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Different developmental profiles of the expression of preprosomatostatin and preprotachykinin-A mRNAs in rat SCN neurons
Developmental Brain Research 127 (2001) 81–86 www.elsevier.com / locate / bres
Short communication
Different developmental profiles of the expression of preprosomatostatin and preprotachykinin-A mRNAs in rat SCN neurons Toru Nakamura a,b,c , Yasufumi Shigeyoshi d , Yoshiro Maebayashi a,e , Shun Yamaguchi a , Kazuhiro Yagita a , Hitoshi Okamura a , * a
Department of Anatomy and Brain Science, Kobe University School of Medicine, 7 -5 -1 Kusunoki-cho, Chuo-ku, Kobe 650 -0017, Japan b Department of Anatomy, Kyoto Prefectural University of Medicine, Kyoto 602 -8566, Japan c Department of Dentistry, Kyoto Prefectural University of Medicine, Kyoto 602 -8566, Japan d Department of Anatomy, Kinki University School of Medicine, Osaka-Sayama, Osaka 589 -8511, Japan e Department of Psychiatry, Kyoto Prefectural University of Medicine, Kyoto 602 -8566, Japan Accepted 19 December 2000
Abstract The suprachiasmatic nucleus (SCN), a central circadian oscillator of mammals, contains various peptides arranged in the compartment specific manner. In the present study, we examined a distinct population of neurons in the central part of the SCN. In situ hybridization histochemistry has demonstrated that these neurons coexpressed both preprosomatostatin (PPSS) and preprotachykinin A (PPT-A) mRNAs, but the developmental expression profiles were different among two. PPSS mRNA first appeared in the SCN at postnatal day 1(P1). The intensity and number of PPSS mRNA signals increased and peaked at P7–P14 and gradually decreased as to adult age (P56). However, PPT-A mRNA-positive appeared late at P7, and gradually increased up to P56. These findings suggest that neurons encoding both the PPSS and PPTA genes first express PPSS and then express PPT-A at a later stage of maturation. 2001 Elsevier Science B.V. All rights reserved. Keywords: Preprosomatostatin; Preprotachykinin A; Coexistence; Suprachiasmatic nucleus; Development; In situ hybridization
In mammals, the suprachiasmatic nucleus (SCN) plays a dominant role of circadian oscillation [12]. One of the characteristic morphological features of this nucleus is that many types of peptidergic neurons are arranged in a compartment specific manner [6]. In addition to the two major peptidergic neurons located in the dorsomedial regions (vasopressin) and the ventrolateral regions (vasoactive intestinal peptide), there is a group of neurons which express somatostatin [6,11] and / or substance P (SP) [14] in the central part of the SCN. Although somatostatin and substance P are produced in a small number of neurons in the central part of the SCN, both peptides have been reported to have significant effects on the circadian oscillation of the SCN. They cause a phase shift of the light pulse type in slice preparations of *Corresponding author. Tel.: 181-78-382-5340; fax: 181-78-3825341. E-mail address: [email protected] (H. Okamura).
