Immunolocalization of a novel, cytoskeleton-associated polypeptide of Mr 230,000 daltons (p230).

Antibodies were raised against a cytoskeleton-associated, nonphosphorylated, 230,000-dalton bovine lens polypeptide (designated p230), and rendered monospecific by using a novel immunoaffinity technique. In immunofluorescence and electron microscopy of cultured fibroblasts, as well as of various other cells (endothelial, epithelial, lenticular, monocytes, neuroblastoma cells) and tissues (human kidney and liver), p230 was localized as a distinct subplasmalemmal layer in the peripheral cytoplasm of the cells. It constituted less than 0.3% of the total cellular protein in cultured fibroblasts and was not extractable with Triton X-100. In detergent-extracted cytoskeletal preparations of cultured fibroblasts, p230 remained as an elaborate peripheral network that showed a distribution distinctly different from that of the major cytoskeletal structures, stress fibers, cortical myosin, vinculin, and intermediate filaments (IF). The distribution was not dependent on the presence of intact stress fibers or microtubules, as shown by double-fluorescence microscopy of cells exposed to cytochalasin B or cultured in the presence of monensin and of cold-treated cells. Upon demecolcine-induced reorganization of intermediate filaments, however, the localization of p230 was rapidly altered to a dense plaque underneath the perinuclear aggregate of intermediate filaments. On the other hand, p230 seemed to colocalize with the detergent-resistant cell surface lamina, visualized in fluorescence microscopy with fluorochrome-coupled wheat germ agglutinin-lectin. The results suggest that p230 is part of a cell surface- and cytoskeleton-associated subplasmalemmal structure that may play an important role in cell surface-cytoskeleton interaction in various cells both in vitro and in vivo.

Spectral characterization of the neuronal pigments of Aplysia juliana.

Spectral analysis at liquid N2 temperature of the circumesophageal ganglia of Aplysia juliana showed that carotenoids and a hemoglobin-like pigment are contained in concentrations of approx. 25 and 3 micronM, respectively, in the whole ganglia. Microspectrophotometrical measurements of Aplysia neurons indicated that the carotenoids reside on lipochondria in a concentration of approx. 38 mM. In addition to lipochondria, two types of pigmented particulate having absorption maxima at about 512 and 525 nm, respectively, were found in the neurons. The neuronal carotenoids consist of violaxanthin, beta-carotene and one minor component; among them the first occupies approx. 77% of total carotenoids. Two principal absorption maxima of the carotenoids, when existing in both ganglial homogenates and Triton X-100 extracts, show a red shift of 10 nm compared with those of free pigments in hexan. The red shift may be interpreted as due to the solvation of the carotenoids by surrounding lipids.

Ultrastructural differentiation within the central nervous system between indolamines and catecholamines by energy dispersive X-ray analysis following paraformaldehyde pretreatment.

Dense bodies containing high amounts of chrome were localized in the perikarya of substantia nigra and dorsal raphe neurons following the cytochemical reaction of endogenous dopamine and serotonin (respectively) with glutaraldehyde-dichromate (GDC). Energy dispersive X-ray analysis of these bodies revealed chrome levels two to four times higher than those recorded from the cytoplasmic background. Pretreatment with paraformaldehyde blocked the GDC reaction within the dense bodies in the substantia nigra (chrome levels similar to background), while the chrome levels in the dense bodies of the raphe neurons remained elevated. This demonstrates that pretreatment with paraformaldehyde allows selective localization of central nervous system serotonin stores by the GDC technique.

Zinc-aldehyde fixation for light-microscopic immunocytochemistry of nervous tissues.

Two procedures are described for vascular perfusion of the nervous system with a zinc-aldehyde fixative. The procedures, simple and economical, combine the advantages of perfusion fixation with an aldehyde solution and matrix stabilization by a mordating agent, and improve the sensitivity of the peroxidase-antiperoxidase (PAP) method for the immunocytochemical localization of several antigens. Procedure A is intended for the light-microscopic immunostaining of cellular elements containing high concentrations of antigen. Penetration of the immunoreagents is adequate without the use of detergents. Procedure B is particularly advantageous for the light-microscopic immunostaining of cellular elements that contain low concentrations of antigen, and for high-resolution microphotography. With procedure B, the tissue penetration of immunoreagents is more limited than with procedure A; however, neuronal cell bodies and dendrites are more easily penetrated by the immunoreagents than are axons. Neuronal cell bodies and dendrites thus become clearly detectable in the light-microscope, even when they are surrounded by numerous immunoreactive axon terminals, and especially after the blockage of axoplasmic transport by the topical injection of colchicine.

Immunohistochemical observation on the correlation between substance P- and vasoactive intestinal polypeptide-like immunoreactivities in the feline dental pulp.

The correlation between substance P (SP) and vasoactive intestinal polypeptide (VIP)-containing nerve fibres in the pulp was examined by double immunofluorescence. Both SP- and VIP-containing fibres entered the pulp in bundles with the blood vessels and spread throughout the pulp. In the coronal pulp, both SP- and VIP-containing nerve fibres formed networks on the walls of blood vessels, but both peptide-containing nerve fibres were not observed together in relation to vessels. There were more SP-containing fibres than VIP-containing ones. In the odontoblast layer, there were many SP-containing fibres but few VIP-containing ones.

