Transient appearance of tyrosine hydroxylase-immunoreactive non-catecholaminergic neurons in the medial geniculate nucleus of postnatal mice

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ELSEVIER

Neuroscience Letters 211 (1996) 183-186

NHROSCI-BCE IEI'TEHS

Transient appearance of tyrosine hydroxylase-immunoreactive non-catecholaminergic neurons in the medial geniculate nucleus of postnatal mice Ikuko Nagatsu a,*, Terumi Takeuchi a, Masao Sakai a, Nobuyuki Karasawa a, Yoko Yamawakia, Ryohachi Arai ~, Toshiharu Nagatsu b aDepartment of Anatomy, School of Medicine, Fujita Health University, Toyoake, Aichi 470-11, Japan blnstitute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-11, Japan

Received 25 April 1996; revised version received 24 May 1996;accepted 24 May 1996

Abstract

Tyrosine hydroxylase-immunoreactive (TH-ir) cells were found to appear transiently in the medial geniculate nuclear region of mice at postnatal day 7 (P7) by use of an avidin-biotin peroxidase complex (ABC) method for the first time. The numbers of TH-ir cells reached maximum between P14 and P21 and decreased until P29. These cells were GTP cyclohydrolase I-negative, aromatic L-amino acid decarboxylase-negative, and dopamine-negative. Thus, they do not belong to the catecholaminergic neuron system, because they lack dopamine production. The results suggest that TH in the cells in the medial geniculate nuclear region of mice has some new functions besides catecholamine biosynthesis. Keywords: Aromatic L-amino acid decarboxylase; Brain; GTP cyclohydrolase I; Immunocytochemistry; Medial geniculate nucleus; Mouse; NADPH-diaphorase; Tyrosine hydroxylase

Our immunocytochemical studies previously revealed that transient tyrosine hydroxylase-immunoreactive (THir) neurons were not stained with antiserum against aromatic L-amino acid decarboxylase (AADC), dopamine/3hydroxylase, phenylethanolamine-N-methyl transferase, dopamine (DA), or serotonin (5HT) in the anterior olfactory nucleus [9], striatum [5], cerebellum [3], and spinal trigeminal nucleus [16] of postnatal mice. In the present study, we describe another novel TH-only-ir neurons in the medial geniculate nucleus (MG). The aim of this paper is to show the existence of some TH-positive noncatecholaminergic neurons in the mouse brain, suggesting that TH has some new functions besides catecholamine biosynthesis. TH and GTP cyclohydrolase I (GCH), AADC, DA, or 5HT were detected on the serial sections of the brain by use of an avidin-biotin peroxidase complex (ABC) method. Our rabbit polyclonal antibody directed against * Corresponding author. Tel.: +81 562 932430; fax: +81 562 932649 (home fax: +81 52 8771785).

GCH-glutaraldehyde-hemocianin conjugate [8], and against TH [10], A A D C [11], DA [17], or 5HT [11], were used as primary antibodies. The specificity of the anticonjugated GCH antibody [8], anti-TH-antibody [10], anti-AADC antibody [ 11 ], anti-DA antibody [ 17], or anti5HT antibody I l l ] were previously described. Twentyfour DDY and C57BL/6J male mice at various postnatal ages were anesthetized with sodium pentobarbital (50 mg/ kg, i.p.) and perfused intracardially with saline followed by 4% paraformaldehyde, or 5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4, 4°C) for 6-10 min. Fixed tissues were dissected out and immersed overnight in the same fixative at 4°C. After the tissues had been soaked in 10-30% sucrose in the phosphate buffer for 2days, cryostat sections (40/~m thick) were cut in a frontal plane of the whole brain, and collected in 0.1 M phosphate buffer (pH 7.4) for immunocytochemistry as described below. Floating sections were incubated with: (i) 10% normal swine serum in 0.01 M phosphatebuffered saline (PBS) for 1 h at room temperature; (ii) primary antibodies: rabbit anti-conjugated GCH antibody

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L Nagatsu et al./Neuroscience Letters 211 (1996) 183-186

