Regulated Production and Secretion of Immunoreactive Neuropeptide Y by Aggregating Fetal Brain Cells in Cultures

Barnea, Ayalla; Hajibeigi, Asghar; Cho, Gloria; Magni, Paolo

doi:10.1159/000125844

Cited by 17 publications

(13 citation statements)

References 40 publications

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“…Sprague-Dawley female rats (Harlan, Indianapolis, IN) were anesthetized with Nembutal on day 17 of pregnancy (day of sperm = day 0), and the fetuses were removed. The cortex was dissected and cells were dissociated using the procedure previously described in detail (Barnea et al, 1991b(Barnea et al, , 1993b. Briefly, diced tissue from 13 fetuses was incubated at 37°C for 20 min in Ca/Mg-free Hanks solution followed by 30 min incubation in 2 ml 0.125% trypsin; then, 13 ml of DMEM containing 20 pg/ml DNAase I and 10% fetal calf serum were added, and the cells were mechanically dissociated.…”

Section: Materials and Methods Cell Culturementioning

confidence: 99%

Brain‐derived neurotrophic factor induces functional expression and phenotypic differentiation of cultured fetal neuropeptide Y‐producing neurons

Barnea

Cho

Lü

et al. 1995

J of Neuroscience Research

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A series of studies from our laboratory has established an aggregate culture system of fetal rat brain cells that can serve as a model for studying regulatory processes of the developing neuropeptide Y (NPY)-producing neurons. Using aggregate cultures derived from 17-day-old fetal rat cortex, we addressed these questions: 1) Does brain-derived neurotrophic factor (BDNF) stimulate NPY production, and if so, is stimulation a function of the developmental state of the cultured NPY neuron? 2) Does BDNF induce phenotypic differentiation of NPY neurons? BDNF led to an increase in NPY production and the accumulation of NPY-mRNA in a dose dependent manner. BDNF did not alter the stability of NPY-mRNA, judged by the disappearance rate of NPY-mRNA after blockade of RNA synthesis (estimated t1/2 was 6-8 hr). BDNF stimulation of NPY production was dependent on length of exposure to BDNF and on culture-age. A continuous 8-day exposure to BDNF resulted in a significantly higher level of NPY production than a pulse of 2 days (comparing BDNF exposure on days 0-8 vs. 6-8, or days 8-17 vs. 15-17). Moreover, older neurons (age 17 days) produced twice as much NPY as younger (age 8 days) neurons in response to a 2-day pulse of BDNF (50 ng/ml). BDNF was significantly more effective than NT-3 in inducing NPY production, and NGF was ineffective. Immunocytochemical analysis of 8-day NPY neurons revealed that a 2-day pulse of BDNF induced the appearance of an abundance of morphologically well-defined neurons bearing an elaborate network of neurites. This was in contrast to the control-treated NPY neurons, which were morphologically undefined. In summary, the age-dependent effect of BDNF on NPY production is consistent with induction of functional expression, rather than promotion of survival, of cultured NPY neurons. The neurotrophin specificity for stimulation of NPY production, and the lack of effect of BDNF on the stability of NPY-mRNA, implicate the TrkB receptor in mediating transcriptional activation of the NPY gene. Thus, BDNF exerts a dual effect on developing cultured NPY neurons: induction of functional expression, and phenotypic differentiation of immature neurons into mature neurite-bearing neurons.

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Section: Materials and Methods Cell Culturementioning

confidence: 99%

Brain‐derived neurotrophic factor induces functional expression and phenotypic differentiation of cultured fetal neuropeptide Y‐producing neurons

Barnea

Cho

Lü

et al. 1995

J of Neuroscience Research

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“…We followed our published protocols for dissociation and culture of cells derived from rat (Barnea et al, 1991b(Barnea et al, , 1993a and human fetal brains (Aguila-Mansilla and . The only difference in handling rat and human tissues was the larger volumes of reagents used for the latter.…”

Section: Aggregate Culturesmentioning

confidence: 99%

“…NPY and SRIF were extracted from the media and aggregates (Barnea et al, 1991b) and peptides were quantified by radioimmunoassay (RIA) (20 µl aliquots of rat/human samples). NPY was assayed using synthetic hNPY as the reference standard and 125 I pNPY as the tracer (Barnea et al, 1993b); the lowest level of sensitivity (20% displacement of 125 I NPY) was 24 Ϯ 2 pg (mean Ϯ SE, N ϭ 5 assays).…”

