Neuroscience Letters, 127 (1991) 91-95 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100280G

NSL 07797

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Expression in brain sensory neurons of the transgene in transgenic mice carrying human tyrosine hydroxylase gene

I. N a g a t s u 1, K. Y a m a d a 1, N. K a r a s a w a 1, M. Sakai 1, T. Takeuch i 1, N. K a n e d a s, T. Sasaoka s, K. K o b a y a s h i s, M. Y o k o y a m a 3, T. N o m u r a 3, M. Ka t suk i 3~, K. Fuj i ta 2 and T. N a g a t s u s

1Department of Anatomy and 2 Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake (Japan), 3Central Institute for Experimental Animals, Kawasaki (Japan), 4Department of DNA Biology, School of Medicine, Tokai University, Isehara

(Japan) and s Department of Biochemistry, Nagoya University School of Medicine, Nagoya (Japan)

(Received 7 January 1991; Revised version received 8 March 1991; Accepted 8 March 1991)

Key words. Human tyrosine hydroxylase; Transgenic mouse; Gene expression; Immunocytochemistry; In situ hybridization; Sensory neuron; Olfac- tory system; Visual system

We have recently reported the production of transgenic (Tg) mice carrying the human tyrosine hydroxylase (TH) gene, and have described tissue- specific expression of the transgene in catecholaminergic (CAergic) neurons and adrenal glands. This paper describes the transgene expression in non-catecholaminergic (nCAergic) neurons in the brain of Tg mice by immunocytochemistry and in situ hybridization. In adult Tg mice, human TH was atypically expressed in the olfactory (typically, the anterior olfactory nucleus and pyriform cortex) and visual (typically, n. suprachiasmaticus and n. parabigeminalis) systems, in addition to typical CAergic neuron-rich nuclei in the brain. These results suggest the possibility that TH plays some novel roles in sensory systems.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the catecholamine (CA) pathway and essential for the production of CAs, and its expression is necessary for establishment of the catecholaminergic (CAergic) phe- notype. Expression of this gene is restricted to adrenal chromaffin cells and a large number of neuronal cells. We isolated and characterized clones of 4 types of full- length cDNAs and genomic DNA encoding human TH (hTH) [3, 5, 6]. Recently, we have produced transgenic (Tg) mice carrying the hTH gene [4]. In the brain of Tg mice, the transgene mRNA and TH-immunoreactivity were expressed specifically in CAergic neuron-rich re- gions including substantia nigra and ventral tegmental area [4]. In addition to these hTH gene expression in CAergic regions, we have also found atypical hTH gene expression in non-catecholaminergic (nCAergic) regions, where CAergic nerve terminals project, of Tg mouse brain by immunocytochemistry at light and electron microscopic levels and by in situ hybridization.

Construction of the h T H gene and production of Tg mice. Nothern blot analysis, and primer extension analy- sis were described elsewhere [4]. We employed an 11-kb

Correspondence: Ikuko Nagatsu, Department of Anatomy, School of Medicine, Fujita Health University, Toyoake, Aichi 470-11, Japan.

DNA fragment of the TH gene consisting of 2.5 kb of 5'-upstream region containing promoter information, the entire exon-intron structure, and 0.5 kb of the 3'- franking region for the production of Tg mice. Tg mice contained about 60 copies of the transgene [4].

Immunocytochemistry. Tg and non-transgenic (nTg) littermate mice were anesthetized with Nembutal (50 mg/kg, i.p.) and perfused intracardially with saline fol- lowed by 4% paraformaldehyde (PFA) in 0.1 M phos- phate buffer (PB, pH 7.4) for 6 min. Fixed tissues were dissected out and immersed overnight in the same fixa- tive at 4°C. After rinsing with 10-30% sucrose in the PB for 2 days, cryostat sections (40/~m thick) through the sagittal or frontal plane of the whole brain were collected in the PB and processed for immunocytochemistry as de- scribed elsewhere [7-10].

