- Email: [email protected]
Aging selectively suppresses vasoactive intestinal peptide messenger RNA expression in the suprachiasmatic nucleus of the Syrian hamster
Molecular Brain Research 87 (2001) 196–203 www.elsevier.com / locate / bres
Research report
Aging selectively suppresses vasoactive intestinal peptide messenger RNA expression in the suprachiasmatic nucleus of the Syrian hamster Marilyn J. Duncan*, Jana M. Herron, Stephanie A. Hill Department of Anatomy and Neurobiology, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536 -0298, USA Accepted 26 December 2000
Abstract Aging leads to many changes in the expression of circadian rhythms, including reduced amplitude, altered relationship to the environmental illumination cycle, and reduced sensitivity to phase resetting signals. Neuropeptide synthesizing neurons in the suprachiasmatic nucleus (SCN), the principal circadian pacemaker in mammals, play a role in regulating pacemaker function and in coupling the pacemaker to overt circadian rhythms. Aging may alter the activity of neuropeptide neurons in the SCN, which could be reflected in changes in mRNA expression. Therefore, this study investigated whether aging alters the level or rhythm of expression of neuropeptide mRNAs in the SCN of male Syrian hamsters, a well established model for the study of age-related changes in circadian rhythms. Three age groups of hamsters ( young [3–5 months old], middle-aged [12–15 months old] and old [19–22 months old] were sacrificed at five times of day. Their brains were dissected and sections through the suprachiasmatic nucleus were prepared and used for in situ hybridization for mRNAs for vasoactive intestinal peptide (VIP), arginine vasopressin (AVP) and somatostatin (SS). Aging selectively decreased the SCN expression of VIP mRNA without affecting AVP mRNA or SS mRNA. Also, only AVP mRNA expression exhibited a robust 24-h rhythm, in contrast to previous findings in other species that VIP mRNA and SS mRNA, as well as AVP mRNA, exhibit 24-h rhythms in the SCN. The present findings suggest that age-related reductions in VIP mRNA expression may contribute to the alterations in entrainment and attenuated sensitivity to phase resetting signals that are characteristic of aging. Furthermore, the results demonstrate that neuropeptide gene expression in the SCN is differentially regulated by aging and varies among species. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: In situ hybridization; Circadian rhythm; Vasoactive intestinal peptide; Vasopressin; Somatostatin
1. Introduction Circadian rhythms govern the behavior and physiological processes of virtually all organisms. By facilitating adjustments to the daily changes in illumination, temperature and other conditions, circadian rhythms enhance survival. In fact, robust and coordinated circadian rhythms favor good health, longevity and optimal cognitive ability in a variety of species, including humans [10,20,38,39,58]. Aging brings about many changes in circadian rhythms. One age-related change is a reduction in amplitude, a common feature of melatonin rhythms in old rodents *Corresponding author. Tel.: 11-859-323-4718; fax: 11-859-3235946. E-mail address: [email protected] (M.J. Duncan).
[43,44]. During aging, circadian rhythms also become disrupted or fragmented, and their phase relationship to environmental time signals is altered [11,31,40,48,61]. For example, locomotor activity rhythms in old hamsters exhibit fragmentation and an earlier onset of the active phase in relationship to the onset of darkness [11,40,48,61]. Also, circulating cortisol rhythms in old men are dampened and phase-advanced compared to the rhythms in young men [51]. During aging, the circadian pacemaker becomes more resistant to phase resetting signals, such as injections of triazolam or 8-hydroxy-2-(din-propylamino)tetralin (8-OH-DPAT) [41,55]. Finally, aging may induce a dissociation of circadian rhythms within an individual [47]. Studies of circadian rhythms in drinking behavior, body temperature, and neuronal activity in old rats showed that some individuals exhibited a loss of
0169-328X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00015-8
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
all three rhythms, while other experienced a loss of only one or two of these rhythms [47]. Thus, aging disrupts the coordinated expression of circadian rhythms. The neural basis for these age-related alterations in circadian rhythms may involve functional changes in the circadian pacemaker in the hypothalamic suprachiasmatic nucleus (SCN). The SCN responds to timing signals received from afferent pathways and drives overt circadian rhythms through its efferent connections [25,37,57]. The SCN consists of neurons that synthesize a variety of neuropeptides, including vasoactive intestinal peptide (VIP), arginine vasopressin (AVP), and somatostatin (SS), and appear to play multiple roles in circadian timekeeping [7,23,52]. VIP and AVP have been identified in several efferent pathways from the SCN and thus may be involved in the regulation of overt circadian rhythms [25,57]. AVP and SS have been shown to modulate the function of the SCN. AVP neurons provide endogenous excitatory tone to the SCN [34]. Somatostatin administration inhibits SCN neuronal firing in vitro [22]. Somatostatin neurons are also involved in regulating the phase of circadian rhythms. Depletion of somatostatin induces phase advances in locomotor activity rhythms and SCN electrical activity rhythms, and permits rhythmic release of VIP from the SCN in vitro [15,16]. The demonstration that VIP neurons receive synaptic inputs from the retinohypothalamic tract, the geniculohypothalamic tract and the serotonergic projection from the median raphe nucleus suggests that the VIP neurons are involved in entrainment of the circadian pacemaker [18,19,21,29]. This concept is supported by findings that VIP microinjections in the SCN region induce phase-dependent phase shifts in locomotor activity rhythms [1,2,42]. Because neuropeptide neurons play important roles in regulating circadian timekeeping, alterations in their function may contribute to age-related changes in circadian rhythms. For example, aging modulates the VIP mRNA rhythm in the rat SCN [28,30]. However, it remains to be determined if aging changes the rhythmic expression of VIP mRNA or other neuropeptide mRNA in the SCN of the Syrian hamster, a well-characterized model of agerelated changes in behavioral circadian rhythms [45,54,62]. Therefore, the current study tested the hypothesis that aging alters the SCN rhythmic expression of messenger RNA for VIP, AVP or SS in Syrian hamsters. In order to assess each mRNA species at the site of greatest expression within the SCN, a preliminary study was conducted to delineate the regional distribution of VIP mRNA, AVP mRNA and SS mRNA.
2. Materials and methods
2.1. Animals and tissue preparation Male Syrian hamsters obtained from Harlan Labs (Har-
197
lan, HsdHan:AURA) were maintained in the Department of Laboratory Animal Research at the University of Kentucky under a 14-h light, 10-h dark photoperiod (lights on from 06:00 to 20:00 h) for at least 1–2 weeks. Food (Teklad, Amway) and water were available continuously. Three age groups were studied: young (3–5 months old), middle-aged (12–15 months old) and old (19–22 months old). The hamsters were sacrificed at five different times of day, 02:00, 07:00, 12:00, 17:00, and 22:00 h (N56–9 / age per time). A dim red light was used when hamsters were euthanized during the dark phase. The brains were dissected, frozen on crushed dry ice, and stored at 2808C. Coronal sections (12 mm thick) through the SCN were cut on a cryostat and mounted on positively charged slides. The slide-mounted sections were stored at 2808C before in situ hybridization was conducted.
