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Topographic immunocytochemical mapping of monoamine oxidase-A, monoamine oxidase-B and tyrosine hydroxylase in human post mortem brain stem
03~-4522~88$3.00+ 0.00 Pergamoo Press pie 6 1988IBRO
N@~roseje~ce Vol. 26, No. 3, Pp. 791-802, 1988 Prinued in Great Britain
TOPOGRAPHIC IMMUNOCYTOCHEMICAL MAPPING OF MONOAMINE OXIDASE-A, MONOAMINE OXIDASE-B AND TYROSINE HYDROXYLASE IN HUMAN POST MORTEM BRAIN STEM CH. KONRADI,* E. SVOMA,~ K. JELLINGER,? P. RIEDERER,* R. DENNEY~ and J. THIBAULT§ *Clinical Neurochemistry, Department of Psychiatry, University of Wiirzburg, D-8700 Wiirzburg, F.R.G., tLudwig Boltzmann Institute of Clinical Neurobiology, Lainz Hospital, A-l 130 Wien, Austria, IDepartment of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550, U.S.A. and $Laboratoire de Biochimie Cellulaire, College de France, F-75231 Paris cedex 05, France A~~act-Immuno~yto~hemical demonstration of monoamine oxidase-A, monoamine oxidase-B and tyrosine hydroxylase was performed in the human brain stem using monoclonal antibodies to monoamine oxidase-A and monoamine oxidase-B and polyclonal antibodies to tyrosine hydroxylase. In most of the brain areas examined, except the serotonergic dorsal nucleus of raphe, the noradrenergic locus coeruleus and the dorsal efferent nucleus of vagus, tyrosine hydroxylase-positive neurons were in greater number than monoamine oxidase-A-stained or monoamine oxidase-B-stained neurons. The dorsal nucleus of raphe showed no tyrosine hydroxylase immunoreactivity, but reacted positively to serotonin- and monoamine oxidase-B antibodies, while monoamine oxidase-A staining was moderate. In none of the investigated brain areas did neurons exclusively react with monoamine oxidase-B antibodies without expressing monoamine oxidase-A in a few neurons, while in some areas neurons expressed both monoamine oxidase-A and tyrosine hydroxylase (locus coeruleus; dorsal efferent nucleus of vagus). The oculomotor nucleus stained only with monoamine oxidase-A antibodies, substantia nigra neurons reacted only with tyrosine hydroxylase antibodies. Glial staining in most of the brain areas examined seemed, with slight differences, to have the same intensity with monoamine oxidase-A and monoamine oxidase-B antibodies used. No glial staining was obtained with tyrosine hydroxylase antibodies.
reported the discovery of two types of monoamine oxidase (MAO; EC1.4.3.4) based on the biochemical utilization of different substrates and on the specificity of the MAO inhibitor clorgyline.** As bi~hemical analyses of brain homogenates provide little information concerning the cellular distribution of MAO-A and -B,39 histological investigations have been performed on various organs from a variety of species including cats, rats, rabbits and guinea-pigs.‘6.‘9,23,37.42 Thereby several histochemical methods have proven useful for pharmacological purposes. By visualizing the sites of degradation of different MAO substrates and, if desired, by use of different inhibitors, one may gather information concerning the MAO subtype located there. The disadvantages of histochemical techniques are their use of biochemical models, wherefore they cannot verify biochemical models, their broad variations in different animals and tissuesr2-r4 and the dependence for their interpretation on the substrate and inhibitor specificities of MAO-A and MAO-B. And unfortunately, demonstrations of the localization of MAO-A and MAO-B in human brain by histochemical methods have generally been unsuccessful. In 1968 Johnson
Abbreoiarions:
hydroxylase.
MAO, monoamine oxidase; TH, tyrosine
Therefore, immunohistological and immunocytochemical methods have been developed which permit the independent detection of MAO-A and MAO-B by their distinct immunological properties.3,5*27*43 The isolation of a mon~lonal antibody to human platelet MAO-B9 and later of monoclonal antibodies to human placental MAO-A*’ yielded the opportunity to visualize both MAO-A and MAO-B in human brain (Ref. 26, Westlund et al., unpublished observations). We have used MAO-A and MAO-B specific antibodies to stain structures in the human brain stem, especialiy in putamen, substantia nigra, dorsal nucleus of raphe, nucleus of locus coeruleus and dorsal efferent nucleus of vagus,7,‘3 and have compared the distribution of MAO staining with that observed with polyclonal antibodies to rat tyrosine hydroxylase (TH; ECl.t4.16.2).2 EXPERIMENTAL
PROCEDURES
Ten human brains (six female/four male) were obtained at autopsy with posf rnor&r~ time ranging from 3 to 6.5 h. The age range was from 46 to 88 years with a mean of 74 f 18 (S.E.M.). Tissues were dissected and handled immediately for immun~yt~hemistry. The left side of the brain was used for pathohistologi~l examinations, the right one for immunohistology. Clinical and neuropathological data showed no CNS lesions and drug treatment before death did not include psychotropic compounds. Dissected samples were 34mm thick and fixed in an iced solution containing 4% paraformaldehyde in sodium 791
CH. KONRADI ct ul.