the SCN [10,19]. Furthermore, somatostatin and preprosomatostain (PPSS) mRNA in the SCN exhibit circadian rhythms in both LD and DD conditions [16,21]. These findings suggest that these two peptides play important roles in the SCN. To understand the interaction of these peptides more deeply, we here investigated the possible coexistence of these peptides in the SCN, and then the developmental expression of these genes using an in situ hybridization technique. They show the divergence in development, although they are co-stored at the cell level. Pregnant female Wistar rats (Japan Animal Company, Osaka, Japan) were housed in individual cages under standard light / dark (LD) conditions (light on at 7:00 A.M., light off at 7:00 P.M.). The day of mating and the birth were designated as the embryonic day 1 (E1) and the postnatal day 1 (P1), respectively. Animals were killed for in situ hybridization between 4 to 6 h after lights on. For the developmental in situ hybridization study using PPSS and PPT-A probes, E18 animals were used (number of
0165-3806 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 01 )00102-X
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T. Nakamura et al. / Developmental Brain Research 127 (2001) 81 – 86
animals used for each probe: n54), as well as E20 (n54), P1 (n54), P3 (n54), P7 (n54), P14 (n53), P28 (n54) and P56 (adult; n53). For the collocalization study, we used the animals (n55) at P28 and P56. All animals were deeply anesthetized with Nembutal (70 mg / kg) ether before being killed. In the present study, we used a PPSS cRNA probe from PPSS cDNA (a gift from Professor R.S. Goodman, Portland), and a b-PPT-A cRNA probe made from b-PPT-A cDNA (a gift from Professor J. E. Krause, St. Louis). A previous study demonstrated that b-PPT-A probe hybridized with all three alternatively spliced forms of PPT-A mRNA (a-PPT-A, b-PPT-A and g-PPT-A) [13]. All these splicing variants contain sequences encoding SP. Spe-
cificity of these hybridization probes were confirmed in previous reports [20]. The distribution of PPSS and PPT-A mRNA positive structures in the brain was consistent with previous reports [5,8,14,20] For the developmental study, in situ hybridization histochemistry was performed by digoxigenin-labelled cRNA probes for b-PPT-A and g-PPT-A using digoxigenin-UTP (Boehringer Mannheim, Germany) according the previously detailed method [2,20,22]. We used the free-floating section of 40 mm thick made by a cryostat. The reactions were processed according to the protocol of the nucleic acid detection kit (Boehringer-Mannheim, Germany), and were visualized blue by nitroblue tetrazolium [20]. We counted the number of signal-positive
Fig. 1. Double-labeling in situ hybridization of digoxigenin-labeled PPT-A and isotope-labeled PPSS cRNA probes in the anterior hypothalamus at P56. PPT-A mRNAs-containing neurons were colored blue by nitroblue tetraformazan, while PPSS mRNA signal was shown as isotope-hitted silver grains. (a), (b) and (c) are the high magnification photomicrographs of indicated area of (A); (a) suprachiasmatic nucleus (SCN), (b) the periventricular area of the anterior hypothalamus, and (c) the lateral hypothalamic area. oc, optic chiasma; v, third ventricle. Bars in (A)5200 mm; (a), (b), (c) 520 mm.
T. Nakamura et al. / Developmental Brain Research 127 (2001) 81 – 86
neurons for 10 SCN sections in each animal under a bright-field microscope. We analyzed the data for each probe at each postnatal day. Co-localization of two types of mRNAs was detected by double-labelling in situ hybridization using 35 S-CTP (New England Nuclear) labelled-PPSS probe and digoxigeninlabelled b-PPT-A probe. We first performed digoxigeninprobe hybridization as described above, except that we used 20 mm thick sections. After finishing nitroblue tetrazolium reaction, slides were dipped into nuclear track emulsion (Illford K5; dilution 1:1 with distilled water) and
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exposed for 2 weeks at 48C. Following development of the tissue autoradiograms, the sections were analyzed under a bright-field microscope. First we examined the collocalization of PPSS mRNA and PPT-A mRNA in the hypothalamus. In the SCN, PPT-A mRNA expressing neurons were virtually all PPSS mRNA positive: PPT-A -positive blue stained cells (number of cells, 128 at P56, 96 at P28) all expressed dense accumulation of PPSS positive silver grains (Fig. 1, 1a). Periventricular isotope-labeled grain-positive PPSS neurons did not show blue PPT-A staining (Fig. 1b), and
Fig. 2. Bright-field photographs showing the development of PPSS mRNA-containing neurons in the SCN. Boundaries of SCN are encircled with dotted lines. Note that the cell number and the signal intensity of each cell increased, peaked at P7–P14 (C, D), then decreased gradually (E, F). Scale bars5100 mm.
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T. Nakamura et al. / Developmental Brain Research 127 (2001) 81 – 86
blue-stained PPT-A positive cells in the lateral hypothalamic area did not show the dense accumulation of signals (Fig. 1c). Then we examined the developmental features of the expression of these genes. The very weak PPSS mRNA signals existed at E20 at the central part of the SCN (data not shown). At P1, the intensity of the PPSS mRNA signals increased slightly, but still showed faint signals in the SCN (Fig. 2A). At P4, the number of positive neurons and the staining intensity of these neurons were increased.