2.2. In situ hybridization In situ hybridization was performed using oligodeoxyribonucleotides (oligos) which were 39-end labeled with [ 35 S]dATP using terminal deoxynucleotidyl transferase. The oligos selected were based on the sequence of the rat genes for AVP, VIP, and SS, because these genes have not been cloned and sequenced from Syrian hamsters. For each oligo, the sequence shows at most very low complementarity to any other reported genes besides the gene coding for the protein of interest, based on analyses with the BLAST program [3]. Previous in situ hybridization studies of hamster brain sections with these oligos showed that the distribution of the specific signal matches the neuroanatomical distribution of cell bodies synthesizing the peptide and that background labelling is low [12,13,49]. The procedures for in situ hybridization for AVP mRNA or somatostatin mRNA each used one specific oligo and were conducted as described previously [12,13]. The AVP oligo (59-TAG /ACC / CGG / GGC / TTG / GCA / GAA / TCC /ACG / GAC / TCT / TGT / GTC / CCA / GCC /AGC - 39) was complementary to the mRNA encoding the last 16 amino acids of the glycopeptide region of rat AVP [60]. The nucleotide sequence of the somatostatin oligo was: 59-GGA / TGT / GAA / TGT / CTT / CCA / GAA / GAA /ATT / CTT / GCA / GCC /AGC / TTT / GCG / TTC-39). The in situ hybridization procedure for VIP mRNA used a mixture of four oligos, as described previously for detection in the Syrian hamster SCN [49]. This method was used because preliminary studies using a single oligo did not generate a detectable signal. Thus, the VIP mRNA appeared to be in relatively low abundance in the Syrian hamster SCN, and the simultaneous use of four oligos amplified the signal. The nucleotide sequences of the VIP oligos, which corresponded to conserved regions of the VIP gene, were as follows. VIP oligo [1 : 59-ACA / TCA / TAA / TAG / GGC / GTG / TCA / TTC / TCC / GCT /AAG / GCA / TTC / TGC /AAG /ATG / TCA-39; VIP oligo[2 : 59AGT / CTA / CTG / TAG / TCG / CTG / GTG /AAA /ACT /
198
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
CCA / TCA / GCA / TGC / CTG / GCA / TTT-39, VIP oligo [3 : 59-AAG / GCG / GGT / GTA / GTT /ATC / TGT / GAA / GAC / TGC /ATC /AGA / GTG / TCG / TTT / GAC-39, and VIP oligo [4 : 59-CTC / CTC / TTC / CCA / TTT /AGA / ATG / GAG / TTC /AAG / TAT / TTC / TTC /ACA / GCC / ATT / TGC / TTT / CT-39. The in situ hybridization procedure included pretreatment of the slides to reduce background, as described previously [13], followed by hybridization with 3 pmol / ml of a mixture of the [ 35 S]dATP-labeled VIP oligos at 378C in a humid chamber for approximately 20 h. The hyridization buffer (hybridization cocktail, Amresco Inc., Solon, OH) contained 50% formamide, 25 mM sodium phosphate, 60 mM sodium citrate (SSC), 50% Denhardt’s solution (0.02% each of Ficoll, polyvinylpyrolidone, and bovine serum albumin), 10% dextran sulfate, 100 mM dithiothreitol (DTT), 250 mg / ml yeast transfer RNA and 500 mg / ml denatured salmon sperm DNA. Following hybridization, the tissue sections were washed in 13 SSC containing 10 mM sodium thiosulfate at 558C for 1 h (fresh solution every 15 min, agitated every 5 min) and at room temperature for 1 h (agitated every 15 min), dipped briefly in sterile distilled water and 95% ethanol, and quickly air-dried. Autoradiograms of the SCN were generated by exposing the tissue sections to X-ray film (Hyperfilm b-max, Amersham Corp.) for approximately 3 days (VIP mRNA and AVP mRNA) or 10 days (SS mRNA). In order to ascertain that the autoradiographic images did not represent film saturation, radioactive standards ( 14 C, American Radiolabeled Chemicals, St Louis, MO) were included in each cassette. The X-ray films were processed using D-19 developer and rapid fixer (Eastman Kodak, Rochester, NY). In order to be able to assess cellular levels of mRNA expression when significant differences in SCN levels of mRNA expression were evident from the film autoradiograms, the slides were dipped in photographic emulsion (NTB2, Eastman Kodak). The emulsion was developed after 10 (VIP mRNA and AVP mRNA) or 30 days (SS mRNA) and the slides were counterstained with cresyl violet. The hybridization signal of the film autoradiograms was assessed by semi-quantitative analysis using computerassisted microdensitometry, as described previously [13]. The hybridization signal, defined as the difference between the relative optical density over the SCN and an adjacent brain region, was averaged for four replicates for each animal. In order to ascertain that the autoradiographic signal corresponded to labeling of the SCN, the autoradiogram was compared with the Nissl-stained SCN. For assessment of cellular levels of mRNA expression, computerized image analysis was conducted on the video images of the emulsion autoradiograms from the same sections that generated the film autoradiograms. The difference between the mean grain area per cell in the SCN and the mean grain area per cell in an adjacent region was determined.
2.3. Statistical analyses The effect of region within the SCN was determined by one-way analysis of variance (ANOVA). The effects of aging and time of day were assessed by two-way ANOVA. In the case of significant (P,0.05) F-values, post-hoc comparisons were made between groups with the Newman–Keuls test.
3. Results
3.1. Regional distribution of neuropeptide mRNA expression in the SCN The Syrian hamster SCN expressed all three mRNA species investigated: VIP, AVP and SS (Fig. 1). VIP mRNA expression, which was localized to the ventral SCN, exhibited a statistically significant variation throughout the rostral to caudal axis of the SCN, characterized by greatest expression in the middle and no detectable expression at either pole (Fig. 2). The expression of AVP mRNA and SS mRNA expression in the dorsal region of the SCN were homogeneous along the rostral to caudal axis of the SCN (Fig. 2).
3.2. Effect of aging on neuropeptide mRNA expression in the SCN VIP mRNA expression in the SCN was significantly affected by age (P,0.05), but not by time of day (P5 0.84) (Fig. 3). There was no significant interaction effect between age and time of day on VIP mRNA expression (P50.58). Collapsing the data across time of day revealed that VIP mRNA expression in the SCN was decreased in middle-aged and old hamsters by 15 and 28%, respectively, as compared to young hamsters (Fig. 4). Analysis of the emulsion autoradiograms revealed that the average area of grains per cell was significantly reduced by aging (P,0.05), but was not affected by time of day (P50.44), nor was there a significant interaction effect (P50.73). When the data for the various times were combined, the average grain area per cell in the SCN was decreased in the middle-aged and old hamsters by approximately 22 and 37%, respectively, as compared to young hamsters (Fig. 4). AVP mRNA expression in the SCN was not affected by age (P50.97), but varied significantly with time of day (P,0.001, Fig. 5). AVP mRNA expression was highest during the middle of the light phase (12:00 and 17:00 h). There was no significant interaction effect between age and time of day on AVP mRNA expression (P50.37). In contrast to VIP mRNA and AVP mRNA, neither age (P50.24) nor time of day (P,0.51) significantly affected SS mRNA expression in the SCN (Fig. 6).
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
199
Fig. 2. Regional distribution of neuropeptide mRNA expression across the rostral to caudal axis of the SCN. The bars represent the mean6S.E.M. of the hybridization signal (dpms) for each region (N56 each). AVP mRNA and somatostatin mRNA were broadly distributed throughout the SCN; in contrast, VIP mRNA was more restricted.
Fig. 3. Twenty-four hour profiles of VIP mRNA expression in the SCN. The bars depict the mean6S.E.M. of the hybridization signal (dpms). VIP mRNA expression was significantly decreased by aging but was not affected by time of day.
Fig. 1. Prints of X-ray film autoradiograms showing the neuropeptide mRNA expression in the SCN. VIP mRNA expression is restricted to the ventral SCN, while AVP mRNA and SS mRNA are localized to the dorsal SCN.
Fig. 4. The effect of aging on VIP mRNA expression in the SCN. Each bar represents the mean6S.E.M. of the hybridization signal (dpms) for all time points within each age group.
200
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
Fig. 5. Twenty-four hour profiles of AVP mRNA expression in the SCN. Values represent the mean6S.E.M. of the hybridization signal (dpms). AVP mRNA expression varied significantly throughout the day but was not significantly affected by aging.
4. Discussion In order to elucidate the neural basis for age-related changes in circadian rhythms, this study investigated the effect of aging on expression of neuropeptide mRNAs in the SCN, the major mammalian circadian pacemaker. The results showed that aging decreased VIP mRNA expression without affecting AVP mRNA or SS mRNA expression. These findings support the hypothesis that aging modulates SCN expression of neuropeptide messenger RNA. The selectivity of this effect for VIP suggests that it may be caused by some age-related change in the activity of one of the neural pathways that selectively innervate the ventral SCN, the site of the VIP neurons, such as the serotonergic projection from the median raphe nucleus [4,33,36]. Serotonin regulates VIP neurons in the SCN, as shown by the findings that depletion of brain serotonin
Fig. 6. Twenty-four hour profiles of SS mRNA expression in the SCN. Values shown represent the mean6S.E.M. of the hybridization signal (dpms). SS mRNA expression was not significantly affected by aging or time of day.