792 Table
I. Sera and antibodies
TH antibodies Serum Second PAP*
antibody
used
MAO-A/MAO-B
normal swine (1%) swine anti rabbit rabbit anti peroxidase
antibodies
normal rabbit (I %) rabbit anti mouse mouse anti peroxidase
MAO-A antibody (monoclonal): 7BlO (for designation of MAO antibodies see Kochersperger ef a/.“; 5 ~1 were immersed in 2.5 ml I % normal serum; MAO-B antibody (monoclonal): lC2; 5 ~1 were immersed in 2.5 ml I% normal serum; TH antibody (polyclonal; rabbit): 2.5 ~1 were immersed in 2.5 ml I % normal serum *Peroxidase-antiperoxidase.
phosphate buffer (0.1 mol/l NaHzPO,;Na,HPO,, pH 7.4). Fixation was performed for 8-12 h in an ice-bath by slightly stirring. Subsequently the tissues were rinsed three times with sodium phosphate buffer. Sections were cut either with a Vibratome in sodium phosphate buffer (30-60pm-thick) or with a cryocut microtome (20 pm-thick). If they were cut with a cryocut microtome, they were immersed in 15% sucrose at 4°C for 24 h beforehand to avoid freeze-damage. The following incubation steps were carried out with free-floating sections (sera and antibodies used are listed in Table I). After preincubation in 0.3% hydrogen peroxide in methanol at room temperature for 30 min to inhibit endogenous peroxidases, washing twice for I5 min in sodium phosphate buffer and incubation in 1% normal serum at room temperature for 30min, sections were immersed in the antibodies to either MAO-A or -B or tyrosine hydroxylase (method of Thibault et a1.40).
Fig. I. Distribution ATH-immunoreactive
CN CTT DENV DNR HN IC ICP IN ION
RESULTS
The
distribution
brachium conjunctivum (decussation cerebellar peduncle) cuneate nucleus central tegmental tract dorsal efferent nucleus of vagus dorsal nucleus of raphe hypoglossal nucleus inferior colliculus inferior cerebellar peduncle interpcduncular nucleus inferior olivary nucleus
of MAO-A-,
MAO-B-
and
neurons
in substantia nigra. Left side: neurons; AMAO-B-immuno-
used in .figures and &dks
of superior
TH-
in the human brainstem is shown in Figs I-3. A short summary is given in Table 2. In view of previous examinations with TH antibodies,‘5.35.44our results using these antibodies served positive
of MAO-A-, MAO-B- and TH-containing neurons neurons. Right side: AMAO-A-immunoreactive reactive neurons.
Ahhreoiukms BC
The following steps included a previously described peroxidase-antiperoxidase method.28 Diaminobenzidine served as dye reagent. Sections were embedded without dehydration in a solution containing glycerol&sodium phosphate (I : I) buffer. Peroxidase-conjugated antibodies were used in consecutive sections of three brains as additional control. Except for a paler staining, no differences in the number of neurons stained were seen. Some sections were counterstained with hemalum, dehydrated and mounted.
