The microtrabecular lattice of the adrenal medulla revealed by polyethylene glycol embedding and stereo electron microscopy.

Stereo transmission electron microscopy of polyethylene glycol (PEG)-embedded rat adrenal medulla reveals the ground cytoplasm of chromaffin cells and preganglionic nerve endings to be a three-dimensional lattice of microtrabeculae that is contiguous with the surface of chromaffm granules and synaptic vesicles and also with the plasma and presynaptic membranes. The microtrabeculae, because of their association with the granules and vesicles, are implicated with the translocation of these structures as well as in the secretion and release of catecholamines and other neurotransmitters. A current belief is that the release of secretory products and transmitters from endocrine cells and nerve endings occurs after a directed deployment of secretory granules and synaptic vesicles toward the inner surface of the plasma membrane (cf., Pappas and Waxman, 1972; Heuser and Reese, 1977). Moreover, it has been suggested that there is a structural organization in endocrine cells and nerve endings which serves as an internal pathway for the movement of the secretory granules and synaptic vesicles. Although the presence of filamentous networks has been reported in neurons (Ellisman and Porter, 1980; LeBeux and Willemot, 1975a, b), these are poorly defined in conventional electron microscopic images of epoxyembedded preparations. This is due in large part to the electron-scattering properties of the epoxy resin in addition to the inability to add sufficient contrast to the embedded structures. The cytoplasmic ground substance of whole, unembedded, critical-point dried cultured cells has been interpreted to have an organized structure (Wolosewick and Porter, 1976, 1979). This structured cytoplasmic ground substance consists of slender strands, termed microtrabeculae, which, together with cytoskeletal and membranous organelles, form an irregular three-dimensional lattice, i.e., the microtrabecular lattice (MTL; Wolosewick and Porter, 1976, 1979). The microtrabeculae vary from ’ This work was supported by National Institutes of Health Grants NS 16610 and GM 28397. * To whom correspondence should be addressed at Department of Anatomy, University of Illinois at the Medical Center, 808 South Wood Street, Chicago, IL 60612. 3 to 12 nm in diameter and are over 50 nm in length. They are contiguous with the membranous and nonmembranous organelles and are confluent with the cortices of the cytoplast. The microtrabeculae represent the nonrandom organization of the cytoplasmic ground substance of living cells (Wolosewick, 1980). A similar lattice structure was demonstrated in resin-less, thin-sectioned preparations using the polyethylene glycol (PEG)-embedding method which enhances remarkably the appearance of the cytoskeletal structures (microtrabeculae, microtubules, microfilaments, and intermediate filaments; Wolosewick, 1980). Before addressing the possibility that the microtrabecular lattice might be involved in the movement of the granules and vesicles, we sought a description of the MTL in chromaffin cells and preganglionic nerve endings. This study utilized the PEG-embedding method and stereo electron microscopy. A specific, contiguous association of the microtrabeculae with chromaftin granules and synaptic vesicles, as well as the plasma membrane, is described. Materials and Methods Adult albino rats weighing 150 gm were anesthetized by intraperitoneal injection of pentobarbital and fixed by whole body vascular perfusion with 2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate (pH 7.4). The adrenals were removed 10 min after perfusion, minced in fresh fixative, and immersed in the fixative for an additional 2 hr. The tissues were subsequently bathed for 2 l-n in 2.5% glutaraldehyde containing 0.5% tannic acid in the sodium cacodylate buffer. The blocks were washed 58 Kondo et al. Vol. 2, No. 1, Jan. 1982 in the buffer (1 hr), postfixed in 1% buffered osmium tetroxide for 2 hr, and stained (1 hr) en bloc with 1% aqueous uranyl acetate. The blocks were dehydrated in ethyl alcohol. Three separate protocols were subsequently followed. PEG embedding. The blocks in absolute alcohol were transferred to a 50% solution of PEG-6000 (Sigma Chemical Co., St. Louis, MO) in 100% absolute ethanol (v/v) and infiltrated (vials uncovered) for 12 hr at 60°C. The blocks were transferred to pure molten PEG for 2 hr, embedded in PEG contained in well dried gelatin capsules, and rapidly solidified by immersion in swirling liquid nitrogen. Sections were made with glass knives using a Sorvall MT-l ultramicrotome and mounted onto Formvar-coated grids which had been treated with polyL-lysine (0.1% in distilled water). The mounting of the sections and removal of the PEG from the sections was carried out in a solution of 95% ethanol containing 5% PEG. The grids were placed in a muliple grid holder and transferred into 95% ethanol. After exchanging the 95% ethanol with absolute ethanol several times, the grids were dried either by the critical point process from CO2 (Wolosewick, 1980) or by desiccator dried from Freon (Becker et al., 1981). The dried grids were stabilized by carbon evaporation and observed with a JEOL-100 CX electron microscope operated at 100 kV. Epon embedding. Tissues fixed and dehydrated as described above were treated with three changes of propylene oxide (10 min each), infiltrated, and embedded in Epon 812. Thin sections were cut with diamond knives on a Sorvall MT-2 ultramicrotome, mounted on uncoated 300 mesh copper grids, stained with aqueous uranyl acetate and lead citrate, and viewed in the JEOL-100 CX electron microscope. PEG-Epon control. Specimens embedded in PEG were soaked in warm 95% ethyl alcohol for 2 hr. This is sufficient time for the removal of the PEG. The specimens were treated in propylene oxide, infiltrated, embedded in Epon, and sectioned as described above.