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Dopamine deficiency in mice
Brain & Development 22 (2000) S54±S60
www.elsevier.com/locate/braindev
Original article
Dopamine de®ciency in mice Kazuto Kobayashi a,*, Hiromi Sano b a
Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical, University School of Medicine, Fukushima 960-1297, Japan b Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan
Abstract Dopamine is the principal neurotransmitter that mediates a wide range of brain functions, including locomotion, emotion, learning, and neuroendocrine modulation. To clarify the role of dopamine during postnatal development, it is useful to have mutant mice genetically deleting dopamine. In this paper, we describe the mice lacking expression of tyrosine hydroxylase (TH), the ®rst and rate-limiting enzyme of catecholamine biosynthetic pathway, in the dopaminergic neuronal type. In these mice, TH expression in noradrenergic and adrenergic cells was restored. Lack of TH expression in dopaminergic neurons resulted in a marked reduction of dopamine accumulation. This led to multiple behavioral abnormalities at the juvenile stage, which were characterized by a reduction in spontaneous locomotor activity, blockade of methamphetamine-induced hyperactivity, cataleptic behavior, and defect in active avoidance learning. In contrast, development of pituitary gland as well as production and secretion of the pituitary peptide hormones dependent on hypothalamic dopaminergic control were normally maintained in spite of the reduced dopamine synthesis. Our ®ndings provide genetic evidence that dopamine is essential for controlling spontaneous and voluntary movement and emotional learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Dopamine; Tyrosine hydroxylase; Motor control; Emotional learning; Gene targeting; Transgenic mouse
1. Introduction Dopamine is the principal neurotransmitter that mediates a variety of brain functions, including locomotion, emotion, learning, and neuroendocrine modulation [1]. Dysfunction in dopamine neurotransmission is associated with the neurological and neuropsychiatric disorders, such as Parkinson disease, Segawa disease, and schizophrenia [2±4]. The major dopaminergic cell groups are localized in the ventral region of the midbrain, including the substantia nigra (A9 cell group) and ventral tegmental area (A10 cell group) [5]. The A9 neurons project their massive ®bers to the caudate-putamen to form the nigrostriatal pathway, which plays an important role in motor control [6]. The A10 neurons innervate the limbic system including the nucleus accumbens, amygdala, and olfactory tubercle, as well as a subset of the cerebral cortex to form the mesocorticolimbic pathway. This pathway is involved in emotion, motivation, and memory formation [7]. There are other dopaminergic cell groups (A11±A14) in the mediobasal region of the hypothalamus. The hypothalamic dopaminergic neurons innervate the median eminence and pituitary gland through different axonal pathways [8].
These pathways are known to modulate development of the pituitary gland [9±10] as well as gene expression, production, and release of some pituitary peptide hormones [11±13]. A number of pharmacological and behavioral approaches have been applied to elucidate brain functions mediated by dopamine. However, little is known about the roles of dopamine during postnatal development. To understand the behavioral and physiological signi®cance of dopamine transmission in developing animals, it is useful to have mutant mice genetically deleting dopamine. Recently, we generated mice defective in dopamine synthesis by ablating the expression of tyrosine hydroxylase (TH), which is an initial and rate-limiting enzyme of catecholamine biosynthesis, only in the neuronal types that normally produce dopamine [14]. In this paper, we review the mutant phenotype of dopamine-de®cient mice. We describe an essential role of dopamine in motor control and emotional learning during postnatal development. In addition, we describe maintenance of pituitary development and function in spite of reduced dopamine synthesis.
2. Generation of dopamine-de®cient mice * Corresponding author. Tel.: 181-24-548-2111, ext. 2840; fax: 181-24548-3936. E-mail address: [email protected] (K. Kobayashi).
To generate mice de®cient in dopamine synthesis, we used the transgenic rescue approach. The biosynthetic path-
0387-7604/00/$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S03 87-7604(00)0013 4-0
K. Kobayashi, H. Sano / Brain & Development 22 (2000) S54±S60
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Fig. 2. Appearance of dopamine-de®cient mice. Photograph of 3-week-old mice.
