Temporal profile of circadian clock gene expression in a transplanted suprachiasmatic nucleus and peripheral tissues.

The mammalian hypothalamic suprachiasmatic nucleus (SCN) is the master oscillator that regulates the circadian rhythms of the peripheral oscillators. Previous studies have demonstrated that the transplantation of embryonic SCN tissues into SCN-lesioned arrhythmic mice restores the behavioral circadian rhythms of these animals. In our present study, we examined the clock gene expression profiles in a transplanted SCN and peripheral tissues, and also analysed the circadian rhythm of the locomotor activity in SCN-grafted mice. These experiments were undertaken to elucidate whether the transplanted SCN generates a dynamic circadian oscillation and maintains the phase relationships that can be detected in intact mice. The grafted SCN indeed showed dynamic circadian expression rhythms of clock genes such as mPeriod1 (mPer1) and mPeriod2 (mPer2). Furthermore, the phase differences between the expression rhythms of these genes in the grafted SCN and the locomotor activity rhythms of the transplanted animals were found to be very similar to those in intact animals. Moreover, in the liver, kidney and skeletal muscles of the transplanted animals, the phase angles between the circadian rhythm of the grafted SCN and that of the peripheral tissues were maintained as in intact animals. However, in the SCN-grafted animals, the amplitudes of the mPer1 and mPer2 rhythms were attenuated in the peripheral tissues. Our current findings therefore indicate that a transplanted SCN has the capacity to generate a dynamic intrinsic circadian oscillation, and can also lock the normal phase angles among the SCN, locomotor activity and peripheral oscillators in a similar manner as in intact control animals.

Suprachiasmatic regulation of circadian rhythms of gene expression in hamster peripheral organs: effects of transplanting the pacemaker.

Neurotransplantation of the suprachiasmatic nucleus (SCN) was used to assess communication between the central circadian pacemaker and peripheral oscillators in Syrian hamsters. Free-running rhythms of haPer1, haPer2, and Bmal1 expression were documented in liver, kidney, spleen, heart, skeletal muscle, and adrenal medulla after 3 d or 11 weeks of exposure to constant darkness. Ablation of the SCN of heterozygote tau mutants eliminated not only rhythms of locomotor activity but also rhythmic expression of these genes in all peripheral organs studied. The Per:Bmal ratio suggests that this effect was attributable not to asynchronous rhythmicity between SCN-lesioned individuals but to arrhythmicity within individuals. Grafts of wild-type SCN to heterozygous, SCN-lesioned tau mutant hamsters not only restored locomotor rhythms with the period of the donor but also led to recovery of rhythmic expression of haPer1, haPer2, and haBmal1 in liver and kidney. The phase of these rhythms most closely resembled that of intact wild-type hamsters. Rhythmic gene expression was also restored in skeletal muscle, but the phase was altered. Behaviorally effective SCN transplants failed to reinstate rhythms of clock gene expression in heart, spleen, or adrenal medulla. These findings confirm that peripheral organs differ in their response to SCN-dependent cues. Furthermore, the results indicate that conventional models of internal entrainment may need to be revised to explain control of the periphery by the pacemaker.

Suprachiasmatic nuclei grafts restore the circadian rhythm in the paraventricular nucleus of the hypothalamus.

The mammalian suprachiasmatic nucleus (SCN) controls the circadian rhythm of many physiological and behavioral events by an orchestrated output of the electrical activity of SCN neurons. We examined the propagation of output signals from the SCN into the hypothalamus, especially into the region of the paraventricular nucleus, through multimicroelectrode recordings using acute and organotypic brain slices. Circadian rhythms in spontaneous firing rate with a period close to 24 hr were demonstrated in the SCN, in directly adjacent hypothalamic regions, and in the region of the paraventricular nucleus of the hypothalamus, an important center for the integration of neuroendocrine, homeostatic, and autonomic functions. The activity rhythms recorded from structures outside of the SCN were in phase with the rhythms in the SCN. Cyclic information in the hypothalamus was lost after ablation of the SCN but could be restored by SCN grafts, indicating that a humoral factor is responsible for the restoration of circadian rhythmicity in the absence of neural connections. Periodic application of arginine-vasopressin (AVP) provided evidence that AVP can induce rhythmicity in the hypothalamus. These data indicate that the SCN uses a dual (neuronal and humoral) mechanism for communication with its targets in the brain.

