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D A Rusakov

Publications and source records attributed to D A Rusakov.

At least 37 records · Page 2Linked to original sources
PubMed1995

Quantification of dendritic spine populations using image analysis and a tilting disector.

A series of image analysis routines, stochastic geometry methodology, and a design-based stereological procedure have been developed to quantify objectively the length, layout, and the true density of neuronal dendritic spines observed at the light (or confocal) microscope level. First, the image of a dendritic fragment of interest (in the plane of view) is scaled to a standard brightness scale, and the dendritic profile is separated from the background using a computerized thresholding algorithm that analyzes the histogram of grey levels. Secondly, the resulting binary image of the dendrite is transformed to a midline skeleton that underlies the dendritic geometry. Thirdly, skeletal branch lengths are directly computed (in pixels), thus giving objective measures of visible spine lengths and inter-spine distances along the dendritic stem. These raw data are the basis for (1) an estimation of the distribution of 3D spine lengths, and (2) a nearest neighbour analysis of the spine layout along the dendrite. A design-based stereological routine, the tilting disector, is suggested for unbiased estimation of the true (3D) density of spines along dendrites. The routine involves tilting the dendritic fragment of interest around its longitudinal axis for a known angular sector and scoring the number of spines seen in one angular position and unseen in the other position. Data from a study of neuronal dendrites in the chick forebrain are presented.

Algorithms↗
PubMed1995

3-dimensional morphometry of intact dendritic spines observed in thick sections using an electron microscope.

An experimental technique is described which allows observation of fixed neuronal dendrites at magnifications from 10-12 K. The method uses 4-7-microns-thick sections of Epon-embedded tissue with nerve cells that are first impregnated by the rapid Golgi technique and then stained with gold particles/aggregates using a modified gold-toning procedure. A relatively high acceleration voltage (200 kV) is employed to observe in fine detail the dendritic fragments of interest at different angular positions in space, by using a eucentric goniometer stage with a tilt angle of +/- 45 degrees. Image analysis methodology is proposed which permits estimation of 3-dimensional (3D) lengths and of the volume of observed intact dendritic spines. The advantages of the technique with respect to 3D reconstruction methodology are discussed.

Animals↗
PubMed1995

Repeated confocal imaging of individual dendritic spines in the living hippocampal slice: evidence for changes in length and orientation associated with chemically induced LTP.

Using confocal microscopy in conjunction with microdrop application of Dil, we have imaged and measured individual dendritic spines of living hippocampal CA1 pyramidal neurons in acute brain slices, before and approximately 3 hr after induction of long-term potentiation by chemical means. Statistical analysis of changes in the length of individual spines, and comparison with results of Monte Carlo simulations, suggests that two forms of structural change occur in chemically induced long-term potentiation: growth of a subpopulation of small spines, and angular displacement of spines. These changes could provide a structural basis for the expression of long-term potentiation.

Algorithms↗
PubMed1994

Training in chicks alters PSA-N-CAM distribution in forebrain cell membranes.

The intermediate and medial hyperstriatum ventrale (IMHV) in the chick is involved in memory formation following one-trial passive avoidance training. Possible links between neural cell adhesion molecule (N-CAM) distribution and memory consolidation were examined in an immunoelectron microscope study of IMHV 6 h after training. An antibody against alpha 2,8 polysialic acid (PSA), characteristic for embryonic N-CAM, and one against the protein backbones of N-CAM were labelled (post-embedding), separately, with 15 nm immunogold, and binding to their epitopes was analysed using the statistics of point processes. No difference in labelling levels between control and trained chicks, for either antibodies, was found, both groups of birds showing that cell membrane regions are 10-30 times more enriched in N-CAM isoforms, and have a two-fold greater proportion of PSA-N-CAM, than tissue as a whole. However, 375-400 nm regular arrays of labelled PSA-N-CAM were revealed statistically in cell membranes of control, but not trained, chicks, which may be related to the possible involvement of such membrane PSA domains in memory-related neuroplasticity.

