Brain Research, 167 (1979) 391-395 © Elsevier/North-Holland Biomedical Press
391
Ultrastructural evidence of synaptogenesis in the adult rat dentate gyrus from brain stem implants
B. K. BEEBE; K. MOLLGARD, A. BJORKLUND and U. STENEVI
Department of Histology, University of Lund (Sweden) and Department of Anatomy A, University of Copenhagen (Denmark) (Accepted January 18th, 1979)
Implants of embryonic brain tissue cultured on the vessel-rich pia of the choroidal fissure in adult rats have been shown to mature and differentiate into a wellorganized 'microbrain' structure, which becomes incorporated into the blood and cerebrospinal fluid circulation of the host brainS,~, 11. Such intracerebral implants have light microscopically been shown to form extensive axonal networks in areas of the host brain. In particular, implants of brain stem or septal tissue, placed adjacent to the hippocampal formation, have been found to reestablish monoaminergic or cholinergic terminal patterns in large areas of the previously denervated hippocampus and dentate gyrus 2-4. The laminar distribution of the ingrowing axons is highly specific, suggesting that they are guided to ramify preferentially in the denervated zones. This raises the question as to whether the axons which grow in from the implant have a synaptic mode of termination or whether they end as free terminal arborizations. In the present paper we report some initial findings of an electron microscopical study which provide evidence for the formation of new synaptic contacts in the dentate gyrus by ingrowing axons from brain stem implants. Mid-sagittal slices (approx. 0.5 x 1 × 2 mm in size) were taken from portsmedulla of rat fetuses with a crown-rump length of 15-20 mm (about 16-17 days of gestation). The pieces were placed into a cavity in the occipital cortex of adult recipient rats in contact with the vessel-rich pia overlying the superior colliculus as described previouslylL Female Sprague-Dawley rats, 180-200 g in weight, were used. The normal serotoninergic innervation of the hippocampus had been removed by an injection of 5,7-dihydroxytryptamine (150 #g intraventricularly) 2-3 weeks before transplantation. After a survival time of 1.5-3 months, when the serotoninergic neurons of the implant are known to have formed extensive fiber networks in the dentate gyrus 4, the skull was once again opened and the implant gently sucked out under visual control in the dissection microscope. Only fully successful implants of large size, which could be easily identified in the dissection microscope, were used. Care was taken to avoid any damage to the host brain tissue. The animals were perfused for electron microscopy 2 days after the second operation. Controls consisted of (a) the contralateral, unoperated side of the above specimens; (b) specimens bearing
392 identical brain stem implants which were not removed and (c) an anim:d ~n which, first, a transplantation cavity was made but no implant inserted. Two months later the skull was opened again and a large part of the rostral and lateral margins ~including a significant portion of the hippocampus and dentate gyrus) was removed by suction. This animal was also perfused two days after the second operation. The brains were fixed by perfusion with 2.5'~',i, glutaraldehyde and I" Formaldehyde in cacodylate buffer, pH 7.4. They were postfixed in osmium tetroxide and block stained with uranyl acetate. The dorsal hippocampus, including the dentate gyrus, located rostral to the implant was taken for analysis. Separate animals bearing identical implants were taken for fluorescence histochemistry according to the Falck-Hillarp method I. These latter animals were pretreated with the MAO-inhibitor nialamide (300 mg/kg 3-5 h before killing) in order to improve serotonm wsualization. In agreement with previous findings 4, fluorescence histochemistry of the dentate gyrus revealed growth of serotonm fibers primarily into the outer half of the molecular layer and into the subgranular zone of the hilus. A major route of the serotonin axons was along the hippocampal fissure. Thus, the serotoninergic axons from the implant grew preferentially into the terminal zone of the perforant path fibers, which were lesioned as a consequence of the preparation of the implantation cavity.
Fig. I.
393
Figs. 1 and 2. Electron micrographs from the outer half of the molecular layer of the dentate gyrus showing degenerating boutons in synaptic contact with simple dendritic spines (asterisks). One terminal is also in contact with a dendritic shaft (2D). Note that all degenerating profiles form assymetric synaptic junctions. Some large empty and dense core synaptic vesicles (indicated by triangles) and several small pleomorphic clear vesicles are still visible. Swollen elements of smooth ER are often present (large profile in 2A and arrowhead in 2C). Bars indicate 0.5/~m; Figs. 2 A - D are of same magnification.