with parachlorophenlyalanine reduces VIP mRNA expression in the SCN [27]. Thus, it is possible that the agerelated decrease in SCN VIP mRNA expression observed in the current study is caused by attenuated serotonin neurotransmission, a phenomenon that has been suggested by previous studies. For example, aging decreases serotonin turnover in the rat SCN [9]. Also, in the hamster SCN, aging mimics the effect of serotonin depletion by upregulating serotonin 1B (5-HT 1B ) receptors [14,32]. Age-related changes in the activity of non-serotonergic pathways innervating the ventral SCN, such as the retinohypothalamic tract or the geniculohypothalamic tract [21,35], may also modulate the reduction of SCN VIP mRNA expression. This hypothesis is supported by the previous finding that the SCN content of NPY, which is derived from the geniculohypothalmic tract originating in the intergeniculate leaflet (IGL) of the lateral geniculate complex [35], is decreased in old rats [46]. Also, aging decreases the sensitivity to the phase-resetting effect of benzodiazepines [55], a phenomenon that depends on the geniculohypothalamic tract [5]. Attenuated SCN VIP mRNA expression during aging may contribute to the age-related changes in circadian function, such as reduced sensitivity to phase shifting stimuli and changes in the phase angle of entrainment or the rate of re-entrainment [41,48,54,55,59,61]. The VIP neurons are innervated by pathways conveying timing information, and thus are well suited to participate in environmental resetting of the circadian pacemaker [18,21,26]. Furthermore, the VIP neurons are also involved in output pathways from the SCN that impose circadian rhythmicity on physiological processes, such as hormone secretion [50,53]. For example, SCN VIP neurons project to the medial preoptic nucleus, the site of GnRH neurons that regulate LH secretion [8,25,57]. Reduction of VIP synthesis in the rat SCN by microinjection of antisense oligonucleotides reduced the amplitude and delayed the onset of the estradiol-induced LH surge, mimicking the effects of aging [17]. Also, aging abolishes the 24-h rhythm of SCN VIP mRNA but not the rhythm of SCN AVP mRNA expression in female rats [30]. Thus, agerelated changes in VIP mRNA expression in the SCN contribute to age-related changes in endocrine function in rats, and may serve the same function in hamsters. The results of the present study also showed that neuroanatomical region and time of day differentially regulate neuropeptide mRNA expression in the Syrian hamster SCN. VIP mRNA exhibited variation in expression throughout the rostral to caudal extent of the Syrian hamster SCN, similar to a previous finding in the Siberian hamster SCN [12]. Expression of AVP mRNA, but not VIP mRNA or SS mRNA, showed robust time of day variations in the Syrian hamster SCN. In contrast, previous findings suggest that time of day variations characterize expression of both AVP mRNA and VIP mRNA in the Siberian hamster SCN, and expression of all three neuro-
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
peptide mRNAs in the rat [13,23,63]. Thus, there appears to be species differences in the rhythmic expression of SCN neuropeptide mRNAs. In the rat, the rhythmic expression in the SCN of VIP and AVP mRNAs, in conjunction with other findings, has suggested that rhythmic release of the corresponding peptides in projection fields couples the central pacemaker to output rhythms, such as secretion of LH, melatonin or corticosterone [6,24,50,53]. The present findings in the Syrian hamster SCN do not support this idea, but are more consistent with the idea that VIP and SS release modulates the function of the SCN or its neuronal targets. Studies of the SCN in vitro have shown that SS inhibits the firing rate of rat SCN neurons [22], while VIP administration stimulates AVP release [56]. Therefore, the decreased SCN VIP mRNA expression during aging suggests that the VIP neurons exert less modulation on the AVP and SS neurons in the SCN at later stages of life. In conclusion, the Syrian hamster SCN exhibits an age-related reduction in VIP mRNA expression without any observable alteration in AVP or SS mRNA expression. This reduction in SCN VIP mRNA expression suggests that these neurons are less active during aging. This phenomenon may contribute to age-related changes in circadian function, especially changes in entrainment and phase resetting, in view of the fact that VIP neurons receive input from pathways conveying timing information.
Acknowledgements We thank Anthony Deveraux for assistance with these experiments and Dr Kathryn Scarbrough for helpful consultation concerning in situ hybridization for VIP mRNA expression. NIH Grant AG-13418 supported these studies.
References [1] H.E. Albers, C.F. Ferris, S.E. Leeman, B.D. Goldman, Avian pancreatic polypeptide phase shifts hamster circadian rhythms when microinjected into the suprachiasmatic region, Science 223 (1984) 833–835. [2] H.E. Albers, S-H. Liou, E.G. Stopa, R.T. Zoeller, Interaction of colocalized neuropeptides: Functional significance in the circadian timing system, J. Neurosci. 11 (1991) 846–851. [3] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. [4] E.C. Azmitia, M. Segal, An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat, J. Comp. Neurol. 179 (1978) 641–659. [5] S.M. Biello, M.E. Harrington, R. Mason, Geniculo-hypothalamic tract lesions block chlordiazepoxide-induced phase advances in Syrian hamsters, Brain Res. 552 (1991) 47–52. [6] R.M. Buijs, J. Wortel, J.J. Van Heerikhuize, M.G. Feenstra, Ter, Horst, H.J. Romijn, A. Kalsbeek, Anatomical and functional demon-
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
201
stration of a multisynaptic suprachiasmatic nucleus adrenal (cortex) pathway, Eur. J. Neurosci. 11 (1999) 1535–1544. J.P. Card, N. Brecha, H.J. Karten, R.Y. Moore, Immunocytochemical localization of vasoactive intestinal polypeptide-containing cells and processes in the suprachiasmatic nucleus of the rat: Light and electron microscopic analysis, J. Neurosci. 1 (1981) 1289–1303. J.P. Card, R.Y. Moore, The suprachiasmatic nucleus of the golden hamster: Immunohistochemical analysis of cell and fiber distribution, Neuroscience 13 (1984) 415–431. I.R. Cohen, P.M. Wise, Age-related changes in the diurnal rhythm of serotonin turnover in microdissected brain areas of estradiol-treated ovariectomized rats, Endocrinology 122 (1988) 2626–2633. R.A. Cohen, H. Albers, Disruption of human circadian and cognitive regulation following a discrete hypothalamic lesion: A case study, Neurology 41 (1991) 726–729. F.C. Davis, N. Viswanathan, Stability of circadian timing with age in Syrian hamsters, Am. J. Physiol. Regul. Integr. Comp. Physiol. 275 (1998) R960–R968. M.J. Duncan, Photoperiodic regulation of hypothalamic neuropeptide messenger RNA expression: effect of pinealectomy and neuroanatomical location, Mol. Brain Res. 57 (1998) 142–148. M.J. Duncan, X. Cheng, K.S. Heller, Photoperiodic exposure and time of day modulate the expression of arginine vasopressin mRNA and vasoactive intestinal peptide mRNA in the suprachiasmatic nuclei of Siberian hamsters, Brain Res. 32 (1995) 181–186. M.J. Duncan, C.J. Crafton, D.L. Wheeler, Aging regulates 5-HT 1B receptors and serotonin reuptake sites in the suprachiasmatic nucleus and other brain regions, Brain Res. 856 (2000) 213–219. C. Fukuhara, T. Hamada, S. Shibata, S. Watanabe, K. Aoki, S.-I.T. Inouye, Phase advances of circadian rhythms in somatostatin depleted rats: Effects of cysteamine on rhythms of locomotor activity and electrical discharge of the suprachiasmatic nucleus, J. Comp. Physiol. [A] 175 (1994) 677–685. C. Fukuhara, T. Nishiwaki, F.R.A. Cagampang, S-I. Inouye, Emergence of VIP rhythmicity following somatostatin depletion in the rat suprachiasmatic nucleus, Brain Res. 645 (1994) 343–346. J.P. Harney, K. Scarbrough, K.L. Rosewell, P.M. Wise, In vivo antisense antagonism of vasoactive intestinal peptide in the suprachiasmatic nuclei causes aging-like changes in the estradiol-induced luteinizing hormone and prolactin surges, Endocrinology 137 (1996) 3696–3701. S. Hisano, M. Chaki-Aoyama, S. Katoh, Y. Kagotani, S. Daikoku, K. Chihara, Y. Takahashi, Suprachiasmatic nucleus neurons immunoreactive for vasoactive intestinal peptide have synaptic contact with axons immunoreactive for neuropeptide Y: an immunoelectron microscopic study in the rat, Neuroscience 88 (1988) 145–150. S. Hisano, M. Chiaki-Aoyama, S. Katoh, M. Maigawa, S. Daikoku, Immunohistochemical evidence of serotonergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus, Histochemistry 86 (1988) 573–578. M.W. Hurd, M.R. Ralph, The significance of circadian organization for longevity in the golden hamster, J. Biol. Rhythms 13 (1998) 430–436. Y. Ibata, Y. Takahashi, H. Okamura, F. Kawakami, H. Terubayashi, T. Kubo, N. Yanaihara, Vasoactive intestinal peptide (VIP)-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection, Neurosci. Lett. 97 (1989) 1–5. C.D. Ingram, R. Ciobanu, I.L. Coculescu, R. Tanasescu, R. Mihai, Vasopressin neurotransmission and the control of circadian rhythms in the suprachiasmatic nucleus, in: I.J.A. Urban, J.P.H. Burbach, D. De Weid (Eds.), Progress in Brain Research, Vol. 119, Elsevier, Amsterdam, 2000, pp. 351–364. S-I.T. Inouye, K. Shinohara, K. Tominaga, J. Takeuchi, H. Nagasaki, Y. Isobe, C. Fukuhara, Y. Otori, J. Yang, F.R.A. Cagampang, S. Yamazaki, A. Tokumasu, Circadian rhythms in peptides and their precursor messenger RNAs in the suprachiasmatic nucleus, in: H. Nakagawa, Y. Oomura, K. Nagai (Eds.), New Functional Aspects
