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C Pilgrim

Publications and source records attributed to C Pilgrim.

At least 55 records · Page 3Linked to original sources
PubMed1992

Sex steroids do not alter sex differences in tyrosine hydroxylase activity of dopaminergic neurons in vitro.

In order to distinguish the effects of genetic sex from those of sex hormones on the sexual differentiation of dopaminergic neurons, catecholamine synthesis was studied in gender-specific cultures of embryonic day-14 rat diencephalon. In addition to embryos from normal dams, embryos were used whose mothers had been treated with the estrogen antagonist tamoxifen or the testosterone antagonist cyproterone acetate on days 12 and 13 of gestation. Cultures from embryos of untreated dams were fed daily with a medium containing 17 beta-estradiol or testosterone. After 10 days in vitro, cultures were immunostained for tyrosine hydroxylase and the accumulation of dihydroxyphenylalanine (DOPA) was measured in the presence of the DOPA decarboxylase inhibitor NSD 1015. Rates of DOPA synthesis, unlike the numbers of tyrosine hydroxylase-immunoreactive neurons, were markedly higher in female cultures under all experimental conditions. Treatment of dams with antisteroids prior to removal of the embryos had no influence on these results. Treatment of cultures with both steroids decreased DOPA formation in a dose-dependent manner without altering the sex difference. These results suggest that cultured diencephalic dopaminergic neurons develop sex differences in the activity of tyrosine hydroxylase. This sexual dimorphism is initiated independently on the activity of gonadal steroid hormones. Sex hormones exert an additional modulatory influence on the activity of the enzyme but do not abolish or reverse sex differences. Therefore, the concept of a purely epigenetic mode of sexual differentiation of the mammalian brain needs to be broadened to incorporate other mechanisms, such as the cell-autonomous fulfillment of a sex-specific genetic program.

Animals↗
PubMed1992

Absence of sex differences in size of the genital ducts of the rat prior to embryonic day 15.5-16.0.

Sexual dimorphisms of the rat brain are generally believed to be brought about by the presence of testosterone during a critical period starting at embryonic day (ED) 17/18. In contrast, sex differences of diencephalic and mesencephalic dopaminergic neurons were observed to develop in cell cultures raised from ED 14 rat brains. This was interpreted as evidence indicating that sexual differentiation of certain neural systems may occur independently of gonadal hormones. To substantiate this claim, it was felt necessary to examine the rat embryo for clues to a possible existence of sex differences in hormonal environment prior to ED 17. Morphometry was applied to compare the development of male and female Wolffian and Müllerian ducts, both primary targets of hormones secreted from the male gonad. Diameters of serially cross-sectioned Wolffian and Müllerian ducts were measured in rats of ED 15.0 to ED 16.5. Females had thicker Müllerian ducts from ED 15.5 on. The first step of differentiation in males was the widening of the lumen and a slight increase of the outer diameter of the Wolffian duct at ED 16.0. The size differences of both ducts were most obvious in the vicinity of the lower half of the gonad. Except in Wolffian ducts of ED 16.5, sex differences were absent in the caudal parts of the ducts. It appears that gonadal androgen and Müllerian inhibiting substance do not affect the development of their classical target organs prior to ED 16.0 and ED 15.5, respectively. Furthermore, the first effects are paracrine in nature. There is no evidence for sex differences in systemic androgen environment until ED 16.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Diencephalic GABAergic neurons in vitro respond to prolactin with a rapid increase in intracellular free calcium.

In order to analyze the feedback action of prolactin (PRL) on the hypothalamus on the cellular level, we used primary cultures of rat embryonic diencephalon to measure the calcium response of individual neurons to PRL by means of fast fluorescence photometry. The cultures were subsequently stained with antibodies against the neuronal marker MAP-2, glutamic acid decarboxylase (GAD) or tyrosine hydroxylase (TH). PRL caused a rapid rise of intracellular free Ca2+ in a specific type of GABAergic neuron characterized by a spindle-shaped bipolar morphology and immunoreactivity to MAP-2 and GAD but not to TH. It is concluded that a subpopulation of hypothalamic GABAergic but not dopaminergic neurons react to PRL with a rapid increase in intracellular free Ca2+. These data are compatible with the assumption of a rapid negative feedback regulation of the secretion of PRL from the pituitary mediated by tuberoinfundibular GABAergic neurons.

Animals↗
PubMed1991

Cells from embryonic rat striatum cocultured with mesencephalic glia express dopaminergic phenotypes.

