- Email: [email protected]
Changes in circadian period and morphology of the hypothalamic suprachiasmatic nucleus in fyn kinase-deficient mice
Brain Research 870 (2000) 36–43 www.elsevier.com / locate / bres
Research report
Changes in circadian period and morphology of the hypothalamic suprachiasmatic nucleus in fyn kinase-deficient mice Takaki Shima a , Takeshi Yagi b , Yasushi Isojima a , Nobuaki Okumura a , Masato Okada a , a, Katsuya Nagai * a
Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3 -2 Yamada-Oka, Suita, Osaka 565 -0871, Japan b National Institute for Physiological Sciences, Okazaki, Aichi 444 -8585, Japan Accepted 11 April 2000
Abstract Protein tyrosine phosphorylation is involved in intracellular signal transduction and plays important roles in various physiological events. To understand the role of Fyn, a non-receptor type tyrosine kinase of Src family kinases, in the mechanism of circadian rhythms, we analyzed the circadian locomotor behavior and morphology of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian oscillator in Fyn mutant mice, because Fyn-like immunoreactive substance was observed in the SCN. Under constant dark (DD) condition the Fyn (2 / 2) mutant mice showed a free-running circadian rhythm, and the period of the circadian rhythm of the locomotor activity was significantly longer than that of the control mice. Fyn (2 / 2) mutant mice had abnormal distribution of neurons containing vasoactive intestinal polypeptide (VIP)-like immunoreactive substance in the SCN. These findings suggest that Fyn is involved in the mechanism of circadian oscillation and morphological formation of the SCN. The mechanism of the implication of Fyn discussed with the Fyn’s roles in neural network formation and cellular signal transduction pathway. 2000 Elsevier Science B.V. All rights reserved. Keywords: Locomotor activity; Wheel running; Period; Immunohistochemistry; Vasopressin; Vasoactive intestinal polypeptide
1. Introduction Almost all living things on the earth have circadian rhythms in their biological events, including behaviors, hormone secretion, enzyme activities, etc, and the rhythms are synchronized by environmental time cues such as light–dark cycle. In mammals, a master circadian oscillator is shown to be located in the suprachiasmatic nucleus (SCN) of the hypothalamus [16,23,20,10]. In respect to the molecular mechanism of the circadian oscillator, recently several components of the oscillator in drosophila and mammals were identified. These includes genes of period ( per), timeless (tim), jrk (dclock), cycle (cyc), double-time (dbt) and cryptochrome (cry) in drosophila and per 1–3, tim, clock, bmal1 and cry in mammals [5,8,24,18]. Autoregulatory feed back systems, which include homologous genes between drosophila and mammals, are proposed to be frame works of the mechanisms of the circadian
oscillation in mammals and Drosophila melanogaster, though the precise mechanisms were not fully clarified yet. On the other hand, protein tyrosine kinases are shown to have important roles in intracellular signal transductions which are implicated in various biological phenomena. Fyn, a non-receptor type tyrosine kinase of Src family, is expressed in the central nervous system and suggested to be involved in the mechanism of neuronal signaling cascade [6,26,27,1]. Therefore, we examined whether Fyn knockout mice showed any changes in features of their circadian rhythm and the morphology of the SCN, in order to reveal the role of Fyn in the mechanism of circadian oscillation. In this study we found changes in the circadian period of wheelrunning activity and in the morphology of the SCN of the Fyn mutant mice. Thus, the details of changes are described. 2. Materials and methods
2.1. Animals *Corresponding author. E-mail address: k [email protected] (K. Nagai) ]
A fyn mutant mice were produced by replacing the SH2,
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02399-4
T. Shima et al. / Brain Research 870 (2000) 36 – 43
SH3 and tyrosine kinase domains of fyn by inserting lacZ in the cells of an embryonic stem (ES) cell line (TT2), derived from blastcysts of C57BL / 6 X CBA F1 mice [26]. Homozygous Fyn deficient (2 / 2) mice were obtained by crossing heterozygous (1 / 2) mice. The genotype was analyzed by performing the PCR analysis [11]. Only male animals of wild type (1 / 1), heterozygous (1 / 2) and homozygous (2 / 2) mutant mice were used in this experiment. These mice were maintained in a room illuminated for 12 h (08:00–20:00 h) and kept at 24618C. Food (type MF; Oriental Yeast Co. Tokyo) and water were freely available. Under this 12 h light and 12 h dark (LD) cycle, the time of light on was defined as zeitgeber time 0 (ZT 0) or 24 (ZT 24), and that of light off ZT 12. Some animals were transferred to a room maintained a constant dim red light (wavelength of more than 600 nm: less than 0.1 lx) dark (DD) condition from their age of 8 weeks. Daily patterns of their locomotor activity were observed from the age of 10 weeks.
2.2. Western blot analysis Brains of wild type mice were quickly removed after decapitation, trimmed, and frozen in dry ice. Frozen 300 mm thick coronal sections of several parts of the brain, one section contains the SCN, were cut using a cryostat. Tissue punch (inner diameter of the punch51.10 mm) containing both SCN were obtained from the frozen sections using the optic chiasm as a maker, punches of another parts were obtained in the same methods with the SCN (Fig. 1A). These tissues were homogenized in RIPA buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10% glycerol, 1 mM sodium orthovanadate, 1 mM PMSF, 10 mg / l aprotinin, 10 mg / l leupeptin and 5 mM mercaptoethanol). An equal amount of total protein from each sample was loaded on 10% SDS–polyacrylamide gel, and proteins were separated electrophoretically. Proteins were transferred to nitrocellulose membrane (Immobilon, Millipore, USA) at 100 mA for 1 h. The membranes were incubated in 100 mM tris buffered saline (TBS) containing 0.1% Tween-20 (Tween-TBS) overnight to prevent nonspecific antibody binding. The membrane was incubated with anti-Fyn (Fyn3, Santa Cruz, U.S.A.) or anti neuron specific enolase (ZYMED, USA) antibodies and protein was detected by chemiluminescence (NEN Life Science Products, USA).
2.3. Daily patterns of locomotor activity of mice Daily pattern of locomotor activity of mice was monitored using running wheels. On the age of 10 weeks each animal was housed in a cage equipped to running wheels, which was placed in a photoperiod-controlled environmental chamber. Age-matched male litter mates were tested
37
simultaneously in the same environment. Entrainment of mice by a LD cycle was examined under the LD condition. Free-running periods of the circadian rhythm were determined from the records for more than 15 days of mice maintained under the DD condition. Activity of individual mouse was recorded and plotted by a computer system (U-1200II, Panafacom Co., Tokyo). To determine the freerunning period (t ), a line was fitted manually along the activity onsets of the activity plot for 15 days. The freerunning period (t ) was calculated from the slope of the fitted line. The duration of the active period (a ) under the DD condition was determined as the duration between the initial and final counts that exceeded the mean wheel count in subjective night [21]. Total daily activities were calculated as averages of total counts of wheel revolution.
