- Email: [email protected]
Dopamine-degrading activity of monoamine oxidase in locus coeruleus and dorsal raphe nucleus neurons. A histochemical study in the rat
Neuroscience Letters 250 (1998) 41–44
Dopamine-degrading activity of monoamine oxidase in locus coeruleus and dorsal raphe nucleus neurons. A histochemical study in the rat Ryohachi Arai a ,*, Kihachiro Horiike b, Yoshimi Hasegawa a a
Department of Anatomy, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan b Department of Biochemistry, Shiga University of Medical Science, Ohtsu, Shiga 520-2192, Japan Received 13 March 1998; received in revised form 22 May 1998; accepted 25 May 1998
Abstract Dopamine-degrading activity of monoamine oxidase (MAO) was detected in the rat using a new histochemical method, with dopamine as the substrate. Our new method, designed to minimise the non-enzymatic oxidation of dopamine, was applied in combination with tyrosine hydroxylase (TH) and serotonin immunohistochemistry. We showed that the distribution pattern of MAO neurons was similar to that of TH-immunoreactive neurons (i.e. noradrenergic neurons) in the locus coeruleus (LC) and to that of serotonergic neurons in the dorsal raphe nucleus (DR). Since LC neurons form dopamine during noradrenaline biosynthesis, and DR neurons produce dopamine from exogenously administered L-dopa, our results indicate that dopamine produced in LC and DR neurons may be degraded, at least in part, by MAO. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Monoamine oxidase; Enzyme histochemistry; Dopamine; Locus coeruleus; Dorsal raphe nucleus; Noradrenergic neurons; Serotonergic neurons; Parkinson’s disease
It has been shown in the rat that monoamine oxidase (MAO) type A or type B is present in neurons of the locus coeruleus (LC) or dorsal raphe nucleus (DR), respectively [4,14,15,20]. While biochemical studies have shown that dopamine can be a substrate for both types of MAO in rat-brain homogenates [6], histochemical studies using dopamine as an MAO substrate have been unsuccessful. To histochemically localise dopamine-degrading activity of MAO, we modified a previously-reported reaction mixture [1,12,16]. By omitting nickel ions and using a phosphate buffer, we succeeded in localising MAO activity in rat brains using dopamine as a substrate. Localisation patterns of MAO activity detected by this histochemical method were compared to the distribution of tyrosine hydroxylase(TH) and serotonin-immunoreactive neurons in the LC and DR, respectively, to determine whether dopamine-degrading MAO activity might be localised within these neuronal populations. * Corresponding author. Tel.: +81 562 932443; fax.: +81 562 932649; e-mail: [email protected]
Male Sprague–Dawley rats (180–200 g) were used for the preparation of either fixed (n = 3) or fresh tissue (n = 3). For fixation, rats were anaesthetised with sodium pentobarbital (60 mg/kg body weight, i.p.) and perfused through the ascending aorta with 50 ml of 0.01 M phosphate-buffered saline (pH 7.4, room temperature), followed by 600 ml of a fixative containing 2% formaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4, 4°C). The brains were cut into 50-mm thick frontal sections on a vibratome and collected in 0.1 M phosphate-buffered saline. Fresh tissue was collected from anaesthetised and decapitated rats. Brains were frozen and cut on a cryostat into 30-mm thick frontal sections which were mounted onto gelatincoated glass slides. Sections through similar regions of the DR were processed for MAO histochemistry or serotonin immunohistochemistry. And sections through similar regions of the LC were processed for MAO histochemistry or TH immunohistochemistry. MAO histochemistry was assessed in both fixed and unfixed tissue, while immunohistochemistry was performed in fixed tissue only. Other unfixed sections were
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00429- 7
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R. Arai et al. / Neuroscience Letters 250 (1998) 41–44
stained with 1% neutral red to provide cytoarchitectonic reference. MAO histochemistry was performed using a staining solution containing 0.019% (1 mM) dopamine hydrochloride (Sigma, St. Louis, MO), 0.1% horseradish peroxidase (Grade III, Toyobo, Osaka, Japan), 0.005% 3,3′-diaminobenzidine tetrahydrochloride, 0.065% sodium azide (Sigma) in 0.1 M phosphate buffer (pH 7.4). Fixed sections were incubated in the staining solution for 48 h at 4°C, and fresh sections for 2 h at 37°C. The specificity of the MAO staining was tested by either omitting the dopamine substrate from the incubation, or by inhibiting MAO activity with pargyline, an irreversible MAO inhibitor [1,16]. In the latter case, sections were incubated in 0.01 M phosphatebuffered saline (pH 7.4) containing 0.1 mM pargyline (Sigma) at room temperature for 15 min prior to incubation in the staining solution. Serotonin or TH immunoreactivity was detected using the avidin-biotin-peroxidase method [11] with either a rat antibody against serotonin (Eugene Tech International, Ridgefield Park, NJ) or a mouse antibody against TH (Incstar, Stillwater, MN).