202
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203 of the Suprachiasmatic Nucleus of the Hypothalamus, John Libbey, 1993, pp. 219–233. A. Kalsbeek, R.M. Buijs, J.J. Van Heerikhuize, M. Arts, T.P. Van der Woude, Vasopressin-containing neurons of the suprachiasmatic nuclei inhibit corticosterone release, Brain Res. 580 (1992) 62–67. ´ A. Kalsbeek, R. Teclemariam-Mesbah, P. Pevet, Efferent projections of the suprachiasmatic nucleus in the golden hamster (Mesocricetus auratus), J. Comp. Neurol. 332 (1993) 293–314. F. Kawakami, H. Okamura, K. Fukui, C. Yanaihara, N. Yanaihara, T. Nakajima, Y. Ibata, The influence of serotonergic inputs on peptide neurons in the rat suprachiasmatic nucleus: an immunocytochemical study, Neurosci. Lett. 61 (1985) 273–277. F. Kawakami, H. Okamura, T. Inatomi, Y. Tamada, T. Nakajima, Y. Ibata, Serotonin depletion by p-chlorophenylalanine decreases VIP mRNA in the suprachiasmatic nucleus, Neurosci. Lett. 174 (1994) 81–84. F. Kawakami, H. Okamura, Y. Tamada, Y. Maebayashi, K. Fukui, Y. Ibata, Loss of day–night differences in VIP mRNA levels in the suprachiasmatic nucleus of aged rats, Neurosci. Lett. 222 (1997) 99–102. J. Kiss, C. Leranth, B. Halasz, Serotoninergic endings on VIPneurons in the suprachiasmatic nucleus and on ACTH-neurons in the arcuate nucleus of the rat hypothalamus. A combination of high resolution autoradiography and electron microscopic immunocytochemistry, Neurosci. Lett. 44 (1984) 119–124. K. Krajnak, M.L. Kashon, K.L. Rosewell, P.M. Wise, Aging alters the rhythmic expression of vasoactive intestinal polypeptide mRNA but not arginine vasopressin mRNA in the suprachiasmatic nuclei of female rats, J. Neurosci. 18 (1998) 4767–4774. H. Li, E. Satinoff, Changes in circadian rhythms of body temperature and sleep in old rats, Am. J. Physiol. Regul. Integr. Comp. Physiol. 269 (1995) R208–R214. ´ C. Manrique, A.M. Franc¸ois-Bellan, L. Segu, D. Becquet, M. Hery, ´ M. Faudon, F. Hery, Impairment of serotoninergic transmission is followed by adaptive changes in 5HT 1B binding sites in the rat suprachiasmatic nucleus, Brain Res. 663 (1994) 93–100. E.L. Meyer-Bernstein, L.P. Morin, Differential serotonergic innervation of the suprachiasmatic nucleus and the intergeniculate leaflet and its role in circadian rhythm modulation, J. Neurosci. 16 (1996) 2097–2111. R. Mihai, T.S. Juss, C.D. Ingram, Suppression of suprachiasmatic nucleus neurone activity with a vasopressin receptor antagonist: possible role for endogenous vasopressin in circadian activity cycles in vitro, Neurosci. Lett. 179 (1994) 95–99. R.Y. Moore, J.P. Card, Intergeniculate leaflet: An anatomically and functionally distinct subdivision of the lateral geniculate complex, J. Comp. Neurol. 344 (1994) 403–430. R.Y. Moore, A.E. Halaris, B.E. Jones, Serotonin neurons of the midbrain raphe: ascending projections, J. Comp. Neurol. 180 (1978) 417–438. L.P. Morin, N. Goodless-Sanchez, L. Smale, R.Y. Moore, Projections of the suprachiasmatic nuclei, subparaventricular zone and retrochiasmatic area in the golden hamster, Neuroscience 61 (1994) 391– 410. Y. Ouyang, C.R. Andersson, T. Kondo, S.S. Golden, C.H. Johnson, Resonating circadian clocks enhance fitness in cyanobacteria, Proc. Natl. Acad. Sci. USA 95 (1998) 8660–8664. P.D. Penev, D.E. Kolker, P.C. Zee, F.W. Turek, Chronic circadian desynchronization decreases the survival of animals with cardiomyopathic heart disease, Am. J. Physiol. Heart Circ. Physiol. 275 (1998) H2334–H2337. P.D. Penev, P.C. Zee, F.W. Turek, Quantitative analysis of the age-related fragmentation of hamster 24-h rhythms, Am. J. Physiol. 273 (1997) R2131–R2137. P.D. Penev, P.C. Zee, E.P. Wallen, F.W. Turek, Aging alters the phase-resetting properties of a serotonin agonist on hamster circadian rhythmicity, Am. J. Physiol. Regul. Integr. Comp. Physiol. 268 (1995) R293–R298.
[42] H.D. Piggins, M.C. Antle, B. Rusak, Neuropeptides phase shift the mammalian circadian pacemaker, J. Neurosci. 15 (1995) 5612– 5622. [43] R.J. Reiter, C.M. Craft, J.E. Johnson Jr., T.S. King, B.A. Richardson, G.M. Vaughan, M.K. Vaughan, Age-associated reduction in nocturnal melatonin levels in female rats, Endocrinology 109 (1981) 1295–1297. [44] R.J. Reiter, B.A. Richardson, L.Y. Johnson, B.N. Ferguson, D.T. Dihn, Pineal melatonin rhythm: Reduction in aging Syrian hamsters, Science 210 (1980) 1372–1373. [45] R.S. Rosenberg, P.C. Zee, F.W. Turek, Phase response curves to light in young and old hamsters, Am. J. Physiol. Regul. Integr. Comp. Physiol. 261 (1991) R491–R495. [46] A. Sahu, P.S. Kalra, W.R. Crowley, S.P. Kalra, Evidence that hypothalamic neuropeptide Y secretion decreases in aged male rats: Implications for reproductive aging, Endocrinology 122 (1988) 2199–2203. [47] E. Satinoff, H. Li, T.K. Tcheng, C. Liu, A.J. McArthur, M. Medanic, M.U. Gillette, Do the suprachiasmatic nuclei oscillate in old rats as they do in young ones?, Am. J. Physiol. Regul. Integr. Comp. Physiol. 265 (1993) R1216–R1222. [48] K. Scarbrough, S. Losee-Olson, E.P. Wallen, F.W. Turek, Aging and photoperiod effects on entrainment and quantitative aspects of locomotor behavior in Syrian hamsters, Am. J. Physiol. Regul. Integr. Comp. Physiol. 272 (1997) R1219–R1225. [49] K. Scarbrough, F.W. Turek, Quantitative differences in the circadian rhythm of locomotor activity and vasopressin and vasoactive intestinal peptide gene expression in the suprachiasmatic nucleus of tau mutant compared to wildtype hamsters, Brain Res. 736 (1996) 251–259. [50] R. Teclemariam-Mesbah, A. Kalsbeek, P. Pevet, R.M. Buijs, Direct vasoactive intestinal polypeptide-containing projection from the suprachiasmatic nucleus to spinal projecting hypothalamic paraventricular neurons, Brain Res. 748 (1997) 71–76. [51] E. Van Cauter, L. Plat, R. Leproult, G. Copinschi, Alterations of circadian rhythmicity and sleep in aging: Endocrine consequences, Horm. Res. 49 (1998) 147–152. [52] A.N. van den Pol, K.L. Tsujimoto, Neurotransmitters of the hypothalamic suprachiasmatic nucleus: Immunocytochemical analysis of 25 neuronal antigens, Neuroscience 15 (1985) 1049–1086. [53] E. Van der Beek, Circadian control of reproduction in the female rat, Prog. Brain Res. 111 (1996) 296–320. [54] O. Van Reeth, Y. Zhang, A. Reddy, P. Zee, F.W. Turek, Aging alters the entraining effects of an activity-inducing stimulus on the circadian clock, Brain Res. 607 (1993) 286–292. [55] O. Van Reeth, Y. Zhang, P.C. Zee, F.W. Turek, Aging alters feedback effects of the activity–rest cycle on the circadian clock, Am. J. Physiol. Regul. Integr. Comp. Physiol. 263 (1992) R981–R986. [56] J. Vanecek, K. Watanabe, Melatonin inhibits the increase of cyclic AMP in rat suprachiasmatic neurons induced by vasoactive intestinal peptide, Neurosci. Lett. 252 (1998) 21–24. [57] A.G. Watts, L.W. Swanson, Efferent projections of the suprachiasmatic nucleus: II. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunochemistry in the rat, J. Comp. Neurol. 258 (1991) 230–252. [58] G. Winocur, L. Hasher, Aging and time-of-day effects on cognition in rats, Behav. Neurosci. 113 (1999) 991–997. [59] P.M. Wise, Alterations in proestrous LH, FSH, and prolactin surges in middle-aged rats, Proc. Soc. Exp. Biol. Med. 169 (1982) 348– 354. [60] W.S. Young III, E. Mezey, R.E. Siegel, Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: analysis by quantitative in situ hybridization histochemistry, Mol. Brain Res. 1 (1986) 231–241. [61] P.C. Zee, R.S. Rosenberg, F.W. Turek, Effects of aging on entrainment and rate of resynchronization of circadian locomotor activity, Am. J. Physiol. Regul. Integr. Comp. Physiol. 263 (1992) R1099– R1103.
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203 [62] Y. Zhang, J.M. Kornhauser, P.C. Zee, K.E. Mayo, J.S. Takahashi, F.W. Turek, Effects of aging on light-induced phase-shifting of circadian behavioral rhythms, Fos expression and CREB phosphorylation in the hamster suprachiasmatic nucleus, Neuroscience 70 (1996) 951–961.
203
[63] R.T. Zoeller, B. Broyles, J. Earley, E.R. Anderson, H.E. Albers, Cellular levels of messenger ribonucleic acids encoding vasoactive peptide and gastrin-releasing peptide in neurons of the suprachiasmatic nucleus exhibit distinct 24-h rhythms, J. Neuroendocrinol. 4 (1992) 119–124.
