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Light regulates Homer mRNA expression in the rat suprachiasmatic nucleus
Molecular Brain Research 52 Ž1997. 318–322
Short communication
Light regulates Homer mRNA expression in the rat suprachiasmatic nucleus Hwan Tae Park ) , Eun Kyoung Kang, Ki Won Bae Department of Anatomy, College of Medicine, Inje UniÕersity, 633-165, Gaegum-dong, Pusanjin-Gu, Pusan 614-735, South Korea Accepted 2 September 1997
Abstract The hypothalamic suprachiasmatic nucleus ŽSCN. of the mammal is the circadian pacemaker responsible for generation of circadian rhythms. Several immediate-early genes are expressed in the SCN by light stimuli which induce phase shifts of animal activity rhythms. In the present study, we investigated whether Homer, a PDZ-like protein which is rapidly induced following synaptic activation, mRNA expression is regulated by light in rat SCN. Homer mRNA expression in the SCN of rat killed at 4 h after onset of the light and dark phases was very low. One hour light stimuli during the subjective night dramatically induced Homer mRNA expression in the ventrolateral portion of the SCN, whereas light stimuli during the subjective light phase did not. This finding implies that Homer may be involved in the photic entrainment of the circadian clock. q 1997 Elsevier Science B.V. Keywords: Circadian rhythm; Homer mRNA; Immediate-early gene; In situ hybridization; Suprachiasmatic nucleus
Spatial localization and clustering of neuronal membrane receptors are largely dependent on a family of proteins which have the PDZ domains w26x. The PDZ proteins include postsynaptic density ŽPSD.-95rsynapseassociated protein ŽSAP.-90, chapsyn-110rPSD-93, SAP97 and SAP-102 w26x. Recently, it was reported that Homer, a protein which has a PDZ-like domain, selectively binds to metabotropic glutamate receptor ŽmGluR.1a and mGluR5 w1x, which are couple to the stimulation of phospholipase C w20x. Interestingly, Homer mRNA expression is regulated by synaptic activity, and the maximal expression of Homer mRNA occurs within 1 h after neuronal activation like an immediate-early gene ŽIEG. w1x. The circadian pacemaker which drives diurnal behavioral rhythms in mammals is the hypothalamic suprachiasmatic nucleus ŽSCN. w9x. Light entrains mammalian circadian rhythms through the retinohypothalamic tract ŽRHT. that terminates primarily in the ventrolateral portion of the SCN w8,9x. Light induces phase shifts of activity rhythms only during subjective night, and concomitant expression of IEGs like c-fos within the SCN has been thought to be involved in the photic entrainment of circadian rhythms w10,18,21,22,27,28x. Previous studies have suggested that
)
Corresponding author. Fax: q82-51-894-4500
0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 2 9 2 - 1
excitatory amino acids may mediate the effects of light on the circadian pacemaker w2,5,16x, and several excitatory amino acid receptors including mGluR1a and mGluR5 are known to be expressed in the SCN w5,6,15,16,23,24x. Taking these data into account, we investigated whether light regulates Homer mRNA expression in rat SCN using in situ hybridization. Male Sprague–Dawley rats Ž200–230 g. were housed inside a room in which light and temperature Ž22–248C. were tightly controlled. The animals were exposed to a 12-h lightrdark cycle with free access to food and water, and 150 lux of white fluorescent light Žat the level of the cage floor. was presented during the light period. Thirty animals were used in this study, and three animals were killed at each time schedule. After 2 weeks of adaptation to the lighting cycle, animals were killed at circadian time ŽCT. 4 Ž4 h after onset of light. and CT16 Ž4 h after offset of light.. After 2 days, the lighting cycle was discontinued and the rats were kept in continuous darkness for 2 days. Animals were killed without light stimuli at 4 h into the projected light and dark phases of their previous lighting cycle, and then the remaining animals were exposed to light stimuli Ž150 lux. for 1 h. The animals were killed 1, 2 and 4 h after onset of the light stimuli. The brains were rapidly frozen in dry-ice and stored at y708C. Coronal sections Ž12 mm. were made using a cryocut ŽFrigocut, Reichert-Jung, Germany.,
