Developmental and functional aspects of grafting of the suprachiasmatic nucleus in the Brattleboro and the arrhythmic rat

The development of suprachiasmatic nuclei (SCN) dissected from fetal rats and grafted in adult rat brains has provided additional insights in the normal ontogeny of the SCN. The SCN survives rather easily and develops to its typical adult cytoarchitectonical arrangement of contiguous clusters of vasopressin (VP)-, vasoactive intestinal polypeptide (VIP)- and somatostatin (SOM)- immunoreactive cells. Neither site of implantation, nor the establishment of efferent or afferent connections of the grafted SCN seems to be essential to allow it to develop normally into this distinguishing cytology. This independent maturation does certainly not contradict with its known endogenous and independent potency of circadian pacemaker function in the brain. If the fetal SCN is grafted in such a way that it could merge with the parenchyma of the brain of a VP-deficient Brattleboro rat, the VP neurons of the SCN often establish efferent connections with the genuine target areas of this nucleus as could be shown immunocytochemically. When the fetal SCN is grafted homotopically in the brain of SCN-lesioned rat (or hamster), the surviving SCN neurons are able to reverse the arrhythmicity of these rats. Free-running circadian rhythm of drinking or motor behaviour in constant darkness are induced within weeks after grafting. A correlation between this restorative effect and the immunocytochemical staining pattern of the SCN in the transplant and/or the afferent and efferent connections between graft and host brain, could, however, not be shown conclusively. Transplants with surviving SCN are also seen when arrhythmicity was still present, which made us conclude that there has to be a neural connection between graft and host rather than a neurohumoral control in order to explain the restorative effect of the SCN graft in SCN-lesioned animals.     

Cellular Requirements of Suprachiasmatic Nucleus Transplants for Restoration of Orcadian 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. Cytoarchitectoni-cally, 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 γ-aminobutyric acid.     

Transplantation of Foetal SCN into the Brain of non SCN-Lesioned Rats

Transplantation of foetal SCN tissue into the brain of arrhythmic SCN-lesioned rats and hamsters has shown to be effective in restoring circadian rhythms. Transplantation of the SCN into normal untreated rats has not been described so far as function is concerned. In rats certain demands have to be met for successful grafting of the SCN. Location, age and method of transplantation play an important role in the survival and function of the graft. This paper describes a method for SCN transplantation in normal rats. Results show transplant survival in 95% and successful grafting of SCN tissue in 85% of the treated rats as shown by VP and VIP staining. Disturbed circadian eating, drinking and activity rhythms are noted when grafts are located very near the endogenous SCN. Rhythms of wheel running and body temperature were less affected. The method described seems therefore well suited to do further research with circadian rhythms.     

Transplantation of Foetal SCN into the Brain of non SCN-Lesioned Rats

Transplantation of foetal SCN tissue into the brain of arrhythmic SCN-lesioned rats and hamsters has shown to be effective in restoring circadian rhythms. Transplantation of the SCN into normal untreated rats has not been described so far as function is concerned. In rats certain demands have to be met for successful grafting of the SCN. Location, age and method of transplantation play an important role in the survival and function of the graft. This paper describes a method for SCN transplantation in normal rats. Results show transplant survival in 95% and successful grafting of SCN tissue in 85% of the treated rats as shown by VP and VIP staining. Disturbed circadian eating, drinking and activity rhythms are noted when grafts are located very near the endogenous SCN. Rhythms of wheel running and body temperature were less affected. The method described seems therefore well suited to do further research with circadian rhythms.     

Effects of preparation time on phase of cultured tissues reveal complexity of circadian organization

The phases of central (SCN) and peripheral circadian oscillators are held in specific relationships under LD cycles but, in the absence of external rhythmic input, may damp or drift out of phase with each other. Rats exposed to prolonged constant light become behaviorally arrhythmic, perhaps as a consequence of dissociation of phases among SCN cells. The authors asked whether individual central and peripheral circadian oscillators were rhythmic in LL-treated arrhythmic rats and, if rhythmic, what were the phase relationships between them. The authors prepared SCN, pineal gland, pituitary, and cornea cultures from transgenic Period1-luciferaserats whose body temperature and locomotor activity were arrhythmic and from several groups of rhythmic rats held in LD, DD, and short-term LL. The authors measured mPer1gene expression by recording light output with sensitive photomultipliers. Most of the cultures from all groups displayed circadian rhythms. This could reflect persistent rhythmicity in vivo prior to culture or, alternatively, rhythmicity that may have been initiated by the culture procedure. To test this, the authors cultured tissues at 2 different times 12 h apart and asked whether phase of the rhythm was related to culture time. The pineal, pituitary, and SCN cultures showed partial or complete dependence of phase on culture time, while peak phases of the cornea cultures were independent of culture time in rhythmic rats and were randomly distributed regardless of culture time in arrhythmic animals. These results suggest that in behaviorally arrhythmic rats, oscillators in the pineal, pituitary, and SCN had been arrhythmic or severely damped in vivo, while the cornea oscillator was free running. The peak phases of the SCN cultures were particularly sensitive to some aspect of the culture procedure since rhythmicity of SCN cultures from robustly rhythmic LD-entrained rats was strongly influenced when the procedure was carried out at any time except the 2nd half of the day.     