A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleus.

A mouse bearing a novel transgene encoding the human VPAC2 receptor (hVIPR; Shen et al. (2000) PNAS, 97, 11575-11580) was used to investigate circadian function in the hypothalamic suprachiasmatic nuclei (SCN). Neurons expressing hVPAC2R, detected by a beta-galactosidase (beta-GAL) tag, have a distinct distribution within the SCN, closely matching that of neurophysin (NP) neurons and extending into the region of peptide histidine isoleucine (PHI) cells. In common with NP and PHI cells, neurons expressing hVPAC2R are circadian in nature, as revealed by synchronous rhythmic expression of mPERIOD (mPER) proteins. A population of SCN cells not expressing PHI, NP or hVPAC2R exhibited circadian PER expression antiphasic with the rest of the SCN. Nocturnal light exposure induced mPER1 in the ventral SCN and mPER2 widely across the nucleus. Induction of nuclear mPER2 in hVPAC2R cells confirmed their photic responsiveness. Having established their circadian properties, we tested the utility of SCN neurons expressing the hVIPR transgene as functionally and anatomically explicit markers for SCN tissue grafts. Prenatal SCN tissue from hVIPR transgenic pups survived transplantation into adult CD1 mice, and expressed beta-GAL, PER and PHI. Over a series of studies, hVIPR transgenic SCN grafts restored circadian activity rhythms to 17 of 72 arrhythmic SCN lesioned recipients (23.6%). By using heterozygous hVIPR transgenic grafts on a heterozygous Clock mutant background we confirmed that restored activity rhythms were conferred by the donor tissue. We conclude that the hVIPR transgene is a powerful and flexible tool for examination of circadian function in the mouse SCN.

Suprachiasmatic nucleus grafts restore circadian behavioral rhythms of genetically arrhythmic mice.

The mammalian master clock driving circadian rhythmicity in physiology and behavior resides within the suprachiasmatic nuclei (SCN) of the hypothalamus. SCN neurons contain a molecular oscillator composed of a set of clock genes that acts in intertwined negative and positive feedback loops [1]. In addition, all peripheral tissues analyzed thus far have been shown to contain circadian oscillators [2]. This raises the question of whether the central circadian pacemaker in the SCN is sufficient to evoke behavioral rhythms or whether peripheral circadian clockworks are also required. Mice with a mutated CLOCK protein (a transcriptional activator of E box-containing clock and clock output genes) or lacking both CRYPTOCHROMES, mCRY1 and mCRY2 proteins (inhibitors of E box-mediated transcription), lack circadian rhythmicity in behavior [3,4]. Here, we show that transplantation of mouse fetal SCN tissue into the hypothalamus restores free-running circadian behavioral rhythmicity in Clock mutant or mCry1/mCry2 double knockout mice. The periodicity of the emerged rhythms is determined by the genetic constitution (i.e., wild-type or mCry2 knockout) of the grafted SCN. Since transplanted mCry1/mCry2-deficient mice do not have functional circadian oscillators [5] other than those present in the grafted hypothalamus region, these findings suggest that the SCN can generate circadian behavioral rhythms in the absence of distant peripheral oscillators in the brain or elsewhere.

A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleus.

