Studies of the behaviour of purified rat astrocytes after transplantation into syngeneic adult brain.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
G Raisman
Publications and source records attributed to G Raisman.
Intracerebral transplantation of cultured neurons after reaggregation in a plasma clot.
In order to be able to transplant neural cells which have been either manipulated in vitro or maintained in culture for the purpose of cell-type enrichment, we have developed a novel plasma clot method which permits reaggregation of previously dissociated cells such that they can be implanted as highly localized transplants rather than as dispersion-prone cell suspension grafts. To establish the method, enzymatically dissociated cells prepared from hippocampal primordia of late embryonic rats were immediately reaggregated into plasma clots and transplanted to the hippocampal formation of adult recipients. By using fluorescein-labelled bovine plasma to form the plasma clot grafts of reaggregated cells, the fate of the plasma clot protein matrix was followed at different post-operative survival times. Initially, 4-5 days post-operative, the plasma clot maintained the grafted cells in a loose sponge-like sack at the site of implantation. After 2-3 weeks, the transplanted cells were more compact and fused with the host neuropil, and the plasma clot matrix had largely been degraded. At 1 month or longer survival, there was no distinguishable boundary between transplant and host, and there was little or no evidence of any remaining plasma clot matrix or proteins. The plasma clot method was subsequently applied to the transplantation of cultures enriched in pyramidal cells. Enrichment for pyramidal cells was achieved by eliminating mitotic cells (dentate granule cells and glia) by brief (200 rad) irradiation of hippocampal primordia followed by dissociation and maintenance in monolayer culture for 4-6 days. Fibres from host dentate granule cells grew into the pyramidal cell-enriched transplants and established mossy fibre terminals on the donor cells. In transplants between embryonic and adult rats, donor cells were identified at long survival times by prelabelling donor cells in utero or in vitro with [3H]thymidine prior to transplantation. In transplants between embryonic and adult mice, donor tissue from A Thy-1.1 strain mice was transplanted to congenic A strain (Thy-1.2) mice such that the donor cells bearing the Thy-1.1 cell surface glycoprotein could be later identified by immunocytochemical staining with antibodies specific for the Thy-1.1 antigen. Reaggregation and transplantation of dissociated cells in a plasma clot thus provides a novel method whereby prior manipulation of neural tissue (separation of neurons and glia, enrichment for specific types of neurons, or glia etc.) can be used to great advantage in studying host-transplant connectivity and in assessing those factors which are critical in sustaining the survival of grafted neural tissue.
Animals↗
Membrane specializations and extracellular material associated with host astrocytes in peripheral neural transplants.
The work of Aguayo and colleagues [Aguayo, David and Bray (1981) J. Exp. Biol. 95, 231-240] demonstrates that grafts of peripheral neural tissue are able to induce regenerative elongation of cut axons in the adult central nervous system. Elucidation of the mechanism of this response requires an understanding of the cellular interactions induced by these types of transplant. In previous studies [Zhou, Lawrence, Morris and Raisman (1986) Neuroscience 17, 815-827; Zhou, Lindsay, Lawrence and Raisman (1986) Neuroscience 17, 803-813] we have transplanted decapsulated adult superior cervical sympathetic ganglia or nodose ganglia into either the septal nuclei or the choroid fissure of adult syngeneic rat hosts. We found that host astrocytes invade the transplants along Schwann cell fascicles and around blood vessels. This raises the questions of what form the migrating astrocytes take, what routes they follow, and what is their fate. In the present study we have taken advantage of the fact that at longer survivals astrocytes accumulate as "paravascular cuffs", and we show that they have several specialized ultrastructural features, such as plasmalemmal caveolae, desmosomes, hemidesmosomes and accumulations of extracellular material. The specific stimuli inducing (or enhancing) these astrocytic specializations and their significance in relation to the wider morphogenetic events induced by peripheral neural transplants remain to be elucidated. However, the observations are further evidence of the remarkable mobility and plasticity of central astrocytes in transplantation situations, and in particular emphasize the involvement of the cell surface and its relationship to extracellular matrix.
Animals↗
Specificity in the reinnervation of adult hippocampus by embryonic hippocampal transplants.
Explore the source record for details and available documents.
Animals↗
Application of tissue culture and cell-marking techniques to the study of neural transplants.
Explore the source record for details and available documents.
