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Timm staining of hippocampal nerve cell bodies in the Kyoto rat. A cell marker in allo- and xenografting of rat and mouse brain tissue, revealing neuronal migration
DevelopmentalBrain Research, 29 (1986) 51-59 Elsevier
51
BRD 50438
Timm Staining of Hippocampal Nerve Cell Bodies in the Kyoto Rat. A Cell Marker in Allo- and Xenografting of Rat and Mouse Brain Tissue, Revealing Neuronal Migration BENTE FINSEN and JENS ZIMMER Institute of Anatomy B (Neurobiology), Universityof Aarhus, DK-8000Aarhus C (Denmark) (Accepted March 1lth, 1986) Key words: rat strain - - fascia dentata - - dentate granule cell - - intracerebral transplantation
Using the histochemical Timm sulphide silver method, a strain-specific, increased stainability of the cell bodies of the dentate granule cells and the hippocam'[aalpyramidal cells was observed in the inbred Kyoto rat. The resulting dense staining which also includes neurons in the neocortex and cerebellum can be used as a cell marker in studies of intracerebral allo- and xenotransplantation of rat and mouse hippocampal tissue. Used in such experiments the marker revealed migration of dentate granule cells from Kyoto transplants into the Wistar host fascia dentata.
INTRODUCTION In order to distinguish cells of different origin in chimera and grafting experiments it is important to have good markers for the cells and cell lines in question. In developmental neurobiology this problem of identification was most elegantly solved by Le Douatin lj in her now classical studies of the differentiation of neural crest cells. Following grafting of quail embryonic neural crests to chick embryos she used a difference in the interphase nuclear morphology of quail and chick cells in ordinary cell stains to follow the fate of the quail cell lines within the chick hosts. The need for cell markers to identify donor and recipient nerve cells is also pertinent in the study of intracerebral transplants of immature brain tissue, especially when the transplantations are performed to homologous brain areas of immature recipients. In such cases pieces of accidentally displaced recipient tissue may survive as autografts and be misinterpreted as allo- or xenografts. Several cell and tissue markers have been used in intracerebral grafting. In a study of hypothalamic grafts Charlton et al. 3 made use of immunohistochemical techniques to distinguish between the allelic forms of the Thy-1 antigen on nerve
cells from different strains of mice. The same technique was used by Inoue et al. 9 to distinguish donor mouse tissue from host rat brain tissue in a study of neocortical xenografts. The advantage of this method is that the Thy-1 surface antigen is expressed by all parts of the nerve cell membrane, including cell body, dendrites, axon and axon terminals, allowing it to be distinguished even at the neuropil level 22. Neurons to be transplanted can also be labeled by retrograde axonal transport of fluorescent dyes injected into the donor brains prior to transplantation, provided that the neurons already have developed axons of some length before the transplantation 2~. Fluorescent dyes have also been used to label neurons simply by soaking the donor tissue in a dye solution prior to the transplantation 13. Finally, Lindsay et al. 12 have made use of a D N A hybridization technique to visualize and identify mouse nerve cells from Mus caroli mice 15transplanted to Mus musculus mice. The cell marker to be described in the present study is based on a strain-specific increase in the Timm stainability of the nerve cell bodies in the hippocampus and fascia dentata of the Kyoto rat, an inbred Wistar rat strain. We came across this marker in a study of xenografting of mouse brain tissue to
Correspondence: J. Zimmer, Institute of Anatomy B (Neurobiology), University of Aarhus, DK-8000 Aarhus C, Denmark. 0165-3806/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
Fig. 1. Thionin [a) and Timm staining (b) of the hippocampal region of the Kyoto rat at a midtemporal level. Note the dense staining ot dentate granule (g) and hippocampal pyramidal (p) celt bodies as well as cell bodies in the retrohippocampal areas and the neocortex fcx I. For comparison with Wistar rats and C57 mice. see Fig 2. × 30.
rats. where inbred Kyoto rats were used as recipients 5.28
two animals from each strata were perfused with a sulphide solution ~ and the brains taken out in toto, frozen and cul in 5 parallel series of 30-urn-thick sections in the horizontal and coronal planes on a crvo-
MATERIALS AND METHODS
stat. The different series of sections were stained
Normal material
with thionin for cells, a histochemical m e t h o d h)r acctylcholinesterase v. the Nauta silver method 14 for fi-
sexes from the o u t b r ed Wistar strain and the inbred
ber architectonics and the T i m m sulphide silver method s,2° (one lightly and one darkly stained se-
Kyoto, Ag o u t i . Brown N o r w e g i a n . Lewis. Salt Hy-
ries). Th e latter method which is a histochemicat
pertension-Resistant.
