THE JOURNAL OF COMPARATIVE NEUROLOGY 442:331–347 (2002)
Developmental Expression of Monoamine Oxidases A and B in the Central and Peripheral Nervous Systems of the Mouse TANIA VITALIS,1 CORALIE FOUQUET,2 CHANTAL ALVAREZ,2 ISABELLE SEIF,3 DAVID PRICE,1 PATRICIA GASPAR,2 AND OLIVIER CASES2* 1 Department of Biomedical Sciences, Edinburgh EH8 9XD, Scotland, United Kingdom 2 INSERM U106, Baˆtiment de Pe´diatrie, Hoˆpital de la Pitie´-Salpeˆtrie`re, 75651 Paris Cedex 13, France 3 CNRS, UMR146, Institut Curie, 91405 Orsay, France
ABSTRACT Monoamine oxidases A (MAOA) and B (MAOB) are key players in the inactivation pathway of biogenic amines. Their cellular localization has been well established in the mature brain, but nothing is known concerning the localization of both enzymes during development. We have combined in situ hybridization and histochemistry to localize MAOA and MAOB in the developing nervous system of mice. Our observations can be summarized as five key features. (1) MAOA is tightly linked to catecholaminergic traits. MAOA is expressed in all noradrenergic and adrenergic neurons early on, and in several dopaminergic cell groups such as the substantia nigra. MAOA is also expressed in all the neurons that display a transient tyrosine hydroxylase expression in the brainstem and the amygdala and in neurons with transient dopamine--hydroxylase expression in the cranial sensory ganglia. (2) MAOA and MAOB are coexpressed in the serotoninergic neurons of the raphe from E12 to P7. During postnatal life, MAOA expression declines, whereas MAOB expression remains stable. (3) MAOA is transiently expressed in the cholinergic motor nuclei of the hindbrain, and MAOB is expressed in the forebrain cholinergic neurons. (4) MAOA- and MAOBexpressing neurons are also detected in structures that do not contain aminergic neurons, such as the thalamus, hippocampus, and claustrum. (5) Starting at birth, MAOB expression is found in a variety of nonneuronal cells, the choroid plexus, the ependyma, and astrocytes. These localizations are of importance for understanding the effects of monoaminergic transmission during development. J. Comp. Neurol. 442:331–347, 2002. © 2002 Wiley-Liss, Inc. Indexing terms: serotonin; noradrenaline; dopamine; acetylcholine; amygdala; thalamus; glia; ependyma
Monoamine oxidases A (MAOA) and B (MAOB) are membrane-bound mitochondrial flavoproteins that oxidatively deaminate a broad range of biogenic amines, including monoaminergic neurotransmitters in neurons, glial cells, and other cell types (Weyler et al., 1990; Shih et al., 1997). In the central nervous system (CNS), MAOs are thought to be involved in maintaining low cytosolic and extracellular levels of monoamines and in preventing various natural substrates from accumulating in monoaminergic neurons to act as false neurotransmitters. In the rodent brain, MAOA mainly metabolizes monoaminergic neurotransmitters, such as serotonin (5-HT), dopamine (DA), noradrenaline (NA), and adrenaline (A), whereas © 2002 WILEY-LISS, INC. DOI 10.1002/cne.10093
MAOB mainly metabolizes trace amines, such as tyramine and -phenylethylamine (Strolin-Benedetti et al., 1992). These functions could be particularly important during development, in that the lack of MAOA causes
Grant sponsor: INSERM; Grant sponsor: European Commission; Grant number: BMH4 CT97-2412; Grant sponsor: University of Edinburgh. *Correspondence to: Dr. Olivier Cases, INSERM U106, Batiment de Pe´diatrie, Hoˆpital de la Pitie´-Salpeˆtrie`re, 47 boulevard de l’hoˆpital, 75651 Paris Cedex 13, France. E-mail:
[email protected] Received 15 May 2001; Revised 2 August 2001; Accepted 22 October 2001 Published online the week of December 31, 2001
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enhanced levels of 5-HT and NA during embryonic and early postnatal life (Cases et al., 1995; Lajard et al., 1999) and causes an abnormal development of the somatosensory (Cases et al., 1996; Vitalis et al., 1998) and visual (Upton et al., 1999) systems. MAOA and MAOB have been localized in rodent, cat, primate, and human adult brain by a variety of techniques, including immunohistochemistry (Levitt et al., 1982; Westlund et al., 1985, 1988), histochemistry (Kitahama et al., 1994), enzyme autoradiography (Saura et al., 1992, 1996), and in situ hybridization (Luque et al., 1995; Jahng et al., 1997). Generally, MAOA is most abundant in (nor)adrenergic neurons, moderate in serotoninergic neurons, and very low in histaminergic neurons (Luque et al., 1995; Jahng et al., 1997), whereas MAOB is most abundant in histaminergic and serotoninergic neurons (Levitt et al., 1982; Saura et al., 1992; Luque et al., 1995; Jahng et al., 1997). MAOs are also present in nonaminergic cell populations. Indeed, MAOA has been found in neurons of the cerebral cortices, the hippocampal formation, and the cerebellar granule cell layer, whereas MAOB is largely expressed in nonneuronal cells, such as astrocytes or Bergmann glial cells, or circumventricular organs (Luque et al., 1995). So far no developmental study of these enzymes has been carried out. In the present study, we have used in situ hybridization and histochemistry to study the developmental distribution of MAOA and MAOB in the brain. This analysis was carried out in mice, which are now the species of choice for studying genetically the developmental effects of amines. We found two striking characteristics of MAOA distribution in embryonic and postnatal life. The first is its association with catecholaminergic traits: MAOA is expressed in all neurons that permanently or transiently express dopamine--hydroxylase (DBH), the biosynthetic enzyme of NA and A. Moreover, MAOA is expressed in most transient and permanent dopaminergic cell groups. The second is its early association with a serotoninergic phenotype: MAOA is strongly expressed in embryonic life in all serotoninergic neurons. In these neurons, MAOA expression progressively declines from P0 to P10. In addition, this developmental study allowed us to confirm most of the documented localizations of MAOA and MAOB and to show a novel and interesting localization of MAOB in forebrain cholinergic groups.
