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Differentiation of the suprachiasmatic nucleus in fetal rat anterior hypothalamic transplants in oculo
Developmental Brain Research, 32 (1987) 59-66
59
Elsevier BRD 50507
Differentiation of the suprachiasmatic nucleus in fetal rat anterior hypothalamic transplants in oculo Michael H. Roberts 1, Mary F. Bernstein 1 and Robert Y. M o o r e 1'2 1Department of Neurology, SUNY-Stony Brook, Stony Brook, NYl1794 (U.S.A.) and 2Departmentof Neurobiology and Behavior, SUNY-Stony Brook, Stony Brook, NYl1794 (U.S.A.) (Accepted 19 August 1986)
Key words: Suprachiasmatic nucleus; Immunohistochemistry; Vasopressin; Vasoactive intestinal polypeptide; Brain transplant; Hypothalamus; Neural development
The capacity of the rat anterior hypothalamus, and particularly the suprachiasmatic nucleus (SCN), to develop and differentiate when removed from its normal environment was examined in this study using light and electron microscopy. The hypothalamus from fetuses ranging in age from embryonic day 12 (E 12) to E 16 was transplanted to the anterior chamber of the eye of adult rats. In initial experiments, we found that transplants from E 15 fetuses and older routinely differentiated into fields of neurons with extensive neuropil with an appearance similar to the anterior hypothalamic area. Groups of small, compactly organized neurons were observed only occasionally in this tissue. Ultrastructural analysis of these transplants typically revealed well-differentiated neuronal perikarya and neuropil with a complex synaptic organization similar in appearance to the normal rat anterior hypothalamic area. Occasionally both mature and immature tissue coexisted in some of the transplants. Tissue from young embryos (E 12-14) frequently showed development of a compact, small neuron nucleus with the cytoarchitectonic appearance of the SCN. At least 45 days were required after transplantation for the successful differentiation to occur in this situation. The SCN in these transplants displayed vasoactive intestinal polypeptide-immunoreactive cells and fibers surrounded by vasopressin-immunoreactive cells and fibers, similar to the pattern observed in the normal adult SCN. Our results indicate that the anterior hypothalamus will differentiate normally in oculo and that the phenotypic specification of the SCN occurs prior to the birthdate of its component neurons.
INTRODUCTION Neuronal transplantation is now a widely used m e t h o d for investigating the processes of neural development. Two types of technique have been used to study the differentiation of the fetal brain. First, transplants directly into the adult brain have been used to investigate the interactions of fetal tissue with the surrounding central nervous system 6. Second, transplants into the anterior c h a m b e r of the eye, presumably a 'neutral' ground, have been used to study the d e v e l o p m e n t of the fetal brain in isolation from normal extrinsic influences 1°. These studies have shown that tissue transplanted n e a r the time of the final cell division of the developing neuroblasts are most likely to grow and differentiate 6.
The time course of neuron formation in the rat hypothalamus has been carefully d o c u m e n t e d by Altman and B a y e r 1. Their studies d e m o n s t r a t e that there is a spatial gradient in cell birthdate with neurons whose final position is distant from the third ventricle forming earlier than those near the ventricle. Cells in the lateral preoptic area, for example, undergo their last cell division between embryonic days (E) 12 and 16 (E 12-16) whereas cells of the suprachiasmatic nucleus (SCN) undergo their last division between E 14 and E 17. Synapse d e v e l o p m e n t lags cell birthdate in the anterior hypothalamus and particularly in the SCN 2. The functional d e v e l o p m e n t of the SCN has also been investigated using the 2-deoxyglucose method 11. This nucleus, which functions as a biologi-
Correspondence: M.H. Roberts, Department of Neurology, HSC T12-020, SUNY-Stony Brook, Stony Brook, NY 11794, U.S.A. 0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
60 cal clock in the mammalian brain 9"12, displays a circadian variation in metabolic activity 13'14which appears on embryonic day 19 (ref. 11). Since the SCN is very immature at E 19 with no apparent intrinsic or extrinsic connections 2, these functional studies indicate that synaptic communication between SCN neurons and the formation of its afferent and efferent projections are not requirements for the development of circadian function in the nucleus. The purpose of the present study was to investigate the development of fetal anterior hypothalamic tissue in oculo, isolated from its normal environment, to analyze the morphological differentiation of the SCN. As noted above, the SCN is now identified as a circadian pacemaker and the intent of this investization was to provide a basis for subsequent functional studies of SCN transplants to both brain and eye. The specific issue addressed was whether the SCN differentiates in oculo with a morphological appearance similar to that observed in the adult nervous system. MATERIALS AND METHODS
Surgery The anterior hypothalamus from E 12-E 16 fetuses were used in this study. Timed-pregnant Sprague-Dawtey rats were obtained from Taconic Farms (Germantown, NY) and maintained on light-dark cycles consisting of 12 h of light and 12 h of darkness. The embryos were removed from the uterus of anesthetized (Ketamine/Rompun, 5:1, 1.0 ml/kg) mothers and staged by measuring their crown-rump length according to Seiger and Olson 15. The embryos' heads were sectioned in the horizontal plane at eye level with microknives, which allowed the base of the brain to be easily visualized. A small (ca. 0.5 mm) cube of hypothalamus, anterior to the developing median eminence, was removed with iridectomy scissors and placed in sterile mammalian Ringers solution. The tissue was transplanted to the anterior chamber of the eye as described by Olson et al.l~. Briefly, a small slit was cut in the cornea of an anesthetized young adult rat, through which the embryonic hypothalamus was inserted with a beveled Pasteur pipette. The explanted tissue was moved to the outer surface of the iris by gently prodding the cornea with forceps. Approximately 150 of these operations were performed.