Hypertension-Sensitive.
method for so-called heavy metals, including zinc. ~s
B l a c k - H o o d e d and Buffalo strains, as well as young adult mice from the inbred strains C57 and B A L B / c .
known to visualize the laminar distribution of the terminal fields of the m a j o r hippocampal and dentate af-
The rats were obtained from MOilegaard Breeding Ce nte r . the mice from G1. B o m h o lt g a a r d . A t least
ferent pathways, based on a differentiated stainability of the terminals of these pathways 4 s >
T he material consisted of young adult rats of both
Salt
53
Transplanted material Rat allografts. Blocks of tissue containing the fascia dentata and adjacent hippocampal tissue were taken from the brains of Kyoto rats at the day of birth and transplanted unilaterally into the hippocampal region of newborn Wistar rats by means of a glass cannula mounted on a 50-pl Hamilton syringe 17. Some of the newborn recipient rats were subjected to X-irradiation of their hippocampal regions just prior to the transplantation, in order to prevent the formation of dentate granule cells 1°'18'19. Following a survival time of 9 weeks the recipient rats (n = 8) were perfused and their brains histologically processed as described above. Mouse-to-rat xenografts. Using the transplantation technique presented above, blocks containing the fascia dentata and adjacent hippocampus were taken from 16-17-day mouse embryos (C57 strain) and grafted unilaterally into the hippocampal region of newborn Kyoto rats (n = 80). Following 40-63 and 135-140 days survival the recipient rats were per-
fused and their brains histologically processed as described above. The xenograft material was supplemented by additional, but otherwise similarly processed cases, where E16-17 embryonic C57-mouse dentate tissue had been grafted to the brains of young adult Kyoto rats some of which were either treated or not treated with the immunosuppressive agent Cyclosporin A 5'28. RESULTS
Comparison of different rat and mouse strains The distribution of Timm staining in the hippocampal and dentate neuropil was the same in all the rat strains examined. The staining was distributed in well-known laminae with the same mutual differences in staining type and density (Figs. 1 and 2a). The only exception was a slightly lighter staining of the mossy fiber terminals of the Kyoto rats (dark brown to black compared with jet black; cf. mf in Figs. lb and 2a, and 3c, d). The two mouse strains
Fig. 2. The hippocampus and fascia dentata of the Wistar rat (a) and the C57-mouse (b) in Timm-stainedsections. ×30.
54
Fig. 3. Timm-stained sections from corresponding parts of the dentate granule cell layer t a, b/and the CA3 pyramidal cell laver t c. d l of a Kyoto and a Wistar rat. The difference in the staining of the neuronal cell bodies is mainly due to an increased staining of the nuclei and possibly also the perinuclear parts of the cytoplasm in the Kyoto rats. cf. the CA3 neuron in te I I arrowl. A presumed astrocytc m the dentate molecular layer (arrow in (all does not show increased staining. ×380.
displayed their known s t r a i n - d e p e n d e n t deviations from the rat staining patterns, as described by Fredens 6 and shown for the C57 strain in Fig. 2b. In the staining of the h i p p o c a m p a l and d e n t a t e cell layers the K y o t o rats were unique. While all other strains of rats and mice only displayed a fine punctate precipitate over the cell layers, these layers all stained m o r e darkly in the K y o t o rat due to a m o d e r ate to dense staining of the individual nerve cell bodies (Figs. 1 and 31. The d e n t a t e granule cell bodies in particular stained very densely, being dark brown to black (Fig. 3a. bl, but also the CA3 and CA1 pyramidal cell bodies, which are larger than those of the granule cells, were m o r e heavily stained than their counterparts in the other rat and mouse strains (Fig. 3c. d). In these large cells the staining was seen to include a light b r o w n to b r o w n staining of the nucleus as well as a punctate perinuclear staining which also e x t e n d e d into the proximal parts of the dendrites (Fig. 3c). The dense staining of K y o t o nerve cell bodies was also present outside the fascia d e n t a t a and h i p p o c a m -
pus. as for instance in the other areas ol the hippocampal region (Fig. lb) as well as areas outside this region. T h e r e was thus an increased stainability of pyramidal cells in the neocortex (Fig. l b ) , the granule cells of the cerebellum and the cells in the Islands of Calleja. Staining ofglial cells. The staining o f the nerve cell layers in the h i p p o c a m p a l region of the Kyoto rats was easily attributed to the neuronal cell bodies on the basis of its detailed location, including p r o x i m a l dendrites extending from the cell bodies. Gliat cells are usually m o r e dispersed and m o r e difficult to identify as such solely on the basis of their location and the general a p p e a r a n c e of the cell body. It was characteristic, however, that the cell b o d i e s a r r a n g e d in concentric, but loose rows in the d e n t a t e m o l e c u l a r layer and known to belong to astrocytes 1'2-s did not show the same dense T i m m staining as the granule cell bodies in the K y o t o rat (Fig. 3a). N e i t h e r did cell bodies in the h i p p o c a m p a l white matter. It cannot on this basis be excluded that some g|ial cells stain more intensely in the K y o t o rat than in other rat and m~mse
55
O ¸i i!! ~ililL~: Fig. 4. Adjacent thionin- (a) and Timm-stained (b) sections of a well-integrated fascia dentata transplant from a Kyoto donor, located in the dorsal hippocampus of a Wistar rat. Donor, newborn Kyoto rat; recipient, newborn Wistar rat irradiated over the hippocampal region earlier the same day; survival, 2 months. Note the distinct staining of the transplant dentate granule cells, and the presence of such cells among the unstained cells in the host dentate granule cell layer. ×34. c: higher magnification of framed area in (b), showing the stained cells similar to the transplant Kyoto granule cells in the Wistar host granule cell layer, x 166.
strains, but the principal d i f f e r e n c e b e t w e e n this and
tar rats. the K y o t o tissue survived, d i f f e r e n t i a t e d and
the o t h e r strains e x a m i n e d was clearly r e l a t e d to the
b e c a m e i n t e g r a t e d in the s a m e way a,~ d e s c r i b e d tor
staining of n e r v e cell bodies.
