Christian P. Reboulleau

Social interactions and androgen levels in birds. I. Female characteristics associated with increased plasma androgen levels in the male ring dove (Streptopelia risoria).

Abstract Male ring doves exposed to female conspecifics exhibit a variety of courtship behaviors and show large and relatively long-lasting elevations in circulating androgens. This series of experiments examined whether (1) a females gonadal condition and (2) contact or auditory cues from the mating situation influence these changes in male androgen levels and courtship behavior. Pairs of doves were placed in breeding cages, their behavior observed for the first 15 min of exposure, and blood obtained from the males 24 hr after pairing. Androgen levels were determined by an RIA. Males exposed to ovariectomized females had lower androgen levels than males exposed to intact females (2.3 ± 0.3 vs 4.0 ± 0.8 ng/ml, X ± SEM ), indicating that a females gonadal condition influences a males endocrine response to her. Deaf males exposed to intact females had lower androgen levels (1.4 ± 0.6 ng/ml) than normal males exposed to females (4.0 ± 0.8 ng/ml), indicating that auditory cues from the mating situation influence female-induced elevations in male plasma androgens. Males exposed to females through glass partitions had androgen levels (2.7 ± 0.6 ng/ml) similar to males given free access to females and higher androgen levels than males in isolation, indicating that contact cues from the mating situation do not necessarily influence female-induced elevations in male plasma androgens. The results also indicate that (1) contact between a pair of doves during early courtship facilitates male aggressive courtship displays and reduces male nest-oriented behavior, and (2) auditory cues from the mating situation facilitates male courtship behavior.

Extracellular Calcium‐Induced Neuroblastoma Cell Differentiation: Involvement of Phosphatidylinositol Turnover

Abstract: The rat CNS neuroblastoma B50 cell line is known to differentiate on addition of 1 mM dibutyryl cyclic AMP or on withdrawal of serum. In this report it is shown that high levels of extracellular calcium (10‐25 mM) cause neurite extension, an important component of morphological differentiation. Stimulation of calcium influx with the ionophore A 23187 or blockade of calcium efflux with lanthanum are less efficient than extracellular calcium in stimulating neurite extension. These data suggest that intracellular calcium is not sufficient to cause full expression of a calcium‐dependent differentiated state. Furthermore, phosphatidylinositol turnover is sharply altered as early as 1 h after addition of calcium to the medium while cyclic nucleotide levels remain unaffected. This suggests that activation of the phosphatidylinositol second‐messenger system by calcium at the level of the cell membrane is the initial step in the cascade of events leading to neurite extension. Later events include a decrease in DNA synthesis (6‐10 h after addition of calcium), and increase in intracellular calcium levels (12‐24 h after calcium addition) concurrent with neurite extension. The intracellular increase in calcium levels is facilitated by synergistic action of 1 mM dibutyryl cyclic AMP with high external calcium (10‐25 mM). This combined treatment results in a more complex pattern of neurite formation characterized by many synaptic‐like junctions: this pattern is not obtained when either dibutyryl cyclic AMP or calcium is used as sole inducer.

Social interactions and androgen levels in birds: II. Social factors associated with a decline in plasma androgen levels in male ring doves (Streptopelia risoria)

