Death of oligodendrocytes and microglial phagocytosis of myelin precede immigration of Schwann cells into the spinal cord.

Small, circumscribed electrolytic lesions were made in the upper cervical corticospinal tract in adult rats. In the centre of the lesion, the axons and all other tissue elements were totally destroyed. Surrounding this region of destruction is an area of tissue which is only partially damaged. In this area TUNEL positive staining of contiguous rows of tract glial cells indicates massive oligodendrocytic apoptosis at 1-3 days after operation, but axons, astrocytes and blood vessels survive. From around 4 days, the corticospinal axons in this area are demyelinated, and the microglia contain ingested myelin, identified in electron micrographs as characteristic MBP immunoreactive laminar cytoplasmic bodies. After around 3 weeks, large numbers of Schwann cells, continuous with those on the pial surface of the spinal cord, accumulate along the lesion track and selectively infiltrate the perilesional reactive area, where they mingle intimately with the phagocytic microglia. Electron micrographs show that at this time basal lamina-enclosed Schwann cell processes establish non-myelinated ensheathment of axons. From around 4 weeks after operation, prominent Schwann cell myelination is indicated by P0 immunoreactivity, and peripheral type, one-to-one myelination in electron micrographs. Thus the effect of the selective loss of oligodendrocytes is to first activate microglia, and then to induce a replacement of myelin by Schwann cells.

Regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells.

Precisely localized focal stereotaxic electrolytic lesions were made in the corticospinal tract at the level of the first to second cervical segments in the adult rat. This consistently destroyed all central nervous tissue elements (axons, astrocytes, oligodendrocytes, microglia, and microvessels) in a highly circumscribed area. In a group of these rats immediately after lesioning, a suspension of cultured adult olfactory ensheathing cells was transplanted into the lesion site. Within the first week after transplantation, the cut corticospinal axons (identified by anterograde transport of biotin dextran) extended caudally along the axis of the corticospinal tract as single, fine, minimally branched sprouts that ended in a simple tip, often preceded by a small varicosity. By 3 weeks, the regenerating axons, ensheathed by P0-positive peripheral myelin had accumulated into parallel bundles, which now extended across the full length of the lesioned area and reentered the caudal part of the host corticospinal tract. The transplants contained two main types of cells: (1) p75-expressing S cells, which later formed typical peripheral one-to-one myelin sheaths around individual ensheathed axons, and (2) fibronectin-expressing A cells, which aggregated into tubular sheaths enclosing bundles of myelinated axons. The point of reentry of the axons into the central nervous territory of the caudal host corticospinal tract was marked by the resumption of oligodendrocytic myelination. Thus the effect of the transplant was to form a "patch" of peripheral-type tissue across which the cut central axons regenerated and then continued to grow along their original central pathway.

Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells.

The upper cervical corticospinal tract was transected on one side in adult rats. A suspension of ensheathing cells cultured from adult rat olfactory bulb was injected into the lesion site. This induced unbranched, elongative growth of the cut corticospinal axons. The axons grew through the transplant and continued to regenerate into the denervated caudal host tract. Rats with complete transections and no transplanted cells did not use the forepaw on the lesioned side for directed reaching. Rats in which the transplanted cells had formed a continuous bridge across the lesion exhibited directed forepaw reaching on the lesioned side.

Embryonic tissue induces growth of adult axons from myelinated fiber tracts.

Suspensions of late embryonic hippocampal tissue were microinjected so as to be completely enclosed within the myelinated fiber bundles of the adult rat fimbria. Previous studies have shown that the axons from such transplanted neurons readily cross the graft/host interface and extend rapidly through the host fiber tract. The present study shows that the adult axons from the host fiber tract can also cross this interface in the opposite direction and enter the transplants. Biotin dextran tracing shows that the adult host fimbrial axons traverse the embryonic grafts and also form terminal arborizations within the transplants. Electron microscopy of orthograde electron-dense degeneration confirms that these host axons form synaptic terminals accounting for at least 6.6% of the synapses in the neuropil of the transplant. Thus, contact with embryonic nervous tissue can induce elongative growth by the adult fibers in a myelinated central tract.

Endogenous synaptogenesis in the deafferented dentate gyrus does not exclude synapse formation by embryonic entorhinal transplants.

