Influence of soluble environmental factors on the development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium: comparison with the effects of L-triiodothyronine (L-T3)

In cerebral hemisphere neuronal cultures derived from 15-day-old rat embryos, the addition of L-triiodothyronine (L-T3) or nerve growth factor (NGF) enhanced the expression of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in a dose-dependent manner. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT and AChE activities was significantly greater than the sum of the individual effects. Conversely, when the cultures were exposed to astrocyte conditioned medium grown in the presence or absence of L-T3 (CM + L-T3 or CM-L-T3). laminin and fibroblast growth factor (FGF), ChAT and AChE activities were not stimulated above those of control cultures when added alone or in combination with L-T3. Furthermore, L-T3, NGF, CMs, laminin and FGF did not affect AChE+ cell survival, but significantly increased neurite outgrowth and branching with NGF and L-T3 being the most powerful agents followed by CMs, laminin and FGF. Additionally, the simultaneous addition of L-T3 with either laminin or FGF in culture, caused an additive effect of L-T2 in the neurite density of AChE+ cells with both agents. This study shows that (1) thyroid hormones do not act through the regulation of soluble neurotrophic factors produced by astroglial cells, (2) thyroid hormones interact with the effect of NGF on ChAT and AChE activities, (3) the regulation of ChAT and AChE activities and the neurite outgrowth are independently regulated. and (4) the regulation of ChAT and AChE activities is very specific compared with that of neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triiodothyronine enhancement of neuronal differentiation in aggregating fetal rat brain cells cultured in a chemically defined medium

Triiodothyronine (30 nM) added to serum-free cultures of mechanically dissociated re-aggregating fetal (15–16 days gestation) rat brain cells greatly increased the enzymatic activity of choline acetyltransferase and acetylcholinesterase throughout the entire culture period (33 days), and markedly accelerated the developmental rise of glutamic acid decar☐ylase specific activity. The enhancement of choline acetyltransferase and acetylcholinesterase specific activities in the presence of triiodothyronine was even more pronounced in cultures of telecephalic cells. If triododthyronine treatment was restricted to the first 17 culture days, the level of choline acetyltransferase specific activity at day 33 was 84% of that in chronically treated cultures and 270% of that in cultures receiving triiodothyronine between days 17 and 33, indicating that relatively undifferentiated cells were more responsive to the hormone. Triiodothyronine had no apparent effect on the incorporation of [³H]thymidine at day 5 or on the total DNA content of cultures, suggesting that cellular differentiation, rather than proliferation was affected by the hormone. Our findings in vitro are in good agreement with many observations in vivo, suggesting that rotation-mediated aggregating cell cultures of fetal rat brain provide a useful model to study thyroid hormone action in the developing brain.     

Long-term cultivation of cryopreserved human fetal brain cells in a chemically defined medium

Conditions for long-term cultivation of human fetal brain cells in a chemically defined medium were established using cryopreserved brain fragments obtained from legal abortions. Tissue of the same gestational age was pooled and the cells cultured in a fully defined medium containing insulin-like growth factors (IGF I and II). Primary cultures were kept for 2-4 weeks and secondary or tertiary cultures could be maintained for 3 months. The cultures were characterized by morphological, electrophysiological and biochemical methods. Glial cells were predominant during the first two weeks of culture. In later stages of cultivation, glial cells diminished in number and most cells were neuronal. Voltage-dependent Na+ channels were recorded from neurons. Biochemical studies indicated that the fetal brain cells contained and secreted immunoreactive somatostatin as well as the tachykinins, substance P and neurokinin A. Cultures grown in IGF II- or nerve growth factor-containing medium expressed increased choline acetyltransferase activity.     

Estrogen treatment enhances survival of cultured fetal rat amygdala neurons in a defined medium

Effects of estradiol on the survival of cultured fetal rat amygdala neurons were estimated to assess a possible organizational action of the sex steroid on the developing amygdala tissue. Dissociated 17-day fetal amygdala cells were cultivated initially in a serum-containing and then in a serum-free defined medium. The survival of the cells in the serum-free medium was highly enhanced when supplemented with estradiol at the concentration of 10 ng/ml. Predominant cell populations of the culture were identified as neuronal cells by the tetanus toxin labeling method. The results support the idea that sex steroids play a role in the brain sexual differentiation by enhancing the neuronal survival in the developing amygdala tissue.     