In situ hybridization. Tg and nTg mice were anaesthe- tized with Nembutal and perfused with 4 % PFA as de- scribed above. Brains were dissected out, postfixed in the same fixative for 2-4 h, washed with 20 % sucrose in the PB for overnight. Frozen sections were cut on a cryostat (40/tm), floated in the buffer and processed for in situ hybridization as described previously [4] with slight modifications. Briefly, sections were immersed in 10 mM Tris-5 mM EDTA buffer containing proteinase K (1/~g/ ml) for 20 min at 37°C. They were washed twice in

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2 × SSC and, without further pretreatment, hybridized to a probe complementary to hTHmRNA. The cDNA probe (probe 1:0.25 kb SacI-EcoRI), which hybridizes only with hTHmRNA, or the cDNA probe (probe 2:1.3 kb EcoRI-SacI), which hybridizes with both human and mouse mRNAs was labeled with [~-35S]dCTP~S (1000 Ci/mmol, 1 Ci = 37 GBq, Amersham) by the multiprime DNA labelling method. Following hybridization for 16 h at 37°C in a solution consisting of 50% formamide, 4 x SSC, 1 x Denhardt's solution, 1% sarcosyl, 0.02 M sodium phosphate buffer (pH 7.0), 10% dextran sul- phate, 500/tg/ml yeast tRNA, 250/~g/ml heat-denatured salmon sperm DNA and 80 mM dithiothreitol, sections were rinsed four times in 2 x SSC at room temperature for 2 3 min, washed twice in 0.1 × SSC at 30°C for 1 h, and placed on glass slides. They were exposed to X-ray films (Hyperfilm-flmax, Amersham) for a week, and thereafter, dipped into Kodak NTB2 or Konica NR-M2 liquid emulsion.

Immunocytochemistry at light microscopic level. Speci- fic neuronal expression of hTH by TH immunocyto- chemistry in the brain of Tg mice was schematically

Fig. I. Schematic drawings of TH-immunostaining (TH-I) perikarya in the brains of Tg mice (left, triangle) and nTg mice (right, circle) on

frontal sections. Fine dots represent TH-I fibers.

shown in Fig. 1. The brain sections from Tg mice revealed the expression of TH immunoreactivity in a dis- crete population of cells in the typical CAergic neurons, such as substantia nigra, ventral tegmental area, locus coeruleus, and n. solitary tract. In addition to these re- gions, TH-I cells were detected in some nCAergic re- gions, which include the anterior olfactory nucleus (AON, Figs. 2A,B), pyriform cortex (Fig. 2E), entorhi- hal cortex, anterior cingulate cortex, septum, corporis callosi, amygdaloid complex, hippocampus, lateral habenula, n.suprachiasmaticus (D13) [2], n.pretectalis (D5), n.corpus geniculati lateralis, superior colliculus (data not shown) and n.parabigeminalis (Fig. 2F).

In the AON of adult Tg mice, numerous TH-I cells (Fig. 3A,B) as well as PNMT-I neurons (Fig. 3C) were observed in the median part of the AON. These cells were aromatic L-amino acid decarboxylase (AADC)- negative (Fig. 3D) and dopamine-~-hydroxylase (DBH)- negative (Fig. 3E). In contrast to the Tg AON, only PNMT-I neurons were seen in the AON of adult nTg mice [9], and no immunoreactivity was observed using antiserum to TH, AADC, or DBH (data not shown).

In situ hybridization. In addition to the CAergic neu- rons of Tg mice, hTH mRNA was found to be expressed by in situ hybridization in nCAergic sensory neurons, such as the AON (Figs. 2C, using probe 2: and Fig. 2D, using probe 1 ). The localization of hTH mRNA-positive cells was similar to those stained by immunocyto- chemistry for TH (Figs. 2A and 3A,B).

Cells labeled by in situ hybridization were morpholo- gically comparable to those that contain TH protein in three different tissue preparations. In contrast, hTHmRNA labeled cells were not found in the AON of adult nTg mice.

Immunocytochemisto' at electron microscopic level. Labelled cells were medium-sized and multipolar-shaped neurons. Ultrastructurally, the neurons had TH-labelled perikarya in the AON of adult Tg mice, but TH-negative nuclei, Golgi apparatus, and mitochondria. One of these bipolar TH-I neurons contacted intercellular-bridged YH-negative neurons (Fig. 4).