After partial deafferentation postsynaptic sites are reinnervated by local sprouting of remaining axons. We have investigated whether this process is sufficient to prevent new synapses being formed by transplanted embryonic tissue. We find that after unilateral entorhinal ablation endogenous sprouting by local axons is unable to reinnervate all the postsynaptic sites in the denervated outer dentate molecular layer. Axons from embryonic entorhinal tissue transplanted adjacent to the denervated area are able to reclaim a further proportion of the denervated postsynaptic sites. Thus, after a large lesion, endogenous sprouting is insufficient to preclude reinnervation by axons from embryonic transplants.

Regeneration of cut adult axons fails even in the presence of continuous aligned glial pathways.

The present study tests whether lesions small enough to allow the rapid reestablishment of a normally aligned tract glial framework would provide a permissive environment for the regeneration of cut adult CNS axons. We made penetrating microlesions which cut a narrow beam of axons in the adult rat cingulum, but caused minimal damage to the tract glial framework and no cavitation. The proximal tips of cut axons were identified by enhanced immunoreactivity for low affinity neurotrophin receptor, p75. From 1 day they became expanded into large growth-cone-like structures. At later times some axons turned back and extended in the reverse direction. Up to 14 days (after which time p75 could no longer be used as a marker), no axons advanced beyond the line of the lesion. From 1 to 2 days, OX42 immunostaining and electron microscopy showed that the lesion site was densely infiltrated by macrophages, which disappeared by 3 to 4 days. This was followed by a local hypertrophy of the OX42 immunoreactive resident tract microglial cells and an increase in both GFAP and vimentin immunoreactivity of the tract astrocytes. These responses were greatly reduced by 8 days, when the longitudinal alignment of glial processes across the lesion site was similar to that of an undamaged tract. The large growth-cone-like structures formed at the ends of the cut axons resemble those of developing axons exposed to chemorepulsive factors. This suggests that cellular elements in adult tract lesions may also exert chemorepulsive influences blocking regeneration of axons even in an apparently "open" tract framework.

Connectional specification of regenerating entorhinal projection neuron classes cannot be overridden by altered target availability in postnatal organotypic slice co-culture.

Layer II neurons of the entorhinal cortex project to the dentate gyrus and field CA3 and also send collaterals to the subiculum; layer III neurons project to the subiculum and field CA1, but not to the dentate gyrus; and layer IV neurons project to the perirhinal cortex. We have previously shown that these specific differences between the projections of the layer II and III neurons are maintained and can regenerate in organotypic slice culture. In the present experiments we have confronted Postnatal Day 7 (P7) rat entorhinal cortex with P7 tissue selected from restricted parts of the overall entorhinal projection field. (1) When entorhinal slices were co-cultured with target slices containing only dentate gyrus, extracellular uptake of biotin dextran from crystals placed on the dentate gyrus retrogradely labeled neurons in layer II, and those of layer III were not labeled. (2) When entorhinal slices were co-cultured with target slices that contained only the subiculum and the hippocampal field CA1 (but not dentate gyrus), neurons in both layers II and III of the entorhinal area were labeled. (3) When entorhinal slices were co-cultured with target slices containing the perirhinal area and no hippocampal or dentate tissue, the neurons of entorhinal layer IV were labeled, but (4) when co-cultured with control target slices taken from the rostral parietal neocortex, no entorhinal neurons were labeled. Thus the exclusive relationship of layer II entorhinal neurons to the dentate gyrus has already been established by 1 week of age and is maintained by the regenerating entorhinal axons. Layer III entorhinal neurons cannot be induced to project to the dentate gyrus even when deprived of their own target, and layer IV neurons are specified to project to the perirhinal area and will not project to any part of the hippocampal complex or to the rostral parietal cortex. Thus, deprivation of the normal target tissue and presentation of an incorrect target tissue (even when it is the correct target for one of the other classes of entorhinal neurons) are not sufficient to override the specificity of the entorhinal projection neurons.

Conditionally immortalized neural progenitor cell lines integrate and differentiate after grafting to the adult rat striatum. A combined autoradiographic and electron microscopic study.

