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 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.

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.

Culture and transplantation of the mammalian circadian pacemaker.

In transplantation studies using the tau mutation in the golden hamster, it has been demonstrated that suprachiasmatic nucleus (SCN) pacemaker cells and mechanisms of communication with the host brain are retained even after tissue dissociation and maintenance for many weeks in primary cell culture. Brain grafts of cultured SCN cells are capable of restoring overt rhythms of locomotor activity, and preliminary studies where cells from two tau genotypes are combined in a single graft demonstrate that pacemaker cells may communicate with each other to produce coherent rhythms with intermediate periods. The opportunity is presented, therefore, to study pacemaker-pacemaker communication in circadian chimeras produced by SCN transplantation. Immunocytochemical analysis of graft-host interactions requires the positive identification of host versus donor cells. Although grafted blocks of tissue are easily recognized during immunocytochemical analysis, implants of dissociated and cultured cells may be more diffusely located and are not as readily identified. Unless distinct strain- or species-specific markers are available, it is difficult to identify connections that may carry timing information to the host organism. We have taken an anatomical approach that utilizes cell-labeling techniques for hamster tissue along with foreign protein expression in transgenic mice to identify patterns of communication among graft and host cells, focusing specifically on SCN-SCN communication. The data indicate the usefulness of these transgenes as markers in transplantation studies where communication between graft and host is addressed.

The effect of transplanting one or two suprachiasmatic nuclei on the period of the restored rhythm.

A fundamental property of circadian rhythms is the free-running period expressed by organisms when isolated from environmental periodicity. The physiological determinants of the free-running period, including variation among and within individuals and among species, are not known. The circadian rhythms of mammals are regulated by a circadian pacemaker within the suprachiasmatic nucleus (SCN) of the hypothalamus. To examine possible determinants of the free-running period, one or two SCNs were transplanted into hamsters that had their own SCNs ablated. Wheel-running behavior was measured to estimate the free-running period of restored rhythmicity. Hosts received grafts containing either the left or right SCN from a single fetus or both SCNs from a single fetus. In some cases, both the left and right SCNs from a single fetus restored rhythmicity in different hosts, demonstrating that each of the right and left SCN alone is a competent circadian pacemaker. The average free-running period of the restored rhythms was significantly longer in hamsters that received both of the SCNs from a single fetus. The sizes of grafts were estimated using immunoreactivity for vasoactive intestinal polypeptide as a marker of SCN tissue. Grafts never grew to be larger than an intact SCN, and a graft only 6.5% the size of the combined left and right intact SCNs restored rhythmicity. The average volume of grafted SCN in hamsters that received two SCNs was larger than that in hamsters that received a single SCN. The results demonstrate that SCN graft volume and/or the number of SCNs that comprise the graft influence the free-running period.

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.

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.

Restoration of circadian rhythmicity by transplants of SCN "micropunches".

Although it is widely accepted that the suprachiasmatic nuclei (SCN) of the hypothalamus serve as biological pacemakers regulating circadian rhythmicity, a number of studies suggest that some circadian rhythms may be controlled by extra-SCN structures. Transplantation of fetal anterior hypothalamic tissue containing the SCN restores circadian locomotor rhythms in SCN-lesioned hosts. Such transplants, however, contain substantial extra-SCN hypothalamic tissue. In the present study, the authors examined the recovery of circadian locomotor rhythms in animals implanted with small grafts harvested by taking "micropunches" from vibratome-sectioned brain slices. Micropunches were taken from three areas of the hypothalamus known to receive retinal input: the SCN, the subparaventricular zone, and the supraoptic nucleus. The results indicate that transplants restricted to the SCN region are necessary and sufficient for restoration of circadian locomotor activity rhythms and that micropunches of tissues from other sources are ineffective.

Age-related changes in light-induced Jun-B and Jun-D expression: effects of transplantation of fetal tissue containing the suprachiasmatic nucleus.