A mouse bearing a novel transgene encoding the human VPAC2 receptor (hVIPR; Shen et al. (2000) PNAS, 97, 11575-11580) was used to investigate circadian function in the hypothalamic suprachiasmatic nuclei (SCN). Neurons expressing hVPAC2R, detected by a beta-galactosidase (beta-GAL) tag, have a distinct distribution within the SCN, closely matching that of neurophysin (NP) neurons and extending into the region of peptide histidine isoleucine (PHI) cells. In common with NP and PHI cells, neurons expressing hVPAC2R are circadian in nature, as revealed by synchronous rhythmic expression of mPERIOD (mPER) proteins. A population of SCN cells not expressing PHI, NP or hVPAC2R exhibited circadian PER expression antiphasic with the rest of the SCN. Nocturnal light exposure induced mPER1 in the ventral SCN and mPER2 widely across the nucleus. Induction of nuclear mPER2 in hVPAC2R cells confirmed their photic responsiveness. Having established their circadian properties, we tested the utility of SCN neurons expressing the hVIPR transgene as functionally and anatomically explicit markers for SCN tissue grafts. Prenatal SCN tissue from hVIPR transgenic pups survived transplantation into adult CD1 mice, and expressed beta-GAL, PER and PHI. Over a series of studies, hVIPR transgenic SCN grafts restored circadian activity rhythms to 17 of 72 arrhythmic SCN lesioned recipients (23.6%). By using heterozygous hVIPR transgenic grafts on a heterozygous Clock mutant background we confirmed that restored activity rhythms were conferred by the donor tissue. We conclude that the hVIPR transgene is a powerful and flexible tool for examination of circadian function in the mouse SCN.

Adenoviral vector-mediated expression of neurotrophin-3 increases neuronal survival in suprachiasmatic nucleus grafts.

To improve transplantation results of fetal suprachiasmatic nucleus (SCN) in SCN-lesioned (SCNX) rats, grafts were ex vivo transduced with an adenoviral vector encoding for neurotrophin-3 (AdNT-3) before implantation. Mock- and AdLacZ-transduced grafts were used as controls. First, transplants were evaluated microscopically and by image analysis for the presence of vasopressinergic (VPergic) and vasoactive intestinal polypeptidergic (VIPergic) SCN neurons at 10 weeks or later postgrafting. Ex vivo AdNT-3-transduced transplants displayed increased volume areas of VPergic and VIPergic SCN cells in comparison with those in mock- and AdLacZ-transduced transplants, but significantly improved graft-to-host VPergic and VIPergic SCN fiber growth was not reached (though AdNT-3-transduced transplants tended to grow more VPergic fibers into the brain of VP-deficient SCNX Brattleboro rat recipients, which were chosen as recipients to circumvent the presence of non-SCN VP fiber staining). Second, a small group of arrhythmic Wistar rats received AdNT-3- or control-treated SCN grafts while continuously on-line for the monitoring of overt circadian activities in the pre- and postgrafting periods. The results indicated that ex vivo transduced SCN grafts can still restore arrhythmia, but that the NT-3-mediated anatomical improvements of the grafting results were not sufficient to enhance efficacy of reinstatement of circadian rhythm in SCN-lesioned rats. However, in this group VIP staining volume area, not VP staining volume area, correlated significantly with reinstatement of circadian rhythm.

Localization of a suprachiasmatic nucleus subregion regulating locomotor rhythmicity.

The bilaterally symmetrical suprachiasmatic nuclei (SCN) of the hypothalamus are the loci of the mammalian clock controlling circadian rhythms. Previous studies suggested that all regions of the SCN are equipotential as circadian rhythmicity is sustained after partial ablation, as long as approximately 25% of the nuclei are spared. In contrast to these results, we found that animals bearing partial lesions of the SCN that spared the subregion delimited by cells containing the calcium-binding protein calbindin-D28K (CaBP), sustained circadian locomotor rhythms. Furthermore, there was a correlation between the strength of the rhythm and the number of spared CaBP cells. Partial lesions that destroyed this region but spared other compartments of the SCN resulted in loss of rhythmicity. The next study indicates that transplants of half-SCN grafts that contain CaBP cells restore locomotor rhythms in SCN-lesioned host animals, whereas transplants containing SCN tissue but lacking cells of this subnucleus fail to restore rhythmicity. Finally, there was a correlation between the number of CaBP-positive cells in the graft and the strength of the restored rhythm. Taken together, the results indicate that pacemakers in the region of the CaBP subnucleus are necessary and sufficient for the control of locomotor rhythmicity and that the SCN is functionally heterogeneous.

