Giant axons from the squid, Loligo pealei, were fixed in glutaraldehyde and posttixed in osmium tetroxide. Calcium chloride (5 mM/liter) was added to all aqueous solutions used for tissue processing. Electron-opaque deposits were found along the axonal plasma membranes, within mitochondria, and along the basal plasma membranes of Schwann ceils. X-ray microprobe analysis (EMMA-4) yielded signals for calcium and phosphorus when deposits were probed, whereas these elements were not detected in the axoplasm. I N T R O D U C T I O NMany cellular processes appear to be mediated by reversible interactions of ionized calcium with the plasma membrane. We have been interested in using the electron microscope to locate membrane areas with high affinity for calcium. Addition of calcium ions to the solutions used for fixing and processing tissues for the electron microscope causes opaque deposits to be formed along the plasma membranes of a variety of ceils (I). In insect intestine, for example, deposits occur on apical membranes (microvilli) and along septate but not along gap junctions (2). Apparently, particular regions of the plasma membrane, or some structure closely associated with the membrane, can sequester enough calcium to make the region adjacent to the membrane opaque to electrons. Although we do not know precisely how the deposits form, we suspect that the presence of calcium ions during processing keeps intraceUular binding sites saturated. The deposits were not observed readily in the past because of the widespread use of osmium, which decalcifies tissues unless excess calcium is present in the fixative.To assess the significance of the deposits adjacent to the membranes, one must know their composition and how they form. The present study was undertaken to utilize the rapidly developing technique of electron-probe X-ray microanalysis (3, 4) to study the composition of the deposits. The analytical electron microscope is a transmission electron microscope fitted with X-ray spectrometers that analyze X rays generated when a micro-area of the specimen is bombarded by the electron beam. Since each element produces X rays of characteristic energy and wavelength, information is obtained about the composition of the specimen. Microprobe analysis is well suited for analyzing bound ions, since conventional fixation and embedding techniques can be used.

Flame photometry reveals that glutaraldehyde and buffer solutions in routine use for electron microscopy contain varying amounts of calcium . The presence of electron-opaque deposits adjacent to membranes in a variety of tissues can be correlated with the presence of calcium in the fixative . In insect intestine (midgut), deposits occur adjacent to apical and lateral plasma membranes . The deposits are particularly evident in tissues fixed in glutaraldehyde without postosmication . They are also observed in osmicated tissue if calcium is added to wash and osmium solutions . Deposits are absent when calcium-free fixatives are used, but are present when traces of CaCl 2 (as low as 5 X 10-5 M) are added . The deposits occur at regular intervals along junctional membranes, providing images strikingly similar to those obtained by other workers who have used pyroantimonate in an effort to localize sodium . Other divalent cations (Mg++ Sr++ Ba++ Mn++, Fe++) appear to substitute for calcium, while sodium, potassium, lanthanum, and mercury do not . After postfixing with osmium with calcium added, the deposits can be resolved as patches along the inner leaflet of apical and lateral plasma membranes . The dense regions may thus localize membrane constituents that bind calcium . The results are discussed in relation to the role of calcium in control of cell-to-cell communication, intestinal calcium uptake, and the pyroantimonate technique for ion localization .

The rectum of Periplaneta americana L. is lined with cuticle and has six radially arranged cushion-shaped thickenings, the rectal pads, composed of columnar cells. Narrow strips of simple rectal cells lie between the pads. Tall junctional cells form a thin but continuous collar around the pads where they join the rectal cells. The epithelium is surrounded by a layer composed of circular and longitudinal muscles and connective tissue. This layer of muscle and connective tissue is innervated and tracheated, and is separated from the pad surface by a subepithelial sinus. Fluid flowing through the sinus enters the haemolymph through openings in the muscle layer whre large tracheae penetrate. These openings can be sealed by muscle contractions that appress the muscle around the openings against the pad surface. The tracheae pass on into the pads, following basement membrane-lined indentations of the pad surface. Within the pad tracheolar cells send fine branches between the cells. Near the apical and basal surfaces the lateral membranes of pad cells are bridged by septate desmosomes that form a continuous band around the cells. Between apical and basal septate desmosomes is a n interconnected labyrinthine system of intercellular spaces. There are three kinds of space, dilations and apical sinuses, both of variable size, and narrow communicating channels about 200 A wide. The membranes of the latter have mitochondria closely associated with them. Continuity between the system of spaces and the subepithelial sinus is established by the basement membrane-lined invaginations of the basal surface where tracheae penetrate between pad cells. Apical surfaces of the pad cells are highly infolded and are also associated with mitochondria. However, unlike the lateral membranes facing the narrow channels, the apical membranes have a cytoplasmic coating of particles. Both associations of mitochondria with membranes constitute discrete structural entities that are found in many transporting epithelia, and we have termed them "plasmalemma-mitochondria1 complexes." As the rectal pads are organized into systems of spaces that ultimately open in the direction of fluid movement, existing models of solute-coupled water transport can be applied. However, the rectal pads are structurally more complex than fluid-transporting tissues of vertebrates. This complexity may be related to the ability of the rectum to withdraw water from ion-free solutions in the lumen. We present a structural model involving solute recycling to explain the physiological characteristics of rectal reabsorption.

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This paper describes the different regions of the Malpighian tubules and the associated structures (ampulla, midgut, ileum) in the cockroach, Periplaneta americana. There are about 150 tubules in each insect. Each tubule consists of at least three parts. The short distal region is thinner than the other parts and is highly contractile. The middle region comprises most of the tubule length and is composed of primary and stellate cells. Primary cells contain numerous refractile mineral concretions, while stellate cells have smaller nuclei, fewer organelles, simpler brush border, and numerous multivesicular bodies. Symbiont protozoa are sometimes present within the lumen of the middle region near where it opens into the proximal region of the tubule. The latter is a short region that drains the tubular fluid into one of the six ampullae. These are contractile diverticula of the intestine located at the midgut-hindgut junction. The ampulla is highly contractile, and consists of a layer of epithelial cells surrounding a cavity that opens into the gut via a narrow slit lined by cells of unusual morphology. The proximal region of the tubule and the ampulla resemble the midgut in that they have similar micromal origin and reabsorptive function for the proximal region of the tubule and for the ampulla. A number of inclusions found within the tubule cells are described, including peroxisomes and modified mitochondria. Current theories of fluid transport are evaluated with regard to physiological and morphological characteristics of Malpighian tubules. The possible role of long narrow channels such as those between microvilli and within basal folds is considered, as is the mechanism by which these structures are formed and maintained. Also discussed is the role of peroxisomes and symbionts in the excretory process.