Light microscopy For our light microscopic observations, explants were allowed to develop for 14-70 days after which time the hosts were anesthetized and transcardially perfused with 200 ml of saline and 400 ml of Bouins fixative. We have found this fixative to be an excellent preservatwe for embryonic tissue ~. Following fixation, the eves were taken out, hemisected, and the lenses removed. Based upon the quality of the perfusion, ca. 20% of the transplants were selected for analysis, The anterior portion of the eye, containing the transplants, was maintained m 70% ethanol for o n e - t w o weeks, placed in 95t~ ethanol overnight and transferred to butanol for 2-3 days before embedding in paraffin under vacuum The tissue was sectioned at 10/~m on a Leitz rotar~ microtome and mounted on slides as 5 replicate sets One set of sections was stained with Cresyl violet.
Irnmunohistochemistrv Our methods for immunohistochemistry using the peroxidase-antiperoxidase technique 17 have been described previously5. In this study, slides were deparaffinized in xylene and rehydrated through an alcohol series to water. The slides were washed in two 10-min changes of 0.1 M phosphate buffer (pH 7.2). All incubations were done in humidified chambers constructed from 15-cm plastic Petri dishes containing damp filter paper. Normal goat serum (10%) in buffer was applied to the slides so as io cover the sections. The Petri dishes were covered and the slides incubated in the goat serum for 20 rain at 4 °C. The serum was drained off the slides, which were then covered with antisera diluted with P:BGT (0.1 M phosphate buffer. 1% normal goat serum, and 0.3% Triton X-100). Anti-vasopressin (VP) was diluted 1:750 and anti-vasoactive intestinal polypeptide (VIP) was diluted 1:500. All antisera were obtained from lmmunoNuclear Corporation lStillwater. MNI. We have found that immunoreactivity to these antisera in the rodent brain can be blocked by low (10 zzM) concentrations of the appropriate antigen 4. The slides were incubated in the antisera overnight (18-22 h) at 4 °C. Following the overnight incubation the slides were rinsed twice for 10 min in phosphate buffer. The slides were then incubated at room temperature for 45 min in goat anti-rabbit IgG diluted 1:50 in PBGT.
61
A
Following this incubation the slides were again rinsed in buffer and then incubated for 45 min at room temperature in peroxidase-antiperoxidase diluted 1:100 in PBGT. Following two 10-min buffer rinses, the slides were placed in 200 ml of buffer which contained 100 mg of diaminobenzidine-HC1. The peroxidase reaction was started by adding 220/A of 30% hydrogen peroxide. The reaction was allowed to continue for ca. 10 rain before being stopped by rinsing the slides in 3 changes of phosphate buffer. The sections were then intensified in 0.2% osmium tetroxide for 2-10 s, dehydrated through an alcohol series, cleared in xylene and cover slipped with Permount.
1 8
Electron microscopy For our ultrastructural studies, the explants from E 16 fetuses were allowed to develop in the anterior chamber of the eye for 15-74 days at which time the animals were anesthetized and transcardially perfused with saline followed by 2% paraformaldehyde and 2.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The eyes were removed and placed in the fixative overnight at 4 °C. Implants were then carefully detached from the iris, hand-cut into 1-mm sections, rinsed in buffer and treated in 2% osmium tetroxide in phosphate buffer for 1 h. The tissue was dehydrated in an alcohol series to propylene oxide and embedded in Epon-Araldite. Thin sections were cut. on a Reichert ultramicrotome and collected on Formvar-coated slot grids. The sections were stained with uranyl acetate and lead citrate and examined with a JEOL 100CX electron microscope. RESULTS During the first week to 10 days all the transplants underwent a pronounced shrinkage with the tissue apparent in the anterior chamber decreasing in size by about 30%. This reduction in the size of in oculo transplants has been noted previously by Seiger and Olson 16 who found that embryonic tissue containing the locus coeruleus would shrink by about 50% in the week following transplantation. During the period of shrinkage our transplants are extensively vascularized and blood vessels are seen throughout the tissue after 14 days of development. The prominent immunohistochemical differences between the dorsomedial and ventrolateral SCN
m
Fig. l. Normal rat SCN. A: coronal section through the optic chiasm of an adult rat. The SCNs, indicated by the small black dots, lie at the base of the third ventricle. B: photomicrograph of the SCNs stained with antisera to VP. C: photomicrograph of the SCN stained with antisera to VIP. Bar = 100/~m.
(Fig. 1) can serve as a convenient basis for assessing differentiation of the transplanted SCN. Because the SCN is the only hypothalamic nucleus that displays this distinctive pattern of organization, any compact mass of cells forming within our transplants that shows this pattern of a VIP-immunoreactive region surrounded by a VP immunoreactive capsule can be regarded as SCN. Anterior hypothalamus taken from E 16 rats shows differentiation of several neuronal cell types. On occasion, dense aggregations of
62
Fig. 2. Cresyl violet stain of an E 16 transplant allowed to develop for 42 days. Note the dense aggregations ol neurons. These cells did not show immunoreactivity to VP or VIP antisera. C. cornea: I. iris: T. transplant. Bar = 50 u m
large neurons are evident (Fig. 2), but these do not show a division into VP and V I P i m m u n o r e a c t i v e regions, characteristic of the SCN (Table I). A t the ultrastructural level, E 16 tissue o b s e r v e d TABLE l
Transplants The table displays the n u m b e r of transplants examined and the n u m b e r of transplants containing an SCN for each e m b r y o m c age after development in the anterior c h a m b e r of the eye for mcreasing periods of time.