Vasopressin-deficient suprachiasmatic nucleus grafts re-instate circadian rhythmicity in suprachiasmatic nucleus-lesioned arrhythmic rats.

It was investigated whether grafts of the suprachiasmatic nucleus could re-instate circadian rhythmicity in the absence of its endogenous vasopressin production and whether the restored rhythm would have the long period length of the donor. Grafts of 17-days-old vasopressin-deficient homozygous Brattleboro rat fetuses, homotopically placed in arrhythmic suprachiasmatic nucleus-lesioned Wistar rats, re-instated circadian drinking rhythm within 20-50 days similar as seen for grafts of heterozygous control fetuses. Period length of the recovered rhythm revealed a similar difference (average 24.3 vs. 23.8 h) as reported for the rhythm between the adult Brattleboro genotypes. In all transplants, also those of the two-third non-recovery rats, a surviving suprachiasmatic nucleus was visible as a vasoactive intestinal polypeptide-positive neuronal cell cluster, whereas heterozygous transplants also revealed the complementary vasopressinergic cell part. Explanation of the absence of recovery failed since no undisputable correlation emerged between recovery of rhythm and vasoactive intestinal polypeptide, vasopressin and/or somatostatin immunocytochemical characteristics of the suprachiasmatic nucleus of the transplant. Special focus on the somatostatinergic neurons revealed their presence only occasionally near or in between the vasoactive intestinal polypeptidergic and (in the case of heterozygous grafts) vasopressinergic cell cluster. However their aberrant cytoarchitectural position appeared not to have affected the possibility to restore drinking rhythm of the suprachiasmatic nucleus-lesioned arrhythmic rat. It was concluded that grafted Brattleboro fetal suprachiasmatic nucleus develop their intrinsic rhythm conform their genotype and that vasopressin is not a crucial component in the maintenance nor in the transfer of circadian activity of the biological clock for drinking activity. Vasopressin of the suprachiasmatic nucleus may instead serve modulation within the circadian system.

Circadian rhythmicity of vasopressin levels in the cerebrospinal fluid of suprachiasmatic nucleus-lesioned and -grafted rats.

Transplantation of the fetal suprachiasmatic nucleus (SCN) in arrhythmic SCN-lesioned rats can reinstate circadian drinking rhythms in 40% to 50% of the cases. In the current article, it was investigated whether the failure in the other rats could be due to the absence of a circadian rhythm in the grafted SCN, using a circadian vasopressin (VP) rhythm in the cerebrospinal fluid (CSF) as the indicator for a rhythmic SCN. CSF was sampled in continuous darkness from-intact control rats and SCN-lesioned and -grafted rats. VP could be detected in all samples, with concentrations of 15 to 30 pg/ml in the control rats and 5 to 15 pg/ml in the grafted rats. A circadian VP rhythm with a two- to threefold difference between peak and nadir values was found in all 7 control rats but in only 4 of 13 experimental rats, despite the presence of a VP-positive SCN in all grafts. A circadian VP rhythm was present in 2 drinking rhythm-recovered rats (6 of 13) and in 2 nonrecovery rats. Apparently, in these latter rats, the failure of the grafted SCN to restore a circadian drinking rhythm cannot be attributed to a lack of rhythmicity in the SCN itself. Thus, the presence of a rhythmic grafted SCN, as is deduced from a circadian CSF VP rhythm, appears not to be sufficient for restoration of a circadian drinking rhythm in SCN-lesioned arrhythmic rats.

Effects of suprachiasmatic transplants on circadian rhythms of neuroendocrine function in golden hamsters.