MATERIALS AND METHODS Animals Experiments were carried out on E10, E12, E14, E16, and E18 embryos and P0, P4, P7, P10, P21, and 5-monthold C3H/He and MAOA knockout mice (Cases et al., 1995). The day of the vaginal plug was counted as E1, and the day of birth as P0. Animal procedures were conducted in strict compliance with approved institutional protocols and in accordance with the provisions for animal care and use described in the Scientific Procedures on Living Animals Act, 1986.
In situ hybridization Postnatal mice were anesthetized with 0.1 ml 25% urethane. Brains were immediately removed and frozen in isopentane. Coronal and sagittal sections (20 m) were cut on a cryostat, collected onto SuperFrost slides, and stored at – 80°C.
A partial murine cDNA encoding from exon 1 to exon 8 of MAOA and MAOB was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), cloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA), and verified by sequencing. The plasmid containing MAOA was linearized with HindIII (Amersham, Arlington Heights, IL) for antisense RNA synthesis by T7 RNA polymerase (Roche Diagnostics), and the plasmid containing MAOB was linearized with EcoRV (Amersham) for antisense RNA synthesis by SP6 RNA polymerase (Roche Diagnostics). The in vitro trancription was carried out by using the Promega kit (Promega, Madison, WI), and the probes were labeled with 35S-UTP (⬎1,000 Ci/mmol; Amersham). Tissue sections were postfixed for 15 minutes in 4% paraformaldehyde, washed in PBS, acetylated, dehydrated, air dried, and hybridized with 106 cpm overnight in a humid chamber at 50°C. Sections were washed in 5⫻ SSC, 0.15% dithiothreitol (DTT) at 42°C and for 20 minutes in 50% formamide, 2⫻ SSC, 1.25% DTT at 60°C. Then, sections were treated for 30 minutes at 37°C in 0.02% RNAse A (Roche Diagnostics) in 10 mM Tris-HCl, pH 7.6, 30 mM NaCl, 10 mM EDTA. Sections were next sequentially washed for 15 minutes at 37°C in 10 mM Tris-HCl, pH 7.6, 30 mM NaCl, 10 mM EDTA, in 2⫻ SSC and in 0.1⫻ SSC. The slides were dehydrated and air dried. Autoradiograms were obtained by apposing the sections to hyperfilms (-max; Amersham) for 4 days. Autoradiographic films were developed in D19 (Kodak) for 3 minutes and fixed in Al-4 (Ilford). Some slides were dipped in photographic emulsion (NTB2; Kodak) and exposed for about 10 days. After development of the emulsion, the sections were counterstained in cresyl violet.
Immunocytochemistry To identify monoaminergic neurons, we used antibodies to 5-HT (1:50; rat monoclonal; Harlan), vesicular monoamine transporter type 2 (1/5,000; rabbit polyclonal; Phoenix Pharmaceuticals, Mountain View, CA), and tyrosine hydroxylase (1/5,000; rabbit polyclonal; gift from A. Vigny). Anesthetized animals were transcardially perfused with saline, followed by 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.4 (PB). Whole embryos or brains were postfixed for 2–5 days in the same fixative and cryoprotected in 30% sucrose in PB. Serial coronal sections (50 m) were cut on a freezing microtome and immediately processed for immunocytochemistry as previously described (Cases et al., 1996). In brief, sections were washed in PB, incubated for 1 hour in PBS⫹ (0.1 M PBS with 0.2% gelatin and 0.25% Triton X-100). Sections were incubated sequentially with the primary antibodies (24 hours at 4°C), PBS⫹ (30 minutes), secondary antibodies (biotinylated goat anti-rat; biotinylated swine anti-rabbit; 1:200; Dako, Glostrup, Denmark; 2 hours at room temperature), PBS⫹, and streptavidin-biotin-peroxidase complex (1:200; Amersham; 2 hours at room temperature) and finally were reacted with a solution containing 0.02% diaminobenzidine, 0.6% nickel ammonium sulfate (Carlo Erba), and 0.003% H2O2 in 0.05 M Tris buffer, pH 7.6. All sections were mounted on TESPA-coated slides, air dried overnight, dehydrated, and coverslipped in DePeX.
Histochemistry of MAOA and MAOB activities E10 –E14 embryos were fixed for 2 hours by immersion in an ice-cold mixture of 1.5% paraformaldehyde and 1.5%
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TABLE 1. MAOA Expression in Developing Aminergic Neurons1 Labeling intensity of neurons at pre- and postnatal ages Localization in the brain Noradrenergic groups CNS A6 locus coeruleus, A6s Subcoeruleus A1–A5 PNS Sympathetic division Adrenergic groups C1–C3 Serotoninergic groups Raphe pallidus, obscurus, magnus (B1–B3) Raphe dorsal, median, pontis (B4– B9) Dopaminergic groups A16 olfactory bulb A15v supraoptic n. A14 periventricular hypothalamic n. A14 paraventricular n. A13 zona incerta A10 ventral tegmental area A9 substantia nigra Hindbrain cholinergic motor neurons Oculomotor n. Trochlear n. Trigeminal motor n. Facial n. Ambiguus n. 1
E12 (n ⫽ 3)
E14 (n ⫽ 3)
E16 (n ⫽ 3)
E18 (n ⫽ 3)
P0 (n ⫽ 3)
P4 (n ⫽ 3)
P7 (n ⫽ 4)
P10 (n ⫽ 4)
P21-adults (n ⫽ 4)
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Intensity of the labeling at prenatal and postnatal ages according to localizations in the brain: ⫺, none; ⫺/⫹, very low; ⫹, weak; ⫹⫹, moderate; ⫹⫹⫹, high.
glutaraldehyde in PB. Older embryos were perfused transcardially with 0.9% saline, followed by the same ice-cold fixative and 1 hour of postfixation. Pups and adults were perfused with a mixture of 1% paraformaldehyde and 1% glutaraldehyde. Brains were removed and cryoprotected overnight in 30% sucrose in PB at 4°C. Serial sections were cut (50 – 60 m) on a freezing microtome, collected in cold PB, and processed immediately. MAO activity was revealed according to Dunning et al. (1997). Briefly, sections were rinsed three times for 1 minute each in PB, preincubated for 15 minutes in 50 mM Tris, pH 7.6, incubated for 4 –24 hours at 22–25°C in a solution containing 0.07% tyramine-HCl (Sigma, St. Louis, MO), 0.005% diaminobenzidine-HCl, 0.05% horseradish peroxidase Type II (Sigma), 0.065% sodium azide, and 0.6% nickel sulfamate (Sigma) in 50 mM Tris, pH 7.6. During the entire procedure, sections were kept in the dark and gently agitated. Because the specific inhibitor of MAOB L-deprenyl (Sigma) can partially inhibit MAOA activity, we used MAOA knockouts to determine the minimal concentration of L-deprenyl necessary to inhibit completely the remaining MAOB activity. We found that 10⫺7 M L-deprenyl was sufficient to inhibit MAOB activity (data not shown). We used 10⫺7 L– deprenyl to reveal specific MAOA activity in normal mice. MAOB activity was revealed by incubating sections in 5 ⫻ 10⫺7 clorgyline (Sigma), a selective inhibitor of MAOA activity.