In fixed sections, both DR and LC neurons showed MAO activity using dopamine as the substrate (Fig. 1A,E), which was blocked with pargyline in both regions (Fig. 1C,G). When dopamine was omitted, no staining was found in the DR (Fig. 1B) or the LC (Fig. 1F). In the DR, the distribution pattern of MAO neurons (Fig. 1A) was similar to that of serotonin-immunoreactive neurons (Fig. 1D). In the LC, the distribution pattern of MAO neurons (Fig. 1E) was also similar to that of TH-immunoreactive neurons (Fig. 1H). The density and distribution of MAO neurons seen in the fixed sections were essentially identical to those observed in the unfixed fresh tissue (Fig. 2). The similarity between the distribution of MAO activity and TH, a marker of noradrenergic neurons in the LC [9], suggests that these noradrenaline neurons are capable of degrading dopamine as a result of MAO activity. Thus, the dopamine that is endogenously produced as a precursor in noradrenaline biosynthesis [7] may be susceptible to degradation, at least in part, by MAO in LC neurons. This is in accordance with previous findings which showed that inhibition of MAO activity in the rat LC decreases extracellular concentrations of 3,4-dihydroxyphenylacetic
Fig. 1. Photographs of fixed sections through similar levels of the DR (A–D) and LC (E–H). (A,E) MAO histochemistry. (B,F) Controls without dopamine. (C,G) Controls after MAO inhibition by pargyline treatment. (D) Serotonin immunohistochemistry. (H) TH immunohistochemistry. Scale bar, 100 mm.
R. Arai et al. / Neuroscience Letters 250 (1998) 41–44
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Fig. 2. Photographs of unfixed sections through similar levels of the DR (A–D) and LC (E–H). (A,E) MAO histochemistry. (B,F) Controls without dopamine. (C,G) Controls after MAO inhibition by pargyline treatment. (D,H) Nissl staining with neutral red. Scale bar, 100 mm.
acid, a product of MAO-induced dopamine degradation [17,18]. Furthermore, our results revealed that the distribution pattern of MAO neurons was similar to that of serotonin neurons in the DR, suggesting that MAO has the capability of degrading dopamine in serotonin neurons. This is an intriguing finding in light of previous evidence suggesting the involvement of serotonergic neurons in the therapeutic actions of L-dopa in treating Parkinson’s disease [10]. In fact, we previously showed that serotonergic fibres in the striatum of the rat contain aromatic L-amino acid decarboxylase [2], which converts exogenous L-dopa to dopamine [3]. Therefore, this newly-produced dopamine may be susceptible, at least in part, to MAO degradation in the serotonin neurons. Interestingly, the selective inhibitor of MAO type B, deprenyl, has proven to be an effective adjunct to Ldopa therapy in Parkinson’s disease [5]. MAO type B has been localised in human DR neurons that are presumably serotonin neurons [13,19], and biochemical studies have shown that dopamine is the preferred substrate of MAO type B in human brain mitochondrial fractions [8]. Therefore, the beneficial effects of deprenyl in L-dopa therapy in
Parkinson’s disease patients may be ascribable to the inhibition of dopamine degradation that may occur in L-dopaconverting serotonergic neurons. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. The financial help of Fujita Health University is appreciated. [1] Arai, R., Kimura, H. and Maeda, T., Topographic atlas of monoamine oxidase-containing neurons in the rat brain studied by an improved histochemical method, Neuroscience, 19 (1986) 905– 925. [2] Arai, R., Karasawa, N. and Nagatsu, I., Aromatic L-amino acid decarboxylase is present in serotonergic fibers of the striatum of the rat. A double-labeling immunofluorescence study, Brain Res., 706 (1996) 177–179. [3] Arai, R., Karasawa, N., Geffard, M. and Nagatsu, I., L-DOPA is converted to dopamine in serotonergic fibers of the striatum of the rat: a double-labeling immunofluorescence study, Neurosci. Lett., 195 (1995) 195–198. [4] Arai, R., Kimura, H., Nagatsu, I. and Maeda, T., Preferential localization of monoamine oxidase type A activity in neurons of the locus coeruleus and type B activity in neurons of the