Fig. 1. At PI4, a coronal section through the mesencephalon shows TH-ir neurons in the MG (C). The soma, dendrites, axons and terminal fields are stained, but not the nucleus. Some cells show NADPH-d reaction (B), but are negative for GCH (A). At adult stage (P56), no TH-ir cells can be seen at the MG (F). In the MG, a population of large cells are reactive for NADPH-d (E), but negative for GCH (D), TH (F), and 5HT. In the MG, TH-ir terminals (F) are much denser than that of 5HT. Bar, 100#m. (diluted 1:8000 in Tris, Triton X-100 with 0.1% NAN3), or rabbit TH, A A D C , DA, or 5HT antibody (each diluted 1 : 3 0 0 0 0 in Tris, Triton X-100 with 0.1% NAN3), for 2 7 days at 4°C, and washed in PBS (3 x 5 min); (iii) biotinylated goat anti-rabbit IgG (Vector, 1:1000) for 1 h, washed again in PBS (2 x 5 min), and incubated with A B C (Vector, 1:1000) for 1 h at room temperature. After final washes in PBS (2 x 5 min), the antigen was visualized by reaction with 3,3"-diaminobenzidine-tetrahydrochloride as chromogen ( 2 m g / 1 0 m l of 0 . 0 5 M Tris buffer, pH 7.6) and hydrogen peroxide (0.003%). The sections were then rinsed in PBS and distilled water followed by dehydration, clearing in xylene, and mounting. To demonstrate the nicotinamide adenine dinucleotide phosphate-diaphorase ( N A D P H - d ) reaction, we incubated

the 4% paraformaldehyde-fixed free-floating sections in 0.1 M phosphate buffer (pH 7.4), containing 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium chloride (NBT; Chemapol, Prague), and 1.0 mg/ml f l - N A D P H (Naa-salt; Serva, Heidelberg) at 37°C for 3 0 - 6 0 min [18]. The enzyme activity can be retained after paraformaldehyde fixation. In the ventral and dorsal divisions of the MG, mediumsized and small neural somata with tufted dendrites were TH-negative (Fig. 1C,F), but NADPH-d-positive (Fig. IB,E) at postnatal day 14 (P14) and at adult stage, respectively. The medial division contained large cells corresponding to the magnocellular neurons (Fig. 1C, THnegative), medium-sized stellate cells (Fig. 1C, THpositive, at P14), smaller stellate cells and tufted bushy

I. Na gatsu et al. / Neuroscience Letters 211 (1996) 183-186

Fig. 2. Schematic drawings of TH immunostainingperikarya (circles) from rostral (left) to caudal (right) parts of the lateral geniculatenucleus and MG regions on coronal sections from mouse brain at PI4. Each circle represents three cell bodies. BIC, nucleus (nu) brachium inferior colliculus; DLG, dorsal lateral geniculate nu; IGL, intergeniculateleaf; PBG, parabigeminal nu; SN, substantia nigra; VLG, ventral lateral geniculate nu; ZI, zona incerta. cells. TH-ir cells in the MG of mice transiently appeared at P7. The numbers of TH-ir cells reached maximum between P14 (Fig. 2) and P21, decreased until P29, and mostly disappeared by P56 (Table I). These cells were GCH-negative (Fig. 1A,D), AADC-negative, DA-negative, and 5HT-negative. TH-ir cells were found in the locus coeruleus as well as in the pars compacta of the substantia nigra and the ventral tegmental area [8]. It may be assumed that MG receives TH-ir fibers arising from the mesencephalic dopaminergic neurons, corresponding to the A8, A9, and A10 cell groups of the rat (i.e. retrorubral field, substantia nigra, and ventral tegmental area, respectively) and the noradrenergic perikarya in the neighboring locus coeruleus [4]. Our present results are consistent with the studies of Kitahama et al. [4] on rats. The rat MG complex was subdivided into three major parts: the ventral, dorsal, and medial divisions [2,20]. The medial division of the rat MG projected to the caudate-putamen and nuclei of the amygdala [6,15], where TH-ir cells transiently appeared at early postnatal stage (caudate-putamen in Komori et al. [5]; amygdala in Mezey [7] and Verney et al. [19]). In the present study, a new cell group composed of a small number of neurons immunoreactive to TH was for the first time demonstrated from P7 in the MG. Several Table 1 Relative frequency of TH-ir cells in the MG regions over the time course of development Postnatally transientTH cells