2.4. Histological examination Mice were anesthetized with sodium pentobarbital (35 mg / kg, ip) and their brains were perfused with a saline containing 0.1% sodium nitrate via the left ventricle. All operations were conducted in the middle of light period (about ZT 6). For staining with crecyl violet or for immunohistochemical examination using a rabbit polyclonal anti-vasoactive intestinal peptide (VIP) antibody (Peptide Institute, inc., Minoh, Osaka) and rabbit polyclonal anti-AVP antibody (kind gift from Dr. R.M. Buijs, The Netherland Institute for Brain Research) [19], this fixtative was followed by a Zamboni solution (2% paraformardehyde, 0.2% picric acid in 0.13 M sodium phosphate buffer, pH 7.4). After fixation, the brains were removed and post-fixed in the same fixtative for 2 days. After post-fixation, brains were equilibrated in 0.1 M sodium phosphate buffer containing 15% sucrose for cryoprotection. Frozen coronal brain sections (14 mm in thickness) containing the SCN were prepared with a cryostat using the optic chiasm as a maker. For examining the expressions of VIP- and AVP-like immunoreactive substances in the SCN, the brain sections were blocked by incubating in 1% normal goat serum / phosphate-buffered saline (PBS, 137 mM NaCl, 3 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) containing 0.3% Triton-X (PBST) for 1 h, followed incubation with the anti-VIP antiserum (diluted 1:500 in blocking solution) or anti-AVP antiserum (diluted 1: 500 in blocking solution) at room temperature for overnight. The slices were then incubated with biotinylated anti-rabbit IgG (1:400 dilution, Zymed Inc., USA) at 37 8C for 3 h and with an avidin-biotin-conjugate (1:800 dilution, Vectastain ABC Kit, Vector Lab, USA) at room temperature for 1 h. The immunoreactivity was visualized in 0.05 M Tris–HCl buffer, pH 7.4, containing 0.02% 3,39diaminobenzidine tetrahydrochloride (DAB) and 0.015% H 2 O 2 . Incubation times in the DAB were determined by visual inspection of sections for optimal signal to background ratios. For the staining using the anti-AVP antibody, the DAB solution appended 5% (v / w) of nickel
38
T. Shima et al. / Brain Research 870 (2000) 36 – 43
Fig. 1. Tissue sampling (A) and expression of Fyn protein in the SCN (B). (A) A photomicrograph of coronal brain section showing sites of the SCN (arrow) and outside of the SCN (arrowhead) in the hypothalamus. OC, optic chiasm; 3V, third ventricle. The scale bar corresponds to 200 mm. (B) Western blot analysis of brain extract from several regions of mouse brain using an anti-Fyn and anti-Neuron specific enolase (NSE) antibody. Bands of Fyn and NSE-like immunoreactive substance are indicate by arrows. out of SCN, outside of SCN in the hypothalamus; Fyn (2 / 2), whole brain extract of a Fyn (2 / 2) mouse.
ammonium sulfate was used. A part of brain sections were stained with crecyl violet. These sections were examined under a microscope.
3. Results
3.1. Expression of Fyn protein in the SCN To investigate the expression of Fyn in the SCN, western blot analysis was carried out using a tissue extracts
from the brain. Expression of Fyn-like immunoreactive substance was observed in the SCN as well as, other part of brain, olfactory bulb, cerebral cortex, hippocampus, and cerebellum in western blotting analysis. In Fyn null (2 / 2) mice, band of Fyn protein was not observed as seen in Fig. 1.
3.2. Circadian rhythm of wheel-running activity in fyn mutant mice Fig. 2 shows daily rhythms in wheel-running activity of
T. Shima et al. / Brain Research 870 (2000) 36 – 43
39
Fig. 3. Free-running periods of wheel running activities of Fyn (2 / 2), Fyn (1 / 2) mutant wild type (1 / 1) mice under DD condition. Circadian period was calculated each data obtained from Fyn (2 / 2), Fyn (1 / 2) mutant wild type (1 / 1) mice under DD condition. Numbers of animals used in the experiment are expressed in parentheses.
Fig. 2. . Free-running circadian pattern of wheel running activities of Fyn (2 / 2), Fyn (1 / 2) mutant wild type (1 / 1) mice under DD condition. Wheel running activity every 30 min is shown in a double plot manner. Data of 1 wild type (1 / 1), 1 Fyn (1 / 2) mutant and 2 Fyn (2 / 2) mutant mice are shown. The days of study are plotted side by side as well as vertically to facilitate visual assessment of circadian phase.
homozygous Fyn (2 / 2) and heterozygous Fyn (1 / 2) mutant mice as well as wild type (1 / 1) mice under the constant dark (DD) condition. All of these wild type and mutant mice showed free-running daily rhythms in their wheel-running activity under the DD condition. However, the heterozygous Fyn (1 / 2) and homozygous Fyn (2 / 2) mutant mice had longer circadian periods in their wheelrunning activity than those of wild type (1 / 1) mice (Fig. 2). Fig. 3 shows the circadian periods (t) of these mice calculated from the activity plots of their circadian wheelrunning activity. Free-running circadian periods (mean6S.E.M.) of the wild type (1 / 1) mice, hetero-
zygous Fyn (1 / 2) and homozygous Fyn (2 / 2) mutant mice were 23.7860.09 h, 24.0260.13 h and 24.2260.08 h, respectively (Fig. 3). The period of Fyn null (2 / 2) mutant was significantly (P,0.01 by unpaired t-test) longer than that of wild type (1 / 1) mice and tended to be longer than that of heterozygous Fyn (1 / 2) mutant mice (Fig. 3). The average duration of the period in which mouse was active was expanded in Fyn null (2 / 2) mutant (P,0.01 by unpaired t-test). The duration of activity (mean6S.E.M.) of wild type (1 / 1) mice was 11.4260.67 h and homozygous Fyn (2 / 2) mutant mice was 14.8360.87 h. Next, we examined the wheel running activity under a 12 h light and 12 h dark (LD) cycle. All of these mice showed well-entrained circadian rhythms in their wheelrunning activity under the LD cycle (Fig. 4). Under the LD and DD condition, the total daily wheel-running activity of some Fyn null (2 / 2) mutant mice was lower than that of wild type (1 / 1) mice (Figs. 2D and 4D).