Fos and Jun mRNA and peptide exhibit a daily light-induced rhythm in the suprachiasmatic nucleus (SCN). The authors previously have reported that Fos expression in the SCN is elevated prematurely during the dark, light-induced Fos expression is attenuated in middle-aged rats, and transplantation of fetal SCN tissue into the third ventricle of rats of this age restores the daily pattern of Fos expression to that of the young. Using immunocytochemistry, the authors performed the present study to determine whether Jun-B and Jun-D expression in the SCN is altered at the same stage during aging and, if so, whether transplantation of fetal tissue containing the SCN can restore the light-induced rhythms of these two immediate early genes. All groups of rats were transcardially perfused 90 min prior to and after light onset. In young rats, light induced a robust increase in the number of Jun-B positive cells in the SCN, whereas very few cells were labeled before light onset. In middle-aged rats, the light-induced increase in the number of Jun-B positive cells was significantly attenuated. Transplantation of fetal SCN tissue into the middle-aged rats successfully restored light-induced Jun-B expression to the levels of young rats. By contrast, Jun-D exhibited a constitutively high level of expression in the SCN both before and after light onset, and light induced only a slight but significant increase. No age-related changes were detected in the expression of Jun-D either before or after light onset. Transplantation of fetal SCN tissue did not alter the daily pattern of Jun-D expression in the middle-aged rats. These data suggest that (1) light-induced activation of SCN neural activity is blunted during aging, (2) fetal SCN tissue can provide the critical support to allow the host to respond properly to light cues, and (3) the age-related change in Jun-B expression in the middle-aged host SCN can be rescued by fetal SCN transplants.

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.

Regulation of the phase and period of circadian rhythms restored by suprachiasmatic transplants.

The influence of exogenous signals on circadian rhythms restored by transplants of the suprachiasmatic nucleus (SCN) of the hypothalamus has received little study. The authors tested the responsiveness of hamsters bearing SCN transplants to photic and pharmacological treatments. Light intensities as high as 6,500 lux were insufficient to produce entrainment, although masking was observed frequently. Triazolam failed to produce statistically significant phase shifts when administered during the subjective day, but 2 animals bearing functional SCN grafts responded to this benzodiazapine during the subjective night. The authors next tested the hypothesis that the host can retain circadian aftereffects that influence the period of the circadian system reconstituted by the graft. Intact hamsters were entrained to light:dark cycles of short (23.25-h) and long (25-h) period (T) for at least 3 months. Control hamsters released into constant darkness exhibited profound and long-lasting aftereffects of entrainment to T cycles. Hamsters that received SCN lesions after exposure to these T cycles and SCN grafts 3 weeks later exhibited marginal but statistically significant aftereffects that disappeared within 3 months. On subsequent transfer to constant light, tau lengthened by 0.25 +/- 0.6 h in hamsters with intact SCN (p < .05). Animals bearing SCN grafts continued to free run in constant light but differed from intact animals in that circadian period did not lengthen. Functional SCN grafts contained vasoactive intestinal polypeptide (VIP), neurophysin (NP), and cholecystokinin (CCK) immunoreactive (ir) cells. Inputs of neuropeptide Y-and serotonin-ir fibers from the host brain to grafted SCN peptide cell clusters were variable. Limited observations using retrograde and anterograde tracers do not support the existence of extensive input to the graft. Retinal input overlapped only rarely with clusters of VIP-ir, CCK-ir, or NP-ir cells. The authors conclude that the circadian system reinstated by SCN transplants is relatively impervious to photic influences that exert parametric and nonparametric influences in intact hamsters. The transient expression of aftereffects induced in the host before transplantation indicates that extra-SCN systems of the host can influence the period of the reconstituted circadian system to at least a limited degree.

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.

Organization and efferent connections of transplanted suprachiasmatic nuclei.

