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C Pilgrim

Publications and source records attributed to C Pilgrim.

At least 73 records · Page 4Linked to original sources
PubMed1989

Expression of synaptophysin and neuron-specific enolase during neuronal differentiation in vitro: effects of dimethyl sulfoxide.

Neural development in dissociated cell cultures of fetal rat brain can be expected to depend on synaptic interactions between cultured neurons. Therefore, an attempt was made to obtain a quantitative measure of the time course of synaptogenesis in such a culture system by assessing the level of the secretory vesicle-associated protein synaptophysin (p38). The developmental schedule of p38 was compared to that of neuron-specific enolase (NSE), an established marker of neuronal differentiation. Cultures were raised from dissociated 14 day-old fetal rat diencephalon. In cultures grown for 1-2 days in vitro (DIV), p38-immunoreactivity was preferentially located in neuronal perikarya. After 10-16 DIV, neurons in culture had formed a dense neuritic network, and almost all of the p38-immunoreactivity occurred in the form of fine punctate deposits associated with neuronal processes that often outlined neuronal cell bodies in a basket-like fashion. Electron-microscopic immunocytochemistry proved the punctate deposits to be presynaptic elements, mostly in the form of axonal varicosities. Quantitative immunoblotting showed that levels of p38 increased from the start of cultivation to DIV 4, stayed fairly constant from DIV 4 to DIV 8, and rose again steeply to peak at DIV 12. In contrast, levels of NSE rose continuously up to DIV 12. After DIV 12, levels of both p38 and NSE fell again. Treatment of cultures with dimethyl sulfoxide (DMSO), an agent known to induce differentiation in various normal and malignant cell types, resulted in a significant increase of p38 levels and in a decrease of NSE levels. The amount of p38 continued to increase beyond DIV 12, whereas NSE diminished after having reached a maximum at DIV 12.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1989

Changes in immunostaining for oxytocin in the forebrain of the female rat during late pregnancy, parturition and early lactation.

Serial brain sections of female rats at late pregnancy, parturition or early lactation were immunostained for oxytocin. Immunoreactive perikarya were visible in the magnocellular nuclei in all experimental animals as well as in ovariectomized, nulliparous controls. During late pregnancy and at parturition additional immunostaining appeared in groups of perivascular neurons in the preoptic region, the lateral subcommissural nucleus, the perifornical region and scattered throughout the ventral portion of the hypothalamus. Immunostaining of almost all of these perivascular neurons disappeared by day two postpartum, while another population of oxytocin neurons, without association with blood vessels, appeared in these brain regions after parturition. Immunostaining of processes from oxytocinergic neurons in the periventricular nucleus increased markedly near parturition. Many of these processes projected toward the third ventricle. Oxytocinergic neuronal systems that are activated in late pregnancy and early postpartum may contribute to several physiological changes associated with parturition and lactation including the onset of maternal behavior.

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PubMed1989

Early sexual differentiation of diencephalic dopaminergic neurons of the rat in vitro.

Development of dopaminergic neurons was investigated in dissociated cell cultures raised from the diencephalon of male and female rat fetuses from days 14 and 17 of gestation. Striking differences in morphology and function of male and female dopaminergic neurons were observed. Outgrowth of tyrosine hydroxylase-immunoreactive processes initially proceeded at a faster rate in female than in male cultures. Morphological differences disappeared in cultures of gestational day 17. Irrespective of the age of the cultures and of the length of cultivation, the uptake capacity for (3H)dopamine per immunoreactive neuron was twice as high in female than in male cultures. Treatment of the cultures with sex steroids did not influence morphology, numbers or transmitter uptake of tyrosine hydroxylase-immunoreactive neurons. The results suggest that diencephalic dopaminergic systems exhibit a sexual dimorphism that develops unexpectedly early in ontogeny and is independent of the action of gonadal hormones.

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PubMed1989

Rapid maturation of synaptic functions of prenatal serotoninergic neurons in short-term cultures: absence of sex differences and hormone effects.

