Histochemically demonstrable catecholamines in the sympathetic nervous system of trisomic 16 and normal fetal mice

Mechanisms of Ageing and Development, 57 (1991) 101--110

101

ElsevierScientificPublishers Ireland Ltd.

H I S T O C H E M I C A L L Y D E M O N S T R A B L E C A T E C H O L A M I N E S IN T H E S Y M P A T H E T I C NERVOUS SYSTEM OF TRISOMIC 16 AND NORMAL F E T A L MICE

JARI K O I S T I N A H O a'b, A N T T I STANLEY I. R A P O P O R T a

H E R V O N E N b,

HEINZ

WINKING c and

*Laboratory of Neurosciences, National Institute on Aging, NIH, Bethesda, MD 20892 (U.S.A.), nDepartment of Public Health, Section of Gerontology, University of Tampere, P.O. Box. 607, 33101Tampere (Finland) and clnstitutefor Biology, Medical University of L~beck, L~tbeck(F.R. G.)

(Received 11 January 1989) SUMMARY The formaldehyde-induced fluorescence (FIF) and micro-spectrofluorometric techniques were used to study catecholamines in the sympathetic nervous system of normal and trisomy 16 fetal mice with a gestation age of 15 days, an animal model for human trisomy 21 (Down's syndrome). FIF intensity in the stellate sympathetic ganglion of trisomic embryos did not differ from that of controls, whereas in the adrenal medulla the FIF intensity was 38°70 less in trisomic than in control embryos. When adrenal medullary cells from embryos with a gestational age of 15 days were maintained in culture for 7m10 days, a difference in FIF intensity between groups was still evident. The rate of noradrenaline uptake in cultured adrenal medullary ceils also was significantly less in trisomic than in control fetal mice. The results suggest that the development of adrenal medulla is slowed in trisomic 16 mice and that uptake o f noradrenaline by trisomic adrenal medullary cells is impaired. K e y words: Sympathetic ganglion; Adrenal medulla; Down's syndrome; Mouse;

Trisomy 16; Catecholamines

INTRODUCTION Down's syndrome (DS), a disorder caused by a trisomy 21 karyotype [1,2], is characterized by mental retardation, hypotension and Alzheimer's disease [I--4]. • Neurochemical studies have indicated abnormalities both in central and peripheral adrenergic markers. Hypothalamic norepinephrine concentration in post-mortem DS brains is reduced [3]. In young DS adults, norepinephrine levels in lumbar cereCorrespondence to: Dr. J. Koistinaho, Laboratory of Neurosciences,National Institute on Aging, NIH, Bcthesda, MD 20892, U.S.A.