394 Fiber degeneration was observed in the ipsilateral dentate gyrus both in the toluidine blue-stained 1 a m survey sections and in the thin sections from the animals in which the implant had been removed 2 days before the perfusion. Fiber degeneration was inconspicuous on the contralateral side and m animals where the implant had been left intact. Consistent with Nafstad's 10 observations, degenerating fibers were seen as small darkly-stained dots in the toluidine blue-stained sections. They were distributed along the hippocampal fissure and in the molecular and subgranular zones of the dentate, The highest density of degeneration was along the fissure in the stratum lacunosum-moleculare of CA I. in the outer half of the dentate molecular layer and in the subgranular zone, thus conforming to the distribution of the ingrowing serotonm axons as seen in the fluorescence microscope. In the thin sections taken from the dorsal blade of the dentate both myelinated and non-myelinated axons were found to undergo dark degeneration. Consistent with the observations from the toluidine blue-stained sections the degenerating axons were found in significant numbers along the hippocampat fissure, in the dentate molecular layer (particularly in the outer zone) and within and just below the granule cell layer. Degenerating synaptic boutons were frequent. Virtually all degenerating terminals formed asymmetric Gray's type I synaptic j unctions. Degenerating symmetric (Gray's type H) synapses were not identified with certainty and only a minor portion of the degenerating terminals were not engaged in the formation of synaptic contacts. The majority of the degenerating boutons were found in synaptic contact with simple dendritic spines (Figs. 1 and 2) but a fair proportion contacted shafts or branches of small and large dendrites (Fig. 2D). They contained numerous pleomorphic synaptic vesicles and both large agranular vesicles as well as dense core vesicles were regularly encountered at a distance from the presynaptic density (triangles in Fig. 2 A, B and C). Degenerating profiles with more than one synaptic junction were frequently observed (Fig. 2A and D). The length of the postsynaptic thickenings was about the double of what has been described in the entorhinallesioned reinnervated outer zone of the molecular layer of the dentate gyrus ~. Although when removing the implant, care was taken not to damage adjacent brain tissue it seemed nevertheless possible that the second operation in these rats could cause some secondary, e.g. vascular or edematic, trauma responsible for the observed axonal degeneration. For this reason an implantation cavity was made in one rat. without inserting any implant into the cavity. Two months later the cavity was opened and a significant portion of the remaining hippocampus and dentate gyrus was sucked out. This rat showed, as expected, both axonal and cellular degeneration in the remaining part of the dorsal hippocampus and dentate gyrus. However. in contrast to the animals in which the implant had been removed, the resulting fiber degeneration was both less abundant and had a diffuse distribution. Thus it seems safe to conclude that the bulk of the axons degenerating in the implanted rats indeed originate in the implant. The adult rat dentate gyrus has previously been shown to be capable of forming new synaptic contacts through collateral sprouting or reactive synaptogenesis after removal of the entorhinal or commissu ral afferents 6-s. The protracted time-course of
395 the reactive synaptogenesis6, 7 implies that the f o r m a t i o n o f synapses by the ingrowing i m p l a n t axons in the dentate m o l e c u l a r layer, which was denervated o f its e n t o r h i n a l afferents by the t r a n s p l a n t a t i o n lesion, occurs c o n c o m i t a n t with the terminal proliferation o f the r e m a i n i n g intact afferents. The present study shows that axons growing into the denervated dentate f r o m a distance do form synapses with n o r m a l ultrastructural characteristics, thus successfully c o m p e t i n g with the intact afferents a l r e a d y present in the area in reinnervating the denervated terminal zone. This indicates that p r o x i m i t y to the d e n e r v a t e d target (cf. ref. 12) is n o t the sole factor d e t e r m i n i n g which fiber system will reinnervate a denervated region. N o r does the occurrence o f extensive collateral s p r o u t i n g or reactive synaptogenesis necessarily prevent distant axons from reinnervating a d e n e r v a t e d b r a i n region. In conclusion, the present findings are the first to d e m o n s t r a t e that neural t r a n s p l a n t s can f o r m synaptic connections with the host brain, indicating that i n t r a c e r e b r a l implants m a y indeed form functional contacts with the m a t u r e n e u r o n s o f the host central nervous tissue. The study was s u p p o r t e d by a grant f r o m the Swedish M R C (04X-3874). W e t h a n k Mr. O. N e e r g a a r d for skillful p h o t o g r a p h i c assistance.