Serotonin is believed to modulate neuronal differentiation during early stages of brain development. In order to assess basic functional requirements for such a role, it was investigated how early serotoninergic neurons mature with respect to transmitter storage and stimulus-secretion coupling. Dissociated cell cultures were raised from embryonic rat rhombencephalon obtained at gestational day 14 and cultured for 3-8 days, which may roughly correspond to the prenatal period in vivo. Because of a possible involvement of serotonin in processes leading to sexual differentiation of the brain, gender-specific cultures were raised in addition and treated with sex steroids. Sensitivity of [3H]serotonin uptake to fluoxetine could already be observed at 3 days in vitro. Vesicular storage as probed with reserpine and nigericin, and the capability of releasing preaccumulated serotonin in a Ca2+-dependent manner were also present as early as 3 days in vitro. Seven per cent of the pre-accumulated transmitter could be released per minute upon stimulation with 54 mM K+. Immunocytochemical and autoradiographic preparations demonstrated that, after the same short culture period, the neurons had formed large fiber networks. No differences could be detected regarding any of the above parameters between female and male serotonin neurons and between cultures treated with and without estradiol, testosterone and dihydrotestosterone. It is concluded that, in contrast to other neuronal phenotypes, serotoninergic neurons are functionally mature when or shortly after they are taken into culture, i.e. around gestational day 14. The functional competence of prenatal serotonin systems should be a prerequisite for their suspected role in modulating neural development at pre and postsynaptic sites. The present results provide no evidence for the occurrence of a sexual dimorphism of serotonin neurons at this early developmental stage.

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PubMed1989

Sexual differentiation of mesencephalic neurons in vitro: effects of sex and gonadal hormones.

In order to study the influence of gender on the development of transmitter uptake by dopaminergic neurons, dissociated cell cultures were raised separately from male and female midbrains of gestational day 14 rats. It was ascertained by use of specific inhibitors and by autoradiography that the uptake of [3H]dopamine was restricted to neurons immunoreactive for tyrosine hydroxylase and that these neurons have dopaminergic properties. The uptake capacity was higher in male than in female dopaminergic neurons by a factor of 1.5. This sexual dimorphism in dopamine uptake was present in cultures of tissue removed before the perinatal rise of testosterone occurs in males, and was present even in the absence of hormonal additives to the culture medium. It therefore appears to be independent of the presence of gonadal steroids. It occurred likewise in cultures raised with serum-supplemented and serum-free medium, which may indicate that glia are not decisive in generating these differences. In addition, sexual differences were found regarding hormone responsiveness. Whereas testosterone and dihydrotestosterone were ineffective, estradiol was seen to promote dopamine uptake in female but not in male neurons. The results would suggest that mesostriatal and/or mesolimbic dopaminergic systems assume an early role in the development of some sexual dimorphisms of the brain.

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PubMed1989

Detection and partial characterization of a developmentally regulated nuclear antigen in neural cells in vitro and in vivo.

We report that a monoclonal antibody directed against phosphorylated neurofilaments (SMI 31) recognizes nuclear antigens present in embryonic but not in adult neural cells. On Western blots, the antibody reacts with four proteins of apparent MW 35, 37, 52/54, and 250 KD which are found exclusively in developing brain tissue. These nuclear antigens are expressed by glial and neuronal cells. Both nuclear staining and immunoreactive proteins decrease with ongoing in vitro differentiation. A computer search for proteins that share the epitope recognized by antibody SMI 31 did not yield any proteins of known nuclear localization that exhibit the same molecular weights and solubility characteristics as the above immunoreactive proteins. We conclude that antibody SMI 31 recognizes hitherto unknown nuclear proteins which, in neural cells, are developmentally regulated.

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PubMed1988

Developmental effect of dimethyl sulfoxide on hypothalamo-neurohypophysial neurons in vitro.

