounds also showed different developmental changes in different regions of the brain. Fish brain contained a relatively high concentration of NA‐Asp, but only a trace amount of NA‐Asp‐Glu. By contrast, a 10 times higher concentration of NA‐Asp‐Glu than NA‐Asp was found in frog brain. Reptilian brain contained similar amounts of each compound. Avian and mammalian brain had NA‐Asp at a roughly 10 times higher concentration than NA‐Asp‐Glu. β‐CG occurred at the highest concentration in the immature brain of rat and guinea pig, but disappeared in the mature brains. The adult frog brain, however, contained a large amount of β‐CG. In the adult rat, testis contained the highest concentration of β‐CG.

High affinity choline uptake: An early index of cholinergic innervation in rat brain

The uptake of [3H]choline was investigated in nuclei-free homogenates or crude synaptosomal fractions (P2) from rat brain under various stages of development. A comparable sensitivity of uptake to treatment by hyposmotic shock suggested the involvement of synaptosomal populations in choline uptake in immature as well as in adult brains. However, significant changes in the apparent Km for the high affinity transport system and quantitative differences in the Na ion requirement for maximal uptake at 0.43 muM choline concentration were found during development; facts which suggested a greater contribution of the low affinity system in the more immature brains. Assuming that the uptake with high and low sensitivity to Na+ reduction reflected that via the high and low affinity system reslectively, we have attempted to obtain real Km values for the high affinity system. These Km values changed less than those measured directly, suggesting that the affinity constant for the high affinity system does not change during development. On these assumptions, the developmental changes of cholinergic synaptogenesis were examined in 5 distinct regions of the brain. It was found that the synaptogenesis begins several days earlier than the increase of choline acetyltransferase (ChAc) level in the frontal cortex, the hippocampus, the superior colliculus and the cerebellum. These regions may be included among the terminal-rich regions according to available evidence related to cholinergic systems. On the other hand, synaptogenesis accompanied the concomitant ChAc increase in the striatum, where the cholinergic interneurons are present. It is concluded that the increase of ChAc in the terminal-rich regions is delayed by the axoplasmic flow; therefore, the earlier index of cholinergic synaptogenesis in these regions is the high affinity uptake activity rather than the enzyme activity.

Cholinergic and GABAergic fibers in the stria medullaris of the rabbit

Abstract After placing uni- and bilateral electrolytic lesions in the habenula (Hb), stria medullaris (SM), septum (Sep) and lateral preoptic area (LPA), activities of choline acetyltransferase (ChAc), acetylcholinesterase (AChE) and L-glutamic acid decarboxylase (GAD), which are the synthesizing and degradating enzymes of acetylcholine and the synthesizing enzyme of γ-aminobutyric acid (GABA) respectively, were analysed in the interpeduncular nucleus (Ipd) and Hb. Confirming earlier results in the rat [5,9,15,16], ChAc activity in the Ipd was markedly reduced after destruction of the Hb and to lesser extent after placing lesions in the Sep or LPA. When lesions were made in the SM, Sep, or LPA, ChAc activity in the Hb was decreased to 25–40% of the control values one week after the operation. Activity of GAD values, on the other hand, was practically not altered in the structures studied, except that the activity decreased in the Hb after placing lesions in the SM. On the basis of these findings, it is suggested that the SM contains Cholinergic afferent fibers to the Hb, that these fibers possibly arise in the forebrain structures including the Sep and LPA, and that some of the Cholinergic fibers in the SM course through the Hb toward the Ipd. The SM might also contain GABAergic afferent fibers to the Hb.

Isolation and identification of β-citryl-L-glutamic acid from newborn rat brain

Abstract Ab unknwon compound containing glutamic acid residue was found in newborn rat brain. The compound occurred predominantly in brain. Its concentration was approx. 1 μmol/g tissue at birth and decreased to one-tenth 24 days after birth. The compound was isolated from newborn rat brains, and subjected to elementary analysis and to infrared and mass spectrometric analysis. Glutamic acid and citric acid were formed from the compound on acid hydrolysis. The compound was presumed to be a citryglutamic acid. Two isomers, α- and β-citrylglutamic acid, were sunthesized. The unknown compound was identified as β-citryl- L -glutamic acid. The occurrence of this compound has not been reported in nature.