Animals↗
Extent of survival and vascularization of adult superior cervical sympathetic or nodose ganglia transplanted into the septal nuclei or choroid fissure of adult rats.
Adult superior cervical sympathetic ganglia were auto-transplanted, and adult nodose ganglia were homografted into the septal nuclei or the choroid fissure of adult Wistar rats. At times from 4 h to 9 weeks after operation, the distribution of surviving transplanted neurons was compared with the development of the transplant vascularization, as visualized by transcardial Indian ink filling of the host vascular system. Within 24 h, the ganglionic neurons and Schwann cells of the interior of the transplants in both sites were necrotic. The surviving neurons and Schwann cells formed a shell, occupying those areas of the transplant periphery which were in direct contact with the host circulation. Occasional ink-filled vessels were evident at this time in transplants in the choroid fissure, but there were none in the septal nuclei, where vessels did not appear until the next day. Blood vessels reached the centre of the ganglia by 3-4 days in the choroid fissure and one week in the septal nuclei, the finest diameter capillaries forming last. At longer survivals there was a slow loss of neurons, notable between 1 and 2 months, and leading progressively (especially in the septal transplantation site) to the disappearance of all but a very small number of ganglionic neurons. The general findings were similar for both types of ganglion, and in both sites, but the initial cell loss was much greater for both types of ganglia in the septum (over 90%) as compared with about a 50% loss in the choroid fissure. The initial rapid cell loss was probably a result of ischaemia. The subsequent, slow progressive loss may be associated with failure to make or receive neuronal connections, or the absence of appropriate growth factors.
Animals↗
Migration of host astrocytes into superior cervical sympathetic ganglia autografted into the septal nuclei or choroid fissure of adult rats.
Adult astrocytes and their processes, identified by glial fibrillary acidic protein immunohistochemistry and by electron microscopy, migrate into superior cervical ganglia auto-transplanted into the choroid fissure or septal nuclei of adult rats. Migration routes were along the blood vessels, and along the Schwann cell bundles of the transplant. Ultrastructurally, astrocytic processes could be seen to lie in direct contact with Schwann cell processes within the basal lamina enwrapping the Schwann cell and its associated axons. Around the region of the host/transplant interface, the astrocytes were transformed into flattened cells with many short, irregular, fringe-like processes, but within the depths of the transplant mass they resumed a more stellate configuration. Glial fibrillary acidic protein immunoreactivity was present within the intrinsic satellite and Schwann cells of the grafted ganglia, but at a much lower level than in the host astrocytes. It is concluded that reactive astrocytes from adult host central nervous system migrate into peripheral ganglionic transplants, where they differentiate and establish organized arrangements with the ganglionic elements.
Animals↗
The density of reinnervation of adult rat superior cervical sympathetic ganglionic neurons is limited by the number of available postsynaptic sites.
The adult rat superior cervical ganglion has about 27,000 neurons and is innervated by about 9000 preganglionic axons which make a total of nearly 11 million synapses. Surgical removal of the upper part of the ganglion, reducing the number of neurons to about 20%, causes an overall reduction of the number of synapses to about 30%, but has no effect on the numbers of preganglionic axons. Thus, a 5-fold increase in the axon/neuron ratio causes an increase of only about 50% in the number of synapses per cell. Axotomy followed by regeneration of the preganglionic axons causes no further increase in the number of synapses per cell, even though the average number of synapses per axon is reduced to about one-quarter of the normal. This suggests that the ganglionic neurons can only accept a limited number of synapses, and that in the normal situation there is only possibility for a relatively minor increase before this limit is reached. This study is complementary to a previous one in which the numbers of preganglionic axons were surgically reduced and it was found that, when allowed to regenerate into an entire denervated ganglion, the remaining axons could not increase their numbers of synapses. Thus, in the normal rat superior cervical sympathetic ganglion the total number of synapses is such that while the preganglionic axons are probably expressing close to their full synaptogenic potential, the ganglionic neurons express only about two-thirds of their ability to receive synapses.
Animals↗
Early stages of Purkinje cell maturation demonstrated by Thy-1 immunohistochemistry on postnatal rat cerebellum.