Reduced Neurophysin Immunoreactivity in Rat Suprachiasmatic Nucleus Parallels Dissociation of Circadian Feeding Rhythm in Constant Light

Several distinct neuronal populations can be outlined in the suprachiasmatic nucleus (SCN) by employing immunohistochemistry. Understanding their interaction may serve as the key to the processes involved in the generation of circadian rhythms by the SCN. 15 adult rats were exposed to constant dim light (LL) and 3 animals as controls to an LD 12:12 light schedule over 140 days. When sacrificed 10 of the LL-animals had lost their circadian feeding rhythm while 5 were free-running and the controls kept an entrained rhythm. The brains were immunohistochemically stained for myelin basic protein, neurophysin (NPH), vasoactive intestinal peptide, neuropeptide Y, synaptophysin and the leucocyte epitopes FAL and HNK-1. Demarcation of intensely and very intensely stained NPH-positive areas by subjective gray-level-discrimination and computerized area measurement revealed that in rhythmic rats (n=8) the areas containing the stained material were twice as large (0.06 ± 0.03 mm2 vs. 0.028 ± 0.027 mm2; p=0.05) than in arrhythmic animals. It is hypothesized that low NPH-contents in arrhythmic animals reflect arrest of the 'clockwork' in the SCN at circadian time 12:00.     

Reduced Neurophysin Immunoreactivity in Rat Suprachiasmatic Nucleus Parallels Dissociation of Circadian Feeding Rhythm in Constant Light

Several distinct neuronal populations can be outlined in the suprachiasmatic nucleus (SCN) by employing immunohistochemistry. Understanding their interaction may serve as the key to the processes involved in the generation of circadian rhythms by the SCN. 15 adult rats were exposed to constant dim light (LL) and 3 animals as controls to an LD 12:12 light schedule over 140 days. When sacrificed 10 of the LL-animals had lost their circadian feeding rhythm while 5 were free-running and the controls kept an entrained rhythm. The brains were immunohistochemically stained for myelin basic protein, neurophysin (NPH), vasoactive intestinal peptide, neuropeptide Y, synaptophysin and the leucocyte epitopes FAL and HNK-1. Demarcation of intensely and very intensely stained NPH-positive areas by subjective gray-level-discrimination and computerized area measurement revealed that in rhythmic rats (n=8) the areas containing the stained material were twice as large (0.06 ± 0.03 mm2 vs. 0.028 ± 0.027 mm2; p=0.05) than in arrhythmic animals. It is hypothesized that low NPH-contents in arrhythmic animals reflect arrest of the ‘clockwork’ in the SCN at circadian time 12:00.     

In vivo monitoring of peripheral circadian clocks in the mouse

The mammalian circadian system is comprised of a central clock in the suprachiasmatic nucleus (SCN) and a network of peripheral oscillators located in all of the major organ systems. The SCN is traditionally thought to be positioned at the top of the hierarchy, with SCN lesions resulting in an arrhythmic organism. However, recent work has demonstrated that the SCN and peripheral tissues generate independent circadian oscillations in Per1 clock gene expression in vitro. In the present study, we sought to clarify the role of the SCN in the intact system by recording rhythms in clock gene expression in vivo. A practical imaging protocol was developed that enables us to measure circadian rhythms easily, noninvasively, and longitudinally in individual mice. Circadian oscillations were detected in the kidney, liver, and submandibular gland studied in about half of the SCN-lesioned, behaviorally arrhythmic mice. However, their amplitude was decreased in these organs. Free-running periods of peripheral clocks were identical to those of activity rhythms recorded before the SCN lesion. Thus, we can report for the first time that many of the fundamental properties of circadian oscillations in peripheral clocks in vivo are maintained in the absence of SCN control.     