Photography and preparation of illustrations Tissue sections were photographed by using Ilford PanF50 black-and-white film. An Agfa StudioScanII was used to import photographic images into Adobe Photoshop v.6 for preparation of the illustrations.
RESULTS The localization of MAOA and MAOB expression was carried out during embryonic and postnatal development. The nomenclature is taken from Paxinos et al. (1994) for embryonic stages and from Franklin and Paxinos (1994) for postnatal ages. The precise course of expression for the different structures is described in Tables 1– 4.
MAOA and MAOB mRNA expression in monoaminergic neurons Noradrenergic and adrenergic cell groups (Table 1). The most conspicuous MAOA mRNA expression was observed in the noradrenergic and adrenergic neurons throughout the CNS and peripheral nervous system (PNS). In the CNS, noradrenergic and adrenergic nuclei are found in two main localizations: (1) in the region of the pons, within the locus coeruleus (LC) and subcoeruleus (A4, A5, A6, and A7); and (2) in the medulla in the reticular nuclei (A1, A2 and C1, A2) and the solitary tract (A3, C3; Moore and Bloom, 1981). By E12, a strong MAOA mRNA expression was detected in areas that coincide with the localization of these different cell groups. In the medulla, labeled cells are found in a ventrolateral area that corresponds to the A1/C1 cell groups (Fig. 5B–F), and in a dorsomedial localization that corresponds to the region where A2/C2 adrenergic neurons are found (Fig. 5D–F). MAOA expression is observed very early in the locus coeruleus (A6) and the locus subcoeruleus (A6s; Fig. 1A). MAOA mRNA expression remained very high throughout embryonic and postnatal life (Fig. 1B,C). From E12, all noradrenergic neurons of the sympathetic ganglia already displayed a strong MAOA mRNA expression (Fig. 1D). MAOB mRNA was not detected in noradrenergic and adrenergic neurons or cells at any age studied.
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Fig. 1. Developmental expression of MAOA in noradrenergic neurons. A: Coronal section of an E12 embryo showing strong MAOA expression in the locus coeruleus (LC) and moderate expression in serotoninergic neurons of the dorsal raphe nucleus (DR). B: Coronal brain section of a P0 pup showing high levels of MAOA expression in LC and DR. C: Coronal brain section of an adult mouse showing
MAOA expression in the LC and the nucleus subcoeruleus (SubLC). D: Coronal brain section of an E18 embryo showing MAOA expression in the superior cervical ganglion (SCGn) and in the ganglion of the 9 and 10 nerves (9/10Gn). Scale bars ⫽ 170 m in A, 260 m in B, 500 m in C, 700 m in D.
Serotoninergic cell groups (Tables 1, 2). Serotoninergic neurons constitute the raphe nuclei and are classically distributed in nine groups (B1–B9), where B1 is the most posterior group and B9 the most anterior (Steinbusch, 1981). Most of the serotoninergic neurons are localized in two main nuclei, the dorsal raphe (B6, B7) and the median raphe (B5, B8). Strong MAOA and weak MAOB mRNA expressions were first detected by E12 close to the midline, where the serotoninergic neurons are normally localized. At E14, all serotoninergic cell groups displayed strong MAOA and MAOB mRNA expressions (Fig. 2A,D). MAOA mRNA expression remained high until P0 –P4 (Fig. 2B), then declined progressively to reach a minimal stable level by P21 (Fig. 2C). The decrease of expression was heterogeneous among serotoninergic cell groups; scattered neurons in the dorsal raphe maintained MAOA mRNA expression, whereas neurons in caudal raphe nuclei stopped expressing MAOA. MAOB mRNA expression in the serotoninergic neurons increased rapidly and remained stable from E14 onward (Fig. 2D–F).
Dopaminergic cell groups (Tables 1, 2). Dopaminergic neurons are classically distributed in eight cell groups: the retrorubral field (A8), the substantia nigra (A9), the ventral tegmental area (A10), the tuberoinfundibular system (A11–A12), the incertohypothalamic system (A13– A14), the dorsal and ventral preoptic areas (A15), and the olfactory bulb (A16). A low level of MAOA mRNA expression was detected at the level of the A9 –A10 complex by P0, which increased progressively to reach a maximum by P10. This expression remained stable during all postnatal life in the SNVTA neurons (Fig. 3A). Low to moderate levels of MAOA mRNA expression were also detected in several other dopaminergic cell groups: in the ventral thalamus at the level of the zona incerta from E16 (A13; Fig. 3B), in the hypothalamus at the level of the periventricular region from E18 (A14 Periv; Fig. 3C), in the paraventricular nucleus from P4, and in the supraoptic nucleus (A15v) from P7. In the olfactory bulb, dopaminergic external tufted cells and periglomerular interneurons (A16) dis-
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TABLE 2. MAOB Expression in Developing Aminergic Neurons1 Labeling intensity of neurons at pre- and postnatal ages Localization in the brain Serotoninergic groups Raphe pallidus, obscurus, magnus (B1–B3) Raphe dorsal, median, pontis (B4–B9) Histaminergic cell groups Melatoninergic cell groups Dopaminergic group A15v, preoptic area Cholinergic groups Ch1–Ch4 Ch5–Ch6 Striatum Ventral pallidum 1
E12 (n ⫽ 3)
E14 (n ⫽ 3)
E16 (n ⫽ 3)
E18 (n ⫽ 3)
P0 (n ⫽ 3)
P4 (n ⫽ 3)
P7 (n ⫽ 4)
P10 (n ⫽ 4)
P21-adults (n ⫽ 4)
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Intensity of the labeling at prenatal and postnatal ages according to localizations in the brain: ⫺, none; ⫺/⫹, very low; ⫹, weak; ⫹⫹, moderate; ⫹⫹⫹, high.