H.T. Park et al.r Molecular Brain Research 52 (1997) 318–322
thaw-mounted onto gelatin-coated slides and stored at y708C. In situ hybridization with 35 S-labeled riboprobe was used to examine the expression of Homer transcripts in the SCN. We also tested the expression of c-fos mRNA in the SCN as control. For making the cRNA probes, we cloned cDNA fragments of rat Homer and c-fos using reversetranscription polymerase chain reaction ŽRT-PCR.. The first strand of cDNA was made from 0.5 mg of rat brain mRNA using a reverse transcription system ŽPromega, USA.. Thirty-five cycles of amplification with specific primers were done. The primers were as follows: y36 to y16 bp and 495 to 516 bp of rat Homer cDNA w1x, 253 to 277 bp and 598 to 620 bp of rat c-fos cDNA w4x. Each reaction cycle consisted of incubations at 948C for 1 min, 558C ŽHomer. or 598C Žc-fos. for 1 min, 728C for 1 min and a final extension at 728C for 5 min. The amplified DNA fragment was subcloned into pGEM-T vector using a TA cloning system ŽPromega., and recombinant clones were sequenced by the dideoxy chain termination method using Sp6 primer ŽUBI, UK.. The plasmids were linearized at SalI or NcoI sites located in the polylinker for the antisense and sense probes, respectively. The antisense and sense cRNA probes were generated by an in vitro transcription kit ŽAmbion, USA. in the presence of w a 35 Sxthio-
319
UTP Ž1000–1300 Cirmmol, Amersham, UK. following manufacturer’s instructions. In situ hybridization was done as previously described w19x. Briefly, after the prehybridization procedure, sections were hybridized with hybridization buffer Ž50% formamide, 0.6 M NaCl, 1 = Denharts, 10 mM Tris, 1 mM EDTA, 10 mM DTT, 0.2 mgrml tRNA, 10% dextran sulfate. containing 1 = 10 7 cpm proberml, coverslipped and incubated overnight at 568C. The next morning, the coverslips were removed, and slides were washed in 2 = SSC followed by digestion in RNase Ž10 mgrml. for 30 min at 368C. The slides were washed to a final stringency of 0.1 = SSC at 548C for 30 min. The sections were dehydrated, dried, and exposed to a Hyperfilm-bmax film ŽAmersham. for 7 days. Slides were then dipped in emulsion solution ŽHypercoat emulsion LM-1, Amersham., stored at 48C for 20 days, developed in Kodak D-19 for 4 min and fixed. The sections were counter-stained with thionin or Harris hematoxylin, coverslipped with Polymount, and examined under light- or darkfield illumination with an Olympus-BX50 microscope. In accordance with the previous report w1x, the antisense cRNA probes to Homer mRNA produced strong hybridization signals in the cerebral cortex, piriform cortex and striatum ŽFig. 1B. whereas the sense probes did not ŽFig.
Fig. 1. Representative autoradiograms illustrating Homer mRNAs expression. A,B: film autoradiograms of a section hybridized with sense ŽA. or antisense probes ŽB. for Homer mRNA. Scale bar: 1 mm. C,D: darkfield photomicrographs showing Homer hybridization signals in the SCN Ždemarcated by white dots. of the rats killed at CT4 ŽC. and CT16 ŽD.. V, 3rd ventricle; OC, optic chiasm. Scale bar: 200 mm.
320
H.T. Park et al.r Molecular Brain Research 52 (1997) 318–322
Fig. 2. Representative autoradiograms illustrating the expression of Homer and c-fos mRNA in the SCN. A,E: darkfield photomicrographs showing Homer hybridization signals in the SCN Ždemarcated by white dots. of the rats killed at subjective dark ŽSD, A. and light ŽSL, E. time without light stimuli. V, 3rd ventricle; OC, optic chiasm. B–D: darkfield micrographs illustrating Homer mRNA expression in the SCN of rat killed at 1 h ŽB., 2 h ŽC. and 4 h ŽD. after onset of light stimuli during the subjective dark phase. F: darkfield micrograph illustrating Homer transcripts in the SCN of rat killed at 1 h after onset of light stimuli during the subjective light phase. Scale bar: 200 mm. G,H: film autoradiograms showing c-fos hybridization signals in a coronal section of the rats receiving 1 h light stimuli during the subjective light ŽG. and dark phases ŽH.. Arrowheads indicate the SCN. Scale bar: 1 mm.