At P7–P14, the intensity of the signal was strong, and the number of positive cells reached the peak (Fig. 2C, 2D). At P28 the signal intensity decreased slightly but still showed intense expression (Fig. 2E). At P56 the number of positive cells and the signal intensity apparently decreased compared with the peak level of P7–14 (Fig. 2F; see also Fig. 4). In contrast to PPSS, PPT-A mRNA-containing neurons appeared in the later stages of the developmental SCN (Fig. 3A–C). At P7 the intensity of PPT-A mRNA-positive
Fig. 3. Bright-field photographs showing the development of PPT-A mRNA-containing neurons in the rat SCN. Boundaries of SCN are encircled with dotted lines. Note that the signal first appeared at P7 (C), and then the intensity and number of cells increased gradually to the adult level (D–F). Scale bars5100 mm.
T. Nakamura et al. / Developmental Brain Research 127 (2001) 81 – 86
cells were faint, then gradually increased up to P56 (Fig. 3C–F). At P56, the signal intensity peaked (Fig. 3F). To clarify the abundance of the positive cells more quantitatively, we counted the number of PPSS and PPT-A mRNA-containing neurons in the SCN at days P1, P4, P7, P14, P28 and P56 (Fig. 4). In SCN, the total number of PPSS mRNA positive-cells increased from P1 (33.4610.2) (mean6S.E.M.) to P7 (116.2611.5), followed by a gradual reduction to P56 (63.863.3) (Fig. 4). However, at P7, when PPT-A mRNA signals were first detected in the SCN, the numbers of PPT-A positive neurons were 21.263.1. After that time the numbers of PPT-A mRNA positive-cells increased consistently up to P56 (55.268.2), which was nearly equal to the numbers of PPSS mRNA positive-cells at P56 (Fig. 4). Thus, PPSS-positive neurons demonstrated transient increase, while PPT-A positive neurons in the SCN showed comparatively late appearance and a consistent increase in number. In the present study, we demonstrated that the timecourse of the gene expression of PPSS and PPT-A coexpressed in the central SCN neurons. In the rat SCN, the neurogenesis is completed by E18 [1], and immediately after that, the SCN begins to oscillate [17] being entrained by the maternal circadian clock [24]. The period of the steep intensification of the PPSS mRNA signal noted between P1 and P14 in the SCN is identical to the most active stage of intrinsic synaptogenesis in the SCN [15], whereas the strong afferent projections from external components, such as the retina, raphe nucleus, and the lateral geniculate nucleus are established at later stages [9]. It is already demonstrated that somatostatin plays a role as a neurotrophic factors [18,23] such as neurite outgrowth [4]. Since somatostatin neurons in the SCN are intrinsic neurons [7], it is probable that somatostain may have a trophic role in the development of intrinsic synaptogenesis in the SCN through somatostain receptors in the SCN [3]. In contrast to PPSS mRNA, coexisting PPT-A mRNA signals appeared late at P7, and the cell number and signal
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intensity of PPT-A mRNA gradually increased as to adulthood. The different time courses of the development of two coexisting peptides suggest that their transcriptions in the developmental SCN neurons are regulated differently. There are several maturation steps in these neurons; first, the beginning of the PPSS-phenotype; second, the transient increment of the PPSS-phenotype and the beginning of the PPT-A phenotype; and third, the PPSS / PPT-A maturation. Approximately half of the PPSS-expressing neurons during early postnatal days continuously express PPSS mRNA, and eventually express PPT-A mRNA in adulthood. It is interesting that previous studies demonstrated that both of these peptides cause phase shifts of the light-pulse type when it is applied to SCN cells [10,19]. Somatostatin application increased 2-deoxyglucose (2-DG) uptake of the rat SCN at CT18 but not at CT6 in vitro slice preparations. Moreover, somatostatin induced a phase-shift in the circadian rhythm of the firing rate of SCN neurons in a phase-dependent manner, and this effect was dose-dependently antagonized by the specific antagonist, cyclosomatostatin [10]. Similarly, SP induced phase-dependent phase shifts, and increased the metabolic activity of SCN neurons at subjective night, but not at subjective day, and the effect was completely antagonized by spantide, an antagonist of SP [19]. These findings suggested that both somatostatin and SP-induced phase response curves were similar to the light-pulse-induced PRC of locomotor rhythm. In conclusion, we demonstrated the developmental differences of PPSS and PPT-A mRNA expression in the SCN. The stage-specific change in the gene expression of PPSS and PPT-A in the SCN suggests the different roles of these peptides in the development of the phase-resetting in the circadian pacemaker.