played MAOA mRNA expression from E16 (Fig. 3D). Among these different dopaminergic cell groups, MAOB mRNA expression was detected only in the preoptic area (Fig. 3E), starting at P4. Histaminergic cell group (Tables 1, 2). Histaminergic neurons are strictly confined to the ventral part of the posterior hypothalamus at the level of the tuberomammillary nucleus (Panula et al., 1984). Scattered neurons displaying low MAOB mRNA expression were first detected by P0 in the caudal hypothalamus. At P4, neurons were clearly focused at the level of the tuberomammillary nucleus. MAOB mRNA expression progressively increased and remained very high in the tuberomammillary region (Fig. 4A). This neuronal population likely corresponds to the histaminergic cell population, as shown by the vesicular monoamine transporter type 2 (Fig. 4B; Schutz et al., 1998). Melatoninergic cell group (Table 2). Melatoninergic cells of the pineal gland began to express MAOB at E16. Then, MAOB mRNA expression increased, to reach a maximum by P0, and remained high throughout life. Cholinergic cell groups (Tables 1, 2). Forebrain structures. Cholinergic neurons in the forebrain form seven major projections systems (Ch), and there are at least three major areas containing intrinsic cholinergic neurons. Earlier reports had shown that cholinergic neurons of the pedunculopontine tegmental (Ch5) and laterodorsal tegmental (Ch6; Ikemoto et al., 1999) and small cholinergic interneurons intrinsic to the caudateputamen (Nakamura et al., 1993) displayed MAOB activity. We confirmed these findings by in situ hybridization and extended the notion of MAOB localization in forebrain cholinergic neurons by showing MAOB expression in additional cholinergic groups (Table 2). Starting at P10, a low MAOB expression was found in cholinergic neurons of Ch5 and Ch6 (Fig. 4C,D). In the basal telencephalon, large neurons in the medial septal nucleus (Ch1), vertical limb of the diagonal band of Broca (Ch2), horizontal limb of the diagonal band of Broca (Ch3), and nucleus basalis of Meynert (Ch4) displayed moderate MAOB expression from P21 onward (Fig. 4C,D); they probably correspond to cholinergic neurons, based on cell size and localization. Moderate MAOB expression was also detected in large interneurons of the striatum (Fig. 4E,F) and the ventral pallidum that could correspond to the intrinsic cholinergic interneurons.
Hindbrain motor nuclei. We observed transient MAOA mRNA expression in the motor neurons of the oculomotor, trochlear, trigeminal, abducens, facial, ambiguus, dorsal vagus, and hypoglossal nuclei (Fig. 5A–E). This expression was first detected by E12–E18 and decreased sharply by P0 –P4 in all nuclei, to disappear by P10, except in the facial and ambiguus motor nuclei, where a low-grade MAOA expression was protracted (Fig. 5G).
MAOA mRNA expression in nuclei displaying transient monoaminergic phenotypes: transient TH-expressing neurons (Table 3) We and others have reported that during development discrete neuronal populations express transiently the catecholamine-synthetisizing enzyme tyrosine hydroxylase (TH). Indeed, transient TH-expressing neurons have been localized in the piriform cortex (Vitalis et al., 2000), the amygdala (Verney et al., 1988; Vitalis et al., 2000), and the inferior colliculus (Jaeger and Joh, 1983). We show here that some of these transient TH-expressing neurons probably display a parallel transient MAOA mRNA expression. In the developing mesencephalon, neurons in the inferior colliculus displayed a parallel moderate TH immunoreactivity (-IR) and MAOA expression from E14 to E16 (Fig. 6A–C). In the developing amygdala, neurons of the central nucleus and cortical and medial amygdaloid nuclei displayed a parallel TH-IR and moderate to low MAOA expression (Fig. 6D–F). In the PNS, cranial sensory ganglia serving the trigeminal (V), facial (VII), glossopharyngeal (IX), and vagus (X) nerves have been reported to express transiently during embryonic development selected catecholaminergic traits, such as TH or DBH or NA uptake (Jonakait et al., 1984; Son et al., 1996). We found that these cranial sensory neurons expressed moderate to high levels of MAOA mRNA from E12 until at least birth (Fig. 7A). A low to moderate MAOA mRNA expression was also found from E12 to at least birth in the sensory neurons of the dorsal root ganglia. On the other hand, MAOB mRNA was detected in only subset of neurons of the trigeminal ganglion (Fig. 7B).
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Fig. 2. Developmental time course of MAOA and MAOB in serotoninergic neurons. Alternate coronal brain sections at the level of the brainstem are shown at different ages E14 (A,D), P0 (B,E), and P21 (C,F). At E14, MAOA (A) and MAOB mRNA (D) are already strongly expressed in postmitotic serotoninergic neurons of the dorsal raphe (DR). Note also the expression of MAOB in the tectal glia limitans (Tgl). At P0, all serotoninergic neurons of the median raphe (MnR)
and DR display strong MAOA (B) and MAOB (E) mRNA expression. MAOA mRNA expression was detected in noradrenergic neurons of A5 and MAOB m RNA expression in Tgl. In adult, a residual MAOA mRNA expression was found in DR (C), whereas MAOB mRNA expression displayed its highest level (F). Note the protracted expression of MAOA in A5 (C). Scale bars ⫽ 300 m in A,D, 350 m in B,E, 400 m in C,F.
