Ultrastructural Immunocytochemistry of GABAergic Cells in Neocortical Neurotransplants.

Neural transplantation is a promising regenerative therapy in the treatment of several neurological diseases. Importantly, transplanted tissue should not become a source of pathological functional activity. To assess the possibility of maintaining the balance between excitatory and inhibitory processes, an electron microscopic immunochemical study of the GABAergic system in rat neocortical transplants was performed. Accumulation of GABA-positive label in astrocytes and a relatively insignificant immune reaction to GABA in neurons and synaptic endings were found. These findings suggest that under conditions of impaired differentiation of GABA-containing neurons that generate phasic inhibition through inhibitory synapses, tonic inhibitory influences predominate in neurotransplants due to GABA released from astrocytes.

Intraocular Neurografts as a Model for Studying of Organization of Synaptic Connections in a Denervated Brain Area.

Intraocular neurografts of the septal region of rats were used as the model of deafferentiated brain area where the lack of adequate innervation is compensated for own interneuronal connections. Septum anlage from the brain of a 17-day fetus served as the donor material. The grafts developing in the anterior eye chamber over 3 months represented well-differentiated samples of the nervous tissue. A comparative morphometric study of the tripartite organization of synapses in the grafts and in the septum in situ was conducted. In the grafts, the mean volume and perimeter of synaptic terminals were below the normal. At the same time, postsynaptic densities did not differ from the control. A significant difference was found in the degree of surrounding of presynaptic terminals by astrocytic processes: in the grafts this parameter was higher by 1.8 times. Our results attest to an important role of perisynaptic glia in the formation of functionally active synaptic contacts with unusual neuronal targets.

Microscopic Study of Nervous System Plasticity: Interactions of Sympathetic Nerves with Neurons of Intraocular Hippocampal Transplants.

Functional interactions of sympathetic fibers innervating the iris with the neurons of central origin in intraocular transplants of the rat hippocampus were studied by optic, confocal, and electron microscopy. After formaldehyde fixation, fluorescent dye Dil was applied to the upper cervical ganglion; the dye migrated to the transplant by lateral diffusion via axons. Sympathetic nerves labeled with fluorescent dye grew into the neurotransplants along perivascular membranes of blood vessels. In addition, some fluorescent axons were identified in the transplant parenchyma. Electron microscopy showed large bundles of the peripheral type axons in the vascular adventitia and Schwann-axonal complexes in the transplant neuropil. Autonomic axons formed synaptic contacts with transplanted neurons.

[Phenotypic Differentiation of Neurons in Intraocular Transplants].

Neurochemical differentiation of neurons in transplants developing in rat anterior eye chamber was studied. Pieces of the somatosensory neocortex area, isolated from 17-day fetuses of Wistar rats, were used for the transplantation. The general cytological analysis and immunochemical identification of GABAergic neurons in neocortical transplants and in the appropriate brain area of the recipient rats (control) were carried out after 6 months. Cytoarchitectonics typical for neocortex was not revealed in the transplants. Furthermore, a 1.4-fold decrease in numerical density of the entire neuron population was found compared to the control. The proportion of GABAergic nerve cells in the transplanted tissue was reduced even more dramatically­ by 13.1 times. The dimensions of all types of neurons, especially GABAergic cells, were greater in the transplants in oculo compared to neocortex in situ. The increase in size occurred mostly due to the cytoplasm. Thus, the nuclei of GABA-positive neurons in the transplants were larger by 1.2 times compared to the control and their perikarya were larger by 1.5 times. The obtained results showed that the conditions in the anterior eye chamber the most dramatically affect the differentiation of GABAergic neurons, and cell hypertrophy, probably, is the functional compensation of the decrease in their number. Considering the literature data on the increased excitability and synchronized neuronal activity in the intraocular transplants, it can be assumed that these transplants can be used as a model for studying the cellular mechanisms of nervous tissue epileptization under disinhibition conditions.

Transsynaptic Coordination of the Formation of Morphofunctional Contacts between the Brain and the Neurotransplant: an Ulrastructural Study.