ML MLF MRF ON PG PT RFM RFNG RN SN VTA
medial lemniscus medial longitudinal fasciculus midbrain reticular formation oculomotor nucleus periaqueductal gray matter pyramidal tract reticular formation of mesencephalon reticular formation, nucleus gigantocellularis red nucleus substantia nigra ventral tegmental area
Immunocytochemistry
793
of MAO-A, MAO-B and TH in human brain stem
Fig. 3. Distribution
of MAO-A-,
MAO-B-
and TH-
containing neurons in a transverse section of the medulla
Fig. 2. Distribution of MAO-A-, MAO-B- and THcontaining neurons in a transverse section of the midbrain at the level of dorsal nucleus of raphe. Left side: ATHimmunoreactive neurons. Right side: AMAO-A-immunoreactive neurons; AMA~B-immunoreactive neurons. as additional control for technique and reliability of the method used and the distribution of dopamine- or norepinephrine-containing neurons. Since MAO-A and MAO-B antibodies were prepared from the same species (mouse), it was not possible to apply a double staining (MAO-AlMAO-B) method without affecting the antibodies, Instead we used consecutive serial sections in an attempt to determine whether any cells stained by both MAO-A and MAO-B antibodies. Examinations were performed in three transverse sections of the brain stem, two in midbrain and one in medutla and in the putamen. The distribution of MAO-A-, MAO-B- and THpositive neurons in the midbrain sections at the level of the red nucleus is shown in Fig. 1. Moderate staining with antibodies to both subtypes of MAO and strong reaction with TH antibodies was obtained in the ventral tegmental area and midbrain reticular formation. MAO-A staining of the oculomotor nucleus (Fig. 4) was not expected since this nucleus is known to be cholinergic. The absence of TH staining indicates that these neurons do not contain dopamine or norepinephrine although catecholaminergic cells in this area have been described in rats.$ In substantia nigra, MAO staining of neurons was negative (Fig. S), while TH reactivity was intense (Fig. 6). Figure 2 shows the reactions at the level of dorsal nucleus of raphe. The mesencephalic reticular formation showed neurons with MAO-A-, MAO-B- and a majority with TH antibody staining. Most of the neurons in dorsal nucleus of raphe expressed MAO-B (Fig. 7), a small number MAO-A (Fig. 8) and none TH. Staining with serotonin antibodies gave positive results, neurons and fibres were attached all through this area.
at the level of nucleus giganto~llula~s (midbrain reticular formation). Left side: ATH-immunoreactive neurons. Right side: AMAO-A-immunoreactive neurons; AMAO-B-immunoreactive neurons.
The neurons of locus coeruleus, which are known to contain norepinephrine3’ expressed both MAO-A (Fig. 9) and TH (Fig. IO). In the medulla, shown in Fig. 3, MAO-A- and TH-positive neurons were observed in solitary nucleus and seemed to be identical. Reticular formation with nucleus gigantocellularis was stained by all three antibodies used (Figs I l-13), most intensively with TH (Fig. 13). Medially to the medulla some neurons were recognized by MAO-B antibodies. MAO-A and MAO-B staining of astroglia was observed in areas which are known to contain catecholaminergic or serotonergic neurotransmitters as well as in other brain areas. Distribution and intensity were Iargeiy identical. Stained glial cells of the medulla are shown in Figs 14 and 15.
Table 2. Intensity of neuronal antibody staining in different brain areas Brain area CTT DENV DNR Locus coeruleus MRF ON Putamen* RFM RFNG SN VTA
MAO-A _ +++ rt
MAO-B
+ + + + ++ f + ++ +
f
+++
t + Cf +
TH + +++ +++ ++ +-I-+ ++ +++ +++ +++
*Mainly fibres existing. + + + High density of neurons stained; + +medium density of neurons stained; f moderate density of neurons stained; + staining uncertain (glial staining too dark or cross-reaction of antibodies . _possible); - no stammg ot neurons observed.
794
CH.
KONRADI
et
al.
Fig. 4. MAO-A staining neurons and gliai cells in ON. PAP method. Original ma~ification:
x 63.
Fig. 5. MAO-B staining in SN. Unbleached neuromelanin of neuronal ceils, background staining of glial cells but no staining of neurons or fibres are shown. PAP method. Original magnification: x 63.
Immunocytochemistry
of MAO-A, MAO-B and TH in human brain stem
Fig. 6. TH staining in SN. No background because of unstained glial cells. NeuromeIanin as well as neuronal staining including fibres can be observed. PAP method. Original ma~ifi~tjon: x 63.
Fig. 7. MAO-B staining in DNR. Immunoreactivity of neuronal cells including fibres and glial cells (dark background). PAP method. Original magnification: x 63.
795
CH.
796
Fig. 8. MAO-A-like
Fig. 9. MAO-A
KONRADl ct al.
immunoreactivity in DNR. Moderate staining of neuronal cells, background of glial cells. PAP method. Original magnification: x 63.
staining in locus coeruleus. Immunoreactivity of neuronal ceils including cells (dark background). PAP method. Original magnification: x 63.
staining
fibres and glial
Immunocytochemistry
of MAO-A,
MAO-B
and TH in human
brain
197
stem
Fig. 10. TH staining
in locus coeruleus. lmmunoreactivity of neuronal cells and fibres. No background. PAP method. Original magnification: x 63.