Primary dissociated cultures were established from diencephalic tissue of 14-day-old fetal rats. Neurons exhibiting immunocytochemical staining for neurophysin appeared in these cultures after 6 days of cultivation. Addition of dimethyl sulfoxide (DMSO) to the culture medium resulted in a slight decrease in total neuronal cell mass as assessed by immunocytochemistry and radio-immunometric quantitation of neuron-specific enolase. In contrast, in DMSO-treated cultures the number of neurophysin-immunoreactive neurons was more than doubled as compared to control cultures. [3H]Thymidine labeling and autoradiography in conjunction with immunocytochemistry for neurophysin showed that this was not due to a mitogenic effect of DMSO on precursor cells. Time-course analysis of the action of DMSO revealed a 6-day time lag between the initiation of treatment and the appearance of increased numbers of neurophysin-immunoreactive cells. These findings suggest that DMSO, which has previously been reported to have a differentiation-inducing effect on malignant transformed cells, may also modulate cellular processes that control differentiation in specific types of neurons in primary culture.

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PubMed1988

Abundant GABAergic innervation of rat posterior pituitary revealed by inhibition of GABA-transaminase.

An antibody against gamma-aminobutyric acid (GABA) was used to identify GABAergic elements immunocytochemically in the rat posterior pituitary. In order to increase the intracellular concentration of GABA, rats were treated with the GABA-transaminase inhibitor gamma-vinyl-GABA (GVG). Light-microscopic observations of Vibratome and semithin sections revealed the presence of numerous immunoreactive nerve fibers throughout the neural lobe; the mean number and length of these fibers increased by 90% after GVG treatment. Electron microscopy demonstrated the immunostained axons to be of small diameter. The reaction product was confined to small vesicles. No immunostaining occurred in pituicytes. The richness of the GABAergic innervation of the neural lobe contrasts with previous reports using antibodies against glutamate decarboxylase and supports the idea that GABA participates in the presynaptic control of neurosecretion.

4-Aminobutyrate Transaminase↗
PubMed1988

Topography of basal glucose utilization in rat thalamus and hypothalamus determined with (1-14C)-glucose.

High resolution autoradiography was used to study the basal pattern of glucose-utilization in the rat thalamus and hypothalamus. Rats were injected via chronic jugular catheter with (1-14C)-glucose and sacrificed 30 min later. The high resolution thaw-mount autoradiographic procedure, using 4 micron frozen sections and nuclear emulsion, permitted discrimination of regional variations in glucose-utilization that have not yet been described. Quantitative data were obtained by means of digital image analysis and computerized densitometry. In the thalamus, high activity was present in the anterodorsal, anteroventral, laterodorsal and reticular nuclei, while low activity was found in the mediodorsal and paraventricular nuclei. The autoradiographic pattern of glucose utilization in the thalamus corresponds largely to classical cytoarchitectonic subdivisions. In the hypothalamus, the median eminence, arcuate nucleus, and periventricular nucleus showed the lowest activity, whereas certain parts of the lateral hypothalamus appeared high. Very high activity was present in mammillary nuclei. The described detailed anatomical data of glucose-utilization may provide insights into the functional circuitry of thalamic and hypothalamic systems and serve as a baseline from which experimental manipulations can be assessed.

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PubMed1988

Developmental changes of neuron-specific enolase and neurofilament proteins in primary neural culture.

The expression of neuron-specific enolase (NSE) and neurofilament (NF) proteins in primary dissociated cell cultures derived from 14-day-old fetal rat diencephalon was studied by immunocytochemistry and quantitative western-blot techniques. Both neuronal marker proteins, NSE and NF, can be detected as early as day 2 in vitro. They show pronounced quantitative increases during the time period studied (12 days), the relative change being highest during the first few days in vitro (DIV). The molar ratio of the medium weight NF to the heavy NF polypeptide is 9.1 after 2 DIV and 2.6 after 12 DIV. Phosphorylation of the heavy NF polypeptide increases steadily during cultivation. Comparison of these results to in vivo data reported in the literature suggests that, qualitatively, neuronal development in vitro follows the pattern observed in vivo, but at an accelerated pace.

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PubMed1988

A histochemical study of the regional distribution in the rat brain of enzymatic activity hydrolyzing glucose- and 2-deoxyglucose-6-phosphate.