3.3. Morphological change in the SCN of Fyn mutant mice In order to examine morphological changes of the SCN in Fyn mutant mice, histological examinations were done using cresyl violet and antibodies against VIP and AVP. Fig. 5 shows the results. In respect to cresyl violet staining, SCN neurons were clearly observed as a pair of clusters of neural cells in homozygous Fyn null (2 / 2) mice as well as wild type (1 / 1) mice (Fig. 5A and D). SCN neurons containing VIP-like immunoreactive substance (VIP neurons) were observed mainly at the ventral part of the SCN and only small numbers of the VIP neurons crossed the ventral border of the SCN into the optic chiasm in the control wild type (1 / 1) mice (Fig. 5B). In contrast, many SCN–VIP neurons of Fyn null (2 / 2) mutant mice moved deeply inside the optic chiasm and were seen even near the
40
T. Shima et al. / Brain Research 870 (2000) 36 – 43
Fig. 4. Entrainment of Fyn mice in LD cycle. Data of 1 wild type (1 / 1), 1 Fyn (1 / 2) mutant and 2 Fyn (2 / 2) mutant mice are shown. Mice were housed in LD cycle (lights on 8 a.m., lights off 8 p.m.). The open and filled bars present the time when light was on and off, respectively. Wheel running activity every 30 min is shown in a double plot manner. The days of study are plotted side by side as well as vertically.
ventral border of the optic chiasm (Fig. 5E). Furthermore, the VIP-like immunoreactive substance in neurites extending to the dorsal direction from VIP neurons of the SCN was densely packed in wild type (1 / 1) mice (Fig. 5B), but that was rather diffusely observed in the Fyn null (2 / 2) mutant mice (Fig. 5E). In somas of neurons of the dorsomedial region and neurites of the SCN, AVP-like immunoreactive substance was observed in both wild type (1 / 1) mice and Fyn null (2 / 2) mutant mice (Fig. 5C and F). Although SCN neurons containing AVP-like immunoreactive substance (AVP-neurons) seems to be seen more widely distributed in Fyn null (2 / 2) mutant mice than in wild type (1 / 1) control animals, remarkable change was not observed with respect to AVP-neurons (Fig. 5G and F).
4. Discussion Fyn, a non-receptor type tyrosine kinase, is expressed in the brain and suggested to be involved in the mechanism of intracellular signal transduction concerning cellular proliferation, differentiation and functions of neurons [1,26,27,7]. Fyn enriched in the nerve growth cone membranes [4] and the postsynaptic density fraction [6]. Fyn expresses in the SCN as well as cerebellum, cerebral cortex, olfactory bulb and hippocampus (Fig. 1). Therefore, we analyzed changes in the circadian rhythm in wheel running activity and morphology of the SCN of Fyn mutant mice in this study, to clarify the role of Fyn in the mechanism of circadian oscillation. And we obtained the results summarized as follows. (i) Free-running circadian
T. Shima et al. / Brain Research 870 (2000) 36 – 43
41
Fig. 5. Morphology of Fyn deficient mice in SCN region. Coronal sections containing the SCN of mice were stained with cresylviolet (A, D), polyclonal anti VIP (B, E) and anti AVP (C, F) antibody. Wild type mice (A, B, C) and homozygous Fyn mutant mice (D, E, F). OC, optic chiasm; 3V, third ventricle. The scale bar correspond to 100 mm.
period of wheel-running activity of homozygous Fyn (2 / 2) mutant mice was significantly (P,0.05) longer than that of wild type (1 / 1) mice, and tended to be longer than that of heterozygous Fyn (1 / 2) mutant mice, (ii) Circadian rhythm of wheel-running activity of homozygous Fyn (2 / 2) and heterozygous Fyn (1 / 2) mutant mice was entrained by a 12 h light and 12 h dark cycle.
(iii) In homozygous Fyn (2 / 2) mutant mice many SCN neurons containing VIP-like immunoreactive substance moved deeply into the optic chiasm and VIP-immunoreactivity in neurites from the SCN to the dorsal direction appeared to be diffusely expanded, when compared with those of wild type (1 / 1) mice. It was suggested to be possible that the SCN–VIP neurons often observed in optic
42
T. Shima et al. / Brain Research 870 (2000) 36 – 43
chiasm. However, in this experiment, SCN–VIP neurons in wild type (1 / 1) mice never moved so deep into the optic chiasm. Since, in our Fyn (2 / 2) mice, undulations of the hippocampal region were observed as described by Grant et al. [6] (data not shown), the movement of the VIP neurons might be elicited by the lack of Fyn. The elongation of the circadian period of wheel running activity of Fyn (2 / 2) mutant mice suggests that Fyn might be implicated in the mechanism of the circadian rhythm. The SCN contains autonomous, single-cell circadian oscillators with different circadian periods [25], these oscillators seems to be synchronized by the communication through cell-cell interactions, and a circadian period, which is an average of circadian periods of individual clock cells, is suggested to be dispatched from the SCN [9]. However, the precise mechanism of the synchronization of individual clock cells are not revealed yet. In this respect, it is reported that neural cell adhesion molecule (NCAM) in its highly polysialylated form is expressed in the SCN. NCAM-180 isoform deletion mutant mice exhibited significantly short circadian periods than wild type mice, the circadian rhythm of wheelrunning of the NCAM mutant mice was abolished by the third week under constant dark (DD) condition, and the mutant mice found to have an abnormal increase in number and distribution of VIP neurons in the SCN of the mutant mice [21]. It is also known that Fyn interacts with the NCAM-140 isoform in migrating growth cones and NCAM-dependent neurites outgrowth is selectively inhibited in cultures of cerebellar neurons in Fyn (2 / 2) mutant mice [2,3]. Moreover, undulations were observed in the granule cell layer in the dentate gyrus and pyramidal cell layer in the CA3 hippocampal region of the Fyn mutant mice [6], and the Fyn mutant have behavioral defects in spatial learning and suckling behavior, and enhanced sensitivities to stress responses such as fearinducing stimuli, acoustic stimuli and injection of ethanol [11–15]. Since Fyn (2 / 2) mutant mice have longer periods than those of wild type mice (Fig. 2) and abnormal localization of VIP neurons in the SCN and changes in the distribution of VIP-like immunoreactive neurites in and around the SCN (Fig. 5), it is possible to hypothesized that Fyn has an important role in the formation of functional networks within and outside the SCN which is essential for the generation and / or transduction of circadian signals. Moreover, it is also possible that NCAM might be involved in this network formation. These possibilities must be studied in future. In the previous work done in our laboratory using an immunotoxin, anti-VIP antibody-ricin A fragment, the selective elimination of AVP-neurons in the SCN elicited the disappearance of the circadian rhythm of drinking behavior in rats [22]. This fact suggested that SCN neurons possessing VIP-receptors are importantly implicated in the mechanism of circadian clock mechanism. In other words, SCN–VIP neurons are also important in the mechanism. In
this meaning, the alteration in the location and thus integration of SCN–VIP neurons found in the Fyn (2 / 2) mice might be casually related to the alterations in the circadian rhythm of wheel-running activity in these mice. In this concern, it should be pointed out that SCN–VIP neurons receive the direct retinal input which transfer the information of the environmental light-dark condition [17]. Therefore, it is possible that Fyn tyrosine kinase exists in the VIP neurons, and participates in the signal transduction pathway of photic entrainment of the circadian clock. Under the constant dark conditions, Fyn (2 / 2) mutant mice exhibited expanding duration of the active period (a ) and less wheel-running activity than wild-type animals. The wheel-running of wild type was continuously observed from the onset of subjective dark period to its end, on the other hand, that of Fyn (2 / 2) mice was fragmentary. The reason why Fyn mutant mice showed these behaviors are not known, but it might be caused by the abnormal functional networks in other brain areas than the SCN or reflect a general effect of the gene targeting. The present reports indicate that Fyn is involved in the morphological formation of the SCN and suggest that Fyn might be implicated in the generation and / or transduction of the circadian time signal and in the entrainment of the circadian clock. Functions of Fyn concerning other brain function and morphology have been already reported, but this is the first report of the role of Fyn in the function of circadian clock mechanism. What is the mechanism of the role of Fyn in the function of the SCN is now under investigation.