Fig. Il. MAO-A-like
immunoreactivity in RFNG. Staining of neuronal cells. PAP method. Original magnification:
cells, background x 63.
staining
of ghal
798
CH.
KONRADI
et
al.
Fig. 12. MAO-B-like imm~noreact~~ity in RFNG. Staining of neuronal cells, background staining of glial cells. PAP method. Original magni~cation: x 63.
Fig. 13. TH staining in RFNG. Immunoreacti~ity of neuronal cells and fibres. No background. PAP method. Original magnification: x 63.
Immunocytochemistry
of MAO-A, MAO-B and TH in human brain stem
Fig. 14. Immunoreactivity
of astroglia in medullar reticular formation. MAO-A, PAP method. Original magnification: x 63.
Fig. 15. Immunoreactivity
of astroglia in medullar reticular formation. MAO-B, PAP method. Original magnification: x 63.
799
800
Of.
K~NRA~Iet al.
DISCUSSIQN
Specificity of MAO antibodies: preparation of antibodies and specification for each form of MAO have been described by Denney et al.’ and Kochersperger et al. 25 Our immunohistologi~al investigations exclude cross-reactivity of MAO-B antibodies with MAO-A because in some brain areas the neurons were only reacting with MAO-A antibodies (e.g. locus coeruleus). Cross-reactivity of MAO-A antibodies with high levels of MAO-B cannot be totally excluded. However, in dorsal nucleus of raphe more MAO-B than MAO-A immunoreactive neurons could be observed. The lack of MAO staining of nerve cells in the dopaminergic substantia nigra was astonishing since TH immunoreactivity of nigral neurons was very strong. On the other hand, glial cells in this region showed intense reaction with both MAO-A and MAO-B antibodies. Biochemical examinations of human brain substantia nigra led to the assumption that MAO-B is located in those neurons.‘7,32 Earlier investigations in primates which showed slight MAO-A staining in neurons of substantia nigra43 could not be confirmed in humans by our study. Presumptive MAO-A association with nigrostriatal dopaminergic neurons in rats was also described.8 in putamen, fibres of the projection areas of substantia nigra showed positive TN immunor~activity whereas only a small number of fibres stained for MAO. In addition, strong glial MAO-A and MAO-B staining in putamen contrasted with weakly stained fibres. Another surprising result was the intense MAO-B antibody staining in the serotonergic dorsal nucleus of raphe, although in the biochemical models serotonin is an MAO-A substrate. However, the immunocytochemistry may be consistent with biochemical data. For example, the intrasynaptosomal deamination of serotonin by MAO-B has been described in rat brain” and in cat brain.14 Furthermore, in citw decrease of serotonin decomposition also depends on inhibition of both MAO-A and MAO-B.” In addition, our data are consistent with recent
immunocytochemical demonstration of MAO-B in serotinergic neurons of rats,” primates” and humans.‘* However, the prior investigations in primate and human brain were carried out with the same antibodies as used in our study. It seems that, in relation to TH-positive neurons, more catecholaminergic neurons exist than MAO-Aor MAO-B-positive ones. This is most striking in substantia nigra. By disregarding the remote possibility of the adrenergic origin of TH-positive/MAOnegative neurons, these findings suggest that the role of MAO activity in neurons might be one other than the degradation of extravesicular neurotransmitt~r generated within the neuron. The available data are consistent with the hypothesis that MAO protects against extraneuronal monoamines, which do not represent intra~llular metabolit~ and which enter the cells by uptake. This hypothesis is supported by the existence of more MAO-B-positive neurons in serotonergic areas than MAO-A-positive neurons, although from biochemical studies it is well known that serotonin has a higher affinity for MAO-A than for MAO-B.‘“,‘” A further possibility is the existence of a third enzyme or enzyme subtype involved in the intraneuronal degradation of biogenic amines without the paratope for MAO-A or MAO-B antibodies.“0~41 However, it is also possible that MAO in the mitochondri~i membrane of neurons of different brain areas (e.g. substantia nigra) could be less accessible to antibodies than MAO in other brain areas (e.g. locus coeruieus, dorsal nucleus of rdphe). In particular, these results require reconsideration of the roles of MAO-A and MAO-B in human brain and their possible involvement in degenerative and mental disorders of catecholaminergic systems1.“‘.‘4as well as a possible role in the generation of neuromelanin.2y These data also need to be considered in understanding the mode of action of drugs affecting monoaminergic metabolism.4,“” In this context, astroglia could be more important than has heretofore been recognized.‘O Drug effects in the central nerv(~us system could be due to changes in both neuronai activity and the physiology of glial cells.