Grafts of fetal tissue including the suprachiasmatic nucleus (SCN) of the hypothalamus restore locomotor rhythmicity to behaviorally arrhythmic, SCN-lesioned Syrian hamsters. We sought to determine whether such transplants also reinstate endocrine rhythms in SCN-lesioned hamsters. In Exp 1, SCN lesions interrupted estrous cycles in a 14 h light, 10 h dark photoperiod and locomotor rhythms in constant dim red light (DD). SCN grafts that reinstated behavioral circadian rhythms consistently failed to reestablish estrous cycles. After ovariectomy, estradiol implants triggered LH surges at approximately circadian time 8 in 10 of 12 brain-intact control females and 0 of 9 SCN-lesioned, grafted females. Daily rhythms of the principal urinary melatonin metabolite, 6alpha-sulfatoxymelatonin, were not reestablished by behaviorally functional grafts. In Exp 2, SCN lesions eliminated locomotor rhythmicity in adult male hamsters maintained in DD. Seven to 12 weeks after restoration of locomotor activity rhythms by fetal grafts, hosts and sham-lesioned controls were decapitated at circadian times 4, 8, 12, 16, 20, or 24. Clear circadian rhythms of both serum corticosterone and cortisol were seen in sham-lesioned males, with peaks in late subjective day. No circadian rhythms in either adrenal hormone were evident in serum from lesioned-grafted males. Testicular regression, observed in intact and sham-lesioned males maintained in DD, was absent not only in arrhythmic SCN-lesioned hamsters given grafts of cerebral cortex, but also in animals in which hypothalamic grafts had reinstated locomotor rhythmicity. The pineal melatonin concentration rose sharply during the late subjective night in control hamsters, but not in SCN-lesioned animals bearing behaviorally effective transplants. Even though circadian rhythms of locomotor activity are restored by SCN transplants, circadian endocrine rhythms are not reestablished. Endocrine rhythms may require qualitatively different or more extensive SCN outputs than those established by fetal grafts.

Restoration of circadian behavior by anterior hypothalamic grafts containing the suprachiasmatic nucleus: graft/host interconnections.

Destruction of the hypothalamic suprachiasmatic nucleus (SCN) disrupts circadian behavior. Transplanting SCN tissue from fetal donors into SCN-lesioned recipients can restore circadian behavior to the arrhythmic hosts. In the transplantation model employing fetal hamster donors and SCN-lesioned hamsters as hosts, the period of the restored circadian behavior is hamster-typical. However, when fetal rat anterior hypothalamic tissue containing the SCN is implanted into SCN-lesioned rats, the period of the restored circadian rhythm is only rarely typical of that of the intact rat. The use of an anterior hypothalamic heterograft model provides new approaches to donor specificity of restored circadian behavior and with the aid of species-specific markers, provides a means for assessing connectivity between the graft and the host. Using an antibody that stains rat and mouse neuronal tissue but not hamster neurons, it has been demonstrated that rat and mouse anterior hypothalamic heterografts containing the SCN send numerous processes into the host (hamster) neuropil surrounding the graft, consistent with graft efferents reported in other hypothalamic transplantation models in which graft and host tissue can be differentiated (i.e., Brattleboro rat and hypogonadal mouse). Moreover, SCN neurons within anterior hypothalamic grafts send an appropriately restricted set of efferent projections to the host brain which may participate in the functional recovery of circadian locomotor activity.

Output signals of the SCN.

The suprachiasmatic nucleus (SCN) of the hypothalamus controls circadian rhythmicity in mammals (for reviews, see Refs. 33 and 59). Responses modulated by the SCN are numerous and include rhythms in sleep/wake cycles, locomotor, gnawing and general activity, temperature, ingestive behavior, and rhythms of hormonal and peptide secretions. Though a great deal is known about the neuroanatomical organization of the SCN, many elements of the structure-function relationships remain to be discovered. For example, it is not known which cellular components of the SCN function as driving pacemakers or which output signal(s) of these pacemakers are important for each of its functions. While some signals from pacemaker cells reach target regions by neural efferents, there is also evidence that rhythmic responses can be controlled by diffusible signals. This article reviews output signals from the SCN. The data available suggest that neural efferents are not necessary for the control of locomotor activity rhythms. Evidence that a diffusible signal is sufficient to restore activity rhythms in SCN-lesioned animals is described. Finally, possible physiological mechanisms for diffusible signals are suggested.

Cellular requirements of suprachiasmatic nucleus transplants for restoration of circadian rhythm.