A modified Wachstein-Meisel lead salt method using glucose-6-phosphate or 2-deoxyglucose-6-phosphate as substrates was employed at the light microscopic level to map the rat brain for glucose-6-phosphatase (G-6-Pase). As has been described, most of the activity of the enzyme resided in neuronal cell bodies and dendritic stems. No differences were found between the results obtained with the two substrates. Two categories of brain structures with heavy and with moderate staining could be distinguished while the majority of brain regions contained only barely discernible neurons. Structures displaying very high enzyme activity included nuclei of cranial nerves, nuclei of the reticular formation, Purkinje cells, and some parts of the limbic system, e.g., CA 3 and CA 4 pyramidal fields of the hippocampus. It is pointed out that accurate biochemical determinations of G-6-Pase activity will critically depend on painstaking microdissection of nuclei and cell layers. The histochemical results may be pertinent to the interpretation of the 2-deoxyglucose method for assessment of regional glucose utilization rates in brain. The present observations make it unlikely that regional variations in G-6-Pase activity account for differences in uptake and retention of radioactivity from (1-14C)glucose and (14C)2-deoxyglucose reported previously by our group.

Animals↗
PubMed1988

Autoradiographic studies on glucose utilization in individual zones of the rat thymus.

Wistar rats were injected intraperitoneally with 2-(14C)deoxyglucose and their thymuses were processed for thaw-mount autoradiography after 5, 10 or 35 min. Highest levels of radioactivity were demonstrated in the thymic medulla (5-fold higher than in the cortex). Scanning of autoradiograms for regional differences in grain densities indicated particularly intense glucose utilization in the cortico-medullary zone. Differences in glucose utilization between individual thymic zones seem to reflect differences in cellular composition, i.e., ratio of stroma cells to thymocytes.

Animals↗
PubMed1988

Topography of basal glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose.

The activity of the pentose phosphate shunt was assessed under basal conditions in subregions of the hippocampus by measuring the uptake and retention of [1-14C]glucose and [6-14C]glucose and their 14C-labelled metabolites. The relative and absolute retention of carbon-14 from each of the two compounds was nearly identical in all regions examined. For each compound, the highest accumulation of 14C occurred in the granule cell layer of the dentate gyrus and in the pyramidal cell layer. Relatively high retention of radioactivity was also found in the molecular layer of dentate gyrus and in the stratum lacunosum-molecular. The stratum radiatum and stratum oriens contained the lowest levels of radioactivity among hippocampal regions. The equal retention of radioactivity from [1-14C]glucose and [6-14C]glucose implies that pentose phosphate shunt activity is very low throughout the hippocampus under the conditions of this study. The uptake and retention of radioactivity was evaluated in different hippocampal regions 10 or 30 min following intravenous injection of [1-14C]glucose. Although there was significantly more radioactivity at 30 min than at 10 min, the same topographic pattern of radioactivity within the hippocampus was observed in rats after both survival periods, indicating that an equal fraction of the [1-14C]glucose utilized in different hippocampal regions is oxidized to 14CO2 under these conditions. Most regions of high glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose correspond to regions of intense histochemical staining for cytochrome oxidase reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1987

Cerebral metabolic mapping at the cellular level with dry-mount autoradiography of [3H]2-deoxyglucose.

The uptake and retention of radioactivity from [3H]2-deoxyglucose [( 3H]2-DG) was assessed in certain regions of the rat brain under basal conditions using dry-mount autoradiography, a procedure which affords the best available conditions to accurately localize diffusable radiolabeled compounds at cellular and subcellular levels. The overall amount of radioactivity accumulated in neuropil and in neuronal cell bodies was similar in most brain regions examined. Of the regions assessed, the CA3 pyramidal cell field of the hippocampus was the only region in which the radioactivity in cell bodies was notably greater than that of neuropil. In the somatosensory cortex and in the lateral hypothalamus, a wide range of radioactivity was found among individual neurons and among different areas of neuropil. In all brain regions examined, a subpopulation of small cells, with morphological characteristics of glial cells, accumulated [3H]2-DG to a much greater extent than other glial cells or neurons. That finding suggests that certain glial cells are in a markedly higher metabolic state than other brain cells.

