Demonstration of monoamine oxidase-A and -B in the human brainstem by a histochemical technique

0306-4522/89 $3.00+ 0.00 PergamonPressplc IBRO

Neuroscience Vol. 33, No. 2, pp. 383400, 1989 Printed in Great Britain

DEMONSTRATION IN THE HUMAN

OF MONOAMINE OXIDASE-A AND -B BRAINSTEM BY A H~STOCHEMICAL TECHNIQUE

C. KomADr,*t

J. KORNHUBER,*L. FROELICH,*J. FRITZE,* H. HEINSEN,*H. BECKMANN,* E. SCHULZ$ and P. RIEDERER* *Clinical Neurochemistry, Department of Psychiatry, University of Wiirzburg, 8700 Wiirzburg, F.R.G. iInstitute of Forensic Medicine, University of Wiirzburg, 8700 Wiirzburg, F.R.G.

Abstract-The distribution of both monoamine oxidase subtypes, monoamine oxidase-A and -B, is demonstrated in brainstems from 16 humans by use of a histochemical technique. The results presented here, focus primarily upon the aminergic areas of the substantia nigra, the locus coeruleus and the raphe nuclei. While dopaminergic neurons of the substantia nigra revealed no staining for monoamine oxidase, noradrener~c neurons of the locus coeruleus stained positively with the monoamine oxidase-A substrate serotonin, and serotonergic neurons of the raphe nuclei were stained by the monoamine oxidase-B substrate ~-phenylethylamine. In addition, data are presented showing that glial cells stain predomifrantly for monoamine oxidase-B.

Monoamine oxidase (MAO) is the chief intraneuronal metabolic enzyme for the biogenic amines dopamine (DA), serotonin (5”HT) and norepinephrine. 35Two forms of MAO, MAO-A and -B, distinguished by their specific affinities for several substrates and inhibitors, have been described.‘4~i9 At higher concentrations, the substrate specificities for these two forms have been demonstrated to overlap. Nevertheless, in biochemical investigations of human brain tissue, MAO-A preferentially deaminates norepinephrine and 5-HT, while MAO-B preferentially oxidizes B-phenylethylamine and DA.‘2,33,35Some substrates like tyramine are metabolized by both MAO subtypes.‘O It has been proposed that each form of MAO and its corresponding substrate are compartmentai~zed into specific sections of neurons.36 There is, however, only a paucity of data avaifable to support this assumption. In a previous investigation we employed MAO-A and MAO-B specific monoclonal antibodies to stain structures in the human brainstem, and compared the distribution of MAO staining with that observed using polyclonal antibodies to tyrosine hydroxylase.2’*23The results obtained in that study contrasted sharply with the biochemical expectations. Neurons in the serotonergic raphe nuclei were discovered to contain p~ma~ly MAO-B, while the neurons in the dopaminergic substantia nigra did not react with either MAO antibodies (but see also Ref. 32). l_l_ tTo

whom correspondence should be addressed at: Clinical Neurochemistry, Department of Psychiatry, University of Wiirzburg, Fiichsleinstrak 15, 8700 Wiirzburg, F.R.G. Abhrrviations: DA, dopamine; SHT, serotonin; MAO, monoamine oxidase; MAOI, monoamine oxidase inhibitor.

Here we present data on the distribution of MAO in the human brainstem utilizing a histochemical technique, which simultaneously serves as control for the quality of the previously used antibodies.23 in addition, this technique enabled us to stain the actual locus of oxidation of specific MAO substrates and to study the effect of MAO inhibitors (MAOIs) in well defined brain areas. Histochemical investigations on MAO have been performed on various organs from a variety of species including mongolian gerbils, guinea-pigs, rats, rabbits and cats.1~‘1~‘3~‘6~‘7~‘*~22*27~34 Most of these investigations were performed using a perfusion fixation technique which excludes the use of human brain material. In our studies, we employed the MAO-A substrate 5-HT, the MAO-B substrate ~-phenylethylamine, and the substrate tyramine, which is oxidized by both MAO subtypes. To localize centers of inhibition of MAO activity in the human brainstem, we exploited both reversible (moclobemide, brofaromine) and irreversible (clorgyline, I-deprenyl, LY 51641) MAOIs. EXPERIMENTALPROCEDURES Investigations were performed

on human brainstems from 16 subjects (IO male/six female) ranginn - - in age from three months to 70 years including thke brains of neonates