Acknowledgements This research is supported by a Grant-in-Aid for Scientific Research (No. 10044283 and No. 07670171) from the Ministry of Education, Science, Sports and Culture of Japan.
References [1] D.J. Bare, J.M. Lauder, M.B. Wilkie, P.F. Maness, p59fyn in rat brain is localized in developing axonal tarcts and subpopulations of adult neurons and glia, Oncogene 8 (1993) 1429–1436. [2] H.E. Beggs, P. Soriano, P.F. Maness, NCAM-dependent neurite outgrowth is inhibition in neurons from Fyn-minus mice, J. Cell Biol. 127 (1994) 825–833. [3] H.E. Beggs, S.C. Baragona, J.J. Hemperly, P.F. Maness, NCAM140 interacts with the focal adhesion kinase p125fak and the SRCrelated tyrosine kinase p59fyn, J. Biol. Chem. 272 (1997) 8310– 8319. [4] J.L. Bixby, P. Jhabvala, Tyrosine phosphorylation in early embryonic growth cones, J. Neurosci. 13 (1993) 3421–3432. [5] J.C. Dunlop, Molecular Bases for Circadian Clocks, Cell 96 (1999) 271–290. [6] S.G. Grant, T.J. O’ Dell, K.A. Karl, P.L. Stein, P. Sariano, E.R. Kandel, Impaired long-term potentiation, spatial learning, and
T. Shima et al. / Brain Research 870 (2000) 36 – 43
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
[15]
[16]
[17]
hippocampal development in fyn mutant mice, Science 258 (1992) 1903–1910. M. Inomata, Y. Takayama, H. Kiyama, S. Nada, M. Okada, H. Nakagawa, Regulation of Src family kinases in the developing rat brain; correlation with their regulator kinase, Csk., J. Biochem. 116 (1994) 386–392. K. Kume, M.J. Zylka, S. Sriram, L.P. Shearman, D.R. Weaver, X. Jin, E.S. Maywood, M.H. Hastings, S.M. Reppert, mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop, Cell 98 (1999) 193–205. C. Liu, D.R. Weaver, S.H. Strogatz, S.M. Reppert, Cellular construction of a circadian clock; period determination in the suprachiasmatic nuclei, Cell 91 (1997) 855–860. J.H. Meijer, W.J. Rietveld, Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Rev. 69 (1989) 671–707. T. Miyakawa, T. Yagi, S. Watanabe, H. Niki, Increased fearfulness of Fyn tyrosine kinase deficient mice, Brain Res. Mol. Brain Res. 27 (1994) 179–182. T. Miyakawa, T. Yagi, M. Taniguti, H. Matsuura, K.L. Takeishi, H. Niki, Enhanced susceptibility of audiogenic seizures in Fyn-kinase deficient mice, Brain Res. Mol. Brain Res. 28 (1995) 349–352. T. Miyakawa, T. Yagi, K. Tateishi, H. Niki, Susceptibility to drug-induced seizures of Fyn tyrosine kinase-deficient mice, Neuroreport 7 (1996) 2723–2726. T. Miyakawa, T. Yagi, A. Kagiyama, H. Niki, Radial maze performance, open-field and elevated plus-maze behaviors in Fynkinase deficient mice; further evidence for increased fearfulness, Brain Res. Mol. Brain Res. 37 (1996) 145–150. T. Miyakawa, T. Yagi, H. Kitazawa, M. Yasuda, N. Kawai, K. Tsuboi, H. Niki, Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function, Science 278 (1997) 698–701. R.Y. Moore, V.B. Eichler, Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat, Brain Res. 42 (1972) 201–206. S. Okamoto, H. Okamura, M. Miyake, Y. Takahashi, S. Takagi, Y. Akagi, K. Fukui, H. Okamoto, Y. Ibata, A diural variation of vasoactive intestinal peptide (VIP) mRNA under a daily light-dark cycle in the rat suprachiasmatic nucleus, Histochemistry 95 (1991) 525–528.
43
[18] J.L. Price, J. Blau, A. Rothenfluh, M. Abodeely, B. Kloss, M.W. Young, Double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation, Cell 94 (1998) 83–95. [19] H.J. Romijn, A.A. Sluiter, J. Wortel, J.F. Van Uum, R.M. Buijs, Immunocytochemical evidence for a diural rhythm of neurons showing colocalization of VIP with GRP in the rat suprachiasmatic nucleus, J. Comp. Neurol. 391 (1998) 397–405. [20] B. Rusak, I. Zucker, Neural reguration of circadian rhythms, Physiol. Rev. 59 (1979) 449–526. [21] H. Shen, M. Watanabe, H. Tomasiewicz, U. Rutishauser, T. Magnuson, J.D. Glass, Role of neural cell adhesion molecule and polysialic acid in mouse circadian clock function, J. Neurosci. 17 (1997) 5221–5229. [22] K. Shimizu, K. Nagai, H. Nakagawa, An immunotoxin, anti-VIP anyibody-ricin A chain cojugate eliminates neurons in the hypothalamic suprachiasmatic nucleus selectively and abolishes the circadian rhythm of water intake, Brain Res. Bull. 41 (1996) 369–378. [23] F.K. Stephan, I. Zucker, Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions, Proc. Natl. Acad. Sci. USA 69 (1972) 1583–1586. [24] G.T.J. van der Horst, M. Muijtjens, K. Kobayashi, R. Takano, S. Kanno, M. Takao, J. de Wit, A. Verkerk, A.P. Eker, D. Leenen, R. Buijs, D. Bootsma, J.H. Hoeijmakers, A. Yasui, Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms, Nature 398 (1999) 627–630. [25] D.K. Welsh, D.E. Logothetis, M. Meister, S.M. Reppert, Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms, Neuron 14 (1995) 697–706. [26] T. Yagi, S. Aizawa, T. Tokunaga, Y. Shigetani, N. Takeda, Y. Ikawa, A role for Fyn tyrosine kinase in the suckling behaviour of neonatal mice, Nature 366 (1993) 742–745. [27] T. Yagi, Y. Shigetani, N. Okado, T. Tokunaga, Y. Ikawa, S. Aizawa, Regional localization of Fyn in adult brain; studies with mice in which fyn gene was replaced by lacZ, Oncogene 8 (1993) 3343– 3351.