MAOA and MAOB mRNA expression in nonaminergic neuronal populations (Table 4)
amygdala (Fig. 7A).These expressions were maintained in the mature brain (Fig. 7C). We confirmed the specificity of MAOA expression in the hippocampus by showing in situ hybridization with sense probes (Fig. 8B). MAOB mRNA expression was found throughout the ventricular and the subventricular zone corresponding to the anlage of the cortex, hippocampus (Fig. 7B), and amygdala from E18 to P0. Later, moderate MAOB mRNA expression was restricted to layer VIb of the cerebral cortex, the claustrum, and the dorsal endopiriform nucleus and to a small
Telencephalon. A weak MAOA mRNA expression was detected in the cerebral cortex at the level of layer V from E18 (Fig. 7A). In the hippocampal formation, MAOA was observed in the CA1–CA3 fields, with the strongest expression in CA3 (Figs. 7A, 8A). MAOA mRNA expression was also detected in the piriform cortex and in the anterior amygdala, mainly in the central nucleus of the
Fig. 3. MAOA expression in dopaminergic cell groups. A: Coronal brain section of the mesencephalon at P21 showing a moderate level of MAOA mRNA expression in neurons of the ventral tegmental area (A10-VTA) and of the substantia nigra (A9-SN). B,C: Coronal brain sections through the diencephalon at P21 showing moderate to weak MAOA expression in discrete dopaminergic populations at the level of the zona incerta (A13-ZI), the paraventricular nucleus (A14Pavh), and the hypothalamic periventricular region (A14Periv). In addition,
moderate MAOA expression was detected in the rhomboid (Rh) and reuniens thalamic nuclei (Re). D: Rostral coronal brain section of the olfactory bulb at P0 showing strong MAOA expression in the external tufted cells (A16Tuf) and in the periglomerular interneurons (A16Pgl). Tuf and Pgl cells constitute the A16 population. E: Coronal section through the anterior hypothalamus showing MAOB expression in the median preoptic area (MPO). AC, anterior commissure. Scale bars ⫽ 525 m in A–C, 140 m in D, 70 m in E.
Fig. 4. MAOB mRNA expression in histaminergic and forebrain cholinergic neuronal populations. A–G: Coronal brain sections of a P21 pup. A,B: Coronal sections showing MAOB expression (A) and VMAT2 immunoreactivity (B) at the level of the tuberomammilar region. A: Strong mRNA expression was detected in scattered neurons of the tuberomammilary region (location of the histaminergic cell group; 3V, third ventricle). B: At the level of the tuberomammilary region, VMAT2 immunoreactivity is present in histaminergic neurons. C–F: MAOB expression in the forebrain cholinergic neurons. C: Moderate MAOB mRNA expression is detected in the nucleus basalis of Meynert (Mey). Note also the MAOB expression in the dorsal endopiriform nucleus (DEn), the intermediate dorsal thalamic
nuclei (itn), and the ependyma of the third ventricle (Ep). D: Rostral brain section showing MAOB mRNA expression in neurons of the medial septum (MS), the horizontal limb of the diagonal band of Broca (HDB). E: In the caudate putamen, arrows point to discrete MAOBpositive interneurons. These neurons are shown at higher magnification in E. F: MAOB mRNA is present in large cells (arrows) corresponding to striatal interneurons. G: Caudal section of the brainstem showing MAOB mRNA expression in cholinergic neurons of the laterodorsal tegmental nucleus (LDTg). Compare with the MAOB mRNA expression in the dorsal raphe nucleus (DRc). Scale bars ⫽ 130 m in A,B,G, 500 m in D,F, 1 mm in C, 90 m in E.
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Fig. 5. MAOA mRNA expression in motor neurons of the hindbrain. A,B: Coronal brain sections of an E12 embryo showing strong MAOA expression in the facial nucleus (7) and the dorsal motor vagus (10) and hypoglossal nucleus (12). On these sections, the MAOA mRNA expression in serotoninergic neurons of the raphe pallidus (RPa) and in noradrenergic neurons of the A1–C1 complex (A1-C1) is also visible. LC, locus coeruleus. C–F: Coronal brain sections of P0 pup at the level of the brainstem. C: MAOA expression at the level of the trigeminal motor nucleus (m5) and the facial nucleus (7). MAOA mRNA expression is also detected in serotoninergic neurons of the raphe pontis (RPn) and in the raphe magnus (RMg). D: MAOA expression at the level of the hypoglossal nucleus (12) and the nucleus
ambiguus (Amb). Note also the MAOA mRNA expression in the adrenergic group C1 (C1), the noradrenergic group A1 (A1), and the serotoninergic neurons of the raphe pallidus (RPa). E: Section showing MAOA expression in the hypoglossal nucleus (12) and the dorsal motor vagus (10). In addition, MAOA mRNA expression was found in the noradrenergic neurons of the A2 group (A2) and in serotoninergic neurons of the raphe obscurus (ROb). F: At the same level, TH immunoreactivity identified A1/C1 and A2/C2 cell groups. G: Coronal brain section of P21 mouse showing a protracted MAOA expression in the facial nucleus (7). Scale bars ⫽ 300 m in A, 180 m in B, 350 m in C–F, 150 m in G.
population of neurons in the field CA2 of the hippocampus (Figs. 7D, 8C). We confirmed the specificity of MAOB expression in the claustrum and layer VIb by showing in situ hybridization with sense probes (Fig. 8D).
Diencephalon. The epithalamus and the medial habenula contained low to moderate levels of MAOA and MAOB transcripts early on (E14 –E16; Fig. 7A,B). In the dorsal thalamus, MAOA and MAOB mRNA were detected
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T. VITALIS ET AL. TABLE 3. MAOA Expression in Neurons Transiently Displaying a Catecholaminergic Phenotype1 Labeling intensity of neurons at pre- and postnatal ages
Localization in the brain TH-positive Central n. of amygdala Medial amygdaloid n. Cortical amygdaloid n. Inferior colliculus TH-positive and DBH-positive Cranial sensory ganglia V, VII, IX, X Dorsal root ganglia 1
E12 (n ⫽ 3)
E14 (n ⫽ 3)
E16 (n ⫽ 3)
E18 (n ⫽ 3)
P0 (n ⫽ 3)
P4 (n ⫽ 3)
P7 (n ⫽ 4)
P10 (n ⫽ 4)
Adults (n ⫽ 2)
⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫹⫹
⫹ ⫺ ⫺ ⫹⫹
⫹ ⫹⫹ ⫹ ⫺
⫹ ⫹⫹ ⫹ ⫺
⫹ ⫺ ⫺ ⫺
⫹ ⫺ ⫺ ⫺
⫹ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺
⫹⫹ ⫹
⫹⫹⫹ ⫹⫹
⫹⫹⫹ ⫹⫹
⫹⫹⫹ ⫹⫹
⫹⫹⫹ ⫹⫹
⫹⫹ ⫹⫹
nd nd
nd nd
nd nd
Intensity of the labeling at prenatal and postnatal ages according to localizations in the brain: ⫺, none; ⫹, weak; ⫹⫹, moderate; ⫹⫹⫹, high; nd, not determined.