Developmental features of rat cerebellar neural cells cultured in a chemically defined medium

We studied some aspects of the differentiation of rat cerebellar neural cells obtained from 8-day postnatal animals and cultured in a serum-free, chemically defined medium (CDM). The ability of the cells to take up radioactive transmitter amino acids was analyzed autoradiographically. The L-glutamate analogue 3H-D-aspartate was taken up by astroglial cells, but not by granule neurons, even in late cultures (20 days in vitro). This is in agreement with the lack of depolarization-induced release of 3H-D-aspartate previously observed in this type of culture. In contrast, 3H-(GABA) was scarcely accumulated by glial-fibrillary-acidic-protein (GFAP)-positive astrocytes, but taken up by glutamate-decarboxylase-positive inhibitory interneurons and was released in a Ca2+-dependent way upon depolarization: 3H-GABA evoked release progressively increased with time in culture. Interestingly, the expression of the vesicle-associated protein synapsin I was much reduced in granule cells cultured in CDM as compared to those maintained in the presence of serum. These data would indicate that in CDM the differentiation of granule neurons is not complete, while that of GABAergic neurons is not greatly affected. Whether the diminished differentiation of granule cells must be attributed only to serum deprivation or also to other differences in the composition of the culture medium remains to be established. 3H-GABA was avidly taken up also by a population of cells which were not recognized by antibodies raised against GFAP, glutamate decarboxylase, and microtubule-associated protein 2. These cells exhibited a stellate morphology, were stained by the monoclonal antibody A2B5, and have been characterized elsewhere [Levi et al, 1986] as bipotential precursors of oligodendrocytes and of a subpopulation of astrocytes bearing a stellate shape and capable of high-affinity 3H-GABA uptake.     

Adverse effects of phenobarbital on morphological and biochemical development of fetal mouse spinal cord neurons in culture

The effects of chronic phenobarbital treatment on neuronal development were assessed in cell cultures of fetal rodent spinal cord. The activity of the principal enzyme involved in acetylcholine synthesis, choline acetyltransferase (CAT), and counts of large spinal cord neurons were used as indicators of neuronal development. Phenobarbital (30 to 120 m?g/ml) added to cultures for two- or six-week periods produced dose-dependent decreases in both CAT activity and neuron counts. Barbituric acid at doses equimolar to those at which phenobarbital produced these decreases had no effect.     

Effects of amyloid-beta on cholinergic and acetylcholinesterase-positive cells in cultured basal forebrain neurons of embryonic rat brain

The neurotoxic effects of amyloid-beta(1-42) and amyloid-beta(25-35) (A beta) on cholinergic and acetylcholinesterase-positive neurons were investigated in primary cultures derived from embryonic 18-day-old rat basal forebrain. After various time intervals, the cultures were treated with 1, 5, 10 or 20 microM A beta for different time periods. The cholinergic neurons and their axon terminals were revealed by vesicular acetylcholine transporter immunohistochemistry and the cholinoceptive cells by acetylcholinesterase histochemical staining. To assess the toxic effects of these A beta peptides on the cholinergic neurons, image analysis was applied for quantitative determination of the numbers of axon varicosities/terminals and cells. The results demonstrate that, following treatment with 1 or 5 microM A beta for 5, 10, 30, 60 or 120 min, no changes in vesicular acetylcholine transporter immunohistochemical staining were observed. However, after treatment for 30 min with 10 or 20 microM A beta, the number of stained axon varicosities was reduced, and treatment for 2 h they had disappeared. In contrast, vesicular acetylcholine transporter-positivity could be seen in some of the neuronal perikarya even after 3 days after treatment. The acetylcholinesterase staining was homogeneously distributed in the control neurons. After A beta treatment, the histochemical reaction end-product was detected in some of the neuronal perikarya or in the dendritic processes near to the soma. It is concluded that the neurotoxic effects of A beta appear more rapidly in the cholinergic axon terminals than in the cholinergic and acetylcholinesterase-positive neuronal perikarya.     

Nerve outgrowth, synaptogenesis and bioelectric activity in fetal rat cerebral cortex tissue cultured in serum-free, chemically defined medium

Dissociated and non-dissociated cerebral cortex of fetal rat was successfully cultured in a serum-free, chemically defined medium for at least 18 days without any preincubation in serum-supplemented medium. Neurite outgrowth, synapse formation and spontaneous bioelectric activity, in its qualitative and quantitative aspects, were essentially the same as was observed in medium containing 20% heat-inactivated horse serum.     