H.T. Park et al.r Molecular Brain Research 52 (1997) 318–322
1A.. The Homer hybridization signals in the anterior hypothalamic regions including the SCN were very weak ŽFig. 1B., and the signals in the SCN were not clearly discernible to that in the optic chiasm at CT4 ŽFig. 1C. and CT16 ŽFig. 1D.. There were no remarkable changes of Homer hybridization signals in the SCN of rat killed at 4 h into the subjective light ŽFig. 2A. and dark phases ŽFig. 2E.. One hour light stimuli during the subjective dark phase dramatically induced Homer mRNA expression in the SCN ŽFig. 2B.. At mid to caudal levels of the SCN, the Homer hybridization signals were primarily found in its ventrolateral portion, whereas at rostral SCN, Homer mRNA expression was found in the ventral portion. The distinct expression of Homer mRNA in the SCN continued at 2 h after onset of the light stimuli ŽFig. 2C.. At 4 h after onset of the light stimuli, Homer mRNA expression was returned to the basal level ŽFig. 2D.. However, the increase of Homer mRNA expression in the SCN was not observed at 1, 2, and 4 h after onset of the light stimuli during the subjective light phase ŽFig. 2F.. As a molecular marker of cellular activation by light stimuli in the SCN, we investigated c-fos mRNA expression in adjacent sections. c-fos mRNA expression was rapidly induced in the SCN by 1 h light stimuli during the subjective dark phase, and there was no c-fos mRNA induction in other conditions ŽFig. 2G,H.. Previous studies have demonstrated that light induces the expression of several IEGs in the SCN only during the circadian time when light is capable of shifting the circadian rhythms w10,18,21,22,27x. In addition, it was reported that the blockage of c-fos and jun-B expression in the SCN by antisense oligonucleotides inhibits light-induced phase shifts w28x. Therefore, it seems that the transcriptional modulation of IEGs might be an important element of circadian timekeeping mechanisms. In the present study, we clearly have demonstrated that Homer mRNA expression is rapidly induced in the SCN by light stimuli which cause phase shifts of activity rhythms, and that its expression is primarily localized in the ventrolateral subdivision of the SCN where induction of c-fos mRNA expression is found. This finding indicates that the photic induction of Homer mRNA expression in the SCN may be involved in light-induced phase shifts. The prerequisite involvement of Homer induction in the photic entrainment could be further ascertained by specifically blocking Homer expression in the SCN with antisense oligonucleotides. Homer, a putative member of the PDZ proteins, is likely to play a role in the spatial targeting of mGluR1a and mGluR5 by binding to the C-terminals of the receptors w1x. The mGluR1 and mGluR5 are known as type I mGluR w20x. The type I mGluR is functionally coupled to the stimulation of phosphatidylinositol hydrolysis, which induces the mobilization of calcium from intracellular calcium stores w20x. Several previous studies have demonstrated that type I mGluR and their mRNAs are found in
321
the SCN w15,23,24x, and that the signals through mGluRs modulate the cellular excitability and calcium signaling within the SCN w7,25x. In addition, the basal phosphoinositide turnover within the SCN has been thought to be important in the maintenance of spontaneous neuronal activities w17x, because the disruption of the phosphoinositide turnover by lithium lengthens the period of free-running activity rhythms w11,13x and suppresses neuronal firings in the SCN w12x. Therefore, it may be possible that Homer induction by light in the SCN increases the membrane targeting of type I mGluR, and long-lasting changes in cellular excitability and calcium level may occur by Homer. These presumptive changes of cellular signaling through phosphoinositide-linked mGluRs within the SCN by light-induced Homer may affect the spontaneous neuronal firings, and this may be involved in the phase resetting of the circadian pacemaker. It has been reported that c-fos gene expression is not induced by non-photic stimuli which cause phase shifts w3,14x. For example, carbachol, an cholinergic agonist, which has similar phase dependence to light for phase shifts induces phase advances or delays without accompanying c-fos induction in the SCN w3x. Therefore, it is not likely that c-fos expression in the SCN is a necessary component of all phase-shifting processes. Elucidating the association between non-photic stimuli which induce phase shifts and Homer mRNA induction may provide further insight into the roles of Homer in the circadian pacemaker. The majority of light-induced IEGs in the SCN is transcription factors, whereas the induction of genes which directly alters characteristics of membrane receptors has not been reported. As far as we know, Homer might be the first example in the SCN. Additional studies are necessary to know the role of Homer within the mammalian biological clock.