To study region-specific transmitter phenotype expression, cells of embryonic day 14 (E14) rat neostriatum (ganglionic eminence plus cortical plate) or of the substantia nigra (ventral mesencephalon) were cultured on glial cells either from substantia nigra or neostriatum (E21). By antityrosine hydroxylase immunocytochemistry, immunoblotting of tyrosine hydroxylase protein and quantitation of dopamine and its metabolites by HPLC, dopaminergic cells were revealed in nigral and neostriatal cultures plated on nigral glial cells. No dopaminergic cells were found among neostriatal neurons plated on neostriatal glial cells. It is concluded that glia from substantia nigra but not glia from neostriatum is capable of inducing development or promoting survival of dopaminergic cells.

Animals↗
PubMed1991

The influence of genetic sex on sexual differentiation of diencephalic dopaminergic neurons in vitro and in vivo.

Soma sizes of embryonic male and female tyrosine hydroxylase-immunoreactive neurons were measured in 3 diencephalic regions in situ and in diencephalic dissociated cell cultures. Male neurons were about 30% larger than female neurons both in vitro and in situ. Treatment of cultures with sex steroids did not affect the sex differences. It is concluded that sexual differentiation of dopaminergic neurons may be under primary genetic control.

Animals↗
PubMed1991

Sexual differentiation of monoaminergic neurons--genetic or epigenetic?

It is currently believed that sexual differentiation of the brain is mediated entirely by the epigenetic action of gonadal steroids during a critical period of development. Ingrid Reisert and Christoph Pilgrim review sexual dimorphisms of monoaminergic systems, which also appear to be generated by sex steroids. However, there are a number of observations that are not explainable by the 'androgen theory of sexual differentiation'. Results obtained from cultures of embryonic rat brain tissue appear to indicate that dopaminergic neurons may develop morphological and functional sex differences in the absence of sex steroids. Hormone-independent and -dependent developmental processes may affect diencephalic and mesencephalic dopaminergic neurons in a regionally diverse fashion. Factors other than sex steroids need to be examined. It is possible that some sexual dimorphisms in the nervous system may develop under primary genetic control.

Animals↗
PubMed1991

Sex-specific schedule in steroid response of rhombencephalic catecholaminergic neurons in vitro.

Morphological differentiation of tyrosine hydroxylase-immunoreactive neurons was investigated in dissociated cell cultures of rhombencephalon of male and female day 14 rat embryos grown in the presence or absence of sex steroids. Numbers of cells were counted and morphometrical measurements carried out of soma size and length of tyrosine hydroxylase-immunoreactive neurites (processes). Subtle sex differences in length of stained neurites, which were not yet present after 3 days in vitro, were observed after 6 days in cultures grown in the absence of sex steroids. Female tyrosine hydroxylase-immunoreactive neurites could be traced over longer distances than male ones. Daily treatment of cultures with testosterone or 17 beta-estradiol resulted in an increase of lengths of stained neurites of female neurons after 3 days and of male neurons after 6 days in vitro. Regarding cell numbers or soma size, there were no differences between genders or between controls and hormone-treated cultures. It is concluded that sex steroids promote the outgrowth of neurites from noradrenergic neurons within a gender-specific time frame. It appears that the critical period for developmental effects of sex steroids differs between males and females.

Animals↗
PubMed1991

Plasticity of the interface between oxytocin neurons and the vasculature in late pregnant rats: an ultrastructural morphometric study.

Increased numbers of oxytocin-immunoreactive perivascular neurons have been shown to occur in the preoptic region and lateral hypothalamus of female rats around parturition. In the present study, electronmicroscopical immunocytochemistry and morphometry were used to examine such perivascular oxytocinergic neurons as well as those in the classical magnocellular nuclei in late pregnant and lactating rats. In 22 d pregnant animals and in rats killed after 2 d of lactation, numerous oxytocinergic neurons were found in direct apposition to the outer basement membrane of arterioles, venules, and capillaries. The distance between immunoreactive neurons and blood vessels was significantly lower in these animals than in 9 d lactating rats and in ovariectomized controls. It is likely that, around parturition, oxytocinergic perivascular neurons are uncovered by active retraction of glial elements. This plasticity is perhaps facilitated by changing hormonal conditions around parturition. The observed changes seem to be transitory and might reflect altered secretory properties of perivascular oxytocinergic neurons.

Animals↗
PubMed1991

Regulation of vasopressin expression in cultured diencephalic neurons by glucocorticoids.