transplants o f Wistar tissue >->)->' -~ In g e n e r a l the
The cell staining in transplant experiments Allotransplants. F o l l o w i n g t r a n s p l a n t a t i o n of im-
sections based on f e a t u r e s like their e e n e r a l l o c a t i o n
m a t u r e K y o t o rat d e n t a t e tissue to the brain of Wis-
u o n s the iden tification of the transplants was. howcx-
transplants c o u l d be i d e n t i f i e d in the t h i o m n - s t a m e d and a p p e a r a n c e (Fig. 4al, in the t i m m - s t a i n e d sec-
Fig. 5. Well-integrated xenograft of mouse C57 dentate tissue located to the medial blade of the host Kyoto fasci a dentata. Donor, E~7 C57 mouse embryo; recipient, newborn Kyoto rat; survival, 2months. In the Timm staining (a) thehost and the xenotransplant granule cells are distinguished by their difference in stainability (interface at arrow), while the distinction in the adjacent thionin-staincd section (b) is more difficult (interface at arrow), a, x 130: b, x 150.
57
Fig. 6. Xenograft of C57 mouse dentate tissue next to the Kyoto host fascia dentata. Donor, E l 7 C57 mouse embryo; recipient, newborn Kyoto rat; survival. 2 months. What appears as a pure xenograft in thionin staining (a), actually contains autografted host CA1 pyramidal cells, as disclosed in the adjacent Timm-stained section (arrowhead, in (b)). A Timm-stained terminal field extends from the autograft into the molecular layer of the xenograft (arrow). ×39.
er, evident. The dentate transplants of Kyoto tissue thus stood out in clear contrast to the host tissue, due to its content of densely stained granule cell bodies (Fig. 4b). Also the transplant hippocampal pyramidal cells displayed the characteristic increased stainability of their cell bodies. In the cases where the transplants at certain levels passed through the dentate and pyramidal cell layers of the host fascia dentata and hippocampus we found areas with complete merging of transplant and host tissue (Fig. 4). In some cases darkly stained cell bodies similar to Kyoto dentate granule cell bodies were found in the Wistar host fascia dentata up to 800 ktm away from the dentate granule cell layers of the transplants (Fig. 4b, c). This suggested that migration of transplant neurons into homologous layers of the host had occurred. This phenomenon was observed both after transplantation of Kyoto dentate tissue to normal and X-irradiated newborn rats and we are presently examining in detail whether it is more extensive in the granule cell deprived X-irradiated recipients than the normal ones.
Xenotransplants.
Surviving xenografts of em-
bryonic C57-mouse hippocampi retained the lack of Timm stainability of their dentate and hippocampal neuronal cell bodies, both in the neonatal and the adult Kyoto rat recipient brains. In cases with very well-integrated transplants like the one shown in Fig. 5, this allowed a distinction to be made between transplant and host parts of the jointly composed fascia dentata (Fig. 5a). This was not possible with the same certainty and precision in any of the other stained series of sections, like the thionin-stained ones (Fig. 5b). Stained cells identical to the host Kyoto cells were sometimes found in what otherwise appeared to be pure xenografts. In some cases the presence of these ceils suggested a migration of Kyoto host cells into the transplants. In others, like the case shown in Fig. 6, it was more likely that the cells were present in host tissue that had been dislocated at the time of transplantation. These cells would not have been identified as host cells without the cell marker (cf. Fig. 6a, b) and the additional connective host-transplant interactions that these cells were involved in would accordingly not have been revealed.
5> I)ISCUSSION The basis for the unique T i m m stainability of the cell bodies, in particular the nuclei, of neurons in the Kvoto rat brain is unknown. The T i m m m e t h o d stains histochemically the so-called heavy metal, i.e., the metals listed in group l i b of the periodic system s2° including zinc. It is so far unknown w h e t h e r any of these metals are present in the Kyoto nerve cell bodies in higher amounts than in other strains of rats or mice, but preliminary experiments with staining of histological sections from K y o t o rats with the zinc-binding fluorescent dye 2-methyl-8-hydroxyquinoline: have not revealed any increased zinc content in the Kyoto granule cell bodies. The stainable metals may instead be present in a form that m a k e them more accessible to the sulphide at the time of perfusion. This would increase the amounts of metallosulphides formed onto which silver subsequently is deposited by the physical d e v e l o p m e n t ~. The functional significance is also unknown so far, as it still is for most metals visualized in the h i p p o c a m p a l region by the T i m m method. This does not, however, invalidate the use of the increased stainability of the K y o t o nerve cell bodies as a cell marker. It is thus not known either what is the functional significance of the difference in the nuclear m o r p h o l o g y b e t w e e n the chick and quail ll, or the difference in D N A plasraids b e t w e e n the M u s caroli and the c o m m o n Mus rmIs(/t,tlus |5 A crucial p r o p e r t y of a cell m a r k e r is specificity and stability. With regard to specificity we have demonstrated that the increased T i m m stainability of the Kyoto nerve cell bodies is strain-specific. The cell body staining of the dentate granule cells and the hipp o c a m p a l p y r a m i d a l cells was not observed in any other of the rat and mouse strains examined. The possibility that the staining later might be found in strains not examined here would not affect the use of
the m a r k e r in transplantations b e t w e e n the presently examined strains. With regard to stability, the staining characteristics of for instance the Kyoto d e n t a t e granule cells did not a p p e a r to change after transplantation and m o r e than two months survival m the brains of Wistar rats. In addition it appears from preliminary tissue culture experiments, using the slice culture technique 24. that the characteristic stainability persists for at least 5 weeks m vm'o in cultures ol h i p p o c a m p a l tissue o b t a i n e d from 7-dav-old rats. Concerning the stability and specificity of the cell staining there is also the possibility that the transplanted Kvoto tissue or the host K y o m brain might be able to induce a Kyoto-like stainability in the nonKvoto host or transplant nerve cells We have no ewdence, however, that such induction occurs. In many cases where host and transplant tissue were in innmate contact, even with exchange of nerve connecnons. the distribution of stained cell bodies, within the one or the other still in most ca~es coincided with what was expected from careful anal'~sis of the adiacent series of thionin-stained sccuons (Figs. 4-61 Seen in relation to these observations it is most likely that the stained cells o b s e r v e d among the unstained host granule cells shown in Fig. 4b. c are cells that have m~grated from the transplam rather than host cells that have had their staining characteristics transformed. The definite p r o o f for neuronal migration will be sought in future studies, where the characteristic stainabilitv of the Kvoto d e n t a t e granule cells is to be c o m b i n e d by a second m a r k e r , like [3H]th3 mldin or fluorochromes. ACKNOWLEDGEMENTS This study was s u p p o r t e d m part by the Danish M R C . the Danish F o u n d a t i o n for the A d v a n c e ot Medical Sciences, and the International Spinal Research Trust. U.K.