Acknowledgements We thank to Dr. Sik Yoon ŽDong-A University., Sun Yong Baek ŽPusan National University. for technical help and discussion on this manuscript.
References w1x P.R. Brakeman, A.A. Lanahan, R. O’Brien, K. Roche, C.A. Barnes, R.L. Huganir, P.F. Worley, Homer: a protein that selectively binds metabotropic glutamate receptors, Nature 386 Ž1997. 284–287. w2x G.M. Cahill, M. Menaker, Effects of excitatory amino acid receptor antagonists and agonists on suprachiasmatic nucleus response to retinohypothalamic tract volleys, Brain Res. 479 Ž1989. 76–82. w3x C.S. Colwell, C.M. Kaufman, M. Menaker, Phase-shifting mechanisms in the mammalian circadian system: new light on the carbachol paradox, J. Neurosci. 13 Ž1993. 1454–1459. w4x T. Curran, M.B. Gordon, K.L. Rubino, L.C. Sambucetti, Isolation and characterization of the c-fos cDNA and analysis of post-translational modification in vitro, Oncogene 2 Ž1987. 79–84.
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w5x F.J.P. Ebling, The role of glutamate in the photic regulation of the suprachiasmatic nucleus, Prog. Neurobiol. 50 Ž1996. 109–132. w6x R.L. Gannon, M.A. Rea, In situ hybridization of antisense mRNA oligonucleotides for AMPA, NMDA and metabotropic glutamate receptor subtypes in the rat suprachiasmatic nucleus at different phases of the circadian cycle, Mol. Brain Res. 23 Ž1994. 338–344. w7x L.L. Haak, H.C. Heller, A.N. Van den pol, Metabotropic glutamate receptor activation modulates kainate and serotonin calcium response in astrocytes, J. Neurosci. 17 Ž1997. 1825–1837. w8x R.F. Johnson, L.P. Morin, R.Y. Moore, Retinohypothalamic projections in the hamster and rat demonstrated using cholera-toxin, Brain Res. 462 Ž1988. 301–312. w9x D.C. Klein, R.Y. Moore, S.M. Reppert, Suprachiasmatic Nucleus: The Mind’s Clock, Oxford University Press, New York, 1991. w10x J.M. Kornhauser, D.E. Nelson, K.E. Mayo, J.S. Takahashi, Regulation of jun-B messenger RNA and AP-1 activity by light and a circadian clock, Science 255 Ž1992. 1581–1584. w11x D.F. Kripke, V.G. Wyborney, Lithium slows rat circadian activity rhythms, Life Sci. 26 Ž1980. 1319–1321. w12x R. Mason, S.M. Biello, A neurophysiological study of a lithium-sensitive phosphoinositide system in the hamster suprachiasmatic ŽSCN. biological clock in vitro, Neurosci. Lett. 144 Ž1992. 135–138. w13x D.L. McEachron, D.F. Kripke, R. Hawkins, E. Haus, D. Pavlinac, L. Deftos, Lithium delays biochemical rhythms in rats, Neuropsychobiology 8 Ž1982. 12–29. w14x S. Mead, F.J.P. Ebling, E.S. Maywood, T. Humby, T. Herbert, M.H. Hastings, A nonphotic stimulus causes instantaneous phase advances of the light-entrainable circadian oscillator of the syrian hamster but does not induce the expression of c-fos in the suprachiasmatic nuclei, J. Neurosci. 12 Ž1992. 2516–2522. w15x G. Mick, R. Yoshimura, K. Ohno, H. Kiyama, M. Tohyama, The messenger RNAs encoding metabotropic glutamate receptor subtypes are expressed in different neuronal subpopulations of the rat suprachiasmatic nucleus, Neuroscience 66 Ž1995. 161–173. w16x J.D. Mikkelsen, P.J. Larsen, G. Mick, N. Vrang, F.J. Ebling, E.S. Maywood, M.H. Hasting, M. Moller, Gating of retinal inputs through the suprachiasmatic nucleus: role of excitatory transmission, Neurochem. Int. 27 Ž1995. 263–272.
