scholarly journals Demonstration of Extracellular Space by Freeze-Drying in the Cerebellar Molecular Layer

1966 ◽  
Vol 1 (2) ◽  
pp. 223-228
Author(s):  
A. VAN HARREVELD ◽  
S. K. MALHOTRA
Keyword(s):  
Tight Junctions ◽  
Electron Micrographs ◽  
Extracellular Space ◽  
Freeze Drying ◽  
Circulatory Arrest ◽  
Molecular Layer ◽  
Freeze Substitution ◽  
Appreciable Amount ◽  
Nerve Fibres ◽  
Cerebellar Molecular Layer

In electron micrographs of the molecular layer of the mouse cerebellum frozen within 30 sec of circulatory arrest and subsequently dried at -79 °C an appreciable extracellular space was found between the axons of the granular cells. Tight junctions were regularly observed between pre- and postsynaptic structures and the enveloping glia cells. In micrographs of cerebellum frozen 8 min after decapitation the space between the axons was absent and tight junctions between the nerve fibres were almost exclusively encountered. The extracellular space of asphyxiated and non-asphyxiated tissue in electron micrographs of frozen-dried material is similar to the space in comparable tissues treated by freeze-substitution. These observations suggest that there is an appreciable amount of extracellular material in oxygenated, living tissue whichis taken up by cellular elements during asphyxiation.

scholarly journals A STUDY OF EXTRACELLULAR SPACE IN CENTRAL NERVOUS TISSUE BY FREEZE-SUBSTITUTION

1965 ◽  
Vol 25 (1) ◽  
pp. 117-137 ◽  
Author(s):  
A. Van Harreveld ◽  
Jane Crowell ◽  
S. K. Malhotra
Keyword(s):  
Tight Junctions ◽  
Extracellular Space ◽  
Nervous Tissue ◽  
Water Distribution ◽  
Circulatory Arrest ◽  
Freeze Substitution ◽  
Synaptic Cleft ◽  
Uranyl Acetate ◽  
Central Nervous Tissue ◽  
Silver Mirror

It was attempted to preserve the water distribution in central nervous tissue by rapid freezing followed by substitution fixation at low temperature. The vermis of the cerebellum of white mice was frozen by bringing it into contact with a polished silver mirror maintained at a temperature of about -207°C. The tissue was subjected to substitution fixation in acetone containing 2 per cent OsO4 at -85°C for 2 days, and then prepared for electron microscopy by embedding in Maraglas, sectioning, and staining with lead citrate or uranyl acetate and lead. Cerebellum frozen within 30 seconds of circulatory arrest was compared with cerebellum frozen after 8 minutes' asphyxiation. From impedance measurements under these conditions, it could be expected that in the former tissue the electrolyte and water distribution is similar to that in the normal, oxygenated cerebellum, whereas in the asphyxiated tissue a transport of water and electrolytes into the intracellular compartment has taken place. Electron micrographs of tissue frozen shortly after circulatory arrest revealed the presence of an appreciable extracellular space between the axons of granular layer cells. Between glia, dendrites, and presynaptic endings the usual narrow clefts and even tight junctions were found. Also the synaptic cleft was of the usual width (250 to 300 A). In asphyxiated tissue, the extracellular space between the axons is either completely obliterated (tight junctions) or reduced to narrow clefts between apposing cell surfaces.

The Microenvironment of Neurons

1970 ◽  
Vol 28 ◽  
pp. 136-137
Author(s):  
William Bondareff
Keyword(s):  
Electron Micrographs ◽  
Extracellular Space ◽  
Brain Volume ◽  
Freeze Substitution ◽  
Electron Microscopic ◽  
Morphological Studies ◽  
Microscopic Studies ◽  
Distribution Composition ◽  
And Function ◽  
The Brain

Neurons in the central nervous system are separated by extracellular spaces, the distribution, composition and function of which are not unequivocally known. Earlier electron microscopic studies of chemically-fixed tissues demonstrated extracellular spaces composed of uniformly narrow, apparently empty channels constituting 3-5% of the brain volume. The results of more recent morphological and non-morphological studies support the existence of less uniform intercellular channels, varying in dimension from 100 Å to almost 1 μ, and constituting 20-25% of the brain volume. Although this space is not revealed in electron micrographs of chemicallyfixed nervous tissues, it is demonstrated readily in specimens fixed by freeze-drying or freeze-substitution (Fig. 1). The dynamic nature of those extracellular spaces, as visualized in electron micrographs of nervous tissue fixed by freeze-substitution, was demonstrated in studies of normalbrain maturation. An extracellular space of 40% became gradually smaller as development proceeded to reach the smaller extracellular space characteristic of mature animals.

scholarly journals Perfusion Fixation With Glutaraldehyde and Post-Fixation With Osmium Tetroxide for Electron Microscopy

1968 ◽  
Vol 3 (4) ◽  
pp. 579-594
Author(s):  
A. VAN HARREVELD ◽  
F. I. KHATTAB
Keyword(s):  
Cerebral Cortex ◽  
Extracellular Space ◽  
Water Distribution ◽  
Osmium Tetroxide ◽  
Circulatory Arrest ◽  
Freeze Substitution ◽  
Tissue Conductivity ◽  
Initial Drop ◽  
Previously Treated ◽  
Perfusion Fixation

