Journal of Neuroscience Methods, 31 (1990) 65-73
65
Elsevier
N S M 01027
Post-fixation horseradish peroxidase tracing in rat fetus and pups C. v a n der T o g t * and H . K . P . F e i r a b e n d Department of Physiologd,, UnwersiO, of Leiden, Faculty of Medicine, Leiden (The Netherlands) (Received 25 April 1989) (Revised version received 15 August 1989) (Accepted 28 August 1989)
Kev words: Postmortem H R P tracing; Fixation effects; Development; Vestibulo-cerebellar connections Horseradish peroxidase (HRP) has been shown to be applicable to fixed tissue in tracing projections in adult animals. This technique has several advantages in studying short projections in difficult attainable brain areas, especially with fetuses in utero. We have used this method to trace developing fibers (e.g., vestibulo-cerebellar connections) in rat fetuses and pups. It has been possible to make discrete and precise injections in any location. The quality of labeling by this method is distinctly related to the grade of fixation. Fixation grade determines how well H R P is anchored inside fibers and cell bodies. In optimally fixed preparations fibers and cell bodies are intensively stained with H R P up to over 3 nun from the center of an injection site. H R P is probably only taken up by damaged fibers so that the amount of fibers labeled is determined by the extent of the lesion caused by the injection pipette.
Introduction
An important objective of m a n y neuroscience studies is the tracing of developing fiber systems. Studying fiber systems in very early stages of development presents a number of serious problems, one of them being the exceptional difficulty in applying neuronal tracers to embryos in vivo. Recently, horseradish peroxidase (HRP) has been shown to label fibers in fixed tissue (Kageyama and Meyer, 1986, 1987). There are several advantages in using neuronal tracers in this manner. After fixation, preparations can be handled much more easily, because the survival of the animal is no longer an overriding criterion. Furthermore, the brain can be removed from the animal giving
* Present address: The Netherlands Ophthalmic Research Institute, Department of Morphology, P.O. Box 12141, 1100 AC Amsterdam, The Netherlands. Correspondence: Dr. H.K.P. Feirabend, Dept. of Physiology, University of Leiden, Faculty of Medicine, Wassenaarseweg 62. P.O. Box 9604, 2300 RC Leiden, The Netherlands.
direct access to the injection locus through identification of surface landmarks. This makes the precision of an injection much greater, and even a stereotaxic approach possible. An important restriction to this technique has been the limited distance labeled fibers can be followed, usually only a few millimeters. In embryos and fetuses, however, neuronal structures are still very close to each other so that one can expect the distances over which fibers project to be short. These considerations make the technique of post-fixation H R P tracing ideal for use in embryos and fetuses. The present study describes the use of this method to trace olivoand vestibulo-cerebellar connections in rat fetuses and pups ranging in age from embryonic day 17 to postnatal day 5.
Materials and methods
Rat fetuses and neonatal rats were obtained from a colony bred in this institute. The fetuses were removed from the uterus and perfused
0165-0270/90/$03.50 ~c, 1990 Elsevier Science Publishers B.V. (Biomedical Division)
66 t h r o u g h the heart. A p p r o x i m a t e l y 70 fetuses a n d 15 p u p s were used in this study.
Fixation of tissue Perfusion consisted of a flush of saline until the h e a d of the fetus a p p e a r e d clear of b l o o d , foll o w e d b y a p p r o x . 20 ml fixative, a m i x t u r e of 1% p a r a f o r m a l d e h y d e a n d 1.25% g l u t a r a l d e h y d e in 0.1 M p h o s p h a t e buffer, p H 7.3 at r o o m t e m p e r a ture. The h e a d o f the e m b r y o was then r e m o v e d a n d its b r a i n e x p o s e d or dissected out, a n d imm e r s e d in the s a m e fixative for 0 - 3 6 h. As an a l t e r n a t i v e we have also used 4% p a r a f o r m a l d e h y d e in 0.1 M p h o s p h a t e b u f f e r b u t this d i d n o t give s a t i s f a c t o r y results. U s i n g the first m e n t i o n e d fixative with p h o s p h a t e - b u f f e r e d saline i n s t e a d of p h o s p h a t e buffer, u n e x p e c t e d l y , also gave inferior results.