The cell surface glycoprotein, Thy-1, is present on Purkinje cells at birth, so allowing Thy-1 immunohistochemistry to demonstrate the final stage of migration and the transition to dendritic growth of these cells. In the most caudal lobule of the cerebellar cortex of the newborn rat, migrating Purkinje cells are found. These have a prominent process (up to 50 micron long) from which fine filopodia project, presumably sensing the environment in front of the cell. These cells are orientated tangentially, at right angles to the radial orientation they assume for dendritic growth. Strong Thy-1 labelling is found not only on their surface, but also on a cytoplasmic cap above the presumed leading pole of the nucleus. More rostrally in the cerebellar cortex, Purkinje cells arrive up to 3 days before birth and are quiescent until the postnatal development of their dendritic tree. At birth and during early postnatal periods a rounded cell is found with little cytoplasm; Thy-1 staining labels its surface and the fine processes which emanate from it. Such cells coexist with other Thy-1-positive Purkinje cells with more developed surface orientated processes. Even as early as the day of birth these fine processes cross the molecular layer and contact the lower level of the external granule layer. Orientated dendritic growth appears to occur by a selective thickening of these processes and a massive apical protrusion of intensely Thy-1-positive cytoplasm. The whole of the Purkinje cell surface membrane exhibits high levels of Thy-1 throughout dendritic growth and synaptogenesis, and cytoplasmic antigen is prominent during the period of greatest growth. Thy-1 is also found on the neurons of the deep cerebellar nuclei, and is seen transiently on Golgi interneurons. High levels of the antigen are present on blood vessels and choroid plexus at birth but are lost from these structures over the first 2 postnatal weeks.
Animals↗
Late emergence of Thy-1 on climbing fibres demonstrates a gradient of maturation from the fissures to the folial convexities in developing rat cerebellum.
In the third week of postnatal life, Thy-1 staining of Purkinje cells starts to decrease, first in the depths of the fissures and then progressively, over the next 10 weeks, up the walls of the fissures to the convexities of the folia. This is accompanied by a far more striking appearance of high levels of Thy-1 on a network of fibres whose distribution strongly suggests they are climbing fibres. They acquire the antigen in the same topographical gradient of maturation, and at the same time, as Purkinje cells lose it. That these are climbing fibres was confirmed by destroying the inferior olive with 3-acetylpyridine, which also eliminated the intense Thy-1 staining in the cerebellum. At a stage (18 days) when only some climbing fibres in the molecular layer are seen to be Thy-1-positive, only a proportion of inferior olivary neurons are Thy-1-positive with intense antigen labelling over the Nissl substance. The possibility that Thy-1 also appears on mossy fibres, and at relatively low levels on parallel fibres, is discussed.
Afferent Pathways↗
Specific patterns of fibre outgrowth from transplants to host mice hippocampi, shown immunohistochemically by the use of allelic forms of Thy-1.
Foetal mouse hippocampal primordia from mice homozygous for the Thy-1.1 allele were transplanted into the hippocampal region of adult histocompatible mice, homozygous for the Thy-1.2 allele. After survival periods of two months to one year the transplants consisted of a discrete tissue mass, well fused with the host, and distinguished from it by an intense Thy-1.1 immunoreactivity of the neuropil. The host hippocampus was entirely negative for Thy-1.1 immunoreactivity except for well defined projections arising from the transplant. These projections had three different patterns of distribution in the host: (1) a mossy-fibre-like distribution (to the stratum lucidum of field CA3); (2) a fimbria-like distribution (to the stratum oriens and stratum radiatum of fields CA1 and CA3), and (3) a commissural/association-like distribution (to the inner stratum moleculare of the dentate gyrus). Analysis of the position of the transplants and their component cell types indicated that, for each of the three distribution patterns, three conditions had to be fulfilled: (1) the transplant had to contain the appropriate type of cell (granule cells for mossy fibre projections, and larger pyramid-like cells for the other two); (2) the transplant neurons giving rise to the projection had to lie in direct contact with the host field to be innervated, and (3) the terminal field had to be specifically denervated. Thus, for three fibre systems the patterns of transplant-to-host projections observe rules of organization comparable to those of the normal hippocampal circuitry. This implies that in the adult host brain there remain (or there can be elicited) the molecular cues capable of establishing patterns of neuronal connectivity comparable to those formed during normal development.
Animals↗
Specialized neuroglial arrangement may explain the capacity of vomeronasal axons to reinnervate central neurons.