EXOCYTOTIC RELEASE OF CATECHOLAMINES AND DOPAMINE‐β‐HYDROXYLASE FROM THE PERFUSED ADRENAL GLAND OF THE RABBIT AND CAT

1 Secretion of catecholamines (CA) and dopamines‐hydroxylase (DBH) activity from the perfused rabbit and cat adrenal gland was studied following stimulation by a number of substances, including the physiological transmitter, acetylcholine (ACh), added to the perfusion fluid. 2 Stimulation caused a proportional secretion of DBH and CA from the untreated rabbit adrenal. The ratio of DBH/CA was 11.2 + 0.9 (mean + s.e.) which was close to that found in the crude granule fraction of the contralateral gland (11.4 ± 0.7). 3 After treatment with insulin (40 u/kg) for 3 h or with reserpine (0.25 mg/kg) for 24 h, CA and DBH contents decreased in a parallel fashion in the granule fraction, thus resulting in a similar ratio of DBH/CA in the gland. The ratio in the effluents after stimulation was similar to that found in the untreated gland. 4 Higher doses of reserpine (0.7 to 2.5 mg/kg) increased the ratio of DBH/CA in the granule fraction and was dependent on the doses employed. The ratio in the effluents from these glands after stimulation paralleled these increased values. 5 Concomitant secretion of CA and DBH in response to stimulation was also observed in the perfused cat adrenal. However, the ratio of DBH/CA in the effluents tended to be lower than that found in the granule fraction. 6 These results support the concept of exocytotic secretion of CA in the adrenal medulla.

Developmental change of choline acetyltransferase and acetylcholinesterase in the ciliary and the superior cervical ganglion of the chick.

Abstract The neonatal and early postnatal development of choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) activities, possibly the neurochemical correlates for the formation of cholinergic neurons or synapses, was studied using the ciliary and the superior cervical ganglion of the chicks from the 7th day (stage 31 of Hamburger and Hamilton 10 ) and the 13th day (stage 39) of incubation, respectively, to the 29th day after hatching. In the ciliary ganglion, where both the pre- and the postganglionic nerves are cholinergic, a sharp increase in the ChAc activity was observed from the 14th day of incubation and reached a maximum on the 29th day after hatching where the value amounted to 450-fold (per pair of ganglia) or 47-fold (per unit weight of protein content) the initial value. On the other hand, the total activity of AChE increased only 7-fold and the specific activity remained practically unchanged. In the superior cervical ganglion, where only the preganglionic nerves are cholinergic, the ChAc and AChE activities began to increase at the 15th day in the embryo, exhibiting an almost similar time course and reached a maximum on the 29th day after hatching. The alteration of both enzyme activities in the ciliary ganglion during development is discussed in connection with the morphological and electrophysiological findings reported to date.

Changes in noradrenaline and its synthesizing enzymes in the nucleus locus ceruleus after intraventricular administration of colchicine and vinblastin

Abstract Concentrations of noradrenaline and its synthesizing enzymes were measured in the locus ceruleus of rats after intraventricular injection of colchicine and vinblastin to determine whether or not neurotubules are involved in the axonal transport of noradrenaline storage vesicles in the central nervous system. Significant increases in the noradrenaline concentration and dopamine-β-hydroxylase activity were evident after 1 day. This together with other available evidence suggested a close relationship between the neurotubules and axonal transport. An increase in tyrosine hydroxylase activity was observed 2 to 4 days after colchicine or, in some cases, vinblastin administration, but a concomitant increase in adrenal tyrosine hydroxylase activity suggests that this increase is due not to the blockage of axonal flow, but to the increased synthesis of enzyme protein in response to stress. On the other hand, the dopamine-β-hydroxylase concentration was later reduced after initial accumulation and marked morphologic changes (chromatolysis) were concomitantly observed. Two possibilities for these changes are discussed.