Neurotransplantation of the Biological Clock in Mammals, Importance and Perspectives

The tool of neurotransplantation has been successfully introduced in the chronobiology of mammals. Grafting of the foetal suprachiasmatic nucleus (SCN) in the IIIrd ventricle of the brain of SCN-lesioned arhythmic rodents restored free-running circadian activity patterns. This ultimately proves the SCN to be the central circadian pacemaker system. However, recovery is not seen in all animals with a surviving SCN implant and the rhythm is usually not as robust as seen for the intact system. Moreover, the grafted foetal SCN has a partially deviant development, whereas the structure-function relationship after restoration of circadian rhythm was reported to differ in the various studies. This has led to two possible mechanisms of graft action: the one a circadian humoral signal diffusing into the SCN-lesioned host brain, and the other a neuritic afferent outgrowth into the brain. There is, moreover, doubt about the integration of the 'new' SCN in terms of afferent input. Given the fact that the in situ SCN has an extensive efferent and afferent system in the intact brain, the SCN grafting experiments seem to indicate that only limited aspects of the SCN can drive circadian physiological rhythms. However, on the basis of current knowledge on grafting results the present paper recommends performing more sophisticated SCN grafting experiments to contribute to the knowledge on the SCN clock system.     

Neurotransplantation of the Biological Clock in Mammals,Importance and Perspectives

The tool of neurotransplantation has been successfully introduced in the chronobiology of mammals. Grafting of the foetal suprachiasmatic nucleus (SCN) in the IIIrd ventricle of the brain of SCN-lesioned arhythmic rodents restored free-running circadian activity patterns. This ultimately proves the SCN to be the central circadian pacemaker system. However, recovery is not seen in all animals with a surviving SCN implant and the rhythm is usually not as robust as seen for the intact system. Moreover, the grafted foetal SCN has a partially deviant development, whereas the structure-function relationship after restoration of circadian rhythm was reported to differ in the various studies. This has led to two possible mechanisms of graft action: the one a circadian humoral signal diffusing into the SCN-lesioned host brain, and the other a neuritic afferent outgrowth into the brain. There is, moreover, doubt about the integration of the ‘new’ SCN in terms of afferent input. Given the fact that the in situ SCN has an extensive efferent and afferent system in the intact brain, the SCN grafting experiments seem to indicate that only limited aspects of the SCN can drive circadian physiological rhythms. However, on the basis of current knowledge on grafting results the present paper recommends performing more sophisticated SCN grafting experiments to contribute to the knowledge on the SCN clock system.     

Restoration of Orcadian 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 arhythmic 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.     

Suprachiasmatic nuclear lesions eliminate circadian rhythms of drinking and activity, but not of body temperature, in male rats

Male Long-Evans rats were maintained in light proof cabinets while drinking, activity, and telemetered body temperature (Tb) data were collected. After suprachiasmatic nuclear (SCN) lesions, the rats were exposed to a 12:12 light-dark cycle, a 6-hr delay in the lighting cycle, and constant dark. Lesions that abolished the drinking and activity rhythms did not eliminate the Tb rhythm. However, the amplitude, phase, and free-running period of the Tb rhythm were altered. Lesions that only partially damaged the SCN had similar, though lesser effects. In some cases, Tb rhythms remained normal, activity rhythms were only temporarily disrupted, and drinking rhythms were eliminated in the same animals. These results support the conclusion that Tb can remain rhythmic after lesions that permanently or temporarily disrupt other circadian rhythms. Of the three rhythms, it appears that drinking rhythms are most easily and Tb rhythms least easily disrupted by SCN lesions.     

Darkness during early postnatal development is required for normal circadian patterns in the adult rat

Early light experience influences the brain during development. Perinatal light exposure has an important effect on the development of the circadian system, although the role of quantity versus quality of light in this process is still unclear. We tested the development of the circadian rhythm of locomotor activity under constant bright light from the day of weaning, of six groups of rats raised under different light conditions during suckling. Results indicated that when rats received daily darkness during suckling (rats reared under constant darkness or light-dark cycles with dim or bright light) became arrhythmic when exposed to continuous bright light after weaning. However, those rats reared in the absence of darkness (constant dim or bright light, or alternating dim and bright light) developed a circadian rhythm, which was stronger and had a shorter period depending on the quantity of light received during suckling. Vasointestinal polypeptide immunoreactivity in the suprachiasmatic nucleus (SCN) was higher in those rats with weaker rhythms. However, no apparent differences among these groups were found in the melanopsin-expressing retinal ganglion cells, which provide the SCN with light input in the photoentrainment process. When bright light was shifted to dim light in three of the groups on day 57 after weaning, all of them generated a circadian rhythm with a longer period in those rats previously arrhythmic. Our results indicate the importance of the amount of light received at the early stages of life in the development of the circadian system and suggest that darkness is needed for the normal development of circadian behaviour.     