way of catecholamines is summarized in Fig. 1A. In mice lacking TH gene, the expression of human TH transgene in noradrenergic and adrenergic cells was rescued with the speci®city of the dopamine b-hydroxylase (DBH) gene promoter (Fig. 1B) [14]. Transgenic mice were produced that carry a human TH cDNA downstream of the 5 0 upstream region of the human DBH gene. Transgenic offspring were mated with mice heterozygous for the TH mutation introduced by gene targeting [15]. The offspring carrying the transgene and heterozygous mutation were further crossed with TH heterozygotes. At the next generation, we could obtain the rescued mutant mice that were homozygous for the TH mutation and had the transgene. The histological examination of the mouse embryos exhibited the absence of TH immunoreactive signals in the ventral region in the midbrain of the rescued mutant mice,
indicating a loss of TH expression in the neuronal types that normally produce dopamine (Fig. 1C). TH expression in noradrenergic and adrenergic cells was recovered in the mutant due to the tissue-speci®c expression of the transgene (Fig. 1C). In consistent with the TH expression pattern, dopamine accumulation was remarkably reduced in the forebrain, midbrain, hindbrain, and pituitary gland of the mutant (Fig. 1D), whereas noradrenaline and adrenaline accumulation was restored to the normal levels. 3. Life of dopamine-de®cient mice Dopamine-de®cient mice grew normally up to around 10 days after birth, but at postnatal day 10±14 (P10±14) they were distinguishable from other littermates owing to their
Fig. 1. Generation of dopamine-de®cient mice by the transgenic rescue procedure.(A) Catecholamine biosynthetic pathway. Dopamine (DA), noradrenaline (NA), and adrenaline (AD), are sequentially synthesized from l-tyrosine by four kinds of enzymes, including tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), dopamine b-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). The cell type-speci®c and coordinated expression of the genes encoding these enzymes is involved in determination of the catecholamine phenotypes. TH catalyzes the formation of L-3,4dihydroxyphenylalanine (L-DOPA) from l-tyrosine, and then AADC synthesizes dopamine with L-DOPA as a substrate. These two enzymes are localized in dopamine-, noradrenaline-, and adrenaline-producing cells. DBH catalyzes the hydroxylation of dopamine to noradrenaline. This enzyme is localized in noradrenaline- and adrenaline-producing cells. Finally, PNMT catalyzes the transmethylation of noradrenaline to adrenaline, and exists only in adrenalineproducing cells. (B) Creation of mice defective in dopamine synthesis. The expression pattern of catecholamine-synthesizing enzymes, TH, AADC, DBH, and PNMT, in the mutant mice is shown. 1, expression of AADC, DBH, or PNMT in each cell type; 2, deletion of endogenous TH expression due to the mutation; 1*, expression of TH transgene driven by the DBH gene promoter. Phenotypes of catecholamine-producing cells in the mutant are shown in the panel. (See the text for the procedure.) (C) Immunocytochemical localization of TH-expressing cells. Sagittal sections prepared from E14.5 embryos were stained with antiTH antibody. Light microscopic images of the midbrain (substantia nigra), hindbrain (locus coeruleus), and thoracic ganglion of wild-type and mutant mice are shown. In the mutant, TH expression is lacking only in the midbrain dopaminergic neurons (arrow) but is recovered in the noradrenergic and adrenergic cell types. Bars: 200 mm. (D) Dopamine accumulation in tissues at P21. Dopamine levels in brain regions and pituitary glands of the wild-type and mutant mice are shown. Values denote mean ^ SE of the data obtained from six mice. Open columns, wild-type; closed columns, mutant. The asterisks indicate a signi®cant difference (*P , 0:05, **P , 0:01) from the wild-type value (Student's t-test).
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Fig. 3. Motor function of dopamine-de®cient mice. (A) Spontaneous locomotor activity of 3-week-old mice. Locomotion activity (upper panel) and rearing activity (lower panel), de®ned as total number of beam breaks by horizontal and vertical movements, respectively, of the wild-type and mutant mice are shown. Values represent mean ^ SE of the data. Open columns, wild-type (n 13); closed columns, mutant (n 12). The asterisks indicate a signi®cant difference (*P , 0:01) from the wild- type value (Student's t-test). (B) Photograph of dopamine-de®cient mice during the parallel bar test. Mice were forced to take a bizarre posture setting forepaws on a parallel bar and hindpaws on the ¯oor. The time was recorded until the animals removed one forepaw from the bar to evaluate of the degree of catalepsy. (C) Cataleptic behavior of the mice monitored by the parallel bar test. Mice were categorized into four groups (,1 s, 1±10 s, 10±30 s, and .30 s) depending on the degree of catalepsy. The percentage of the number of animals in each group is indicated (wild type, n 26; mutant, n 21).
