We Have Shown That the Injection of Semicarbazide (SC), an Antivitamin B6 (Anti-B6) Into the Superior Colliculus of the Mouse Induces Running Fits (1)

J. Nutr. Sci. Vitaminol., 25, 15-22, 1979

RUNNING FITS AND ƒÁ-AMINOPUTYRIC ACID OF THE SUPERIOR COLLICULUS OF THE MOUSE1 Junko YAMASHITA2 and Yukio HIRATA3

Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neurosciences, Musashidai, Tokyo 183, Japan (Received October 27, 1978)

Summary The present experiment was performed to examine the relationship between the content of ƒÁ-aminobutyric acid of the superior colliculus and running fits induced by injection of semicarbazide (SC), an antivitamin B6, into the superior colliculus of the mouse.

(1) The distribution of 4-aminobutyrate: 2-oxoglutarate aminotransferase [EC 2.6.1.19] of the superior colliculus was studied histochemically. A high activity was observed in the superficial and medial gray layers of the superior colliculus. Pretreatment with aminooxyacetic acid at a dose which inhibits running fits induced by intracollicular injection of SC depressed the activity. (2) The contents of ƒÁ-aminobutyric acid in superior colliculi of mice which received an intracollicular injection of SC were 60 per cent of the control, when measured immediately after the first running fit. Administration of pyridoxine together with SC inhibited the fall. (3) Intracollicular injection of thiosemicarbazide and 4-deoxypyridoxine, other antivitamins B6, also induced running fits. These results suggest that the running fits by SC injected intracollicularly are induced by the decrease of ƒÁ-aminobutyrate due to insufficient vitamin B6 in the superior colliculus.

Keywords semicarbazide, running fit, superior colliculus, ƒÁ-aminobu tyric acid, vitamin B6

We have shown that the injection of semicarbazide (SC), an antivitamin B6 (anti-B6) into the superior colliculus of the mouse induces running fits (1). These fits are prevented by aminooxyacetic acid (AOAA) which is more effective when administered 5 hr before SC injection than at 1-60 min beforehand (1). Since AOAA is a potent inhibitor of 4-aminobutyrate: 2-oxoglutarate aminotransferase

1 Abbreviations used: SC , semicarbazide; anti-B6, antivitamin B6; AOAA, aminooxyacetic acid; GABA-T, 4-aminobutyrate: 2-oxoglutarate aminotransferase [EC 2.6.1.19]; GABA, ƒÁ-aminobutyric acid; TSC, thiosemicarbazide; DPN, 4-deoxypyridoxine; PN, pyridoxine. 2 山下洵子 , Present address: Department of Nutritian, Jikei University School of Medicine, Nishi- shinbashi, Tokyo 105, Japan. 3 平田幸男

15 16 J. YAMASHITA and Y. HIRATA

[GABA-T; EC 2.6.1.19] (2, 3), we assume that the prevention of running fits by AOAA may be related to the decreased activity of GABA-T in the brain. We have

histochemically examined the differences in distribution of this enzyme in the brain,

particularly in the superior colliculus 30 min, and 5 hr after the administration of AOAA. If an antidotal effect of AOAA on the induction of running fits is related to the

inhibition of GABA-T resulting in the increase of ƒÁ-aminobutyric acid (GABA) in the superior colliculus, running fits will be caused by a decrease there in the GABA

content. We, therefore, have measured the GABA content of the superior colliculus immediately after the first running fit induced by an intracollicular injection of SC. In addition, we have studied whether other anti-B6 injected intracollicularly

also induce running fits. For this study, thiosemicarbazide (TSC) and 4 - deoxypyridoxine (DPN) have been chosen for the following reasons. TSC is a

derivative of SC. DPN, though a convulsant, hardly induces running fits when administered systemically, differing from many other anti-B6 compounds.