1 Bj6rklund, A., Falck, B. and Owman, Ch., Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In J. E. Rail and 1. Kopin (Eds.), Methods. of Investigative and Diagnostic Endocrinology, Vol. 1, North-Holland, Amsterdam, 1972, pp. 318-368. 2 Bj6rklund, A., Segal, M. and Stenevi, U., Functional reinnervation of rat hippocampus by locus coeruleus implants, Brain Research, (1979) in press. 3 Bj6rklund, A. and Stenevi, U., Reformation of the severed septohippecampal cholinergic pathway in the adult rat by transplanted septal neurons, Cell and Tiss. Res., 185 (1977) 289-302. 4 Bj6rklund, A., Stenevi, U. and Svendgaard, N.-A., Growth of transplanted moneaminergic neurones into the adult hippocampus along the perforant path, Nature (Lond.), 262 (1976) 787-790. 5 Kromer, L. F., Bj6rklund, A. and Stenevi, U., Intracephalic implants - - a technique for studying neuronal interactions, Science, (1979) in press. 6 Lee, K. S., Stanford, E. J., Cotman, C. W. and Lynch, G. S., Ultrastructural evidence for bouton proliferation in the partially deafferented dentate gyrus of the adult rat, Exp. Brain Res., 29 (1977) 475-485. 7 Matthews, D. A., Cotman, C. and Lunch, G., An electron microscopic study of lesion-induced synaptogenesis in the dentate gyrus of the adult rat. II. Reappearance of morphologically nermal synaptic contacts, Brain Research, 115, (1976) 23-41. 8 McWilliams, R. and Lynch, G., Terminal proliferation and synaptogenesis following partial deafferentation: The reinnervation of the inner molecular layer of the dentate gyrus following removal of its commissural afferents, J. Comp. Neurol., 180 (1978) 581 616. 9 Mollg',Ttrd,K., Lundberg, J. J., Beebe, B. K., Bj6rklund, A. and Stenevi, U., The intracerebralty cultured 'microbrain': A new tool in developmental neurobiology, Neurosci. Lett., 8 (1978), 295 301. 10 Nafstad, P. H. J., An electron microscope study on the termination of the perforant path fibres in the hippocampus and the fascia dentata, Z. Zellforsch., 76 (1967) 532-542. 11 Stenevi, U., Bj6rklund, A. and Svendgaard, N.-Aa., Transplantation of central and peripheral monoamine neurons to the adult rat brain : techniques and conditions for survival, Brain Research, 114 (1976) 1-20. 12 Zimmer, J., Proximity as a factor in the regulation of aberrant axonal growth in postnatally deafferented fascia dentata, Brain Research, 72 (1974) 137 142.
391
Ultrastructural evidence of synaptogenesis in the adult rat dentate gyrus from brain stem implants
B. K. BEEBE; K. MOLLGARD, A. BJORKLUND and U. STENEVI
Department of Histology, University of Lund (Sweden) and Department of Anatomy A, University of Copenhagen (Denmark) (Accepted January 18th, 1979)
Implants of embryonic brain tissue cultured on the vessel-rich pia of the choroidal fissure in adult rats have been shown to mature and differentiate into a wellorganized 'microbrain' structure, which becomes incorporated into the blood and cerebrospinal fluid circulation of the host brainS,~, 11. Such intracerebral implants have light microscopically been shown to form extensive axonal networks in areas of the host brain. In particular, implants of brain stem or septal tissue, placed adjacent to the hippocampal formation, have been found to reestablish monoaminergic or cholinergic terminal patterns in large areas of the previously denervated hippocampus and dentate gyrus 2-4. The laminar distribution of the ingrowing axons is highly specific, suggesting that they are guided to ramify preferentially in the denervated zones. This raises the question as to whether the axons which grow in from the implant have a synaptic mode of termination or whether they end as free terminal arborizations. In the present paper we report some initial findings of an electron microscopical study which provide evidence for the formation of new synaptic contacts in the dentate gyrus by ingrowing axons from brain stem implants. Mid-sagittal slices (approx. 0.5 x 1 × 2 mm in size) were taken from portsmedulla of rat fetuses with a crown-rump length of 15-20 mm (about 16-17 days of gestation). The pieces were placed into a cavity in the occipital cortex of adult recipient rats in contact with the vessel-rich pia overlying the superior colliculus as described previouslylL Female Sprague-Dawley rats, 180-200 g in weight, were used. The normal serotoninergic innervation of the hippocampus had been removed by an injection of 5,7-dihydroxytryptamine (150 #g intraventricularly) 2-3 weeks before transplantation. After a survival time of 1.5-3 months, when the serotoninergic neurons of the implant are known to have formed extensive fiber networks in the dentate gyrus 4, the skull was once again opened and the implant gently sucked out under visual control in the dissection microscope. Only fully successful implants of large size, which could be easily identified in the dissection microscope, were used. Care was taken to avoid any damage to the host brain tissue. The animals were perfused for electron microscopy 2 days after the second operation. Controls consisted of (a) the contralateral, unoperated side of the above specimens; (b) specimens bearing