Physiological and anatomic evidence for regulation of the heart by suprachiasmatic nucleus in rats

The suprachiasmatic nucleus (SCN) is the mammalian biological clock that generates the daily rhythms in physiology and behavior. Light can phase shift the rhythm of the SCN but can also acutely affect SCN activity and output, e.g., output to the pineal. Recently, multisynaptic SCN connections to other organs were also demonstrated. Moreover, they were shown to affect those organs functionally. The aim of the present study was to investigate the role of the SCN in the regulation of the heart. First, we demonstrated that heart rate (HR) in SCN-intact, but not SCN-lesioned (SCNx), male Wistar rats had a clear circadian rhythm, which was not caused by locomotor activity. Second, we demonstrated that light at night reduces HR in intact but not in SCNx rats. Finally, we demonstrated the presence of a multisynaptic autonomic connection from SCN neurons to the heart with the retrograde pseudorabies virus tracing technique. Together, these results demonstrate that the SCN affects the heart in rats and suggest that this is mediated by a neuronal mechanism.     

Expressions of <Emphasis Type="Italic">per1</Emphasis> Clock Gene and Genes of Signaling Peptides Vasopressin,Vasoactive Intestinal Peptide,and Oxytocin in the Suprachiasmatic and Paraventricular Nuclei of Hypertensive TGR[mREN2]27 Rats

Hypertensive rats with multiple extra copies of the renin gene (TGR) exert an inverted circadian blood pressure (BP) profile. We investigated whether circadian oscillations in the hypothalamic suprachiasmatic nucleus (SCN), a main circadian oscillator, and the paraventricular nucleus (PVN), involved in BP control, are influenced in TGR rats. The expression of the clock gene per1, a marker of circadian timing, was measured in the SCN and PVN. Moreover, the expression of genes encoding vasopressin (AVP), vasoactive intestinal peptide (VIP) in the SCN, and AVP and oxytocin (OXT) in the PVN were studied by in situ hybridization. Expression of the per1 gene showed a distinct circadian rhythm in both the SCN and PVN with no differences observed between the TGR and control Sprague–Dawley (SD) rats. The expression of avp in the SCN was rhythmic in both strains and moderately higher in TGR than in SD rats while no significant changes were found in the PVN. The expression of vip in the SCN and oxt in the PVN did not differ between both strains. Our results may indicate that changes occurring downstream to the SCN are responsible for the development of the inverted BP rhythm in TGR hypertensive rats.     

Phase Relations between Host and Grafted SCN Depend on Graft Location in Rats

Fetal SCN grafts into intact rats were used as a model to study coupling among circadian oscillators. The phase relation in metabolic activity was analyzed between host SCN and fetal SCN grafted into the third ( n = 24) or lateral ( n = 18) ventricles. Host and third ventricle SCN grafts showed metabolic activity rhythms with peak values at CT 6, as extrapolated from drinking rhythmicity. In lateral ventricle grafted animals only the host SCN showed rhythmicity, although peak values oc curred as indicated by the drinking rhythms at CT 9. The present data suggest asymmetrical communication between host and grafted SCN depending on graft location, which may be related to different types of signal transmission.     

Phase Relations between Host and Grafted SCN Depend on Graft Location in Rats

Fetal SCN grafts into intact rats were used as a model to study coupling among circadian oscillators. The phase relation in metabolic activity was analyzed between host SCN and fetal SCN grafted into the third (n = 24) or lateral (n = 18) ventricles. Host and third ventricle SCN grafts showed metabolic activity rhythms with peak values at CT 6, as extrapolated from drinking rhythmicity. In lateral ventricle grafted animals only the host SCN showed rhythmicity, although peak values oc curred as indicated by the drinking rhythms at CT 9. The present data suggest asymmetrical communication between host and grafted SCN depending on graft location, which may be related to different types of signal transmission.     