in all intralaminar nuclei: the paraventricular, the centrolateral, the centromedian, the rhomboid, and the reuniens (Fig. 7A–D). MAOB mRNA expression was also detected in other thalamic nuclei, the paracentral, the ventromedian, the ventrolateral, and the parafascicular. MAOB expression increased, to reach a maximum by P21 (Fig. 7D). In the hypothalamus, the ventromedial hypothalamic nucleus displayed a strong MAOA mRNA expression from P0 onward (Fig. 7A,C). Nonneuronal cells. Cells localized at the level of the tectal glia limitans displayed strong MAOB mRNA expression early on (E12; Figs. 2D, 9D). Astrocytes displayed low to moderate MAOB mRNA expression in all brain regions from P0 to P4. MAOB mRNA expression was detected in the choroid plexus by E18 (Fig. 7B). Then, MAOB expression increased, to reach a maximum by P7 (Fig. 7D,E). A moderate level of MAOB mRNA expression was also detected from E18 throughout ventricular ependyma (Figs. 7D, 8C). Vascular cells surrounding arteries displayed strong MAOB mRNA expression (Fig. 7B), whereas vascular cells surrounding cerebral capillaries displayed MAOA mRNA expression.
Histochemical activity To investigate whether MAOs mRNA are processed into active proteins during development, we turned to histochemistry of MAO activity. We used clorgyline, a selective inhibitor of MAOA, and deprenyl, a selective inhibitor of MAOB, to determine the specific pattern of MAOA and MAOB activity during development. We used MAOA knockouts to determine the minimal concentration of deprenyl necessary to inhibit completely the remaining MAOB activity. We found that 10⫺7 M deprenyl was sufficient to inhibit MAOB activity. We found also that 10⫺7 M clorgyline was sufficient to isolate MAOB activity. On the whole, we found a good spatiotemporal correlation between MAO activity and MAO mRNA expression, especially with noradrenergic (Fig. 9A), serotoninergic (Fig. 9A–D), histaminergic (Fig. 9A,E), melatoninergic, cholinergic, and nonaminergic (Fig. 9B,E) neurons.
DISCUSSION We provide here the first description of the developmental localization of MAOA and MAOB. In general, we found that MAOA and MAOB had complementary patterns of expression in monoaminergic neurons, except in the serotoninergic neurons, where they were similarly expressed during embryonic life. In addition, we observed that MAOs are widely expressed in other neuronal cell groups,
in particular in all cholinergic nuclei, the thalamus, and the hippocampus. In all these areas, MAOA and MAOB have a distinctive spatiotemporal pattern of expression with generally complementary domains of expression. In addition, we show transient developmental localizations of MAOA in neurons with transient catecholaminergic cell groups and of MAOB in the ventricular proliferative zone of the telencephalon. These expressions correspond to the presence of functional active proteins as indicated by histochemical analyses.
MAOA expression is linked to the catecholaminergic phenotype Previous localization studies in the adult have shown that, in all species studied, humans (Westlund et al., 1985), cats (Kitahama et al., 1994), and rats (Luque et al., 1995), MAOA is most abundantly expressed in noradrenergic neurons of the CNS and PNS. In this study, we show that in mice the typical noradrenergic (A1–A6) and adrenergic (C1–C3) neurons express MAOA soon after they differentiate by E12. As previously reported for adult rats, we found that MAOA is also expressed in most of the dopaminergic cell groups, the A9 –10 complex (Luque et al., 1995), A13, A14Pavh (Luque et al., 1995), A14Periv, and A15v hypothalamic cell groups and the A16 cell groups in the olfactory bulb. This expression begins between E16 and P7 according to the cell groups. In addition, we find that most of the neurons that transiently express a catecholaminergic phenotype also express MAOA. In the CNS this is the case for the inferior colliculus and the amygdala, which have both been shown to express TH (Jaeger and Joh, 1983; Verney et al., 1988). In the PNS the Vth, VIIth, IXth, and Xth cranial sensory ganglia that express the NA-synthesizing enzyme DBH (Tiveron et al., 1996) also express the MAOA gene during the same period. This suggests that the DBH and MAOA genes could be similarly regulated. Interestingly, recent studies have implicated several transcription factors, MASH1, Phox2a and Phox2b, in the control of (nor)adrenergic differentiation (Morin et al., 1997; Hirsch et al., 1998; Pattyn et al., 1999). In particular, Phox2a and Phox2b have been shown to be positive regulators of the DBH gene (Yang et al., 1998), and inactivation of Phox2b leads to a complete agenesis of neurons that display either a permanent or a transient noradrenergic phenotype: the LC and parasympathetic ganglia are absent, and the cranial sensory ganglia and the superior cervical ganglion are atrophic or altered (Pattyn et al., 1999, 2000). It is tempting to speculate that, in addition to its control of the DBH
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Fig. 6. MAOA mRNA expression in neurons displaying a transient TH immunoreactivity in the inferior colliculus and the amygdala. A,B: Coronal sections of E14 pups showing MAOA expression (A) and TH immunoreactivity (B) at the level of the caudal inferior colliculus. Arrows point to individual neurons at a similar level. C: Higher magnification of boxed area in B; arrows show small and lightly stained TH-immunoreactive neurons. D–F: Coronal sections of P0 pups showing MAOA expression (D) and TH immunoreactivity (E,F)
in the developing amygdala. D: MAOA expression is localized at the level of the medial cortical amygdaloid nucleus (single arrow), the central nucleus (arrowhead), and the basal cortical amygdaloid nucleus (double arrow). E,F: Numerous TH-immunoreactive neurons are localized in the central nucleus (E; arrowhead), the medial cortical amygdaloid nucleus (F; single arrow), and the cortical amygdaloid nucleus (F; double arrow). Scale bar ⫽ 2 mm for A,B,E,F, 500 m for C, 3 mm for D.
gene, Phox2b could be a positive regulator of the MAOA gene in (nor)adrenergic cell groups.