w17x J.J. Nadakavukaren, D.K. Welsh, S.M. Reppert, Aluminum fluoride reveals a phosphoinositide system within the suprachiasmatic region of rat hypothalamus, Brain Res. 507 Ž1990. 181–188. w18x H.T. Park, S.Y. Baek, B.S. Kim, J.B. Kim, J.J. Kim, Profile of Fos-like immunoreactivity induction by light stimuli in the intergeniculate leaflet is different from that of the suprachiasmatic nucleus, Brain Res. 610 Ž1993. 334–339. w19x H.T. Park, S.Y. Baek, B.S. Kim, J.B. Kim, J.J. Kim, Developmental expression of RZRb, a putative nuclear-melatonin receptor mRNA in the suprachiasmatic nucleus of the rat, Neurosci. Lett. 217 Ž1996. 17–20. w20x J.P. Pin, R. Duvoisin, The metabotropic glutamate receptors: structure and functions, Neuropharmacology 34 Ž1995. 1–26. w21x B. Rusak, L. McNaughton, H.A. Robertson, S.P. Hunt, Circadian variation in photic regulation of immediate-early gene mRNAs in rat suprachiasmatic nucleus cells, Mol. Brain Res. 14 Ž1992. 124–130. w22x B. Rusak, H.A. Robertson, W. Wisden, S.P. Hunt, Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus, Science 248 Ž1990. 1237–1240. w23x A.N. Van den pol, L. Kogelman, P. Ghosh, P. Liljelund, C. Blackstone, Developmental regulation of the hypothalamic metabotropic glutamate receptor mGluR1, J. Neurosci. 14 Ž1994. 3816–3834. w24x A.N. Van den pol, C. Romano, P. Ghosh, Metabotropic glutamate receptor mGluR5: subcellular distribution and developmental expression in hypothalamus, J. Comp. Neurol. 362 Ž1995. 134–150. w25x G. Scott, B. Rusak, Activation of hamster suprachiasmatic neurons in vitro via metabotropic glutamate receptor, Neuroscience 71 Ž1996. 533–541. w26x M. Sheng, PDZs and receptorrchannel clustering: rounding up the latest suspects, Neuron 17 Ž1996. 575–578. w27x J. Takeuchi, W. Shannon, N. Aronin, W.J. Schwartz, Compositional changes of AP-1 DNA-binding proteins are regulated by light in a mammalian circadian clock, Neuron 11 Ž1993. 825–836. w28x F. Wollnik, W. Brysch, E. Uhlmann, F. Gillardon, R. Bravo, M. Zimmermann, K.H. Schlingensiepen, T. Herdegen, Block of c-fos and jun-B expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock, Eur. J. Neurosci. 7 Ž1995. 388–393.