Fetal neurografts containing the suprachiasmatic nucleus (SCN) can restore the circadian locomotor and drinking rhythm of SCN-lesioned (SCNX) rat and hamster. This functional outcome finally proves that the endogenous biological clock autonomously resides in the SCN. Observations on the cellular requirements of the "new" SCN for restoration of the arrhythmic SCNX animals have led to some new insights and confirmed findings from other studies. A critical mass of SCN neurons appeared necessary for functional effects, whereas the temporal profile of reinstatement of rhythm correlated with the delayed maturation of the grafted SCN. Cytoarchitectonically, the grafted SCN does not seem to develop normally for all anatomical aspects. Complementary clusters of vasoactive intestinal polypeptide(VIP)- and vasopressin(VP)ergic neurons are formed, but somatostatin(SOM)ergic neurons do not always "join" this group, as is normally seen in situ. Nevertheless, these new SCNs can restore the ablated functions. As the period length of restored rhythms tends to vary, it might be that the grafted SCN underwent an altered or impaired maturation that resulted in a different setting of its clock mechanism. A prominent role of VIPergic neurons seems indicated by their presence in all functional grafts, but, although they may be required, these cells do not appear to be a sufficient condition for restoration of rhythm. Many grafts exhibit the presence of VIPergic cells without counteracting the arrhythmia, whereas VP- and SOMergic SCN neurons are usually present as well. Findings with VP-deficient Brattleboro rat grafts indicated that VP is not the primary obligatory signal of circadian activity. It is argued that perhaps the role of SOMergic neurons in the clock function of the (grafted) SCN has been insufficiently considered. However, one should keep in mind that the peptides of the various types of SCN neurons may function only as cofactors, mutually modulating molecular or bioelectrical cellular activities within the nucleus or the message of the main transmitter gamma-aminobutyric acid.

The significance of circadian organization for longevity in the golden hamster.

While functional roles for biological clocks have been demonstrated in organisms throughout phylogeny, the adaptive advantages of circadian organization per se are largely matters of conjecture. It is generally accepted, though without direct experimental evidence, that organisms derive primary benefits from the temporal organization of their physiology and behavior, as well as from the anticipation of daily changes in their environment and their own fluctuating physiological requirements. However, the consequences of circadian dysfunction that might demonstrate a primary adaptive advantage and explain the natural origins and apparent ubiquity of circadian systems have not been documented. The authors report that longevity in hamsters is decreased with a noninvasive disruption of rhythmicity and is increased in older animals given suprachiasmatic implants that restore higher amplitude rhythms. The results substantiate the importance of the temporal organization of physiology and behavior provided by the circadian clock to the health and longevity of an organism.

Melatonin entrains the restored circadian activity rhythms of syrian hamsters bearing fetal suprachiasmatic nucleus grafts.

A circadian pacemaker consists of at least three essential features: the ability to generate circadian oscillations, an output signal, and the ability to be entrained by external signals. In rodents, ablation of the suprachiasmatic nucleus (SCN) results in the loss of circadian rhythms in activity. Rhythmicity can be restored by transplanting fetal SCN into the brain of the lesioned animal, demonstrating the first two of the essential pacemaker features within the grafts. External signals, such as the light/dark cycle, have not, however, been shown to entrain the restored rhythms. Melatonin injections are an effective entraining stimulus in fetal and neonatal Syrian hamsters of the same developmental ages used to provide donor tissue for transplantation. Therefore, melatonin was used to test the hypothesis that SCN grafts contain an entrainable pacemaker. Daily injections of melatonin were given to SCN-lesioned hosts beginning on the day after transplantation of fetal SCN. Two groups that received melatonin at different times of day 12 hr apart each showed significantly clustered phases but with average phases that differed by 8.67 hr. Thus melatonin was able to entrain the restored circadian activity rhythms. In contrast to these initial injections, injections given 6 weeks after transplantation were unable to entrain or phase shift the rhythms. The results demonstrate that SCN grafts contain an entrainable circadian pacemaker. In addition, the results also indicate that the fetal SCN is directly sensitive to melatonin and, as with intact hamsters, sensitivity to melatonin is lost during SCN development.