pression begins soon after serotoninergic neurons differentiate and start producing 5-HT. Serotoninergic neurons start producing 5-HT when neurons are still in the process of migrating to their final destination (E10 –E12 in mice, present study; E12–E14 in rats, Lauder and Bloom, 1974; Lidov and Molliver, 1982; Wallace and Lauder, 1983). Concomitantly, serotoninergic neurons express the sero-
MAOA in serotoninergic neurons during early development We found that MAOA is strongly expressed in all serotoninergic neurons during early development. This ex-
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Fig. 7. MAOA and MAOB mRNA expression in nonaminergic cells of the forebrain. A–D: MAOA (A,C) and MAOB (B,D) in situ hybridization in E18 embryos (A,B) and in adult mice (C,D). A: At E18 MAOA is expressed in the medial thalamic nuclei, central (CL), paraventricular (PV), rhomboid (Rh), and reuniens (Re), and CA2–CA3 pyramidal fields of the hippocampus, medial habenula (mHb), and amygdala, with higher levels of expression in the central (Ce) and the anterior amygdala. Note the MAOA expression in the trigeminal ganglion (V). AA, anterior amygdala. B: Moderate to low MAOB expression was found in the paraventricular (PV) and the ventral medial thalamic (VM) nuclei, the cortical and hippocampal (Hi) subventricular zone, the fimbria, the plexus choroid (chp), the medial habenula (mHB), the pineal recess (pnr) and the arcuate nucleus (Arc). In addition, MAOB mRNA expression was detected in the trigeminal ganglion (V), mast cells (mtc), internal carotid artery (ictd),
T. VITALIS ET AL.
and pterygopalatine artery (ptgpal). C: MAOA expression was found in the intermediate dorsal thalamic nuclei (itn), the CA3 and CA2 formation of the hippocampus, the piriform cortex (Pir), the anterior amygdala (AA), and the ventral medial hypothalamic nucleus (vmh). D: Strong MAOB expression was found in the pineal gland (Pin), the intermediate thalamic nuclei (itn), the plexus choroid (chp), the CA2 formation of the hippocampus, the remaining subplate of the cerebral cortex (Subp), the claustrum (Cl), and the dorsal endopiriform nucleus (DEn). E: MAOB mRNA expression in the cerebellum at the level of the Bergman glia (Bg) and in circumventricular organs of the fourth ventricle (chp 4V). F: Cellular localization of MAOB mRNA in putative Bergmann glial cells (arrows) and Purkinje cells (arrowhead). GCL, granule cell layer. Scale bars ⫽ 600 m in A,B, 700 m in C,D, 250 m in E, 60 m in F.
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TABLE 4. MAOA and MAOB Expression in Nonaminergic Cells1 Labeling intensity of neurons at pre- and postnatal ages Localization in the brain MAOA Telencephalon Neocortical layer V Piriform cortex CA3 hippocampal field CA1 hippocampal field Anterior amygdala Diencephalon Medial habenula Intralaminar thalamic nuclei Ventromedial hypothalamic n. Brainstem Solitary tract n. MAOB Telencephalon Neocortical and hippocampal ventricular and subventricular zones Subplate CA2 hippocampal field Claustrum Dorsal endopirirform Diencephalon Medial habenula Central, intralaminar, intermediary thalamic nuclei Nonneuronal cells Glia limitans Astroglial cells and astrocytes Choroid plexus and ependymal cells 1
E12 (n ⫽ 2)
E14 (n ⫽ 2)
E16 (n ⫽ 3)
E18 (n ⫽ 3)
P0 (n ⫽ 3)
P4 (n ⫽ 3)
P7 (n ⫽ 4)
P10 (n ⫽ 4)
Adults (n ⫽ 2)
⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺ ⫺
⫹ ⫹ ⫹⫹ ⫹ ⫹⫹
⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹
⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹
⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
⫺ ⫺ ⫺
⫹ ⫺ ⫺
⫹⫹ ⫺ ⫺
⫹⫹ ⫹ ⫺
⫹⫹ ⫹ ⫹⫹
⫹⫹ ⫹⫹ ⫹⫹
⫹⫹ ⫹⫹ ⫹⫹⫹
⫹⫹ ⫹⫹ ⫹⫹⫹
⫹⫹ ⫹⫹ ⫹⫹⫹
⫺
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
⫹⫹
⫹
⫺
⫺
⫺
⫺
⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫹ ⫹
⫺ ⫺ ⫹ ⫹
⫹ ⫹ ⫹⫹ ⫹⫹
⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
⫺ ⫺
⫺ ⫺
⫹ ⫺
⫹⫹ ⫹⫹
⫹⫹ ⫹⫹
⫹⫹ ⫹⫹
⫹⫹ ⫹⫹
⫹⫹ ⫹⫹
⫹⫹ ⫹⫹⫹
⫹ ⫺ ⫺
⫹ ⫺ ⫺
⫹⫹ ⫺ ⫺
⫹⫹ ⫺ ⫹
⫹⫹ ⫾ ⫹
⫹⫹ ⫹⫹ ⫹⫹
⫹⫹ ⫹⫹ ⫹⫹⫹
⫹⫹ ⫹⫹ ⫹⫹⫹
⫹⫹ ⫹⫹ ⫹⫹⫹
Intensity of the labeling at prenatal and postnatal ages according to localizations in the brain: ⫺, none; ⫹, weak; ⫹⫹, moderate; ⫹⫹⫹, high.
tonin transporter (SERT; Schroeter and Blakely, 1996) and VMAT2 (Hansson et al., 1998; Lebrand et al., 1998). Thus, soon after they are generated, developing serotoninergic neurons are already able to produce, store, release, take up, and degrade 5-HT. The finding that serotoninergic neurons strongly express MAOA during embryogenesis and early postnatal life is unexpected, with respect to previous observations in adults from which weak or moderate MAOA expression was reported in the raphe (Luque et al., 1995; Jahng et al., 1997). In mice, we find that the shift toward the adult pattern begins by P0, when the MAOA gene decreases to reach low levels in adult serotoninergic neurons, whereas MAOB gene expression remains strong. In that MAOA degrades 5-HT more efficiently than MAOB, the presence of MAOA in serotoninergic neurons during embryonic life could be important for regulating the levels of this amine. Indirect evidence for this interpretation is the high levels of 5-HT during embryonic development and the progressive decrease of these high levels during early postnatal development in mice lacking MAOA (Cases et al., 1995; Lajard et al., 1999), whereas 5-HT levels are unchanged in mice lacking MAOB (Grimsby et al., 1997). It will be important to determine whether similar mechanisms occur in humans. In support of this possibility, it has been shown that MAOA deficiency is associated with mild mental retardation, behavioral abnormalities, and abnormal 5-HT metabolism (Brunner et al., 1993a,b), whereas MAOB deficiency has not been associated with behavioral alterations. This suggests that MAOA is the main effector of 5-HT degradation during development and throughout life in humans, as in rodents.