Neural progenitor cell lines, generated by conditional immortalization from the embryonic CNS, have previously been shown to survive and integrate after transplantation to the adult brain. The present study was designed to investigate the in vivo differentiation and morphological features of grafted neural progenitors using combined autoradiography and transmission electron microscopy of two temperature-sensitive neural progenitor cell lines, HiB5 and ST14A, labeled with 3H-thymidine prior to grafting. Two weeks after transplantation to the striatum the cells were found dispersed over an area extending about 1.5 mm from the injection site. Labeled cells located within the myelinated fiber bundles of the internal capsule were closely associated with myelinated axons and presented profiles similar to oligodendrocytes, while most of the grafted cells in the grey matter had morphological features of astroglia. Some labeled cells occurred also in close association with small blood vessels, morphologically resembling host pericytes. The results show that the immortalized neural progenitors can differentiate into mature glial cells, including astrocytes, oligodendrocytes and pericytes, after implantation into the adult striatum. The ability of the cells to become fully integrated with the resident glial population suggests that they will be highly useful as vehicles for intracerebral transgene expression in ex vivo gene transfer.

Failure of axon regeneration in postnatal rat entorhinohippocampal slice coculture is due to maturation of the axon, not that of the pathway or target.

Horizontal slices which included the entorhinal area in continuity with the hippocampus were taken from the ventral levels of the cerebral hemispheres of rat pups from two age groups, from the 6th to the 8th postnatal days ('young') and the 12th to the 15th days ('old'). The slices were divided into an entorhinal part and a hippocampal part (which consisted of the hippocampus proper, dentate gyrus and subiculum) by a knife cut passing through the deep white matter of the entorhinal area. The slices were recombined in their normal orientation by matching the cut edges in the following age combinations: young/young, old/old, young/old and old/young. After 14 days in culture, crystals of biocytin were placed on the superficial layers of the entorhinal area. In the young/young combination the same placement of biocytin simultaneously labelled projections passing in both directions across the interface, i.e. (i) orthograde transport of biocytin taken up by entorhinal projection neurons resulted in labelling of axons passing from the entorhinal area across the interface between the cocultures to reach the correct terminal zone in the outer molecular layer of the dentate gyrus, and (ii) retrograde transport of biocytin taken up by axons and their terminals in the entorhinal area labelled the slender subicular and adjacent hippocampal field CA1 pyramidal cells whose axons project to the entorhinal area. In the old/old cocultures there were no projections in either direction. In the mixed age combinations, young entorhinal cortical tissue projected correctly across the interface to old dentate gyrus, but old entorhinal tissue did not project to young dentate gyrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Axons regenerate with correct specificity in horizontal slice culture of the postnatal rat entorhino-hippocampal system.

We have used slice culture of the entorhino-hippocampal system to investigate (1) whether nerve fibres which are cut postnatally are able to regenerate and (2) whether the regenerating fibres are able to establish correct selective target specificity in the formation of their terminal fields. Slices of tissue were taken in the horizontal plane through the caudo-ventral pole of the cerebral hemisphere of 9- to 10-day-old rats. Such slices maintain the entorhinal cortex in continuity with the hippocampus and intervening retrohippocampal areas. However, because of the dorsal inclination of the entorhino-hippocampal projection fibres in situ, the segments of the entorhinal cortex and hippocampus contained within each individual horizontal slice were disconnected from each other. During subsequent culture, the formation of fibre connections between the entorhinal area and the hippocampal complex was studied by the extracellular and intracellular anterograde transport of biocytin or biotin dextran, the retrograde transport of biotin dextran or carbocyanine dyes, and by electrical stimulation and recording. For the first 24 h after taking the slice, there were no entorhinal projections beyond the deep white matter, and no fibres reached the hippocampus or dentate gyrus. After 3 days in culture a small number of growing fibres had perforated the subiculum and entered the target areas. Between 6 and 14 days these projections increased and matured. As in the normal adult brain, entorhinal layer II stellate cells projected correctly to the dentate gyrus and hippocampal field CA3, whereas layer III pyramidal cells projected to hippocampal field CA1 and the subiculum. The new fibres grew along both alvear and perforant pathways. Anterograde and retrograde labelling showed that the reciprocal projections from the pyramidal cells of the subiculum and CA1 to the entorhinal area had also been severed at the time of taking the slices, and had similarly regenerated. Our results demonstrate that by taking tissue slices in appropriate planes it is possible to study the regeneration of axons in the tissue environment through which they normally run. This approach avoids the use of coculture and the concomitant difficulties associated with the need for fibres to cross a coculture interface. In horizontal slices of postnatal tissue, severed fibre projections between the entorhinal cortex and the hippocampal complex can regenerate in both directions and re-establish their correct laminar, pathway and target specificity.

Long interfascicular axon growth from embryonic neurons transplanted into adult myelinated tracts.