We studied the role of neurotransmitter signaling mediated by synaptic vesicles in the formation of aberrant functional connections between fascia dentata grafts and the somatosensory neocortex in adult rats. Quantitative analysis of the different populations of synaptic vesicles in the ectopic giant axonal endings of granular neurons was performed and the results were compared with the normal. Two pools of small clear vesicles (rapidly releasable pool and pool of reserve vesicles circulating in the active zone) and one pool of large dense-core vesicles were analyzed. Significant differences from the control suggest that synaptic integration of the transplants into the recipient brain is coordinated by transsynaptic signaling and mediated by different populations of synaptic vesicles.

Structural organization of glial cells at the border between the neurotransplant and recipient brain.

Integration of fetal hippocampal dentate fascia neurotransplants with the neocortical somatosensory region in adult rats was studied by electron microscopy. The growth of nerve fiber through the neurotransplant/brain border formed by the glial cells was studied. The interface zone was organized by various astrocyte subpopulations and ependymocytes forming multilamellar accumulations in some sites of the interface. These conglomerations of the glial cells and their processes did not prevent the growth of axonal and axodendritic bundles; moreover, fibrous astrocyte axons accompanied them. Under conditions of immature nervous tissue transplantation to the focus of mature brain damage, the glial cells created a substrate in the interface permeable for nerve fibers, thus promoting the functional integration of the neurotransplant.

Structural signs of dynamic state of synaptic contacts between neurotransplant and brain.

We studied ultrastructure of synaptic connections between long-living dentate fascia transplants and somatosensory area of the neocortex in rats. Giant synaptic terminals of granular neurons upon contact with unusual neuronal targets in the neocortex reproduced their specific constitutive morphological features. At the same time, developing synapses with signs of active structural and metabolic reorganization were revealed. This is indicative of a dynamic state and instability of functional connections between the transplant and brain despite long time after transplantation.

[Inductive role of mossy fibers of hippocampus in the development of dendritic spines in aberrant synaptogenesis at neurotransplantation].

The dentate fascia of the hippocampal formation isolated from 20-day-old Wistar rat fetuses was subjected to heterotopic transplantation into the somatosensory area of the neocortex of adult rats of the same strain. Five months after surgery, neurotransplantates, together with neighboring area of the neocortex, were studied using light and electron microscopy. We carried out a detailed study of the ultrastructure of the ectopic synaptic endings formed by the axons of granular neurons of the dentate fascia (mossy fibers) with neurons of the neocortex unusual for them in a normal state. Ultrastructural analysis revealed that most ectopic synaptic endings produce its determinant morphological features: giant sizes ofpresynaptic knobs, active zones with branched dendritic spines, and adherens junctions with the surface of dendrites. The data indicate that the mossy fibers growing from neurotransplantates induce structural and chemical reorganization of dendrites of the neocortex using transmembrane adherens junctions, such as puncta adherentia junctions. This results in the differentiation of active zones and development of dendritic spines typical for giant synaptic endings that are invaginated into presynaptic endings. Thus, the ability of neurons of the dentate fascia to form aberrant synaptic connections at transplantation results from the inductive synaptogenic properties of mossy fibers.

[Participation of puncta adherentia junctions in the formation of synaptic connections between a dentate fascia graft and the host brain].

The fetal dentate fascia of Wistar rats on the 20th day of gestation was heterotopically grafted into the somatosensory neocortex of adult rats. Granule cells of a graft projected their axons (mossy fibers) to the host brain and established synaptic contacts with inappropriate targets. The organization of ectopic mossy fiber synapses was studied by electron microscopy. It was shown that ectopic synapses reproduce the structural determinants of hippocampal giant synapses and induce a subcellular reorganization of postsynaptic neocortex dendrites. Using morphometric analysis, a significant increase was found in the number of discrete puncta adherentia junctions and their total length in ectopic synapses as compared with the control group. The data obtained indicate that puncta adherentia contacts participate in the structural and chemical adaptation of neuronal targets to alien axons growing from transplants.

Morphofunctional interactions of peripheral nerve fibers of the iris with neurons developing in the anterior chamber of the eye in rats.