A large difference was observed between brains of Tg and nTg mice both by immunocytochemistry and in situ hybridization. In the brain of Tg mice, TH immunoreac- tivity was increased not only in CAergic neurons but also in nCAergic neurons. In situ hybridization of the brain of Tg mice also clearly demonstrated the expression of hTHmRNA in some nCAergic neurons, in addition to the CAergic neurons. Relatively high-level TH expres- sion in nCAergic neurons was observed mainly in the ol- factory system such as prefrontal (anteromedial, ante- rior cingulate, suprarhinal) and limbic (pyriform and entorhinal) cortices. TH-I cells were also observed

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Fig. 2. The micrographs display the low magnification appearance of the AON (A,B), the pyriform cortex (E), and n.parabigeminalis (F) of adult Tg (A,E,F) and nTg (B) mice, demonstrated by immunocytochemistry against TH antiserum. In situ hybridization for human and mouse THmRNA

(C) and hTHmRNA (D) are shown in the AON of the Tg mice. Arrows indicate immunoreactive neurons. A-D x 13; E x 37; F ×65.

within the neocortical areas such as the dorsolateral frontal cortex (Krieg's area 10), the parietotemporal cor- tex (area 1, 2, 40, 41 and 20), the occipital cortex (area 17 and 18a) and the hippocampal formation especially in its temporal part as well as restricted neocortical field (sensorimotor, visual and retrosplenial cortices). More- over, in adult Tg mice, TH-I cells were detected in the visual system (n.suprachiasmaticus, n.pretectalis, n.cor- poris geniculati lateralis, n.parabigeminalis, superior

colliculus). The distribution of these atypical TH-I cells is very characteristic, compared with that of endogenous mouse TH. In nTG mice, TH-I cells were not observed in olfactory and visual systems, but TH-I fibers were clearly found as dopaminergic terminals [1, 11]. In adult Tg mice, TH-I cells were found along these sensory tracts. The TH-I cells in sensory systems may not pro- duce dopamine because AADC immunoreactivity was absent, and therefore, may not belong to the dopaminer-

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nism between h u m a n and mouse T H genes. Second,

some regu la to ry element(s) would be absent in the t rans-

gene cons t ruc t or masked by some u n k n o w n mecha-

nisms, which would be required for a strict spacial and

t empora l T H gene expression. Thi rd , h T H express ion

could be enhanced in Tg mice, and these nCAerg ic neu-

rons would no t be able to a t t enua te T H gene expression

even at adu l t stage.

W e have shown here tha t h T H transgene is expressed

in o l fac tory and visual sensory neurons in add i t i on to

typical CAerg ic neurons , which gives some new clues to

s tudy regu la to ry mechanisms o f T H expression.

The au thors are very grateful to Mrs. K. Katsuk i ,

Messrs. T. Hasegawa , R. Takahash i , K. N a k a o , K.

Kaseda , K. K o m o r i , T. Fuj i i and Misses A. Mor i t a , M.

H a t a n a k a for their excellent technical assistance. This

work was suppo r t ed in par t by a G r a n t - i n - A i d for Scien-

tific Research on Pr ior i ty Areas , Japanese Minis t ry o f

Educa t ion , Science and Cul ture (01623001, 01570035), and by a G r a n t - i n - A i d f rom Fu j i t a Hea l th Univers i ty ,

Japan .

Fig. 3. Immunohistochemical micrographs of the AON at adult stage of Tg (A-E) mouse, after incubation with antiserum to TH (A,B), AADC (D), DBH (E), and PNMT (C). B-E show adjacent 40-/tm- thick tissue sections. Numerous TH-positive cells (A,B) are seen in the median part of the AON, as well as PNMT-positive neurons (C). These cells are AADC-negative (D) and DBH-negative (E). Arrows indicate

immunoreactive neurons. A ×25; B-E x65.

gic neuron system. We have previously r epor ted the

t rans ient expression o f T H in the A O N dur ing late

embryon ic and pos tna t a l stages [8]. In Tg mice, T H - I

neurons were observed in the a rea o f A O N even at adu l t

stage. T H t ransgene was expressed in nCAerg ic a rea in

spacial ly and t empora l ly a typ ica l manner . There are

several poss ible exp lana t ions for presence o f T H - I cells

in sensory systems. Fi rs t , t ransgene express ion in nCAer -

gic cells wou ld be due to difference in regu la tory mecha-

I Berger, B., Verney, C., Alvarez, C., Vigny, A. and Helle, K.B., New dopaminergic terminal fields in the motor, visual (area 18b) and retrosplenial cortex in the young and adult rat, Immunocytochemi- cal and catecholamine histochemical analyses, Neuroscience, 15 (1985) 983-998.