Short communication
Light regulates Homer mRNA expression in the rat suprachiasmatic nucleus Hwan Tae Park ) , Eun Kyoung Kang, Ki Won Bae Department of Anatomy, College of Medicine, Inje UniÕersity, 633-165, Gaegum-dong, Pusanjin-Gu, Pusan 614-735, South Korea Accepted 2 September 1997
Abstract The hypothalamic suprachiasmatic nucleus ŽSCN. of the mammal is the circadian pacemaker responsible for generation of circadian rhythms. Several immediate-early genes are expressed in the SCN by light stimuli which induce phase shifts of animal activity rhythms. In the present study, we investigated whether Homer, a PDZ-like protein which is rapidly induced following synaptic activation, mRNA expression is regulated by light in rat SCN. Homer mRNA expression in the SCN of rat killed at 4 h after onset of the light and dark phases was very low. One hour light stimuli during the subjective night dramatically induced Homer mRNA expression in the ventrolateral portion of the SCN, whereas light stimuli during the subjective light phase did not. This finding implies that Homer may be involved in the photic entrainment of the circadian clock. q 1997 Elsevier Science B.V. Keywords: Circadian rhythm; Homer mRNA; Immediate-early gene; In situ hybridization; Suprachiasmatic nucleus
Spatial localization and clustering of neuronal membrane receptors are largely dependent on a family of proteins which have the PDZ domains w26x. The PDZ proteins include postsynaptic density ŽPSD.-95rsynapseassociated protein ŽSAP.-90, chapsyn-110rPSD-93, SAP97 and SAP-102 w26x. Recently, it was reported that Homer, a protein which has a PDZ-like domain, selectively binds to metabotropic glutamate receptor ŽmGluR.1a and mGluR5 w1x, which are couple to the stimulation of phospholipase C w20x. Interestingly, Homer mRNA expression is regulated by synaptic activity, and the maximal expression of Homer mRNA occurs within 1 h after neuronal activation like an immediate-early gene ŽIEG. w1x. The circadian pacemaker which drives diurnal behavioral rhythms in mammals is the hypothalamic suprachiasmatic nucleus ŽSCN. w9x. Light entrains mammalian circadian rhythms through the retinohypothalamic tract ŽRHT. that terminates primarily in the ventrolateral portion of the SCN w8,9x. Light induces phase shifts of activity rhythms only during subjective night, and concomitant expression of IEGs like c-fos within the SCN has been thought to be involved in the photic entrainment of circadian rhythms w10,18,21,22,27,28x. Previous studies have suggested that
)
Corresponding author. Fax: q82-51-894-4500
0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 2 9 2 - 1
excitatory amino acids may mediate the effects of light on the circadian pacemaker w2,5,16x, and several excitatory amino acid receptors including mGluR1a and mGluR5 are known to be expressed in the SCN w5,6,15,16,23,24x. Taking these data into account, we investigated whether light regulates Homer mRNA expression in rat SCN using in situ hybridization. Male Sprague–Dawley rats Ž200–230 g. were housed inside a room in which light and temperature Ž22–248C. were tightly controlled. The animals were exposed to a 12-h lightrdark cycle with free access to food and water, and 150 lux of white fluorescent light Žat the level of the cage floor. was presented during the light period. Thirty animals were used in this study, and three animals were killed at each time schedule. After 2 weeks of adaptation to the lighting cycle, animals were killed at circadian time ŽCT. 4 Ž4 h after onset of light. and CT16 Ž4 h after offset of light.. After 2 days, the lighting cycle was discontinued and the rats were kept in continuous darkness for 2 days. Animals were killed without light stimuli at 4 h into the projected light and dark phases of their previous lighting cycle, and then the remaining animals were exposed to light stimuli Ž150 lux. for 1 h. The animals were killed 1, 2 and 4 h after onset of the light stimuli. The brains were rapidly frozen in dry-ice and stored at y708C. Coronal sections Ž12 mm. were made using a cryocut ŽFrigocut, Reichert-Jung, Germany.,