Research report
Aging selectively suppresses vasoactive intestinal peptide messenger RNA expression in the suprachiasmatic nucleus of the Syrian hamster Marilyn J. Duncan*, Jana M. Herron, Stephanie A. Hill Department of Anatomy and Neurobiology, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536 -0298, USA Accepted 26 December 2000
Abstract Aging leads to many changes in the expression of circadian rhythms, including reduced amplitude, altered relationship to the environmental illumination cycle, and reduced sensitivity to phase resetting signals. Neuropeptide synthesizing neurons in the suprachiasmatic nucleus (SCN), the principal circadian pacemaker in mammals, play a role in regulating pacemaker function and in coupling the pacemaker to overt circadian rhythms. Aging may alter the activity of neuropeptide neurons in the SCN, which could be reflected in changes in mRNA expression. Therefore, this study investigated whether aging alters the level or rhythm of expression of neuropeptide mRNAs in the SCN of male Syrian hamsters, a well established model for the study of age-related changes in circadian rhythms. Three age groups of hamsters ( young [3–5 months old], middle-aged [12–15 months old] and old [19–22 months old] were sacrificed at five times of day. Their brains were dissected and sections through the suprachiasmatic nucleus were prepared and used for in situ hybridization for mRNAs for vasoactive intestinal peptide (VIP), arginine vasopressin (AVP) and somatostatin (SS). Aging selectively decreased the SCN expression of VIP mRNA without affecting AVP mRNA or SS mRNA. Also, only AVP mRNA expression exhibited a robust 24-h rhythm, in contrast to previous findings in other species that VIP mRNA and SS mRNA, as well as AVP mRNA, exhibit 24-h rhythms in the SCN. The present findings suggest that age-related reductions in VIP mRNA expression may contribute to the alterations in entrainment and attenuated sensitivity to phase resetting signals that are characteristic of aging. Furthermore, the results demonstrate that neuropeptide gene expression in the SCN is differentially regulated by aging and varies among species. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: In situ hybridization; Circadian rhythm; Vasoactive intestinal peptide; Vasopressin; Somatostatin
1. Introduction Circadian rhythms govern the behavior and physiological processes of virtually all organisms. By facilitating adjustments to the daily changes in illumination, temperature and other conditions, circadian rhythms enhance survival. In fact, robust and coordinated circadian rhythms favor good health, longevity and optimal cognitive ability in a variety of species, including humans [10,20,38,39,58]. Aging brings about many changes in circadian rhythms. One age-related change is a reduction in amplitude, a common feature of melatonin rhythms in old rodents *Corresponding author. Tel.: 11-859-323-4718; fax: 11-859-3235946. E-mail address: [email protected] (M.J. Duncan).
[43,44]. During aging, circadian rhythms also become disrupted or fragmented, and their phase relationship to environmental time signals is altered [11,31,40,48,61]. For example, locomotor activity rhythms in old hamsters exhibit fragmentation and an earlier onset of the active phase in relationship to the onset of darkness [11,40,48,61]. Also, circulating cortisol rhythms in old men are dampened and phase-advanced compared to the rhythms in young men [51]. During aging, the circadian pacemaker becomes more resistant to phase resetting signals, such as injections of triazolam or 8-hydroxy-2-(din-propylamino)tetralin (8-OH-DPAT) [41,55]. Finally, aging may induce a dissociation of circadian rhythms within an individual [47]. Studies of circadian rhythms in drinking behavior, body temperature, and neuronal activity in old rats showed that some individuals exhibited a loss of
0169-328X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00015-8
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
all three rhythms, while other experienced a loss of only one or two of these rhythms [47]. Thus, aging disrupts the coordinated expression of circadian rhythms. The neural basis for these age-related alterations in circadian rhythms may involve functional changes in the circadian pacemaker in the hypothalamic suprachiasmatic nucleus (SCN). The SCN responds to timing signals received from afferent pathways and drives overt circadian rhythms through its efferent connections [25,37,57]. The SCN consists of neurons that synthesize a variety of neuropeptides, including vasoactive intestinal peptide (VIP), arginine vasopressin (AVP), and somatostatin (SS), and appear to play multiple roles in circadian timekeeping [7,23,52]. VIP and AVP have been identified in several efferent pathways from the SCN and thus may be involved in the regulation of overt circadian rhythms [25,57]. AVP and SS have been shown to modulate the function of the SCN. AVP neurons provide endogenous excitatory tone to the SCN [34]. Somatostatin administration inhibits SCN neuronal firing in vitro [22]. Somatostatin neurons are also involved in regulating the phase of circadian rhythms. Depletion of somatostatin induces phase advances in locomotor activity rhythms and SCN electrical activity rhythms, and permits rhythmic release of VIP from the SCN in vitro [15,16]. The demonstration that VIP neurons receive synaptic inputs from the retinohypothalamic tract, the geniculohypothalamic tract and the serotonergic projection from the median raphe nucleus suggests that the VIP neurons are involved in entrainment of the circadian pacemaker [18,19,21,29]. This concept is supported by findings that VIP microinjections in the SCN region induce phase-dependent phase shifts in locomotor activity rhythms [1,2,42]. Because neuropeptide neurons play important roles in regulating circadian timekeeping, alterations in their function may contribute to age-related changes in circadian rhythms. For example, aging modulates the VIP mRNA rhythm in the rat SCN [28,30]. However, it remains to be determined if aging changes the rhythmic expression of VIP mRNA or other neuropeptide mRNA in the SCN of the Syrian hamster, a well-characterized model of agerelated changes in behavioral circadian rhythms [45,54,62]. Therefore, the current study tested the hypothesis that aging alters the SCN rhythmic expression of messenger RNA for VIP, AVP or SS in Syrian hamsters. In order to assess each mRNA species at the site of greatest expression within the SCN, a preliminary study was conducted to delineate the regional distribution of VIP mRNA, AVP mRNA and SS mRNA.
2. Materials and methods
2.1. Animals and tissue preparation Male Syrian hamsters obtained from Harlan Labs (Har-
197
lan, HsdHan:AURA) were maintained in the Department of Laboratory Animal Research at the University of Kentucky under a 14-h light, 10-h dark photoperiod (lights on from 06:00 to 20:00 h) for at least 1–2 weeks. Food (Teklad, Amway) and water were available continuously. Three age groups were studied: young (3–5 months old), middle-aged (12–15 months old) and old (19–22 months old). The hamsters were sacrificed at five different times of day, 02:00, 07:00, 12:00, 17:00, and 22:00 h (N56–9 / age per time). A dim red light was used when hamsters were euthanized during the dark phase. The brains were dissected, frozen on crushed dry ice, and stored at 2808C. Coronal sections (12 mm thick) through the SCN were cut on a cryostat and mounted on positively charged slides. The slide-mounted sections were stored at 2808C before in situ hybridization was conducted.