Electron microscopic studies were performed on intraocular transplants of embryonic septal and hippocampal tissue developing in the anterior chamber of the eye in rats for 3-4 months. The aim of the study was to seek ultrastructural identification of peripheral nerve fibers entering transplants from the iris, and to assess their ability to establish true synaptic contacts with transplanted CNS neurons. Bundles of myelinated and unmyelinated axons surrounded by Schwann cell cytoplasm were seen within the perivascular spaces of ingrowing blood vessels. Both types of peripheral fiber were also identified in the neuropil areas of transplants. At the ultrastructural level, unmyelinated axons were found to be free of glial Schwann cell sheaths and to form typical asymmetrical synapses with the dendrites and dendritic spines of transplant neurons. These results provide evidence of the high morphofunctional plasticity of both parts (central, peripheral) of the nervous system.

[Morpho-functional interactions of the iris peripheral nervous fibers with the neurons developing in the rat anterior eye chamber].

The intraocular grafts of the septal or hippocampal embryonic tissues developing in the rat anterior eye chamber for three to four months were investigated by electron microscopy. The aim of this study was both the ultrastructural identification of the peripheral nervous fibers entering the grafts from host iris and the estimation of their capacity to establish true synaptic contacts with the central nervous system neurons of the grafts. The bundles of myelinated and unmyelinated axons, surrounded by the Schwann cell cytoplasm, were observed within the perivascular spaces of the ingrowing blood vessels. In the neuropil areas of the grafts, both types of the peripheral nervous fibers were also identified. It was demonstrated on the ultrastructural level that the unmyelinated axons lost their glial envelope of the Schwann cell and formed the typical asymmetric synapses with the dendrites and dendritic spines of the grafted neurons. The results are indicative of the high morpho-functional plasticity of both parts of the nervous system.

Involvement of neuropeptide mechanisms in the process of integration of heterotopic dental fascia transplants with recipient brains.

Embryonic dentate fascia was transplanted into the somatosensory area of the neocortex of adult rats. Ultrastructural and morphometric analyses of giant synapses formed by the granule neurons of transplants with inappropriate neuronal targets in the recipient brains were performed after nine months. As compared with intact synaptic terminals in the control hippocampus, there were differences in the quantity and distribution of large synaptic vesicles with electron-dense centers storing neuropeptide cotransmitters. The proportion of peptidergic vesicles (of the total number of vesicles) in ectopic giant synapses was 5.8 +/- 0.6%, compared with 3.3 +/- 0.6% in controls. Accumulations of large, dense vesicles close to the active zones of aberrant connections were seen almost 7.9 times more often than in controls. These results show that neuropeptide transmitters are critical for maintaining synaptic connections between heterotopic dentate fascia transplants and recipient brains.

[Involvement of neuropeptide mechanisms in the integration of heterotopic dentate fascia grafts and recipient brain].

Embryonic dentate fascia was grafted into the somatosensory neocortex of adult rats. Nine months post-grafting, the ultrastructural and morphometric analysis of the giant synapses established between the grafted granular neurons and inappropriate targets in the recipient brain was performed. As compared to the intact synaptic endings in the control hippocampus, differences were found in both the number and distribution of large dense-core synaptic vesicles, which store the neuropeptide co-transmitters. The peptidergic vesicle proportion (of total vesicle pool) within the ectopic giant synapses was 5.8 +/- 0.6% (versus 3.3 +/- 0.6% in the control). Clusters of large dense-core vesicles near the active zones of aberrant connections were observed almost 7.9 times more frequently than that of normal contacts. These data provide evidence that neuropeptide transmitters are critical for the maintenance of synaptic connections between the heterotopic dentate grafts and host brain.

The hippocampus and neurotransplantation.