Section: Assaysmentioning

confidence: 99%

Opposite effects of astrocyte-derived soluble factor(s) on the functional expression of fetal peptidergic neurons in aggregate cultures: Enhancement of neuropeptide Y and suppression of somatostatin

et al. 1997

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Previous studies established that fetal rat and human neuropeptide Y (NPY) cortical neurons in aggregate cultures are differentially regulated. Whereas brain-derived neurotrophic factor (BDNF) or phorbol 12-myristate-13-acetate (PMA) induces NPY production in rat cultures, only PMA does so in human cultures. We addressed these questions: 1) Do soluble products of rat or human astrocytes (conditioned medium; rCM and hCM, respectively) enhance the functional expression of cultured NPY neurons and if so, do they enhance the expression of somatostatin (SRIF) neurons as well? 2) Is the NPY-enhancing activity (EA) in the CM species specific? rCM enhanced (approximately 2-fold) both basal and BDNF-stimulated production of NPY and coculture of rat aggregates and astrocytes did not prevent this NPY-EA. Likewise, the hCM enhanced (approximately 2.5-fold) basal and PMA-stimulated production of NPY by human aggregates. Moreover, the hCM enhanced NPY production by rat aggregates and rCM enhanced NPY production by human aggregates. In addition, rCM and hCM each enhanced BDNF-, forskolin-, or PMA-stimulated NPY production by rat aggregates. Under each of the above conditions, the rCM/hCM suppressed (approximately 50%) production of SRIF by rat aggregates. In summary, secretory products of rat and human astrocytes exert opposite effects on the functional expression of NPY and SRIF neurons in culture: enhancement of NPY and suppression of SRIF. By the criteria evaluated in this study, these astrocyte-derived activities do not exhibit species specificity.

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“…Aggregates were processed for DNA analysis as described. 7 The DNA was assayed fluorimetrically, ~9 using salmon testes DNA as the standard.…”

Section: Dna Assaymentioning

confidence: 99%

Human fetal brain cells in aggregate culture: A model system to study regulatory processes of the developing human neuropeptide Y (NPY)‐producing neuron

Aguila-Mansilla

Barnea

1996

Intl J of Devlp Neuroscience

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Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the brain and it has been implicated in a wide range of brain functions, including mentation. The aim of this study was to establish a culture system of human fetal brain cells expressing NPY in a regulated manner. The NPY production in response to forskolin and phorbol 12-myristate 13-acetate (PMA) was taken as a criterion for regulated expression of NPY. Aggregates were formed from dissociated cells derived from the cerebral hemispheres of human fetuses (12.5-19 weeks' gestation) by constant rotation and were maintained in serum-free medium. A 24 hr exposure to 10 microM forskolin + 20 nM PMA led to a 2-6-fold increase in NPY content of the cultures, most of which (80-90%) was secreted into the medium. The latter consisted of two substances differing in size: one corresponding in size to proNPY and the other to NPY. Thus, forskolin + PMA led to an increased production of NPY. Exposure to PMA alone led to an increase in NPY production comparable to that seen after forskolin + PMA and this effect of PMA was dose-dependent. In contrast, forskolin alone did not induce NPY production. Conditioned medium, derived from monolayer cultures enriched with human astrocytes, enhanced NPY production in response to forskolin+PMA in an age-dependent manner. The NPY production by aggregates derived from a 12.5 week-, 14-week- and 18-week-old fetus was enhanced 3-3.6-fold, 1.6-2-fold and 1.1-fold, respectively. Thus, expression of the NPY neurons in this culture system is a regulated process. The NPY production is enhanced markedly by activation of the protein kinase C pathway and by an astrocyte-derived soluble substance(s). Based on these results, we propose that this culture system can serve as a model for the study of regulatory processes of the human developing NPY neuron.

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Regulated Production and Secretion of Immunoreactive Neuropeptide Y by Aggregating Fetal Brain Cells in Cultures

Cited by 17 publications

References 40 publications

Brain‐derived neurotrophic factor induces functional expression and phenotypic differentiation of cultured fetal neuropeptide Y‐producing neurons

Brain‐derived neurotrophic factor induces functional expression and phenotypic differentiation of cultured fetal neuropeptide Y‐producing neurons

Opposite effects of astrocyte-derived soluble factor(s) on the functional expression of fetal peptidergic neurons in aggregate cultures: Enhancement of neuropeptide Y and suppression of somatostatin

Human fetal brain cells in aggregate culture: A model system to study regulatory processes of the developing human neuropeptide Y (NPY)‐producing neuron

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