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N@~roseje~ce Vol. 26, No. 3, Pp. 791-802, 1988 Prinued in Great Britain
TOPOGRAPHIC IMMUNOCYTOCHEMICAL MAPPING OF MONOAMINE OXIDASE-A, MONOAMINE OXIDASE-B AND TYROSINE HYDROXYLASE IN HUMAN POST MORTEM BRAIN STEM CH. KONRADI,* E. SVOMA,~ K. JELLINGER,? P. RIEDERER,* R. DENNEY~ and J. THIBAULT§ *Clinical Neurochemistry, Department of Psychiatry, University of Wiirzburg, D-8700 Wiirzburg, F.R.G., tLudwig Boltzmann Institute of Clinical Neurobiology, Lainz Hospital, A-l 130 Wien, Austria, IDepartment of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550, U.S.A. and $Laboratoire de Biochimie Cellulaire, College de France, F-75231 Paris cedex 05, France A~~act-Immuno~yto~hemical demonstration of monoamine oxidase-A, monoamine oxidase-B and tyrosine hydroxylase was performed in the human brain stem using monoclonal antibodies to monoamine oxidase-A and monoamine oxidase-B and polyclonal antibodies to tyrosine hydroxylase. In most of the brain areas examined, except the serotonergic dorsal nucleus of raphe, the noradrenergic locus coeruleus and the dorsal efferent nucleus of vagus, tyrosine hydroxylase-positive neurons were in greater number than monoamine oxidase-A-stained or monoamine oxidase-B-stained neurons. The dorsal nucleus of raphe showed no tyrosine hydroxylase immunoreactivity, but reacted positively to serotonin- and monoamine oxidase-B antibodies, while monoamine oxidase-A staining was moderate. In none of the investigated brain areas did neurons exclusively react with monoamine oxidase-B antibodies without expressing monoamine oxidase-A in a few neurons, while in some areas neurons expressed both monoamine oxidase-A and tyrosine hydroxylase (locus coeruleus; dorsal efferent nucleus of vagus). The oculomotor nucleus stained only with monoamine oxidase-A antibodies, substantia nigra neurons reacted only with tyrosine hydroxylase antibodies. Glial staining in most of the brain areas examined seemed, with slight differences, to have the same intensity with monoamine oxidase-A and monoamine oxidase-B antibodies used. No glial staining was obtained with tyrosine hydroxylase antibodies.
reported the discovery of two types of monoamine oxidase (MAO; EC1.4.3.4) based on the biochemical utilization of different substrates and on the specificity of the MAO inhibitor clorgyline.** As bi~hemical analyses of brain homogenates provide little information concerning the cellular distribution of MAO-A and -B,39 histological investigations have been performed on various organs from a variety of species including cats, rats, rabbits and guinea-pigs.‘6.‘9,23,37.42 Thereby several histochemical methods have proven useful for pharmacological purposes. By visualizing the sites of degradation of different MAO substrates and, if desired, by use of different inhibitors, one may gather information concerning the MAO subtype located there. The disadvantages of histochemical techniques are their use of biochemical models, wherefore they cannot verify biochemical models, their broad variations in different animals and tissuesr2-r4 and the dependence for their interpretation on the substrate and inhibitor specificities of MAO-A and MAO-B. And unfortunately, demonstrations of the localization of MAO-A and MAO-B in human brain by histochemical methods have generally been unsuccessful. In 1968 Johnson
Abbreoiarions:
hydroxylase.
MAO, monoamine oxidase; TH, tyrosine
Therefore, immunohistological and immunocytochemical methods have been developed which permit the independent detection of MAO-A and MAO-B by their distinct immunological properties.3,5*27*43 The isolation of a mon~lonal antibody to human platelet MAO-B9 and later of monoclonal antibodies to human placental MAO-A*’ yielded the opportunity to visualize both MAO-A and MAO-B in human brain (Ref. 26, Westlund et al., unpublished observations). We have used MAO-A and MAO-B specific antibodies to stain structures in the human brain stem, especialiy in putamen, substantia nigra, dorsal nucleus of raphe, nucleus of locus coeruleus and dorsal efferent nucleus of vagus,7,‘3 and have compared the distribution of MAO staining with that observed with polyclonal antibodies to rat tyrosine hydroxylase (TH; ECl.t4.16.2).2 EXPERIMENTAL
PROCEDURES
Ten human brains (six female/four male) were obtained at autopsy with posf rnor&r~ time ranging from 3 to 6.5 h. The age range was from 46 to 88 years with a mean of 74 f 18 (S.E.M.). Tissues were dissected and handled immediately for immun~yt~hemistry. The left side of the brain was used for pathohistologi~l examinations, the right one for immunohistology. Clinical and neuropathological data showed no CNS lesions and drug treatment before death did not include psychotropic compounds. Dissected samples were 34mm thick and fixed in an iced solution containing 4% paraformaldehyde in sodium 791
CH. KONRADI ct ul.