The present article is a review of our own results from histological and electron microscopic studies of hippocampal neurotransplants with different levels of integration with recipient brains. A model providing complete isolation from the brain was obtained using transplants developing in the anterior chamber of the eye. The growth, development, and cytological composition of transplanted tissue was found to depend on factors such as the age of the donor embryo tissue, the genetic compatibility between the donor and recipient, and the level of integration with the brain. Ultrastructural analysis of intraocular and intracortical transplants showed that overall, nerve and glial cells have the characteristics of highly differentiated, mature elements; the numerical density and structures of synaptic contacts were similar to those in normal conditions. However, transplanted tissues contained morphological features providing evidence of continuing growth of several nerve processes and increases in non-synaptic and transport-metabolic intercellular interactions. The ultrastructural deviations observed here are regarded as the manifestations of compensatory-adaptive changes during the development of tissues in conditions deficient in natural afferent synaptic influences. It is also demonstrated that the axons of transplanted neurons lacking adequate cellular targets can establish functional synaptic contacts with neuronal elements in the recipient brain which are not their normal targets.

[The hippocampus and neuro-transplantation]. / Gippokamp i neirotransplantatsiia.

A review of the author's histological and electron-microscopic studies of differentiation of hippocampal transplants with different levels of the graft/host integration. The grafts developing in the anterior eye chamber were the experimental model of complete isolation from the brain. The effects of various factors (age of the donor fetal tissue, host age and strain, degree of the integration with the recipient brain) on the growth and neural organization of grafts were studied. Analysis of fine structure of intraocular and intracortical grafts, as a rule, showed mature highly differentiated neurons and glia and normal density of typical synaptic contacts. However, morphological features suggesting both hyperactivity of some neurons and continuous growth of some neural processes were observed. The expression of nonsynaptic and transport-metabolic interactions between the cells was increased. The observed ultrastructural deviations can be regarded as a compensatory adaptation of the tissue to the deficit of specific afferent signals. It was shown that in the absence of normal cellular targets, axons of the grafted neurons establish functional synaptic contacts with improper neural elements in the host brain.

Intercellular Transport-metabolic interactions in the nervous tissue developing under conditions of afferentation deficit (transplants in the anterior eye chamber).

Intercellular transport-metabolic interactions were investigated at the ultrastructural level in the grafts of the embryonic rat hippocampus and septum developing for 3 to 12 months in the anterior eye chamber of adult rats. The signs of highly increased transport from the blood capillaries ingrowing from the host's iris into the grafts (multiple pinocytotic vesicles in the endothelium and pericytes) were observed. The glial cells, which were accumulated at the graft surface, had pinocytotic invaginations and microvilli, which indicated their possible participation in the active transport of metabolites from the surrounding intraocular fluid. An increased level of direct communications, manifested in pinocytoses and large gap junctions between apposed nerve and glial cells was also present within the grafts. Moreover, microphagocytosis, as internalization of surface membrane fragments with adjacent cytoplasm of the neighbouring structure (including dendrites and axons), was often observed in the grafted tissue. It is suggested that the observed communications between neuronal and glial cells may participate in both trophic and functional interactions. An increase in the level of nonsynaptic interactions in the grafted nervous tissue may be regarded as a manifestation of the compensatory adaptation to the absence of normal tissue surrounding afferent connections and efferent targets.

[Differentiation of neurons and synapses of mossy fibers in transplants of fascia dentata developing in the rat neocortex]. / Differentsirovka neironov i sinapsov mshistykh volokon v transplantatakh zubchatoi fastsii, razvivaiushchikhsia v neokortekse krys.

Embryonic fascia dentata tissue isolated from the hippocampus was transplanted heterotopically into the neocortex of adult rats. Ultrastructural characteristics of neurons and synapses in transplants were studied nine months later. It has been found that the main types of neurons present in fascia dentata undergo differentiation in the transplants, and a dense neuropile containing various types of synapses is produced. A characteristic feature of the transplanted neurons is the presence of additional microspines on somatic and dendrite surfaces; this appears to be due to a deficiency of external and internal afferents. Gigantic synaptic terminals of granule cell axons (mossy fibers) in transplants possess unique morphological characteristics, which allow their identification in a complex neuropile. Just as in situ, they form two types of contacts: chemical asymmetric contacts with dendrite spines and desmosome-like ones with dendrite surface characteristics. However, accumulations of large vesicles with electron-dense centers can often be observed near the active zones of the synapses, and desmosome-like connections are more prominent. The most important feature is that gigantic synapses in transplants use midsize and small dendrites as postsynaptic targets up to terminal branches, and they contact with spines of the usual shape and size, whereas in situ terminal synaptic contacts of mossy fibers are formed only with gigantic processes of initial segments of the large apical dendrites. Thus, in the absence of normal synaptic targets, mossy fibers can produce contacts having all features of functional synapses, but with atypical postsynaptic structures.