Research report
Changes in circadian period and morphology of the hypothalamic suprachiasmatic nucleus in fyn kinase-deficient mice Takaki Shima a , Takeshi Yagi b , Yasushi Isojima a , Nobuaki Okumura a , Masato Okada a , a, Katsuya Nagai * a
Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3 -2 Yamada-Oka, Suita, Osaka 565 -0871, Japan b National Institute for Physiological Sciences, Okazaki, Aichi 444 -8585, Japan Accepted 11 April 2000
Abstract Protein tyrosine phosphorylation is involved in intracellular signal transduction and plays important roles in various physiological events. To understand the role of Fyn, a non-receptor type tyrosine kinase of Src family kinases, in the mechanism of circadian rhythms, we analyzed the circadian locomotor behavior and morphology of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian oscillator in Fyn mutant mice, because Fyn-like immunoreactive substance was observed in the SCN. Under constant dark (DD) condition the Fyn (2 / 2) mutant mice showed a free-running circadian rhythm, and the period of the circadian rhythm of the locomotor activity was significantly longer than that of the control mice. Fyn (2 / 2) mutant mice had abnormal distribution of neurons containing vasoactive intestinal polypeptide (VIP)-like immunoreactive substance in the SCN. These findings suggest that Fyn is involved in the mechanism of circadian oscillation and morphological formation of the SCN. The mechanism of the implication of Fyn discussed with the Fyn’s roles in neural network formation and cellular signal transduction pathway. 2000 Elsevier Science B.V. All rights reserved. Keywords: Locomotor activity; Wheel running; Period; Immunohistochemistry; Vasopressin; Vasoactive intestinal polypeptide
1. Introduction Almost all living things on the earth have circadian rhythms in their biological events, including behaviors, hormone secretion, enzyme activities, etc, and the rhythms are synchronized by environmental time cues such as light–dark cycle. In mammals, a master circadian oscillator is shown to be located in the suprachiasmatic nucleus (SCN) of the hypothalamus [16,23,20,10]. In respect to the molecular mechanism of the circadian oscillator, recently several components of the oscillator in drosophila and mammals were identified. These includes genes of period ( per), timeless (tim), jrk (dclock), cycle (cyc), double-time (dbt) and cryptochrome (cry) in drosophila and per 1–3, tim, clock, bmal1 and cry in mammals [5,8,24,18]. Autoregulatory feed back systems, which include homologous genes between drosophila and mammals, are proposed to be frame works of the mechanisms of the circadian
oscillation in mammals and Drosophila melanogaster, though the precise mechanisms were not fully clarified yet. On the other hand, protein tyrosine kinases are shown to have important roles in intracellular signal transductions which are implicated in various biological phenomena. Fyn, a non-receptor type tyrosine kinase of Src family, is expressed in the central nervous system and suggested to be involved in the mechanism of neuronal signaling cascade [6,26,27,1]. Therefore, we examined whether Fyn knockout mice showed any changes in features of their circadian rhythm and the morphology of the SCN, in order to reveal the role of Fyn in the mechanism of circadian oscillation. In this study we found changes in the circadian period of wheelrunning activity and in the morphology of the SCN of the Fyn mutant mice. Thus, the details of changes are described. 2. Materials and methods
2.1. Animals *Corresponding author. E-mail address: k [email protected] (K. Nagai) ]
A fyn mutant mice were produced by replacing the SH2,
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02399-4
T. Shima et al. / Brain Research 870 (2000) 36 – 43
SH3 and tyrosine kinase domains of fyn by inserting lacZ in the cells of an embryonic stem (ES) cell line (TT2), derived from blastcysts of C57BL / 6 X CBA F1 mice [26]. Homozygous Fyn deficient (2 / 2) mice were obtained by crossing heterozygous (1 / 2) mice. The genotype was analyzed by performing the PCR analysis [11]. Only male animals of wild type (1 / 1), heterozygous (1 / 2) and homozygous (2 / 2) mutant mice were used in this experiment. These mice were maintained in a room illuminated for 12 h (08:00–20:00 h) and kept at 24618C. Food (type MF; Oriental Yeast Co. Tokyo) and water were freely available. Under this 12 h light and 12 h dark (LD) cycle, the time of light on was defined as zeitgeber time 0 (ZT 0) or 24 (ZT 24), and that of light off ZT 12. Some animals were transferred to a room maintained a constant dim red light (wavelength of more than 600 nm: less than 0.1 lx) dark (DD) condition from their age of 8 weeks. Daily patterns of their locomotor activity were observed from the age of 10 weeks.
2.2. Western blot analysis Brains of wild type mice were quickly removed after decapitation, trimmed, and frozen in dry ice. Frozen 300 mm thick coronal sections of several parts of the brain, one section contains the SCN, were cut using a cryostat. Tissue punch (inner diameter of the punch51.10 mm) containing both SCN were obtained from the frozen sections using the optic chiasm as a maker, punches of another parts were obtained in the same methods with the SCN (Fig. 1A). These tissues were homogenized in RIPA buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10% glycerol, 1 mM sodium orthovanadate, 1 mM PMSF, 10 mg / l aprotinin, 10 mg / l leupeptin and 5 mM mercaptoethanol). An equal amount of total protein from each sample was loaded on 10% SDS–polyacrylamide gel, and proteins were separated electrophoretically. Proteins were transferred to nitrocellulose membrane (Immobilon, Millipore, USA) at 100 mA for 1 h. The membranes were incubated in 100 mM tris buffered saline (TBS) containing 0.1% Tween-20 (Tween-TBS) overnight to prevent nonspecific antibody binding. The membrane was incubated with anti-Fyn (Fyn3, Santa Cruz, U.S.A.) or anti neuron specific enolase (ZYMED, USA) antibodies and protein was detected by chemiluminescence (NEN Life Science Products, USA).
2.3. Daily patterns of locomotor activity of mice Daily pattern of locomotor activity of mice was monitored using running wheels. On the age of 10 weeks each animal was housed in a cage equipped to running wheels, which was placed in a photoperiod-controlled environmental chamber. Age-matched male litter mates were tested
37
simultaneously in the same environment. Entrainment of mice by a LD cycle was examined under the LD condition. Free-running periods of the circadian rhythm were determined from the records for more than 15 days of mice maintained under the DD condition. Activity of individual mouse was recorded and plotted by a computer system (U-1200II, Panafacom Co., Tokyo). To determine the freerunning period (t ), a line was fitted manually along the activity onsets of the activity plot for 15 days. The freerunning period (t ) was calculated from the slope of the fitted line. The duration of the active period (a ) under the DD condition was determined as the duration between the initial and final counts that exceeded the mean wheel count in subjective night [21]. Total daily activities were calculated as averages of total counts of wheel revolution.