In a previous study we used the species-specific marker M6 to demonstrate that transplanted mouse embryonic hippocampal neurons grow axons at a rate of at least 1 mm/d for a distance of at least 10 mm along the longitudinal axis of the fimbria in immunosuppressed adult rat hosts. We now show that hippocampal neurons are able to grow comparably long interfascicular axons in two other myelinated adult fiber tracts, the corpus callosum and the cingulum. Moreover, suspensions of cells from embryonic neocortex and superior colliculus transplanted into each of these three adult host sites also give interfascicular axon growth whose speed, intensity, and pattern of distribution are identical to those of transplanted hippocampal neurons. The axons of the donor cells grow in both directions along the longitudinal axis of the host tracts, where they are interspersed in parallel among the normal host axons, the rows of host interfascicular glial nuclei, and the longitudinal processes of host tract astrocytes. Serial section analysis through the complex trajectories of the host fiber bundles of the fimbria and corpus callosum shows that the course of the donor axons conforms to the underlying orientation of the axonal and glial structures of the host fiber tract. These observations indicate that long interfascicular axon growth can occur in several different adult myelinated fiber tracts. The donor axons become integrated with the host tract fibers and glia, and they respect intertract boundaries. Growth is not restricted to the types of axons normally present in the tracts.

Long fibre growth by axons of embryonic mouse hippocampal neurons microtransplanted into the adult rat fimbria.

We have described a method for the microtransplantation of a suspension of a few thousand cells from mid to late embryonic mouse hippocampi into the fimbria of immunosuppressed adult rat hosts. There was close graft-to-host contact, across a non-scarred interface. The transplanted cells included CA3 type pyramids, and were enclosed within the host myelinated fibre tract, whose glial framework was largely undisturbed. Immunohistochemistry of two species-specific markers (M6 and Thy-1.2) showed that the donor mouse neurons grew fine (< 0.5 micron diameter) axons which extended singly or in fascicles through the rat host fimbria for a maximum distance of at least 10 mm. The donor axons were intimately integrated among and closely aligned to the host tract axons and to the interfascicular glial rows of the host tract. The axons travelled (i) laterally through the ipsilateral fimbria, (ii) medially across the midline in the ventral hippocampal commissure to reach the contralateral fimbria and alveus, and (iii) rostro-medially to the septum. On approaching the terminal fields appropriate to hippocampal CA3 pyramidal cell axons, the transplant axons gave rise to fine preterminal branches which were continuous with a reticular or amorphous immunoreactivity in the stratum oriens and stratum pyramidale of the ipsilateral hippocampus, and in the lateral and triangular septal nuclei. The donor axons extended along the host fimbria at a rate of approximately 1 mm per day, reaching their terminal field destinations by approximately 1-2 weeks. At 7 weeks the projections were maintained, but with little further extension. These observations indicate that the microenvironment of myelinated adult fibre tracts is permissive for an abundant and rapid growth of axons from transplanted embryonic cell suspensions. These axons can leave host tracts to invade appropriate terminal fields.

Entorhinal axons project to dentate gyrus in organotypic slice co-culture.

We have demonstrated the formation of entorhinodentate projections by axons arising from explants of embryonic mouse entorhinal cortex or slices of postnatal rat entorhinal area co-cultured in contact with slices of postnatal rat hippocampus in roller tube and static culture. Species-specific markers (Thy-1 alleles and M6) showed that the most dense part of the projection was to the outer part of the molecular layer of the dentate gyrus (i.e. excluding the commissural-association zone). Retrograde axonal transport of fluorescent tracers placed in the dentate gyrus labelled a densely packed superficial layer of stellate cells in the entorhinal cortex. Anterograde axonal transport of biocytin placed in the entorhinal cortex showed that the entorhinodentate fibres formed typical parallel bundles oriented at right angles to the dentate granule cell dendrites and had short-stalked boutons. The formation of entorhinodentate synapses was confirmed in the electron microscope by electron-dense degeneration after cutting the previously formed connection between the co-cultures. Synaptic transmission was demonstrated by extracellular recording of postsynaptic field potentials after entorhinal stimulation. The entorhinal fibres also projected to the hippocampal stratum lacunosum-moleculare of fields CA1 and CA3, and were present in the outer part of the stratum oriens of the subiculum; in some cases they perforated the pyramidal cell layer of the subiculum. We conclude that the necessary molecular and tissue organizational signals for the formation of an entorhinodentate projection are present in tissues maintained in organotypic slice co-culture, and remain effective in the cross-species mouse-to-rat situation.

