The maturation of monoamine oxidase and 5-hydroxyindole acetic acid in regions of the mouse brain

Brain Research, 65 (1974) 255-264

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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

THE MATURATION OF MONOAMINE OXIDASE AND 5-HYDROXYINDOLE ACETIC ACID IN REGIONS OF THE MOUSE BRAIN

PETER C. BAKER, K E N N E T H M. H O F F AND M. DEBORAH SMITH

Department of Biology, Cleveland State University, Cleveland, Ohio 44115 (U.S.A.) (Accepted June 19th, 1973)

SUMMARY

Monoamine oxidase (MAO) activity and 5-hydroxyindole acetic acid (5-HIAA) amount have been measured in 4 subdivisions of the mouse brain during various stages of postnatal maturation. Each region and each indoleamine pathway component (MAO and 5-HIAA) demonstrated an individual pattern of maturation. MAO increased rapidly from day 1 postpartum and reached adult-like specific activity by 2 weeks postpartum except in the cerebellum where increase continued after week 6. 5-HIAA levels exceeded adult-like levels by day 3 postpartum, continued to rise during the first week and reached adult level by week 6. Comparison of these data to previously reported maturational patterns of 5-hydroxytryptamine (5-HT) and 5hydroxytryptophan decarboxylase (5-HTPD), measured upon a similar regional basis, indicate that the components of the indoleamine pathway in the brain do not mature in a harmonious way.

INTRODUCTION

Investigative interest in the development of indoleamine metabolism in the mammalian brain appears to be increasing. A variety of approaches have been employed in an attempt to define the biochemical, histological and physiological characteristics of the serotonergic system as it grows toward the mature state4, 7,17. Although various laboratory animals have been used in these kinds of studies the animal of recent preference is the rat. Within the context of the rat brain model there is a divergence of opinion concerning the underlying control mechanisms of the system during ontogeny. Some investigators have focused their attention upon the pathway's initial enzyme, tryptophan-5-hydroxylase (T5-H) (E.C. 1.14.16.4), and consider its increase in activity during the postnatal period to be the regulatory agent of pathway maturation ~,6,2°. Other investigators consider that the critical element in the matura-

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tional scheme is at the microanatomical level and can be followed by the histochemical visualization of axonal outgrowth and terminal establishment by the 5-hydroxytryptamine (5-HT) containing neurons11,lz, 17. The process however is complex and there are indications that alternative trophic functions 15, effects of peripheral indoleamine metabolism 9, and multiple forms of at least one enzyme19, zt may be additional factors involved in the control and regulation of the brain's serotonergic system during the course of its establishment. Our laboratory has been involved in the study of the postnatal maturation of the components of the indoleamine pathway upon a regional basis in the brain of the mouse. We have found that 5-HT and its formative enzyme, 5-hydroxytryptophan decarboxylase (5-HTPD) (E.C. 4.1.1.28), show wide regional variation during the period from birth to adulthood2, 3. We have noted as well that the maturational patterns of the enzyme and its product did not resemble each other in any but the most cursory way during the juvenile period of life. We have continued our study into the postnatal differentiation of the pathway and can now report upon the regional maturational patterns of monoamine oxidase (MAO) (E.C. 1.4.3.4), the enzyme for 5-HT destruction, and 5-hydroxyindole acetic acid (5-HIAA) the primary indoleamine endproduct. As before, we find that there is considerable regional variation and lack of similarity in pattern. MATERIALS A N D M E T H O D S

Mice of the CFW strain from Carworth Farms with a lighting regime of 10 h of light and 14 h of darkness were assayed between 1 day postpartum and adulthood. Immature animals of l, 2, 3, 5, 7, 14, 28, and 42 days of age were used, as well as adult animals between 4.5 and 5 months of age. All animals were killed during the fourth hour of their light period. Following decapitation, the brain case was split parasagittally and the roof removed. The hypophysis and olfactory bulbs were discarded and the brains were dissected into 4 parts in 0.1 M NaC1. The cerebellum was separated at its point of attachment. The cerebral hemispheres were removed after bisecting the corpus callosum, lifting the hemispheres forward and cutting the connection to the underlying tissue just anterior to the optic chiasma. The mesencephalondiencephalon fraction which included the pineal, was isolated from the medulla-pons fraction by cutting obliquely from the dorsal side in the groove caudal to the posterior colliculus to the ventral side just rostral to the pons. The parts were each blotted on filter paper to remove any excess fluid and quickly weighed to the nearest 0.1 rag. MAO was assayed by a previously described modification 1 of the method of Wurtman and Axelrod 2s. Tissues were homogenized on ice by hand in glass homogenizers containing cold 0.1 M phosphate buffer, pH 7.4. Aliquots of 0.2 ml from each preparation were transferred to a series of incubation tubes on ice. Each tube received 10 #1 [14C]5-HT solution (6.25 nM, 3 mCi/mmole) and were incubated at 37 °C in a shaking water bath for 30 min. The reaction was halted by the addition of 0.15 ml 1.0 N HC1. Each tube then received 3 ml of diethyl ether, was stoppered, shaken and then centrifuged to separate the phases. 2.5 ml of each organic phase were then trans-