792 Table
I. Sera and antibodies
TH antibodies Serum Second PAP*
antibody
used
MAO-A/MAO-B
normal swine (1%) swine anti rabbit rabbit anti peroxidase
antibodies
normal rabbit (I %) rabbit anti mouse mouse anti peroxidase
MAO-A antibody (monoclonal): 7BlO (for designation of MAO antibodies see Kochersperger ef a/.“; 5 ~1 were immersed in 2.5 ml I % normal serum; MAO-B antibody (monoclonal): lC2; 5 ~1 were immersed in 2.5 ml I% normal serum; TH antibody (polyclonal; rabbit): 2.5 ~1 were immersed in 2.5 ml I % normal serum *Peroxidase-antiperoxidase.
phosphate buffer (0.1 mol/l NaHzPO,;Na,HPO,, pH 7.4). Fixation was performed for 8-12 h in an ice-bath by slightly stirring. Subsequently the tissues were rinsed three times with sodium phosphate buffer. Sections were cut either with a Vibratome in sodium phosphate buffer (30-60pm-thick) or with a cryocut microtome (20 pm-thick). If they were cut with a cryocut microtome, they were immersed in 15% sucrose at 4°C for 24 h beforehand to avoid freeze-damage. The following incubation steps were carried out with free-floating sections (sera and antibodies used are listed in Table I). After preincubation in 0.3% hydrogen peroxide in methanol at room temperature for 30 min to inhibit endogenous peroxidases, washing twice for I5 min in sodium phosphate buffer and incubation in 1% normal serum at room temperature for 30min, sections were immersed in the antibodies to either MAO-A or -B or tyrosine hydroxylase (method of Thibault et a1.40).
Fig. I. Distribution ATH-immunoreactive
CN CTT DENV DNR HN IC ICP IN ION
RESULTS
The
distribution
brachium conjunctivum (decussation cerebellar peduncle) cuneate nucleus central tegmental tract dorsal efferent nucleus of vagus dorsal nucleus of raphe hypoglossal nucleus inferior colliculus inferior cerebellar peduncle interpcduncular nucleus inferior olivary nucleus
of MAO-A-,
MAO-B-
and
neurons
in substantia nigra. Left side: neurons; AMAO-B-immuno-
used in .figures and &dks
of superior
TH-
in the human brainstem is shown in Figs I-3. A short summary is given in Table 2. In view of previous examinations with TH antibodies,‘5.35.44our results using these antibodies served positive
of MAO-A-, MAO-B- and TH-containing neurons neurons. Right side: AMAO-A-immunoreactive reactive neurons.
Ahhreoiukms BC
The following steps included a previously described peroxidase-antiperoxidase method.28 Diaminobenzidine served as dye reagent. Sections were embedded without dehydration in a solution containing glycerol&sodium phosphate (I : I) buffer. Peroxidase-conjugated antibodies were used in consecutive sections of three brains as additional control. Except for a paler staining, no differences in the number of neurons stained were seen. Some sections were counterstained with hemalum, dehydrated and mounted.
