Pavel V. Belichenko

Morphological study of neocortical areas in Rett syndrome.

Abstract Various neocortical areas from four females aged 16–24 years with Rett syndrome (RS) were investigated and compared with brains of therapy-resistant partial epilepsy (TRPE) patients (18–25 years), infantile autism (IA), and control brains (24 and 58 years). The cytoarchitecture of area 10 (frontal), area 21 (temporal), area 4 (primary motor cortex), and area 17 (primary visual cortex) was studied by the combined Klüver-Barrera (luxol fast blue and cresyl violet) standard procedure. Autofluorescence of lipofuscin, immunofluorescence of synaptic vesicle proteins [synaptophysin (p38)] and lectin-stained (Wisteria floribunda agglutinin) perineuronal nets (PNs) were studied in the cortices using dual-channel confocal laser scanning microscopy. The brains of RS females show various types of morphological/cytoarchitectonical abnormalities of single pyramidal neurons in layers II–III, and V–VII of different cortical areas. The abnormalities include mild losses of pyramidal neurons, more pronounced in layers II and III than in layers V and VII, and more evident in frontal and temporal areas than in the visual cortex. Microdysgenesis, including abnormalities due to neuronal migration disorders, was not found in RS, in contrast to the observations in TRPE patients, strongly indicating that RS is not a neuronal migration disorder. Lipofuscin distribution was normal but amounts were lower in RS cases than in control and TRPE brains. PNs were less expressed in cortices of the IA case, but were clearly overexpressed in the motor cortex of RS. Quantitative analysis of p38 showed a decrease in the area occupied by p38 immunoreactivity by 20–40% in RS compared with controls. It is concluded that RS could best be explained by a postnatal synaptogenic developmental deficiency; the basic defect, however, is still completely unknown.

Calretinin-positive Cajal-Retzius cells persist in the adult human neocortex

The occurrence of Cajal–Retzius (CjRe) cells was studied in four cortical areas of five normal adult humans using antibodies against calretinin. Calretinin immunofluorescence and autofluorescence of lipofusin granules in CjRe cells were visualized by dual channel confocal laser scanning microscopy. Three types of CjRe cells existed in the adult human cortex: horizontal, triangular and multipolar, and their number did not decrease with ageing. Horizontal CjRe cells were found in all cortical areas; they contained no lipofusin or less than other cells. Triangular CjRe cells with descending dendrites were less numerous. Multipolar CjRe cells were rare and contained more lipofuscin. We conclude that calretinin-immunoreactivity can be used to study CjRe cell morphology in normal and diseased adult human brain.

Dendritic changes of the pyramidal neurons in layer V of sensory-motor cortex of the rat brain during the postresuscitation period

Experiments were performed on 40 Wistar rats. Total brain ischemia was induced by 10 min clamping of the cardiac blood vessels. The brains were examined in control rats, after 90 min and after 1, 3, 7, 30 and 90 days during the postresuscitation period. Histological sections were stained with the Golgi method. Morphometrical parameters, 12, of dendritic changes of the pyramidal neurons in layer V of sensory motor cortex (SMC) in rat brain were studied at different intervals of the postresuscitation period. Reduction of the dendrites of the pyramidal neurons due to the loss of the terminal branches of the oblique apical dendrites in layer III-IV was detected from the first day after ischemia. The maximal dendritic change was detected 3 and 7 days after ischemia. Decrease the volume of dendritic territory (54, 4%), the total dendritic length of the whole dendritic territory (56, 0%) and branching of dendrites, and decrease in the number of dendritic spines on apical dendrites in layers I-II (46, 1%) were the main changes during this period. Reduction of the total length of dendrites occurred mostly due to disappearance of the 2nd and 3rd order branches of the apical and basal dendrites. The change of dendrites neurons had returned to control levels after 30 days. By that time the diameter of the dendrites had increased, the varicosities on oblique apical and basal dendrites had disappeared, and the number of 2nd and 3rd order dendrites and of dendritic spines had increased.

Dual channel confocal laser scanning microscopy of lucifer yellow-microinjected human brain cells combined with Texas red immunofluorescence

A method for visualization of individual human brain cells and their dendritic extensions in combination with immunofluorescence is described. Microinjection of Lucifer Yellow was used to reveal the dendritic morphology of cortical brain cells. Indirect immunofluorescence with Texas Red as label was used to investigate the distribution of 3 different groups of immunogens: enzymes (monoamine oxidase A and B), receptors (beta-adrenoceptor protein), and synaptic vesicle proteins (synapsin I and synaptophysin) in each cortical slice. A dual-channel confocal laser scanning microscope with an argon/krypton laser was used for imaging these double-stained fluorescent specimens. Lucifer Yellow and Texas Red were recorded simultaneously or separately, taking advantage of the different activating lines (488 lambda and 568 lambda) of the laser and using the two filter blocks (K1 and K2) supplied with the instrument (BioRad MRC-600) for recording the emission of either fluorophore. Using this technique we have demonstrated the localization of immunoreactive material in relation to the dendritic morphology of cortical cells.