2.4. Histological examination Mice were anesthetized with sodium pentobarbital (35 mg / kg, ip) and their brains were perfused with a saline containing 0.1% sodium nitrate via the left ventricle. All operations were conducted in the middle of light period (about ZT 6). For staining with crecyl violet or for immunohistochemical examination using a rabbit polyclonal anti-vasoactive intestinal peptide (VIP) antibody (Peptide Institute, inc., Minoh, Osaka) and rabbit polyclonal anti-AVP antibody (kind gift from Dr. R.M. Buijs, The Netherland Institute for Brain Research) [19], this fixtative was followed by a Zamboni solution (2% paraformardehyde, 0.2% picric acid in 0.13 M sodium phosphate buffer, pH 7.4). After fixation, the brains were removed and post-fixed in the same fixtative for 2 days. After post-fixation, brains were equilibrated in 0.1 M sodium phosphate buffer containing 15% sucrose for cryoprotection. Frozen coronal brain sections (14 mm in thickness) containing the SCN were prepared with a cryostat using the optic chiasm as a maker. For examining the expressions of VIP- and AVP-like immunoreactive substances in the SCN, the brain sections were blocked by incubating in 1% normal goat serum / phosphate-buffered saline (PBS, 137 mM NaCl, 3 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4) containing 0.3% Triton-X (PBST) for 1 h, followed incubation with the anti-VIP antiserum (diluted 1:500 in blocking solution) or anti-AVP antiserum (diluted 1: 500 in blocking solution) at room temperature for overnight. The slices were then incubated with biotinylated anti-rabbit IgG (1:400 dilution, Zymed Inc., USA) at 37 8C for 3 h and with an avidin-biotin-conjugate (1:800 dilution, Vectastain ABC Kit, Vector Lab, USA) at room temperature for 1 h. The immunoreactivity was visualized in 0.05 M Tris–HCl buffer, pH 7.4, containing 0.02% 3,39diaminobenzidine tetrahydrochloride (DAB) and 0.015% H 2 O 2 . Incubation times in the DAB were determined by visual inspection of sections for optimal signal to background ratios. For the staining using the anti-AVP antibody, the DAB solution appended 5% (v / w) of nickel
38
T. Shima et al. / Brain Research 870 (2000) 36 – 43
Fig. 1. Tissue sampling (A) and expression of Fyn protein in the SCN (B). (A) A photomicrograph of coronal brain section showing sites of the SCN (arrow) and outside of the SCN (arrowhead) in the hypothalamus. OC, optic chiasm; 3V, third ventricle. The scale bar corresponds to 200 mm. (B) Western blot analysis of brain extract from several regions of mouse brain using an anti-Fyn and anti-Neuron specific enolase (NSE) antibody. Bands of Fyn and NSE-like immunoreactive substance are indicate by arrows. out of SCN, outside of SCN in the hypothalamus; Fyn (2 / 2), whole brain extract of a Fyn (2 / 2) mouse.
ammonium sulfate was used. A part of brain sections were stained with crecyl violet. These sections were examined under a microscope.
3. Results
3.1. Expression of Fyn protein in the SCN To investigate the expression of Fyn in the SCN, western blot analysis was carried out using a tissue extracts
from the brain. Expression of Fyn-like immunoreactive substance was observed in the SCN as well as, other part of brain, olfactory bulb, cerebral cortex, hippocampus, and cerebellum in western blotting analysis. In Fyn null (2 / 2) mice, band of Fyn protein was not observed as seen in Fig. 1.
3.2. Circadian rhythm of wheel-running activity in fyn mutant mice Fig. 2 shows daily rhythms in wheel-running activity of
T. Shima et al. / Brain Research 870 (2000) 36 – 43
39
Fig. 3. Free-running periods of wheel running activities of Fyn (2 / 2), Fyn (1 / 2) mutant wild type (1 / 1) mice under DD condition. Circadian period was calculated each data obtained from Fyn (2 / 2), Fyn (1 / 2) mutant wild type (1 / 1) mice under DD condition. Numbers of animals used in the experiment are expressed in parentheses.
Fig. 2. . Free-running circadian pattern of wheel running activities of Fyn (2 / 2), Fyn (1 / 2) mutant wild type (1 / 1) mice under DD condition. Wheel running activity every 30 min is shown in a double plot manner. Data of 1 wild type (1 / 1), 1 Fyn (1 / 2) mutant and 2 Fyn (2 / 2) mutant mice are shown. The days of study are plotted side by side as well as vertically to facilitate visual assessment of circadian phase.
homozygous Fyn (2 / 2) and heterozygous Fyn (1 / 2) mutant mice as well as wild type (1 / 1) mice under the constant dark (DD) condition. All of these wild type and mutant mice showed free-running daily rhythms in their wheel-running activity under the DD condition. However, the heterozygous Fyn (1 / 2) and homozygous Fyn (2 / 2) mutant mice had longer circadian periods in their wheelrunning activity than those of wild type (1 / 1) mice (Fig. 2). Fig. 3 shows the circadian periods (t) of these mice calculated from the activity plots of their circadian wheelrunning activity. Free-running circadian periods (mean6S.E.M.) of the wild type (1 / 1) mice, hetero-
zygous Fyn (1 / 2) and homozygous Fyn (2 / 2) mutant mice were 23.7860.09 h, 24.0260.13 h and 24.2260.08 h, respectively (Fig. 3). The period of Fyn null (2 / 2) mutant was significantly (P,0.01 by unpaired t-test) longer than that of wild type (1 / 1) mice and tended to be longer than that of heterozygous Fyn (1 / 2) mutant mice (Fig. 3). The average duration of the period in which mouse was active was expanded in Fyn null (2 / 2) mutant (P,0.01 by unpaired t-test). The duration of activity (mean6S.E.M.) of wild type (1 / 1) mice was 11.4260.67 h and homozygous Fyn (2 / 2) mutant mice was 14.8360.87 h. Next, we examined the wheel running activity under a 12 h light and 12 h dark (LD) cycle. All of these mice showed well-entrained circadian rhythms in their wheelrunning activity under the LD cycle (Fig. 4). Under the LD and DD condition, the total daily wheel-running activity of some Fyn null (2 / 2) mutant mice was lower than that of wild type (1 / 1) mice (Figs. 2D and 4D).