H.T. Park et al.r Molecular Brain Research 52 (1997) 318–322
thaw-mounted onto gelatin-coated slides and stored at y708C. In situ hybridization with 35 S-labeled riboprobe was used to examine the expression of Homer transcripts in the SCN. We also tested the expression of c-fos mRNA in the SCN as control. For making the cRNA probes, we cloned cDNA fragments of rat Homer and c-fos using reversetranscription polymerase chain reaction ŽRT-PCR.. The first strand of cDNA was made from 0.5 mg of rat brain mRNA using a reverse transcription system ŽPromega, USA.. Thirty-five cycles of amplification with specific primers were done. The primers were as follows: y36 to y16 bp and 495 to 516 bp of rat Homer cDNA w1x, 253 to 277 bp and 598 to 620 bp of rat c-fos cDNA w4x. Each reaction cycle consisted of incubations at 948C for 1 min, 558C ŽHomer. or 598C Žc-fos. for 1 min, 728C for 1 min and a final extension at 728C for 5 min. The amplified DNA fragment was subcloned into pGEM-T vector using a TA cloning system ŽPromega., and recombinant clones were sequenced by the dideoxy chain termination method using Sp6 primer ŽUBI, UK.. The plasmids were linearized at SalI or NcoI sites located in the polylinker for the antisense and sense probes, respectively. The antisense and sense cRNA probes were generated by an in vitro transcription kit ŽAmbion, USA. in the presence of w a 35 Sxthio-
319
UTP Ž1000–1300 Cirmmol, Amersham, UK. following manufacturer’s instructions. In situ hybridization was done as previously described w19x. Briefly, after the prehybridization procedure, sections were hybridized with hybridization buffer Ž50% formamide, 0.6 M NaCl, 1 = Denharts, 10 mM Tris, 1 mM EDTA, 10 mM DTT, 0.2 mgrml tRNA, 10% dextran sulfate. containing 1 = 10 7 cpm proberml, coverslipped and incubated overnight at 568C. The next morning, the coverslips were removed, and slides were washed in 2 = SSC followed by digestion in RNase Ž10 mgrml. for 30 min at 368C. The slides were washed to a final stringency of 0.1 = SSC at 548C for 30 min. The sections were dehydrated, dried, and exposed to a Hyperfilm-bmax film ŽAmersham. for 7 days. Slides were then dipped in emulsion solution ŽHypercoat emulsion LM-1, Amersham., stored at 48C for 20 days, developed in Kodak D-19 for 4 min and fixed. The sections were counter-stained with thionin or Harris hematoxylin, coverslipped with Polymount, and examined under light- or darkfield illumination with an Olympus-BX50 microscope. In accordance with the previous report w1x, the antisense cRNA probes to Homer mRNA produced strong hybridization signals in the cerebral cortex, piriform cortex and striatum ŽFig. 1B. whereas the sense probes did not ŽFig.
Fig. 1. Representative autoradiograms illustrating Homer mRNAs expression. A,B: film autoradiograms of a section hybridized with sense ŽA. or antisense probes ŽB. for Homer mRNA. Scale bar: 1 mm. C,D: darkfield photomicrographs showing Homer hybridization signals in the SCN Ždemarcated by white dots. of the rats killed at CT4 ŽC. and CT16 ŽD.. V, 3rd ventricle; OC, optic chiasm. Scale bar: 200 mm.
320
H.T. Park et al.r Molecular Brain Research 52 (1997) 318–322
Fig. 2. Representative autoradiograms illustrating the expression of Homer and c-fos mRNA in the SCN. A,E: darkfield photomicrographs showing Homer hybridization signals in the SCN Ždemarcated by white dots. of the rats killed at subjective dark ŽSD, A. and light ŽSL, E. time without light stimuli. V, 3rd ventricle; OC, optic chiasm. B–D: darkfield micrographs illustrating Homer mRNA expression in the SCN of rat killed at 1 h ŽB., 2 h ŽC. and 4 h ŽD. after onset of light stimuli during the subjective dark phase. F: darkfield micrograph illustrating Homer transcripts in the SCN of rat killed at 1 h after onset of light stimuli during the subjective light phase. Scale bar: 200 mm. G,H: film autoradiograms showing c-fos hybridization signals in a coronal section of the rats receiving 1 h light stimuli during the subjective light ŽG. and dark phases ŽH.. Arrowheads indicate the SCN. Scale bar: 1 mm.