Application of HRP and storage of tissue A f t e r fixation the b r a i n s were rinsed in 0.1 M p h o s p h a t e buffer, p H 7.3, 4 ° C and left for ]2-3f, h in this buffer. 30% H R P in distilled water was recrystallized on to the tips of glass m i c r o p i p e t t e s ( H R P type VI, S i g m a P-8375). T h e p i p e t t e tips were then inserted into the desired aret~ of the b r a i n using a m i c r o m a n i p u l a t o r under microscopic visual control. Care was taken not to touch the b r a i n surface with the p i p e t t e tip toc~ long. As the b r a i n surface is wet, H R P on the tip dissolves r a p i d l y on c o n t a c t even before the tip is inserted. So if the tip is not quickly inserted a large a r e a of brain surface is c o n t a m i n a t e d with H R P . Surface c o n t a m i n a t i o n of H R P resulted in an extensive a n d diffuse b a c k g r o u n d labeling of the tissue imm e d i a t e l y u n d e r the surface without H R P being taken up in fibers or cell bodies.
Fig. 1. Sagittal section of an El9 rat fetus medulla oblongata and cerebellum (lower part). The injection has been made from the ventral side in the pontine area. The injection site is approx. 1 mm in diameter. Discrete fiber bundles (arrow-heads) can be seen arising from the injection site opposite to a small laceration caused by the injection pipette. Caudally labelled fibers (arrows) can be followed around the flexura cervicalis up to a distance of approx. 3.5 mm. Bar = 0.5 ram.
67 small, closed plastic or glass c o n t a i n e r s either immersed in p h o s p h a t e buffer or on p h o s p h a t e buffer-soaked pieces of tissue paper. These were stored in a refrigerator at 4 ° C.
!~ ~ i!!iii~'!!ii! ~
Histological procedures
Fig. 2. An enlargement of the box in Fig. 1. It illustrates the intense labeling of fibers and the complete absence of diffused HRP in the background. Bar = 100/~m. Mixing the H R P with D M S O or N o n i d e t did n o t e n h a n c e H R P u p t a k e a n d transport. Following application of H R P the b r a i n s were placed in
' I
~
After 1 - 1 2 days the b r a i n s were i m m e r s e d in 10-20% s u c r o s e / 0 . 1 M p h o s p h a t e buffer. After 4 - 6 h, they were placed in 12% g e l a t i n / 0 . 1 M p h o s p h a t e buffer a n d left for several hours or overnight in a 3 7 ° C i n c u b a t o r . T h e b r a i n s were then e m b e d d e d in gelatin blocks, which were stored in 4% p a r a f o r m a l d e h y d e for 3 . 5 - 4 h. Sagittal or transverse 40-/~m sections were cut on a freezing m i c r o t o m e a n d stored in 0.1 M p h o s p h a t e buffer. Sections were stained with the metal intensified D A B p r o c e d u r e as described by A d a m s (1981), m o u n t e d on c h r o m e - a l u m slides, air-dried, alternately c o u n t e r s t a i n e d with cresyl violet, dehydrated in a s c e n d i n g series of ethanol, cleared in xylene, a n d coverslipped with P e r m o u n t .
_~
s
•
Fig. 3. Transversal section through the medulla oblongata of an E20 rat fetus. In the upper part obex and cerebellum are seen. The injection site is approx. 0.8 mm caudal to this section. A fiber system projecting rostrally has been heavily stained. Two smaller projections are seen arising from this system to the ipsilateral and contralateral side. Bar = 0.5 ram.
68
Results General observations The size of the injection site usually ranged from 1-2 m m in diameter with a small laceration or hole in the center caused by the insertion of the glass pipette. The laceration varied from 0.1 to 0.5 m m in diameter. Around the injection site the intraceUular diffusion of H R P diminishes promptly giving a clear view of stained fibers and cell bodies (Figs. 1, 2). Fibers and cells were uniformly filled, but the intensity of staining weakened with distance from the injection site. In some cases an outstanding morphological staining of neurons and fiber projections was obtained (Figs. 3, 4). Retrograde and anterograde transport was identified by the fact that both cell bodies and growth cones were observed millimeters from the injection site. In exceptional cases stained fibers could be followed up to 3.5 m m from the center of an injection (Fig. 1), but usually transport was restricted to 1.5 and 2.5 mm. Retrogradely stained cells were sometimes found up to 3 m m from the center of an injection site. If reasonable uptake and transport of H R P had taken place, discrete fiber bundles could be seen arising from the injection site in several directions. An interesting observation is that the width of
Fig. 5. Transversal section through cerebellum and medulla oblongata of a 5-day-old postnatal rat. The injection was made medially and dorsally through the cerebellum. Note the small injection site that was realized (0.5 mm in diameter}. Bar = 0.5 mm.
these bundles was always proportional to the width of the laceration m a d e by the glass pipette rather than the extent of the injection site (Figs. 1, 5, 6). This indicates that H R P is preferably, if not exclusively, taken up by damaged fibers. This is in accordance with the fact that in areas where H R P was mistakenly deposited, as with surface contamination where there is no fiber damage, only a general diffusion and no uptake of H R P in fibers was found. Fig. 4. An enlargement of the ipsilateral projection in the box in Fig. 3. A small group of cell bodies has been retrogradely stained via very fine fibers. These cells can be well characterized and distinguished from the background, Bar =100 p,m.
