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.

[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.

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 effect of chronic irradiation with intermittent unmodulated microwaves on the functional status of the rabbit]. / Vliianie khronicheskogo oblucheniia preryvistymi nemodulirovannymi mikrovolnami na funktsional'noe sostoianie krolika.

A group of 10 chinchilla male rabbits were exposed to 800 MHz alternative microwave fields. The data obtained revealed changes in the EEG, ECG and respiration in only 2 rabbits after 5-7 experiments.

[Changes in the functions and biorhythms of the normal rabbit ECoG]. / Ob izmeneniiakh sostoianii i bioritmov EKoG krolika v norme.

The rabbit ECoG is characterised by alternating active and inhibitory signs. This suggests the necessity to take into consideration individual features and initial states of the rabbit CNS and the dynamics of their changes when studying the effects of various stimuli.

[Dynamics of conditioned reflex preservation in young and adult rats]. / Dinamika sokhraneniia pishchevogo uslovnogo refleksa u molodykh i vzroslykh krys.

Experimental data are presented on the formation and retention during 24 hours of a motor alimentary conditioned reflex (MCR) in a T-maze, in rats 4--5 months and 1,5--2 months old. In adult rats the formation of MCR required a significantly smaller number of runs and less time, than in young ones. In young animals reflex retention is better as compared to adults. Traces of MCR change regularly throughout 24 hours in both animal groups which may be due to development of memory in several stages, in young animals this process being slower.