Transgene expression in rat fetal brain grafts is maintained for 7 months after ex vivo adenoviral vector-mediated gene transfer.

Fetal brain tissue fragments containing the suprachiasmatic nucleus were infected with an adenoviral vector containing the marker gene LacZ encoding for beta-galactosidase, and subsequently cultured or transplanted in the third ventricle of SCN-lesioned adult Wistar rats. In previous studies we optimized the infection procedure and characterized the immunological response directed against the viral vector in this model. The present study reports on beta-gal expression for at least 7 months in neuronal and glial cells. Maturation of the transplanted fetal SCN with respect to immunoreactivity for vasoactive intestinal polypeptide and C-terminal propressophysin was not hampered by the viral infection.

Fetal grafts containing suprachiasmatic nuclei restore the diurnal rhythm of CRH and POMC mRNA in aging rats.

We assessed whether fetal tissue containing the suprachiasmatic nuclei (SCN) can restore age-related changes in the diurnal rhythm of hypothalamic corticotropin-releasing hormone (CRH) and anterior pituitary proopiomelanocortin (POMC) mRNA. Young, middle-aged, and middle-aged SCN-transplanted rats were killed at seven times of day. In young rats, CRH mRNA exhibited a diurnal rhythm in the dorsomedial paraventricular nuclei but not in other subdivisions of the nuclei. No rhythm was detected in aging rats. SCN transplants restored a rhythm in CRH mRNA, but the timing was not precisely the same as in young animals. POMC mRNA exhibited a daily rhythm in young rats. Aging abolished the rhythm and decreased the average mRNA level; fetal transplants restored the rhythm, but the amplitude remained attenuated. These data are the first demonstration that fetal tissue can restore the diurnal rhythm of a neuroendocrine axis that is driven by the SCN. We conclude that the neuroendocrine substrate from the aging host remains capable of responding to diurnal cues to express diurnal rhythmicity in CRH/POMC mRNA when fetal SCN transplants confer the appropriate signals.

Attachment site of grafted SCN influences precision of restored circadian rhythm.

Fetal hypothalamic grafts containing the suprachiasmatic nucleus (SCN) restore circadian locomotor rhythmicity when implanted into the third ventricle of SCN-lesioned hamsters. However, the quality of restored rhythms is variable, and the locomotor rhythms of grafted animals are generally less robust than those of intact animals. The present study explored whether anatomical features of the graft predict the quality of the recovered rhythm and whether such information might provide insight as to the target of the signal from the SCN that controls locomotor rhythmicity. The following graft parameters were assessed: distance between the attachment site of the graft and potential targets for the output signal from the SCN, number and overall size of SCN clusters, the size of the cluster closest to the SCN lesion site, and extent of vasoactive intestinal polypeptide (VIP) and vasopressin-associated neurophysin (NP) positive fiber outgrowth from the graft. The restored circadian activity rhythm was assessed by quantifying the precision of activity onset and the amount, period, and robustness of rhythmicity. The results indicate a significant positive correlation between the precision of activity onset and the proximity of the closest SCN cluster to the site of the lesioned host SCN. A more detailed analysis of the spatial location of the graft indicates that proximity of the graft in the dorsal and caudal directions, but not the rostral direction, is positively correlated with the precision of the recovered rhythm. This suggests two possibilities: the coupling signal may act on a site very near the SCN and travel preferentially in a rostro-caudal direction. Alternatively, the coupling signal may act on a site rostral to the SCN. That the site is not far rostral to the SCN was suggested by the lack of a correlation between the precision of the restored rhythm and the rostrally lying anterior medial preoptic nucleus. Finally, evaluation of NP- and VIP-ergic fibers in nuclei known to receive input from the SCN indicates that the extent of such innervation by graft efferents does not predict either the occurrence of recovery or the precision of the recovered rhythm. Overall, these results suggest that the target(s) of SCN pacemakers regulating locomotor rhythmicity lie in the hypothalamus, close to or rostral to the SCN.