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Fig. 1. Monoamine oxidase activity in various regions of the mouse brain during postnatal maturation. Enzyme activity is expressed as nmoles 5-HIAA formed/h/brain region. Brackets around plotted points represent S.E.M. Unbracketed points have S.E.M. smaller than the symbol used to mark the point. ferred to a series of counting vials which then received 10 ml P O P O P - P P O phosphor solution and 1 ml ethanol. The vials were counted in a Nuclear Chicago Unilux II. 'Heated enzyme' blanks were used to determine blank correction values. 5-HIAA was assayed by the method of Quay z6. Tissues were homogenized in 1 ml of 0.1 N HC1 containing 0.5 ~ (w/v) ascorbic acid. The homogenates were transferred to stoppered tubes which then received 0.2 ml of 1 ~ (w/v) E D T A ; 0.25 ml of borate buffer, p H 10; 600 mg of NaCI; and 3 ml diethyl ether. The tubes were then shaken, centrifuged and the ether was aspirated. Then 0.15 ml o f 1.0 N HC1 and 3 ml of diethyl ether were added and the tubes were again shaken and centrifuged. The ether was then transferred to a second series of tubes containing 0.5 ml o f 0.1 N HCI containing 0.5 700 (w/v) of ascorbic acid and 8 ml of heptane. Following shaking and centrifugation, 0.25 ml of the aqueous phase were transferred to a microcuvette and mixed with 0.075 ml o f concentrated HC1. The samples were then read in an Aminco Bowman spectrophotofluorometer at 540-550 nm with activation at 295 nm. Blanks and standards were carried through the whole procedure and read along with the samples. For M A O 10 animals were used for each preadult age and 15 for the adult. For 5-HIAA 8-10 animals were used for preadult ages and 11 for the adult. Student t-test was used to test each region at any given age against the next oldest age and against the adult. The wet tissue weights used to determine M A O specific activity and 5-HIAA level were essentially the same as those previously reportedL The whole brain

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maturation. Enzyme specific activity is expressed as nmoles 5-HIAA formed/h/nag wet wt. tissue. Brackets around plotted points represent S.E.M. Unbracketed points have S.E.M. smaller than the symbol used to mark the point. values presented here are composites derived by summation of values from the 4 brain fractions of each animal and are not actual measurements of whole brain. RESULTS

Except in the cerebellum, MAO activity (Fig. l) increases quickly and significantly between each successive age in all regions and in whole brain between day 1 and the second week of postnatal life. Following that time, no age is significantly different from any older age. However, at week 2 medulla-pons and whole brain are still significantly lower than adult, while hemispheres and mesencephalon-diencephalon are not. Only by week 4 do medulla-pons and whole brain become statistically indistinguishable from adult, and at that time hemispheres are actually significantly higherthan adult. In the cerebellum significant differences were found between successive ages from day 2 to week 4 and between week 6 and adult. Thus, MAO activity per region between day 1 postpartum and adulthood increases as much as 50-fold in the cerebellum, as little as 6-fold in the mesencephalon-diencephalon, and 10-fold in the whole brain. When these data are considered as activity/h/unit tissue weight, i.e., specific activity (Fig. 2), the increases between day 1 and adult are by no means as dramatic, ranging from 2-fold in the medulla-pons to 4-fold in the cerebellum with about a 2.4fold increase in whole brain. Except in the cerebellum, there are rapid and significant

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Fig. 3. 5-Hydroxyindole acetic acid amount in various regions of the mouse brain during postnatal maturation. Values expressed as ng 5-HIAA/brain region. Brackets around plotted points represent S.E.M. Unbracketed points have S.E.M. smaller than the symbol used to mark the point.