Cognitive Performance as a Zeitgeber: Cognitive Oscillators and Cholinergic Modulation of the SCN Entrain Circadian Rhythms

The suprachiasmatic nucleus (SCN) is the primary circadian pacemaker in mammals that can synchronize or entrain to environmental cues. Although light exerts powerful influences on SCN output, other non-photic stimuli can modulate the SCN as well. We recently demonstrated that daily performance of a cognitive task requiring sustained periods of attentional effort that relies upon basal forebrain (BF) cholinergic activity dramatically alters circadian rhythms in rats. In particular, normally nocturnal rats adopt a robust diurnal activity pattern that persists for several days in the absence of cognitive training. Although anatomical and pharmacological data from non-performing animals support a relationship between cholinergic signaling and circadian rhythms, little is known about how endogenous cholinergic signaling influences SCN function in behaving animals. Here we report that BF cholinergic projections to the SCN provide the principal signal allowing for the expression of cognitive entrainment in light-phase trained animals. We also reveal that oscillator(s) outside of the SCN drive cognitive entrainment as daily timed cognitive training robustly entrains SCN-lesioned arrhythmic animals. Ablation of the SCN, however, resulted in significant impairments in task acquisition, indicating that SCN-mediated timekeeping benefits new learning and cognitive performance. Taken together, we conclude that cognition entrains non-photic oscillators, and cholinergic signaling to the SCN serves as a temporal timestamp attenuating SCN photic-driven rhythms, thereby permitting cognitive demands to modulate behavior.     

Light/dark‐induced effects on behavioral rhythms in suprachiasmatic nucleus‐lesioned rats irrespective of the presence of functional suprachiasmatic nucleus brain implants

Summary

Suprachiasmatic nucleus (SCN)‐lesioned rats which had received a fetal SCN graft were kept in constant red light for three months. After this period it was examined whether those rats that showed a recovered free‐running circadian rhythm could be entrained to light/dark cycles. To this end, they were subjected to a 12 h light/12 h dark schedule, followed by a 12 h light shift and again to dark conditions. In addition, the same regime was imposed on SCN‐grafted rats without recovered circadian rhythms and on sham‐grafted animals with a lesion, which were studied as controls. The presence of an SCN graft was identified immunocytochemically by the presence of vasopressin, vasoactive intestinal polypeptide and somatostatin cells.

Drinking, eating and wheel‐running rhythms were found to synchronize to the light/dark cycles in all rats, not with standing the presence of an SCN graft was. A 12 h light shift was immediately followed by a shift in the three rhythms. Under final dark conditions, free‐running patterns reappeared in rhythm‐recovered animals, without any convincing evidence for entrainment of the rhythms in the pattern of transition.

Behavioral rhythms in SCN‐lesioned rats are apparently masked by 12 h light/dark schedules via other visual pathways than the direct projection from the retina to the SCN.     

Further evaluation of the tetrodotoxin-resistant circadian pacemaker in the suprachiasmatic nuclei.

We previously reported the results of an experimental paradigm in which tetrodotoxin (TTX) was chronically infused by miniosmotic pump into the rat suprachiasmatic nuclei (SCN) (Schwartz et al., 1987). Although TTX reversibly blocked photic entrainment and overt expression of the circadian drinking rhythm, the circadian pacemaker in the SCN continued to oscillate unperturbed by the toxin, and we concluded that Na(+)-dependent action potentials are not a part of the SCN pacemaker's internal timekeeping mechanism. In the research reported in the present paper, we used our paradigm to chronically infuse other agents, in order to evaluate the validity of this interpretation further. (1) Infusion of 50% procaine into the SCN of blinded rats resulted in a disorganized circadian drinking rhythm during the infusion, after which behavioral rhythmicity returned without apparent phase shift. In intact rats, procaine reduced the phase-resetting action of a reversed light-dark cycle imposed during the infusion. Thus, the effects of voltage-dependent Na+ channel blockade by a local anesthetic resemble those produced by TTX. (2) Infusion of high (20 mM) K+ or 100 microM veratridine into the SCN of blinded rats resulted in an apparent phase advance of the circadian drinking rhythm by over 4 hr. The phase-shifting effect of veratridine was blocked by simultaneous infusion of 1 microM TTX. Thus, membrane depolarization or direct activation of voltage-dependent Na+ channels can affect the pacemaker's oscillation. Our infusion paradigm can detect alterations of rhythm phase, and the lack of phase shift after TTX or procaine infusion is not an artifact of an insensitive method.