Animals↗
PubMed1987

Hypothalamo-neurohypophysial neurons in vitro: developmental potentials depend on the donor rat stock.

Primary dispersed cell cultures were established from fetal diencephalic tissue derived from two stocks of outbred rats. The overall growth pattern, as followed by phase-contrast microscopy and visualized by immunocytochemistry for neurofilament proteins and neuron specific enolase, depended on the age of the donor animal. Stock differences were not revealed by these methods. In contrast, when the cultures were immunostained in order to assess the yield of a specific type of cell, i.e., magnocellular hypothalamic neurons, striking differences were observed between the two stocks of rats. Whereas neurophysin-immunoreactive cells could be grown in cultures established from fetal Chbb: Thom rats at embryonic days 14, 15, and 18, no such cells could be detected in cultures derived from Crl: CD (SD) BR rats older than embryonic day 14. These findings may reflect differences in the differentiation schedule and/or requirements for certain epigenetic and trophic factors between magnocellular neuroblasts derived from the two stocks of rats.

Animals↗
PubMed1987

High resolution autoradiography at the regional topographic level with [14C]2-deoxyglucose and [3H]2-deoxyglucose.

After injection of 2-deoxyglucose (2-DG) labeled with tritium or carbon-14, autoradiograms were produced by thaw-mounting 4 micron frozen sections of rat brains on nuclear emulsion-coated slides. The results show that the distribution of radioactivity among different brain regions was similar and that the resolution at the regional topographical level was virtually identical for both compounds. The resolution obtained with the thaw-mounting of thin frozen sections onto nuclear emulsion was considerably greater than the resolution demonstrated in published results in the literature, when carbon-14 or tritium-labeled 2-deoxyglucose were used with 20 micron frozen sections and X-ray film or tritium-sensitive film. The results indicate that section thickness, detection medium and intimacy of contact between section and photographic emulsion influence resolution. At the regional level, the detection medium apparently influences resolution to a greater extent than the energy differences of the beta particles emitted from 14C or 3H. Although diffusion of radiolabeled 2-deoxyglucose and metabolites during the thaw-mounting process precludes single cell resolution of these autoradiograms, the improvement of visualizing regional topographic detail demonstrates that the described technique is a valuable approach with which to study regional 2-DG uptake.

Animals↗
PubMed1987

Sex steroids promote neurite growth in mesencephalic tyrosine hydroxylase immunoreactive neurons in vitro.

The influence of steroid hormones on the differentiation of catecholaminergic and serotonergic (5-HT) neurons was studied in dissociated cell cultures from embryonic day 14 (E14) rat diencephalon, mesencephalon and metencephalon treated for 6 days with 17 beta-estradiol (E), testosterone (T), 5 alpha-dihydrostestosterone (DHT), progesterone (P), dexamethasone (DEX), or E + T. The effects of these hormones on morphologic differentiation were determined by morphometric measurements of total length of neurites of immunocytochemically identified neurons in culture, which were stained with antisera against tyrosine hydroxylase (TH) or 5-HT. A significant increase in neurite length was observed in cultures of TH-immunoreactive (TH-IR) neurons from the mesencephalon treated with E, T, E + T, but not with P, DHT or DEX. Based on labeling with [3H]dopamine (DA) uptake and competition with specific inhibitors, these mesencephalic TH-IR cells appear to represent DA neurons of the A8-A10 groups (which includes the substantia nigra). No statistically significant effects of these steroids were observed on TH-IR neurons from the diencephalon (assumed to be precursors of the tuberoinfundibular and incertohypothalamic dopaminergic groups). The 5-HT neurons of the raphe nuclei (metencephalon) showed no statistically significant response to steroids. We conclude that during the early fetal period, sex steroids can affect the morphologic differentiation of mesencephalic DA neurons in vitro, indicating that these hormones are capable of selectively influencing the development of a specific population of monoamine neurons during this critical period.

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