MATERIALS AND METHODS

In all series of experiments male mice of the ddY strain, obtained commercially,

were used at the age of about 5 weeks. AOAA hemihydrochloride, ƒ¿-ketoglutaric acid, nitroblue tetrazolium and phenazine methosulphate were obtained from Sigma

Chemical Co., U.S.A., and ƒÀ-NAD+ from Oriental Yeast Co., Japan. Pyridoxine

(PN) hydrochloride was distributed by the National Institute of Hygienic Sciences of Japan. All reagents used were neutralized with NaOH solution, if necessary. The activity of GABA-T was investigated principally according to the method

of HYDE and ROBINSON(4); GABA-T activity was visualized as deep purplish deposits of formazan. Six mice were injected with 40 ƒÊg/g body weight of AOAA

intraperitoneally (i.p.), and two mice with saline alone as controls. They were anesthetized with Nembutal 30min or 5hr after the injection, and perfused

transcardially with a formaldehyde/glutaraldehyde (1%/1%) fixative, buffered with 0.1M phosphate (pH 7.5). The brains were quickly taken out and frozen on a freezing microtome stage by dry ice. Cryostat sagittal sections (20 ƒÊm) were attached

to the slide, kept at -20•Ž for one hour, and incubated at 37•Ž for 30min in the same medium as HYDE and ROBINSON'S (4). Intracollicular injection of anti-B6 was performed as described previously (1). For the determination of the content of GABA mice were killed by decapitation immediately after the first running fit. The brains were removed on ice within one minute to lessen postmortem changes in the GABA content. The contents of the whole brains were measured colorimetrically by the method of ABE(5). In other mice treated similarly the left and right superior colliculi were dissected out separately, put into 0.5ml of 75% ethyl alcohol and homogenized by a sonicator. After centrifugation the pellets were washed once. Supernatants were concentrated by the method of TsUKADAand NAGATA(6) to measure the content of GABA; RUNNING FITS AND GABA 17

GABA was determined by the method combining the use of a bacterial enzyme, GABA®T and succinic semialdehyde dehydrogenase (7). The bacterial enzyme was kindly supplied by Dr. Y. Okada, Department of Neurochemistry of this institute. Pellets were used for protein concentration assay by the method of LOWRY et al. (8) using crystalline bovine serum albumin as the standard. The content of GABA in the tissue was shown as nmole per mg protein contained in the pellet, The protein content per wet weight of the four untreated superior colliculi was 76.4•}6.5 ƒÊg/mg

(mean•}S. D.).

RESULTS

GABA-T activity Control animals, either untreated, or treated with saline alone, showed a high activity of GABA-T in the superior colliculus, especially neuropil of the lateral half of the superficial and medial gray layers (Fig. 1C) but not in neuronal perikarya (Fig. 2). A high activity, comparable to that of the superior colliculus, was observed in the nucleus accumbens septi, preoptic area, ventral anterior thalamic nucleus, pretectum, lateral habenular nucleus, zona incerta, subthalamic nucleus, substantia nigra, mammillary body, central gray substance, dorsal and ventral tegmental nuclei, cerebellar nuclei and interpeduncular nucleus (Fig. 1C). AOAA-treated mice showed a decreased activity of GABA-T, when compared with the controls. They showed a weak activity in the superficial and medial gray layers of the superior colliculus and some other regions 30min after AOAA treatment (Fig. 1B), but a very weak activity was found only in the superficial gray layer of the superior colliculus and a few other regions 5 hr after A®AA (Fig. 1A).

Content of GABA in the whole brain immediately after the first running fit Mice injected with 10 ƒÊg/animal of SC hydrochloride into the left superior colliculus showed the first running fit 6 to 8min later. The GABA content of the whole brains of these animals did not differ significantly from those of the control mice which were killed 6 to 8 min after the administration of saline (Table 1).

Content of GABA in the superior colliculus immediately after the first running fit Mice in group 1 were injected with l0 ƒÊg/g animal of SC hydrochloride into the left superior colliculus, mice in group 2 with SC hydrochloride at the same dose together with 5 ƒÊg/animal of PN hydrochloride, and mice in group 3 with NaCl at the same molarity as that of group 1. In mice of group 1 the first running fit was induced 5 to 8 min after the treatment of SC, while in mice of groups 2 and 3 no fits occurred. The content of GABA in the superior colliculi obtained from mice which were killed immediately after the first running fit was about 60 per cent of those of 18 J. YAMASHITA and Y. HRATA

Fig. 1. Change of GABA-T[EC 2.6.1.19]activityin a sagittalsection of the mouse brain following administrationof aminooxyacetic acid (AOAA). A. 5 hr afterAOAA. Very weak activityis found in the superficialgray layer(arrow) of the RUNNING FITS AND GABA 19

Fig. 2. Superior colliculus-pretectum region of an untreated mouse. The precipitates of formazan were formed in neuropil, but not in neuronal perikaiya. HIPP, hipocampus; CC, cerebral cortex: PRT, pretectum; SG, MG, DG, superficial, medial, deep gray layers of the superior colliculus; INF COL, inferior colliculus.