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small size and hypokinesia. During subsequent development, they further lost the body weight, and gradually weakened (Fig. 2). Finally, all of them died by P30. The brain weight of the mutant was reduced to 87% of the wild type weight at P21, but the gross morphology of the mutant brain appeared to be normal. The phenotypic abnormalities of the mutant revealed that dopamine is essential for animal development and survival during juvenile stage. It appeared unlikely that dopamine is required for the patterning and morphogenesis of the brain. 4. Role of dopamine in motor control Dopamine-de®cient mice exhibited impairment in motor control functions during juvenile stage past P14. First, the spontaneous locomotor activity of the mutant mice displayed a signi®cant reduction relative to the wild type; the horizontal and vertical movements were decreased to about 43 and 30% of the controls, respectively, (Fig. 3A). Second, the mutant mice exhibited the cataleptic behavior, which is de®ned as the absence of voluntary movement. When the mice were assayed by the parallel bar test, the mutant maintained a bizarre posture on the bar much longer than the wild type (Fig. 3B,C). Third, the mutant appeared to be insensitive to the methamphetamine treatment, which normally induces the hyperactivity of locomotion. Normally, the aforementioned motor control functions are mediated by the nigrostriatal or mesocorticolimbic dopaminergic pathway. Chemical lesion with selective neurotoxins of the nigrostriatal dopaminergic neurons impairs spontaneous movement and drug-induced behavior, providing an animal model for parkinsonism [16]. Also, lesion of the dopaminergic terminals in the mesolimbic region produces similar behavioral defects [17,18]. The two pathways are assumed to participate in a neuronal network loop for modulating spontaneous and drug-induced locomotor activity. In contrast, it appears that the primary site of cataleptogenesis by dopamine receptor blockers is the caudate-putamen, the terminal region of the nigrostriatal pathway [19]. The abnormalities of motor responses in dopamine-de®cient mice are considered to result from a combined loss of the actions through the two major dopaminergic pathways. 5. Role of dopamine in emotional learning In addition to the motor dysfunctions, dopamine-de®cient mice exhibited defects in a certain emotional learning paradigm. Active avoidance is the paradigm that monitors the performance to escape an aversive stimulus (a footshock) associated with a conditioned stimulus (a tone). The task requires the amygdala and its linking pathways. When the mice were explored to the tone-dependent active avoidance (Fig. 4), the mutant learned the task much more slowly than the wild type. In the ®nal session of the training, the success
of avoidance in the mutant was only 60%. The difference in the shape of the learning curve between the two genotypes reveals that dopamine is essential for the acquisition of the active avoidance paradigm. In early studies the impaired avoidance response due to dopamine depletion has been explained as an inability to initiate movement [20,21]. However, we found no difference in the escape latency, which de®nes the time taken between onset of the footshock and initiation of the response between the wild type and mutant mice. Also, there were no difference in the pain sensitivity measured by the tail-¯ick test between the two genotypes. Our observations suggest that the mutant mice can normally initiate movement in response to the footshock. Thus, the impairment in the active avoidance in the mutant seems to be attributable to de®cits in the learning process. Recent studies with a different active avoidance task using the lever press paradigm suggest that the avoidance response is disrupted by dopamine depletion in nucleus accumbens [22]. The nucleus accumbens is reported to be an important route, in which the associative information in the amygdala accesses to the emotional response [23]. The abnormalities of emotional learning in dopamine-de®cient mice can be explained predominantly by the de®cits in the mesolimbic dopaminergic pathway projecting the nucleus accumbens.
Fig. 4. Emotional learning of dopamine-de®cient mice. Mice (3-week-old) were tested for the acquisition of active avoidance response triggered by a tone stimulus. The shuttle box apparatus consisted of two compartments with a grid ¯oor including one light and one dark chamber, which were separated by a guillotine door. Each trial was started by placing the animals in the dark compartment after which a tone stimulus was given for 5 s. Unless the mouse moved into the light compartment within 5 s of the tone stimulus, an electric shock (0.2 mA) for 5 s was delivered to the feet with a scrambled shock generator. When the mouse moved into the light compartment before the onset of the footshock, such action was counted as an avoidance. Each animal was given ten trials/day for 7 consecutive days. Percentage of successful avoidances in ten trials per day is plotted. Values indicate mean ^ SE of the data. Open squares, wild-type (n 10); closed squares, mutant (n 6). Two-way analysis of variance (ANOVA) indicated signi®cant main effects of genotypes (F(1,14) 13.77, P , 0:01) and trial days (F(6,84) 22.04, P , 0:01).