Embrvonic age Davs in oculo No. examined
No. with SCN
13
14 3(t 45 6(I +
2 1 3 3
0 0 2 1
14
15 30 45 60+
1 1 1 2
o 0 I) 1
16
16 24 30 42
2 5 2 2
0 0 0 0
60+
after 16 days of d e v e l o p m e n t in oculo appears immature. There are many closely a p p o s e d immature perikarya with little neuropil and few synapses. G r o w t h cones also are evident throughout the developing transplant at this age. At 24 days after transplantation. there are regions of mature tissue characterized bv well-differentiated cell bodies and a complex neuropil (Fig. 3t. The synapses primarily contain small. lucent vesicles although occasional dense-core and flattened vesicles are seen. Interestingly, adjacent to these regions of differentiated tissue there are regions of immature tissue, perhaps indicating a continuous gene ration of neurons or glia, Tissue examined after 74 days in oculo appears m a t u r e throughout. Neuronal p e r i k a r y a are well differentiated and there is an extenstve neuropil with a complex synaptic organization and m a n y m y e l i n a t e d axons. Transplants taken from younger e m b r y o s (E 14~ and examined by light microscopy after t5 or 30 days of d e v e l o p m e n t in oculo, show neuronal differentiation but similar to E 16 tissue, no morphological evidence of SCN-like cell groups is present. H o w e v e r , after longer periods of d e v e l o p m e n t (72 days), compact groups of small neurons are seen with a core of
63
Fig. 3 Electron micrograph of a transplant allowed to develop for 24 days in oculo. Region of the transplant distant from the ependymal layer. The large cell in the center of the figure (N) lies within a network of fibers. Several synapses can be seen, most containing small clear vesicles (sc), although occasional dense core vesicles are present (dc). Bar = 1/am.
VIP-immunoreactive cells and fibers surrounded by a capsule of VP-immunoreactive cells and fibers. No other easily identifiable hypothalamic cell groups, such as the paraventricular or supraoptic nuclei, are evident in the transplant. Anterior hypothalamus from E 13 fetuses shows the same differentiation of an SCN-like structure. Similar to transplants from E 14 embryos, there is no differentiation of a compact cell group with VP- and VIP-immunoreactive regions after 14 or 32 days, but an SCN-like structure is present after 45 days (Fig. 4). Table I summarizes the resuits of our immunohistochemicai studies. We conclude that after long periods in oculo the transplants do display differentiation of cell groups which fulfill criteria for designation as SCN.
DISCUSSION The capacity of the anterior hypothalamus and the SCN to develop after removal from its normal environment was examined in this study. Although explants from E 15 or older embryos never show the differentiation of a compact, parvocellular cell group with the appearance of the SCN, differentiation into complex neuronal fields with extensive neuropil and a variety of neuronal sizes and staining density was common. Ultrastructural observations on these explants indicate that the embryonic anterior hypothalamus will develop and differentiate perikarya, axons and axon bundles, myelinated axons, and a neuropil with a variety of synaptic complexes. The ultrastructural appearance of the explants in oculo is very similar to that of adult anterior hypothalamus.
64
Fig. 4. Two E 13 transplants allowed to develop for 47 days in oculo. A and B show VP immunoreactivlty and panels C and D show adjacent sections processed for VIP immunoreactivity. In both cases the VP immunoreactive region surrounds the region of VIP immunoreactivity. Bar = 50 urn.
The normal rat SCN shows a distinctive pattern of immunoreactivity with antisera to VP and VIP. VIPpositive cells and fibers are found in the ventrolateral SCN 5 and VP-positive cells and fibers are found in the dorsomedial SCN l~. VP fibers also extend ventrally to form a capsule a r o u n d the region of VIP immunoreactivity. This pattern of VIP immunoreactivity s u r r o u n d e d by VP-immunoreactive cells and fibers was found in several of our explants. However, the donor tissue had to be young; E 15 or E 16 explants did not develop the typical pattern whereas explants from E 13 or E 14 embryos did differentiate a compact cell group with VP and VIP immunoreactivity.