(mean f SD. = 29.5 + 24.9 veard. Brains were obtained between 12 and 48 h post k&m from individuals who died suddenly and without findings of neuropsychiatric disorders, The substrates S-HT (creatinine sulfate complex), b-

phenylethylamine-HCl and tyramine-HC1 were purchased from Sigma (U.S.A.). I-Deprenyl was from Chinoin (Hungary), clorgyline from May and Baker (U.K.), LY 51641 from Lilly Company (U.S.A.), brofaromine from Ciba Geigy (Switzerland) and moclobemide from Roche (Switzerland). Horseradish peroxidase (type I) and 3,3’diami~o~n~dine.te~ahydr~hlo~de were obtained from Sigma. 383

Table I. Staining of the main aminergic areas in the human raphe

brainstem:

substantia

mgra. locus coeruleus

LIIIL’

nuclei inhibitor

Control

TYR

S-HT

,!I-phenylethylamine

L-Deprenyl

Clorgyiine

LY 51641

Brofaromine Moclobemide

SN LC NR

i” +

SN LC NR

+ -

SN LC NR

+

SN LC NR

-+

SN LC NR

+

SN

-

SN

-

SN

-

SrJ

-

SN

LC NR SN LC NR

+ (4) +

LC NR SN LC NR

+ -

LC NR SN LC NR

+

LC NR SN LC NR

--f

LC NR SN LC NR

SN LC

.-+

--

NR SN

+ -

(+)

LC NR SN LC NR

+ (+) -+

LC, locus coeruleus; NR, raphe nuclei; SN, substantia nigra; TY R, tyramine; + , stained neurons observed; -> no neurons stained, f &), weak staining in some preparations observed; (+), staining extremely reduced; substrate concentration: 1 mmol/l; inhibitor concentration: 100 pmol/i of I-deprenyl, clorgyline or LY 51641; 1 mmol/i of brofaromine or moclobemide. Brainstem was dissected at the levels of the locus coeruieus, the dorsal and median raphe nuclei and the substantia nigra. Dissected tissue was S-?-mm-thick and fixed in an iced solution containing 0.2% paraformaidehyde and 0.1% giutaraidehyde in sodium phosphate buffer (0.1 moi/i, pH 7.4). Fixation was performed for 16-18 h at 4°C by gently stirring. Subsequently the tissues were rinsed three times with sodium phosphate buffer. Samples were immersed in 15% sucrose at 4-6°C for 24 h and cut in 80-IOO-pm sections with a freezing microtome (Jung Co., Heidelberg). Slices were washed and transferred to the incubation media. The free-floating sections were carefully agitated for 1 h at room temperature and afterwards stored in a refrigerator for 24 to 48 h. The incubation medium consisted of 0.1% horseradish peroxidase, 3 mmoi/l sodium azide, 1 mmoi/i substrate (5-HT, /?-phenylethylamine, tyramine), 1 mmol/i reversible MAOI (brofaromine, moclobemide) or iOO~mol/l irreversible MAOI (ciorgyline, I-deprenyi, LY 51641), 0.01% 3,~-~~ino~n~~ne.tetrahydr~hio~de and 0.3% nickel ammonium sulfate in 0.05 mol/i Tris-HCi buffer (pH 7.6). Preincubation time with the MAOI lasted 10 min. Staining was performed on consecutive slices, for additional routine

staining gailocyanine was used.16After staining, slices were mounted on slides, dehydrated and embedded in !&kit@ (Kindler, F.R.G.). No staining was observed when the substrate was omitted. The definition of brain areas and the schematic anatomical drawings were according to De Armond er al.’ RESULTS

The results of the staining in three transverse brain sections including aminergic areas is shown in Table 1. Figure la-c gives schematic drawings of the distribution of both MAO subtypes in these three sections. Particular attention was paid to the locus coeruleus, the raphe nuclei and the substantia ni8ra. Routine staining with gallocyanine in the brainstem at the level of the locus coeruleus and the dorsal and median raphe nuclei is exemplified in Fig. 2. The noradrenergic locus coeruleuszs stained positively with the MAO-A substrate S-HT (Fig. 3) and the unselective substrate tyramine (Fig. 4). Staining by both substrates was abolished when the irreversible MAO-AIs LY 51641,‘* (Fig. 5) or clorgyline’4 as well as the reversible MAO-AI brofaromine,*TTM (Figs 6 and 7) were present in the