The neurosensory cells of the primary olfactory and vomeronasal projections are in a state of continuous replacement throughout adult life. Since their axons form synaptic terminals with neurons in the olfactory and accessory olfactory bulbs, this system is an apparent exception to the rule that peripheral axons cannot grow into the central nervous system of adult mammals. Electron microscopy of sections (especially in a plane tangential to the surface of the accessory olfactory bulb) shows a unique glial arrangement. By virtue of their greater electron density and "secretory-type" organelle content (Golgi apparatus and dense-core vesicles) the glial cells of the superficial layers of the accessory olfactory bulb are distinguished both from the glia of the vomeronasal nerves and from the astrocytes of the deeper bulbar layers. The synapses between the vomeronasal axons and the postsynaptic elements are formed in glomeruli which are encapsulated by an inner layer of glial cytoplasm derived from the superficial glia, and an outer layer derived from the astrocytes. The principle of the organization is that the superficial glial processes are reflected off the axons before they reach the synaptic terminal zone. Conversely, for the postsynaptic elements, the astrocytic processes are reflected off the dendrites of the accessory olfactory bulb neurons before they enter the core of the glomeruli. In effect, the synapses are formed in a "no-man's-land" between the two glial cell types. This peculiar glial arrangement may be important for the unique regenerative capacity of this system.
Animals↗
Solitary magnocellular neurons in the homozygous Brattleboro rat have vasopressin and glycopeptide immunoreactivity.
A small but distinctive population (about 1 in 600) of magnocellular neurosecretory neurons in homozygous Brattleboro rats are immunoreactive for vasopressin, and a similar number for the carboxy-terminal glycopeptide of the vasopressin prohormone. These solitary cells are found in all animals and in all parts of the magnocellular system, but not in the suprachiasmatic or other hypothalamic nuclei. The majority of the solitary cells do not differ morphologically from the remainder of the magnocellular neurons. The immunoreactivity is markedly denser in the Nissl bodies than in the Golgi region. Serial sections show that the vasopressin and glycopeptide immunoreactive material is co-localized in the same cells, and that these cells are not immunoreactive for oxytocin. A published sequence for the Brattleboro vasopression gene mutation indicates a base-deletion upstream from the glycopeptide-encoding portion, and implies a frameshift that would cause translation of incorrect protein continuing into the poly-A tail of the mRNA. Although this could apply to the majority of the Brattleboro presumptive vasopressin neurons, the co-localization in our solitary cells of material immunoreactive with antibodies to both the amino- and carboxy-terminals of the vasopressin prohormone suggest that in these cases an additional mechanism may be operating.
Animals↗
Is Thy-1 expressed only by ganglion cells and their axons in the retina and optic nerve?
The distribution of Thy-1 in the retina and optic nerve has been examined immunohistochemically, and compared to that of the astrocytic marker glial fibrillary acidic protein. The axons and cell bodies of ganglion cells were found to be Thy-1 positive as were processes within the inner plexiform layer. Transection of the optic nerve in the neonatal rat results in the rapid degeneration of the ganglion cells but some Thy-1 staining remains in the inner plexiform layer. We have estimated using an immunoassay of normal and optic nerve transected retinae that about 70% of the Thy-1 in the retina is on ganglion cells and their axons and the remainder is on cells which contribute processes to the inner plexiform layer, presumably amacrine, bipolar or Müller cells. In the optic nerve the Thy-1 was found to be limited to the fascicles of optic nerve fibres and the intrafascicular spaces, containing astrocytes and their processes, were not stained. Axotomy of the adult nerve, which produced axonal degeneration and astrocytic proliferation, led to a loss of over 95% of the Thy-1 from the nerve. We found no evidence that the astrocytes of the retina or optic nerve were Thy-1 positive in normal animals or during degeneration.
Animals↗
An autoradiographic study of neuronal development, vascularization and glial cell migration from hippocampal transplants labelled in intermediate explant culture.