In a trltiated thvmidine study of hypothalamic neurogenesis, A l t m a n and Bayer ~ demonstrated that SCN neurons are formed between 12; 14 and E 17. Taken together with these data. our results indicate that the neuroepithelial cells which give rise to ~tlc SCN are committed to form the nucleus prior to their final cell division. Similarb,. HofI)er el al. ~ have shown that embryonic cerebettar tissue transplanted in oculo prior to the final division will form normal cerebellar structures and intrinsic connections. In contrast to our results. Wiegand and G a s h 19 have reported in abstract, that the anterior hypothalamus from day 17 fetuses will form an SCN with connections to hosl tissue when transplanted into the
65 third ventricle. Although we did observe perikarya similar in morphology to parvocellular SCN neurons in tissue obtained from E 15 or older fetuses, we never observed VP or VIP immunoreactive neurons organized into a compact cell group. The discrepancy between our results and those of Wiegand and Gash may be due to differences in the transplant sites. The failure of our older transplants to differentiate may result from the inability of the neurons already formed to make connections with the brain. In addition to the age of the donor tissue affecting the formation of the SCN, we also found that the tissue had to be maintained in the anterior chamber of the eye for extended periods of time. This differs from the studies of Hoffer et al. 8, cited above, who find that there is only a 5-day difference between in oculo and in situ cerebellar development. Although there is early differentiation of neuronal fields with the appearance of anterior hypothalamus in our material, we did not observe the differentiation of an immunohistochemically identifiable SCN earlier than 45 days following the transplantation of the tissue into the anterior chamber of the eye. It is difficult to account for the extremely long time required for the formation of this hypothalamic nucleus in oculo. In normal fetal development the nucleus is readily recognized with Cresyl violet staining at E 18, and VP immunoreactivity appears shortly after birth at postnatal day 2 (ref. 7). On that basis, we would expect to observe an immunohistochemically identifiable SCN ca. two weeks after transplantation. While a developmental delay was observed in our tissue as assayed by the relatively late appearance of myelin in our electron micrographs after 24 days in oculo, as opposed to post natal day 10 in normal development 2, the pronounced delay in the appearance of the SCN seems extreme. One explanation is that the developmental lag is an artifact of our criteria for identifying an SCN. The nucleus may develop earlier than our immunohistochemical studies indicate by virtue of the explant's inability to produce detectable VP or VIP until 45 days after transplantation. If this were the case, we would expect to see dense aggregations of small neurons in our young transplants that failed to stain with antisera to VP or VIP. We did not
observe, however, any of these structures in our 'unsuccessful' transplants. Two additional explanations for the developmental delay are that the anterior chamber of the adult eye contains markedly lower concentrations of trophic substances, present in the embryo, that promote the rapid proliferation of neural tissue, or that interactions between the transplant and the contents of the anterior chamber of the eye, particularly the iris, inhibit neuronal differentiation. Our transplants, in either case, would develop more slowly. However, neither of these explanations are supported by the studies of Hoffer et al. 8. Finally, it may be the case that the formation of the SCN requires the migration of neurons away from the ependyma which could be retarded by the initial degeneration of the transplant or by physical barriers formed by the rapid outgrowth of fiber tracts observed in our electron micrographs. Further studies may allow us to distinguish between all these possibilities. In summary, we can draw 3 conclusions from this study. First, the anterior hypothalamus and the SCN will develop in an in vivo explant system removed from its usual environment. Second, the SCN differentiate normally as expressed by the organization of VP- and VIP-containing neurons. Finally, the cells of the germinal epithelium that give rise to the SCN are committed to form the nuclei as early as day 13 of embryonic development, prior to the previously established birthdates of SCN neurons. It is not known whether transplants which develop morphologically into SCN-iike cell groups exhibit spontaneous circadian rhythms or are capable of functioning as circadian pacemakers. Electrophysiologicai studies on these transplants may provide eivdence for the functional, as well as the morphological development of the SCN in oculo.
ACKNOWLEDGEMENTS We would like to thank J. Speh for technical assistance and V. Cassone, E. Gustafson and S. Dewey for critical review of an early version of this manuscript. Supported by USPHS Grant NS-16304 to R.Y.M.