Acknowledgements This work was supported in part by grants from the Special Coordination Funds of the Science and Technology Agency of Japan, Grants-in-Aid for the Scientific Research and for the Scientific Research on Priority Areas of the Ministry of Education, Science, Sports and Culture of Japan, Uehara Memorial Foundation, and SRF.
References
Fig. 4. Statistics on the number of PPSS mRNA and PPT-A mRNApositive cells at various postnatal days in the SCN. Mean6S.E.M. (n54 at each postnatal day, except P14 and P56 being n53).
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[15] R.Y. Moore, M.E. Bernstein, Synaptogenesis in the rat suprachiasmatic nucleus demonstrated by electron microscopy and synapsin 1 immunoreactivity, J. Neurosci. 9 (1989) 2151–2162. [16] T. Nishiwaki, H. Okamura, K. kanemasa, T. Inatomi, Y. Ibata, C. Fukuhara, S.I.T. Inouye, Differences of somatostatin mRNA in the rat suprachiasmatic nucleus under light dark and constant dark conditions: an analysis by in situ hybridization, Neurosci. Lett. 197 (1995) 231–234. [17] S.M. Reppert, W.J. Schwartz, The suprachiasmatic nuclei of the fetal rat: characterization of a functional circadian clock using 14 Clabeled deoxyglucose, J. Neurosci. 4 (1984) 1677–1682. [18] J.P. Schwartz, Z. Ji, J. Epelbaum, Somatostatin as a neurotrophic factor. Which receptor / second messenger transduction system is involved?, Perspect. Dev. Neurobiol. 5 (1998) 427–435. [19] S. Shibata, A. Tsuneyoshi, T. Hamada, K. Tominaga, S. Watanabe, Effect of substance P on circadian rhythms of firing activity and 2-deoxyglucose uptake in the rat suprachiasmatic nucleus in vitro, Brain Res. 597 (1992) 257–263. [20] Y. Shigeyoshi, Y. Maebayashi, H. Okamura, Co-localization of preprosomatostatin mRNA and preprotachykinin A mRNA in neurons of the rat suprachiasmatic nucleus, Mol. Brain Res. 48 (1997) 159–163. [21] J. Takeuchi, H. Nagasaki, K. Shinohara, S.-I.T. Inouye, A circadian rhythm of somatostatin messenger RNA levels, but not of vasoactive intestinal polypeptide / peptide histidine isoleucine messenger RNA levels in rat suprachiasmatic nucleus, Mol. Cell. Neurosci. 3 (1992) 29–35. [22] M. Tanaka, H. Okamura, T. Matsuda, Y. Shigeyoshi, Y. Hisa, K. Chihara, Y. Ibata, Somatostatin neurons share a unique peptidergic neuronal group in the rat suprachiasmatic nucleus: A double-labeling in situ hybridization study, Neurosci. Lett. 215 (1996) 119–122. [23] T. Taniwaki, J.P. Schwartz, Somatostatin enhances neurofilament expression and neurite outgrowth in cultured rat cerebellar granule cells, Dev. Brain Res. 88 (1995) 109–116. [24] D.R. Weaver, S.M. Reppert, Direct in utero perception of light by the mammalian fetus, Brain Res. Dev. Brain Res. 47 (1989) 151– 155.