superior colliculus (SUP COL), lateral habenular nucleus, dorsal tegmental nucleus and interpeduncular nucleus. B. 30 min after AOAA. Weak activityis found, in addition to the regions shown in Fig. 1A, in the medial layer (arrow) of the superior colliculus,nucleus accumbens, mamillary body and central gray substance. C. Untreated. 20 J. YAMASHITA and Y. HIRATA

Table 1. Effect of intracollicular injection of semicarbazide (SC) on the GABA content of the whole brain of the mouse. There were 5 mice in each group.

Table 2. Effect of intracollicular injection of semicarbazide (SC) with or without pyridoxine (PN) on the GABA content of the superior colliculus. In each group there were five mice (ten superior colliculi).

* Differs from NaCI solution's group by p<0 .01; ** differs from SC's group by p<0.02 (Student's t test). group 3 which were killed 5 to 8min after the injection. The GABA level from group 2 was higher than that from group 1, but lower than that from group 3 (Table 2). Since there was no difference between the GABA content of the left and right superior colliculi, data from each side are not considered separately in the table. Effect of intracollicular injection of TSC and DPN TSC or DPI also induced running fits when injected into the superior colliculus (Table 3). Though the latency of the first running fit in TSC-treated mice

Table 3. Effect of intracollicular injection of thiosemicarbazide (TSC) and 4-deoxypyridoxine (DPN).

was longer than that by the same molarity of SC, manifestations before and after running fits were similar to those caused by SC (1): 4hyperkinesis, hyperacusis, and/or tonic and chronic convulsions were often observed. Mice injected with DPN RUNNING FITS AND GABA 21

turned round and round very quickly within one minute after the injection , and subsequently showed running fits.

DISCUSSION

VAN GELDER (9) studied the regional distribution of GABA-T activity in unfixed

mouse nervous tissues by a histochemical method similar to ours . He found a strong reaction on a number of nuclei in the brain stem and the spinal cord . There is,

however, no detailed description about the superior colliculus , though our present study confirmed many of his findings. AOAA inhibits GABA-T activity, and consequently raises the level of GABA

in the whole brain of various animal species (2 , 3). It has an anticonvulsive effect against convulsions induced by some anti-B6 compounds (10) . In the previous paper we showed that administration of AOAA, 5 to 8 hr before intracollicular injection of

SC, prevents running fits, while that within one hour was less effective (1) . The

increase of the GABA content in the mouse brain stem after AOAA (25 ƒÊg/g; i .p.) is biphasic: the first rise occurs during the first 3 hr , a subsequent secondary rise then occurs between 4 and 6 hr, and the values attain a maximum at 6 hr (10) . In the present experiment sections from the case of 5 hr post-AOAA exhibited very weak GABA-T activity in the superficial gray layer of the superior colliculus , compared with that of 30min post-AOAA . The preventive effect of AOAA on the fits therefore must be related to an almost complete depletion of GABA-T activity from the superior colliculus. A high content of GABA has been shown in the superior colliculus of many

species of animals (11). NITSCH and ORADA (12) measured the contents of GABA in various structures of the brain after the injection of methoxypyridoxine , another anti-B6, and reported that in GABA-rich structures , including the superior colliculus, the GABA content drops more steeply pre-ictally than in GABA -poor

ones. In the present experiment , the content of GABA in the superior colliculus of SC-treated mice decreased when assayed immediately after the first running fit , whereas that of the whole brain did not differ from the saline -treated control . Though we did not try to follow the time course , the content in the superior colliculus is supposed to have decreased before the first running fit . It is of interest that the decrease in GABA content , which must be intermediately induced by the suppression of vitamin B6 caused by anti-B6 , occurred within a remarkably short latency as compared with systemic administration , and that the decrease of GABA is inhibited by concomitant administration of PN . There is generally no association between GABA and GABA -T distribution in the brain areas (11), but the superior colliculus has both a high content of GABA and a high activity of GABA-T. From the present experiments we now consider that

the some GABA-mediated system of the superior colliculus modifies the locomotor activity by incoming multimodal sensory information (13) and that the derangement of this system produces such a disordered behavior as running fits . 22 J. YAMASHITA and Y. HIRATA

The authors wish to thank Dr. M. Matsuda for his helpful discussion.

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