Vestibulo-cerebellar and cerebello-vestibular .fiber connections With this technique it has been possible to make precise injections in the vestibular nuclei and the cerebellar cortex. After injections in the
69
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II
Fig. 6. In an enlargement of Fig. 5 a small fiber bundle can be seen directly in apposition to a small rupture caused by the pipette Bar = 100/zm.
vestibular nuclei at E21 (Fig. 7), retrograde labeling was found in the Purkinje cells, which at this stage are still organized in sagittal clusters (Fig. 7, arrows). In addition, labeling was also found in the cells of the central cerebellar nuclei (Fig. 7, asterisk). Injections in the cerebellar cortex at P5 led to retrograde filling of vestibular cells. These observations in the developing cerebellum are in accordance with the adult sagittal organization of the afferent and efferent fiber connections (the concept of cerebellar modules) as described by Voogd and co-workers (Voogd and Bigarr, 1980; Feirabend and Voogd, 1986). The results indicate that precise tracing of developing fiber systems involved in the formation of the sagittal cerebeUar organization has been made possible by the postfixation technique.
Effect of fixation The severity and type of fixation of tissue arc possibly the most critical factors in this procedure. Alternative fixatives to a mixture of 1.25% glutaraldehyde and 1.0% paraformaldehyde in phosphate buffer gave unsatisfactory results. These phenomena are not well understood and require further study. Kageyama and Meyer (1987) have described the use of considerable long post-fixation periods in adult tissue. We have alternatively found that the duration of the immersion- or post-fixation following the perfusion may have a deleterious effect on uptake and transport of HRP. Naturally one must take into account the differences in maturation of adult versus embryonic tissue and the amount of tissue used. After the perfusion, which resulted in a very light fixation in
70
Fig. 7. Transversal section s h o w i n g one side of the c e r e b e l l u m a n d c o r p u s restiforme of an E21 rat fetus. ~\ large injecti~m ,,~as made in the v e s t i b u l a r area of the m e d u l l a o b l o n g a t a . There are projections in all d i r e c t i o n s a n d a very obvious one e n d i n g in the nuclear area (asterisk). A r r o w s i n d i c a t e labeled Purkinje cell clusters. Bar = 0.25 ram. Inset: e n l a r g e m e n t of a field of r e t r o g r a d e i y labeled Purkinje cells. N o t e the rather blurred a p p e a r a n c e of the cells due to diffusion of H R P across the cell m e m b r a n e as a result o{' the m i n i m a l posl-fixation.
our series, we immersed the preparations in fixative over various periods of time extending from 0 to 2 days. Series in which the post-fixation was prolonged up to 1.5-2 days usually showed no uptake and transport. In these cases H R P was dispersed diffusely around the injection site. When the post-fixation was approx. 12 h (overnight for E21 fetuses) our results were similar to those reported by Kageyama and Meyer (1987). In these cases intensely stained fiber bundles could be seen (Fig. 8). Cell bodies were also stained, but tended to be found in the neighborhood of the injection site as illustrated in Fig. 3. A salient feature in these series is that well-stained fibers
and cell bodies were restricted to the central parts of the preparation, and that in the periphery fiber bundles had a blurred appearance with H R P diffusely around them. Series in which the post-fixation was omitted showed exceptionally good labeling of cells and fibers in the periphery as well as in the center. Surprisingly, the amount of retrograde labelled cells seemed greater than in post-fixed preparations (Fig. 9). We also had the impression that the distance H R P was transported away from the injection site was greater than in post-fixed preparations. These results are comparable to those obtained by Haber (1988) and Beach and McGeer (1988). In both studies H R P tracing had been
71 In cases where the post-fixation was very light or omitted, fibers and cells were well stained with storage periods less than 24 h. When the storage period was extended up to three or more days fiber and cell labeling attenuates until retrogradely filled cell bodies are barely distinguishable. This indicates that H R P is gradually lost, possibly by diffusion, from fibers and cells. In mild to well fixed preparations (12 h immersion post-fixation for an E21 embryo) this phenomenon is not seen, even with storage times of 4 - 1 4 days, apparently because H R P is tightly bound inside axons preventing diffusion over the membrane. Less clear differences have been noted when comparing different ways of storing preparations: (1) immersed or (2) placed in a container on paper tissue soaked in phosphate buffer. Storing the preparations in the latter method gave very satisfactory results. It seemed that extracellular diffusion was less pronounced than after immersion storage.
