Robert E. Hausman

Determination of fractal dimension of physiologically characterized neurons in two and three dimensions

Although there is a growing interest in the application of fractal analysis in neurobiology, questions about the methodology have restricted its wider application. In this report we discuss some of the underlying principles for fractal analysis, we propose the cumulative-mass method as a standard method and we extend the applicability of fractal analysis to both 2 and 3 dimensions. We have examined the relationship between the method of log-log Sholl analysis and fractal analysis and have found that they correlate well. Measurements of physiologically characterized retinal ganglion cells indicate that different cell types can have significantly different fractal dimensions. Such differences may allow the correlation of the physiological type of a neuron with its morphological fractal dimension.

Immunologic detection of retina cognin on the surface of embryonic cells

Abstract The retina cell-aggregating glycoprotein, referred to as the retina cognin, has been demonstrated to be located at the surface of embryonic neural retina cells. The term cognin is used to indicate its postulated role in the mechanism of mutual recognition and morphogenetic association of embryonic cells. Antiserum was prepared to the highly purified retina cognin derived from isolated cell membranes of chick embryo retina, and it was used to detect the cognin on cells from chick embryos by means of complement-mediated cell lysis. Retina cells (from 10-day embryos) freshly dissociated with trypsin showed little—if any—lysis by the cognin antiserum; this is consistent with the sensitivity of the cognin to trypsin. However, the cells became susceptible to immunolysis after a period of incubation at 37 °C, which indicates regeneration of the cognin at the cell surface during the recovery period. This regeneration required protein synthesis. Immunofluorescence tests showed binding of the antiserum to the surface of the recovered cells, thereby further demonstrating the surface location of the cognin. The presence, availability or ability to regenerate the cognin, as assayed here, declined sharply with the embryonic age of the cells. Addition of exogenous cognin to freshly trypsin-dissociated retina cells (from 10-day embryos) markedly increased their susceptibility to immunolysis by the cognin antiserum, which indicates that the added cognin becomes associated with the surface of these cells. In contrast, addition of retina cognin to cells freshly trypsinized from 10-day embryo optic tectum and cerebrum, or from 14-day retina did not increase their susceptibility to immunolysis by the cognin antiserum. These results are consistent with earlier findings that enhancement of cell aggregation by the retina cognin is tissue-specific and stage-specific. Cells from non-neural tissues of the chick embryo were not lysed by the retina cognin antiserum. However, neural tissues, such as optic tectum, were found to contain cells which showed surface cross-reaction with the retina cognin antiserum.

Visualization of a cell surface glycoprotein, the retina cognin, on embryonic cells by immuno-latex labeling and scanning electron microscopy

Abstract Antiserum prepared against the retina cell-aggregating glycoprotein (referred to as the retina cognin) was used for immunolabeling the antigen on the surface of chick embryo cells and its detection by scanning electron microscopy (SEM). The cognin was isolated from membranes of neural retina cells of 10-day chick embryos and was highly purified prior to its use as antigen in rabbits. Cells dissociated from embryonic neural retina and from other neural and nonneural tissues (of 10-day chick embryos) were surveyed in vitro for the presence on their surface of binding sites specific for antibodies to retina cognin. Cells were treated with the antiserum (or with control nonimmune serum), and then labeled with latex-GAR (polystyrene latex microbeads coated with goat anti-rabbit immunoglobulins) to visualize the binding sites for detection by SEM. Retina cells freshly dissociated with trypsin showed no labeling because of disruption of the antigen sites by the protease; however, if the cells were first incubated at 37°C to allow recovery of their surface and of their capacity for morphogenetic cell reaggregation, then virtually all the cells labeled with the cognin antiserum-latex GAR. Similarly tested cells from several nonneural tissues showed no labeling with this antiserum, which indicates that the retina cognin is not present on their surface. However, neural tissues (particularly optic tectum) were found to contain a proportion of cells which labeled with the retina cognin antiserum after recovery from dissociation; it remains to be determined whether the labeling of these cells is due to the presence of retina cognin, or of other surface molecules which share partial antigenic similarity with this cognin.