Glucocorticoids have long been recognized as playing a major role in the regulation of vasopressin synthesis. However, the factors determining cellular specificity and molecular mechanisms of glucocorticoid action on the vasopressin gene are not understood. In the present investigation, we used primary cell cultures derived from 14-day-old fetal rat diencephalon to investigate the regulation of vasopressin expression under controlled conditions. The experimental paradigm used ensured that only magnocellular, but not parvocellular neurons grew in the cultures. The following criteria were used to establish this phenotype. (1) Cultures were derived from fetal brain well before the time parvocellular neurons are generated, and neuronal precursors did not proliferate in vitro. (2) Vasopressinergic neurons measured some 18 x 25 microns, being conspicuously larger than the average neuronal population in vitro, and clearly larger than parvocellular neurons in vivo. (3) Neurons did not express corticotropin releasing factor in vitro. Selective neutralization of glucocorticoids contained in the serum-supplemented culture medium by the drug RU 38 486 resulted in an about 2-fold increase of numbers of vasopressinergic cells and about 4-fold increase in vasopressin mRNA, but did not affect numbers of oxytocinergic neurons or expression of general neuronal marker proteins. The effects of RU 38,486 were not dependent on synaptic communication between cultured cells, as the drug was still effective when cells were synaptically isolated by growth is in 14 mM Mg2+. RU was not mitogenic for vasopressinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Dopamine content and metabolism in mesencephalic and diencephalic cell cultures: sex differences and effects of sex steroids.

Sexual differentiation of dopaminergic neurons was studied in gender-specific cultures. Dissociated cell cultures were prepared from di- or mesencephalon of gestational day 14 rat embryos and raised in the absence or presence of 17 beta-estradiol or testosterone for up to 13 days in vitro (DIV). Developmental profiles of levels of dopamine (DA) and metabolites as well as capacity for vesicular storage of the transmitter were determined by HPLC. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted. Higher levels of DA were measured in female than in male cultures of both brain regions. In mesencephalic cultures, the differences in DA levels were fully accounted for by sex differences in numbers of TH-IR cells, whereas no sex differences in cell numbers were found in diencephalic cultures. Dihydroxyphenylacetic acid (DOPAC) levels and vesicular storage capacity matured faster in mesencephalic than in diencephalic cultures, but no sex differences were observed. Homovanillic acid (HVA) could not be detected except in 13-DIV mesencephalic cultures. Hormonal treatment did not erase sexual differentiation of dopaminergic neurons. Irrespective of the gender, however, both steroids decreased DA and DOPAC contents in diencephalic cultures but not in mesencephalic cultures. It is proposed that sexual differentiation of dopaminergic systems proceeds in a region-specific fashion and that neurogenesis and development of various parameters of dopaminergic activity may be differentially affected. Sexual differentiation of dopaminergic neurons may be initiated independently of the action of gonadal steroid hormones and may subsequently be modified by differences in hormonal environment.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1991

[How thick is the glycocalyx of human erythrocytes?].

The aim of the experiment was to determine the thickness of the glycocalyx of human erythrocytes of the blood group A. For that purpose, the distance between the electron dense contrasted lipid layer of the plasmalemma and gold sol particles loaded with Helix pomatia lectin was determined. The mean thickness of the glycocalyx under this conditions was 5.9 nm.

Antibodies↗
PubMed1990

Prenatal development of mesencephalic and diencephalic dopaminergic systems in the male and female rat.

Previous results had suggested mesencephalic dopaminergic neurons in cultures of gestational day (GD) 14 rat embryonic brains to be characterized by an early maturation and acquisition of sex-related differences in transmitter uptake. Therefore development of dopaminergic systems was reexamined in the rat in vivo with special emphasis on the prenatal period, mesencephalo-hypothalamic relationships, and possible sex differences. Perfusion-fixed brains of GD 14, 17, 20, 21 and newborn rats were sectioned or processed as whole-mounts and immunostained for tyrosine hydroxylase (TH). Total numbers of mesencephalic TH-immunoreactive cell bodies as assessed by a stereological method rose between GD 14 and 17 and fell again between GD 17 and 21. As early as GD 14, a prominent mesencephalo-hypothalamic projection was observed coming off the medial forebrain bundle and terminating in the retrochiasmatic region. Two additional TH-immunoreactive fiber bundles leaving the medial forebrain bundle, one rostral and one caudal to the former, and terminating in the paraventricular and premammillary region, respectively, were noticed on GD 17. Careful examination of developing TH-immunoreactive neurons in the lower brainstem ascertained that there was no interference from ascending catecholaminergic fibers other than dopaminergic of mesencephalic origin during this early prenatal period. All 3 mesencephalo-hypothalamic projections had largely disappeared by GD 20 and were no longer detectable as distinct fiber bundles thereafter. There were subtle sex differences in numbers and distribution of both mesencephalic and diencephalic TH-immunoreactive neurons present at GD 17, which thus occurred prior to manifestation of other well-known sexual dimorphisms of the rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Region- and sex-related differences in maturation of astrocytes in dissociated cell cultures of embryonic rat brain.