Short communication
Different developmental profiles of the expression of preprosomatostatin and preprotachykinin-A mRNAs in rat SCN neurons Toru Nakamura a,b,c , Yasufumi Shigeyoshi d , Yoshiro Maebayashi a,e , Shun Yamaguchi a , Kazuhiro Yagita a , Hitoshi Okamura a , * a
Department of Anatomy and Brain Science, Kobe University School of Medicine, 7 -5 -1 Kusunoki-cho, Chuo-ku, Kobe 650 -0017, Japan b Department of Anatomy, Kyoto Prefectural University of Medicine, Kyoto 602 -8566, Japan c Department of Dentistry, Kyoto Prefectural University of Medicine, Kyoto 602 -8566, Japan d Department of Anatomy, Kinki University School of Medicine, Osaka-Sayama, Osaka 589 -8511, Japan e Department of Psychiatry, Kyoto Prefectural University of Medicine, Kyoto 602 -8566, Japan Accepted 19 December 2000
Abstract The suprachiasmatic nucleus (SCN), a central circadian oscillator of mammals, contains various peptides arranged in the compartment specific manner. In the present study, we examined a distinct population of neurons in the central part of the SCN. In situ hybridization histochemistry has demonstrated that these neurons coexpressed both preprosomatostatin (PPSS) and preprotachykinin A (PPT-A) mRNAs, but the developmental expression profiles were different among two. PPSS mRNA first appeared in the SCN at postnatal day 1(P1). The intensity and number of PPSS mRNA signals increased and peaked at P7–P14 and gradually decreased as to adult age (P56). However, PPT-A mRNA-positive appeared late at P7, and gradually increased up to P56. These findings suggest that neurons encoding both the PPSS and PPTA genes first express PPSS and then express PPT-A at a later stage of maturation. 2001 Elsevier Science B.V. All rights reserved. Keywords: Preprosomatostatin; Preprotachykinin A; Coexistence; Suprachiasmatic nucleus; Development; In situ hybridization
In mammals, the suprachiasmatic nucleus (SCN) plays a dominant role of circadian oscillation [12]. One of the characteristic morphological features of this nucleus is that many types of peptidergic neurons are arranged in a compartment specific manner [6]. In addition to the two major peptidergic neurons located in the dorsomedial regions (vasopressin) and the ventrolateral regions (vasoactive intestinal peptide), there is a group of neurons which express somatostatin [6,11] and / or substance P (SP) [14] in the central part of the SCN. Although somatostatin and substance P are produced in a small number of neurons in the central part of the SCN, both peptides have been reported to have significant effects on the circadian oscillation of the SCN. They cause a phase shift of the light pulse type in slice preparations of *Corresponding author. Tel.: 181-78-382-5340; fax: 181-78-3825341. E-mail address: [email protected] (H. Okamura).
the SCN [10,19]. Furthermore, somatostatin and preprosomatostain (PPSS) mRNA in the SCN exhibit circadian rhythms in both LD and DD conditions [16,21]. These findings suggest that these two peptides play important roles in the SCN. To understand the interaction of these peptides more deeply, we here investigated the possible coexistence of these peptides in the SCN, and then the developmental expression of these genes using an in situ hybridization technique. They show the divergence in development, although they are co-stored at the cell level. Pregnant female Wistar rats (Japan Animal Company, Osaka, Japan) were housed in individual cages under standard light / dark (LD) conditions (light on at 7:00 A.M., light off at 7:00 P.M.). The day of mating and the birth were designated as the embryonic day 1 (E1) and the postnatal day 1 (P1), respectively. Animals were killed for in situ hybridization between 4 to 6 h after lights on. For the developmental in situ hybridization study using PPSS and PPT-A probes, E18 animals were used (number of
0165-3806 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 01 )00102-X
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T. Nakamura et al. / Developmental Brain Research 127 (2001) 81 – 86
animals used for each probe: n54), as well as E20 (n54), P1 (n54), P3 (n54), P7 (n54), P14 (n53), P28 (n54) and P56 (adult; n53). For the collocalization study, we used the animals (n55) at P28 and P56. All animals were deeply anesthetized with Nembutal (70 mg / kg) ether before being killed. In the present study, we used a PPSS cRNA probe from PPSS cDNA (a gift from Professor R.S. Goodman, Portland), and a b-PPT-A cRNA probe made from b-PPT-A cDNA (a gift from Professor J. E. Krause, St. Louis). A previous study demonstrated that b-PPT-A probe hybridized with all three alternatively spliced forms of PPT-A mRNA (a-PPT-A, b-PPT-A and g-PPT-A) [13]. All these splicing variants contain sequences encoding SP. Spe-
cificity of these hybridization probes were confirmed in previous reports [20]. The distribution of PPSS and PPT-A mRNA positive structures in the brain was consistent with previous reports [5,8,14,20] For the developmental study, in situ hybridization histochemistry was performed by digoxigenin-labelled cRNA probes for b-PPT-A and g-PPT-A using digoxigenin-UTP (Boehringer Mannheim, Germany) according the previously detailed method [2,20,22]. We used the free-floating section of 40 mm thick made by a cryostat. The reactions were processed according to the protocol of the nucleic acid detection kit (Boehringer-Mannheim, Germany), and were visualized blue by nitroblue tetrazolium [20]. We counted the number of signal-positive
Fig. 1. Double-labeling in situ hybridization of digoxigenin-labeled PPT-A and isotope-labeled PPSS cRNA probes in the anterior hypothalamus at P56. PPT-A mRNAs-containing neurons were colored blue by nitroblue tetraformazan, while PPSS mRNA signal was shown as isotope-hitted silver grains. (a), (b) and (c) are the high magnification photomicrographs of indicated area of (A); (a) suprachiasmatic nucleus (SCN), (b) the periventricular area of the anterior hypothalamus, and (c) the lateral hypothalamic area. oc, optic chiasma; v, third ventricle. Bars in (A)5200 mm; (a), (b), (c) 520 mm.