44
[5]
[6]
[7]
[8]
[9]
[10] [11]
[12]
R. Arai et al. / Neuroscience Letters 250 (1998) 41–44 dorsal raphe nucleus of the rat: a detailed enzyme histochemical study, Brain Res., 745 (1997) 352–356. Berry, M.D., Juorio, A.V. and Paterson, I.A., Possible mechanisms of action of (−)deprenyl and other MAO-B inhibitors in some neurologic and psychiatric disorders, Prog. Neurobiol., 44 (1994) 141–161. Fowler, C.J. and Strolin Benedetti, M., The metabolism of dopamine by both forms of monoamine oxidase in the rat brain and its inhibition by cimoxatone, J. Neurochem., 40 (1983) 1534– 1541. Geffard, M., Buijs, R.M., Seguela, P., Pool, C.W. and Le Moal, M., First demonstration of highly specific and sensitive antibodies against dopamine, Brain Res., 294 (1984) 161–165. Glover, V., Sandler, M., Owen, F. and Riley, G.J., Dopamine is a monoamine oxidase B substrate in man, Nature, 265 (1977) 80–81. Ho¨kfelt, T., Ma˚rtensson, R., Bjo¨rklund, A., Kleinau, S. and Goldstein, M., Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In A. Bjo¨rklund and T. Ho¨kfelt (Eds.), Classical Transmitters in the CNS, Part I. Handbook of Chemical Neuroanatomy, Vol. 2, Elsevier, Amsterdam, 1984, pp. 277–379. Hornykiewicz, O., The mechanisms of action of L-dopa in Parkinson’s disease, Life Sci., 15 (1974) 1249–1259. Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577–580. Kitahama, K., Maeda, T., Denney, R.M. and Jouvet, M., Monoamine oxidase: distribution in the cat brain studied by enzymeand immunohistochemistry: recent progress, Prog. Neurobiol., 42 (1994) 53–78.
[13] Konradi, C., Kornhuber, J., Froelich, L., Fritze, J., Heinsen, H., Beckmann, H., Schulz, E. and Riederer, P., Demonstration of monoamine oxidase-A and -B in the human brainstem by a histochemical technique, Neuroscience, 33 (1989) 383–400. [14] Levitt, P., Pintar, J.E. and Breakefield, X.O., Immunocytochemical demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurons, Proc. Natl. Acad. Sci. (USA), 79 (1982) 6385–6389. [15] Luque, J.M., Kwan, S.-W., Abell, C.W., Da Prada, M. and Richards, J.G., Cellular expression of mRNAs encoding monoamine oxidases A and B in the rat central nervous system, J. Comp. Neurol., 363 (1995) 665–680. [16] Maeda, T., Imai, H., Arai, R., Tago, H., Nagai, T., Sakumoto, T., Kitahama, K., Onteniente, B. and Kimura, H., An improved coupled peroxidatic oxidation method of MAO histochemistry for neuroanatomical research at light and electron microscopic levels, Cell. Mol. Biol., 33 (1987) 1–11. [17] Milon, H. and McRae-Degueurce, A., Pharmacological investigation on the role of dopamine in the rat locus coeruleus, Neurosci. Lett., 30 (1982) 297–301. [18] Ortemann, C., Robert, F., Renaud, B. and Lamba´s-Sen˜as, L., In vivo microdialysis study of the extracellular 3,4-dihydroxyphenylacetic acid in the rat locus ceruleus: topographical and pharmacological aspects, J. Neurochem., 61 (1993) 594–601. [19] Westlund, K.N., Denney, R.M., Rose, R.M. and Abell, C.W., Localization of distinct monoamine oxidase A and monoamine oxidase B cell populations in human brainstem, Neuroscience, 25 (1988) 439–456. [20] Willoughby, J., Glover, V. and Sandler, M., Histochemical localisation of monoamine oxidase A and B in rat brain, J. Neural Transm., 74 (1988) 29–42.