The conductivity of cerebral cortex drops during perfusion with glutaraldehyde in 5 min to about 60% of the original value, to remain unchanged during the subsequent 10-15 min of perfusion. Circulatory arrest causes a similar drop in the tissue conductivity. Perfusion of asphyxiated tissue with glutaraldehyde does not produce additional major changes in the conductivity. Perfusion of the cortex with an osmium tetroxide solution causes an initial drop in conductivity. However, after about 3 min this trend is reversed and the conductivity increases again to close to the pre-perfusion value. Perfusion of asphyxiated cortex with OsO4 causes a marked increase of the conductivity. So does perfusion with an OsO4 solution of tissue previously treated with glutaraldehyde. One interpretation of these impedance changes is that glutaraldehyde perfusion causes, like asphyxiation, a transport of extracellular material into the intracellular compartment and that during OsO4 perfusion an extracellular space is again created. This possibility is supported by electron micrographs made of this material. Cerebral cortex perfused with glutaraldehyde and post-fixed with OsO4 shows electron-transparent dendritic elements and to a lesser extent pre-synaptic terminals, which seem to be swollen. When the cortex is flooded with a salt solution during glutaraldehyde perfusion the dendrites exhibit ballooning in the surface layer of the cortex, suggesting that the fluid on the cortex participates in the swelling. The tissue elements in the glutaraldehyde-perfused and OsO4 post-fixed cortex are separated by narrow extracellular spaces. The latter may have been produced by the OsO4 perfusion as is suggested by a comparison of micrographs prepared by freeze substitution (which tends to preserve the water distribution) of glutaraldehyde-perfused but not post-fixed cortex with micrographs of cortex treated with OsO4 after the glutaraldehyde perfusion. In accordance with the conductivity changes, the former micrographs showed very little extracellular space, and in many places tight junctions, whereas the latter showed clefts between the tissue elements.

Ca-Histochemistry in slime mold plasmodia

1978 ◽  
Vol 36 (2) ◽  
pp. 130-131
Author(s):  
Ulrich Dierkes
Keyword(s):  
Physarum Polycephalum ◽  
Carbon Coating ◽  
Freeze Drying ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Slime Mold ◽  
Staining Procedure ◽  
Protoplasmic Streaming ◽  
Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

High-Resolution Scanning Electron Microscopy of Biological Specimens

1988 ◽  
Vol 46 ◽  
pp. 186-187
Author(s):  
M. Müller ◽  
R. Hermann
Keyword(s):  
High Resolution ◽  
Freeze Drying ◽  
Room Temperature ◽  
Structural Information ◽  
Freeze Substitution ◽  
Critical Point Drying ◽  
Sem Study ◽  
Major Factors ◽  
Structural Preservation ◽  
Preparation Techniques

Three major factors must be concomitantly assessed in order to extract relevant structural information from the surface of biological material at high resolution (2-3nm).Procedures based on chemical fixation and dehydration in graded solvent series seem inappropriate when aiming for TEM-like resolution. Cells inevitably shrink up to 30-70% of their initial volume during gehydration; important surface components e.g. glycoproteins may be lost. These problems may be circumvented by preparation techniques based on cryofixation. Freezedrying and freeze-substitution followed by critical point drying yields improved structural preservation in TEM. An appropriate preservation of dimensional integrity may be achieved by freeze-drying at - 85° C. The sample shrinks and may partially collapse as it is warmed to room temperature for subsequent SEM study. Observations at low temperatures are therefore a necessary prerequisite for high fidelity SEM. Compromises however have been unavoidable up until now. Aldehyde prefixation is frequently needed prior to freeze drying, rendering the sample resistant to treatment with distilled water.

Chemical and histological space determinations in rabbit heart

1962 ◽  
Vol 202 (3) ◽  
pp. 589-592 ◽  
Author(s):  
John A. Johnson ◽  
Mary A. Simonds
Keyword(s):  
Weight Change ◽  
Extracellular Space ◽  
Chemical Space ◽  
Freeze Substitution ◽  
Rabbit Heart ◽  
Quantitative Agreement ◽  
Tissue Water ◽  
Histological Techniques ◽  
Chemical Technique ◽  
Osmotic Activity

The extracellular space of perfused rabbit heart ventricles was estimated by chemical and histological techniques. Sucrose and thiocyanate were used for the chemical space estimations. The method proposed by Chalkley following fixation by freeze substitution was used for the histological extracellular space determination. The extracellular space fraction was altered by using perfusing solutions of different osmotic activity to shrink or swell the cells. The magnitude of the tissue water loss or gain was determined by weighing the heart. Using the assumption that the cells alone changed in volume during an osmotically induced weight change, an alteration in the fractional extracellular space was predicted from the original chemical or histological space determination and the magnitude of the weight change. This predicted change when compared to the observed change in the histological and chemical spaces always agreed in direction with the measured values of both the histological and chemical spaces but was in better quantitative agreement with the values from the chemical technique.

scholarly journals Nitric Oxide Signaling Strengthens Inhibitory Synapses of Cerebellar Molecular Layer Interneurons through a GABARAP-Dependent Mechanism

2020 ◽  
Vol 40 (17) ◽  
pp. 3348-3359 ◽  
Author(s):  
Erik A. Larson ◽  
Michael V. Accardi ◽  
Ying Wang ◽  
Martina D'Antoni ◽  
Benyamin Karimi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Molecular Layer ◽  
Inhibitory Synapses ◽  
Nitric Oxide Signaling ◽  
Cerebellar Molecular Layer ◽  
Dependent Mechanism
Sign in / Sign up

Export Citation Format

Share Document