66 REFERENCES 1 Altman, J. and Bayer, S.A., Development of the diencephalon in the rat: II. Correlation of the embryonic development of the hypothalamus with the time of origin of the neurons, J. Comp. Neurol., 182 (1982) 973-994. 2 Bernstein, M.F. and Moore, R.Y., Early development of the suprachiasmatic nucleus of the rat: an ultrastructural study, Soc. Neurosci. Abstr., 8 (1982) 215. 3 Bernstein, M.F. and Moore, R.Y., Synapsin I immunohistochemistry demonstrates synaptic development in the rat suprachiasmatic nucleus, Soc. Neurosci. Abstr., 11 (1985) 990. 4 Card, J.P. and Moore, R.Y., The suprachiasmatic nucleus of the golden hamster: immunohistochemical analysis of cell and fiber distribution, Neuroscience, 13 (1984) 415-431. 5 Card. J.P., Brecha, N., Karten, H.J. and Moore, R.Y., Immunocytochemical localization of vasoactive intestinal polypeptide containing cells and processes in the suprachiasmatic nucleus of the rat: light and electron microscopic analysis, J. Neurosci., 1 (1981) 1289-1303. 6 Das, G.D., Hallas, B.H., and Das, K.G.. Transplantation of brain tissue in the brain of rat: I. Growth characteristics of neocortical transplants from embryos of different ages, Am. J. Anat., 158 (1980) 135-145. 7 De Vries, G.J., Buijs, R.M. and Swaab, D.F., Ontogeny of the vasopressinergic neurons of the suprachiasmatic nucleus and their extra-hypothalamic projections in the rat brain: presence of a sex difference in the lateral septum, Brain Res., 218 (1981) 67-78. 8 Hoffer, B., Seiger, A., Ljungberg, T. and Olson, L., Electrophysiology and cytological studies of brain homografts in the anterior chamber of the eye: maturation of cerebellar cortex in oculo, Brain Res., 79 (1974) 165-184. 9 Moore, R.Y., Organization and function of a central nervous system circadian oscillator: the suprachiasmatic nu-
10
If
12 13
14
15
16
17 18
19
cleus, Fed. Proc. Fed. A m . Soc. Exp. Biot, 42 t 19831 27832789. Olson. L.. Seiger, A. and Stromberg. I., tntraocular transplantation in rodents: a detailed account of the procedure and examples of its use in neurobiology with special reference to brain tissue grafting. In. S. Fedoroff and L. Hertz (Eds. I, Advances in Cellular Neurobiology, Vol. 4. Academic Press. New York. 1983 Reppert. S.M. and Schwartz. W.J.. l]le suprachlasmauc nuclei of the fetal rat: characterization of a functional circadian clock using 14-C labeled deoxyglucose. J. Neurosci.. 4 1984 ~ 1677-1682. Rusak. B. and Zucker. 1.. Neural regulation of circadian rhythms, Physiol. Rev.. 5911979) 449--526 Schwartz. W.J. and Gainer. H.. Suprachiasmatic nucleus: use of ~4C-tabeled deoxyglucose uptake as a functional marker. Science. 197 119771 1089-1091. Schwartz W.J.. Davidsen. L.C. and Smith. C.B.. In vwo metabolic activity of a putative circadian oscillator, the rat suprachiasmatic nucleus. J. C o m p '~;eurol.. 189 (1980) 157-167. Seiger. A. and Olson. L.. Late prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemistry. Z. Anat. Entwickl.-Gesch.. t40 (1973)281-318. Seiger. A and Olson. k.. Quantitation of fiber growth m transplanted central monoamine neurons. Cell Tissue Res . 179 (19771 285-316. Sternberger~ L.A.. lmmunohistochem~strv. 2nd edn.. Wiley, New York. 1979, pp. 104-169 Vandesande. F.. Dierickx. K and DeMey, 1 . Identification of the vasopressin-neurophysin producing neurons ot the rat suprachiasmatic nucleus. Cell Fi,sue Res.. t56 ~19751 377-380. Wiegand, S.T, and Gash. D.M., Orgamzation of efferent projections of suprachiasmatic nucleus contained in intraventricular transplants of fetal anterior hypothalamus. Soc. Neurosci Abstr I1 (1985 607.
59
Elsevier BRD 50507
Differentiation of the suprachiasmatic nucleus in fetal rat anterior hypothalamic transplants in oculo Michael H. Roberts 1, Mary F. Bernstein 1 and Robert Y. M o o r e 1'2 1Department of Neurology, SUNY-Stony Brook, Stony Brook, NYl1794 (U.S.A.) and 2Departmentof Neurobiology and Behavior, SUNY-Stony Brook, Stony Brook, NYl1794 (U.S.A.) (Accepted 19 August 1986)
Key words: Suprachiasmatic nucleus; Immunohistochemistry; Vasopressin; Vasoactive intestinal polypeptide; Brain transplant; Hypothalamus; Neural development
The capacity of the rat anterior hypothalamus, and particularly the suprachiasmatic nucleus (SCN), to develop and differentiate when removed from its normal environment was examined in this study using light and electron microscopy. The hypothalamus from fetuses ranging in age from embryonic day 12 (E 12) to E 16 was transplanted to the anterior chamber of the eye of adult rats. In initial experiments, we found that transplants from E 15 fetuses and older routinely differentiated into fields of neurons with extensive neuropil with an appearance similar to the anterior hypothalamic area. Groups of small, compactly organized neurons were observed only occasionally in this tissue. Ultrastructural analysis of these transplants typically revealed well-differentiated neuronal perikarya and neuropil with a complex synaptic organization similar in appearance to the normal rat anterior hypothalamic area. Occasionally both mature and immature tissue coexisted in some of the transplants. Tissue from young embryos (E 12-14) frequently showed development of a compact, small neuron nucleus with the cytoarchitectonic appearance of the SCN. At least 45 days were required after transplantation for the successful differentiation to occur in this situation. The SCN in these transplants displayed vasoactive intestinal polypeptide-immunoreactive cells and fibers surrounded by vasopressin-immunoreactive cells and fibers, similar to the pattern observed in the normal adult SCN. Our results indicate that the anterior hypothalamus will differentiate normally in oculo and that the phenotypic specification of the SCN occurs prior to the birthdate of its component neurons.
INTRODUCTION Neuronal transplantation is now a widely used m e t h o d for investigating the processes of neural development. Two types of technique have been used to study the differentiation of the fetal brain. First, transplants directly into the adult brain have been used to investigate the interactions of fetal tissue with the surrounding central nervous system 6. Second, transplants into the anterior c h a m b e r of the eye, presumably a 'neutral' ground, have been used to study the d e v e l o p m e n t of the fetal brain in isolation from normal extrinsic influences 1°. These studies have shown that tissue transplanted n e a r the time of the final cell division of the developing neuroblasts are most likely to grow and differentiate 6.