Animals↗
PubMed1994

Interactions between brain mitochondria and cytoskeleton: evidence for specialized outer membrane domains involved in the association of cytoskeleton-associated proteins to mitochondria in situ and in vitro.

The surface distribution of several proteins (porin, hexokinase, and two proteins associated with microtubules or actin filaments) on the outer membrane of brain mitochondria was analyzed by immunogold labelling of purified mitochondria in vitro. The results suggest the existence of specialized domains for the distribution of porin in the outer mitochondrial membrane. Similarities between the distribution of porin and the distribution of microtubule-associated proteins bound in vitro to mitochondria suggested that mitochondria and microtubules interact by binding microtubule-associated proteins to porin-containing domains of the outer membrane. This hypothesis was supported by biochemical studies on outer mitochondrial proteins involved in in vitro binding of cytoskeleton elements. In vitro interactions between mitochondria and microtubules or neurofilaments were analyzed by electron microscopy. These studies revealed cross-bridging between the outer membrane of mitochondria and the two cytoskeleton elements. Cross-bridging was influenced by ATP hydrolysis and by several proteins associated with the surface of mitochondria or with microtubules. In addition, unidentified proteins which were recognized by antibodies to all intermediate filaments subunits were associated either with the mitochondrial surface or with microtubules. This data suggest the participation of additional cytoplasmic proteins in the interactions between cytoskeleton elements and mitochondria.

Animals↗
PubMed1994

Statistical analysis of the surface distribution of microtubule-associated proteins (MAPs) bound in vitro to rat brain mitochondria and labelled by 10 nm gold-coupled antibodies.

Purified mitochondria from rat brain were incubated in vitro which microtubule-associated proteins (MAPs) that are known to bind specifically on sites present on the outer membrane. The bound molecules were detected by immunoelectron microscopy and the linear distribution of the label along mitochondrial profiles was analyzed by statistical methods. The results demonstrate that gold-conjugated antibodies are distributed in a non-random fashion on the surface of mitochondria, suggesting regional concentrations of MAPs-binding sites. This finding argue for the existence of specialized domains on mitochondria that are involved in the association of the organelles to microtubules in situ.

Animals↗
PubMed1993

Quantal behaviour of synaptic transmission can be statistically examined using the Fourier line spectrum of the histogram of synaptic potentials.

A statistical approach to reveal the quantal behaviour of postsynaptic potentials (PSPs) is described. This includes: (1) obtaining the Fourier line spectrum (decomposition into harmonics) of the PSP histogram; and (2) testing the null hypothesis, 'the spectrum is that of white noise', using an ANOVA. The harmonic that rejects the hypothesis will indicate the regular peaks in the histogram, i.e., the quantal size of PSPs. The method was tested using published results of three experimental studies in central synapses and yielded the quantal sizes close to those derived from other methods. However, using three examples of published simulation studies (where the quantal model of synaptic transmission was known a priori), it was shown that the approach can estimate quantal sizes of PSPs more reliably than other methods.

Analysis of Variance↗
PubMed1993

Spatial re-arrangement of the vesicle apparatus in forebrain synapses of chicks 30 min after passive avoidance training.

A quantitative ultrastructural study of synapses was carried out in the forebrain IMHV (intermediate and medial hyperstriatum ventrale) of 1-day-old chicks 30 min after training to avoid pecking at a bead coated with methyl anthranilate. In 10 birds (5 control and 5 trained), the length, curvature and the number of the synaptic active zone profiles were measured, and the active zone profile length was observed to increase in trained chicks. The spatial arrangement of synaptic vesicles with respect to the active zone was examined using a statistical stereological approach. This showed that vesicles are not located uniformly but accumulate in two spatial pools which appear to rearrange following training, with a greater number of vesicles near the active zone. These data may reflect subtle changes in the functional efficacy of synapses in the IMHV in the initial phases of memory formation.