Dopamine-degrading activity of monoamine oxidase in locus coeruleus and dorsal raphe nucleus neurons. A histochemical study in the rat Ryohachi Arai a ,*, Kihachiro Horiike b, Yoshimi Hasegawa a a
Department of Anatomy, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan b Department of Biochemistry, Shiga University of Medical Science, Ohtsu, Shiga 520-2192, Japan Received 13 March 1998; received in revised form 22 May 1998; accepted 25 May 1998
Abstract Dopamine-degrading activity of monoamine oxidase (MAO) was detected in the rat using a new histochemical method, with dopamine as the substrate. Our new method, designed to minimise the non-enzymatic oxidation of dopamine, was applied in combination with tyrosine hydroxylase (TH) and serotonin immunohistochemistry. We showed that the distribution pattern of MAO neurons was similar to that of TH-immunoreactive neurons (i.e. noradrenergic neurons) in the locus coeruleus (LC) and to that of serotonergic neurons in the dorsal raphe nucleus (DR). Since LC neurons form dopamine during noradrenaline biosynthesis, and DR neurons produce dopamine from exogenously administered L-dopa, our results indicate that dopamine produced in LC and DR neurons may be degraded, at least in part, by MAO. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Monoamine oxidase; Enzyme histochemistry; Dopamine; Locus coeruleus; Dorsal raphe nucleus; Noradrenergic neurons; Serotonergic neurons; Parkinson’s disease
It has been shown in the rat that monoamine oxidase (MAO) type A or type B is present in neurons of the locus coeruleus (LC) or dorsal raphe nucleus (DR), respectively [4,14,15,20]. While biochemical studies have shown that dopamine can be a substrate for both types of MAO in rat-brain homogenates [6], histochemical studies using dopamine as an MAO substrate have been unsuccessful. To histochemically localise dopamine-degrading activity of MAO, we modified a previously-reported reaction mixture [1,12,16]. By omitting nickel ions and using a phosphate buffer, we succeeded in localising MAO activity in rat brains using dopamine as a substrate. Localisation patterns of MAO activity detected by this histochemical method were compared to the distribution of tyrosine hydroxylase(TH) and serotonin-immunoreactive neurons in the LC and DR, respectively, to determine whether dopamine-degrading MAO activity might be localised within these neuronal populations. * Corresponding author. Tel.: +81 562 932443; fax.: +81 562 932649; e-mail: [email protected]
Male Sprague–Dawley rats (180–200 g) were used for the preparation of either fixed (n = 3) or fresh tissue (n = 3). For fixation, rats were anaesthetised with sodium pentobarbital (60 mg/kg body weight, i.p.) and perfused through the ascending aorta with 50 ml of 0.01 M phosphate-buffered saline (pH 7.4, room temperature), followed by 600 ml of a fixative containing 2% formaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4, 4°C). The brains were cut into 50-mm thick frontal sections on a vibratome and collected in 0.1 M phosphate-buffered saline. Fresh tissue was collected from anaesthetised and decapitated rats. Brains were frozen and cut on a cryostat into 30-mm thick frontal sections which were mounted onto gelatincoated glass slides. Sections through similar regions of the DR were processed for MAO histochemistry or serotonin immunohistochemistry. And sections through similar regions of the LC were processed for MAO histochemistry or TH immunohistochemistry. MAO histochemistry was assessed in both fixed and unfixed tissue, while immunohistochemistry was performed in fixed tissue only. Other unfixed sections were
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00429- 7
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R. Arai et al. / Neuroscience Letters 250 (1998) 41–44
stained with 1% neutral red to provide cytoarchitectonic reference. MAO histochemistry was performed using a staining solution containing 0.019% (1 mM) dopamine hydrochloride (Sigma, St. Louis, MO), 0.1% horseradish peroxidase (Grade III, Toyobo, Osaka, Japan), 0.005% 3,3′-diaminobenzidine tetrahydrochloride, 0.065% sodium azide (Sigma) in 0.1 M phosphate buffer (pH 7.4). Fixed sections were incubated in the staining solution for 48 h at 4°C, and fresh sections for 2 h at 37°C. The specificity of the MAO staining was tested by either omitting the dopamine substrate from the incubation, or by inhibiting MAO activity with pargyline, an irreversible MAO inhibitor [1,16]. In the latter case, sections were incubated in 0.01 M phosphatebuffered saline (pH 7.4) containing 0.1 mM pargyline (Sigma) at room temperature for 15 min prior to incubation in the staining solution. Serotonin or TH immunoreactivity was detected using the avidin-biotin-peroxidase method [11] with either a rat antibody against serotonin (Eugene Tech International, Ridgefield Park, NJ) or a mouse antibody against TH (Incstar, Stillwater, MN).