Printed and Published in Ireland

102 brospinal fluid are elevated but plasma levels after venipuncture are unchanged [2,4]. Urinary levels of epinephrine are reported to be low in DS [5]. Serum dopamine-beta-hydroxylase, the final enzyme in the synthesis of norepinephrine, is reduced in DS patients [ 1,6--9]. The activity of catecholamine-O-methyl-transferase (COMT), an extraneuronal degradative enzyme, is increased in erythrocytes of DS individuals [10], whereas platelet monoamine oxidase activity is decreased [5]. In addition, beta-receptor sensitivity in fibroblasts and alpha-receptor sensitivity in platelets are increased in patients with DS [11,12]. The mouse with trisomy 16 (Ts 16) is an animal model of DS [13--15], as the distal part of murine chromosome 16 contains genes also localized in the region of human chromosome 21 reported to be responsible for DS [14]. Furthermore, the expression of adrenergic function in the brain of Ts 16 fetal mice is reported to be altered [13]. Tyrosine hydroxylase activity, dopamine-betahydroxylase activity and norepinephrine are all decreased in the Ts 16 fetal brain, whereas DOPA-decarboxylase activity and catechol-O-methyl transferase activity are increased [ 13]. It has been suggested that patients with DS have a defect in the sympathetic nervous response to various forms of stress [1,4,7]. Systolic pressure has been reported to be lower in young DS patients [2,4]. However, no histochemical studies of the sympathetic nervous system in DS patients or in Ts 16 fetal mice have been reported. We therefore thought it of interest to apply a microspectrofluorometric technique to characterize the development of sympathetic ganglia and of the adrenal medulla in normal and Ts 16 fetal mice and to compare also catecholamine contents and noradrenaline uptakes of adrenal medullary cells cultured from these two groups. MATERIALSAND METHODS Embryos were obtained by breeding normal female mice C57BL/6N (NIH stock) with heterozygous double Robertsonian translocation males, Rb(16.17)32Lub/ Rh(11.16)2H, as described previously [15]. Experiments were carried out with fetuses with a gestation age of 15 days, which was determined by considering the day of vaginal plug formation as day zero. Ten to 15% of the embryos showed edema and microphthalmia and were considered to be trisomic for chromosome 16, as has been reported [ 13,15,16]. Normal litter mates were taken as diploic controls. Tissue culture Adrenals of each fetus were treated in separate plastic dishes with IX crystallized trypsin (Biofluids, RockviUe, MD) in Ca-Mg-free Puck's saline solution (Colorado Serum, Denver, CO), for 35 min at 37°C. They were dissociated by four series of 15 passages each through a Pasteur pipette, with l-mm diameter tip. Cells were plated in plastic culture chamber/slides (Lab-Tek, Miles Scientific, Naperville, IL), coated with poly-D-lysine (Sigma Chemical, St. Louis, MO) and were maintained in Eagle's minimum essential medium (MEM) (GIBCO, Grand Island, NY) with 3.7 g/liter

103 NaHCO 3, 6 g/liter glucose, 10eTa (v/v) horse serum (GIBCO) and 5e70 fetal calf serum (GIBCO). Nerve growth factor (Calbiochem, Behring Diagnostics, La Jolla, CA) was included at a final concentration of 30 nM. Cells were incubated at 37°C in humidified 10% CO2-90e70 air v/v. After 24 h, 5fluoro-2'deoxyuridine (15/~g/ml) and uridine (35/~g/ml) were added to inhibit proliferation of glia and fibroblasts. Experiments were carried out after 7--10 days of culture.

Histochemistry Whole trisomic and diploid embryos and adrenal medulla cultures were prepared for fluorescence microscopy according to Hervonen [17]. Cultures were washed fre¢ from culture medium by rinsing twice for 15 s with Dulbecco's salt solution (Quality Biological, Gaithersburg, MD), dried under cool air for 2 min and immersed in liquid nitrogen. Six cultures of control and 6 of trisomic adrenal chromaffin cells were treated with 0.1 mM L-noradrenaline-D-bitartrate (Sigma) for 15 and 30 min. The drug was dissolved in the culture medium before rinsing. Together with whole embryos, the cultures were freeze-dried in vacuo at - 40 °C for 7 days using phosphorus pentoxide in excess as a water trap. After treatment with paraformaldehyde vapour (60 min at 80 °C) the whole embryos were embedded '~ in paraffin under a vacuum and 10-/~m thick sections were cut with a microtome (Spencer, American Optical, Oxford, CA). All specimens were mounted with xylene and covered with a glass coverslip. Fluorescence microscopy A modified MPV 2 microspectrofluorometer (Leitz, F.R.G.) was used. The light source was a stabilized HBO 100 mercury lamp (Osram). The lamp housing conrained 2 mm KG1 and 4 mm BG 38 filters. The specimens were excited with the 405 nm HG peak obtained through the Leitz filter set D (BG 3, KP 425, TK 455 and K 460). The diameter of the circular field for measuring the intensity of formaldehyde induced fluorescence (FIF) was 3/~m. After rapid identification of the tissue under low magnification, FIF intensity was measured starting at a random point from 100 cells of stellate ganglion and adrenal medulla of each sectioned embryo and from 25 to 30 adrenal medullary cells of each separate culture. Quantitation of FIF intensity was performed according to the method of Alho et al. [18]. Data were analyzed statistically by using a two-tailed Student's t-test. RESULTS