Animals↗
PubMed1993

Changes in the neural cell adhesion molecule patterns on the rat glial cell surfaces with development and contact formation in vitro.

In monolayer cultures of newborn rat hippocampal cells, immunogold-labelling at the electron microscope level was used to study quantitatively the neural cell adhesion molecule (N-CAM) arrangement on the surface of glial soma and processes on 5 and 12 days in vitro (DIV). Four corresponding samples of micrographs were formed. To quantify the labelling, a stochastic geometry approach was used. Spectra of lateral distances between labels as well as simulated images of the surface label arrangement (invisible in micrographs) were derived and compared. The data show that, on both 5 and 12 DIV, N-CAM density on the surface of processes is approximately 2 times higher than that in somata; 12-DIV cells showing a lower (approximately 25%) N-CAM surface density as compared with the 5-DIV cells. This suggests that N-CAM expression in glia surfaces decreases while the cells form contacts, and N-CAM sorting between soma and processes remains stable. The simulated topographies of the lateral N-CAM arrangement might highlight fundamental mechanisms that underlie formation of the neural network.

Animals↗
PubMed1993

Tridimensional organization of Purkinje neuron cisternal stacks, a specialized endoplasmic reticulum subcompartment rich in inositol 1,4,5-trisphosphate receptors.

Stacks of regularly spaced, flat, smooth-surfaced endoplasmic reticulum cisternae frequently observed in both the cell body and dendrites of cerebellar Purkinje neurons, were previously shown by immunocytochemistry to be highly enriched in receptors for the second messenger, inositol 1,4,5-trisphosphate. Morphometric analyses have been carried out on randomly selected thin section images of rat Purkinje neurons to reveal the tridimensional organization of these structures. Individual stacked cisternae (on the average approximately 3.5 per stack) were shown to be separated from each other by a 23.5 nm space occupied by perpendicular bridges, approximately 20 nm in diameter, most probably composed by two apposed receptor homotetramer molecules, inserted into the parallel membranes in their hydrophobic domains. In the stacked membranes the density of the bridges was approximately 500 microns -2, corresponding to approximately 15% of the surface area. The lateral distribution of bridges was not random, but revealed regular distances that might correspond to unoccupied receptor slots. In each stack, the external cisternae were often in direct lumenal continuity with conventional elements of the endoplasmic reticulum, whereas the internal cisternae were not. Since continuities between stacked cisternae were never observed, the results indicate that the internal cisternae are at least transitorily discrete, i.e. they are not in permanent lumenal continuity with the rest of the endoplasmic reticulum. To our knowledge this is the first demonstration of a physical subcompartmentalization of the latter endomembrane system in a non-mitotic cells. A model for the biogenesis of cisternal stacks, based on the head-to-head binding and lateral interaction of the inositol 1,4,5-trisphosphate receptor molecules in the plane of the interacting membranes, is proposed and critically discussed.

Animals↗
PubMed1993

Cytoskeleton-mediated, age-dependent lateral topography of lectin-gold-labelled molecules on the plasma membrane of cultured neurons: a statistical view.

In dissociated spinal cord neurons (12-day-old mouse embryo, monolayer culture), an electron microscopic study was carried out to examine quantitatively the rearrangement of wheat-germ agglutinin-gold-labelled molecules on the neuronal somatic surface at two developmental stages (on the fifth and 15th days in vitro), and after cytoskeletal interruptions. In tests, before labelling the cultures were incubated with colchicine or cytochalasin in order to affect microtubules or mostly actin filaments, respectively. Samples of electron micrographs that display soma membrane (profile) fragments were quantified. A set of stochastic geometry approaches was accomplished, which allowed statistical and stereological analysis of labelling. Images that illustrate the lateral (surface) patterns of label were simulated. On the fifth day in vitro, both colchicine and cytochalasin were found to cause an increase in the surface density and aggregation of wheat-germ agglutinin label relative to controls, the effect of cytochalasin being significantly more profound. By the 15th day in vitro, treatment with both drugs led to a similar tendency towards heavy aggregation of wheat-germ agglutinin labels. In contrast, neuron processes showed an opposite tendency of label rearrangement, which suggests lateral migration of labelled molecules, as a result of drug action. Possible molecular mechanisms involved in the phenomena are discussed.