3.3. Morphological change in the SCN of Fyn mutant mice In order to examine morphological changes of the SCN in Fyn mutant mice, histological examinations were done using cresyl violet and antibodies against VIP and AVP. Fig. 5 shows the results. In respect to cresyl violet staining, SCN neurons were clearly observed as a pair of clusters of neural cells in homozygous Fyn null (2 / 2) mice as well as wild type (1 / 1) mice (Fig. 5A and D). SCN neurons containing VIP-like immunoreactive substance (VIP neurons) were observed mainly at the ventral part of the SCN and only small numbers of the VIP neurons crossed the ventral border of the SCN into the optic chiasm in the control wild type (1 / 1) mice (Fig. 5B). In contrast, many SCN–VIP neurons of Fyn null (2 / 2) mutant mice moved deeply inside the optic chiasm and were seen even near the
40
T. Shima et al. / Brain Research 870 (2000) 36 – 43
Fig. 4. Entrainment of Fyn mice in LD cycle. Data of 1 wild type (1 / 1), 1 Fyn (1 / 2) mutant and 2 Fyn (2 / 2) mutant mice are shown. Mice were housed in LD cycle (lights on 8 a.m., lights off 8 p.m.). The open and filled bars present the time when light was on and off, respectively. Wheel running activity every 30 min is shown in a double plot manner. The days of study are plotted side by side as well as vertically.
ventral border of the optic chiasm (Fig. 5E). Furthermore, the VIP-like immunoreactive substance in neurites extending to the dorsal direction from VIP neurons of the SCN was densely packed in wild type (1 / 1) mice (Fig. 5B), but that was rather diffusely observed in the Fyn null (2 / 2) mutant mice (Fig. 5E). In somas of neurons of the dorsomedial region and neurites of the SCN, AVP-like immunoreactive substance was observed in both wild type (1 / 1) mice and Fyn null (2 / 2) mutant mice (Fig. 5C and F). Although SCN neurons containing AVP-like immunoreactive substance (AVP-neurons) seems to be seen more widely distributed in Fyn null (2 / 2) mutant mice than in wild type (1 / 1) control animals, remarkable change was not observed with respect to AVP-neurons (Fig. 5G and F).
4. Discussion Fyn, a non-receptor type tyrosine kinase, is expressed in the brain and suggested to be involved in the mechanism of intracellular signal transduction concerning cellular proliferation, differentiation and functions of neurons [1,26,27,7]. Fyn enriched in the nerve growth cone membranes [4] and the postsynaptic density fraction [6]. Fyn expresses in the SCN as well as cerebellum, cerebral cortex, olfactory bulb and hippocampus (Fig. 1). Therefore, we analyzed changes in the circadian rhythm in wheel running activity and morphology of the SCN of Fyn mutant mice in this study, to clarify the role of Fyn in the mechanism of circadian oscillation. And we obtained the results summarized as follows. (i) Free-running circadian
T. Shima et al. / Brain Research 870 (2000) 36 – 43
41
Fig. 5. Morphology of Fyn deficient mice in SCN region. Coronal sections containing the SCN of mice were stained with cresylviolet (A, D), polyclonal anti VIP (B, E) and anti AVP (C, F) antibody. Wild type mice (A, B, C) and homozygous Fyn mutant mice (D, E, F). OC, optic chiasm; 3V, third ventricle. The scale bar correspond to 100 mm.
period of wheel-running activity of homozygous Fyn (2 / 2) mutant mice was significantly (P,0.05) longer than that of wild type (1 / 1) mice, and tended to be longer than that of heterozygous Fyn (1 / 2) mutant mice, (ii) Circadian rhythm of wheel-running activity of homozygous Fyn (2 / 2) and heterozygous Fyn (1 / 2) mutant mice was entrained by a 12 h light and 12 h dark cycle.
(iii) In homozygous Fyn (2 / 2) mutant mice many SCN neurons containing VIP-like immunoreactive substance moved deeply into the optic chiasm and VIP-immunoreactivity in neurites from the SCN to the dorsal direction appeared to be diffusely expanded, when compared with those of wild type (1 / 1) mice. It was suggested to be possible that the SCN–VIP neurons often observed in optic
42
T. Shima et al. / Brain Research 870 (2000) 36 – 43
chiasm. However, in this experiment, SCN–VIP neurons in wild type (1 / 1) mice never moved so deep into the optic chiasm. Since, in our Fyn (2 / 2) mice, undulations of the hippocampal region were observed as described by Grant et al. [6] (data not shown), the movement of the VIP neurons might be elicited by the lack of Fyn. The elongation of the circadian period of wheel running activity of Fyn (2 / 2) mutant mice suggests that Fyn might be implicated in the mechanism of the circadian rhythm. The SCN contains autonomous, single-cell circadian oscillators with different circadian periods [25], these oscillators seems to be synchronized by the communication through cell-cell interactions, and a circadian period, which is an average of circadian periods of individual clock cells, is suggested to be dispatched from the SCN [9]. However, the precise mechanism of the synchronization of individual clock cells are not revealed yet. In this respect, it is reported that neural cell adhesion molecule (NCAM) in its highly polysialylated form is expressed in the SCN. NCAM-180 isoform deletion mutant mice exhibited significantly short circadian periods than wild type mice, the circadian rhythm of wheelrunning of the NCAM mutant mice was abolished by the third week under constant dark (DD) condition, and the mutant mice found to have an abnormal increase in number and distribution of VIP neurons in the SCN of the mutant mice [21]. It is also known that Fyn interacts with the NCAM-140 isoform in migrating growth cones and NCAM-dependent neurites outgrowth is selectively inhibited in cultures of cerebellar neurons in Fyn (2 / 2) mutant mice [2,3]. Moreover, undulations were observed in the granule cell layer in the dentate gyrus and pyramidal cell layer in the CA3 hippocampal region of the Fyn mutant mice [6], and the Fyn mutant have behavioral defects in spatial learning and suckling behavior, and enhanced sensitivities to stress responses such as fearinducing stimuli, acoustic stimuli and injection of ethanol [11–15]. Since Fyn (2 / 2) mutant mice have longer periods than those of wild type mice (Fig. 2) and abnormal localization of VIP neurons in the SCN and changes in the distribution of VIP-like immunoreactive neurites in and around the SCN (Fig. 5), it is possible to hypothesized that Fyn has an important role in the formation of functional networks within and outside the SCN which is essential for the generation and / or transduction of circadian signals. Moreover, it is also possible that NCAM might be involved in this network formation. These possibilities must be studied in future. In the previous work done in our laboratory using an immunotoxin, anti-VIP antibody-ricin A fragment, the selective elimination of AVP-neurons in the SCN elicited the disappearance of the circadian rhythm of drinking behavior in rats [22]. This fact suggested that SCN neurons possessing VIP-receptors are importantly implicated in the mechanism of circadian clock mechanism. In other words, SCN–VIP neurons are also important in the mechanism. In
this meaning, the alteration in the location and thus integration of SCN–VIP neurons found in the Fyn (2 / 2) mice might be casually related to the alterations in the circadian rhythm of wheel-running activity in these mice. In this concern, it should be pointed out that SCN–VIP neurons receive the direct retinal input which transfer the information of the environmental light-dark condition [17]. Therefore, it is possible that Fyn tyrosine kinase exists in the VIP neurons, and participates in the signal transduction pathway of photic entrainment of the circadian clock. Under the constant dark conditions, Fyn (2 / 2) mutant mice exhibited expanding duration of the active period (a ) and less wheel-running activity than wild-type animals. The wheel-running of wild type was continuously observed from the onset of subjective dark period to its end, on the other hand, that of Fyn (2 / 2) mice was fragmentary. The reason why Fyn mutant mice showed these behaviors are not known, but it might be caused by the abnormal functional networks in other brain areas than the SCN or reflect a general effect of the gene targeting. The present reports indicate that Fyn is involved in the morphological formation of the SCN and suggest that Fyn might be implicated in the generation and / or transduction of the circadian time signal and in the entrainment of the circadian clock. Functions of Fyn concerning other brain function and morphology have been already reported, but this is the first report of the role of Fyn in the function of circadian clock mechanism. What is the mechanism of the role of Fyn in the function of the SCN is now under investigation.
