Biological tissues are passed through numerous procedures before they can be studied at the ultrastructural level with the electron microscope. Chemical fixation is widely used as a method for preserving structural detail and can be performed by simple immersion or total body vascular perfusion. A 2 to 4% solution of glutaraldehyde buffered with 0.1 M sodium phosphate, or a combination of similarly buffered glutaraldehyde and paraformaldehyde, can be used successfully to preserve the fine structure of biological tissues. The material next is washed briefly in the buffer vehicle and then secondarily fixed in 1% osmium tetroxide (osmic acid), which also is buffered with sodium phosphate. The tissue then is thoroughly dehydrated in solutions of ethanol at increasing concentrations of 50%, 70%, 95%, and 100%. After dehydration, tissues are infiltrated for a prescribed time interval with an epoxy embedding medium. After infiltration, specimens are transferred into fresh epoxy resin and polymerized at 60 to 70 degrees C for several hours. This orderly process ultimately yields fixed tissues that are encased in hardened blocks that can be thin-sectioned with an ultramicrotome. The thin sections are counterstained with solutions of heavy metals to add contrast. The material then can be subjected to the electron beam in an electron microscope to produce useful images for ultrastructural study. This overall procedure has been used successfully since the advent of biological electron microscopy to define the minute details of cells and tissues.

No abstract

Radioiodinated alpha-bungarotoxin (alpha-Bgt) was used to localize alpha-Bgt-acetylcholine receptors in the carotid body of the rat. The gamma spectrometer analyses indicated a high uptake of [125I] alpha-Bgt in carotid bodies incubated in vitro (1.51 fmole per organ). Incorporation of the isotope was effectively blocked by pretreatment of carotid bodies with d-tubocurarine and unlabeled alpha-Bgt, but not by atropine. Light microscopic autoradiography showed a heavy labeling of some parenchymal cells. Electron-microscopic autoradiography revealed that labeling was localized along the interface between parenchymal cells, especially where their cytoplasmic processes engage in complex interdigitations. The silver grain counts on electron-microscopic autoradiographs suggest that labelings are preferentially associated with the plasma membrane of certain Type I cells. It is suggested that these Type I cells in the rat's carotid body probably are provided with nicotinic acetylcholine receptors on their plasma membranes.

Male and female A/Jax mice 10-18 days of age were given one, two, or three daily subcutaneous injections of Serpasil (Reserpine USP; 2.5 mg/ kg) and sacrificed 24 hours after the last injection. Abdominal extra-adrenal tissue was processed for electron microscopy to determine the effects of this catecholamine depleting drug on the dense cared cytoplasmic granules of the parenchymal chief cells. Electron microscopic investigations of sympathetic paraganglia from treated animals revealed a marked decrease in granule opacity as compared to that seen in cells from control animals. The cells with granules reduced in opacity following reserpine treatment could be consistently distinguished from those of nontreated animals which led us to assume that the drug depleted the amine content from its storage site in the granule without completely destroying the granule structure. These results further substantiate our earlier speculations that the granules in abdominal paraganglion chief cells of the mouse contain catecholamines.

Abdominal extra-adrenal chromaffin tissue, or paraganglia, was examined at the ultrastructural level to elucidate the innervation of this adrenal medullary homologue. Paraganglia display unmyelinated nerve fibers surrounded by Schwann cell cytoplasm. These nerves are separated from the paraganglion Type I (granule-containing) cells by cytoplasmic projections of paraganglion Type I1 (satellite) cells. However, serial sections show that the nerves eventually make synaptic contact with the Type I cell. At the axon-chromaffin cell junction, only the outer aspect of the nerve is covered by the satellite cell. The presynaptic endings contain numerous synaptic vesicles, mitochondria and glycogen particles. The vesicles are predominantly of the clear-cored variety, but a few possess centers which are electron opaque. The pre-and postsynaptic membranes are separated by a subsynaptic space and occasionally exhibit the membranal densities usually associated with synaptic areas. These ultrastructural studies establish definite evidence that abdominal paraganglion cells are innervated.The ultrastructure and probable function of abdominal extra-adrenal chromaffin tissue, or paraganglia, have been reported by several investigators (Viragh and Both, '67; Coupland and Weakley, '68; Mascorro and Yates, '70), and detailed morphological studies indicate that paraganglionic tissue is essentially the same as that of the adrenal medulla (Mascorro and Yates, in press). While a preganglionic sympathetic innervation for the adrenal medulla has been described (Cummings, '69), few ultrastructural reports have dealt with this aspect of the paraganglia, and synaptic contacts on these cells have not been reported.Earlier studies with various staining techniques and silver impregnation methods demonstrated unmyelinated nerve fibers within the paraganglia and suggested that extra-adrenal chromaffin tissue receives a preganglionic sympathetic innervation (Pines, '24; Kofmann, '35; Hollinshead, '37). Also, studies on the chromaffin cells in the ganglia of the sympathetic trunks indicated that these receive a direct innervation via the anterior roots of the spinal cord (Karpenko, '64). Recent work has failed to demonstrate an innervation of paraganglion cells (Hervonen, '71 ; Mascor-J. MORPH., 142: 153-164, ro and Yates, '72a). The present study provides evidence that such cells are innervated. MATERIALS AND METHODS Syrian hamsters (Mesocricetus auratus;Lakeview Hamster Colony, Newfield, N J.) 1 to 13 days of age and young squirrel monkeys (Saimiri sciureus; Tarpon Zoo, Florida) were anesthetized with sodium amobarbital (Amytal; 1 mg/kg) and perfused through the heart with a solution of 3.0% glutaraldehyde buffered with 0.2 M sodium phosphate to pH 7.2-7.4. The retroperitoneal area from the renal vessels caudal to the abdominal aortic bifurcation was removed, divided into several transverse slices and fixed in the perfusion fluid an additional two hours. After a two hour wash in phosphate buffer containing 10 % sucrose, the tissue slices we...