K. Kobayashi, H. Sano / Brain & Development 22 (2000) S54±S60
6. Pituitary development and hormone metabolism The dopaminergic pathway originating from the hypothalamus is known to negatively regulate cell proliferation and maintenance of the pituitary lactotroph and melanotroph [9,10] as well as their neuroendocrine functions [11±13]. However, dopamine-de®cient mice never displayed remarkable changes in gross morphology of the pituitary gland, cell density of the lactotroph and melanotroph, or serum levels of prolactin and a-melanocyte stimulating hormone [14]. Our results suggest the presence of compensatory mechanisms in the pituitary for dopamine depletion. One of the possible mechanisms is an up-regulation of dopamine receptors in response to the decreased dopamine level [24,25]. Another compensatory mechanism may be the involvement of different factors that activate pituitary functions. Estrogen is thought to act positively on lactotroph proliferation in an opposite fashion to dopaminergic activity [26]. In addition to estrogen, several hypothalamic peptide hormones, including thyrotropin-releasing factor, and vasoactive intestinal peptide, possess a stimulating activity for prolactin release [11]. Also, corticotrophin-releasing factor is a potent stimulator of a-melanocyte stimulating hormone secretion [27]. The reduced activity of these positive regulators may compensate defects in dopaminergic input into the pituitary. 7. Conclusion Dopamine-de®cient mice were generated by targeted expression of the human TH transgene in noradrenergic and adrenergic cell types in the TH knockout mice. This resulted in a lack of TH in the cells that normally produce dopamine. The mutant mice exhibited the impairments in motor control at the juvenile stage, including the reduction in spontaneous locomotor activity, cataleptic behavior, and blockade of methamphetamine-induced hyperactivity. They also exhibited the impairments in the acquisition of the associative learning. On the other hand, the pituitary development and function in the mutant were apparently normal in spite of the reduced dopamine level in the pituitary gland. Our ®ndings demonstrate that dopamine plays an essential role on movement control and emotional learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways. These mutant mice also provide an experimental model for studying the pathogenesis based on the de®cits in dopamine synthesis. References [1] Riederer P, So®c E, Konradi C, Kornhuber J, Beckmann H, Dietl M, et al. The role of dopamine in the control of neurobiological functions. In: FluÈckiger E, MuÈller EE, Thorner MO, et al., editors. The role of brain dopamine. Berlin: Springer-Verlag; 1985. pp. 1±17. [2] Nagatsu T, Yamaguchi T, Rahman MK, Trocewicz J, Oka K, Hirata Y, et al. Catecholamine-related enzymes and the biopterin cofactor in
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Parkinson's disease and related extrapyramidal diseases. In: Hassler RG, Christ JF, et al., editors. Advances in neurology, vol. 40. New York: Raven Press; 1984. pp. 467±473. Segawa M, Nomura Y, Kase M. Diurnally ¯uctuating hereditary progressive dystonia. In: Vinken PJ, Bruyn GW, Klawans HL, editors. Handbook of clinical neurology. New York: Elsevier; 1986. pp. 529± 539. Davidson M, Keefe RSE, Mohs RC, Siever LJ, Losonczy MF, Horvath TB, Davis KL. L-DOPA challenge and relapse in schizophrenia. Am J Psychiatry 1987;122:509±522. Lindvall O, BjoÈklund A. Dopamine- and norepinephrine-containing neuron systems: their anatomy in the rat brain. In: Emson PC, editor. Chemical neuroanatomy. New York: Raven Press; 1983. pp. 229± 255. Gerfen CR. The neostriatal mosaic: multiple levels of compartmental organization in the basal ganglia. Annu Rev Neurosci 1992;15:285± 320. Le Moal M, Simon H. Mesocorticolimbic dopaminergic network: functional and regulatory roles. Physiol Rev 1991;71:155±234. Moore KE, Lookingland KJ. Dopaminergic neuronal systems in the hypothalamus. In: Bloom FE, Kupfer DJ, editors. Psychopharmacology. New York: Raven Press; 1995. pp. 245±256. Bevan JS, Webster J, Burke CW, Scanlon MF. Dopamine agonists and pituitary tumor shrinkage. Endocr Rev 1992;13:220±240. Gary KA, Chronwall BM. The onset of dopaminergic innervation during ontogeny decreases melanotrope proliferation in the intermediate lobe of the rat pituitary. Int J Dev Neurosci 1992;10:131±142. Ben-Jonathan N, Arbogast LA, Hyde JF. Neuroendocrine regulation of prolactin release. Prog Neurobiol 1989;33:399±447. Beaulieu M, Goldman ME, Miyazaki K, Frey EA, Eskay RL, Kebabian JW, Cote TE. Bromocriptine-induced changes in the biochemistry, physiology, and histology of the intermediate lobe of the rat pituitary gland. Endocrinology 1984;114:1871±1884. Loeffer J-P, Demeneix BA, Kley NA, HoÈllt V. Dopamine inhibition of proopiomelanocortin gene expression in the intermediate lobe of the pituitary. Neuroendocrinology 1988;47:95±101. Nishii K, Matsushita N, Sawada H, Sano H, Noda Y, Mamiya T, et al. Motor and learning dysfunction during postnatal development in mice defective in dopamine neuronal transmission. J Neurosci Res 1998;54:450±464. Kobayashi K, Morita S, Sawada H, Mizuguchi T, Yamada K, Nagatsu I, et al. Targeted disruption of the tyrosine hydroxylase locus results in severe catecholamine depletion and perinatal lethality in mice. J Biol Chem 1995;270:27235±27243. Hu