Monoclonal antibodies reveal molecular differences between terminal fields in the rat dentate gyrus.

We have derived a number of monoclonal antibodies which detect molecular differences correlating with the afferent inputs to the molecular layer of the adult rat hippocampal dentate gyrus. One group, dubbed OM-1 to OM-4, strongly stain the outer zone of the molecular layer, which receives its major innervation from the ipsilateral entorhinal cortex. A second group, IM-1 and IM-2, show a complementary pattern and preferentially stain the inner molecular layer, which receives inputs from the ipsilateral and contralateral hippocampus. These antigens are not, however, restricted to these layers, being found outside the hippocampus in several other areas of neuropil in the adult brain. In the developing brain the IM-1 antigen appears ubiquitously from the earliest age studied, embryonic day 12. Within the dentate gyrus, its restriction to the inner terminal field of the molecular layer only occurs during the second postnatal week. In contrast, OM staining appears only sparsely and late in the prenatal brain, appearing in developing cortical white matter between embryonic days 18 and 20. The outer dentate molecular layer becomes OM-positive from birth onwards, corresponding to the time of arrival of entorhinal axons during the first postnatal week. These two groups of monoclonal antibodies recognize a number of different glycoproteins. Ultrastructural immunohistochemistry shows they are cell surface molecules, and as such may be involved in the recognition events required for the establishment of specific patterns of neuronal connectivity.

Selective innervation of embryonic hippocampal transplants by adult host dentate granule cell axons.

Fragments containing different cytoarchitectonic fields were dissected out of late embryonic rat hippocampal primordia and transplanted into the hippocampus or septum of adult syngeneic hosts. Field CA3 transplants contained clusters of large, angular (pyramidal) cell bodies surrounded by a radiating corona of dendrites. These cells stained selectively with our monoclonal antibody Py, and a proportion were labelled by [3H]thymidine administered on the 15th day of embryonic life. Field CA1 transplants contained smaller, angular, Py-negative cells, which formed elongated laminae rather than globular clusters. The ability of the host dentate granule cells to project to the transplants was examined by (1) the Timm stain for mossy fibres, (2) electron microscopy of Golgi-impregnated CA3 pyramidal neurons in the transplants, and (3) quantitative electron microscopic assessment of the proportions of large mossy fibre terminals in the synaptic population of the transplants. The Timm stain showed that CA3 transplants received a projection from host dentate granule cells when the transplants were placed in direct contact with the axons in the host mossy fibre pathway. As in the normal host field CA3, the ingrowing mossy fibres terminated selectively on the juxtacellular regions of the dendritic tree and ignored the major part of the dendrites in the radiating corona. The electron micrographs showed that within this territory the host mossy fibres formed synaptic terminals with all the complex features typical of normal mossy fibres, and were presynaptic to complex spines arising from the juxtacellular region of Golgi-impregnated donor CA3 pyramidal cells. The quantitative electron microscopic study demonstrated that the mossy fibre-innervated juxtacellular regions of the field CA3 transplants had up to 20% of the normal density of mossy fibre synapses found in the stratum lucidum of field CA3 in situ. CA3 transplants which were placed in the septum, remote from the host mossy fibres, had either trivial numbers of mossy fibre synapses or none. This confirmed that the abundant mossy fibre terminals in the intrahippocampal CA3 transplants were of host origin, and not due to donor dentate granule cells inadvertently included in the grafts. The selectivity of the host dentate projection for field CA3 transplants was demonstrated by the observation that CA1 transplants in the same locations received only slight mossy fibre projections in the Timm stain, and in electron micrographs their synaptic population had only insignificant numbers of large mossy fibre terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

Synapse formation in the adult brain after lesions and after transplantation of embryonic tissue.