Prostaglandin binding activity and myoblast fusion in aggregates of avian myoblasts

Myoblast aggregates provide a system for studying cell interactions which have several advantages over standard, stationary cultures. In gyrotory rotation, aggregate size can be controlled and is independent of cell migration. In muscle aggregates, fibroblasts are excluded, yet myoblast differentiation and fusion occur in a highly synchronous fashion. Specific PG binding occurs in chick or quail myoblast aggregates: in chick the peak of binding is at 35-36 hr. Aggregation is complete 16 hr before PG binding activity appears. This suggests either that gyrotory aggregation is not identical to myoblast recognition, or that PG binding activity occurs subsequent to myoblast recognition. Myoblast aggregates begin to release PG before 18 hr. The amount detected remains constant until binding begins at 34 hr when PG binding to the aggregates begins. Thus, both the release of PG and PG receptor activity are characteristics of the myoblasts and release of prostaglandin precedes appearance of the binding activity. As a first step in identifying the PG receptor and determining its appearance on the myoblast cell surface, we have prepared antisera against myoblast surfaces which blocks receptor-ligand interaction and have absorbed it against both peripheral and intrinsic membrane fractions. The results indicate that the PG receptor is a myoblast peripheral membrane macromolecule.

Light-microscopic immunocytochemical localization of fibronectin in the developing rat lung

SummaryThe development of the rat lung is a process of continuing morphological change. Indications from work in other mammalian systems suggest that fibronectin may be important in the control of this process. The present study has examined embryonic, neonatal, and adult lung tissue of the rat by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate fibronectin at the light-microscopic level. Positive reaction was observed with anti-fibronectin serum in all stages examined. Control sections treated with pre-immune serum or no primary serum gave negative results in each case. Fibronectin in adult tissue was localized to the alveolar surface and alveolar basal lamina. Neonatal tissue showed fibronectin on pulmonary tubule walls and in basal lamina while embryonic tissue revealed localization of the protein in the basal lamina and in association with small groups of cells at the base of septal buds. These findings suggest a role for fibronectin in the control of rat lung development. The results are discussed in terms of the known functions of fibronectin as a preliminary matrix for the subsequent deposition of collagenous connective tissue, as a cellular adhesion protein, and as surface-bound material for cellular migration.

Ocular extracellular matrices in development

The extracellular matrix is known to play important roles in regulating tissue development and cell differentiation. At a basic level the matrix is the substratum to which cells adhere and over which they migrate during morphogenesis. The recognition and adhesion of cells to matrices, especially to specialized matrices such as basal laminae, in developing tissues plays an important role in their decision to adhere or move on, to continue mitosis, to differentiate or to undergo apoptosis. The matrix is also the milieu from which growth factors and morphogens interact with the cell. Recent work demonstrates that matrix molecules are often active participants in these signaling processes, regulating the cellular response. Our understanding of the complexity of different ocular matrices continues to grow with the characterization of new proteins that modify collagen fibrils or serve as ligands for cell surface adhesion receptors. This review looks at those aspects of development in major tissues of the vertebrate eye where the extracellular matrix is known to participate, or is likely to do so based on similar roles in the development of the embryonic forebrain or other tissues. It concludes by highlighting some of the major developmental questions about the roles of the matrix in development in several ocular tissues.

Effect of viscosity on neurite outgrowth and fractal dimension

The growth mechanism by which neurons achieve their characteristic ramified morphology has long been of interest, but determining whether physical parameters, such as viscosity, are important has been difficult due to a lack of useful hypotheses and standard reproducible techniques. We have recently shown that neurons exhibit fractal behavior and that their fractal dimension (df) is consistent with a physical process called diffusion-limited aggregation (DLA). We suggested that this DLA behavior might stem from viscosity differences, chemical gradients or electrical fields (Caserta et al., Phys. Rev. Lett., 64 (1990) 95-98). DLA is a model for a large family of growth processes. In order for a process to fit the DLA model, the growth rate must be proportional to the gradient of a field at a point on the growing structure (Feder, Plenum, New York, 1988, Ch. 4). Chemical, electrical, or fluid pressure fields can fit the model depending on the particular physical system under study. Here, we studied growth of retinal neurons from chick embryos in culture media of various fluid viscosities. Thus, we test whether DLA in this system was based on a fluid pressure field. As viscosity was increased from 1 to 4.3 cps, the number of neurite branches decreased 98%. However, there was no effect on df. Over this range of viscosities, total cellular protein synthesis decreased only 17%. The results indicate that, while differences in viscosity between the interior and exterior of the cell affect neurite outgrowth, they do not affect the fractal behavior of neurons. Thus, viscosity differences are not the basis for the DLA pattern of neuronal arborization.