ML MLF MRF ON PG PT RFM RFNG RN SN VTA
medial lemniscus medial longitudinal fasciculus midbrain reticular formation oculomotor nucleus periaqueductal gray matter pyramidal tract reticular formation of mesencephalon reticular formation, nucleus gigantocellularis red nucleus substantia nigra ventral tegmental area
Immunocytochemistry
793
of MAO-A, MAO-B and TH in human brain stem
Fig. 3. Distribution
of MAO-A-,
MAO-B-
and TH-
containing neurons in a transverse section of the medulla
Fig. 2. Distribution of MAO-A-, MAO-B- and THcontaining neurons in a transverse section of the midbrain at the level of dorsal nucleus of raphe. Left side: ATHimmunoreactive neurons. Right side: AMAO-A-immunoreactive neurons; AMA~B-immunoreactive neurons. as additional control for technique and reliability of the method used and the distribution of dopamine- or norepinephrine-containing neurons. Since MAO-A and MAO-B antibodies were prepared from the same species (mouse), it was not possible to apply a double staining (MAO-AlMAO-B) method without affecting the antibodies, Instead we used consecutive serial sections in an attempt to determine whether any cells stained by both MAO-A and MAO-B antibodies. Examinations were performed in three transverse sections of the brain stem, two in midbrain and one in medutla and in the putamen. The distribution of MAO-A-, MAO-B- and THpositive neurons in the midbrain sections at the level of the red nucleus is shown in Fig. 1. Moderate staining with antibodies to both subtypes of MAO and strong reaction with TH antibodies was obtained in the ventral tegmental area and midbrain reticular formation. MAO-A staining of the oculomotor nucleus (Fig. 4) was not expected since this nucleus is known to be cholinergic. The absence of TH staining indicates that these neurons do not contain dopamine or norepinephrine although catecholaminergic cells in this area have been described in rats.$ In substantia nigra, MAO staining of neurons was negative (Fig. S), while TH reactivity was intense (Fig. 6). Figure 2 shows the reactions at the level of dorsal nucleus of raphe. The mesencephalic reticular formation showed neurons with MAO-A-, MAO-B- and a majority with TH antibody staining. Most of the neurons in dorsal nucleus of raphe expressed MAO-B (Fig. 7), a small number MAO-A (Fig. 8) and none TH. Staining with serotonin antibodies gave positive results, neurons and fibres were attached all through this area.
at the level of nucleus giganto~llula~s (midbrain reticular formation). Left side: ATH-immunoreactive neurons. Right side: AMAO-A-immunoreactive neurons; AMAO-B-immunoreactive neurons.
The neurons of locus coeruleus, which are known to contain norepinephrine3’ expressed both MAO-A (Fig. 9) and TH (Fig. IO). In the medulla, shown in Fig. 3, MAO-A- and TH-positive neurons were observed in solitary nucleus and seemed to be identical. Reticular formation with nucleus gigantocellularis was stained by all three antibodies used (Figs I l-13), most intensively with TH (Fig. 13). Medially to the medulla some neurons were recognized by MAO-B antibodies. MAO-A and MAO-B staining of astroglia was observed in areas which are known to contain catecholaminergic or serotonergic neurotransmitters as well as in other brain areas. Distribution and intensity were Iargeiy identical. Stained glial cells of the medulla are shown in Figs 14 and 15.
Table 2. Intensity of neuronal antibody staining in different brain areas Brain area CTT DENV DNR Locus coeruleus MRF ON Putamen* RFM RFNG SN VTA
MAO-A _ +++ rt
MAO-B
+ + + + ++ f + ++ +
f
+++
t + Cf +
TH + +++ +++ ++ +-I-+ ++ +++ +++ +++
*Mainly fibres existing. + + + High density of neurons stained; + +medium density of neurons stained; f moderate density of neurons stained; + staining uncertain (glial staining too dark or cross-reaction of antibodies . _possible); - no stammg ot neurons observed.
794
CH.
KONRADI
et
al.
Fig. 4. MAO-A staining neurons and gliai cells in ON. PAP method. Original ma~ification:
x 63.
Fig. 5. MAO-B staining in SN. Unbleached neuromelanin of neuronal ceils, background staining of glial cells but no staining of neurons or fibres are shown. PAP method. Original magnification: x 63.
Immunocytochemistry
of MAO-A, MAO-B and TH in human brain stem
Fig. 6. TH staining in SN. No background because of unstained glial cells. NeuromeIanin as well as neuronal staining including fibres can be observed. PAP method. Original ma~ifi~tjon: x 63.
Fig. 7. MAO-B staining in DNR. Immunoreactivity of neuronal cells including fibres and glial cells (dark background). PAP method. Original magnification: x 63.
795
CH.
796
Fig. 8. MAO-A-like
Fig. 9. MAO-A
KONRADl ct al.
immunoreactivity in DNR. Moderate staining of neuronal cells, background of glial cells. PAP method. Original magnification: x 63.
staining in locus coeruleus. Immunoreactivity of neuronal ceils including cells (dark background). PAP method. Original magnification: x 63.
staining
fibres and glial
Immunocytochemistry
of MAO-A,
MAO-B
and TH in human
brain
197
stem
Fig. 10. TH staining
in locus coeruleus. lmmunoreactivity of neuronal cells and fibres. No background. PAP method. Original magnification: x 63.