392 identical brain stem implants which were not removed and (c) an anim:d ~n which, first, a transplantation cavity was made but no implant inserted. Two months later the skull was opened again and a large part of the rostral and lateral margins ~including a significant portion of the hippocampus and dentate gyrus) was removed by suction. This animal was also perfused two days after the second operation. The brains were fixed by perfusion with 2.5'~',i, glutaraldehyde and I" Formaldehyde in cacodylate buffer, pH 7.4. They were postfixed in osmium tetroxide and block stained with uranyl acetate. The dorsal hippocampus, including the dentate gyrus, located rostral to the implant was taken for analysis. Separate animals bearing identical implants were taken for fluorescence histochemistry according to the Falck-Hillarp method I. These latter animals were pretreated with the MAO-inhibitor nialamide (300 mg/kg 3-5 h before killing) in order to improve serotonm wsualization. In agreement with previous findings 4, fluorescence histochemistry of the dentate gyrus revealed growth of serotonm fibers primarily into the outer half of the molecular layer and into the subgranular zone of the hilus. A major route of the serotonin axons was along the hippocampal fissure. Thus, the serotoninergic axons from the implant grew preferentially into the terminal zone of the perforant path fibers, which were lesioned as a consequence of the preparation of the implantation cavity.
Fig. I.
393
Figs. 1 and 2. Electron micrographs from the outer half of the molecular layer of the dentate gyrus showing degenerating boutons in synaptic contact with simple dendritic spines (asterisks). One terminal is also in contact with a dendritic shaft (2D). Note that all degenerating profiles form assymetric synaptic junctions. Some large empty and dense core synaptic vesicles (indicated by triangles) and several small pleomorphic clear vesicles are still visible. Swollen elements of smooth ER are often present (large profile in 2A and arrowhead in 2C). Bars indicate 0.5/~m; Figs. 2 A - D are of same magnification.
394 Fiber degeneration was observed in the ipsilateral dentate gyrus both in the toluidine blue-stained 1 a m survey sections and in the thin sections from the animals in which the implant had been removed 2 days before the perfusion. Fiber degeneration was inconspicuous on the contralateral side and m animals where the implant had been left intact. Consistent with Nafstad's 10 observations, degenerating fibers were seen as small darkly-stained dots in the toluidine blue-stained sections. They were distributed along the hippocampal fissure and in the molecular and subgranular zones of the dentate, The highest density of degeneration was along the fissure in the stratum lacunosum-moleculare of CA I. in the outer half of the dentate molecular layer and in the subgranular zone, thus conforming to the distribution of the ingrowing serotonm axons as seen in the fluorescence microscope. In the thin sections taken from the dorsal blade of the dentate both myelinated and non-myelinated axons were found to undergo dark degeneration. Consistent with the observations from the toluidine blue-stained sections the degenerating axons were found in significant numbers along the hippocampat fissure, in the dentate molecular layer (particularly in the outer zone) and within and just below the granule cell layer. Degenerating synaptic boutons were frequent. Virtually all degenerating terminals formed asymmetric Gray's type I synaptic j unctions. Degenerating symmetric (Gray's type H) synapses were not identified with certainty and only a minor portion of the degenerating terminals were not engaged in the formation of synaptic contacts. The majority of the degenerating boutons were found in synaptic contact with simple dendritic spines (Figs. 1 and 2) but a fair proportion contacted shafts or branches of small and large dendrites (Fig. 2D). They contained numerous pleomorphic synaptic vesicles and both large agranular vesicles as well as dense core vesicles were regularly encountered at a distance from the presynaptic density (triangles in Fig. 2 A, B and C). Degenerating profiles with more than one synaptic junction were frequently observed (Fig. 2A and D). The length of the postsynaptic thickenings was about the double of what has been described in the entorhinallesioned reinnervated outer zone of the molecular layer of the dentate gyrus ~. Although when removing the implant, care was taken not to damage adjacent brain tissue it seemed nevertheless possible that the second operation in these rats could cause some secondary, e.g. vascular