Micromapping of the human brain: Three‐dimensional imaging of immunofluorescence and dendritic morphology using dual‐channel confocal laser scanning microscopy

A strategy for investigating neuronal networks at the microscopic level is described. The Lucifer Yellow microinjection technique was combined with immunofluorescence on human brain material, which was then studied in a confocal laser scanning microscope with dual‐channel scanning and equipped with an argon/krypton laser. The three‐dimensional architecture of the Lucifer Yellow‐injected neurons was investigated after transfer of the scanned frames in file format to a Silicon Graphics IRIS computer, using VoxelView software from Vital Images Inc. Microinjection of Lucifer Yellow revealed the dendritic morphology of various types of cells in different brain areas. Indirect immunofluorescence, with Texas Red as the secondary label, was used to determine the distribution of various categories of macromolecules (enzymes, receptor protein, and synaptic vesicle proteins) in the brain slices. We used single‐ as well as dual‐channel confocal laser scanning microscopy for imaging these double‐stained fluorescent specimens. Using these techniques in combination, we have created and saved three‐dimensional confocal images of detailed morphology (axons and dendrites with spines and varicosities) of individual cells, together with the localization of immunofluorescence. These three‐dimensional confocal images will be collected in a database for probable future use in human brain mapping.

Relationship between oligodendrocytes and axons.

Oligodendrocytes can myelinate a variable number of axons in their surroundings; however, the mechanisms underlying axon—oligodendrocyte associations are unknown. We tested the hypothesis that single oligodendrocytes exclusively myelinate axons belonging to the same functional system. Carbocyanine dyes (DiI, FAST DiI) were applied to the sternomastoid muscle of the rat and allowed to transport retrogradely for 4 weeks within the motor axons. Using fluorescence microscopy and iontophoretic injection of Lucifer Yellow (LY), oligodendrocytes were injected in the proximity of retrogradely carbocyanine dye-labeled axons. Using dual channel confocal laser scanning microscopy (CLSM), the three-dimensional relationship between axons and glia was studied. The data indicate that a single oligodendrocyte can myelinate retrogradely labeled axons concomitantly with other, unlabeled axons belonging to apparently different functional systems.

Quantitative analysis of synaptophysin immunoreactivity in human neocortex after cardiac arrest: confocal laser scanning microscopy study.

Transient global ischaemia caused by cardiac arrest results in lesions that involve all brain structures. The aim of this study was to investigate the condition of synapses in patients surviving, but remaining in a persistent vegetative state, following resuscitation after cardiac arrest. We performed a quantitative analysis of the distribution and density of elements containing a synaptic vesicle protein--synaptophysin (p38)--in human neocortex in cases which survived for 1 week, 2 months, and 1 year after the cardiac arrest. Neurologically healthy cases that died following an accident served as control. Dual-channel confocal laser scanning microscopy (CLSM) was used to image p38-immunoreactivity (IR) and lipofuscin autofluorescence. In control cases no statistically significant differences were found for p38-IR between layers II-III and V-VII. However, the temporal cortex had a higher density of p38-immunoreactive structures than the motor cortex. In postischaemic cases a reduction in the density of p38-IR elements was apparent, mainly in the frontal and motor cortices and less pronounced in the temporal cortex. The least decrease compared with controls was observed in the visual cortex. In the 1 week survival case, a maximal decrease in p38-IR (35% below control) was found. In this case, the number of p38-IR elements per visual field was decreased, and big aggregates of p38-IR structures were observed. In general, the amounts of p38-IR structures were higher in all of the control cases compared with the postischaemic cases.

The cellular distribution of GAP-43 immunoreactivity in human neocortical areas using immunofluorescence and confocal microscopy: post-ischemic influence.

The distribution of growth associated protein-43 (GAP-43) immunoreactive (IR) neurons were studied in human neocortical areas, using immunofluorescence and confocal microscopy. The GAP-43-IR cells were generally localised close to blood vessels, and contained fewer lipofuscin granules than GAP-43 negative cells. Quantification of the relative number of GAP-43-IR cells in control cases showed that the highest number of GAP-43-IR cells were present in layers III and V in the motor and visual cortices, fewer in the temporal cortex, and the lowest number in the frontal cortex. After general ischemia, GAP-43-IR cells were significantly reduced at various survival times, with the counts being lowest in the 1 week surviving case, and higher, but still subcontrol, in the 1 year post-ischemic case.

Application of Confocal Microscopy for the Study of Neuronal Organization in Human Cortical Areas after Microinjection of Lucifer Yellow

The relationship between 2-D or 3-D neuronal morphology and the intrinsic organization of different cortical areas in the human brain was investigated after intracellular microelectrode injection of Lucifer Yellow (the Lucifer Yellow-method). In some areas pyramid and nonpyramidal neurons were differentiated by the presence of autofluorescent lipofuscin granules. The Lucifer Yellow-method allows the visualization in a brain specimen obtained from the neurosurgery clinic of a large number (200–300) of single neurons including the dendrites and axons with collaterals in a small area of tissue. The method has been tested in the study of frontal, temporal, parietal, and occipital cortices, and in the hippocampus in normal and pathological human brain areas. The number of neurons is sufficient to allow all necessary morphometrical investigations needed for each patient. Since neurons are inherently 3-D in their organization, 3-D reconstructions and analysis of neurons using confocal laser scanning microscopy (CLSM) in combination with VoxelViewTM software from Vital Images Inc. gives additional information to the classical 2-D representation on neuronal network organization. Data can be demonstrated using 3-D pseudocoloured moving reconstructions on video tapes. These new data represent a starting point for contributions from our laboratories to the Human Brain Mapping Project.