ABBREVIATIONS IN THE FIGURES ca (_'At CA3 cx EA f FD g h Ky
commissural-associationalzone hippocampal subfield CA 1, regio superior hippocampal subfield CA3, regio inferior ncocortex entorhinal area fimbria fascia dentata granule cell layer hilus Kyoto
lpp m rnl mpp p Prs Ps Sb l'r Wi
latcral pcrtorant path zone dentate molecular layer mossy fiber layer medial perforant path zone p~ ramidal cell layer presubiculum para~ubiculum sublculunl transplant Wistar
NC
XC llO~I'[t I'l
59 REFERENCES 1 Bignami, A. and Dahl, D., Astrocyte°specific protein and neuroglial differentiation. An immunofluorescence study with antibodies to the glial fibrillary acidic protein, J. Comp. Neurol., 153 (1974) 27-38. 2 Blockus, M.L., McCourt, J.D., Kasarakia, E.J. and Frederickson, C.J., Histofluorescent method for hippocampal zinc, Soc. Neurosci. Abstr., 11 (1985) 438. 3 Charlton, H.M., Barclay, A.N. and Williams, A.F., Detection of neuronal tissue from brain grafts with anti-Thy-l.1 antibody, Nature (London), 305 (1983) 825-827. 4 Danscher, G. and Zimmer, J., An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues, Histochemistry, 55 (1978) 27-40. 5 Finsen, B. and Zimmer, J., Nerve connections between mouse and rat brain tissue: observations after intracerebral xenotransplantation and tissue culture, Neurosci. Lett., Suppl. 22 (1985) $529. 6 Fredens, K., Genetic variation in the histoarchitecture of the hippocampal region of mice, Anat. Embryol., 161 (1981) 265-281. 7 Geneser-Jensen, F.A. and Blackstad, T.W., Distribution of acetylcholinesterase in the hippocampal region of the guinea pig. I. Entorhinal area, parasubiculum, and pr'esubiculum, Z. Zellforsch., 114 (1971) 460-481. 8 Haug, F.-M.S., Heavy metals in the brain, A light microscope study of the rat with Timm's sulphide silver method. Methodological considerations and cytological and regional staining patterns, Adv. Anat. Embryol. Cell Biol., 47 (1973) 1-71. 9 Inoue, H., Kohsaka, S., Yoshida, K., Ohtani, M., Toya, S. and Tsukada, Y., Cyclosporin A enhances the survivability of mouse cerebral cortex grafted into the third ventricle of rat brain, Neurosci. Leg., 54 (1985) 85-90. 10 Laurberg, S. and Hjorth-Simonsen, A., Growing central axons deprived of normal target neurons by neonatal X-irradiation still terminate in a precisely laminated fashion, Nature (London), 269 (1977) 158-160. 11 Le Douarin, N.M., A biological cell labelling technique and its use in experimental embryology, Dev. Biol., 30 (1973) 217-222, 12 Lindsay, R.M., Raisman, G. and Seeley, P.J., Neural tissue transplants: studies using tissue culture manipulation, cell marking techniques and a plasma clot method to follow the development of grafted neurons and glia. In W. Seifert and H. Althaus (Eds.), Proceedings of Advanced Research Workshop on Glial-Neuronal Communication in Development and Regeneration, in press. 13 McConnell, S.K., Migration and differentiation of cerebral cortical neurons after transplantation into the brains of ferrets, Science, 229 (1985) 1268-127l. 14 Nauta, W.J.H., fiber die sogenannte terminale Degeneration in Centralnervensystem und ihre Darstellung durch Silberimpr~ignation, Arch. Neurol. Psychiat., 66 (1950) 353-376. 15 Rossant, J., Vijh, M., Siracusa, L.D. and Chapman, V.M.,