Discussion k;
\ }
Fig. 8. Preparation showing intensely stained fibers 12 h after fixation of E21 rat fetus. Note the complete absence of retrogradely stained cell bodies. Bar = 100 ~m.
done on postmortem material that had not been prefixed. Their illustrations show that although fibers appear less intensely stained, there is an extensive labeling of retrogradely filled cells.
Storage of tissue The effects of variations in tissue storage after H R P injection have not been extensively studied. In storing the preparations in dark and at 4 ° C we have simply followed Kageyama and Meyer's (1987) protocol. On the other hand, we have noted clear differences in the intensity of labeling, although not in the distance labeled fibers and cell bodies could be found away from the injection site, when comparing the outcome of variations in storage time.
How H R P is transported in fixed tissue remains unclear. The most acceptable idea is that this is by passive diffusion. However, the differences caused by fixation raise new questions about the mechanism of labeling in this procedure. Kageyama and Meyer (1987) proposed that in their preparations HRP, diffusing through an axon, binds to fixed intracellular structures. If this is so, then it could well be that HRP is impeded in reaching the cell body or endings because it is bound on its way. On the other hand~ in cases where the fixation is much lighter (e.g., no postfixation) so that intracellular structures are less radically altered, we propose that HRP is not anchored to intracellular structures, and therefore can reach cell bodies and endings more effectively. Indeed, one may even expect that in lightly fixed preparations labelling of cell bodies will be obtained over greater distances as Haber (1988) has reported. However, as H R P is less tightly bound inside cells and fibers of lightly fixed tissue this may explain the loss of HRP from fibers and cell
72
Fig. 9. Illustration showing a large group of retrogradely stained cell bodies. Fibers are barely stained in this preparation which was post-fixed for less than 1 h. Otherwise both preparations in Figs. 8 and 9 are comparable. They were taken from the same area and the storage time in both cases was approx. 1 week. Bar = 100 ~tm.
bodies seen after longer storage times ( > 3 days). Better fixed preparations will result in more intensely stained fibers and less retrogradely-filled cell bodies since HRP is bound and restricted inside axons. Loss of H R P is less possible, even over long periods (14 days) of storage, because of the greater affinity of H R P to fixed intracelhilar structures. In preparations fixed for prolonged periods of time (1.5-2 days), no uptake and labeling of fibers occurred. There even seemed to be no preferential binding of H R P to cell membranes, as can be seen in less well fixed preparations. This indicates that above a certain degree of fixation binding sites for H R P are lost. Although labeling of neurons is attained over
far greater distances with in vivo H R P tracing, use of the above described method has many advantages. There can be no loss of embryos or fetuses as a result of a surgical intervention or the injection of the tracer. A great advantage is the possibility of direct viewing of the brain making precise injections attainable. In comparison with H R P tracing in in vitro tissue slabs there are also advantages. Although a high injection precision is possible much tissue must be cut away in preparing the slab thus excluding efferent and afferent pathways from analysis. The degree of fixation is obviously very critical and the optimal fixation period can not be precisely predicted, since it will depend on the age and size of the preparation used.
73
This technique may be an ideal way to study short projections in the prenatal period of development of any part of brain.
References Adams, J.C. (1981) Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem., 29: 6. Beach, T.G. and McGeer, E.G. (1987) Tract tracing with HRP in postmortem human brain. Neurosci. Lett., 76: 37-41. Beach, T.G. and McGeer, E.G. (1988) Retrograde filling of pyramidal neurons in postmortem human cerebral cortex using HRP. J. Neurosci. Meth., 23: 187-193. Feirabend, H.K.P. and Voogd, J. (1986) Myeloarchitecture of the cerebellum of the chicken (Gallus domesticus): an atlas of the compartmental subdivision of the cerebellar white matter. J. Comp. Neurol., 251: 44-66.
Haber, S.N. and Eskin, T. (1986) HRP fiber labeling in the basal ganglia: a postmortem study. Soc. Neurosci. Abstr., 12: 1540. Haber, S.N. (1988) Tracing intrinsic fiber connections in postmortem human brain with WGA-HRP. J. Neurosci. Meth., 23: 15-22. Kageyama, G.H. and Meyer, R.L. (1986) Dense filling of neurons and glia cells and their processes with HRP applied to pre-fixed tissue. Soc. Neurosci. Abstr., 12: 1566. Kageyama, G.H. and Meyer, R.L. (1987) Dense HRP filling in prefixed brain tissue for light and electron microscopy. J. Histochem. Cytochem., 35: 10. Voogd, J. and F. Bigar6 (1980) Topographical distribution of olivary and corticonuclear fibers in the cerebellum: a review. In: J. Courville and Y. Lamarre (Eds.), The Inferior Olivary Nucleus, Anatomy and Physiology, Raven Press, New York, pp. 207-234.