2.2. In situ hybridization In situ hybridization was performed using oligodeoxyribonucleotides (oligos) which were 39-end labeled with [ 35 S]dATP using terminal deoxynucleotidyl transferase. The oligos selected were based on the sequence of the rat genes for AVP, VIP, and SS, because these genes have not been cloned and sequenced from Syrian hamsters. For each oligo, the sequence shows at most very low complementarity to any other reported genes besides the gene coding for the protein of interest, based on analyses with the BLAST program [3]. Previous in situ hybridization studies of hamster brain sections with these oligos showed that the distribution of the specific signal matches the neuroanatomical distribution of cell bodies synthesizing the peptide and that background labelling is low [12,13,49]. The procedures for in situ hybridization for AVP mRNA or somatostatin mRNA each used one specific oligo and were conducted as described previously [12,13]. The AVP oligo (59-TAG /ACC / CGG / GGC / TTG / GCA / GAA / TCC /ACG / GAC / TCT / TGT / GTC / CCA / GCC /AGC - 39) was complementary to the mRNA encoding the last 16 amino acids of the glycopeptide region of rat AVP [60]. The nucleotide sequence of the somatostatin oligo was: 59-GGA / TGT / GAA / TGT / CTT / CCA / GAA / GAA /ATT / CTT / GCA / GCC /AGC / TTT / GCG / TTC-39). The in situ hybridization procedure for VIP mRNA used a mixture of four oligos, as described previously for detection in the Syrian hamster SCN [49]. This method was used because preliminary studies using a single oligo did not generate a detectable signal. Thus, the VIP mRNA appeared to be in relatively low abundance in the Syrian hamster SCN, and the simultaneous use of four oligos amplified the signal. The nucleotide sequences of the VIP oligos, which corresponded to conserved regions of the VIP gene, were as follows. VIP oligo [1 : 59-ACA / TCA / TAA / TAG / GGC / GTG / TCA / TTC / TCC / GCT /AAG / GCA / TTC / TGC /AAG /ATG / TCA-39; VIP oligo[2 : 59AGT / CTA / CTG / TAG / TCG / CTG / GTG /AAA /ACT /
198
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
CCA / TCA / GCA / TGC / CTG / GCA / TTT-39, VIP oligo [3 : 59-AAG / GCG / GGT / GTA / GTT /ATC / TGT / GAA / GAC / TGC /ATC /AGA / GTG / TCG / TTT / GAC-39, and VIP oligo [4 : 59-CTC / CTC / TTC / CCA / TTT /AGA / ATG / GAG / TTC /AAG / TAT / TTC / TTC /ACA / GCC / ATT / TGC / TTT / CT-39. The in situ hybridization procedure included pretreatment of the slides to reduce background, as described previously [13], followed by hybridization with 3 pmol / ml of a mixture of the [ 35 S]dATP-labeled VIP oligos at 378C in a humid chamber for approximately 20 h. The hyridization buffer (hybridization cocktail, Amresco Inc., Solon, OH) contained 50% formamide, 25 mM sodium phosphate, 60 mM sodium citrate (SSC), 50% Denhardt’s solution (0.02% each of Ficoll, polyvinylpyrolidone, and bovine serum albumin), 10% dextran sulfate, 100 mM dithiothreitol (DTT), 250 mg / ml yeast transfer RNA and 500 mg / ml denatured salmon sperm DNA. Following hybridization, the tissue sections were washed in 13 SSC containing 10 mM sodium thiosulfate at 558C for 1 h (fresh solution every 15 min, agitated every 5 min) and at room temperature for 1 h (agitated every 15 min), dipped briefly in sterile distilled water and 95% ethanol, and quickly air-dried. Autoradiograms of the SCN were generated by exposing the tissue sections to X-ray film (Hyperfilm b-max, Amersham Corp.) for approximately 3 days (VIP mRNA and AVP mRNA) or 10 days (SS mRNA). In order to ascertain that the autoradiographic images did not represent film saturation, radioactive standards ( 14 C, American Radiolabeled Chemicals, St Louis, MO) were included in each cassette. The X-ray films were processed using D-19 developer and rapid fixer (Eastman Kodak, Rochester, NY). In order to be able to assess cellular levels of mRNA expression when significant differences in SCN levels of mRNA expression were evident from the film autoradiograms, the slides were dipped in photographic emulsion (NTB2, Eastman Kodak). The emulsion was developed after 10 (VIP mRNA and AVP mRNA) or 30 days (SS mRNA) and the slides were counterstained with cresyl violet. The hybridization signal of the film autoradiograms was assessed by semi-quantitative analysis using computerassisted microdensitometry, as described previously [13]. The hybridization signal, defined as the difference between the relative optical density over the SCN and an adjacent brain region, was averaged for four replicates for each animal. In order to ascertain that the autoradiographic signal corresponded to labeling of the SCN, the autoradiogram was compared with the Nissl-stained SCN. For assessment of cellular levels of mRNA expression, computerized image analysis was conducted on the video images of the emulsion autoradiograms from the same sections that generated the film autoradiograms. The difference between the mean grain area per cell in the SCN and the mean grain area per cell in an adjacent region was determined.
2.3. Statistical analyses The effect of region within the SCN was determined by one-way analysis of variance (ANOVA). The effects of aging and time of day were assessed by two-way ANOVA. In the case of significant (P,0.05) F-values, post-hoc comparisons were made between groups with the Newman–Keuls test.
3. Results
3.1. Regional distribution of neuropeptide mRNA expression in the SCN The Syrian hamster SCN expressed all three mRNA species investigated: VIP, AVP and SS (Fig. 1). VIP mRNA expression, which was localized to the ventral SCN, exhibited a statistically significant variation throughout the rostral to caudal axis of the SCN, characterized by greatest expression in the middle and no detectable expression at either pole (Fig. 2). The expression of AVP mRNA and SS mRNA expression in the dorsal region of the SCN were homogeneous along the rostral to caudal axis of the SCN (Fig. 2).
3.2. Effect of aging on neuropeptide mRNA expression in the SCN VIP mRNA expression in the SCN was significantly affected by age (P,0.05), but not by time of day (P5 0.84) (Fig. 3). There was no significant interaction effect between age and time of day on VIP mRNA expression (P50.58). Collapsing the data across time of day revealed that VIP mRNA expression in the SCN was decreased in middle-aged and old hamsters by 15 and 28%, respectively, as compared to young hamsters (Fig. 4). Analysis of the emulsion autoradiograms revealed that the average area of grains per cell was significantly reduced by aging (P,0.05), but was not affected by time of day (P50.44), nor was there a significant interaction effect (P50.73). When the data for the various times were combined, the average grain area per cell in the SCN was decreased in the middle-aged and old hamsters by approximately 22 and 37%, respectively, as compared to young hamsters (Fig. 4). AVP mRNA expression in the SCN was not affected by age (P50.97), but varied significantly with time of day (P,0.001, Fig. 5). AVP mRNA expression was highest during the middle of the light phase (12:00 and 17:00 h). There was no significant interaction effect between age and time of day on AVP mRNA expression (P50.37). In contrast to VIP mRNA and AVP mRNA, neither age (P50.24) nor time of day (P,0.51) significantly affected SS mRNA expression in the SCN (Fig. 6).
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
199
Fig. 2. Regional distribution of neuropeptide mRNA expression across the rostral to caudal axis of the SCN. The bars represent the mean6S.E.M. of the hybridization signal (dpms) for each region (N56 each). AVP mRNA and somatostatin mRNA were broadly distributed throughout the SCN; in contrast, VIP mRNA was more restricted.
Fig. 3. Twenty-four hour profiles of VIP mRNA expression in the SCN. The bars depict the mean6S.E.M. of the hybridization signal (dpms). VIP mRNA expression was significantly decreased by aging but was not affected by time of day.
Fig. 1. Prints of X-ray film autoradiograms showing the neuropeptide mRNA expression in the SCN. VIP mRNA expression is restricted to the ventral SCN, while AVP mRNA and SS mRNA are localized to the dorsal SCN.
Fig. 4. The effect of aging on VIP mRNA expression in the SCN. Each bar represents the mean6S.E.M. of the hybridization signal (dpms) for all time points within each age group.
200
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
Fig. 5. Twenty-four hour profiles of AVP mRNA expression in the SCN. Values represent the mean6S.E.M. of the hybridization signal (dpms). AVP mRNA expression varied significantly throughout the day but was not significantly affected by aging.
4. Discussion In order to elucidate the neural basis for age-related changes in circadian rhythms, this study investigated the effect of aging on expression of neuropeptide mRNAs in the SCN, the major mammalian circadian pacemaker. The results showed that aging decreased VIP mRNA expression without affecting AVP mRNA or SS mRNA expression. These findings support the hypothesis that aging modulates SCN expression of neuropeptide messenger RNA. The selectivity of this effect for VIP suggests that it may be caused by some age-related change in the activity of one of the neural pathways that selectively innervate the ventral SCN, the site of the VIP neurons, such as the serotonergic projection from the median raphe nucleus [4,33,36]. Serotonin regulates VIP neurons in the SCN, as shown by the findings that depletion of brain serotonin
Fig. 6. Twenty-four hour profiles of SS mRNA expression in the SCN. Values shown represent the mean6S.E.M. of the hybridization signal (dpms). SS mRNA expression was not significantly affected by aging or time of day.