The hypothalamic suprachiasmatic nucleus (SCh) is the principal brain structure involved in the generation of circadian rhythms. In the present study, we have employed immunohistochemical techniques to evaluate the development of the fetal SCh following its transplantation to the brain of adult host animals. Donor hypothalami were obtained from normal Long-Evans fetuses and transplanted to the lateral, third, or fourth ventricle of Brattleboro rats. Neuronal aggregations exhibiting the organotypic features of the SCh were present in over 90% of the grafts recovered at each transplantation site. Like the normal endogenous SCh, SCh-like cell groups identified within the transplants contained a prominent population of parvicellular (9-13 micron), neurophysin-containing neurons that were immunopositive for vasopressin (VP) but not oxytocin. These SCh-like cell groups also invariably contained similar small neurons that were immunoreactive for vasoactive intestinal polypeptide (VIP). Typically, VP and VIP immunoreactive perikarya were concentrated in contiguous, complementary parts of the grafted SCh, but fibers immunoreactive for either peptide were distributed throughout the extent of the nucleus. Because the brain of the Brattleboro rat is deficient in vasopressin, it was possible to evaluate the projection of the vasopressinergic component of the transplanted SCh to the host brain. Although SCh were identified in grafts recovered from each intraventricular transplantation site, an appreciable input to the host brain could be identified only when the fetal tissue was grafted to the third ventricle. Here, grafted SCh established efferent connections with periventricular diencephalic structures which ordinarily receive a projection from the in situ SCh. Specifically, VP immunoreactive fibers originating from transplanted SCh were identified in the medial preoptic area, the periventricular and dorsomedial hypothalamic nuclei, the paraventricular nuclei of the thalamus and hypothalamus, and in the retrochiasmatic area, arcuate nucleus, and suprachiasmatic nucleus of the host brain. These results demonstrate that the fetal SCh not only survives transplantation but also retains its distinguishing cytological features and the capacity to form an appropriately restricted set of efferent connections with the brain of adult host animals.

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.

Cellular communication in the circadian clock, the suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus functions as the circadian clock in the mammalian brain. Communication between the cells of the suprachiasmatic nucleus is likely to be responsible for the generation and accuracy of this biological clock. Communication between many cells of the brain is mediated by action potentials that pass down the axon and cause release of neurotransmitters at the neuronal synaptic junction. Additional mechanisms of cellular communication appear to operate in the suprachiasmatic nucleus. Several lines of evidence point to multiple modes of cellular communication: these include the continuing operation of the clock after Na(+)-mediated action potentials have been blocked, the orchestrated metabolic rhythms of suprachiasmatic nucleus cells prior to synaptogenesis, the entrainment of fetal to maternal rhythms, and the rapid recovery of function after suprachiasmatic nucleus transplants into arrhythmic rodents. Possible alternative means of intercellular communication in the suprachiasmatic nucleus are examined, including calcium spikes in presynaptic dendrites, ephaptic interaction, paracrine communication, glial mediation, and gap junctions. This paper identifies and examines some of the unanswered questions related to intercellular communication of suprachiasmatic nucleus cells.

How do fetal grafts of the suprachiasmatic nucleus communicate with the host brain?

Fetal grafts containing the hypothalamic suprachiasmatic nucleus (SCN), the site of an endogenous circadian pacemaker, can reinstate behavioral rhythms in lesioned recipients but the precise routes of communication between the graft and the host brain remain unknown. Grafts containing the SCN may convey temporal information to the host brain via neural efferents, diffusible factors, or a combination of both. We examined graft-host connections in anterior hypothalamic homografts (hamster-to hamster) and heterografts (rat-to hamster) implanted in the third ventricle by: (a) applying the carbocyanine dye, diI, directly onto homo- and heterografts in fixed tissue sections; and (b) using a donor-specific neurofilament (NF) antibody to immunocytochemically visualize heterograft efferents. DiI applied onto either homografts or heterografts labeled relatively few graft efferents which could be followed only short distances into the host brain. In contrast, NF-labeled heterograft efferents were both more numerous and extended for longer distances into the host brain than anticipated on the basis of diI tract tracing. The results suggest that anterior hypothalamic grafts implanted in the third ventricle provide substantial input to the adjacent host hypothalamus although it is not known whether these projections arise from SCN cells or from other extra-SCN hypothalamic tissue within these grafts. Nor is it known whether these projections are functional. To determine if neural efferents are required for the restoration of rhythmicity after grafting, we have encapsulated fetal anterior hypothalamus in a permselective polymer which prevents neurite outgrowth but allows diffusible signals to reach the host brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation of the neonatal suprachiasmatic nuclei into rats with complete bilateral suprachiasmatic lesions.