Abstract In male ring doves, androgen levels rise within hours of exposure to a female mate, but decline to baseline values by Day 1 of incubation (7–9 days after pairing). These experiments examined the role of social stimuli in the maintenance and decline of plasma androgens. In Experiment 1, males were exposed for 9 consecutive days to either a novel female or the same female and androgen levels were compared on the ninth day. There are no significant differences in androgen levels between these groups (3.0 ± 0.7 vs 1.8 ± 0.3 ng/ml, X ± SEM ), indicating that the presence of a novel female is not sufficient to maintain elevated androgen levels in male doves. In Experiment 2, males were paired with females and (1) were allowed to proceed uninterrupted to incubation, (2) had their nest destroyed daily, or (3) were given no nest material. All males were bled on Day 8 after pairing. Males given nest material and left undisturbed exhibited low androgen levels (1.3 ± 0.3 ng/ml) characteristic of the incubation phase. In contrast, males whose nests were destroyed daily or those given no nest material had significantly higher androgen levels than undisturbed males (3.3 ± 0.3 and 3.6 ± 1.0 ng/ml, respectively). It appears that interference with nest construction (and presumably incubation activity) prevents a decline in circulating androgens, and this suggests that the beginning of incubation rather than time since pairing regulates the decline in male androgens. In Experiment 3, androgen levels were compared in males taken from courtship with one female and exposed to either an incubating female or to another courting female. Courting males exposed to incubating females exhibited low androgen levels typical of incubation (2.0 ± 0.6 ng/ml), while courting males exposed to other courting females exhibited high androgen levels typical of courtship (4.7 ± 0.7 ng/ml). We conclude that the decline in male circulating androgens is strongly influenced by the behavior and stimuli presented by the female partner.

Inositol Metabolism During Neuroblastoma B50 Cell Differentiation: Effects of Differentiating Agents on Inositol Uptake

Inositol uptake was studied in the rat CNS neuroblastoma B50 cell line. Eadie‐Hofstee analysis of the uptake pattern reveals two defined modes of inositol entry into the cell. The high‐affinity uptake component requires the presence of extracellular sodium and is inhibited by phloridzin. Analysis of the uptake velocities of the high‐affinity uptake component provided the following apparent kinetic parameters: Km= 13.7 μM and Vmax= 14.7 pmol/mg of protein/min (without correcting for residual diffusion) and Km= 12.9 μM and Vmax= 12.3 pmol/mg of protein/min (with correction). At physiological concentrations, the high‐affinity . transport process contributes approximately 70% to total uptake; the remainder is due to a low‐affinity diffusion‐like process. Uptake inhibition studies reveal that the uptake process is sensitive to ouabain, amiloride, and dichlorobenzamil inhibition but relatively insensitive to cytochalasin B or phloretin. When neuroblastoma B50 cells are induced to differentiate morphologically with high extracellular calcium or with dibutyryl cyclic AMP, a significant decrease in inositol uptake is observed. The dibutyrl cyclic AMP‐mediated inhibition of uptake affects only the high‐affinity uptake component and is noncompetitive in nature. The high extracellular calcium‐mediated inhibition is less specific; it involves “disappearance” of the high‐affinity process, some inhibition of the low‐affinity process, and an increase of inositol efflux. The significance of these observations is discussed in the context of neuroblastoma B50 cell differentiation.

A cell line with decreased sensitivity to the methyl mercury-induced stimulation of α-amanitin sensitive RNA synthesis in isolated nuclei☆

1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on alpha-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275-6279). 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.

Multiple types of neurotransmitter binding sites in the rat neuroblastoma B 50 cell line. II. Response of second messenger systems to physiological stimuli in proliferating and differentiated cells

The functionality of the alpha 1-, beta-adrenergic and muscarinic cholinergic binding sites of neuroblastoma B 50 is investigated under proliferating and differentiating conditions. In proliferating cells, the stimulation of the alpha 1-adrenergic and muscarinic cholinergic binding sites by their respective agonists causes an increase in both extracellular calcium association with the cells and phosphatidylinositol (PI) turnover; effects usually associated with functional receptors. When the cells are induced to differentiate morphologically with dibutyryl cyclic AMP (db-cAMP), extracellular calcium or a combination of both, the activity of the muscarinic receptor-coupled PI turnover is strictly correlated with the binding affinity of the receptor. This is not the case for the alpha 1-adrenergic receptor stimulation of PI turnover. The latter result, however, may be explained in terms of the intrinsic properties of the inducing agents used to cause neurite extension. The stimulation of the beta-adrenergic binding site with isoproterenol in proliferating cells, both with and without a phosphodiesterase inhibitor present, does not result in cellular cAMP accumulation. In morphologically differentiated cells, only the db-cAMP-induced state exhibits an increase in [3H]adenosine incorporation into cellular cAMP upon isoproterenol stimulation. This happens only in the presence of a phosphodiesterase inhibitor. The data presented in this study are discussed in terms of the affinity of the receptors for their respective ligands and in terms of the intrinsic properties of the inducing agents.