Some years ago it was demonstrated that when the adult rat septal nuclei are partially deafferented the remaining afferent fibres form new connections. The conclusion that new synaptic connections form in the adult central nervous system (CNS) was greeted initially with much scepticism, later with over-enthusiasm and unwarranted generalisation to all lesion situations, together with even less warranted attribution of various beneficial functional properties. Today, as the pendulum swings into a more reasonable position, some of the original observations, which at the time attracted little attention, have become more interesting. (1) The observation that in the normal septal nuclei the ratio of spine to shaft synapses is extraordinarily constant (to an accuracy better than 1%) from one animal to another. How could such almost crystalline rigidity of structure be produced in normal development and maintained in the face of major lesion-induced changes in connectivity? (2) The observation that synaptic re-occupation by sprouting axons restores exactly the normal number of synapses, presumably indicating that the neurones have a fixed number (as well as spine/shaft distribution) of postsynaptic sites. Thus, the septal lesion paradigm is as strong a method for investigating synaptic rigidity as for investigating plasticity. In the intervening years, the use of embryo to adult transplantation has made it obvious that considerable reconstruction of adult brain synaptology is possible, and that many of the normal rules of connectivity are maintained (most prominently for the 'point-to-point' axonal systems). What could lead to further fruitful investigation is the extent to which the observations (e.g. relating to hierarchies of axonal preference, the need for denervation, and the involvement of glial cells) in partially deafferented adult systems, such as the septal nuclei, are retained, or modified, in face of the ingrowing fibres from embryonic transplants.

Fetal medial habenula transplants: innervation of the rat interpeduncular nucleus.

The effects of donor age and site of placement on the survival of fetal medial habenula (MH) transplants into adult rats hosts were examined. The innervation of the interpeduncular nucleus (IPN) in such cases was also examined. Explants of MH consisting of the medial-dorsal lip of the third ventricle were held in vitro for 1-2 days. Colloidal gold conjugated to wheat germ agglutinin was added for the last 18 hours to label the cells. Four of 16 cases with E19 derived transplants contained donor neurons. Markedly larger transplants were present in 95% of 20 cases with E16 derived transplants. Sites in the ventral midbrain were successful, while limited or no survival occurred at sites more remote from IPN. Retrograde labeling of transplant neurons was present in each case studied with HRP injection into host IPN. Colloidal gold-labeled macrophages, some oriented capillaries and GFAP-positive processes marked the donor-host interface. In EM the interface was evident only by the difference in tissue elements in the transplant versus host. Numerous synapses of Gray types I and II were present in the transplant. Excellent survival of MH neurons, donor/host interfaces, innervation of IPN by the transplant and fine structure in and around the transplants, all suggest that such preparations are suitable for further experimental analysis of the habenulo-interpeduncular system.

Use of colloidal gold complexes of wheat germ agglutinin as a label for neural cells.

We have made stable complexes between wheat germ agglutinin and either 5 or 10 nm particles of colloidal gold. These complexes were phagocytosed by neuronal and glial cells in embryonic rat hippocampal cultures and the incorporated gold gave intense, low-background staining in the light microscope either directly, for the most heavily labelled cells, or after intensification by physical development of silver. Cells were labelled in a punctate fashion over perikarya and processes. In the electron microscope, particles of gold were observed in lysosomal vesicles, frequently in an aggregated form. Gold complex incorporated into cells in culture was retained by those cells over periods up to 20 days. Embryonic hippocampal cells were labelled in suspension culture by incorporation of wheat germ agglutinin-gold complexes and transplanted into the brains of syngeneic adult host rats. Grafted neurons and glia were observed in the electron microscope to retain high levels of gold label over periods up to 30 days. Receipt of synaptic connections by transplanted neurones was observed. Complexes of wheat germ agglutinin with 10 nm gold particles were injected unilaterally into field CA3 of the hippocampus of adult rats. Specific retrograde transport of gold was observed in the light and electron microscopes to pyramidal and hilar neurones of the contralateral hippocampus and to neurones of the medial septal nucleus. Colloidal gold-wheat germ agglutinin complexes appear to be useful cellular markers that can be visualized at both light and electron microscope levels.

A software package for the manipulation, display and analysis of electron and light microscope data.

Quantitative light and electron microscopy is an expanding field which has found applications in many biological disciplines. Computer-enhanced data-acquisition has led to increased speed and accuracy, and combined with computational analysis, stereological parameters can be quickly derived and spatial hypotheses can be easily tested. A software package is described, which controls the manipulation, display and analysis of two-dimensional microscope data. Features include alignment of specimens, rotations, translations, deletions, recoding, perimeter and area measurement, point-counts, tests of dispersion and tests of spatial distributions. The programme is graphics-orientated and supports various point-plot modes and histograms. It includes linear and non-linear curve-fitting options and has been specifically designed to encourage new analysis procedures, which automatically benefit from existing graphics options.

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.