MAOA and MAOB in cholinergic cell groups We describe for the first time the expression of MAOA and MAOB genes in a complementary pattern in known cholinergic cell groups. MAOA is expressed in the hindbrain cholinergic motoneurons, whereas MAOB is expressed in the forebrain cholinergic cell groups, in the septum, and in the nucleus basalis of Meynert. These localizations suggest that MAOs could be present in cholinergic neurons. Although acetylcholine has not been reported to be a substrate for MAOs, it is possible that MAOs in these cell groups could serve to eliminate biogenic amines (monoaminergic neurotransmitters or trace amines) that could interfere with acetylcholine synthesis or storage in vesicles. This could be the case in hindbrain cholinergic motoneurons, which transiently express display the vesicular monoamine transporter type 2 (Hansson et al., 1998) during the same developmental period as MAOA. Cholinergic neurons could thus store and degrade monoamines despite the fact that they do not synthesize them. Earlier reports had shown the existence of MAOB in subsets of cholinergic neurons (Nakamura et al., 1993; Ikemoto et al., 1997, 1999). Our study extends this by showing the expression of MAOB in all forebrain cholinergic neurons. Although the role of MAOB in forebrain cholinergic neurons remains speculative, its inhibition could be of importance in neurodegenerative conditions involving cholinergic neurons, such as Alzheimer’s disease or even aging. In Alzheimer’s disease, several clinical trials have shown the benefit of the MAOB inhibitor deprenyl used at a low dosage (Birks
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Fig. 8. MAOA and MAOB mRNA expression in the telencephalon. MAOA (A,B) and MAOB (C,D) in situ hybridization with antisense (A,C) and sense (B,D) probes in adult mice. A: Coronal brain section showing MAOA expression in the hippocampus; note that MAOA expression was stronger in the CA3 than in the CA1 hippocampal
fields. DG, dentate gyrus. B: An adjacent section was hybridized with MAOA sense probe controls. C: Coronal brain section showing MAOB expression in the claustrum (Cl), layer VIb of the cerebral cortex, and ependyma (Ep). D: An adjacent section was hybridized with MAOB sense probe controls. Scale bars ⫽ 90 m.
and Flicker, 2000), and a 40% decrease of MAOB activity has been found in the brains of smokers, who appear to be protected to some extent against Alzheimer’s disease (Fowler et al., 1996). The effects of MAOB inhibition in this system have not been elucidated but could involve a delay in cell death (Magyar et al., 1998; Paterson and Tatton, 1998).
MAOA-KO mice, there is an accumulation of 5-HT in all the SERT-expressing neurons (Cases et al., 1998). This study shows that with the exception of the CA3 hippocampal field, the MAOA gene is not expressed simultaneously with SERT in nonaminergic neurons. This suggests that the 5-HT taken up from the extracellular space by SERT is not degraded but could be immediately stored into synaptic vesicles by the vesicular monoamine transporter type 2, which is coexpressed in most cases (Lebrand et al., 1998). Taken together with our previous observations, the present result argues in favor of a role for 5-HT as a borrowed neurotransmitter by immature developing circuits, such as the thalamocortical and visual systems.
Serotonin as a borrowed neurotransmitter Previous studies using in situ hybridization and immunochemistry (Hansson et al., 1998; Lebrand et al., 1998) have shown that SERT is transiently expressed in nonmonoaminergic structures such as the cortex, the hippocampal formation, and the dorsal thalamus. SERTpositive neurons in these structures are thus able to take up 5-HT, although they do not produce it. Several neurons that transiently express SERT do not contain detectable 5-HT levels, but, when MAOA is inhibited pharmacologically with clorgyline or genetically inactivated in
MAOB in the ventricular zone, ependyma, and astroglia A particularly interesting localization of MAOB during development is the expression in the ventricular zone. Our present investigation does not allow us to characterize the
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Fig. 9. MAOA and MAOB activity in the developing brain. A: Sagittal brain section of an E13 embryo showing MAO activity in different serotoninergic cell groups, the dorsal raphe (DR), the median raphe (MnR), the raphe pontis (Rpn), the raphe magnus (Rmg), and the raphe pallidus (Rpa). MAO activity is also observed in histaminergic cell groups of the caudal hypothalamus (Hist), in noradrenergic neurons of the locus coeruleus (LC), in the plexus choroides (chp), and in the inferior colliculus (IC). B: Coronal brain section of an E14 embryo through the brainstem showing MAOA activity in serotoninergic neurons of MnR and Rpa, in cholinergic neurons of the motor
trigeminal (mo5) and facial (7) nuclei, and in noradrenergic neurons of A5. C,D: Consecutive coronal sections from P0 to show the strong MAOA (C) and MAOB (D) activity in the serotoninergic neurons of MnR and DR. Note in addition the strong MAOB activity in the aqueductal ependyma and in the tectal glia limitans (Tgl). E: Coronal section of an adult showing MAOB activity in the intralaminar thalamic nuclei (CL, CM, VM), in the histaminergic cell groups of the caudal hypothalamus (Hist), and in the medial habenula (MHb). Scale bars ⫽ 450 m in A, 150 m in B, 500 m in C,D, 180 m in E.
identity of the labeled cells. One possibility is that MAOB is present in proliferating cells. The other possibility is that MAOB is an early marker of ependymal cells. Indeed, MAOB expression appears coincidentally with the genesis of ependymal cells around E18 (Das, 1979). Furthermore, at birth MAOB becomes confined to the ependymal system. This finding is in agreement with previous observations in the adult brain showing MAOB activity in the ependymal system and the circumventricular organs (choroid plexus, area postrema, and pineal gland; Saura et al., 1992; Luque et al., 1995). However, it remains to be determined whether MAOB can be used as a marker of ependymocytes, insofar as MAOB is also present in the astroglial cells. The physiological role of MAOB in the ependymal systems is to protect the CNS from trace amines present in the cerebrospinal fluid (Levitt et al., 1982; Willoughby et al., 1988; Saura et al., 1992). In astrocytes the role of MAOB might be dual: 1) In the vicinity of monoaminergic nerve terminals, it could degrade trans-
mitters taken up by the glial cells, and, 2) throughout the brain, it could inactivate trace amines in the extracellular space. Recent reports have shown that astrocytes are capable of high-affinity 5-HT uptake (Bel et al., 1997) and possess a facilitated diffusion system for dopamine (Hosli and Hosli, 1997).
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