Hormonal Aspects of the Morphological Differentiation of Neuronal Clonal Cell Lines

The biochemical processes that control cell differentiation during brain development can be studied at several levels. The whole-animal model is primarily used to investigate the biochemical changes resulting from interactive processes that are mediated by various tissues and organs. In this case, overall, final changes are measured and it is often difficult to identify the various mechanisms that cause certain cells to differentiate the way they do. In an effort to understand the changes mediated by certain functionally important biomolecules in whole animals, one can try to replicate these changes in vitro using tissue explants maintained in culture medium. Although this technique allows one to investigate the role of a specific molecule suspected of playing an important role in the process of development, its drawback is that tissue culture explants are generally heterogeneous in nature and therefore the cell type on which a particular chemical may act is open to question. Similarly, while biochemical changes can easily be measured in these systems, morphological change cannot be measured readily. To remedy this problem, primary cultures of dissociated tissues are often used. These systems allow the monitoring of cell morphology, but they too are often heterogeneous and the number of cells that one can use and their life span in culture are often limited. A different level of investigation, making use of theoretically homogeneous clonal cell lines, is available. Since such cell lines permit the collection of large numbers of cells that can multiply indefinitbly, from a practical point of view, this model is more convenient to study minute macromolecular changes that take place during morphological differentiation. All these approaches are useful and complementary. The following discussion, however, will be limited to work done on clonal cell lines and will focus on the biochemical changes that arise as morphological differentiation proceeds in cell lines of neuronal origin. Clonal cell lines of neuronal origin have been obtained in several ways: (1) by cloning single cells isolated from spontaneous tumors such as neuroblastoma C 1300 of neural crest origins or pheochromocytoma PC 12, a sympathetic ganglion-like cell line3; (2) by cloning single cells from isolated normal embyronic tissue4s5; (3) through viral transformation and cloning of cells isolated from embryonic brain tissue6; and (4) by cloning single cells isolated from chemically induced tumors such as rat central nervous system neuroblastomas B35, B50, B65, B103, and B104. It must be remem-

Multiple types of neurotransmitter binding sites in the rat neuroblastoma B 50 cell line. I. Characterization and binding affinity changes during various differentiation paradigms

In this study, it is shown that the neuroblastoma B 50 cell line of rat central nervous system origin possesses both adrenergic and cholinergic binding sites. The adrenergic binding site population comprises a major alpha 1-adrenergic component (using [3H]prazosin as ligand; KD = 0.55 nM; 19.3 fmol/mg protein), and a minor beta-adrenergic species (KD = 0.45 nM; 3.4 fmol/mg protein using [125I]cyanopindolol as ligand). The cholinergic binding site is of a muscarinic type. Binding studies with quinuclidinyl benzylate (QNB) indicate the presence of two binding sites with different affinities. This is supported by competition studies with agonists. However, in view of this unusual binding behavior of the antagonist, the possibility of a one-site model for QNB is also considered and affords the following parameters: KD = 0.247 nM, Bmax = 29.6 fmoles/mg protein. When the cells are induced to differentiate with dibutyryl cyclic AMP (db-cAMP) or high extracellular calcium, all of these binding sites undergo a variety of changes in their affinities for their respective ligands. Db-cAMP increases the affinity of the alpha 1-site for prazosin while high extracellular calcium lowers it. In both cases the number of binding sites remains unchanged. The affinity of the beta-adrenergic component is markedly enhanced upon induction of differentiation with either extracellular calcium or/and db-cAMP. Finally, the muscarinic cholinergic binding sites exhibit a decrease in receptor number upon db-cAMP treatment and an apparent loss of one of the binding sites upon calcium treatment. The significance of these affinity changes is discussed for each type of binding site.