Sympathetic nervous system in vivo Distinct ganglia consisting of weakly fluorescent sympathoblasts and strongly fluorescent cells half as large were observed throughout the sympathetic chains of both normal and trisomic embryos. FIF intensity varied slightly from cell to cell

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105 TABLE I D A T A O B T A I N E D F R O M S T E L L A T E G A N G L I O N A N D A D R E N A L M E D U L L A OF TRISOMY 16 F E T A L MICE

Formaldehyde-inducedfluorescence (arbitrary units) Stellate ganglion

Control Trisomy 16

74.8 _+ 8.2 78.6 ± 7.6

Adrenal medulla In vivo (15 days gestation)

In vitro (+ 7--10 days cultured)

194.4 + 20.1 119.5 ± 8.0*

176 _+ 14.2 101.6 _+ 6.2*

Each value represents the mean ± S.D. of six animals. *Significant difference ( P < 0.001) in formaldehyde-induced fluorescence intensity from control value.

within each ganglion (Figs. 1 and 2), but no dramatic difference in intensity between different ganglia was seen. Occasionally, fluorescent varicose fibers surrounding the ganglion cells were distinguished in the embryo (Fig. 3) and fluorescent postganglionic fibers were found along arteria subclavia in both control and trisomic groups. The adrenals displayed medullary islets of brightly fluorescent cells with a yellowgreen color (Fig. 4). FIF intensity of these medullary cells appeared slightly weaker in trisomic embryos than in controls (Figs. 5 and 6). Ventral to the adrenals, paraganglia composed of fluorescent cells similar to those in the adrenal medulla, were observed in both control and trisomic embryos (Fig. 7).

Microfluorometry Measurements of intracellular FIF intensity were carried out on the stellate ganglion and the adrenal medulla. In the ganglion, only sympathoblasts were examined. FIF intensity (arbitrary mean values) of sympathoblasts in stellate ganglion and of adrenal medullary cells are presented in Table I. Mean FIF intensity in the adrenal

Figs.l--3. Fluorescencemicrographs of control sympathetic ganglia (Mag. × 650). Fig. 1. FIF o f sympathoblasts varies slightly from cell to cell within each ganglion. Fig. 2. Sympathetic ganglia are already separated from each other. In the upper ganglion a group o f small, intensively fluorescent cells are seen. Fig. 3. Fluorescent nerve fibers in control stellate ganglion. Fig. 4. A fluorescence micrograph of the control adrenal medulla. (Mag. x 260). Fig. 5. A fluorescence micrograph showing adrenal medullary ceils of the trisomic fetal mouse. (Mag. X 650). Fig. 6. A fluorescence micrograph showing adrenal medullary cells of the control fetal mouse. (Mag. x 650).

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medulla was significantly higher than in the stellate ganglion in both trisomic and control groups (P < 0.001). Further, the mean FIF intensity of adrenal medullary cells was significantly less (119.5 _ 8.0, arbitrary units) in trisomic than in control embryos (194.4 __. 20.1, arbitrary units), whereas ganglion cells did not show a significant difference in FIF.

© Fig. 7. Fluorescence micrograph which shows paraganglion of the control fetal mouse. (Mag. x 1500). Figs. 8 and 9. Fluorescence micrographs showing differentiated adrenal medullary ceils of the trisomlc (Fig. 8) and control (Fig. 9) fetal mouse, maintained in culture for 10 days. (Mag. X 650).

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Fig. I0. The formaldehyde-induced fluorescence intensity of cultured adrenal medullary cells from trisomy 16 and control fetal mice, after 15 and 30 min treatment with 0.1 mM noradrenaline. Each point represents mean ± S.D. of measurements from 25 to 30 adrenal medullary cells of three dishes.

