Paramembranous specialized formations of the synaptic cytoskeleton -dense projections and postsynaptic condensation of axospinous synapses of the molecular layer of white rat sensorimotor and cerebellar cortex -in health and acute total ischemia are studied by selective contrast staining with phosphotungstic acid. A direct relationship is revealed between the pattern and degree of deformation of the contact plane, on the one hand, and the postsynaptic condensation and ratio of the volumes of pre-and postsynaptic accumulations of paramembranous fdaments, on the other. Key Words: neocortex; cerebellum; interneuronal synapse; ischemiaSynaptic deformation is regarded as the basis of morphological characterization of synapse shape [3]. Experimental findings attest to a relationship between the pattern of deformation of the plane of a synaptic contact and its functional state [2,3,7]. The factors determining the type (positive or negative) and degree of deformation include various external and internal factors (state of the synaptic cytoskeleton, type of transmitter metabolism, size of the synapse, complexity of arrangement of the contact, its localization on the neuron, and its appurtenance to a particular brain compartment) [2,3,5,6]. Discrepancies in the data, however, leave unsolved the problem of the basic structural mechanisms of synapse formation in health and disease.In this study we assessed the effect of the spatial organization of the system of paramembranous fdamentous formations (dense projections, postsynaptic condensation) on the degree of changes in the deformation of the synaptic contact plane in health and ischemia. MATERIALS AND METHODSThe objects of this study were the axospinous synapses of the molecular layer of the sensorimotor cortex (SMC) and the cerebellar cortex, which differ appreciably in biochemical composition and in the spatial arrangement of postsynaptic condensation [4]. Experiments were carried out with 6 male white rats weighing 190 to 210 g under ether narcosis. Acute total ischemia of the brain was induced by 10-rain clamping of the cardiac vascular bundle [1]. The brain was fixed by perfusion of a mixture of 1% glutaraldehyde, 4% paraformaldehyde, and 5% sucrose in phosphate buffer (pH 7.4) through the left ventricle of the heart for 15 min, and then postfixed for 2 h in the same solution at 4"C. Oriented pyramidal fragments of SMC and cerebellar cortex were contrast-stained in ethyl solution of phosphotungstic acid as described elsewhere [7]. The fragments were embedded in an epon-araldite mixture. Ultrathin slices were prepared in a tangential plane at the level of the molecular layer of the SMC and cerebeUar cortex. The slices were examined under an EVM 100AK electron microscope; 30 random visual fields of the neuropil were photographed at the standard 15,000
Disturbances in the structural and functional organization of different parts of the brain due to acute interruption of the blood supply, as well as post-ischemic compensatory and restorative processes, have a diffuse/focal character [3][4][5]. The causes underlying selective focal damage to different parts of the brain, to structural-functional modules, and to neurons during ischemia remain unclear [ 12]. Data on the post-ischemic plasticity of surviving structural-functional systems of different orders (neurons, modules, brain regions) are incomplete, contradictory, and often fail to reflect the focal nature of compensatory and restorative processes [5-7, 9, 12].It was thus of interest to carry out a comparative analysis of interneuron interactions in regions with different extents of destructive change and compensatory/restorative processes during the post-ischemic period. The most convenient model for this study was the molecular layer of the cerebellar cortex, where the dendritic territories of each pear-shaped cell (Purkinje cell) have mutually nonoverlapping distribution zones [I0].The aim of the present work was to assess the structural features of focal changes in the synaptic pool and to carry out a comparative analysis of the zones of different types of changes in the synaptic architectonics of the molecular layer of the cerebellar cortex in white rats in the post-ischemic period. MATERIALS AND METHODSExperiments were carried out on 22 white male rats (190-210 g) under ether anesthesia. Systemic blood flow was interrupted by clamping the vascular bundle of the heart for 10 min. Subsequent restoration of blood flow was ensured by external cardiac massage and mechanical ventilation of the lungs [1]. Specimens for electron microscopic studies of synapses were.collected from control animals (four rats), at the end of ischemia (10 min), and at 90 min, and 1, 3, 7, and 30 days (three rats at each time point) after re-establishment of systemic blood flow. Brains were fixed by perfusion with a mixture of 4% paraformaldehyde, 1% glutaraldehyde, and 5% sucrose in phosphate buffer pH 7.4 via the left ventricle of the heart for 15 rain; after sectio.ning, pieces of brain tissue were prefixed in the same mixture for 2 h at 47C. Pyramidal pieces of cortex from _ the lateral surface of the cerebellar gyrus were contrasted with two mutually additive methods: treatment with OsO 4 and contrasting with phosphotungstic acid. Material was embedded in Epon-Araldite by a parallel plane technique [5]. In each case, series of 10 adjacent (sequentially located) blocks were prepared. Ultrathin tangential sections were prepared from the region corresponding to the upper third of the molecular layer, where axodendritic synapses between the parallel fibers of granule cells and the distal segments of dendrites and Purkinje cells predominate [10]. An I~VM-100AK electron microscope was used to photomicrograph random areas of the neuropile (the area of the field of view was 48 ~m 2) at a standard magnification of 15,000• Quantitative a...
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