Identification of embryonic cell lineages in histological sections of M. musculus-M, caroli chimeras, J. Embryol. Exp. Morphol. 73 (1983) 163-178. 16 Sunde, N. and Zimmer, J., Transplantation of central nervous tissue. An introduction with results and implications, Acta Neurol. Scand., 63 (1981) 323-335. 17 Sunde, N. and Zimmer, J., Cellular, histochemical and connective organization of the hippocampus and fascia dentata transplanted to different regions of immature and adult rat brains, Dev. Brain Res., 8 (1983) 165-191. 18 Sunde, N., Laurberg, S. and Zimmer, J., Brain grafts can restore irradiation-damaged neuronal connections in newborn rats, Nature (London), 310 (1984) 51-53. 19 Sunde, N., Zimmer, J. and Laurberg, S., Repair of neonatal irradiation-induced damage to the rat fascia dentata. Effects of delayed intracerebral transplantation. In A. Bj6rklund and U. Stenevi (Eds.), Neural Grafting in the Mammalian CNS, Elsevier. Amsterdam, 1985, pp. 301-308. 20 Timm, F., Zur Histochemie der Schwermetalle. Das SulfidSilber-Verfahren, Dtsch. Z. Ges. Ger. Med., 47 (1958) 428-481. 21 Tender, N., Gaarskj~er, F.B., Sunde, N. and Zimmer, J., Labeling of hippocampal neurons by retrograde axonal transport of Granular Blue prior to intracerebral transplantation, Neurosci. Lett., Suppl. 22 (1985) $426. 22 Zhou, C.-F., Morris, R.J., Lindsay, R.M., Seeley, P. and Raisman, G., Projection patterns of hippocampal transplants in adult host hippocampus visualized by Thy-1.1 immunohistochemistry, Soc. Neurosci. Abstr., 11 (1985) 585. 23 Zimmer, J., Development of the hippocampus and fascia dentata: morphological and histochemical aspects, Prog. Brain Res., 48 (1978) 171-189. 24 Zimmer, J. and G~ihwiler, B.H., Cellular and connective organization of slice cultures of the rat hippocampus and fascia dentata, J. Comp. Neurol., 228 (1984) 432-446. 25 Zimmer, J. and Sunde, N., Neuropeptides and astroglia in intracerebral hippocampal transplants: an immunohistochemical study in the rat, J. Comp. Neurol., 227 (1984) 331-347. 26 Zimmer, J., Sunde, N., Sorensen, T., Jensen, S., M011er, A.G. and G~ihwiler, B.H., The hippocampus and fascia dentata. An anatomical study of intracerebral transplants and intraocular and in vitro cultures. In A. Bj6rklund and U. Stenevi (Eds.), Neural Grafting in the Mammalian CNS, Elsevier, Amsterdam, 1985, pp. 285-299. 27 Zimmer, J., Sunde, N. and S0rensen, T., Reorganization and restoration of central nervous connections after injury: a lesion and transplant study of the rat hippocampus. In B.E. Will, P. Schmitt and J.C. Dalrymple-Alford (Eds.), Brain Plasticity, Learning, and Memory, Plenum, 1985, pp. 505-518. 28 Zimmer, J,, Finsen, B., Sorensen, T. and Sunde, N., Hippocampal transplants: synaptic organization, their use in repair of neuronal circuits and mouse to rat xenografting. In W. Seifert and H. Althaus (Eds.), Proceedings of Advanced Research Workshop on Glial-Neuronal Communication in Development and Regeneration, in press.
51
BRD 50438
Timm Staining of Hippocampal Nerve Cell Bodies in the Kyoto Rat. A Cell Marker in Allo- and Xenografting of Rat and Mouse Brain Tissue, Revealing Neuronal Migration BENTE FINSEN and JENS ZIMMER Institute of Anatomy B (Neurobiology), Universityof Aarhus, DK-8000Aarhus C (Denmark) (Accepted March 1lth, 1986) Key words: rat strain - - fascia dentata - - dentate granule cell - - intracerebral transplantation
Using the histochemical Timm sulphide silver method, a strain-specific, increased stainability of the cell bodies of the dentate granule cells and the hippocam'[aalpyramidal cells was observed in the inbred Kyoto rat. The resulting dense staining which also includes neurons in the neocortex and cerebellum can be used as a cell marker in studies of intracerebral allo- and xenotransplantation of rat and mouse hippocampal tissue. Used in such experiments the marker revealed migration of dentate granule cells from Kyoto transplants into the Wistar host fascia dentata.