Learning, using natural reinforcements, in insect preparations that permit cellular neuronal analysis.

A general paradigm is described that permits testing the ability of an arthropod to learn (by operant conditioning) to alter the position of a single leg segment in order to relate to behaviorally appropriate reinforcement. The paradigm was designed so that intracellular recording from identified neurons involved would be possible during the training of a locust or grasshopper, for which extensive neuron maps are available. As a prelude to such studies, electromyograms were made from the antagonistic muscles that move the conditioned limb, which in the present experiments was the tibia of the metathoracic leg. Negative (aversive) reinforcement was provided by a loud sound/vibration and positive (reward) reinforcement by food in the form of sugar-water or fresh-growing grass. In the aversive reinforcement experiments the sound, which reflexly caused flexion, was on continually except when the tibia of one hind leg was voluntarily placed in an electronically set position "window" displaced, in extension, away from the preferred position. In feeding experiments, food was brought automatically to the mouth by a motor-driven arm when the tibia was held within a position window set away from the preferred position in either extension or flexion. Whole or headless insects learned to turn off the sound permanently, except for sporadic brief interruptions, by tonic shifting of tibial position. Insects learned to bring food to the mouth by modifying the plateau phase of a position displacement lasting for a few minutes, that was found to occur from time to time also in controls. In aversive learning, minimum times to turn off the sound were 22 sec for the easiest position and 4 min for the most difficult. The longest time in the easiest position was 1 min 40 sec and in the most difficult 39 min; excluding measurement for individuals that did not learn. In reward learning, the minimum time in the easiest position was just under 1 min, and 12 min in the most difficult position. The longest times were about 8 hr regardless of difficulty.

Neurofilament protein abnormalities in PC12 cells: comparison with neurofilament proteins of normal cultured rat sympathetic neurons.

The studies described here characterize abnormalities in the expression of neurofilament (NF) proteins in a clonal rat pheochromocytoma (PC12) cell line as compared with normal NF proteins in cultured rat sympathetic neurons (RSNs). Cytoskeletal extracts from PC12 cells grown in the presence (PC12+ cells) or absence (PC12- cells) of nerve growth factor (NGF) and from RSNs grown in the presence of NGF were analyzed in nitrocellulose replicas of one- and two-dimensional polyacrylamide gels by the immunoblot method using monoclonal antibodies and antiserum to individual NF subunits. RSNs failed to express the high molecular weight NF subunit (NF200) for the first 10 days in culture although both lower molecular weight NF subunits (NF68 and NF150) were expressed by these cells. At later times in culture, all three NF subunits were present in immunoblots of RSNs. The immunoblot profile of NF proteins in PC12- cells was identical to that of RSNs grown in culture for up to 10 days. Growth of PC12 cells in NGF for up to 3 weeks did not alter this immunoblot profile except that no immunoband corresponding in NF200 was seen and the immunobands corresponding to NF68 and NF150 became more prominent. These data suggest that abnormalities in NF protein expression in PC12 cells are due to a paucity of NF200 or to the presence of immunochemically altered NF200. PC12 cells are an attractive model system for probing abnormal NF metabolism.

Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. The use of liposome-inserted radioiodinated N-CAM to study its transbilayer orientation.

The transmembrane orientation of the polypeptide chains present in preparations of adult and neonatal mouse N-CAM was studied using, as a model system, liposome-inserted purified N-CAM preparations. N-CAM purified from adult or neonatal mouse brain was 125I-labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides that remained associated with the liposomes were isolated by floatation of the vesicles on sucrose gradients. In control experiments the liposomes were lysed before trypsin treatment. Large, overlapping peptides were obtained after this treatment, several of which were protected by the liposome membrane. Sialic-acid-bearing peptides were revealed by their sensitivity to neuraminidase. To distinguish between peptides corresponding to intracellular or extracellular domains use was made of the P61 and H28.123 monoclonal antibodies, which recognize determinants located on the cytoplasmic and the extracellular part of the molecules respectively. There was no indication that the N-CAM chains were inserted in an inside-out configuration. Peptides protected from trypsin attack by the liposomes and recognized only by P61 had Mr values of 92 000, 42 000 and 35 000. The H28.123 determinant could be mapped to a 32 000-Mr peptide located close to the membrane at the vesicle's exterior. The bulk of the sialic acid seemed to be carried by a rather short sequence distal to the H28.123-reactive peptide but at some distance from the N terminus. Fragments of very similar Mr were generated from young and adult material. However, a 45 000-Mr peptide from neonatal N-CAM appeared to migrate in the higher-Mr region of sodium dodecyl sulfate/polyacrylamide gels in its fully sialylated form. It is concluded that (a) identical polypeptide chains are present in young and adult preparation, (b) the 180 000-Mr, 140 000-Mr and 120 000-Mr chains differ by the length of their cytoplasmic extensions and (c) the largest cytoplasmic sequences have a Mr close to 90 000. A tentative linear model of the transmembrane topography of the N-CAM polypeptides is presented.