increases in specific activity in all regions, including whole brain, between day 1 and day 2 postpartum. Between day 2 and week 2 the increases for these regions are still rapid but somewhat subdued. Although not all successive ages show significant increases in these regions, especially between days 2 and 5, we find that significant increases do occur between day 7 and week 2. After week 2 a static period ensues until adulthood, except in the hemispheres where there is actually a decline, since the week 2 value is significantly higher than the adult. Cerebellum specific activity shows no significant increase until day 5; but following day 5 significant increases are found between day 7 and week 2, week 2 and week 6, and week 6 and adult. If, as is often done, mature MAO status were to be defined as adult-like specific activity then all regions but cerebellum are mature by week 2. 5-HIAA changes during maturation are quite different from MAO. Considered as amount of 5-HIAA/brain region (Fig. 3) values in all regions are essentially static between day 1 and day 2 but rise rapidly and significantly by day 3. Maximal values are reached in cerebellum by day 5 and remain static until week 4. In all other regions, including whole brain, significant increases continue with maximal values being reached by week 4. All regions then fall to essentially adult values by week 6. Although day 1 values are more adult-like than was seen for MAO, they are still below the adult amount in all regions. However, adult values are reached in the cerebellum, mesencephalon-diencephalon and medulla-pons by day 3; in the whole brain by day 5 and in

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the hemispheres by day 7. By week 4, 5-HIAA values are as much as 8-fold above day 1 in the hemispheres and mesencephalon-diencephalon, and as much as 3-fold above the adult in the cerebellum. The week 4 maximal values are significantly higher than both day 1 and adult values in all regions. When these data are considered as amount of 5-H|AA/unit tissue weight, i.e., level (Fig. 4), the patterns are quite interesting. No region at day 1 is significantly different from adult except cerebellum which is significantly higher. By day 3 all regions, including whole brain, are significantly higher than the adult with the elevation being as much as 15-fold in the cerebellum although not nearly as dramatic in the other regions. Except for a rapid fall in level in cerebellum between day 5 and week 2, all regions sustain levels well in excess of adult until week 6 when the adult status is reached. Defining maturity as the time at which adult-like levels are attained would not appear to be a useful device here for 5-HIAA. DISCUSSION

Previous measurements of 5-HT ~ and 5-HTPD 3 in mouse brain indicated that maturation occurred in a unique way for each region and for each pathway component. However, the internal relationships between regions were similar, with 5-HT level and 5-HTPD specific activity being highest in mesencephalon-diencephalon, decreasing through medulla-pons and hemispheres, and being lowest in cerebellum. The enzyme, 5-HTPD, and its product, 5-HT, show limited demonstrable relationship to each other in their patterns of maturation. This kind of situation has been

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reported before in whole rat brain studies 5,22 and could be considered as supportive evidence for the view that T5-H controls pathway maturation while 5-HTPD is an essentially passive participant in the process 5,6,2°. The patterns reported here for MAO and 5-HIAA bear out the unique nature of each pathway component and brain region. It should be noted however that, except for cerebellum, regional specific activity patterns of MAO maturation are rather similar despite relative differences in amount. Internal relationships between regions for 5-HIAA are ordered in the same way as 5-HT 2 and 5-HTPD 3, however for MAO they are different, with cerebellum being the highest and hemispheres the lowest. In general, the cerebellum undergoes the greatest variation during its indoleamine maturation while the hemispheres are the most conservative2,L 5-HT 2 and 5-HTPD 3 were found to be much higher in the mesencephalon-diencephalon than in the medullapons, a situation of similarity between product and enzyme. This relationship is also reflected in 5-HIAA and MAO. In general, for 5-HTPD, 5-HT and MAO the first 2 weeks postpartum represent the period of maximal change, and in many cases adult-like status is reached by that point in maturation2,L 5-HIAA is exceptional in this respect and extreme alterations occur within the first week. Despite these possible similarities in the maturation of indoleamine pathway components, it must be admitted that each is an entity alone and so too is each brain region. The nature of the maturational program underlying such seemingly unrelated patterns does not seem to manifest itself in enzyme activities and metabolite levels. Maturational studies of MAO are not nearly as numerous as similar studies of 5-HT, however a number of whole brain rat studies do indicate a sharp rise in MAO activity during the first 2 weeks postpartum 5,1°,1a. There are conflicting data here 1s,26, but most reports do support the view of an early and rapid MAO increase. When such a MAO pattern of maturation is compared with similar studies of whole rat brain 5-HTS, 1°,22 and 5-HTPD 5,22, it becomes apparent that MAO is maturing more quickly and attaining adult-like status sooner than either 5-HT or 5-HTPD. Even in the guinea pig 8,22,23 and the rabbiP 3, which have indoleamine maturational patterns very different from the rat, the reports indicate the same early increase in brain MAO when compared to 5-HT and 5-HTPD. Our data here for mouse brain MAO are therefore in broad agreement with the relationships seen in rat, guinea pig and rabbit. Maturational studies of 5-HIAA in brain are severely limited in number. Tyce e t al. 25 have reported that newborn whole rat brain 5-HIAA levels were very high right after birth and then exceeded adult levels during the second week postpartum. Tissari and Pekkarinen 24 have described very high 5-HIAA levels on day 1 postpartum. Dynamic studies by Kellogg and Lundborg 9 with labeled 5-hydroxytryptophan (5-HTP) have demonstrated that young rat brains (1 and 4 days postpartum) have greater 5-HIAA retention than older brains (21 days postpartum). Kellogg and Lundborg 9 have also described a rapid change in 5-HIAA retention occurring in the brain stem during the first day postpartum. This is supportive of Tissari and Pekkarinen's 24 finding o f a large 5-HIAA increase in brain stem between the last day prepartum and the first day postpartum. Studies by Kellogg and Lundborg 9 with peripheral decarboxylase inhibitors have demonstrated that an understanding of brain 5-HIAA content