Morphometric analysis of hippocampal pyramidal neurons in situ and in grafts developing in the anterior eye chambers of young and aged Wistar rats.

We performed a morphometric analysis of the somatic and nuclear areas in the pyramidal neurons of the hippocampal fields CA1 and CA3 in situ and in grafts developing for six weeks in the anterior eye chambers of young (3-to-9 wk.) and of aged (18-to-19.5 mos.) Wistar rats. The mean areas of the CA1 pyramidal somata and nuclei were significantly decreased in the aged animals in situ. The mean parameters of the CA3 pyramidal neurons were not changed, although their distribution was different (bimodal versus unimodal in the young animals). In both groups of recipients, the areas of CA1 neurons and of their nuclei were significantly larger in the grafted tissue than those found in situ. The areas of CA3 neurons did not show any difference in aged recipients and demonstrated only slight hypertrophy in young recipients. We concluded that the area sizes of the pyramidal cell bodies and nuclei in CA1 neurons are more sensitive than those of CA3 neurons to both aging and transplantation. The age of recipients did not significantly influence the growth and development of grafted pyramidal cells.

Intracortical dentate fascia grafts: mossy fiber synapses in the host neocortex.

Embryonic dentate fascia was grafted into a cavity in the area of the adult rat neocortex which represents the vibrissae (barrel field). We wished to test the possibility of development of connections between the two brain areas which do not have synaptic or tissue contacts in situ. The unique characteristics of the giant synaptic boutons of the dentate mossy fibers were used for detection of the dentate synaptic contacts with neocortical neurons at the electron microscopic level. Ultrastructural analysis nine months postgrafting has shown that the bundles of mossy axons enter the host neocortex and develop multiple terminal and en passant contacts with typical characteristics. Neuronal perikarya, large dendritic trunks and fine caliber terminal dendritic branches were used by the mossy fibers as postsynaptic targets, as well as spines of various complexity and configurations. The subsynaptic dendrites seemed to be modified by synapsing giant boutons. Accumulation of cytoplasmic organelles was observed at these sites. Various bumps and protuberances were formed by the subsynaptic dendrite surface. The contents of these appendages were variable; some of them contained organelles typical of dendroplasm, while others were more spine-like, often with inclusion of ribosomes. It is concluded that mossy fibers growing into the host neocortex can develop typical contacts with inappropriate targets with all the ultrastructural features of functional synapses.

Ultrastructural signs of regenerative-degenerative processes in long-term dentate fascia grafts.

An ultrastructural investigation of embryonic (E20) dentate fascia grafts transplanted into an acute cavity in the somatosensory neocortex of adult rats revealed a continuous dynamic state of the tissue nine months postgrafting. The grafts consisted mainly of typical granular cells with some admixture of hippocampal pyramidal neurons and polymorph hilar cells with a normal, mature ultrastructure. Many features of the transplanted tissue suggested continuing development and growth. Dendritic branches with growth tips, axonal growth cones, synaptic boutons with growth vesicles, immature myelin sheaths and myelin-producing cells were observed. In contrast, ultrastructural signs of degeneration were present in some axons, and, less often, in dendrites. These processes, as well as some of the terminal synapses, contained various amounts of lysosomes and lipofuscine granules. In many such terminals the signs of degenerative change were combined with the presence of multiple mitochondria, polymorph vesicles and tubular reticulum, indicating simultaneous reparative processes. It is suggested that continuous recycling of neuronal processes occurs in long-term dentate grafts. This morphological instability may depend on the paucity of synaptic targets within the dentate tissue transplanted with a minimal quantity of hippocampal pyramidal cells and on the limitation of the afferent input. However, the observed features of the grafted dentate tissue are not qualitatively different from those observed in normal dentate with its protracted development and active compensatory reorganization.