65
Elsevier
N S M 01027
Post-fixation horseradish peroxidase tracing in rat fetus and pups C. v a n der T o g t * and H . K . P . F e i r a b e n d Department of Physiologd,, UnwersiO, of Leiden, Faculty of Medicine, Leiden (The Netherlands) (Received 25 April 1989) (Revised version received 15 August 1989) (Accepted 28 August 1989)
Kev words: Postmortem H R P tracing; Fixation effects; Development; Vestibulo-cerebellar connections Horseradish peroxidase (HRP) has been shown to be applicable to fixed tissue in tracing projections in adult animals. This technique has several advantages in studying short projections in difficult attainable brain areas, especially with fetuses in utero. We have used this method to trace developing fibers (e.g., vestibulo-cerebellar connections) in rat fetuses and pups. It has been possible to make discrete and precise injections in any location. The quality of labeling by this method is distinctly related to the grade of fixation. Fixation grade determines how well H R P is anchored inside fibers and cell bodies. In optimally fixed preparations fibers and cell bodies are intensively stained with H R P up to over 3 nun from the center of an injection site. H R P is probably only taken up by damaged fibers so that the amount of fibers labeled is determined by the extent of the lesion caused by the injection pipette.
Introduction
An important objective of m a n y neuroscience studies is the tracing of developing fiber systems. Studying fiber systems in very early stages of development presents a number of serious problems, one of them being the exceptional difficulty in applying neuronal tracers to embryos in vivo. Recently, horseradish peroxidase (HRP) has been shown to label fibers in fixed tissue (Kageyama and Meyer, 1986, 1987). There are several advantages in using neuronal tracers in this manner. After fixation, preparations can be handled much more easily, because the survival of the animal is no longer an overriding criterion. Furthermore, the brain can be removed from the animal giving
* Present address: The Netherlands Ophthalmic Research Institute, Department of Morphology, P.O. Box 12141, 1100 AC Amsterdam, The Netherlands. Correspondence: Dr. H.K.P. Feirabend, Dept. of Physiology, University of Leiden, Faculty of Medicine, Wassenaarseweg 62. P.O. Box 9604, 2300 RC Leiden, The Netherlands.
direct access to the injection locus through identification of surface landmarks. This makes the precision of an injection much greater, and even a stereotaxic approach possible. An important restriction to this technique has been the limited distance labeled fibers can be followed, usually only a few millimeters. In embryos and fetuses, however, neuronal structures are still very close to each other so that one can expect the distances over which fibers project to be short. These considerations make the technique of post-fixation H R P tracing ideal for use in embryos and fetuses. The present study describes the use of this method to trace olivoand vestibulo-cerebellar connections in rat fetuses and pups ranging in age from embryonic day 17 to postnatal day 5.
Materials and methods
Rat fetuses and neonatal rats were obtained from a colony bred in this institute. The fetuses were removed from the uterus and perfused
0165-0270/90/$03.50 ~c, 1990 Elsevier Science Publishers B.V. (Biomedical Division)
66 t h r o u g h the heart. A p p r o x i m a t e l y 70 fetuses a n d 15 p u p s were used in this study.
Fixation of tissue Perfusion consisted of a flush of saline until the h e a d of the fetus a p p e a r e d clear of b l o o d , foll o w e d b y a p p r o x . 20 ml fixative, a m i x t u r e of 1% p a r a f o r m a l d e h y d e a n d 1.25% g l u t a r a l d e h y d e in 0.1 M p h o s p h a t e buffer, p H 7.3 at r o o m t e m p e r a ture. The h e a d o f the e m b r y o was then r e m o v e d a n d its b r a i n e x p o s e d or dissected out, a n d imm e r s e d in the s a m e fixative for 0 - 3 6 h. As an a l t e r n a t i v e we have also used 4% p a r a f o r m a l d e h y d e in 0.1 M p h o s p h a t e b u f f e r b u t this d i d n o t give s a t i s f a c t o r y results. U s i n g the first m e n t i o n e d fixative with p h o s p h a t e - b u f f e r e d saline i n s t e a d of p h o s p h a t e buffer, u n e x p e c t e d l y , also gave inferior results.