Ultrastructural immunocytochemical localization of fibronectin in the developing rat lung

SummaryIn a previous study changes in the macrodistribution of fibronectin during rat-lung development were examined. Using the peroxidase-antiperoxidase immunocytochemical technique, we have demonstrated the presence of fibronectin in embryonic, neonatal, and adult rat lung at the ultrastructural level. In the embryo, fibronectin is found both in an intra-and extracellular association with isolated pneumoblasts, and in a periodic distribution along the basal lamina. The neonate displays fibronectin in an intracellular association with early type-I cells and on their basal and luminal surfaces, but not in association with type-II cells. Neonatal basal lamina is diffusely labeled by anti-fibronectin antiserum. Fibronectin in adult tissue is found both intracellularly and on the basal and luminal surfaces of type-I cells but not in type-II cells. The basal lamina and interstitial connective tissue are slightly or non-reactive. These observations confirm and extend our initial suggestion that fibronectin is involved in rat-lung development.

Initial cholinergic differentiation in embryonic chick retina is responsive to insulin and cell-cell interactions

Previous work [Kyriakis et al., Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7463-7467] had shown that insulin, when added during a window of binding from embryonic days 9-11, stimulates the normal developmental increase in choline acetyltransferase (ChAT) activity (a marker for cholinergic differentiation) in cultured embryonic chick retinal neurons. Here, we investigated the effect of insulin and IGF 1 on embryonic chick retinal neurons at the stage of development (embryonic day 6) when ChAT activity is first expressed. We investigated insulin peptide effects in retinal tissue developing in vitro as well as in cultures of retinal cells. We show that insulin also stimulated the initial embryonic increase in ChAT activity but had no stimulatory effect on glutamic acid decarboxylase activity (a marker for GABAergic differentiation), an enzyme whose activity also increases developmentally in the same retinal neurons. In fact, insulin inhibited the expression of GAD activity in the retina. The insulin-mediated increase in ChAT activity was independent of normal cell-cell interactions but could not replace them. Insulin also stimulated choline uptake but only after a two day delay, suggesting that the normal program for cholinergic differentiation in the chick retina was induced by insulin. IGF 1 did not have any effect on either cholinergic or GABAergic differentiation. We conclude that cholinergic differentiation in chick embryo retinal neurons is dependent on both insulin- and cell contact-mediated signals.

Effect of insulin on GABAergic development in the embryonic chick retina

We investigated the role of insulin in GABAergic differentiation in the embryonic chick retina at different embryonic ages using glutamate decarboxylase (GAD) and high-affinity GABA uptake as developmental markers. Both these GABAergic markers exhibit developmentally programmed increases in activity during retinogenesis that also occur in culture. Insulin stimulated GABA uptake in retina neurons at all embryonic ages in a dose-dependent manner and GAD activity by 30% in embryonic retina neurons after 11 days of development. The stimulation of GABA uptake by insulin was blocked by addition of ouabain suggesting a role for the Na+,K+ ATPase. The same concentration of insulin caused a 76% stimulation of protein synthesis in these retinal cells, and previous work demonstrated that insulin also stimulates cholinergic differentiation in the chick retina (Hausman et al., Dev. Brain. Res. 59, (1991) 31-37). Thus, there was no selective stimulation of GABAergic differentiation by insulin but likely a neurotrophic effect. The increase in GAD activity in neurons from post-11-day embryonic neurons contrasts with our previous findings at embryonic days 6-7 where there is little change in GAD activity after addition of insulin. It is possible that the failure of insulin to stimulate GAD activity during early retina development is due to the increased accumulation of GABA in the presence of insulin. GABA levels were increased more than two-fold by 100 ng/ml insulin.