The Journal of Neuroscience, August 1989. g(8): 2671-2677 Specificity of Circadian Function Transplants of the Fetal Suprachiasmatic Nucleus David J. Earnest, Celia D. Sladek, Don M. Gash, and Stanley J. Wiegand Department of Neurobiology and Anatomy, University of Rochester School of Medicine, Rochester, New York 14642 Fetal tissues obtained from specific regions of the develop- and locomotor activity (Drucker-Colin et al., 1984; DeCoursey ing hypothalamus were transplanted to determine wheth- and Buggy, 1986; Lehman et al., 1987). However, little is known er the precursor neurons of the suprachiasmatic nucleus about the mechanismsby which thesetransplants restore rhyth- (SCN) can be distinguished from those of the presumptive micity. One possibility is that the grafted neuronsrestore rhyth- paraventricular nucleus (PVN) on the basis of the functional micity by providing a specific trophic factor(s) which induces capacity to generate circadian rhythms. The presumptive plasticity in the neural organization of the circadian systemand SCN, the PVN, and a portion of the neocortical primordium enablesother neurons in the host brain to assumethis time- were dissected from the developing forebrains of normal keepingfunction in the absenceofthe in situ SCN. Alternatively, Long-Evans fetuses, separated, and selectively transplant- the transplanted hypothalamic neurons may intrinsically func- ed into the periventricular-third ventricle region of adult, tion as a circadian pacemakerand restore rhythmicity in SCN- vasopressin (VP)-deficient Srattleboro rats. In host animals lesioned hosts through neurohumoral signalsand/or the estab- that received grafts containing the precursor population of lishment of neuralconnections with the host brain. In this regard, SCN neurons, the temporal profile of VP levels in the cere- if the transplanted neuronsare functioning as a circadian pace- brospinal fluid (CSF) oscillated with a circadian periodicity maker, then endogenousbiological activities cxpresscdby these in a manner similar to that observed in normal Long-Evans neurons should oscillate with a circadian periodicity. This hy- rats. CSF collected serially from animals with grafts of the pothesis is basedon observations that indices of SCN activity presumptive PVN also contained VP, but no circadian vari- such as neuronal firing rate and secretory activity oscillate in a ation was manifested in peptide levels. VP was undetectable circadian fashion and that the oscillatory nature of these activ- in CSF samples obtained from Srattleboro rats with cortical ities is an endogenousfeature of SCN neurons (Green and Gil- grafts. In association with their circadian functional capacity, lette, 1982; Inouyc and Kawamura, 1982; Earnest and Sladek, grafts of the SCN primordium were characterized by clusters 1987; Gillette and Reppert, 1987). Consequently, the present of parvicellular neurons immunopositive for VP or vasoactive investigation was conducted to determine whether grafts of the intestinal polypeptide (VIP) that resembled the cell groups primordial SCN develop the distinctive capacity to function as of the in situSCN. In contrast, transplants of the presumptive a circadian clock. PVN did not contain neurons immunoreactive for VIP, and Since the circadian patterns of vasopressin (VP) secretion the VP neurons in these grafts resembled the neurosecretory observed in vivo and in vitro (Schwartz and Reppert, 1985; cells of the PVN. These results demonstrate that grafts con- Earnest and Sladek, 1987; Gillette and Reppert, 1987) appear taining VP neurons derived from the primordial SCN develop to directly reflect the intrinsic activity ofa prominent population not only the cytological and neurochemical features which of VP neurons located in the SCN (Vandesandeet al., 1975; distinguish this population from the VP neurons of the PVN Moore, 1983), the presentstudy utilized VP releaseas an index in situ but also the unique functional capacity of a circadian of the activity of transplanted SCN neurons. Experiments were pacemaker to generate endogenous rhythms in secretory designedto determine whether grafts containing the precursor activity. populations of SCN neuronsdevelop the capacity to releaseVP into the cerebrospinalfluid (CSF’) in a circadian fashion. Brat- The suprachiasmatic nucleus (SCN) of the hypothalamus is an tleboro rats were utilized as transplant recipients becausethe integral neural locus for the generation of circadian rhythms in genetic deficiency in brain VP in this strain (Sokol et al., 1976) mammals. Recent applications of the neural transplantation allows unequivocal identification of vasopressinergicneural technique to the study of the mammalian circadian organization processesand VP in the CSF asemanating from grafts of normal have served to corroborate evidence for this function of the tissue. SCN. Specifically, transplants of fetal hypothalamus containing Based on reports indicating that grafts of the fetal anterior the SCN have been reported to restore circadian rhythms in hypothalamus frequently contain 2 or more cytologically dis- behavioral activities of SCN-lesioned hosts, such as drinking tinct populations of VP neurons that resemblethe normal com- plements found in the in situ paraventricular, supraoptic and suprachiasmaticnuclei (Boer et al., 1985; Wiegand and Gash, Received July 29, 1988; revised Nov. 8, 1988; accepted Jan. 18, 1989. 