SC, Chang FW, Sung YJ, Hsu WH, Lee EHY. Neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine in the substantia nigra and the locus coeruleus in BALB/c mice. J Pharmacol Exp Ther 1991;259:1379±1387. Kelly PH, Iversen SD. Selective 6OHDA-induced destruction of mesolimbic dopamine neurons: abolition of psychostimulant-induced locomotor activity in rats. Eur J Pharmacol 1976;40:45±56. Koob GF, Riley SJ, Smith SC, Robbins TW. Effects of 6-hydroxydopamine lesions of the nucleus accumbens septi and olfactory tubercle on feeding, locomotor activity, and amphetamine anorexia in the rat. J Comp Physiol Psychol 1978;92:917±927. Dunstan R, Broekkamp CL, Lloyd KG. Involvement of caudate nucleus, amygdala, or reticular formation in neuroleptic and narcotic catalepsy. Pharmacol Biochem Behav 1980;14:168±174. Fibiger HC, Zis AP, Phillips AG. Haloperidol-induced disruption of conditioned avoidance responding: attenuation by prior training or by anticholinergic drugs. Eur J Pharmacol 1975;30:309±314. Koob GF, Simon H, Herman JP, Le Moal M. Neuroleptic-like disruption of the conditioned avoidance response requires destruction of both the mesolimbic and nigrostriatal dopamine systems. Brain Res 1984;303:319±329. McCullough LD, Sokolowski JD, Salamone JD. A neurochemical and behavioral investigation of the involvement of nucleus accumbens
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dopamine in instrumental avoidance. Neuroscience 1993;52:919± 925. [23] Everitt B, Morris K, O'Brien A, Robbins TW. The basolateral amygdala-central striatal system and conditioned place preference: further evidence of limbic-striatal interactions underlying reward-related processes. Neuroscience 1991;42:1±18. [24] Neve KA, Loeschen S, Marshall JF. Denervation accelerates the reappearence of neostriatal D-2 receptors after irreversible receptor blockade. Brain Res 1985;329:225±231.
[25] Martres MP, Sokoloff P, Giros B, Schwartz JC. Effects of dopaminergic transmission interruption on the D2 receptor isoforms in various cerebral tissues. J Neurochem 1992;58:673±679. [26] Ellias KA, Weiner RI. Inhibition of estrogen-induced anterior pituitary enlargement and arteriogenesis by bromocriptine in Fischer 344 rats. Endocrinology 1987;120:617±621. [27] Proulx-Ferland L, Labrie F, Dumont D, CoÃte J, Coy DH, Sveiraf J. Corticotropin-releasing factor stimulates secretion of melanocytestimulating hormone from the rat pituitary. Science 1982;217:62±63.
www.elsevier.com/locate/braindev
Original article
Dopamine de®ciency in mice Kazuto Kobayashi a,*, Hiromi Sano b a
Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical, University School of Medicine, Fukushima 960-1297, Japan b Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan
Abstract Dopamine is the principal neurotransmitter that mediates a wide range of brain functions, including locomotion, emotion, learning, and neuroendocrine modulation. To clarify the role of dopamine during postnatal development, it is useful to have mutant mice genetically deleting dopamine. In this paper, we describe the mice lacking expression of tyrosine hydroxylase (TH), the ®rst and rate-limiting enzyme of catecholamine biosynthetic pathway, in the dopaminergic neuronal type. In these mice, TH expression in noradrenergic and adrenergic cells was restored. Lack of TH expression in dopaminergic neurons resulted in a marked reduction of dopamine accumulation. This led to multiple behavioral abnormalities at the juvenile stage, which were characterized by a reduction in spontaneous locomotor activity, blockade of methamphetamine-induced hyperactivity, cataleptic behavior, and defect in active avoidance learning. In contrast, development of pituitary gland as well as production and secretion of the pituitary peptide hormones dependent on hypothalamic dopaminergic control were normally maintained in spite of the reduced dopamine synthesis. Our ®ndings provide genetic evidence that dopamine is essential for controlling spontaneous and voluntary movement and emotional learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Dopamine; Tyrosine hydroxylase; Motor control; Emotional learning; Gene targeting; Transgenic mouse
1. Introduction Dopamine is the principal neurotransmitter that mediates a variety of brain functions, including locomotion, emotion, learning, and neuroendocrine modulation [1]. Dysfunction in dopamine neurotransmission is associated with the neurological and neuropsychiatric disorders, such as Parkinson disease, Segawa disease, and schizophrenia [2±4]. The major dopaminergic cell groups are localized in the ventral region of the midbrain, including the substantia nigra (A9 cell group) and ventral tegmental area (A10 cell group) [5]. The A9 neurons project their massive ®bers to the caudate-putamen to form the nigrostriatal pathway, which plays an important role in motor control [6]. The A10 neurons innervate the limbic system including the nucleus accumbens, amygdala, and olfactory tubercle, as well as a subset of the cerebral cortex to form the mesocorticolimbic pathway. This pathway is involved in emotion, motivation, and memory formation [7]. There are other dopaminergic cell groups (A11±A14) in the mediobasal region of the hypothalamus. The hypothalamic dopaminergic neurons innervate the median eminence and pituitary gland through different axonal pathways [8].