Morphological and biochemical maturation of rat astroglial cells grown in a chemically defined medium: Influence of an astroglial growth factor

Astroglial cells from cerebral hemispheres of newborn rats were cultured for 5 days in Waymouth's MD 705/1 medium containing 10% fetal calf serum. Thereafter, cells were grown in a chemically defined medium consisting of basal Waymouth's medium supplemented with insulin (5 μg/ml) and fatty acid free bovine serum albumin (0.5 mg/ml). The cells underwent morphological and biochemical development over a period of 28 days. The changes in the amount of glial fibrillary acidic protein indicated a development of gliofilaments. The level of S100 protein increased during the entire culture period, while glutamine synthetase activity remained low and relatively constant. The addition of an astroglial growth factor, partially purified from bovine brain soluble extract, stimulated the morphological maturation of the astroglial cells. The cells extended cytoplasmic processes and resembled mature astrocytes. At the ultrastructural level an increase in free ribosomes was observed and the intermediate filaments became organized into large bundles. The amount of glial fibrillary acidic protein was not significantly increased, but the level of S100 protein and the glutamine synthetase activity were greatly enhanced. Our results indicate that astroglial cells undergo limited maturation in the chemically defined medium and that this process is positively affected by the astroglial growth factor.     

Synapse formation and development of neurotransmitter functions in neuronal cells from chick brain cultured in a serum-free, defined medium

Cells dissociated from cerebral hemispheres of 8-day-old chick embryos were seeded on poly-L-lysine coated Petri dishes in serum-containing medium. After 24 hr the culture medium was switched to a serum-free, chemically defined medium. These cultures contain mainly neuronal cells until day 14, characterized by the presence of acetylcholinesterase activity and neurofilament proteins. After 2 weeks glial cells progressively contaminated the neuronal culture. Cultures were maintained for a period of 4 weeks. From day 6 on numerous synapses with clear vesicles were observed. The activity of choline acetyltransferase remained low throughout the culture period, while GABA levels increased in parallel with synaptogenesis. Our observations indicate that chick cerebral hemisphere neuronal cultures grown in serum-free, chemically defined medium contain GABAergic neurons that undergo maturation.     

Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium

Cultures obtained from early postnatal rat cerebellum, grown in either chemically defined or in serum-supplemented medium containing 25 mM K+, contained predominantly (greater than 90%) small interneurones, mostly granule cells, with good and comparable viability (assessed by the retention of preloaded 51Cr). Neuronal survival was prolonged in the chemically defined medium, nerve cells living up to two weeks longer than in serum-supplemented medium, although the proportion of non-neuronal cells was not greatly increased. In the serum-supplemented medium neurones became organised into clumps connected by thick, fasciculated bundles of neurites by about one week in vitro. In comparison, in the chemically defined medium aggregation of neurones and fasciculation of neurites was markedly reduced even after 4 weeks in culture. The possible relationship between the organisation of neurones and the nature of the substratum, chemical factors in the medium as well as the surface properties of the cells is discussed.     

A novel way of removing quiescent astrocytes in a culture of subcortical neurons grown in a chemically defined medium

A new method has been described for removing a very small number of contaminating astrocytes in neuronal cultures (derived from the septal-diagonal band region of 17-day-old embryonic rat brain) grown in a chemically defined medium. The proportion of these glial fibrillary acidic protein (GFAP)-positive cells was usually less than 1.5% up to 10 days, but thereafter their number increased rapidly reaching 10-15% by 22 days in vitro. A prolonged exposure to normally used concentration of cytosine arabinoside (Ara-C; 10 microM) was toxic to both astroglial and neuronal cells, while a brief treatment (48 h) with a low level (4 microM) of Ara-C failed to eliminate these astrocytes, as judged by glutamine synthetase activity and GFAP-positive cell count. However, these quiescent astroglial cells could be easily eliminated if they were induced to proliferate by epidermal growth factor before exposure to Ara-C. The combined treatment with these agents had no effect on the number of acetylcholinesterase-positive cells, and on the development of cholinergic and GABA-ergic neurons, as measured in terms of choline acetyltransferase and glutamate decarboxylase activity, respectively.     

Culture of rat cerebral oligodendrocytes in a serum-free, chemically defined medium

Oligodendrocytes were isolated from the cerebra of young rats (5-10 days old) by trypsinization of the tissue followed by cell separation on Percoll gradients. The isolation was carried out in physiological, isotonic media. The cell yield was 2-4 X 10(6) cells per brain; the plating efficiency was greater than or equal to 70%. Isolated cells were seeded on poly-L-lysine-coated culture dishes and maintained in a serum-free, chemically defined medium for at least 30 days. After 10 days in culture 67 +/- 10% of the surviving cells were oligodendrocytes, as judged by immunocytochemical and morphological criteria, whereas most of the other cells reacted positively with antiserum against glial fibrillary acidic protein. The expression of typical oligodendrocyte markers (2':3'-cyclic-nucleotide 3'-phosphodiesterase, galactocerebrosides and myelin basic protein) was greatly enhanced under these serum-free conditions as compared with cultures in serum-containing medium. The antigenic markers (galactocerebrosides, myelin basic protein) were absent in the freshly isolated cells but could be detected after 3 days in culture by immunocytochemistry. The activity of 2':3'-cyclic-nucleotide 3'-phosphodiesterase increased from 75 nmol min-1 mg-1 protein on day 4 to 400 nmol min-1 mg-1 protein on day 14 in culture.     