Aging↗
PubMed1993

Lateral patterns of the neural cell adhesion molecule on the surface of hippocampal cells developing in vitro.

In monolayer cultures of hippocampal neurons from newborn rats, an immunocytochemical quantitative study was carried out to investigate age-dependent arrangement of the neural cell adhesion molecules in different parts of cell membranes. On the fifth and 12th day in vitro, neural cell adhesion molecules were labelled with specific antibodies and protein A conjugated to colloidal gold particles. Samples of randomly selected electron micrographs that displayed labelled membrane fragments of cell bodies, growth cones, and axons were numerically analysed for the five- and 12-day in vitro neurons. Neural cell adhesion molecules surface topography was quantitatively described and compared, using a statistical stereological approach. The mean surface density of labelled neural cell adhesion molecules was found to be approximately 2.5 times higher in growth cone membranes relative to somatic and axonal membranes in five-day in vitro neurons. By the 12th day in vitro, this density decreases in somatic membranes (approximately 18%) and increases in axonal membranes (approximately 60%). Representative spectra of lateral intervals between labels as well as images that show typical topography of label on membrane surfaces were simulated. The results revealed regular patterns of neural cell adhesion molecules on the somatic surface and allowed consideration of neural cell adhesion molecules arrangement in a view of membrane adhesion properties. Participation of cytoskeleton in neural cell adhesion molecules rearrangement is discussed.

Animals↗
PubMed1991

The spatial pattern of the synaptic vesicular apparatus as a correlate of transmitter storage models.

A statistical stereological approach which allows one to derive a three-dimensional pattern of synaptic vesicle accumulation in relation to the active zone from an analysis of electron micrograms (random sections) of synapses is described. This approach is illustrated with a study of presynaptic terminals from the dorsal horn of the cat spinal cord, based on the morphometrical treatment of 105 micrographs containing 5190 synaptic vesicles. The spatial pattern obtained was found to have a bimodal shape, which can be considered a possible structural correlate of the two-pool model of transmitter storage. The connection of similar quantitative estimates with physiological data is discussed.

Animals↗
PubMed1991

[Quantitative characteristics and reconstruction of the surface topography of histochemical markers on the nerve cell plasmalemma].

An electron microscopic study of topography of colloidal gold-bound lectins conjugating to glycocalix carbohydrate residues on the somatic membrane surface of the cultivated spinal neurons has been carried out. The quantitative procedures are suggested for analyzing the surface pattern of the markers: two stochastic functions are considered to correspond properly to the particle distribution observed in the electron micrograms. The analysis of these functions permits obtaining required numerical characteristics. The Monte Carlo reconstructing model is described, and results of its work (on the basis of the above experimental data) are demonstrated in the form of "averaged" surface topography of the studied markers within the membrane fragments bordered. Possible connection of the obtained data with cooperative properties of the membrane is discussed.

Animals↗
PubMed1990

[An evaluation of the spatial morphofunctional characteristics of the presynaptic endings in the lateral area of the base of the dorsal horn].

A statistical stereological pattern allowing estimation of populational three-dimensional characteristics of cell elements on the basis of the analysis of their plain sections has been developed. Using such an approach a morphometrical electron microscopic study of presynaptic terminals (PTs) within the lateral areas of the dorsal horn of the cat spinal cord was carried out. The estimated distribution of PT volumes, PT axolemma areas, sizes of active zones (AZ) have been obtained. The mean number of AZs per a PT, parameters of AZ localization with respect to the terminal axonal expansion have been estimated. The data obtained are compared with the morphometrical estimations for the another PT population studied previously. The connection between the parameters estimated and functional features of the studied synapses are discussed.

Animals↗