INTRODUCTION In order to distinguish cells of different origin in chimera and grafting experiments it is important to have good markers for the cells and cell lines in question. In developmental neurobiology this problem of identification was most elegantly solved by Le Douatin lj in her now classical studies of the differentiation of neural crest cells. Following grafting of quail embryonic neural crests to chick embryos she used a difference in the interphase nuclear morphology of quail and chick cells in ordinary cell stains to follow the fate of the quail cell lines within the chick hosts. The need for cell markers to identify donor and recipient nerve cells is also pertinent in the study of intracerebral transplants of immature brain tissue, especially when the transplantations are performed to homologous brain areas of immature recipients. In such cases pieces of accidentally displaced recipient tissue may survive as autografts and be misinterpreted as allo- or xenografts. Several cell and tissue markers have been used in intracerebral grafting. In a study of hypothalamic grafts Charlton et al. 3 made use of immunohistochemical techniques to distinguish between the allelic forms of the Thy-1 antigen on nerve
cells from different strains of mice. The same technique was used by Inoue et al. 9 to distinguish donor mouse tissue from host rat brain tissue in a study of neocortical xenografts. The advantage of this method is that the Thy-1 surface antigen is expressed by all parts of the nerve cell membrane, including cell body, dendrites, axon and axon terminals, allowing it to be distinguished even at the neuropil level 22. Neurons to be transplanted can also be labeled by retrograde axonal transport of fluorescent dyes injected into the donor brains prior to transplantation, provided that the neurons already have developed axons of some length before the transplantation 2~. Fluorescent dyes have also been used to label neurons simply by soaking the donor tissue in a dye solution prior to the transplantation 13. Finally, Lindsay et al. 12 have made use of a D N A hybridization technique to visualize and identify mouse nerve cells from Mus caroli mice 15transplanted to Mus musculus mice. The cell marker to be described in the present study is based on a strain-specific increase in the Timm stainability of the nerve cell bodies in the hippocampus and fascia dentata of the Kyoto rat, an inbred Wistar rat strain. We came across this marker in a study of xenografting of mouse brain tissue to
Correspondence: J. Zimmer, Institute of Anatomy B (Neurobiology), University of Aarhus, DK-8000 Aarhus C, Denmark. 0165-3806/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
Fig. 1. Thionin [a) and Timm staining (b) of the hippocampal region of the Kyoto rat at a midtemporal level. Note the dense staining ot dentate granule (g) and hippocampal pyramidal (p) celt bodies as well as cell bodies in the retrohippocampal areas and the neocortex fcx I. For comparison with Wistar rats and C57 mice. see Fig 2. × 30.
rats. where inbred Kyoto rats were used as recipients 5.28
two animals from each strata were perfused with a sulphide solution ~ and the brains taken out in toto, frozen and cul in 5 parallel series of 30-urn-thick sections in the horizontal and coronal planes on a crvo-
MATERIALS AND METHODS
stat. The different series of sections were stained
Normal material
with thionin for cells, a histochemical m e t h o d h)r acctylcholinesterase v. the Nauta silver method 14 for fi-
sexes from the o u t b r ed Wistar strain and the inbred
ber architectonics and the T i m m sulphide silver method s,2° (one lightly and one darkly stained se-
Kyoto, Ag o u t i . Brown N o r w e g i a n . Lewis. Salt Hy-
ries). Th e latter method which is a histochemicat
pertension-Resistant.
Hypertension-Sensitive.
method for so-called heavy metals, including zinc. ~s
B l a c k - H o o d e d and Buffalo strains, as well as young adult mice from the inbred strains C57 and B A L B / c .
known to visualize the laminar distribution of the terminal fields of the m a j o r hippocampal and dentate af-
The rats were obtained from MOilegaard Breeding Ce nte r . the mice from G1. B o m h o lt g a a r d . A t least
ferent pathways, based on a differentiated stainability of the terminals of these pathways 4 s >
T he material consisted of young adult rats of both
Salt
53
Transplanted material Rat allografts. Blocks of tissue containing the fascia dentata and adjacent hippocampal tissue were taken from the brains of Kyoto rats at the day of birth and transplanted unilaterally into the hippocampal region of newborn Wistar rats by means of a glass cannula mounted on a 50-pl Hamilton syringe 17. Some of the newborn recipient rats were subjected to X-irradiation of their hippocampal regions just prior to the transplantation, in order to prevent the formation of dentate granule cells 1°'18'19. Following a survival time of 9 weeks the recipient rats (n = 8) were perfused and their brains histologically processed as described above. Mouse-to-rat xenografts. Using the transplantation technique presented above, blocks containing the fascia dentata and adjacent hippocampus were taken from 16-17-day mouse embryos (C57 strain) and grafted unilaterally into the hippocampal region of newborn Kyoto rats (n = 80). Following 40-63 and 135-140 days survival the recipient rats were per-
fused and their brains histologically processed as described above. The xenograft material was supplemented by additional, but otherwise similarly processed cases, where E16-17 embryonic C57-mouse dentate tissue had been grafted to the brains of young adult Kyoto rats some of which were either treated or not treated with the immunosuppressive agent Cyclosporin A 5'28. RESULTS
Comparison of different rat and mouse strains The distribution of Timm staining in the hippocampal and dentate neuropil was the same in all the rat strains examined. The staining was distributed in well-known laminae with the same mutual differences in staining type and density (Figs. 1 and 2a). The only exception was a slightly lighter staining of the mossy fiber terminals of the Kyoto rats (dark brown to black compared with jet black; cf. mf in Figs. lb and 2a, and 3c, d). The two mouse strains
Fig. 2. The hippocampus and fascia dentata of the Wistar rat (a) and the C57-mouse (b) in Timm-stainedsections. ×30.