1987, 1988a), this study was designedto provide for the trans- Correspondence should be addressed IO David J. Earnest, Department of Neu- robiology and Anatomy, University of Rochester School of Medicine, 601 Elm- plantation of a more homogeneouspopulation of SCN-like VP wood Ave., Rochester, NY 14642. neurons. Our approach entailed the selective transplantation of Copyright 0 1989 Society for Neuroscience 0270-6474/89/082671-07$02.00/O topographically discrete regionsof the developing hypothalamus 2672 Earnest et al. - Circadian Activity of Transplanted Suprachiasmatic Neurons containing the anlagen of either the SCN or paraventricular at least 2 times greater than the nadir value for the cycle. Using this nucleus (PVN; Altman and Bayer, 1978a, b) so as to evaluate phase reference point, the time interval between consecutive reference points was measured so as to determine period length and, ultimately, specificity in the capacity of the grafts to generate circadian VP whether the peak in CSF VP recurred with a circadian periodicity. rhythms. The relationship between the functional capacities and Statistical analvsis was oerformed on the means for VP levels usina a the snecific cvtoloaical and immunohistochemical features of one-way analysis of valance with repeated measures to determine Lhe grafts was also evaluated. significance of sampling time. Group means in this analysis were ob- tained by arbitrarily aligning the highest value for CSF VP in individual animals because the individual profiles expressed bv SCN (or PVN) Materials and Methods grafts were not phase-coordinated with each other. Differences in VP levels between sampling intervals within a given circadian day were Animals and housing conditions. Young adult male rats of the Brattle- tested post hoc for significance using the Newman-Keuls sequential boro strain (Blue Spruce Farms), homozygous for diabetes insipidus, range test. were utilized as transplant recipients. Age-matched male Long-Evans Histological procedures. Following the completion of CSF sampling, rats (Charles Rivers Laboratories) served as nontransplanted controls. animals were killed with a lethal dose of sodium uentobarbital and Throughout this study, experimental and control animals were housed perfused with fixative solution (4% paraformaldehyhe or 2% parafor- individually and maintained in a temperature-controlled environment maldehyde and 0.1% glutaraldehyde), and the brains were sectioned and (21°C) with food and water provided ad libitum. Except where noted prepared for immunohistochemical processing as described previously otherwise, the animals were exposed to a 12-hr: 12-hr light-dark cycle (Watson et al., 1986). A few of the animals with neural grafts were (lights on 06.00 to 18.00). Donor tissue of known gestational age was perfused and subsequently injected with opaque gelatin-ink solutions obtained from the fetuses of Long-Evans females (Charles Rivers Lab- for the visualization of microvasculature (Wiegand and Gash, 1988a). oratories) that had been bred with males of the same strain. The preg- A l-in-6 series of 30-wrn sections was processed for localization of nancies were timed by considering the morning of sperm detection in arginine VP. Additional series were processed for localization of va- the vaginal lavage as day 0 of gestation. soactive intestinal polypeptide (VIP) or were stained with thionin. The Preparation and transplantation ofdonor tissue. Fetuses were removed remaining series of sections were reserved for further immunohisto- from Long-Evans dams, killed on days 15, 16, or 17 of gestation and chemical analysis. Peptides were detected by means of an avidin-biotin placed in an ice-cold chamber moistened with Eagle’s medium. The immunoperoxidase procedure (Watson et al., 1986; Wiegand and Gash, fetal brain was removed and a coronal section containing the anterior 1988b). The primary antiseraagainst VP (ICN Immunobiologicals)and hypothalamus was obtained by making incisions just anterior to the VIP (ICN Immunobiologicals) were raised in rabbits and used at di- optic chiasm and at the midpoint between the chiasm and the developing