TH(+)/GCH(-)

PI

P7

PI4

P21

P29

P56

-

+/-

++

++

+/-

-

The signs represent the abundance of TH-ir-positive/GCH-negative cells in the MG regions: ++++, +++, areas where most neurons showed strong reactivity; ++, some cells showed strong reactivity;+, a few cells displayed strong reactivity; +/-, rare cell reactivity of any type; -, no reactivity in any cells.

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studies [7,19] reported transient expression of TH in a subpopulation of neurons, which were of small to medium size and often displayed a typical bipolar configuration, in the bed nucleus of stria terminalis of preadolescent rats. By the use of a combination of experimental tracer techniques and immunocytochemical methods, Beltramino et al. [1] demonstrated that these TH-ir neurons received a significant number of amygdaloid afferents, which established mostly symmetric synaptic contacts on the cell bodies and sparsely spined dendritic shafts of the TH neurons. These neurons also received a small number of TH-positive terminals of unspecified origin. Many extensive immunocytochemical studies by us [12-14] revealed that most TH-ir neurons lacked additional catecholamine- or DA-synthesizing enzymes. TH may have a new function different from catecholamine biosynthesis in these TH-ir neurons. This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, Japan (to I.N.), Grantsin-Aid for Specially Promoted Research from the Ministry of Education, Science and Culture of Japan (to T.N.), and by a grant-in-aid from Fujita Health University, Japan. The authors are grateful to Dr. L.D. Frye for helping us to improve the readability of the manuscript. [1] Beltramino,C.A., Forbes, M.S., Swanson, D.J., Alheid, G.F. and Heimer, L., Amygdaloidinput to transientlytyrosine hydroxylase immunoreactiveneurons in the bed nucleus of the stria terminalis of the rat, Brain Res., 706 (1996) 37-46. [2] Clerici, W.J. and Coleman, J.R., Anatomyof the rat medial geniculate body, I: cytoarchitecture, myeloarchitecture, and neocortical connectivity,J. Comp. Neurol., 297 (1990) 14-31. [3] Fujii,T., Sakai, M. and Nagatsu, 1., Immunohistochemicaldemonstration of expression of tyrosine hydroxylase in cerebellar Purkinje cells of the human and mouse, Neurosci. Lett., 165 (1994) 161-163. [4] Kitahama,K., Nagatsu, I. and Pearson, J., Catecholaminesystems in mammalianmidbrain and hindbrain: theme and variations. In W.J.A.J. Smeets and A. Reiner, (Eds.), Phylogeny and Development of CatecholamineSystems in the CNS of Vertebrates, Cambridge UniversityPress, Cambridge, 1994, pp. 183-205. [5] Komori,K., Sakai, M., Karasawa, N., Yamada, K. and Nagatsu, 1., Evidence for transient expression of tyrosine hydroxylase immunoreactivityin the mouse striatum and the effect of colchieine, Acta Histochem. Cytochem., 24 (1991) 223-231. [6] LeDoux, J.E., Ruggiero, D.A. and Reis, D.L, Projections to the subcortical forebrain from anatomically defined regions of the medial geniculate body in the rat, J. Comp. Neurol., 242 (1985) 182-213. [7l Mezey, E., PhenylethanolamineN-methyltransferase-containing neurons in the limbic system of the young rat, Proc. Natl. Acad. Sci. USA, 86 (1989) 347-351. [8] Nagatsu, I., Ichinose, H., Sakai, M., Titani, K., Suzuki, M. and Nagatsu, T., Immunocytochemicallocalization of GTP cyclohydrola.~e I in the brain, adrenal gland, and liver of mice, J. Neural Transm. (Gen Sect.), 102 (1995) 175-188. [9] Nagatsu,I., Komori, K., Takeuchi, T., Sakai, M., Yamada, K. and Karasawa, N., Transient tyrosine hydroxylase-immunoreactive neurons in the region of the anterior olfactory nucleus of pre- and

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