In fixed sections, both DR and LC neurons showed MAO activity using dopamine as the substrate (Fig. 1A,E), which was blocked with pargyline in both regions (Fig. 1C,G). When dopamine was omitted, no staining was found in the DR (Fig. 1B) or the LC (Fig. 1F). In the DR, the distribution pattern of MAO neurons (Fig. 1A) was similar to that of serotonin-immunoreactive neurons (Fig. 1D). In the LC, the distribution pattern of MAO neurons (Fig. 1E) was also similar to that of TH-immunoreactive neurons (Fig. 1H). The density and distribution of MAO neurons seen in the fixed sections were essentially identical to those observed in the unfixed fresh tissue (Fig. 2). The similarity between the distribution of MAO activity and TH, a marker of noradrenergic neurons in the LC [9], suggests that these noradrenaline neurons are capable of degrading dopamine as a result of MAO activity. Thus, the dopamine that is endogenously produced as a precursor in noradrenaline biosynthesis [7] may be susceptible to degradation, at least in part, by MAO in LC neurons. This is in accordance with previous findings which showed that inhibition of MAO activity in the rat LC decreases extracellular concentrations of 3,4-dihydroxyphenylacetic
Fig. 1. Photographs of fixed sections through similar levels of the DR (A–D) and LC (E–H). (A,E) MAO histochemistry. (B,F) Controls without dopamine. (C,G) Controls after MAO inhibition by pargyline treatment. (D) Serotonin immunohistochemistry. (H) TH immunohistochemistry. Scale bar, 100 mm.
R. Arai et al. / Neuroscience Letters 250 (1998) 41–44
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Fig. 2. Photographs of unfixed sections through similar levels of the DR (A–D) and LC (E–H). (A,E) MAO histochemistry. (B,F) Controls without dopamine. (C,G) Controls after MAO inhibition by pargyline treatment. (D,H) Nissl staining with neutral red. Scale bar, 100 mm.
acid, a product of MAO-induced dopamine degradation [17,18]. Furthermore, our results revealed that the distribution pattern of MAO neurons was similar to that of serotonin neurons in the DR, suggesting that MAO has the capability of degrading dopamine in serotonin neurons. This is an intriguing finding in light of previous evidence suggesting the involvement of serotonergic neurons in the therapeutic actions of L-dopa in treating Parkinson’s disease [10]. In fact, we previously showed that serotonergic fibres in the striatum of the rat contain aromatic L-amino acid decarboxylase [2], which converts exogenous L-dopa to dopamine [3]. Therefore, this newly-produced dopamine may be susceptible, at least in part, to MAO degradation in the serotonin neurons. Interestingly, the selective inhibitor of MAO type B, deprenyl, has proven to be an effective adjunct to Ldopa therapy in Parkinson’s disease [5]. MAO type B has been localised in human DR neurons that are presumably serotonin neurons [13,19], and biochemical studies have shown that dopamine is the preferred substrate of MAO type B in human brain mitochondrial fractions [8]. Therefore, the beneficial effects of deprenyl in L-dopa therapy in
Parkinson’s disease patients may be ascribable to the inhibition of dopamine degradation that may occur in L-dopaconverting serotonergic neurons. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. The financial help of Fujita Health University is appreciated. [1] Arai, R., Kimura, H. and Maeda, T., Topographic atlas of monoamine oxidase-containing neurons in the rat brain studied by an improved histochemical method, Neuroscience, 19 (1986) 905– 925. [2] Arai, R., Karasawa, N. and Nagatsu, I., Aromatic L-amino acid decarboxylase is present in serotonergic fibers of the striatum of the rat. A double-labeling immunofluorescence study, Brain Res., 706 (1996) 177–179. [3] Arai, R., Karasawa, N., Geffard, M. and Nagatsu, I., L-DOPA is converted to dopamine in serotonergic fibers of the striatum of the rat: a double-labeling immunofluorescence study, Neurosci. Lett., 195 (1995) 195–198. [4] Arai, R., Kimura, H., Nagatsu, I. and Maeda, T., Preferential localization of monoamine oxidase type A activity in neurons of the locus coeruleus and type B activity in neurons of the