Application of HRP and storage of tissue A f t e r fixation the b r a i n s were rinsed in 0.1 M p h o s p h a t e buffer, p H 7.3, 4 ° C and left for ]2-3f, h in this buffer. 30% H R P in distilled water was recrystallized on to the tips of glass m i c r o p i p e t t e s ( H R P type VI, S i g m a P-8375). T h e p i p e t t e tips were then inserted into the desired aret~ of the b r a i n using a m i c r o m a n i p u l a t o r under microscopic visual control. Care was taken not to touch the b r a i n surface with the p i p e t t e tip toc~ long. As the b r a i n surface is wet, H R P on the tip dissolves r a p i d l y on c o n t a c t even before the tip is inserted. So if the tip is not quickly inserted a large a r e a of brain surface is c o n t a m i n a t e d with H R P . Surface c o n t a m i n a t i o n of H R P resulted in an extensive a n d diffuse b a c k g r o u n d labeling of the tissue imm e d i a t e l y u n d e r the surface without H R P being taken up in fibers or cell bodies.
Fig. 1. Sagittal section of an El9 rat fetus medulla oblongata and cerebellum (lower part). The injection has been made from the ventral side in the pontine area. The injection site is approx. 1 mm in diameter. Discrete fiber bundles (arrow-heads) can be seen arising from the injection site opposite to a small laceration caused by the injection pipette. Caudally labelled fibers (arrows) can be followed around the flexura cervicalis up to a distance of approx. 3.5 mm. Bar = 0.5 ram.
67 small, closed plastic or glass c o n t a i n e r s either immersed in p h o s p h a t e buffer or on p h o s p h a t e buffer-soaked pieces of tissue paper. These were stored in a refrigerator at 4 ° C.
!~ ~ i!!iii~'!!ii! ~
Histological procedures
Fig. 2. An enlargement of the box in Fig. 1. It illustrates the intense labeling of fibers and the complete absence of diffused HRP in the background. Bar = 100/~m. Mixing the H R P with D M S O or N o n i d e t did n o t e n h a n c e H R P u p t a k e a n d transport. Following application of H R P the b r a i n s were placed in
' I
~
After 1 - 1 2 days the b r a i n s were i m m e r s e d in 10-20% s u c r o s e / 0 . 1 M p h o s p h a t e buffer. After 4 - 6 h, they were placed in 12% g e l a t i n / 0 . 1 M p h o s p h a t e buffer a n d left for several hours or overnight in a 3 7 ° C i n c u b a t o r . T h e b r a i n s were then e m b e d d e d in gelatin blocks, which were stored in 4% p a r a f o r m a l d e h y d e for 3 . 5 - 4 h. Sagittal or transverse 40-/~m sections were cut on a freezing m i c r o t o m e a n d stored in 0.1 M p h o s p h a t e buffer. Sections were stained with the metal intensified D A B p r o c e d u r e as described by A d a m s (1981), m o u n t e d on c h r o m e - a l u m slides, air-dried, alternately c o u n t e r s t a i n e d with cresyl violet, dehydrated in a s c e n d i n g series of ethanol, cleared in xylene, a n d coverslipped with P e r m o u n t .
_~
s
•
Fig. 3. Transversal section through the medulla oblongata of an E20 rat fetus. In the upper part obex and cerebellum are seen. The injection site is approx. 0.8 mm caudal to this section. A fiber system projecting rostrally has been heavily stained. Two smaller projections are seen arising from this system to the ipsilateral and contralateral side. Bar = 0.5 ram.
68
Results General observations The size of the injection site usually ranged from 1-2 m m in diameter with a small laceration or hole in the center caused by the insertion of the glass pipette. The laceration varied from 0.1 to 0.5 m m in diameter. Around the injection site the intraceUular diffusion of H R P diminishes promptly giving a clear view of stained fibers and cell bodies (Figs. 1, 2). Fibers and cells were uniformly filled, but the intensity of staining weakened with distance from the injection site. In some cases an outstanding morphological staining of neurons and fiber projections was obtained (Figs. 3, 4). Retrograde and anterograde transport was identified by the fact that both cell bodies and growth cones were observed millimeters from the injection site. In exceptional cases stained fibers could be followed up to 3.5 m m from the center of an injection (Fig. 1), but usually transport was restricted to 1.5 and 2.5 mm. Retrogradely stained cells were sometimes found up to 3 m m from the center of an injection site. If reasonable uptake and transport of H R P had taken place, discrete fiber bundles could be seen arising from the injection site in several directions. An interesting observation is that the width of
Fig. 5. Transversal section through cerebellum and medulla oblongata of a 5-day-old postnatal rat. The injection was made medially and dorsally through the cerebellum. Note the small injection site that was realized (0.5 mm in diameter}. Bar = 0.5 mm.
these bundles was always proportional to the width of the laceration m a d e by the glass pipette rather than the extent of the injection site (Figs. 1, 5, 6). This indicates that H R P is preferably, if not exclusively, taken up by damaged fibers. This is in accordance with the fact that in areas where H R P was mistakenly deposited, as with surface contamination where there is no fiber damage, only a general diffusion and no uptake of H R P in fibers was found. Fig. 4. An enlargement of the ipsilateral projection in the box in Fig. 3. A small group of cell bodies has been retrogradely stained via very fine fibers. These cells can be well characterized and distinguished from the background, Bar =100 p,m.