The time course of neuron formation in the rat hypothalamus has been carefully d o c u m e n t e d by Altman and B a y e r 1. Their studies d e m o n s t r a t e that there is a spatial gradient in cell birthdate with neurons whose final position is distant from the third ventricle forming earlier than those near the ventricle. Cells in the lateral preoptic area, for example, undergo their last cell division between embryonic days (E) 12 and 16 (E 12-16) whereas cells of the suprachiasmatic nucleus (SCN) undergo their last division between E 14 and E 17. Synapse d e v e l o p m e n t lags cell birthdate in the anterior hypothalamus and particularly in the SCN 2. The functional d e v e l o p m e n t of the SCN has also been investigated using the 2-deoxyglucose method 11. This nucleus, which functions as a biologi-
Correspondence: M.H. Roberts, Department of Neurology, HSC T12-020, SUNY-Stony Brook, Stony Brook, NY 11794, U.S.A. 0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
60 cal clock in the mammalian brain 9"12, displays a circadian variation in metabolic activity 13'14which appears on embryonic day 19 (ref. 11). Since the SCN is very immature at E 19 with no apparent intrinsic or extrinsic connections 2, these functional studies indicate that synaptic communication between SCN neurons and the formation of its afferent and efferent projections are not requirements for the development of circadian function in the nucleus. The purpose of the present study was to investigate the development of fetal anterior hypothalamic tissue in oculo, isolated from its normal environment, to analyze the morphological differentiation of the SCN. As noted above, the SCN is now identified as a circadian pacemaker and the intent of this investization was to provide a basis for subsequent functional studies of SCN transplants to both brain and eye. The specific issue addressed was whether the SCN differentiates in oculo with a morphological appearance similar to that observed in the adult nervous system. MATERIALS AND METHODS
Surgery The anterior hypothalamus from E 12-E 16 fetuses were used in this study. Timed-pregnant Sprague-Dawtey rats were obtained from Taconic Farms (Germantown, NY) and maintained on light-dark cycles consisting of 12 h of light and 12 h of darkness. The embryos were removed from the uterus of anesthetized (Ketamine/Rompun, 5:1, 1.0 ml/kg) mothers and staged by measuring their crown-rump length according to Seiger and Olson 15. The embryos' heads were sectioned in the horizontal plane at eye level with microknives, which allowed the base of the brain to be easily visualized. A small (ca. 0.5 mm) cube of hypothalamus, anterior to the developing median eminence, was removed with iridectomy scissors and placed in sterile mammalian Ringers solution. The tissue was transplanted to the anterior chamber of the eye as described by Olson et al.l~. Briefly, a small slit was cut in the cornea of an anesthetized young adult rat, through which the embryonic hypothalamus was inserted with a beveled Pasteur pipette. The explanted tissue was moved to the outer surface of the iris by gently prodding the cornea with forceps. Approximately 150 of these operations were performed.
Light microscopy For our light microscopic observations, explants were allowed to develop for 14-70 days after which time the hosts were anesthetized and transcardially perfused with 200 ml of saline and 400 ml of Bouins fixative. We have found this fixative to be an excellent preservatwe for embryonic tissue ~. Following fixation, the eves were taken out, hemisected, and the lenses removed. Based upon the quality of the perfusion, ca. 20% of the transplants were selected for analysis, The anterior portion of the eye, containing the transplants, was maintained m 70% ethanol for o n e - t w o weeks, placed in 95t~ ethanol overnight and transferred to butanol for 2-3 days before embedding in paraffin under vacuum The tissue was sectioned at 10/~m on a Leitz rotar~ microtome and mounted on slides as 5 replicate sets One set of sections was stained with Cresyl violet.
Irnmunohistochemistrv Our methods for immunohistochemistry using the peroxidase-antiperoxidase technique 17 have been described previously5. In this study, slides were deparaffinized in xylene and rehydrated through an alcohol series to water. The slides were washed in two 10-min changes of 0.1 M phosphate buffer (pH 7.2). All incubations were done in humidified chambers constructed from 15-cm plastic Petri dishes containing damp filter paper. Normal goat serum (10%) in buffer was applied to the slides so as io cover the sections. The Petri dishes were covered and the slides incubated in the goat serum for 20 rain at 4 °C. The serum was drained off the slides, which were then covered with antisera diluted with P:BGT (0.1 M phosphate buffer. 1% normal goat serum, and 0.3% Triton X-100). Anti-vasopressin (VP) was diluted 1:750 and anti-vasoactive intestinal polypeptide (VIP) was diluted 1:500. All antisera were obtained from lmmunoNuclear Corporation lStillwater. MNI. We have found that immunoreactivity to these antisera in the rodent brain can be blocked by low (10 zzM) concentrations of the appropriate antigen 4. The slides were incubated in the antisera overnight (18-22 h) at 4 °C. Following the overnight incubation the slides were rinsed twice for 10 min in phosphate buffer. The slides were then incubated at room temperature for 45 min in goat anti-rabbit IgG diluted 1:50 in PBGT.
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A
Following this incubation the slides were again rinsed in buffer and then incubated for 45 min at room temperature in peroxidase-antiperoxidase diluted 1:100 in PBGT. Following two 10-min buffer rinses, the slides were placed in 200 ml of buffer which contained 100 mg of diaminobenzidine-HC1. The peroxidase reaction was started by adding 220/A of 30% hydrogen peroxide. The reaction was allowed to continue for ca. 10 rain before being stopped by rinsing the slides in 3 changes of phosphate buffer. The sections were then intensified in 0.2% osmium tetroxide for 2-10 s, dehydrated through an alcohol series, cleared in xylene and cover slipped with Permount.