The suprachiasmatic nuclei (SCN) obtained from neonatal day 1 rats were transplanted into the third ventricle of SCN lesioned rats which had shown circadian arrhythmicity in wheel-running activity for more than 2 months. In 8 out of 16 rats that received SCN transplantation, appearance of circadian rhythms in wheel-running activity was observed between 2 and 9 weeks after transplantation. Histological examination revealed ingrowth of the grafts into the periventricular zone, caudal from the lesioned SCN. These findings suggest that the recovery of circadian rhythmicity was the result of functional reinnervation of the periventricular zone by efferent fibers from the SCN.

Adenoviral vector-mediated gene transfer and neurotransplantation: possibilities and limitations in grafting of the fetal rat suprachiasmatic nucleus.

Several studies have reported on the use of primary neural cells transduced by adenoviral vectors as donor cells in neurotransplantation. In the present investigation, we examined whether adenoviral vector-mediated gene transfer could be used to introduce and express a foreign gene in solid neural transplants of fetal suprachiasmatic nucleus (SCN) tissue. A recombinant adenoviral vector containing the reporter gene LacZ encoding for beta-galactosidase (Ad-LacZ) was used in order to establish the optimal procedure for ex vivo gene transfer. Expression of beta-galactosidase was dependent on the duration of the infection and on the vector concentration. Infection for a short period (< 4 h) with a high concentration of Ad-LacZ (3.4 x 10(9) pfu/ml), or for 18 h with a lower vector concentration (2 x 10(8) pfu/ml), resulted in expression of beta-galactosidase in a large number of neurons and glial cells up to 21 days in vitro. When infected fetal SCN tissue was implanted in the third ventricle of adult Wistar rats, expression was high after 8 days. After 21 days, the number of beta-galactosidase expressing cells had clearly declined, but expression remained present for at least 70 days. The method described in this paper might be applicable to introduce trophic factor genes in SCN grafts in order to support graft survival and to stimulate neurite outgrowth.

Fetal suprachiasmatic nucleus transplants: diurnal rhythm recovery of lesioned rats.

The diurnal rhythm organization of drinking behavior was determined prior to and after suprachiasmatic nucleus (SCN) lesions. Five weeks after SCN lesions which produced total loss of the diurnal rhythm, the anterior hypothalamus including the SCN of fetal rats was transplanted into the floor of the 3rd ventricle of the lesioned rats. Eight weeks after the graft, rats recovered their rhythm. The results show that grafts allow animals to recover lost functional properties due to lesions, and further support the notion that the SCN is a pacemaker for certain behaviors.

Suprachiasmatic nucleus grafts restore circadian function in aged hamsters.

The regulation of circadian rhythms changes with age. In humans, changes in the timing of sleep and wakefulness are especially common. In Syrian hamsters Mesocricetus auratus the free running period of the activity/rest rhythm shortens with age. The present study tested the hypothesis that critical age-related changes occur within the hypothalamic suprachiasmatic nucleus (SCN), known to contain a circadian pacemaker. Fetal SCN were transplanted into the brains of younger (20 weeks) and older (81 weeks) hamsters which had had their own SCNs ablated. The restoration of rhythmicity and the free running period of the rhythmicity were determined from continuous records of wheel-running activity. Transplantation restored rhythmicity in hosts of both ages. In older hamsters, the mean free running period after transplantation was longer than that measured before SCN ablation, but a similar lengthening of period was not observed after transplantation to younger hamsters. In addition, the mean period after transplantation was the same for both younger and older hosts even when there was a difference between the groups before SCN ablation. When the grafts were allowed to age, the mean free running period of the restored rhythms became shorter, indicating that the grafts can also undergo age-related changes. The results indicate that age-related changes specifically in the SCN are responsible for an age-related change in free running period.