with parachlorophenlyalanine reduces VIP mRNA expression in the SCN [27]. Thus, it is possible that the agerelated decrease in SCN VIP mRNA expression observed in the current study is caused by attenuated serotonin neurotransmission, a phenomenon that has been suggested by previous studies. For example, aging decreases serotonin turnover in the rat SCN [9]. Also, in the hamster SCN, aging mimics the effect of serotonin depletion by upregulating serotonin 1B (5-HT 1B ) receptors [14,32]. Age-related changes in the activity of non-serotonergic pathways innervating the ventral SCN, such as the retinohypothalamic tract or the geniculohypothalamic tract [21,35], may also modulate the reduction of SCN VIP mRNA expression. This hypothesis is supported by the previous finding that the SCN content of NPY, which is derived from the geniculohypothalmic tract originating in the intergeniculate leaflet (IGL) of the lateral geniculate complex [35], is decreased in old rats [46]. Also, aging decreases the sensitivity to the phase-resetting effect of benzodiazepines [55], a phenomenon that depends on the geniculohypothalamic tract [5]. Attenuated SCN VIP mRNA expression during aging may contribute to the age-related changes in circadian function, such as reduced sensitivity to phase shifting stimuli and changes in the phase angle of entrainment or the rate of re-entrainment [41,48,54,55,59,61]. The VIP neurons are innervated by pathways conveying timing information, and thus are well suited to participate in environmental resetting of the circadian pacemaker [18,21,26]. Furthermore, the VIP neurons are also involved in output pathways from the SCN that impose circadian rhythmicity on physiological processes, such as hormone secretion [50,53]. For example, SCN VIP neurons project to the medial preoptic nucleus, the site of GnRH neurons that regulate LH secretion [8,25,57]. Reduction of VIP synthesis in the rat SCN by microinjection of antisense oligonucleotides reduced the amplitude and delayed the onset of the estradiol-induced LH surge, mimicking the effects of aging [17]. Also, aging abolishes the 24-h rhythm of SCN VIP mRNA but not the rhythm of SCN AVP mRNA expression in female rats [30]. Thus, agerelated changes in VIP mRNA expression in the SCN contribute to age-related changes in endocrine function in rats, and may serve the same function in hamsters. The results of the present study also showed that neuroanatomical region and time of day differentially regulate neuropeptide mRNA expression in the Syrian hamster SCN. VIP mRNA exhibited variation in expression throughout the rostral to caudal extent of the Syrian hamster SCN, similar to a previous finding in the Siberian hamster SCN [12]. Expression of AVP mRNA, but not VIP mRNA or SS mRNA, showed robust time of day variations in the Syrian hamster SCN. In contrast, previous findings suggest that time of day variations characterize expression of both AVP mRNA and VIP mRNA in the Siberian hamster SCN, and expression of all three neuro-
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203
peptide mRNAs in the rat [13,23,63]. Thus, there appears to be species differences in the rhythmic expression of SCN neuropeptide mRNAs. In the rat, the rhythmic expression in the SCN of VIP and AVP mRNAs, in conjunction with other findings, has suggested that rhythmic release of the corresponding peptides in projection fields couples the central pacemaker to output rhythms, such as secretion of LH, melatonin or corticosterone [6,24,50,53]. The present findings in the Syrian hamster SCN do not support this idea, but are more consistent with the idea that VIP and SS release modulates the function of the SCN or its neuronal targets. Studies of the SCN in vitro have shown that SS inhibits the firing rate of rat SCN neurons [22], while VIP administration stimulates AVP release [56]. Therefore, the decreased SCN VIP mRNA expression during aging suggests that the VIP neurons exert less modulation on the AVP and SS neurons in the SCN at later stages of life. In conclusion, the Syrian hamster SCN exhibits an age-related reduction in VIP mRNA expression without any observable alteration in AVP or SS mRNA expression. This reduction in SCN VIP mRNA expression suggests that these neurons are less active during aging. This phenomenon may contribute to age-related changes in circadian function, especially changes in entrainment and phase resetting, in view of the fact that VIP neurons receive input from pathways conveying timing information.
Acknowledgements We thank Anthony Deveraux for assistance with these experiments and Dr Kathryn Scarbrough for helpful consultation concerning in situ hybridization for VIP mRNA expression. NIH Grant AG-13418 supported these studies.
References [1] H.E. Albers, C.F. Ferris, S.E. Leeman, B.D. Goldman, Avian pancreatic polypeptide phase shifts hamster circadian rhythms when microinjected into the suprachiasmatic region, Science 223 (1984) 833–835. [2] H.E. Albers, S-H. Liou, E.G. Stopa, R.T. Zoeller, Interaction of colocalized neuropeptides: Functional significance in the circadian timing system, J. Neurosci. 11 (1991) 846–851. [3] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389–3402. [4] E.C. Azmitia, M. Segal, An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat, J. Comp. Neurol. 179 (1978) 641–659. [5] S.M. Biello, M.E. Harrington, R. Mason, Geniculo-hypothalamic tract lesions block chlordiazepoxide-induced phase advances in Syrian hamsters, Brain Res. 552 (1991) 47–52. [6] R.M. Buijs, J. Wortel, J.J. Van Heerikhuize, M.G. Feenstra, Ter, Horst, H.J. Romijn, A. Kalsbeek, Anatomical and functional demon-
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
201
stration of a multisynaptic suprachiasmatic nucleus adrenal (cortex) pathway, Eur. J. Neurosci. 11 (1999) 1535–1544. J.P. Card, N. Brecha, H.J. Karten, R.Y. Moore, Immunocytochemical localization of vasoactive intestinal polypeptide-containing cells and processes in the suprachiasmatic nucleus of the rat: Light and electron microscopic analysis, J. Neurosci. 1 (1981) 1289–1303. J.P. Card, R.Y. Moore, The suprachiasmatic nucleus of the golden hamster: Immunohistochemical analysis of cell and fiber distribution, Neuroscience 13 (1984) 415–431. I.R. Cohen, P.M. Wise, Age-related changes in the diurnal rhythm of serotonin turnover in microdissected brain areas of estradiol-treated ovariectomized rats, Endocrinology 122 (1988) 2626–2633. R.A. Cohen, H. Albers, Disruption of human circadian and cognitive regulation following a discrete hypothalamic lesion: A case study, Neurology 41 (1991) 726–729. F.C. Davis, N. Viswanathan, Stability of circadian timing with age in Syrian hamsters, Am. J. Physiol. Regul. Integr. Comp. Physiol. 275 (1998) R960–R968. M.J. Duncan, Photoperiodic regulation of hypothalamic neuropeptide messenger RNA expression: effect of pinealectomy and neuroanatomical location, Mol. Brain Res. 57 (1998) 142–148. M.J. Duncan, X. Cheng, K.S. Heller, Photoperiodic exposure and time of day modulate the expression of arginine vasopressin mRNA and vasoactive intestinal peptide mRNA in the suprachiasmatic nuclei of Siberian hamsters, Brain Res. 32 (1995) 181–186. M.J. Duncan, C.J. Crafton, D.L. Wheeler, Aging regulates 5-HT 1B receptors and serotonin reuptake sites in the suprachiasmatic nucleus and other brain regions, Brain Res. 856 (2000) 213–219. C. Fukuhara, T. Hamada, S. Shibata, S. Watanabe, K. Aoki, S.-I.T. Inouye, Phase advances of circadian rhythms in somatostatin depleted rats: Effects of cysteamine on rhythms of locomotor activity and electrical discharge of the suprachiasmatic nucleus, J. Comp. Physiol. [A] 175 (1994) 677–685. C. Fukuhara, T. Nishiwaki, F.R.A. Cagampang, S-I. Inouye, Emergence of VIP rhythmicity following somatostatin depletion in the rat suprachiasmatic nucleus, Brain Res. 645 (1994) 343–346. J.P. Harney, K. Scarbrough, K.L. Rosewell, P.M. Wise, In vivo antisense antagonism of vasoactive intestinal peptide in the suprachiasmatic nuclei causes aging-like changes in the estradiol-induced luteinizing hormone and prolactin surges, Endocrinology 137 (1996) 3696–3701. S. Hisano, M. Chaki-Aoyama, S. Katoh, Y. Kagotani, S. Daikoku, K. Chihara, Y. Takahashi, Suprachiasmatic nucleus neurons immunoreactive for vasoactive intestinal peptide have synaptic contact with axons immunoreactive for neuropeptide Y: an immunoelectron microscopic study in the rat, Neuroscience 88 (1988) 145–150. S. Hisano, M. Chiaki-Aoyama, S. Katoh, M. Maigawa, S. Daikoku, Immunohistochemical evidence of serotonergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus, Histochemistry 86 (1988) 573–578. M.W. Hurd, M.R. Ralph, The significance of circadian organization for longevity in the golden hamster, J. Biol. Rhythms 13 (1998) 430–436. Y. Ibata, Y. Takahashi, H. Okamura, F. Kawakami, H. Terubayashi, T. Kubo, N. Yanaihara, Vasoactive intestinal peptide (VIP)-like immunoreactive neurons located in the rat suprachiasmatic nucleus receive a direct retinal projection, Neurosci. Lett. 97 (1989) 1–5. C.D. Ingram, R. Ciobanu, I.L. Coculescu, R. Tanasescu, R. Mihai, Vasopressin neurotransmission and the control of circadian rhythms in the suprachiasmatic nucleus, in: I.J.A. Urban, J.P.H. Burbach, D. De Weid (Eds.), Progress in Brain Research, Vol. 119, Elsevier, Amsterdam, 2000, pp. 351–364. S-I.T. Inouye, K. Shinohara, K. Tominaga, J. Takeuchi, H. Nagasaki, Y. Isobe, C. Fukuhara, Y. Otori, J. Yang, F.R.A. Cagampang, S. Yamazaki, A. Tokumasu, Circadian rhythms in peptides and their precursor messenger RNAs in the suprachiasmatic nucleus, in: H. Nakagawa, Y. Oomura, K. Nagai (Eds.), New Functional Aspects