Fig. Il. MAO-A-like
immunoreactivity in RFNG. Staining of neuronal cells. PAP method. Original magnification:
cells, background x 63.
staining
of ghal
798
CH.
KONRADI
et
al.
Fig. 12. MAO-B-like imm~noreact~~ity in RFNG. Staining of neuronal cells, background staining of glial cells. PAP method. Original magni~cation: x 63.
Fig. 13. TH staining in RFNG. Immunoreacti~ity of neuronal cells and fibres. No background. PAP method. Original magnification: x 63.
Immunocytochemistry
of MAO-A, MAO-B and TH in human brain stem
Fig. 14. Immunoreactivity
of astroglia in medullar reticular formation. MAO-A, PAP method. Original magnification: x 63.
Fig. 15. Immunoreactivity
of astroglia in medullar reticular formation. MAO-B, PAP method. Original magnification: x 63.
799
800
Of.
K~NRA~Iet al.
DISCUSSIQN
Specificity of MAO antibodies: preparation of antibodies and specification for each form of MAO have been described by Denney et al.’ and Kochersperger et al. 25 Our immunohistologi~al investigations exclude cross-reactivity of MAO-B antibodies with MAO-A because in some brain areas the neurons were only reacting with MAO-A antibodies (e.g. locus coeruleus). Cross-reactivity of MAO-A antibodies with high levels of MAO-B cannot be totally excluded. However, in dorsal nucleus of raphe more MAO-B than MAO-A immunoreactive neurons could be observed. The lack of MAO staining of nerve cells in the dopaminergic substantia nigra was astonishing since TH immunoreactivity of nigral neurons was very strong. On the other hand, glial cells in this region showed intense reaction with both MAO-A and MAO-B antibodies. Biochemical examinations of human brain substantia nigra led to the assumption that MAO-B is located in those neurons.‘7,32 Earlier investigations in primates which showed slight MAO-A staining in neurons of substantia nigra43 could not be confirmed in humans by our study. Presumptive MAO-A association with nigrostriatal dopaminergic neurons in rats was also described.8 in putamen, fibres of the projection areas of substantia nigra showed positive TN immunor~activity whereas only a small number of fibres stained for MAO. In addition, strong glial MAO-A and MAO-B staining in putamen contrasted with weakly stained fibres. Another surprising result was the intense MAO-B antibody staining in the serotonergic dorsal nucleus of raphe, although in the biochemical models serotonin is an MAO-A substrate. However, the immunocytochemistry may be consistent with biochemical data. For example, the intrasynaptosomal deamination of serotonin by MAO-B has been described in rat brain” and in cat brain.14 Furthermore, in citw decrease of serotonin decomposition also depends on inhibition of both MAO-A and MAO-B.” In addition, our data are consistent with recent
immunocytochemical demonstration of MAO-B in serotinergic neurons of rats,” primates” and humans.‘* However, the prior investigations in primate and human brain were carried out with the same antibodies as used in our study. It seems that, in relation to TH-positive neurons, more catecholaminergic neurons exist than MAO-Aor MAO-B-positive ones. This is most striking in substantia nigra. By disregarding the remote possibility of the adrenergic origin of TH-positive/MAOnegative neurons, these findings suggest that the role of MAO activity in neurons might be one other than the degradation of extravesicular neurotransmitt~r generated within the neuron. The available data are consistent with the hypothesis that MAO protects against extraneuronal monoamines, which do not represent intra~llular metabolit~ and which enter the cells by uptake. This hypothesis is supported by the existence of more MAO-B-positive neurons in serotonergic areas than MAO-A-positive neurons, although from biochemical studies it is well known that serotonin has a higher affinity for MAO-A than for MAO-B.‘“,‘” A further possibility is the existence of a third enzyme or enzyme subtype involved in the intraneuronal degradation of biogenic amines without the paratope for MAO-A or MAO-B antibodies.“0~41 However, it is also possible that MAO in the mitochondri~i membrane of neurons of different brain areas (e.g. substantia nigra) could be less accessible to antibodies than MAO in other brain areas (e.g. locus coeruieus, dorsal nucleus of rdphe). In particular, these results require reconsideration of the roles of MAO-A and MAO-B in human brain and their possible involvement in degenerative and mental disorders of catecholaminergic systems1.“‘.‘4as well as a possible role in the generation of neuromelanin.2y These data also need to be considered in understanding the mode of action of drugs affecting monoaminergic metabolism.4,“” In this context, astroglia could be more important than has heretofore been recognized.‘O Drug effects in the central nerv(~us system could be due to changes in both neuronai activity and the physiology of glial cells.
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