These pathways are known to modulate development of the pituitary gland [9±10] as well as gene expression, production, and release of some pituitary peptide hormones [11±13]. A number of pharmacological and behavioral approaches have been applied to elucidate brain functions mediated by dopamine. However, little is known about the roles of dopamine during postnatal development. To understand the behavioral and physiological signi®cance of dopamine transmission in developing animals, it is useful to have mutant mice genetically deleting dopamine. Recently, we generated mice defective in dopamine synthesis by ablating the expression of tyrosine hydroxylase (TH), which is an initial and rate-limiting enzyme of catecholamine biosynthesis, only in the neuronal types that normally produce dopamine [14]. In this paper, we review the mutant phenotype of dopamine-de®cient mice. We describe an essential role of dopamine in motor control and emotional learning during postnatal development. In addition, we describe maintenance of pituitary development and function in spite of reduced dopamine synthesis.
2. Generation of dopamine-de®cient mice * Corresponding author. Tel.: 181-24-548-2111, ext. 2840; fax: 181-24548-3936. E-mail address: [email protected] (K. Kobayashi).
To generate mice de®cient in dopamine synthesis, we used the transgenic rescue approach. The biosynthetic path-
0387-7604/00/$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S03 87-7604(00)0013 4-0
K. Kobayashi, H. Sano / Brain & Development 22 (2000) S54±S60
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Fig. 2. Appearance of dopamine-de®cient mice. Photograph of 3-week-old mice.
way of catecholamines is summarized in Fig. 1A. In mice lacking TH gene, the expression of human TH transgene in noradrenergic and adrenergic cells was rescued with the speci®city of the dopamine b-hydroxylase (DBH) gene promoter (Fig. 1B) [14]. Transgenic mice were produced that carry a human TH cDNA downstream of the 5 0 upstream region of the human DBH gene. Transgenic offspring were mated with mice heterozygous for the TH mutation introduced by gene targeting [15]. The offspring carrying the transgene and heterozygous mutation were further crossed with TH heterozygotes. At the next generation, we could obtain the rescued mutant mice that were homozygous for the TH mutation and had the transgene. The histological examination of the mouse embryos exhibited the absence of TH immunoreactive signals in the ventral region in the midbrain of the rescued mutant mice,
indicating a loss of TH expression in the neuronal types that normally produce dopamine (Fig. 1C). TH expression in noradrenergic and adrenergic cells was recovered in the mutant due to the tissue-speci®c expression of the transgene (Fig. 1C). In consistent with the TH expression pattern, dopamine accumulation was remarkably reduced in the forebrain, midbrain, hindbrain, and pituitary gland of the mutant (Fig. 1D), whereas noradrenaline and adrenaline accumulation was restored to the normal levels. 3. Life of dopamine-de®cient mice Dopamine-de®cient mice grew normally up to around 10 days after birth, but at postnatal day 10±14 (P10±14) they were distinguishable from other littermates owing to their
Fig. 1. Generation of dopamine-de®cient mice by the transgenic rescue procedure.(A) Catecholamine biosynthetic pathway. Dopamine (DA), noradrenaline (NA), and adrenaline (AD), are sequentially synthesized from l-tyrosine by four kinds of enzymes, including tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), dopamine b-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). The cell type-speci®c and coordinated expression of the genes encoding these enzymes is involved in determination of the catecholamine phenotypes. TH catalyzes the formation of L-3,4dihydroxyphenylalanine (L-DOPA) from l-tyrosine, and then AADC synthesizes dopamine with L-DOPA as a substrate. These two enzymes are localized in dopamine-, noradrenaline-, and adrenaline-producing cells. DBH catalyzes the hydroxylation of dopamine to noradrenaline. This enzyme is localized in noradrenaline- and adrenaline-producing cells. Finally, PNMT catalyzes the transmethylation of noradrenaline to adrenaline, and exists only in adrenalineproducing cells. (B) Creation of mice defective in dopamine synthesis. The expression pattern of catecholamine-synthesizing enzymes, TH, AADC, DBH, and PNMT, in the mutant mice is shown. 1, expression of AADC, DBH, or PNMT in each cell type; 2, deletion of endogenous TH expression due to the mutation; 1*, expression of TH transgene driven by the DBH gene promoter. Phenotypes of catecholamine-producing cells in the mutant are shown in the panel. (See the text for the procedure.) (C) Immunocytochemical localization of TH-expressing cells. Sagittal sections prepared from E14.5 embryos were stained with antiTH antibody. Light microscopic images of the midbrain (substantia nigra), hindbrain (locus coeruleus), and thoracic ganglion of wild-type and mutant mice are shown. In the mutant, TH expression is lacking only in the midbrain dopaminergic neurons (arrow) but is recovered in the noradrenergic and adrenergic cell types. Bars: 200 mm. (D) Dopamine accumulation in tissues at P21. Dopamine levels in brain regions and pituitary glands of the wild-type and mutant mice are shown. Values denote mean ^ SE of the data obtained from six mice. Open columns, wild-type; closed columns, mutant. The asterisks indicate a signi®cant difference (*P , 0:05, **P , 0:01) from the wild-type value (Student's t-test).