54
Fig. 3. Timm-stained sections from corresponding parts of the dentate granule cell layer t a, b/and the CA3 pyramidal cell laver t c. d l of a Kyoto and a Wistar rat. The difference in the staining of the neuronal cell bodies is mainly due to an increased staining of the nuclei and possibly also the perinuclear parts of the cytoplasm in the Kyoto rats. cf. the CA3 neuron in te I I arrowl. A presumed astrocytc m the dentate molecular layer (arrow in (all does not show increased staining. ×380.
displayed their known s t r a i n - d e p e n d e n t deviations from the rat staining patterns, as described by Fredens 6 and shown for the C57 strain in Fig. 2b. In the staining of the h i p p o c a m p a l and d e n t a t e cell layers the K y o t o rats were unique. While all other strains of rats and mice only displayed a fine punctate precipitate over the cell layers, these layers all stained m o r e darkly in the K y o t o rat due to a m o d e r ate to dense staining of the individual nerve cell bodies (Figs. 1 and 31. The d e n t a t e granule cell bodies in particular stained very densely, being dark brown to black (Fig. 3a. bl, but also the CA3 and CA1 pyramidal cell bodies, which are larger than those of the granule cells, were m o r e heavily stained than their counterparts in the other rat and mouse strains (Fig. 3c. d). In these large cells the staining was seen to include a light b r o w n to b r o w n staining of the nucleus as well as a punctate perinuclear staining which also e x t e n d e d into the proximal parts of the dendrites (Fig. 3c). The dense staining of K y o t o nerve cell bodies was also present outside the fascia d e n t a t a and h i p p o c a m -
pus. as for instance in the other areas ol the hippocampal region (Fig. lb) as well as areas outside this region. T h e r e was thus an increased stainability of pyramidal cells in the neocortex (Fig. l b ) , the granule cells of the cerebellum and the cells in the Islands of Calleja. Staining ofglial cells. The staining o f the nerve cell layers in the h i p p o c a m p a l region of the Kyoto rats was easily attributed to the neuronal cell bodies on the basis of its detailed location, including p r o x i m a l dendrites extending from the cell bodies. Gliat cells are usually m o r e dispersed and m o r e difficult to identify as such solely on the basis of their location and the general a p p e a r a n c e of the cell body. It was characteristic, however, that the cell b o d i e s a r r a n g e d in concentric, but loose rows in the d e n t a t e m o l e c u l a r layer and known to belong to astrocytes 1'2-s did not show the same dense T i m m staining as the granule cell bodies in the K y o t o rat (Fig. 3a). N e i t h e r did cell bodies in the h i p p o c a m p a l white matter. It cannot on this basis be excluded that some g|ial cells stain more intensely in the K y o t o rat than in other rat and m~mse
55
O ¸i i!! ~ililL~: Fig. 4. Adjacent thionin- (a) and Timm-stained (b) sections of a well-integrated fascia dentata transplant from a Kyoto donor, located in the dorsal hippocampus of a Wistar rat. Donor, newborn Kyoto rat; recipient, newborn Wistar rat irradiated over the hippocampal region earlier the same day; survival, 2 months. Note the distinct staining of the transplant dentate granule cells, and the presence of such cells among the unstained cells in the host dentate granule cell layer. ×34. c: higher magnification of framed area in (b), showing the stained cells similar to the transplant Kyoto granule cells in the Wistar host granule cell layer, x 166.
strains, but the principal d i f f e r e n c e b e t w e e n this and
tar rats. the K y o t o tissue survived, d i f f e r e n t i a t e d and
the o t h e r strains e x a m i n e d was clearly r e l a t e d to the
b e c a m e i n t e g r a t e d in the s a m e way a,~ d e s c r i b e d tor
staining of n e r v e cell bodies.
transplants o f Wistar tissue >->)->' -~ In g e n e r a l the
The cell staining in transplant experiments Allotransplants. F o l l o w i n g t r a n s p l a n t a t i o n of im-
sections based on f e a t u r e s like their e e n e r a l l o c a t i o n
m a t u r e K y o t o rat d e n t a t e tissue to the brain of Wis-
u o n s the iden tification of the transplants was. howcx-
transplants c o u l d be i d e n t i f i e d in the t h i o m n - s t a m e d and a p p e a r a n c e (Fig. 4al, in the t i m m - s t a i n e d sec-
Fig. 5. Well-integrated xenograft of mouse C57 dentate tissue located to the medial blade of the host Kyoto fasci a dentata. Donor, E~7 C57 mouse embryo; recipient, newborn Kyoto rat; survival, 2months. In the Timm staining (a) thehost and the xenotransplant granule cells are distinguished by their difference in stainability (interface at arrow), while the distinction in the adjacent thionin-staincd section (b) is more difficult (interface at arrow), a, x 130: b, x 150.
57
Fig. 6. Xenograft of C57 mouse dentate tissue next to the Kyoto host fascia dentata. Donor, E l 7 C57 mouse embryo; recipient, newborn Kyoto rat; survival. 2 months. What appears as a pure xenograft in thionin staining (a), actually contains autografted host CA1 pyramidal cells, as disclosed in the adjacent Timm-stained section (arrowhead, in (b)). A Timm-stained terminal field extends from the autograft into the molecular layer of the xenograft (arrow). ×39.
er, evident. The dentate transplants of Kyoto tissue thus stood out in clear contrast to the host tissue, due to its content of densely stained granule cell bodies (Fig. 4b). Also the transplant hippocampal pyramidal cells displayed the characteristic increased stainability of their cell bodies. In the cases where the transplants at certain levels passed through the dentate and pyramidal cell layers of the host fascia dentata and hippocampus we found areas with complete merging of transplant and host tissue (Fig. 4). In some cases darkly stained cell bodies similar to Kyoto dentate granule cell bodies were found in the Wistar host fascia dentata up to 800 ktm away from the dentate granule cell layers of the transplants (Fig. 4b, c). This suggested that migration of transplant neurons into homologous layers of the host had occurred. This phenomenon was observed both after transplantation of Kyoto dentate tissue to normal and X-irradiated newborn rats and we are presently examining in detail whether it is more extensive in the granule cell deprived X-irradiated recipients than the normal ones.