1 8
Electron microscopy For our ultrastructural studies, the explants from E 16 fetuses were allowed to develop in the anterior chamber of the eye for 15-74 days at which time the animals were anesthetized and transcardially perfused with saline followed by 2% paraformaldehyde and 2.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The eyes were removed and placed in the fixative overnight at 4 °C. Implants were then carefully detached from the iris, hand-cut into 1-mm sections, rinsed in buffer and treated in 2% osmium tetroxide in phosphate buffer for 1 h. The tissue was dehydrated in an alcohol series to propylene oxide and embedded in Epon-Araldite. Thin sections were cut. on a Reichert ultramicrotome and collected on Formvar-coated slot grids. The sections were stained with uranyl acetate and lead citrate and examined with a JEOL 100CX electron microscope. RESULTS During the first week to 10 days all the transplants underwent a pronounced shrinkage with the tissue apparent in the anterior chamber decreasing in size by about 30%. This reduction in the size of in oculo transplants has been noted previously by Seiger and Olson 16 who found that embryonic tissue containing the locus coeruleus would shrink by about 50% in the week following transplantation. During the period of shrinkage our transplants are extensively vascularized and blood vessels are seen throughout the tissue after 14 days of development. The prominent immunohistochemical differences between the dorsomedial and ventrolateral SCN
m
Fig. l. Normal rat SCN. A: coronal section through the optic chiasm of an adult rat. The SCNs, indicated by the small black dots, lie at the base of the third ventricle. B: photomicrograph of the SCNs stained with antisera to VP. C: photomicrograph of the SCN stained with antisera to VIP. Bar = 100/~m.
(Fig. 1) can serve as a convenient basis for assessing differentiation of the transplanted SCN. Because the SCN is the only hypothalamic nucleus that displays this distinctive pattern of organization, any compact mass of cells forming within our transplants that shows this pattern of a VIP-immunoreactive region surrounded by a VP immunoreactive capsule can be regarded as SCN. Anterior hypothalamus taken from E 16 rats shows differentiation of several neuronal cell types. On occasion, dense aggregations of
62
Fig. 2. Cresyl violet stain of an E 16 transplant allowed to develop for 42 days. Note the dense aggregations ol neurons. These cells did not show immunoreactivity to VP or VIP antisera. C. cornea: I. iris: T. transplant. Bar = 50 u m
large neurons are evident (Fig. 2), but these do not show a division into VP and V I P i m m u n o r e a c t i v e regions, characteristic of the SCN (Table I). A t the ultrastructural level, E 16 tissue o b s e r v e d TABLE l
Transplants The table displays the n u m b e r of transplants examined and the n u m b e r of transplants containing an SCN for each e m b r y o m c age after development in the anterior c h a m b e r of the eye for mcreasing periods of time.
Embrvonic age Davs in oculo No. examined
No. with SCN
13
14 3(t 45 6(I +
2 1 3 3
0 0 2 1
14
15 30 45 60+
1 1 1 2
o 0 I) 1
16
16 24 30 42
2 5 2 2
0 0 0 0
60+
after 16 days of d e v e l o p m e n t in oculo appears immature. There are many closely a p p o s e d immature perikarya with little neuropil and few synapses. G r o w t h cones also are evident throughout the developing transplant at this age. At 24 days after transplantation. there are regions of mature tissue characterized bv well-differentiated cell bodies and a complex neuropil (Fig. 3t. The synapses primarily contain small. lucent vesicles although occasional dense-core and flattened vesicles are seen. Interestingly, adjacent to these regions of differentiated tissue there are regions of immature tissue, perhaps indicating a continuous gene ration of neurons or glia, Tissue examined after 74 days in oculo appears m a t u r e throughout. Neuronal p e r i k a r y a are well differentiated and there is an extenstve neuropil with a complex synaptic organization and m a n y m y e l i n a t e d axons. Transplants taken from younger e m b r y o s (E 14~ and examined by light microscopy after t5 or 30 days of d e v e l o p m e n t in oculo, show neuronal differentiation but similar to E 16 tissue, no morphological evidence of SCN-like cell groups is present. H o w e v e r , after longer periods of d e v e l o p m e n t (72 days), compact groups of small neurons are seen with a core of
63
Fig. 3 Electron micrograph of a transplant allowed to develop for 24 days in oculo. Region of the transplant distant from the ependymal layer. The large cell in the center of the figure (N) lies within a network of fibers. Several synapses can be seen, most containing small clear vesicles (sc), although occasional dense core vesicles are present (dc). Bar = 1/am.
VIP-immunoreactive cells and fibers surrounded by a capsule of VP-immunoreactive cells and fibers. No other easily identifiable hypothalamic cell groups, such as the paraventricular or supraoptic nuclei, are evident in the transplant. Anterior hypothalamus from E 13 fetuses shows the same differentiation of an SCN-like structure. Similar to transplants from E 14 embryos, there is no differentiation of a compact cell group with VP- and VIP-immunoreactive regions after 14 or 32 days, but an SCN-like structure is present after 45 days (Fig. 4). Table I summarizes the resuits of our immunohistochemicai studies. We conclude that after long periods in oculo the transplants do display differentiation of cell groups which fulfill criteria for designation as SCN.
DISCUSSION The capacity of the anterior hypothalamus and the SCN to develop after removal from its normal environment was examined in this study. Although explants from E 15 or older embryos never show the differentiation of a compact, parvocellular cell group with the appearance of the SCN, differentiation into complex neuronal fields with extensive neuropil and a variety of neuronal sizes and staining density was common. Ultrastructural observations on these explants indicate that the embryonic anterior hypothalamus will develop and differentiate perikarya, axons and axon bundles, myelinated axons, and a neuropil with a variety of synaptic complexes. The ultrastructural appearance of the explants in oculo is very similar to that of adult anterior hypothalamus.