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and retention in the early days of maturation may be complicated by extraneuronal enzymatic components of the pathway. Regardless of the extent o( disagreement concerning the maturation of brain MAO and 5-HIAA, there is every indication so far that the postnatal development of indoleamine metabolism in rat brain is independent of MAO development. At the same time the possibility of increased 5-HIAA retention in immature brain makes that metabolite a poor indicator of pathway activity, as indeed one might expect on the basis of published reports of the maturation of other pathway components such as T5-H6,2°, zT, 5-HTPDS, 2'~ and 5-HT~,I°, 14, all of which appear to have a more restrained postpartum increase, especially during the early days after birth. The existence of the two week point of general change that we find for 5-HT '~, 5-HTPD 3 and M A O is in keeping with the histochemical studies of Loizoull, 1'~ who has found that the early weeks of maturation represent a period of outgrowth for 5-HT neurons and their establishment of terminals. Using a MAO inhibitor, an amine depletor, and the pathway precursor, 5-HTP, Loizou 1'~ has been able to determine that the adult complement of 5-HT neurons is present in immature rats but that axonal outgrowth and formation of terminals are still incomplete. Responses to drugs and precursor were adult-like and Loizou therefore concludes that the indoleamine biochemistry of these cells is differentiated at birth. Olson and Seiger ~5 have been able to demonstrate that 5-HT containing neurons are first identifiable in the prenatal brain 12 days after conception. MAO inhibitor injected into the mother increased the fluorescence in the fetal 5-HT neurons, thus indicating a functional biochemistry at this early stage. Since M A O is present in prenatal brain so early in gestation one would assume the existence of 5-HIAA at the same time, however, direct measurement of the metabolite at this point before birth has not been reported. It is clear from our measurements that each region and each pathway component has a scheme of postnatal development that is unique. Whole brain evidence from other sources is supportive of this view '~,7. This is difficult to place into context with the suggestions advanced so far to explain the development and maturation of the indoleamine system in brain. As we discussed above, biochemical evidence is used to support the view that the initial enzyme in indoleamine biosynthesis, T5-H, is a controlling factor in the maturation of the serotonergic neurons ~',6,'°. This hypothesis is contested by Loizou 12 and by Renson iv who believe that the microanatomical changes and events of axon terminal proliferation are the basis for the maturation of the 5-HT neuronal system in the brain. The clear indication of functional 5-HT biochemistry at an early age in conjunction with an unquestionable disharmony of maturational pattern for the various components of the pathway makes it difficult to endorse either hypothesis alone. Other factors may well be involved however. Olson and Seiger 1~ have suggested a broader view of 5-HT synthesis in brain. Their prenatal studies recognize that 5-HT neurons are organized and grow toward an adult configuration, but since 5-HT synthesis occurs well in advance of any adult-like 5-HT action they suggest an earlier trophic and chemotaxic function for such prenatal biochemical activity. Yet another

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variable has been advanced by Kellogg a n d L u n d b o r g 9 who have d e m o n s t r a t e d that the n o r m a l m a t u r a t i o n of 5-HT biochemistry in the y o u n g b r a i n is greatly influenced by e x t r a n e u r o n a l events which themselves change d u r i n g the postnatal period. A further influence which m a y be operating d u r i n g m a t u r a t i o n involves multiple enzyme forms 21. The existence of multiple M A O types n o w seems well established 19 a n d there is i n d i c a t i o n that they change in b r a i n d u r i n g developmentT, 21. The action of just these kinds o f mechanisms, which may change a n d o v e r l a p in time, could be at least partly responsible for the postnatal v a r i a t i o n seen between p a t h w a y c o m p o n e n t s a n d b r a i n regions. ACKNOWLEDGEMENTS This research was supported by U.S.P.H.S. Research G r a n t No. NS 09763. We would like to t h a n k R o b e r t Buda for his technical assistance a n d Paul Dinculescu for his assistance in the a n i m a l quarters.

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