In vitro studies After 7--10 days in culture, only a few adrenal cortical cells containing yellow autofluorescent material and non-fluorescent glial cells were found. FIF revealed catecholamine containing neuroblast-like cells, with bright fluorescent neurites Figs. 8 and 9). As shown in Table I and Fig. 10, the fluorescence intensity of adrenal medullary cells was less from trisomic mice than from control (101.6 - 6.2 in trisomics and 167.1 ± 14.2 in controls, arbitrary units). After 30 min treatment with 0.1 mM noradrenaline, the FIF intensity increased 35~/0 (136.5 ± 15.3) in trisomic cells and 60.6°7o (268.2 ± 41.3) in control cells (Fig. 10). DISCUSSION

The intensity of catecholamine fluorescence in adrenal chromaffin cells of fetal mice with trisomy 16 was reduced significantly below control (P < 0.001). At 15 days of gestation, 90~70 of catecholamines in the adrenal medulla are reported to be noradrenaline, the rest adrenaline and dopamine [19]. Our results agree with the finding by Ozand et al. [13] that expression of catecholaminergic function in brains of trisomy 16 fetal mice is decreased. However, sympathetic ganglia of trisomic embryos did not show less FIF than did control ganglia. The catecholamine fluorescence of mouse sympathetic ganglia decreases from 13 days to 14 days of gestation and remains quite constant from 15 to 17 days of gestation [20]. In the present study, the adrenal medullary cells from 15-day-old mouse embryos were cultured in medium supplemented with NGF. All medullary cells grew neurites

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and contained a considerable amount of catecholamines. Explants from 15-dayold rat embryos rarely grow neurites, with or without NGF in the medium, whereas explants from 17-day-old embryos do spontaneously grow neurites [21]. It is likely that the onset of monoamine synthesis in the sympathetic nervous system starts 1--2 days earlier in the mouse and that the 15th gestation day of the mouse is comparable to the 16th gestation day of the rat [20]. Thus, adrenal medullary cells cultured in the present study may be comparable to those from rat at the 17th day of gestation. As for the in vivo adrenal medulla, cultured adrenal medullary cells from the trisomic adrenal had a significantly lower mean FIF intensity per cell than did cultures from controls. The FIF intensity measurements were, however, performed from whole cells and are therefore not directly comparable to measurements made from 10-/am sections of adrenal medulla in vivo. Cultured rat adrenal glands from 17-dayold embryos show both morphological and biochemical maturation, but the maturational level fails to reach that observed in vivo [21]. In the present work, all adrenal medullary cells in both trisomic and control groups in vitro showed morphological differentiation in the form of neuronal phenotype. This indicates also that more differentiated adrenal medullary cells of trisomy 16 mouse contain less catecholamines than normal adrenal medullary cells in culture. The relation between catecholamine concentration in neurons or adrenal medullary cells and formaldehyde-induced fluorescence is linear [22,23]. Alho et al. [22] have shown that the microspectrofluorometric method is sensitive enough to detect even minute changes induced by reserpine in the catecholamine content of adrenergic cells and that even very high cellular concentrations of catecholamines can be registered. Also, cultured sympathetic neurons incubated with 0.1 mM noraderenaline or dopamine for 30 min clearly show higher FIF intensity compared to controls [17]. Therefore, the method used in the present study should not have sensitivity limitations when applied to catecholamine uptake studies. Due to the small number of trisomy 16 embryos obtained from each dissection, FIF intensity was measured only at two points of incubation time. Nevertheless, we could demonstrate a lower accumulation rate of exogenous noradrenaline into trisomic chromaffin cells than into control cells. Cu/Zn-superoxide dismutase (-SOD), a key enzyme in the metabolism of free oxygen radicals [24], is coded for one mouse chromosome 16 and its activity is 50°7o increased in trisomy 16 fetuses and fetal brains [25]. Noradrenaline and dopamine uptake in PC12 cells overexpressing human Cu/Zn-SOD is decreased by 50--80°/0 [26]. These findings suggest that noradrenaline uptake is impaired in adrenal medullary cells of trisomy 16 fetal mice due to an overdose of Cu/Zn-SOD enzyme. Catecholamines are stored within granules in chromaffin cells [26]. Monoamine oxidase (MAO) is an intraneuronal enzyme responsible for degradation of unstored catecholamines [26]. In the present study no MAO inhibitors were used, so that it remains possible that the difference in catecholamine uptake rate was due to different MAO activities in these two cell populations. However, MAO activities were