64
Fig. 4. Two E 13 transplants allowed to develop for 47 days in oculo. A and B show VP immunoreactivlty and panels C and D show adjacent sections processed for VIP immunoreactivity. In both cases the VP immunoreactive region surrounds the region of VIP immunoreactivity. Bar = 50 urn.
The normal rat SCN shows a distinctive pattern of immunoreactivity with antisera to VP and VIP. VIPpositive cells and fibers are found in the ventrolateral SCN 5 and VP-positive cells and fibers are found in the dorsomedial SCN l~. VP fibers also extend ventrally to form a capsule a r o u n d the region of VIP immunoreactivity. This pattern of VIP immunoreactivity s u r r o u n d e d by VP-immunoreactive cells and fibers was found in several of our explants. However, the donor tissue had to be young; E 15 or E 16 explants did not develop the typical pattern whereas explants from E 13 or E 14 embryos did differentiate a compact cell group with VP and VIP immunoreactivity.
In a trltiated thvmidine study of hypothalamic neurogenesis, A l t m a n and Bayer ~ demonstrated that SCN neurons are formed between 12; 14 and E 17. Taken together with these data. our results indicate that the neuroepithelial cells which give rise to ~tlc SCN are committed to form the nucleus prior to their final cell division. Similarb,. HofI)er el al. ~ have shown that embryonic cerebettar tissue transplanted in oculo prior to the final division will form normal cerebellar structures and intrinsic connections. In contrast to our results. Wiegand and G a s h 19 have reported in abstract, that the anterior hypothalamus from day 17 fetuses will form an SCN with connections to hosl tissue when transplanted into the
65 third ventricle. Although we did observe perikarya similar in morphology to parvocellular SCN neurons in tissue obtained from E 15 or older fetuses, we never observed VP or VIP immunoreactive neurons organized into a compact cell group. The discrepancy between our results and those of Wiegand and Gash may be due to differences in the transplant sites. The failure of our older transplants to differentiate may result from the inability of the neurons already formed to make connections with the brain. In addition to the age of the donor tissue affecting the formation of the SCN, we also found that the tissue had to be maintained in the anterior chamber of the eye for extended periods of time. This differs from the studies of Hoffer et al. 8, cited above, who find that there is only a 5-day difference between in oculo and in situ cerebellar development. Although there is early differentiation of neuronal fields with the appearance of anterior hypothalamus in our material, we did not observe the differentiation of an immunohistochemically identifiable SCN earlier than 45 days following the transplantation of the tissue into the anterior chamber of the eye. It is difficult to account for the extremely long time required for the formation of this hypothalamic nucleus in oculo. In normal fetal development the nucleus is readily recognized with Cresyl violet staining at E 18, and VP immunoreactivity appears shortly after birth at postnatal day 2 (ref. 7). On that basis, we would expect to observe an immunohistochemically identifiable SCN ca. two weeks after transplantation. While a developmental delay was observed in our tissue as assayed by the relatively late appearance of myelin in our electron micrographs after 24 days in oculo, as opposed to post natal day 10 in normal development 2, the pronounced delay in the appearance of the SCN seems extreme. One explanation is that the developmental lag is an artifact of our criteria for identifying an SCN. The nucleus may develop earlier than our immunohistochemical studies indicate by virtue of the explant's inability to produce detectable VP or VIP until 45 days after transplantation. If this were the case, we would expect to see dense aggregations of small neurons in our young transplants that failed to stain with antisera to VP or VIP. We did not
observe, however, any of these structures in our 'unsuccessful' transplants. Two additional explanations for the developmental delay are that the anterior chamber of the adult eye contains markedly lower concentrations of trophic substances, present in the embryo, that promote the rapid proliferation of neural tissue, or that interactions between the transplant and the contents of the anterior chamber of the eye, particularly the iris, inhibit neuronal differentiation. Our transplants, in either case, would develop more slowly. However, neither of these explanations are supported by the studies of Hoffer et al. 8. Finally, it may be the case that the formation of the SCN requires the migration of neurons away from the ependyma which could be retarded by the initial degeneration of the transplant or by physical barriers formed by the rapid outgrowth of fiber tracts observed in our electron micrographs. Further studies may allow us to distinguish between all these possibilities. In summary, we can draw 3 conclusions from this study. First, the anterior hypothalamus and the SCN will develop in an in vivo explant system removed from its usual environment. Second, the SCN differentiate normally as expressed by the organization of VP- and VIP-containing neurons. Finally, the cells of the germinal epithelium that give rise to the SCN are committed to form the nuclei as early as day 13 of embryonic development, prior to the previously established birthdates of SCN neurons. It is not known whether transplants which develop morphologically into SCN-iike cell groups exhibit spontaneous circadian rhythms or are capable of functioning as circadian pacemakers. Electrophysiologicai studies on these transplants may provide eivdence for the functional, as well as the morphological development of the SCN in oculo.
ACKNOWLEDGEMENTS We would like to thank J. Speh for technical assistance and V. Cassone, E. Gustafson and S. Dewey for critical review of an early version of this manuscript. Supported by USPHS Grant NS-16304 to R.Y.M.
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