T. Nakamura et al. / Developmental Brain Research 127 (2001) 81 – 86
neurons for 10 SCN sections in each animal under a bright-field microscope. We analyzed the data for each probe at each postnatal day. Co-localization of two types of mRNAs was detected by double-labelling in situ hybridization using 35 S-CTP (New England Nuclear) labelled-PPSS probe and digoxigeninlabelled b-PPT-A probe. We first performed digoxigeninprobe hybridization as described above, except that we used 20 mm thick sections. After finishing nitroblue tetrazolium reaction, slides were dipped into nuclear track emulsion (Illford K5; dilution 1:1 with distilled water) and
83
exposed for 2 weeks at 48C. Following development of the tissue autoradiograms, the sections were analyzed under a bright-field microscope. First we examined the collocalization of PPSS mRNA and PPT-A mRNA in the hypothalamus. In the SCN, PPT-A mRNA expressing neurons were virtually all PPSS mRNA positive: PPT-A -positive blue stained cells (number of cells, 128 at P56, 96 at P28) all expressed dense accumulation of PPSS positive silver grains (Fig. 1, 1a). Periventricular isotope-labeled grain-positive PPSS neurons did not show blue PPT-A staining (Fig. 1b), and
Fig. 2. Bright-field photographs showing the development of PPSS mRNA-containing neurons in the SCN. Boundaries of SCN are encircled with dotted lines. Note that the cell number and the signal intensity of each cell increased, peaked at P7–P14 (C, D), then decreased gradually (E, F). Scale bars5100 mm.
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T. Nakamura et al. / Developmental Brain Research 127 (2001) 81 – 86
blue-stained PPT-A positive cells in the lateral hypothalamic area did not show the dense accumulation of signals (Fig. 1c). Then we examined the developmental features of the expression of these genes. The very weak PPSS mRNA signals existed at E20 at the central part of the SCN (data not shown). At P1, the intensity of the PPSS mRNA signals increased slightly, but still showed faint signals in the SCN (Fig. 2A). At P4, the number of positive neurons and the staining intensity of these neurons were increased.
At P7–P14, the intensity of the signal was strong, and the number of positive cells reached the peak (Fig. 2C, 2D). At P28 the signal intensity decreased slightly but still showed intense expression (Fig. 2E). At P56 the number of positive cells and the signal intensity apparently decreased compared with the peak level of P7–14 (Fig. 2F; see also Fig. 4). In contrast to PPSS, PPT-A mRNA-containing neurons appeared in the later stages of the developmental SCN (Fig. 3A–C). At P7 the intensity of PPT-A mRNA-positive
Fig. 3. Bright-field photographs showing the development of PPT-A mRNA-containing neurons in the rat SCN. Boundaries of SCN are encircled with dotted lines. Note that the signal first appeared at P7 (C), and then the intensity and number of cells increased gradually to the adult level (D–F). Scale bars5100 mm.