2 Jaeger, C.B., Ruggiero, D.A., Albert, V.R., Joh, T.H. and Reis, D.J., Immunocytochemical localization of aromatic-u-amino acid decarboxylase. In A. Bj6rklund and T. H6kfelt (Eds.), Classical Transmitters in the CNS, Part 1. Handbook of Chemical Neuro- anatomy, Vol. 2, Elsevier, Amsterdam, 1984, pp. 387-408.

3 Kaneda, N., Kobayashi, K., Ichinose, H., Kishi, F., Nakazawa, A., Kurosawa, Y., Fujita, K. and Nagatsu, T., Isolation of a novel cDNA clone for human tyrosine hydroxylase: alternative RNA splicing produces four kinds of mRNA from a single gene, Bio- chem. Biophys. Res. Commun., 146 (1987) 971-975.

4 Kaneda, N., Sasaoka, T., Kobayashi, K., Kiuchi, K., Nagatsu, I., Kurosawa, Y., Fujita, K., Yokoyama, M., Nomura, T., Katsuki, M. and Nagatsu, T., Tissue-specific and high-level expression of human tyrosine hydroxylase gene in transgenic mice, Neuron, in press.

5 Kobayashi, K., Kaneda, N., Ichinose, H., Kishi, F., Nakazawa, A., Kurosawa, Y., Fujita, K. and Nagatsu, T., Isolation of a full-length cDNA clone encoding human tyrosine hydroxylase type 3, Nucleic Acids Res., 15 (1987) 6733.

6 Kobayashi, K., Kaneda, N., Ichinose, H., Kishi, F., Nakazawa, H., Kurosawa, Y., Fujita, K. and Nagatsu, T., Structure of the human tyrosine hydroxylase gene: alternative splicing from a single gene accounts for generation of four mRNA types, J. Biochem., 103 (1988) 907-912.

7 Nagatsu, I., Kobayashi, K., Fujii, T., Komori, K., Sekiguchi, K., Titani, K., Fujita, K. and Nagatsu, T., Antibodies raised to the dif- ferent oligopeptide segments of human dopamine-fl-hydroxylase, Neurosci. Lett., 120 (1990) 141-145.

8 Nagatsu, I., Komori, K., Takeuchi, T., Sakai, M., Yamada, K. and Karasawa, N., Transient tyrosine hydroxylase-immunoreactive

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Fig. 4. Ultrastructure of TH-positive perikarya in the AON at adult stage of Tg mouse shows TH-labeled cytoplasm. The staining is not localized in the round nucleus, mitochondria and Golgi apparatus. One of these bipolar TH-I neurons is observed to contact intercellular-bridged TH-negative

neurons. Bar = 1/tm.

neurons in the region of the anterior olfactory nucleus of pre- and postnatal mice do not contain dopamine, Brain Res., 511 (1990) 55-62.

9 Nagatsu, I., Komori, K., Miura, K., Sakai, M., Karasawa, N. and Yamada, K., Ontogeny of phenylethanolamine-N-methyltransfer- ase and tyrosine hydroxylase-immunoreactive expression in the mouse anterior olfactory nucleus. Biomed. Res., 10 Suppl. 3 (1989) 277-286.

10 Nagatsu, I., Sakai, M., Yoshida, M. and Nagatsu, T., Aromatic L-

amino acid decarboxylase-immunoreactive neurons in and around the cerebrospinal fluid-contacting neurons of the central canal do not contain dopamine or serotonin in the mouse and rat spinal cord, Brain Res., 475 (1988) 91-102.

11 Yoshida, M., Sakai, M., Kani, K., Nagatsu, I. and Tanaka, M., The dopaminergic innervation as observed by immunohisto- chemistry using anti-dopamine serum in the rat cerebral cortex, Experientia, 44 (1988) 700-702.