109 similar in trisomic fetal brain and controls [13]. Furthermore, there is evidence that impaired catecholamine uptake in cells overexpressing C u / Z n - S O D is due to an altered transport mechanism o f the granules [26]. Therefore, it is unlikely that the difference in noradrenaline uptake rate between trisomy 16 and control adrenal medullary cells is affected by changes in catecholamine degradation. In the present study, a reduction in catecholamine fluorescence intensity was found in adrenal medullary cells o f trisomy 16 fetal mice at 15 days of gestation. A similar difference from controls was observed in more differentiated medullary cells in culture, suggesting that the development o f the peripheral adrenergic nervous system is slowed in trisomy 16. Noradrenaline uptake was impaired also in trisomic adrenal medullary cells, which might be linked to an overdose of C u / Z n - S O D in trisomy 16 fetal tissue. In human trisomy 21, the C u / Z n - S O D gene also is overexpressed and the enzyme activies are reported to be elevated by 500/o in trisomy 21 fibroblasts [25]. Systolic blood pressure and plasma dopamine-beta-hydroxylase, an enzyme which converts dopamine to noradrenaline, are significantly reduced in young Down's syndrome patients [4], indicating a decreased sympathetic function in trisomy 21. The present results suggest that diminished catecholamine content and impaired catecholamine uptake in adrenal medullary cells contribute to abnormal sympathetic function in human trisomy 21. REFERENCES 1 L.S. Freedman, M. Goldstein and M, Coleman, Serum dopamine-beta-hydroxylaseactivity in Down's syndrome:A familial study. Res. Commun Chem. Pathol. Pharmacoi., 8 (1974) 543--549. 2 M.B. Shapiro, A.D. Kay, C. May, A.K. Ryker, J.V. Huxby, S. Kanfman, S. Milstien and S.I. Rapoport, Cerebrospinal fluid monoaminesin Down's syndromeadults at different ages. J. Mental Def. Res., 31 (1987)259--269. 3 C.M. Yates, J. Simpson, A. Gordon, A.F.J. Maloney, Y. Allison, I.M. Ritchie and A. Urquhart, Catecbolamines and cholinergic enzymes in presenile and senile Alzheimer-type dementia and Down's syndrome.Brain Res., 280 0983) 119--126. 4 C.R. Lake, M.G. Ziegler, M. Coleman and I.J. Kopin, Evaluation of the sympathetic nervous system in trisomy-21(Down'ssyndrome).J. Psychiat. Res., 15 (1979) 1--6. 5 D.K. Keele, C. Richards, J. Brown and J. Marchal, Catecholamine metabolism in Down's syndrome. Am. J. MentaI Defic., 74 (1969) 125--129. 6 L. Wetterberg, K.H. Gustavson, M. Backstrom, S.B. Ross and O. Froden, Low dopamine-betahydroxylaseactivityin Down's syndrome. Clin. Genet., 3 (1972) 152--157. 7 L. Wettenberg, H. Aberg, S.B. Ross and O. Froden, Plasma dopamine-beta-hydroxylaseactivityin hypertension and various neuropsychiatric disorders. Scand. J. Clin. Lab. Invest., 30 (1972) 283-289. 8 M.Coleman,M. Campbell, L.S. Freedman, M. Roffman, R.P. Ebstein and M. Goldstein, Serum dopamine-beta-hydroxylaselevelsin Down's syndrome. Clin. Genet., 5 (1974) 312--315. 9 M. Goldstein, L.S. Freedman, R.P. Ebstein and D.H. Park, Studies on dopamine-beta-hydroxylase in mental disorders. J. Psychiat. Res., 11 (1974)205--210. 10 K.H. Gustavson, L. Wettenberg, M. Backstrom and S.B. Ross, Catechol-O-methyltransferase activityin Down's syndrome. Clin. Genet., 4 (1973)279--280. 11 J.R. Sheppards, W. Schumacher, J.G. White, K.H. Jakobs and G. Schultz, The alpha adrenergic response of Down's syndromeplatelets. J. Pharmacol. Exp. Ther., 225 (1983) 584--588.

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