Late embryonic and early postnatal rat hippocampal primordia were labelled with [3H]thymidine for varying periods in explant culture before being implanted into hippocampi of adult hosts. The types and distributions of nuclear-labelled cells were determined autoradiographically at 1 month after operation. The labelling of small pyramidal neurons and dentate granule cells was in accordance with their time of normal developmental origin, the dentate granule cells forming distinctive shells with an appropriate gradient of cell accretion. There was a high proportion of labelled glial cells in the transplants, and a massive accumulation around the interface with the host. Labelled glia migrate for up to 2 mm into the host tissue. Labelled endothelial cells occur in vessels in the transplant, in wide-diameter marginal vessels and in two specific types of vascular configuration in the host. Thus the establishment of embryonic hippocampal transplants is associated with a major migration of non-neuronal cells into the host brain, and the formation of specific types of tissue chimaera. This chimaera formation is essential (e.g. in the case of blood vessels) for the survival of the transplant and the routes of glial cell migration may determine the pathways along which transplant nerve fibres can penetrate the host.
Animals↗
Vascular and astrocytic reactions during establishment of hippocampal transplants in adult host brain.
Hippocampal primordia were transplanted into the hippocampus or septum of adult rats, and the development of the transplant vasculature studied by light microscopy after India ink perfusion, by immunohistochemistry of collagen type IV (as a marker for basal lamina), and by electron microscopy. From the basal lamina of all vessel types arise streamers of basal lamina which clothe the external plasmalemmal surfaces of adjacent astrocytic processes and invaginate them, suggesting that the astrocytic processess may be involved in directing the assembly of this basal lamina. A few host blood vessels reach the transplants by one day after operation, and probably re-perfuse existing transplant vessels. Two main types of vascular reaction occur over the next week. The first is the formation of wide-diameter reactive vessels with an expanded perivascular space, engorged with mesodermal cells (such as macrophages). The second is the formation of dilated, thin-walled, marginal vessels, from which numerous fine capillaries arise. The formation of astrocyte-apposed basal laminar streamers may be important in the development of channels along which the endothelial cells of the newly growing capillaries later migrate. By one month after operation, the transplant vasculature consists of small capillaries whose size, distribution and density are comparable to those of the host. This paper describes the vascular changes which occur when embryonic central nervous transplants become established in the brain of adult hosts, and indicates that one of the characteristic features is the formation of basal lamina-lined channels invaginating the perivascular astrocytes.
Animals↗
Relative slowness of heterotypic synaptogenesis in the septal nuclei.
The dorsolateral quadrant of the lateral septal nucleus receives a bilateral projection from the fimbria. When the fimbria of one side is cut, the axons of the remaining fimbria take over its synaptic sites preferentially, but when both fimbrias are cut the sites are reinnervated by non-fimbrial axons. To explore the basis of this preference, the present study plots the time courses of the appearance and disappearance of degenerating synapses, and the loss and recovery of non-degenerating synapses after ipsi-, contra- and bi-lateral fimbrial lesions. A preliminary investigation showed that at any time after these three lesions there was no change in the numerical density per unit area of 'control' structures such as shaft synapses (which do not degenerate) and neuronal perikarya (which neither shrink nor degenerate). This indicates that the changes in the numerical density of fimbrial (spine) synapses can be used as a measure of the processes of deafferentation and reinnervation without the danger of the numerical data being distorted by shrinkage. In the sampled area, the ipsilateral fimbrial axons account for about 45% of the synapses and the contralateral fimbrial axons for 25%. The number of degenerating synapses appearing at any one time underestimates the loss of non-degenerating synapses by about one-third, and a photographic simulation of degeneration suggests that a major factor in this discrepancy is the difficulty in recognizing degenerating synapses. Our main finding is that there is a major delay in the rate of removal of degeneration, and in the rate of reinnervation, after bilateral as opposed to unilateral lesions. This delay cannot be accounted for in any simple way by the greater amounts of degeneration. Thus after unilateral lesions, which cause the turnover of 25% (contralateral) or 45% (ipsilateral) of the synapses, 50% of the degeneration is removed in 1-2 days after the peak, whereas after bilateral lesions, which affect 70% of the synapses, it takes 20 days for 50% of the degeneration to be removed. That the synaptic changes after bilateral lesions involve a qualitatively different mechanism is also suggested by the observations of a much greater proportional increase in the multiple synapse index, and a decreased astroglial response.
Afferent Pathways↗
Formation of mossy fibre connections between hippocampal transplants and the brain of adult host rats.
Hippocampal primordia can survive transplantation into adult rat hippocampi. The transplants differentiate and form mossy fibre connections with host pyramidal cells, while mossy fibre projections from the host can innervate transplants.
Animals↗