or edematic, trauma responsible for the observed axonal degeneration. For this reason an implantation cavity was made in one rat. without inserting any implant into the cavity. Two months later the cavity was opened and a significant portion of the remaining hippocampus and dentate gyrus was sucked out. This rat showed, as expected, both axonal and cellular degeneration in the remaining part of the dorsal hippocampus and dentate gyrus. However. in contrast to the animals in which the implant had been removed, the resulting fiber degeneration was both less abundant and had a diffuse distribution. Thus it seems safe to conclude that the bulk of the axons degenerating in the implanted rats indeed originate in the implant. The adult rat dentate gyrus has previously been shown to be capable of forming new synaptic contacts through collateral sprouting or reactive synaptogenesis after removal of the entorhinal or commissu ral afferents 6-s. The protracted time-course of
395 the reactive synaptogenesis6, 7 implies that the f o r m a t i o n o f synapses by the ingrowing i m p l a n t axons in the dentate m o l e c u l a r layer, which was denervated o f its e n t o r h i n a l afferents by the t r a n s p l a n t a t i o n lesion, occurs c o n c o m i t a n t with the terminal proliferation o f the r e m a i n i n g intact afferents. The present study shows that axons growing into the denervated dentate f r o m a distance do form synapses with n o r m a l ultrastructural characteristics, thus successfully c o m p e t i n g with the intact afferents a l r e a d y present in the area in reinnervating the denervated terminal zone. This indicates that p r o x i m i t y to the d e n e r v a t e d target (cf. ref. 12) is n o t the sole factor d e t e r m i n i n g which fiber system will reinnervate a denervated region. N o r does the occurrence o f extensive collateral s p r o u t i n g or reactive synaptogenesis necessarily prevent distant axons from reinnervating a d e n e r v a t e d b r a i n region. In conclusion, the present findings are the first to d e m o n s t r a t e that neural t r a n s p l a n t s can f o r m synaptic connections with the host brain, indicating that i n t r a c e r e b r a l implants m a y indeed form functional contacts with the m a t u r e n e u r o n s o f the host central nervous tissue. The study was s u p p o r t e d by a grant f r o m the Swedish M R C (04X-3874). W e t h a n k Mr. O. N e e r g a a r d for skillful p h o t o g r a p h i c assistance.
1 Bj6rklund, A., Falck, B. and Owman, Ch., Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In J. E. Rail and 1. Kopin (Eds.), Methods. of Investigative and Diagnostic Endocrinology, Vol. 1, North-Holland, Amsterdam, 1972, pp. 318-368. 2 Bj6rklund, A., Segal, M. and Stenevi, U., Functional reinnervation of rat hippocampus by locus coeruleus implants, Brain Research, (1979) in press. 3 Bj6rklund, A. and Stenevi, U., Reformation of the severed septohippecampal cholinergic pathway in the adult rat by transplanted septal neurons, Cell and Tiss. Res., 185 (1977) 289-302. 4 Bj6rklund, A., Stenevi, U. and Svendgaard, N.-A., Growth of transplanted moneaminergic neurones into the adult hippocampus along the perforant path, Nature (Lond.), 262 (1976) 787-790. 5 Kromer, L. F., Bj6rklund, A. and Stenevi, U., Intracephalic implants - - a technique for studying neuronal interactions, Science, (1979) in press. 6 Lee, K. S., Stanford, E. J., Cotman, C. W. and Lynch, G. S., Ultrastructural evidence for bouton proliferation in the partially deafferented dentate gyrus of the adult rat, Exp. Brain Res., 29 (1977) 475-485. 7 Matthews, D. A., Cotman, C. and Lunch, G., An electron microscopic study of lesion-induced synaptogenesis in the dentate gyrus of the adult rat. II. Reappearance of morphologically nermal synaptic contacts, Brain Research, 115, (1976) 23-41. 8 McWilliams, R. and Lynch, G., Terminal proliferation and synaptogenesis following partial deafferentation: The reinnervation of the inner molecular layer of the dentate gyrus following removal of its commissural afferents, J. Comp. Neurol., 180 (1978) 581 616. 9 Mollg',Ttrd,K., Lundberg, J. J., Beebe, B. K., Bj6rklund, A. and Stenevi, U., The intracerebralty cultured 'microbrain': A new tool in developmental neurobiology, Neurosci. Lett., 8 (1978), 295 301. 10 Nafstad, P. H. J., An electron microscope study on the termination of the perforant path fibres in the hippocampus and the fascia dentata, Z. Zellforsch., 76 (1967) 532-542. 11 Stenevi, U., Bj6rklund, A. and Svendgaard, N.-Aa., Transplantation of central and peripheral monoamine neurons to the adult rat brain : techniques and conditions for survival, Brain Research, 114 (1976) 1-20. 12 Zimmer, J., Proximity as a factor in the regulation of aberrant axonal growth in postnatally deafferented fascia dentata, Brain Research, 72 (1974) 137 142.