Acknowledgements This research is supported by a Grant-in-Aid for Scientific Research (No. 10044283 and No. 07670171) from the Ministry of Education, Science, Sports and Culture of Japan.
References [1] D.J. Bare, J.M. Lauder, M.B. Wilkie, P.F. Maness, p59fyn in rat brain is localized in developing axonal tarcts and subpopulations of adult neurons and glia, Oncogene 8 (1993) 1429–1436. [2] H.E. Beggs, P. Soriano, P.F. Maness, NCAM-dependent neurite outgrowth is inhibition in neurons from Fyn-minus mice, J. Cell Biol. 127 (1994) 825–833. [3] H.E. Beggs, S.C. Baragona, J.J. Hemperly, P.F. Maness, NCAM140 interacts with the focal adhesion kinase p125fak and the SRCrelated tyrosine kinase p59fyn, J. Biol. Chem. 272 (1997) 8310– 8319. [4] J.L. Bixby, P. Jhabvala, Tyrosine phosphorylation in early embryonic growth cones, J. Neurosci. 13 (1993) 3421–3432. [5] J.C. Dunlop, Molecular Bases for Circadian Clocks, Cell 96 (1999) 271–290. [6] S.G. Grant, T.J. O’ Dell, K.A. Karl, P.L. Stein, P. Sariano, E.R. Kandel, Impaired long-term potentiation, spatial learning, and
T. Shima et al. / Brain Research 870 (2000) 36 – 43
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
[15]
[16]
[17]
hippocampal development in fyn mutant mice, Science 258 (1992) 1903–1910. M. Inomata, Y. Takayama, H. Kiyama, S. Nada, M. Okada, H. Nakagawa, Regulation of Src family kinases in the developing rat brain; correlation with their regulator kinase, Csk., J. Biochem. 116 (1994) 386–392. K. Kume, M.J. Zylka, S. Sriram, L.P. Shearman, D.R. Weaver, X. Jin, E.S. Maywood, M.H. Hastings, S.M. Reppert, mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop, Cell 98 (1999) 193–205. C. Liu, D.R. Weaver, S.H. Strogatz, S.M. Reppert, Cellular construction of a circadian clock; period determination in the suprachiasmatic nuclei, Cell 91 (1997) 855–860. J.H. Meijer, W.J. Rietveld, Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Rev. 69 (1989) 671–707. T. Miyakawa, T. Yagi, S. Watanabe, H. Niki, Increased fearfulness of Fyn tyrosine kinase deficient mice, Brain Res. Mol. Brain Res. 27 (1994) 179–182. T. Miyakawa, T. Yagi, M. Taniguti, H. Matsuura, K.L. Takeishi, H. Niki, Enhanced susceptibility of audiogenic seizures in Fyn-kinase deficient mice, Brain Res. Mol. Brain Res. 28 (1995) 349–352. T. Miyakawa, T. Yagi, K. Tateishi, H. Niki, Susceptibility to drug-induced seizures of Fyn tyrosine kinase-deficient mice, Neuroreport 7 (1996) 2723–2726. T. Miyakawa, T. Yagi, A. Kagiyama, H. Niki, Radial maze performance, open-field and elevated plus-maze behaviors in Fynkinase deficient mice; further evidence for increased fearfulness, Brain Res. Mol. Brain Res. 37 (1996) 145–150. T. Miyakawa, T. Yagi, H. Kitazawa, M. Yasuda, N. Kawai, K. Tsuboi, H. Niki, Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function, Science 278 (1997) 698–701. R.Y. Moore, V.B. Eichler, Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat, Brain Res. 42 (1972) 201–206. S. Okamoto, H. Okamura, M. Miyake, Y. Takahashi, S. Takagi, Y. Akagi, K. Fukui, H. Okamoto, Y. Ibata, A diural variation of vasoactive intestinal peptide (VIP) mRNA under a daily light-dark cycle in the rat suprachiasmatic nucleus, Histochemistry 95 (1991) 525–528.
43
[18] J.L. Price, J. Blau, A. Rothenfluh, M. Abodeely, B. Kloss, M.W. Young, Double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation, Cell 94 (1998) 83–95. [19] H.J. Romijn, A.A. Sluiter, J. Wortel, J.F. Van Uum, R.M. Buijs, Immunocytochemical evidence for a diural rhythm of neurons showing colocalization of VIP with GRP in the rat suprachiasmatic nucleus, J. Comp. Neurol. 391 (1998) 397–405. [20] B. Rusak, I. Zucker, Neural reguration of circadian rhythms, Physiol. Rev. 59 (1979) 449–526. [21] H. Shen, M. Watanabe, H. Tomasiewicz, U. Rutishauser, T. Magnuson, J.D. Glass, Role of neural cell adhesion molecule and polysialic acid in mouse circadian clock function, J. Neurosci. 17 (1997) 5221–5229. [22] K. Shimizu, K. Nagai, H. Nakagawa, An immunotoxin, anti-VIP anyibody-ricin A chain cojugate eliminates neurons in the hypothalamic suprachiasmatic nucleus selectively and abolishes the circadian rhythm of water intake, Brain Res. Bull. 41 (1996) 369–378. [23] F.K. Stephan, I. Zucker, Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions, Proc. Natl. Acad. Sci. USA 69 (1972) 1583–1586. [24] G.T.J. van der Horst, M. Muijtjens, K. Kobayashi, R. Takano, S. Kanno, M. Takao, J. de Wit, A. Verkerk, A.P. Eker, D. Leenen, R. Buijs, D. Bootsma, J.H. Hoeijmakers, A. Yasui, Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms, Nature 398 (1999) 627–630. [25] D.K. Welsh, D.E. Logothetis, M. Meister, S.M. Reppert, Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms, Neuron 14 (1995) 697–706. [26] T. Yagi, S. Aizawa, T. Tokunaga, Y. Shigetani, N. Takeda, Y. Ikawa, A role for Fyn tyrosine kinase in the suckling behaviour of neonatal mice, Nature 366 (1993) 742–745. [27] T. Yagi, Y. Shigetani, N. Okado, T. Tokunaga, Y. Ikawa, S. Aizawa, Regional localization of Fyn in adult brain; studies with mice in which fyn gene was replaced by lacZ, Oncogene 8 (1993) 3343– 3351.