44
[5]
[6]
[7]
[8]
[9]
[10] [11]
[12]
R. Arai et al. / Neuroscience Letters 250 (1998) 41–44 dorsal raphe nucleus of the rat: a detailed enzyme histochemical study, Brain Res., 745 (1997) 352–356. Berry, M.D., Juorio, A.V. and Paterson, I.A., Possible mechanisms of action of (−)deprenyl and other MAO-B inhibitors in some neurologic and psychiatric disorders, Prog. Neurobiol., 44 (1994) 141–161. Fowler, C.J. and Strolin Benedetti, M., The metabolism of dopamine by both forms of monoamine oxidase in the rat brain and its inhibition by cimoxatone, J. Neurochem., 40 (1983) 1534– 1541. Geffard, M., Buijs, R.M., Seguela, P., Pool, C.W. and Le Moal, M., First demonstration of highly specific and sensitive antibodies against dopamine, Brain Res., 294 (1984) 161–165. Glover, V., Sandler, M., Owen, F. and Riley, G.J., Dopamine is a monoamine oxidase B substrate in man, Nature, 265 (1977) 80–81. Ho¨kfelt, T., Ma˚rtensson, R., Bjo¨rklund, A., Kleinau, S. and Goldstein, M., Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In A. Bjo¨rklund and T. Ho¨kfelt (Eds.), Classical Transmitters in the CNS, Part I. Handbook of Chemical Neuroanatomy, Vol. 2, Elsevier, Amsterdam, 1984, pp. 277–379. Hornykiewicz, O., The mechanisms of action of L-dopa in Parkinson’s disease, Life Sci., 15 (1974) 1249–1259. Hsu, S.M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577–580. Kitahama, K., Maeda, T., Denney, R.M. and Jouvet, M., Monoamine oxidase: distribution in the cat brain studied by enzymeand immunohistochemistry: recent progress, Prog. Neurobiol., 42 (1994) 53–78.
[13] Konradi, C., Kornhuber, J., Froelich, L., Fritze, J., Heinsen, H., Beckmann, H., Schulz, E. and Riederer, P., Demonstration of monoamine oxidase-A and -B in the human brainstem by a histochemical technique, Neuroscience, 33 (1989) 383–400. [14] Levitt, P., Pintar, J.E. and Breakefield, X.O., Immunocytochemical demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurons, Proc. Natl. Acad. Sci. (USA), 79 (1982) 6385–6389. [15] Luque, J.M., Kwan, S.-W., Abell, C.W., Da Prada, M. and Richards, J.G., Cellular expression of mRNAs encoding monoamine oxidases A and B in the rat central nervous system, J. Comp. Neurol., 363 (1995) 665–680. [16] Maeda, T., Imai, H., Arai, R., Tago, H., Nagai, T., Sakumoto, T., Kitahama, K., Onteniente, B. and Kimura, H., An improved coupled peroxidatic oxidation method of MAO histochemistry for neuroanatomical research at light and electron microscopic levels, Cell. Mol. Biol., 33 (1987) 1–11. [17] Milon, H. and McRae-Degueurce, A., Pharmacological investigation on the role of dopamine in the rat locus coeruleus, Neurosci. Lett., 30 (1982) 297–301. [18] Ortemann, C., Robert, F., Renaud, B. and Lamba´s-Sen˜as, L., In vivo microdialysis study of the extracellular 3,4-dihydroxyphenylacetic acid in the rat locus ceruleus: topographical and pharmacological aspects, J. Neurochem., 61 (1993) 594–601. [19] Westlund, K.N., Denney, R.M., Rose, R.M. and Abell, C.W., Localization of distinct monoamine oxidase A and monoamine oxidase B cell populations in human brainstem, Neuroscience, 25 (1988) 439–456. [20] Willoughby, J., Glover, V. and Sandler, M., Histochemical localisation of monoamine oxidase A and B in rat brain, J. Neural Transm., 74 (1988) 29–42.