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Fig. 3. Motor function of dopamine-de®cient mice. (A) Spontaneous locomotor activity of 3-week-old mice. Locomotion activity (upper panel) and rearing activity (lower panel), de®ned as total number of beam breaks by horizontal and vertical movements, respectively, of the wild-type and mutant mice are shown. Values represent mean ^ SE of the data. Open columns, wild-type (n 13); closed columns, mutant (n 12). The asterisks indicate a signi®cant difference (*P , 0:01) from the wild- type value (Student's t-test). (B) Photograph of dopamine-de®cient mice during the parallel bar test. Mice were forced to take a bizarre posture setting forepaws on a parallel bar and hindpaws on the ¯oor. The time was recorded until the animals removed one forepaw from the bar to evaluate of the degree of catalepsy. (C) Cataleptic behavior of the mice monitored by the parallel bar test. Mice were categorized into four groups (,1 s, 1±10 s, 10±30 s, and .30 s) depending on the degree of catalepsy. The percentage of the number of animals in each group is indicated (wild type, n 26; mutant, n 21).
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small size and hypokinesia. During subsequent development, they further lost the body weight, and gradually weakened (Fig. 2). Finally, all of them died by P30. The brain weight of the mutant was reduced to 87% of the wild type weight at P21, but the gross morphology of the mutant brain appeared to be normal. The phenotypic abnormalities of the mutant revealed that dopamine is essential for animal development and survival during juvenile stage. It appeared unlikely that dopamine is required for the patterning and morphogenesis of the brain. 4. Role of dopamine in motor control Dopamine-de®cient mice exhibited impairment in motor control functions during juvenile stage past P14. First, the spontaneous locomotor activity of the mutant mice displayed a signi®cant reduction relative to the wild type; the horizontal and vertical movements were decreased to about 43 and 30% of the controls, respectively, (Fig. 3A). Second, the mutant mice exhibited the cataleptic behavior, which is de®ned as the absence of voluntary movement. When the mice were assayed by the parallel bar test, the mutant maintained a bizarre posture on the bar much longer than the wild type (Fig. 3B,C). Third, the mutant appeared to be insensitive to the methamphetamine treatment, which normally induces the hyperactivity of locomotion. Normally, the aforementioned motor control functions are mediated by the nigrostriatal or mesocorticolimbic dopaminergic pathway. Chemical lesion with selective neurotoxins of the nigrostriatal dopaminergic neurons impairs spontaneous movement and drug-induced behavior, providing an animal model for parkinsonism [16]. Also, lesion of the dopaminergic terminals in the mesolimbic region produces similar behavioral defects [17,18]. The two pathways are assumed to participate in a neuronal network loop for modulating spontaneous and drug-induced locomotor activity. In contrast, it appears that the primary site of cataleptogenesis by dopamine receptor blockers is the caudate-putamen, the terminal region of the nigrostriatal pathway [19]. The abnormalities of motor responses in dopamine-de®cient mice are considered to result from a combined loss of the actions through the two major dopaminergic pathways. 5. Role of dopamine in emotional learning In addition to the motor dysfunctions, dopamine-de®cient mice exhibited defects in a certain emotional learning paradigm. Active avoidance is the paradigm that monitors the performance to escape an aversive stimulus (a footshock) associated with a conditioned stimulus (a tone). The task requires the amygdala and its linking pathways. When the mice were explored to the tone-dependent active avoidance (Fig. 4), the mutant learned the task much more slowly than the wild type. In the ®nal session of the training, the success
of avoidance in the mutant was only 60%. The difference in the shape of the learning curve between the two genotypes reveals that dopamine is essential for the acquisition of the active avoidance paradigm. In early studies the impaired avoidance response due to dopamine depletion has been explained as an inability to initiate movement [20,21]. However, we found no difference in the escape latency, which de®nes the time taken between onset of the footshock and initiation of the response between the wild type and mutant mice. Also, there were no difference in the pain sensitivity measured by the tail-¯ick test between the two genotypes. Our observations suggest that the mutant mice can normally initiate movement in response to the footshock. Thus, the impairment in the active avoidance in the mutant seems to be attributable to de®cits in the learning process. Recent studies with a different active avoidance task using the lever press paradigm suggest that the avoidance response is disrupted by dopamine depletion in nucleus accumbens [22]. The nucleus accumbens is reported to be an important route, in which the associative information in the amygdala accesses to the emotional response [23]. The abnormalities of emotional learning in dopamine-de®cient mice can be explained predominantly by the de®cits in the mesolimbic dopaminergic pathway projecting the nucleus accumbens.