incubation medium. The irreversible MAO-B inhibitor l-depreoyi,‘9 (Figs 8 and 9) and the reversibie MAO-AI moclobemide5~‘S did not affect the intensity of the staining (Fig. 10). In the raphe nuclei, serotonergic neurons3h29 were found predominantly to contain MAO-B, as indicated by their staining with ~-phenylethyl~ine (Fig. 11) and tyramine (Fig. 4), and also by inhibitor studies (Figs 5, 6, 9, 12, 13). While brofaromine also appeared to reduce p-phenylethylamine metabolization (Figs 12 and 13), moclobemide was totally ineffective. Although the glial cells of the substantia nigra, and some fibers, showed MAO-B-related staining, the perikarya of this area (Fig. 14) were not stained by either of the three substrates. Glial cell staining was more pronounced in the neonates’ brain and exhibited a preponderance of MAO-B in all sections examined. S-HT stained only a few astroglial cells primarily around and in the locus coeruleus. In contrast, vessels and capillaries were stained by 5-HT and tyramine, and seemed to contain predominantly MAO-A. The neurons of the oculomotor nucleus were stained by all three substrates. Since a weak staining was also observed on the control slices and in the presence of MAOIs, staining in this case might be independent of MAO activity. DISCUSSXON

The major advantage of the histochemical technique is that it affords the possibility to visualize the loci of MAO inhibitory activity. This aspect was especially useful for demonstrating the differences between glial and neuronal ~om~~mentation. Since the histochemical technique is based on the biochemical MAO-A/MAO-I3 model, it deals with the differential affinities of both subtypes to distinct substrates and inhibitors. At higher sub&&e as well as inhibitor ~n~ntrations interaction of both MAO subtypes may occur.8.9*36Nevertheless, although the

~monstration

385

of monoamine oxidase-A and -B in the human brainstem

substrate and inhibitor concentrations were very high, a specific reaction was obtained in different brain areas. As predicted by the enzyme-substrate affinity, the noradrener~c neurons of the locus coeruleu8 contained MAO-A. In contrast, the serotonergk neurons of the raphe nuclei3T2’ reacted mainly with B-phenylethylamine, indicating that they contain MAO-B. This was not expected, since in biochemical investigations, 5-HT is a typical MAO-A substrate with low affinity for MAO-B.36 Nevertheless, the compartmentation of S-HT with MAO-B has also been reported for rats,” cats,“,” and in studies of blood platelets.37 The neuronal cell bodies of the substantia nigra showed no reaction with either MAO substrate. In a recent publication,32 small amounts of MAO-A were detected in some neurons of the substantia nigra only. The technique described here might not be sensitive enough to reproduce this finding. But, since the neurons of the substantia nigra contain dramatically less MAO-A than neurons of for example the locus coeruleus, an important question is, whether this con~ntration of MAO-A is sufkient to influence dopamine availability.

Glial ceil staining, mostly MAO-B related, was observed in all brain areas investigated, but was most prominent in the substantia nigra, the periaqueductal gray matter, the pons, the raphe nuclei and the locus coeruleus. MAO in the glial cells of infant brains reacted much better than that in adult brains. This might be due to a better post mortem conservation of infant brains or to higher MAO activity during childhood. Glial MAO subtypes may change depending on the stage of ontogenesis.“.‘* Nevertheless, tyramine oxidation and ~-phenylethylamine oxidation in the glial 41s of all age groups predominated over 5-HT oxidation. The latter was restricted to catecholaminergic areas, predominantly the locus coeruleus. The irreversible MAOIs I-deprenyl (MAO-BI), LY 51641 (MAO-AI) and clorgyline (MAO-AI) successfully ~fferentiat~ MAO subtypes in the presence of each substrate, especially of tyramine. However, the results with the reversible MAO-AIs brofaromine and moclobemide were complicated. While brofaromine, by totally inhibiting 5-HT staining, also inhibited ~-phenylethylamine staining, moclobemide did not affect the staining at all. The difference between both inhibitors may be, that brofaromine is

\I,

=

fibers

,.:::. ..