H.T. Park et al.r Molecular Brain Research 52 (1997) 318–322
1A.. The Homer hybridization signals in the anterior hypothalamic regions including the SCN were very weak ŽFig. 1B., and the signals in the SCN were not clearly discernible to that in the optic chiasm at CT4 ŽFig. 1C. and CT16 ŽFig. 1D.. There were no remarkable changes of Homer hybridization signals in the SCN of rat killed at 4 h into the subjective light ŽFig. 2A. and dark phases ŽFig. 2E.. One hour light stimuli during the subjective dark phase dramatically induced Homer mRNA expression in the SCN ŽFig. 2B.. At mid to caudal levels of the SCN, the Homer hybridization signals were primarily found in its ventrolateral portion, whereas at rostral SCN, Homer mRNA expression was found in the ventral portion. The distinct expression of Homer mRNA in the SCN continued at 2 h after onset of the light stimuli ŽFig. 2C.. At 4 h after onset of the light stimuli, Homer mRNA expression was returned to the basal level ŽFig. 2D.. However, the increase of Homer mRNA expression in the SCN was not observed at 1, 2, and 4 h after onset of the light stimuli during the subjective light phase ŽFig. 2F.. As a molecular marker of cellular activation by light stimuli in the SCN, we investigated c-fos mRNA expression in adjacent sections. c-fos mRNA expression was rapidly induced in the SCN by 1 h light stimuli during the subjective dark phase, and there was no c-fos mRNA induction in other conditions ŽFig. 2G,H.. Previous studies have demonstrated that light induces the expression of several IEGs in the SCN only during the circadian time when light is capable of shifting the circadian rhythms w10,18,21,22,27x. In addition, it was reported that the blockage of c-fos and jun-B expression in the SCN by antisense oligonucleotides inhibits light-induced phase shifts w28x. Therefore, it seems that the transcriptional modulation of IEGs might be an important element of circadian timekeeping mechanisms. In the present study, we clearly have demonstrated that Homer mRNA expression is rapidly induced in the SCN by light stimuli which cause phase shifts of activity rhythms, and that its expression is primarily localized in the ventrolateral subdivision of the SCN where induction of c-fos mRNA expression is found. This finding indicates that the photic induction of Homer mRNA expression in the SCN may be involved in light-induced phase shifts. The prerequisite involvement of Homer induction in the photic entrainment could be further ascertained by specifically blocking Homer expression in the SCN with antisense oligonucleotides. Homer, a putative member of the PDZ proteins, is likely to play a role in the spatial targeting of mGluR1a and mGluR5 by binding to the C-terminals of the receptors w1x. The mGluR1 and mGluR5 are known as type I mGluR w20x. The type I mGluR is functionally coupled to the stimulation of phosphatidylinositol hydrolysis, which induces the mobilization of calcium from intracellular calcium stores w20x. Several previous studies have demonstrated that type I mGluR and their mRNAs are found in
321
the SCN w15,23,24x, and that the signals through mGluRs modulate the cellular excitability and calcium signaling within the SCN w7,25x. In addition, the basal phosphoinositide turnover within the SCN has been thought to be important in the maintenance of spontaneous neuronal activities w17x, because the disruption of the phosphoinositide turnover by lithium lengthens the period of free-running activity rhythms w11,13x and suppresses neuronal firings in the SCN w12x. Therefore, it may be possible that Homer induction by light in the SCN increases the membrane targeting of type I mGluR, and long-lasting changes in cellular excitability and calcium level may occur by Homer. These presumptive changes of cellular signaling through phosphoinositide-linked mGluRs within the SCN by light-induced Homer may affect the spontaneous neuronal firings, and this may be involved in the phase resetting of the circadian pacemaker. It has been reported that c-fos gene expression is not induced by non-photic stimuli which cause phase shifts w3,14x. For example, carbachol, an cholinergic agonist, which has similar phase dependence to light for phase shifts induces phase advances or delays without accompanying c-fos induction in the SCN w3x. Therefore, it is not likely that c-fos expression in the SCN is a necessary component of all phase-shifting processes. Elucidating the association between non-photic stimuli which induce phase shifts and Homer mRNA induction may provide further insight into the roles of Homer in the circadian pacemaker. The majority of light-induced IEGs in the SCN is transcription factors, whereas the induction of genes which directly alters characteristics of membrane receptors has not been reported. As far as we know, Homer might be the first example in the SCN. Additional studies are necessary to know the role of Homer within the mammalian biological clock.
Acknowledgements We thank to Dr. Sik Yoon ŽDong-A University., Sun Yong Baek ŽPusan National University. for technical help and discussion on this manuscript.
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