Vestibulo-cerebellar and cerebello-vestibular .fiber connections With this technique it has been possible to make precise injections in the vestibular nuclei and the cerebellar cortex. After injections in the
69
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Fig. 6. In an enlargement of Fig. 5 a small fiber bundle can be seen directly in apposition to a small rupture caused by the pipette Bar = 100/zm.
vestibular nuclei at E21 (Fig. 7), retrograde labeling was found in the Purkinje cells, which at this stage are still organized in sagittal clusters (Fig. 7, arrows). In addition, labeling was also found in the cells of the central cerebellar nuclei (Fig. 7, asterisk). Injections in the cerebellar cortex at P5 led to retrograde filling of vestibular cells. These observations in the developing cerebellum are in accordance with the adult sagittal organization of the afferent and efferent fiber connections (the concept of cerebellar modules) as described by Voogd and co-workers (Voogd and Bigarr, 1980; Feirabend and Voogd, 1986). The results indicate that precise tracing of developing fiber systems involved in the formation of the sagittal cerebeUar organization has been made possible by the postfixation technique.
Effect of fixation The severity and type of fixation of tissue arc possibly the most critical factors in this procedure. Alternative fixatives to a mixture of 1.25% glutaraldehyde and 1.0% paraformaldehyde in phosphate buffer gave unsatisfactory results. These phenomena are not well understood and require further study. Kageyama and Meyer (1987) have described the use of considerable long post-fixation periods in adult tissue. We have alternatively found that the duration of the immersion- or post-fixation following the perfusion may have a deleterious effect on uptake and transport of HRP. Naturally one must take into account the differences in maturation of adult versus embryonic tissue and the amount of tissue used. After the perfusion, which resulted in a very light fixation in
70
Fig. 7. Transversal section s h o w i n g one side of the c e r e b e l l u m a n d c o r p u s restiforme of an E21 rat fetus. ~\ large injecti~m ,,~as made in the v e s t i b u l a r area of the m e d u l l a o b l o n g a t a . There are projections in all d i r e c t i o n s a n d a very obvious one e n d i n g in the nuclear area (asterisk). A r r o w s i n d i c a t e labeled Purkinje cell clusters. Bar = 0.25 ram. Inset: e n l a r g e m e n t of a field of r e t r o g r a d e i y labeled Purkinje cells. N o t e the rather blurred a p p e a r a n c e of the cells due to diffusion of H R P across the cell m e m b r a n e as a result o{' the m i n i m a l posl-fixation.
our series, we immersed the preparations in fixative over various periods of time extending from 0 to 2 days. Series in which the post-fixation was prolonged up to 1.5-2 days usually showed no uptake and transport. In these cases H R P was dispersed diffusely around the injection site. When the post-fixation was approx. 12 h (overnight for E21 fetuses) our results were similar to those reported by Kageyama and Meyer (1987). In these cases intensely stained fiber bundles could be seen (Fig. 8). Cell bodies were also stained, but tended to be found in the neighborhood of the injection site as illustrated in Fig. 3. A salient feature in these series is that well-stained fibers
and cell bodies were restricted to the central parts of the preparation, and that in the periphery fiber bundles had a blurred appearance with H R P diffusely around them. Series in which the post-fixation was omitted showed exceptionally good labeling of cells and fibers in the periphery as well as in the center. Surprisingly, the amount of retrograde labelled cells seemed greater than in post-fixed preparations (Fig. 9). We also had the impression that the distance H R P was transported away from the injection site was greater than in post-fixed preparations. These results are comparable to those obtained by Haber (1988) and Beach and McGeer (1988). In both studies H R P tracing had been
71 In cases where the post-fixation was very light or omitted, fibers and cells were well stained with storage periods less than 24 h. When the storage period was extended up to three or more days fiber and cell labeling attenuates until retrogradely filled cell bodies are barely distinguishable. This indicates that H R P is gradually lost, possibly by diffusion, from fibers and cells. In mild to well fixed preparations (12 h immersion post-fixation for an E21 embryo) this phenomenon is not seen, even with storage times of 4 - 1 4 days, apparently because H R P is tightly bound inside axons preventing diffusion over the membrane. Less clear differences have been noted when comparing different ways of storing preparations: (1) immersed or (2) placed in a container on paper tissue soaked in phosphate buffer. Storing the preparations in the latter method gave very satisfactory results. It seemed that extracellular diffusion was less pronounced than after immersion storage.