Fig. 4. Emotional learning of dopamine-de®cient mice. Mice (3-week-old) were tested for the acquisition of active avoidance response triggered by a tone stimulus. The shuttle box apparatus consisted of two compartments with a grid ¯oor including one light and one dark chamber, which were separated by a guillotine door. Each trial was started by placing the animals in the dark compartment after which a tone stimulus was given for 5 s. Unless the mouse moved into the light compartment within 5 s of the tone stimulus, an electric shock (0.2 mA) for 5 s was delivered to the feet with a scrambled shock generator. When the mouse moved into the light compartment before the onset of the footshock, such action was counted as an avoidance. Each animal was given ten trials/day for 7 consecutive days. Percentage of successful avoidances in ten trials per day is plotted. Values indicate mean ^ SE of the data. Open squares, wild-type (n 10); closed squares, mutant (n 6). Two-way analysis of variance (ANOVA) indicated signi®cant main effects of genotypes (F(1,14) 13.77, P , 0:01) and trial days (F(6,84) 22.04, P , 0:01).
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6. Pituitary development and hormone metabolism The dopaminergic pathway originating from the hypothalamus is known to negatively regulate cell proliferation and maintenance of the pituitary lactotroph and melanotroph [9,10] as well as their neuroendocrine functions [11±13]. However, dopamine-de®cient mice never displayed remarkable changes in gross morphology of the pituitary gland, cell density of the lactotroph and melanotroph, or serum levels of prolactin and a-melanocyte stimulating hormone [14]. Our results suggest the presence of compensatory mechanisms in the pituitary for dopamine depletion. One of the possible mechanisms is an up-regulation of dopamine receptors in response to the decreased dopamine level [24,25]. Another compensatory mechanism may be the involvement of different factors that activate pituitary functions. Estrogen is thought to act positively on lactotroph proliferation in an opposite fashion to dopaminergic activity [26]. In addition to estrogen, several hypothalamic peptide hormones, including thyrotropin-releasing factor, and vasoactive intestinal peptide, possess a stimulating activity for prolactin release [11]. Also, corticotrophin-releasing factor is a potent stimulator of a-melanocyte stimulating hormone secretion [27]. The reduced activity of these positive regulators may compensate defects in dopaminergic input into the pituitary. 7. Conclusion Dopamine-de®cient mice were generated by targeted expression of the human TH transgene in noradrenergic and adrenergic cell types in the TH knockout mice. This resulted in a lack of TH in the cells that normally produce dopamine. The mutant mice exhibited the impairments in motor control at the juvenile stage, including the reduction in spontaneous locomotor activity, cataleptic behavior, and blockade of methamphetamine-induced hyperactivity. They also exhibited the impairments in the acquisition of the associative learning. On the other hand, the pituitary development and function in the mutant were apparently normal in spite of the reduced dopamine level in the pituitary gland. Our ®ndings demonstrate that dopamine plays an essential role on movement control and emotional learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways. These mutant mice also provide an experimental model for studying the pathogenesis based on the de®cits in dopamine synthesis. References [1] Riederer P, So®c E, Konradi C, Kornhuber J, Beckmann H, Dietl M, et al. The role of dopamine in the control of neurobiological functions. In: FluÈckiger E, MuÈller EE, Thorner MO, et al., editors. The role of brain dopamine. Berlin: Springer-Verlag; 1985. pp. 1±17. [2] Nagatsu T, Yamaguchi T, Rahman MK, Trocewicz J, Oka K, Hirata Y, et al. Catecholamine-related enzymes and the biopterin cofactor in
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