Previous studies using dissociated cell cultures of fetal rat brain have revealed considerable regional diversity as well as sex steroid-independent sex differences in developmental schedules of dopaminergic neurons. Because these phenomena might be related to glial heterogeneity, cultures of dissociated male and female diencephalon, mesencephalon, and rhombencephalon of gestational day 14 rats were investigated with respect to the development of astrocytic markers. Cultures were incubated for 3-8 days in vitro (DIV) in serum-supplemented or serum-free medium. Vimentin and glial fibrillary acidic protein (GFAP) were quantified by counting of immunolabeled cells and immunoblotting. Vimentin and GFAP content rose from DIV 3 to 6 in all cultures. Regional variation of vimentin content was low, but large differences occurred in amounts of GFAP. GFAP reached high levels in rhombencephalon, especially when supplemented with serum, but remained very low or not detectable in mesencephalon. Simultaneous immunostaining for both cytoskeletal proteins revealed the presence of large numbers of vimentin single-labeled and small numbers of vimentin/GFAP double-labeled cells. Numbers of cells expressing GFAP showed similar regional variations as GFAP contents in both serum-free and serum-supplemented medium. They rose steeply from DIV 3 to 8 in rhomb- and diencephalon but not in mesencephalon. Transiently, female diencephalic cultures contained slightly more GFAP-immunoreactive cells than male cultures. The results thus demonstrate considerable regional heterogeneity of astrocytic maturation. However, neither the regional nor the sex differences show a consistent correlation with previous data on development of dopaminergic and other monoaminergic neurons in vitro. It seems likely that the dependence of neurons on glial environment for realization of an inherent developmental program varies among neuronal phenotypes.

Animals↗
PubMed1990

Relations between baseline contingencies and equivalence probe performances.

Following the emergence of two three-member equivalence classes (A1B1C1 and A2B2C2), 5 college students were exposed to one or more changes in the reinforcement contingencies controlling baseline conditional discriminations. AC relations were either reversed (i.e., C2 was reinforced and C1 punished when A1 was the sample; C1 was reinforced and C2 punished when A2 was the sample) or arranged randomly (i.e., C2 and C1 were reinforced and punished equally often in the presence of A1 and A2). In a third condition, the original AB and AC relations were reversed. Results showed that although baseline conditional discrimination performances were under the control of reinforcement contingencies, and performances on symmetry trials varied with baseline responding for 3 of 4 subjects when contingencies were reversed, performances on transitivity probes remained consistent with the initial equivalence class. These inconsistencies between probe and baseline performances were striking because conditional discriminations are thought to be the determinants of equivalence class performance. Similarly, the contrast between performances on symmetry and transitivity probes was of theoretical interest because equivalence classes are defined by congruent patterns of responding on probe trials.

Journal Article↗
PubMed1989

Different developmental potentials of diencephalic and mesencephalic dopaminergic neurons in vitro.

Morphological and functional differentiation of dopamine (DA) neurons was compared in dissociated cultures from gestational day 14 rat mesencephalon and diencephalon. Numbers of tyrosine hydroxylase-immunoreactive (TH-IR) neurons relative to all neurons were 4 and 1.7 times higher in mesencephalic than in diencephalic cultures at 6 and 13 days in vitro (DIV), respectively. Morphological maturation of diencephalic DA neurons was retarded in comparison to mesencephalic DA neurons. There were gross differences in soma size and length of processes between the two types of DA neurons, the appearance of which was strongly reminiscent of DA cell types described in vivo. Functional maturation of DA neurons was quantified by measuring uptake and Ca2+-dependent K+-stimulated release of [3H]DA per TH-IR neuron. As early as 6 DIV, DA uptake by mesencephalic DA neurons was saturable, was sensitive to benztropine and reserpine, and could be displaced by unlabeled DA. Twenty to 30% of the radioactivity accumulated could be released upon depolarization within a period of 5 min. At 6 DIV, influx of [3H]DA into diencephalic DA neurons was almost insensitive to benztropine, reserpine and unlabeled DA. Even after 13 DIV, diencephalic DA uptake was characterized by a markedly lower initial velocity, a longer time needed to reach saturation, a lower uptake capacity, and a lower sensitivity to benztropine than mesencephalic DA uptake. The releasable pool was very small and did not increase between DIV 6 and 13. The results demonstrate that mesencephalic DA neurons in vitro differentiate considerably faster than diencephalic DA neurons and gain functional competence very early in brain development. Comparison with data on adult nigrostriatal and hypothalamic DA systems suggests that the in vitro differences reflect a fundamental regional diversity of DA neurons.

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