Discussion k;
\ }
Fig. 8. Preparation showing intensely stained fibers 12 h after fixation of E21 rat fetus. Note the complete absence of retrogradely stained cell bodies. Bar = 100 ~m.
done on postmortem material that had not been prefixed. Their illustrations show that although fibers appear less intensely stained, there is an extensive labeling of retrogradely filled cells.
Storage of tissue The effects of variations in tissue storage after H R P injection have not been extensively studied. In storing the preparations in dark and at 4 ° C we have simply followed Kageyama and Meyer's (1987) protocol. On the other hand, we have noted clear differences in the intensity of labeling, although not in the distance labeled fibers and cell bodies could be found away from the injection site, when comparing the outcome of variations in storage time.
How H R P is transported in fixed tissue remains unclear. The most acceptable idea is that this is by passive diffusion. However, the differences caused by fixation raise new questions about the mechanism of labeling in this procedure. Kageyama and Meyer (1987) proposed that in their preparations HRP, diffusing through an axon, binds to fixed intracellular structures. If this is so, then it could well be that HRP is impeded in reaching the cell body or endings because it is bound on its way. On the other hand~ in cases where the fixation is much lighter (e.g., no postfixation) so that intracellular structures are less radically altered, we propose that HRP is not anchored to intracellular structures, and therefore can reach cell bodies and endings more effectively. Indeed, one may even expect that in lightly fixed preparations labelling of cell bodies will be obtained over greater distances as Haber (1988) has reported. However, as H R P is less tightly bound inside cells and fibers of lightly fixed tissue this may explain the loss of HRP from fibers and cell
72
Fig. 9. Illustration showing a large group of retrogradely stained cell bodies. Fibers are barely stained in this preparation which was post-fixed for less than 1 h. Otherwise both preparations in Figs. 8 and 9 are comparable. They were taken from the same area and the storage time in both cases was approx. 1 week. Bar = 100 ~tm.
bodies seen after longer storage times ( > 3 days). Better fixed preparations will result in more intensely stained fibers and less retrogradely-filled cell bodies since HRP is bound and restricted inside axons. Loss of H R P is less possible, even over long periods (14 days) of storage, because of the greater affinity of H R P to fixed intracelhilar structures. In preparations fixed for prolonged periods of time (1.5-2 days), no uptake and labeling of fibers occurred. There even seemed to be no preferential binding of H R P to cell membranes, as can be seen in less well fixed preparations. This indicates that above a certain degree of fixation binding sites for H R P are lost. Although labeling of neurons is attained over
far greater distances with in vivo H R P tracing, use of the above described method has many advantages. There can be no loss of embryos or fetuses as a result of a surgical intervention or the injection of the tracer. A great advantage is the possibility of direct viewing of the brain making precise injections attainable. In comparison with H R P tracing in in vitro tissue slabs there are also advantages. Although a high injection precision is possible much tissue must be cut away in preparing the slab thus excluding efferent and afferent pathways from analysis. The degree of fixation is obviously very critical and the optimal fixation period can not be precisely predicted, since it will depend on the age and size of the preparation used.
73
This technique may be an ideal way to study short projections in the prenatal period of development of any part of brain.
References Adams, J.C. (1981) Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem., 29: 6. Beach, T.G. and McGeer, E.G. (1987) Tract tracing with HRP in postmortem human brain. Neurosci. Lett., 76: 37-41. Beach, T.G. and McGeer, E.G. (1988) Retrograde filling of pyramidal neurons in postmortem human cerebral cortex using HRP. J. Neurosci. Meth., 23: 187-193. Feirabend, H.K.P. and Voogd, J. (1986) Myeloarchitecture of the cerebellum of the chicken (Gallus domesticus): an atlas of the compartmental subdivision of the cerebellar white matter. J. Comp. Neurol., 251: 44-66.
Haber, S.N. and Eskin, T. (1986) HRP fiber labeling in the basal ganglia: a postmortem study. Soc. Neurosci. Abstr., 12: 1540. Haber, S.N. (1988) Tracing intrinsic fiber connections in postmortem human brain with WGA-HRP. J. Neurosci. Meth., 23: 15-22. Kageyama, G.H. and Meyer, R.L. (1986) Dense filling of neurons and glia cells and their processes with HRP applied to pre-fixed tissue. Soc. Neurosci. Abstr., 12: 1566. Kageyama, G.H. and Meyer, R.L. (1987) Dense HRP filling in prefixed brain tissue for light and electron microscopy. J. Histochem. Cytochem., 35: 10. Voogd, J. and F. Bigar6 (1980) Topographical distribution of olivary and corticonuclear fibers in the cerebellum: a review. In: J. Courville and Y. Lamarre (Eds.), The Inferior Olivary Nucleus, Anatomy and Physiology, Raven Press, New York, pp. 207-234.