Xenotransplants.
Surviving xenografts of em-
bryonic C57-mouse hippocampi retained the lack of Timm stainability of their dentate and hippocampal neuronal cell bodies, both in the neonatal and the adult Kyoto rat recipient brains. In cases with very well-integrated transplants like the one shown in Fig. 5, this allowed a distinction to be made between transplant and host parts of the jointly composed fascia dentata (Fig. 5a). This was not possible with the same certainty and precision in any of the other stained series of sections, like the thionin-stained ones (Fig. 5b). Stained cells identical to the host Kyoto cells were sometimes found in what otherwise appeared to be pure xenografts. In some cases the presence of these ceils suggested a migration of Kyoto host cells into the transplants. In others, like the case shown in Fig. 6, it was more likely that the cells were present in host tissue that had been dislocated at the time of transplantation. These cells would not have been identified as host cells without the cell marker (cf. Fig. 6a, b) and the additional connective host-transplant interactions that these cells were involved in would accordingly not have been revealed.
5> I)ISCUSSION The basis for the unique T i m m stainability of the cell bodies, in particular the nuclei, of neurons in the Kvoto rat brain is unknown. The T i m m m e t h o d stains histochemically the so-called heavy metal, i.e., the metals listed in group l i b of the periodic system s2° including zinc. It is so far unknown w h e t h e r any of these metals are present in the Kyoto nerve cell bodies in higher amounts than in other strains of rats or mice, but preliminary experiments with staining of histological sections from K y o t o rats with the zinc-binding fluorescent dye 2-methyl-8-hydroxyquinoline: have not revealed any increased zinc content in the Kyoto granule cell bodies. The stainable metals may instead be present in a form that m a k e them more accessible to the sulphide at the time of perfusion. This would increase the amounts of metallosulphides formed onto which silver subsequently is deposited by the physical d e v e l o p m e n t ~. The functional significance is also unknown so far, as it still is for most metals visualized in the h i p p o c a m p a l region by the T i m m method. This does not, however, invalidate the use of the increased stainability of the K y o t o nerve cell bodies as a cell marker. It is thus not known either what is the functional significance of the difference in the nuclear m o r p h o l o g y b e t w e e n the chick and quail ll, or the difference in D N A plasraids b e t w e e n the M u s caroli and the c o m m o n Mus rmIs(/t,tlus |5 A crucial p r o p e r t y of a cell m a r k e r is specificity and stability. With regard to specificity we have demonstrated that the increased T i m m stainability of the Kyoto nerve cell bodies is strain-specific. The cell body staining of the dentate granule cells and the hipp o c a m p a l p y r a m i d a l cells was not observed in any other of the rat and mouse strains examined. The possibility that the staining later might be found in strains not examined here would not affect the use of
the m a r k e r in transplantations b e t w e e n the presently examined strains. With regard to stability, the staining characteristics of for instance the Kyoto d e n t a t e granule cells did not a p p e a r to change after transplantation and m o r e than two months survival m the brains of Wistar rats. In addition it appears from preliminary tissue culture experiments, using the slice culture technique 24. that the characteristic stainability persists for at least 5 weeks m vm'o in cultures ol h i p p o c a m p a l tissue o b t a i n e d from 7-dav-old rats. Concerning the stability and specificity of the cell staining there is also the possibility that the transplanted Kvoto tissue or the host K y o m brain might be able to induce a Kyoto-like stainability in the nonKvoto host or transplant nerve cells We have no ewdence, however, that such induction occurs. In many cases where host and transplant tissue were in innmate contact, even with exchange of nerve connecnons. the distribution of stained cell bodies, within the one or the other still in most ca~es coincided with what was expected from careful anal'~sis of the adiacent series of thionin-stained sccuons (Figs. 4-61 Seen in relation to these observations it is most likely that the stained cells o b s e r v e d among the unstained host granule cells shown in Fig. 4b. c are cells that have m~grated from the transplam rather than host cells that have had their staining characteristics transformed. The definite p r o o f for neuronal migration will be sought in future studies, where the characteristic stainabilitv of the Kvoto d e n t a t e granule cells is to be c o m b i n e d by a second m a r k e r , like [3H]th3 mldin or fluorochromes. ACKNOWLEDGEMENTS This study was s u p p o r t e d m part by the Danish M R C . the Danish F o u n d a t i o n for the A d v a n c e ot Medical Sciences, and the International Spinal Research Trust. U.K.
ABBREVIATIONS IN THE FIGURES ca (_'At CA3 cx EA f FD g h Ky
commissural-associationalzone hippocampal subfield CA 1, regio superior hippocampal subfield CA3, regio inferior ncocortex entorhinal area fimbria fascia dentata granule cell layer hilus Kyoto
lpp m rnl mpp p Prs Ps Sb l'r Wi
latcral pcrtorant path zone dentate molecular layer mossy fiber layer medial perforant path zone p~ ramidal cell layer presubiculum para~ubiculum sublculunl transplant Wistar
NC
XC llO~I'[t I'l
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