202
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203 of the Suprachiasmatic Nucleus of the Hypothalamus, John Libbey, 1993, pp. 219–233. A. Kalsbeek, R.M. Buijs, J.J. Van Heerikhuize, M. Arts, T.P. Van der Woude, Vasopressin-containing neurons of the suprachiasmatic nuclei inhibit corticosterone release, Brain Res. 580 (1992) 62–67. ´ A. Kalsbeek, R. Teclemariam-Mesbah, P. Pevet, Efferent projections of the suprachiasmatic nucleus in the golden hamster (Mesocricetus auratus), J. Comp. Neurol. 332 (1993) 293–314. F. Kawakami, H. Okamura, K. Fukui, C. Yanaihara, N. Yanaihara, T. Nakajima, Y. Ibata, The influence of serotonergic inputs on peptide neurons in the rat suprachiasmatic nucleus: an immunocytochemical study, Neurosci. Lett. 61 (1985) 273–277. F. Kawakami, H. Okamura, T. Inatomi, Y. Tamada, T. Nakajima, Y. Ibata, Serotonin depletion by p-chlorophenylalanine decreases VIP mRNA in the suprachiasmatic nucleus, Neurosci. Lett. 174 (1994) 81–84. F. Kawakami, H. Okamura, Y. Tamada, Y. Maebayashi, K. Fukui, Y. Ibata, Loss of day–night differences in VIP mRNA levels in the suprachiasmatic nucleus of aged rats, Neurosci. Lett. 222 (1997) 99–102. J. Kiss, C. Leranth, B. Halasz, Serotoninergic endings on VIPneurons in the suprachiasmatic nucleus and on ACTH-neurons in the arcuate nucleus of the rat hypothalamus. A combination of high resolution autoradiography and electron microscopic immunocytochemistry, Neurosci. Lett. 44 (1984) 119–124. K. Krajnak, M.L. Kashon, K.L. Rosewell, P.M. Wise, Aging alters the rhythmic expression of vasoactive intestinal polypeptide mRNA but not arginine vasopressin mRNA in the suprachiasmatic nuclei of female rats, J. Neurosci. 18 (1998) 4767–4774. H. Li, E. Satinoff, Changes in circadian rhythms of body temperature and sleep in old rats, Am. J. Physiol. Regul. Integr. Comp. Physiol. 269 (1995) R208–R214. ´ C. Manrique, A.M. Franc¸ois-Bellan, L. Segu, D. Becquet, M. Hery, ´ M. Faudon, F. Hery, Impairment of serotoninergic transmission is followed by adaptive changes in 5HT 1B binding sites in the rat suprachiasmatic nucleus, Brain Res. 663 (1994) 93–100. E.L. Meyer-Bernstein, L.P. Morin, Differential serotonergic innervation of the suprachiasmatic nucleus and the intergeniculate leaflet and its role in circadian rhythm modulation, J. Neurosci. 16 (1996) 2097–2111. R. Mihai, T.S. Juss, C.D. Ingram, Suppression of suprachiasmatic nucleus neurone activity with a vasopressin receptor antagonist: possible role for endogenous vasopressin in circadian activity cycles in vitro, Neurosci. Lett. 179 (1994) 95–99. R.Y. Moore, J.P. Card, Intergeniculate leaflet: An anatomically and functionally distinct subdivision of the lateral geniculate complex, J. Comp. Neurol. 344 (1994) 403–430. R.Y. Moore, A.E. Halaris, B.E. Jones, Serotonin neurons of the midbrain raphe: ascending projections, J. Comp. Neurol. 180 (1978) 417–438. L.P. Morin, N. Goodless-Sanchez, L. Smale, R.Y. Moore, Projections of the suprachiasmatic nuclei, subparaventricular zone and retrochiasmatic area in the golden hamster, Neuroscience 61 (1994) 391– 410. Y. Ouyang, C.R. Andersson, T. Kondo, S.S. Golden, C.H. Johnson, Resonating circadian clocks enhance fitness in cyanobacteria, Proc. Natl. Acad. Sci. USA 95 (1998) 8660–8664. P.D. Penev, D.E. Kolker, P.C. Zee, F.W. Turek, Chronic circadian desynchronization decreases the survival of animals with cardiomyopathic heart disease, Am. J. Physiol. Heart Circ. Physiol. 275 (1998) H2334–H2337. P.D. Penev, P.C. Zee, F.W. Turek, Quantitative analysis of the age-related fragmentation of hamster 24-h rhythms, Am. J. Physiol. 273 (1997) R2131–R2137. P.D. Penev, P.C. Zee, E.P. Wallen, F.W. Turek, Aging alters the phase-resetting properties of a serotonin agonist on hamster circadian rhythmicity, Am. J. Physiol. Regul. Integr. Comp. Physiol. 268 (1995) R293–R298.
[42] H.D. Piggins, M.C. Antle, B. Rusak, Neuropeptides phase shift the mammalian circadian pacemaker, J. Neurosci. 15 (1995) 5612– 5622. [43] R.J. Reiter, C.M. Craft, J.E. Johnson Jr., T.S. King, B.A. Richardson, G.M. Vaughan, M.K. Vaughan, Age-associated reduction in nocturnal melatonin levels in female rats, Endocrinology 109 (1981) 1295–1297. [44] R.J. Reiter, B.A. Richardson, L.Y. Johnson, B.N. Ferguson, D.T. Dihn, Pineal melatonin rhythm: Reduction in aging Syrian hamsters, Science 210 (1980) 1372–1373. [45] R.S. Rosenberg, P.C. Zee, F.W. Turek, Phase response curves to light in young and old hamsters, Am. J. Physiol. Regul. Integr. Comp. Physiol. 261 (1991) R491–R495. [46] A. Sahu, P.S. Kalra, W.R. Crowley, S.P. Kalra, Evidence that hypothalamic neuropeptide Y secretion decreases in aged male rats: Implications for reproductive aging, Endocrinology 122 (1988) 2199–2203. [47] E. Satinoff, H. Li, T.K. Tcheng, C. Liu, A.J. McArthur, M. Medanic, M.U. Gillette, Do the suprachiasmatic nuclei oscillate in old rats as they do in young ones?, Am. J. Physiol. Regul. Integr. Comp. Physiol. 265 (1993) R1216–R1222. [48] K. Scarbrough, S. Losee-Olson, E.P. Wallen, F.W. Turek, Aging and photoperiod effects on entrainment and quantitative aspects of locomotor behavior in Syrian hamsters, Am. J. Physiol. Regul. Integr. Comp. Physiol. 272 (1997) R1219–R1225. [49] K. Scarbrough, F.W. Turek, Quantitative differences in the circadian rhythm of locomotor activity and vasopressin and vasoactive intestinal peptide gene expression in the suprachiasmatic nucleus of tau mutant compared to wildtype hamsters, Brain Res. 736 (1996) 251–259. [50] R. Teclemariam-Mesbah, A. Kalsbeek, P. Pevet, R.M. Buijs, Direct vasoactive intestinal polypeptide-containing projection from the suprachiasmatic nucleus to spinal projecting hypothalamic paraventricular neurons, Brain Res. 748 (1997) 71–76. [51] E. Van Cauter, L. Plat, R. Leproult, G. Copinschi, Alterations of circadian rhythmicity and sleep in aging: Endocrine consequences, Horm. Res. 49 (1998) 147–152. [52] A.N. van den Pol, K.L. Tsujimoto, Neurotransmitters of the hypothalamic suprachiasmatic nucleus: Immunocytochemical analysis of 25 neuronal antigens, Neuroscience 15 (1985) 1049–1086. [53] E. Van der Beek, Circadian control of reproduction in the female rat, Prog. Brain Res. 111 (1996) 296–320. [54] O. Van Reeth, Y. Zhang, A. Reddy, P. Zee, F.W. Turek, Aging alters the entraining effects of an activity-inducing stimulus on the circadian clock, Brain Res. 607 (1993) 286–292. [55] O. Van Reeth, Y. Zhang, P.C. Zee, F.W. Turek, Aging alters feedback effects of the activity–rest cycle on the circadian clock, Am. J. Physiol. Regul. Integr. Comp. Physiol. 263 (1992) R981–R986. [56] J. Vanecek, K. Watanabe, Melatonin inhibits the increase of cyclic AMP in rat suprachiasmatic neurons induced by vasoactive intestinal peptide, Neurosci. Lett. 252 (1998) 21–24. [57] A.G. Watts, L.W. Swanson, Efferent projections of the suprachiasmatic nucleus: II. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunochemistry in the rat, J. Comp. Neurol. 258 (1991) 230–252. [58] G. Winocur, L. Hasher, Aging and time-of-day effects on cognition in rats, Behav. Neurosci. 113 (1999) 991–997. [59] P.M. Wise, Alterations in proestrous LH, FSH, and prolactin surges in middle-aged rats, Proc. Soc. Exp. Biol. Med. 169 (1982) 348– 354. [60] W.S. Young III, E. Mezey, R.E. Siegel, Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: analysis by quantitative in situ hybridization histochemistry, Mol. Brain Res. 1 (1986) 231–241. [61] P.C. Zee, R.S. Rosenberg, F.W. Turek, Effects of aging on entrainment and rate of resynchronization of circadian locomotor activity, Am. J. Physiol. Regul. Integr. Comp. Physiol. 263 (1992) R1099– R1103.
M. J. Duncan et al. / Molecular Brain Research 87 (2001) 196 – 203 [62] Y. Zhang, J.M. Kornhauser, P.C. Zee, K.E. Mayo, J.S. Takahashi, F.W. Turek, Effects of aging on light-induced phase-shifting of circadian behavioral rhythms, Fos expression and CREB phosphorylation in the hamster suprachiasmatic nucleus, Neuroscience 70 (1996) 951–961.
203
[63] R.T. Zoeller, B. Broyles, J. Earley, E.R. Anderson, H.E. Albers, Cellular levels of messenger ribonucleic acids encoding vasoactive peptide and gastrin-releasing peptide in neurons of the suprachiasmatic nucleus exhibit distinct 24-h rhythms, J. Neuroendocrinol. 4 (1992) 119–124.