Electron microscopic and immunocytochemical study of rapidly frozen, freeze-substituted neural lobes of rats

Abstract

Rapid freezing of freshly dissected or incubated neural lobes was explored as a means of obtaining ultrastructural preservation of the more natural state of this tissue. A quantitative assessment of the region of good fixation was made in order to determine the relative fractions occupied by axons, pituicytes and the extracellular space. The immunocytochemical distributions of neurophysins and the glycopeptide portion of the vasopressin precursor were evaluated using the immunogold technique in order to determine the relative numbers of oxytocin and vasopressin fibre types in the fixed region, and the subcellular localization of these antigens. The uncut surface of rat neural lobes was rapidly frozen against a highly polished copper plug and freeze-substituted in osmium-acetone either immediately after dissection (approximately 2 min), or after a 15 min incubation period in vitro in an oxygenated, balanced salt solution. Substituted neural lobes were prepared for either conventional electron microscopy, or for immunogold labelling of neurophysins and the glycopeptide precursor to vasopressin. Membranes, subcellular organelles and extracellular matrix were well preserved 10 microns deep to the contacted surface. The extracellular space accounted for approximately 30% of the cross-sectional area of the neuropil and could be divided into two domains: an extended perivascular space (28-29% of total area); and a narrow (approximately 24 nm; approximately 1% of total) space between closely apposed neurosecretory processes or between these processes and pituicytes. Pituicytes accounted for about 30% of the area and axons 20-25%. Pituicytes occupied close to 60% of the basal lamina at the neurohaemal contact zone, while axons occupied approximately 20%. There were no differences between neural lobes frozen immediately after dissection and those incubated for 15 min in any of these measures, suggesting minimal fluid redistribution. Gold particles were specifically localized over large (100-200 nm) dense core vesicles, and less frequently over multivesicular bodies and lysosomes. No etching of the plastic or reduction of osmium was necessary to achieve labelling. Specific labelling of one set of terminals and axons (about 80%) was observed with the monoclonal antibody previously shown to be specific for oxytocin-neurophysin, while in neighbouring sections the remaining 20% of the processes were labelled with the antiserum to the vasopressin precursor, or with non-specific antibodies to neurophysins. In conclusion, ultrarapid freezing preserves a large extracellular space in the neural lobe and provides for high resolution morphological and immunocytochemical studies of neurohypophysial structure.

Freeze substitution after fast‐freeze fixation in preparation for immunocytochemistry

“…Moreover, etching obviously caused deterioration of the ultrastructural detail. A similar observation has been reported by Tian et al (1991). Conclusion.…”

Neurochemical Regulation of Oxytocin Secretion in Lactation*

“…Although there is considerable overlap in their localization, OT fibers are preferentially distributed in the perimeter of the lobe, while VP fibers tend to localize more centrally (304,305). The extent of branching of individual fibers within the neurohypophysis is not well studied, but it is known that the individual axons have numerous, perhaps thousands, of swellings (106,111,306), some of which are large Herring bodies, which may function primarily as storage rather than release sites (307).…”

“…Panel A, Electron micrograph of a rapidly frozen, freeze-substituted neural lobe from a male rat, showing the neurohemal contact zone. The tissue was labeled for OT-neurophysin using a postembedding, immunogold technique(304). Gold particles are most densely located over the dense core vesicles in nerve profiles, (arrows, arrowheads), but a certain amount of background labeling is apparent.…”

Analysis of K+ accumulation reveals privileged extracellular region in the vicinity of glial cells in situ

“…For the CNS, recent data from electron microscopy techniques that preserve the ECS (Tian et al, 1991), radiotracer methods (Levin et al, 1970), and particularly in vivo measurements with the real‐time iontophoretic method (Nicholson and Philips, 1981) indicate that the average ECS volume is about 20–24% of the brain volume; i.e., the ECS volume fraction, defined as the ratio of the ECS volume to the total volume of the brain, is 0.20–0.24 (for reviews see Nicholson and Syková, 1998; Nicholson, 2001). During pathological situations, such as ischemia or anoxia, however, the ECS volume fraction can decrease dramatically to 0.03–0.05 (for reviews see Syková et al, 2000; Syková, 2001).…”