=

astroglia = PEA and tyra~i~

1. =

stained

Y

= 5-HT and tyramine stained

BC

CTT IN LC ML MRF NR ON

brachium conjunctivum (decussation of superior cerebellar peduncle) central tegmental tract interpeduncular nucleus locus coeruleus medial lemniscus midbrain reticular formation raphe nuclei oculomotor nucleus

PEA PCi RF RFM !lzl VTA IV

fi-phenylethylamine periaqueductal gray matter reticular formation reticular formation of mesencephalon Red Nucleus substantia nigra ventral tegmental area ventricle IV

Fig. lax:. Distribution of SHT, b-phenylethylamine and tyramine metabolism by MAO in three transverse sections of the human brainstem at the level of SN (a), NR (b) and LC (c)(histological drawings according to De Armond et ~1.~).

386

Figs 2-13. Brainstem of a male neonate. age nine months, died on sudden infant death. Fig. 2. Routine staining of the human brainstem at the level of the raphe nuclei (NR) and the IOCUS coeruleus (LC) with gallocyanine. 26Neurons as well as cell nuclei are stained.

Demonstration

387

of monoamine oxidase-A and -B in the human brainstem

Fig. 3. Locations of serotonin turnover in the human locuscoeruleus: microscopical magnification

x 63.

388

Fig. 4. Locations of tyramine turnover in the human brainstem of an infant (no n~oromelanin coeruleus) at the level of the locus coeruleus (LC) and the raphe nuciei (NR).

in the locus

Demonstration

of monoamine oxidase-A and -B in the human brainstem

Fig. 5. Tyramine turnover in the brainstem of a human infant at the level of the locus coeruleus (LC) and the raphe nuclei (NR) in the presence of the MAO-A inhibitor LY 51641. Note: Staining of the neurons of the raphe nuclei, no staining of the neurons of the locus coeruleus. Astroglial staining (arrows).

389

390

Fig. 6. Inhibition of tyramine turnover in the brainstem of a human infant at the level of the locus coeruleus (LC) and the raphe nuclei (NR) in the presence of the MAO-A inhibitor brofaromine. Note: decreased staining of the neurons of the raphe nuclei, no staining of the neurons of the locus coeruleus.

Demonstration

of monoamine oxidase-A and -B in the human brainstem

Fig. 7. Inhibition of serotonin turnover in the brainstem of a human infant at the level of the locus coeruleus (LC) and the raphe nuclei (NR) in the presence of the MAO-A inhibitor brofaromine.

391

392

Fig. 8. Serotonin turnover and the raphe

in the brainstem of a human infant at the level of the locus coeruleus nuclei (NR) in the presence of the MAO-B inhibitor I-deprenyl.

(LC)

Demonstration

of monoamine oxidase-A and -B in the human brainstem

Fig. 9. Inhibition of tyramine turnover in the brainstem of a human infant at the level of the locus coeruleus (LC) and the raphe nuclei (NR) in the presence of the MAO-B inhibitor I-deprenyl. Note: no staining of the neurons of the raphe nuclei, but staining of the neurons of the locus coeruleus.

393

Fig. 10. Serotonin turnover in the brainstem of a human infant at the level of the locus coeruleus (LC) and the raphe nuclei (NR) in the presence of the MAO-A inhibitor moclobemide. Note: no inhibition.

Demonstration

of monoamine oxidase-A and -B in the human brainstem

Fig. 11. Locations of phenylethylamine

turnover in the human dorsal raphe nucleus: microscopical magnification x 63.

395

396

Fig. 12. Phenylethylamine turnover in the brainstem of a human infant at the level of the locus coeruleus (LC) and the raphe nuclei (NR) in the presence of the MAO-A inhibitor LY 51641. Note: staining of astroglia (arrows).

Demonstration

of monoamine oxidase-A and -B in the human brainstem

Fig. 13. Incomplete inhibition of phenylethylamine turnover in the brainstem of a human infant at the level of the locus coeruleus (LC) and the raphe nuclei (NR) in the presence of the MAO-A inhibitor brofaromine. Note: reduced staining in NR and in astroglia.

NSC 3312-F

397

398

Demonstration

of monoamine

oxidase-A

partly irreversible, as suggested in other reports.1513’ Another possibility is, that moclobemide is not an MAO1 per se, but an active metabolite of it.4 These do not seem to be generated in the brain under conditions of short incubation, since otherwise an inhibition would have been observed.

and -B in the human brainstem

399

The present findings might contribute to the understanding of the action of MAO-AIs in depression and MAO-BIs in Parkinson’s disease in terms of localization. Acknowledgement-We reading the manuscript.

thank

R. E. Tanzi

for carefully

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31.

32.

33.

34. 35. 36.

37. 38.

(Accepted

5 June 1989)