T. Nakamura et al. / Developmental Brain Research 127 (2001) 81 – 86
cells were faint, then gradually increased up to P56 (Fig. 3C–F). At P56, the signal intensity peaked (Fig. 3F). To clarify the abundance of the positive cells more quantitatively, we counted the number of PPSS and PPT-A mRNA-containing neurons in the SCN at days P1, P4, P7, P14, P28 and P56 (Fig. 4). In SCN, the total number of PPSS mRNA positive-cells increased from P1 (33.4610.2) (mean6S.E.M.) to P7 (116.2611.5), followed by a gradual reduction to P56 (63.863.3) (Fig. 4). However, at P7, when PPT-A mRNA signals were first detected in the SCN, the numbers of PPT-A positive neurons were 21.263.1. After that time the numbers of PPT-A mRNA positive-cells increased consistently up to P56 (55.268.2), which was nearly equal to the numbers of PPSS mRNA positive-cells at P56 (Fig. 4). Thus, PPSS-positive neurons demonstrated transient increase, while PPT-A positive neurons in the SCN showed comparatively late appearance and a consistent increase in number. In the present study, we demonstrated that the timecourse of the gene expression of PPSS and PPT-A coexpressed in the central SCN neurons. In the rat SCN, the neurogenesis is completed by E18 [1], and immediately after that, the SCN begins to oscillate [17] being entrained by the maternal circadian clock [24]. The period of the steep intensification of the PPSS mRNA signal noted between P1 and P14 in the SCN is identical to the most active stage of intrinsic synaptogenesis in the SCN [15], whereas the strong afferent projections from external components, such as the retina, raphe nucleus, and the lateral geniculate nucleus are established at later stages [9]. It is already demonstrated that somatostatin plays a role as a neurotrophic factors [18,23] such as neurite outgrowth [4]. Since somatostatin neurons in the SCN are intrinsic neurons [7], it is probable that somatostain may have a trophic role in the development of intrinsic synaptogenesis in the SCN through somatostain receptors in the SCN [3]. In contrast to PPSS mRNA, coexisting PPT-A mRNA signals appeared late at P7, and the cell number and signal
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intensity of PPT-A mRNA gradually increased as to adulthood. The different time courses of the development of two coexisting peptides suggest that their transcriptions in the developmental SCN neurons are regulated differently. There are several maturation steps in these neurons; first, the beginning of the PPSS-phenotype; second, the transient increment of the PPSS-phenotype and the beginning of the PPT-A phenotype; and third, the PPSS / PPT-A maturation. Approximately half of the PPSS-expressing neurons during early postnatal days continuously express PPSS mRNA, and eventually express PPT-A mRNA in adulthood. It is interesting that previous studies demonstrated that both of these peptides cause phase shifts of the light-pulse type when it is applied to SCN cells [10,19]. Somatostatin application increased 2-deoxyglucose (2-DG) uptake of the rat SCN at CT18 but not at CT6 in vitro slice preparations. Moreover, somatostatin induced a phase-shift in the circadian rhythm of the firing rate of SCN neurons in a phase-dependent manner, and this effect was dose-dependently antagonized by the specific antagonist, cyclosomatostatin [10]. Similarly, SP induced phase-dependent phase shifts, and increased the metabolic activity of SCN neurons at subjective night, but not at subjective day, and the effect was completely antagonized by spantide, an antagonist of SP [19]. These findings suggested that both somatostatin and SP-induced phase response curves were similar to the light-pulse-induced PRC of locomotor rhythm. In conclusion, we demonstrated the developmental differences of PPSS and PPT-A mRNA expression in the SCN. The stage-specific change in the gene expression of PPSS and PPT-A in the SCN suggests the different roles of these peptides in the development of the phase-resetting in the circadian pacemaker.
Acknowledgements This work was supported in part by grants from the Special Coordination Funds of the Science and Technology Agency of Japan, Grants-in-Aid for the Scientific Research and for the Scientific Research on Priority Areas of the Ministry of Education, Science, Sports and Culture of Japan, Uehara Memorial Foundation, and SRF.
References
Fig. 4. Statistics on the number of PPSS mRNA and PPT-A mRNApositive cells at various postnatal days in the SCN. Mean6S.E.M. (n54 at each postnatal day, except P14 and P56 being n53).
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