Phenotypic plasticity of avian embryonic sympathetic neurons grown in a chemically defined medium: direct evidence for noradrenergic and cholinergic properties in the same neurons.

Avian embryonic sympathetic ganglia possess both catecholaminergic and cholinergic features and can synthesize noradrenaline (NAd) and acetylcholine (ACh) simultaneously. In the present study we sought to determine (1) whether or not this coproduction of NAd and ACh corresponds to the existence of two non-overlapping populations, and (2) to what extent the levels of synthesis are influenced by non-neuronal ganglion cells. We have focused on the correlation between the immunocytochemically demonstrable presence of the noradrenergic and cholinergic enzymes tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively, and the synthesis of the corresponding neurotransmitters in embryonic quail sympathetic neuronal and non-neuronal cells purified by fluorescence-activated cell sorting. We show that (1) freshly sorted neurons synthesize both NAd and ACh, whereas non-neuronal cells produce neither; (2) the overwhelming majority of the sympathetic neurons display TH immunoreactivity; (3) about half of the TH-positive neurons are recognized by an anti-ChAT antibody in an artificial medium that selectively enhances synthesis and/or accumulation of ACh; (4) the non-neuronal cells are important for survival of the neurons and potentiate their synthesis of ACh in this medium, and (5) finally, we present evidence that expression of TH in noradrenergic neurons and in small intensely fluorescent cells of sympathetic ganglia is differentially regulated.     

Effects of maternal hyperphenylalaninemia on fetal brain development: A morphological study

We examined the effects of maternal hyperphenylalaninemia on body and brain growth, and the biochemical maturation of the fetal and neonatal rat brain. Elevated concentrations of plasma phenylalanine were induced in pregnant rats under two experimental conditions from the 14th through the 21st days of gestation. In the first treatment, pregnant rats were injected subcutaneously with alpha-methylphenylalanine (to inhibit maternal liver phenylalanine hydroxylase) at a dosage of 30 mg/100 g body weight plus phenylalanine supplementation (to increase maternal and fetal plasma phenylalanine) at a dosage of 60 mg/100 g body weight two times daily. In the second treatment, pregnant dams were injected with phenylalanine only at a dosage of 65 mg/100 g body weight three times daily. Treatment with alpha-methylphenylalanine/phenylalanine (mPhe/Phe) resulted in a 76% inhibition in the activity of maternal phenylalanine hydroxylase and a 25-fold increase in the mean daily concentration of phenylalanine in the maternal and fetal plasma. Phenylalanine treatment alone resulted in a 15-fold increase in plasma phenylalanine in the maternal and fetal animals. Significant reductions in body and brain weights in the fetal and neonatal rats were found in both treatment groups. Biochemical determinations indicated that the total DNA, RNA, and protein contents of the cerebra were reduced, with the reductions being greater in the mPhe/Phe- than the phenylalanine-treated rats. However, the retardation in body and brain growth of both treatment groups did not appear to be permanent because substantial recovery was noted in the rats after postnatal day 7. These results suggest that exposure of the fetus to high plasma concentrations of phenylalanine caused a delay in the biochemical maturation of the fetal rat brain.     

Expression of adenosine A1 receptors in cultured neurons from fetal rat brain

The expression of adenosine A₁ receptors was investigated using [³H]2-chloro-N⁶ -cyclopentyladenosine (CCPA) in 8-day-old cultured neurons from fetal rat forebrain grown in serum-free medium. [³H]CCPA bound specifically and with high affinity (Kd = 2.9 nM) to a homogeneous population of sites. Displacement of CCPA binding by various adenosine derivatives indicated that A₁ receptors were selectively labeled. The presence of Gpp(NH)p, a GTP analogue, reduced significantly the binding affinity (Kd = 12.2 nM), suggesting that A₁ receptors detected in intact cultured cells are linked to associated G proteins. © 1993 Wiley-Liss, Inc.     

Effect of coenzyme Q(10) on biochemical and morphological changes in experimental ischemia in the rat brain

The aim of the work was to evaluate an influence of CoQ(10) on lactate acidosis, adenosine-5'-triphosphate (ATP) concentrations, oxidized to reduced glutathione ratio and on superoxide dismutase activity in endothelin model of cerebral ischemia in the rat. Light microscopic studies in the central nervous system and morphometric analysis of pyramidal cells in the hippocampus were also performed. Endothelins (ET-1 or ET-3; 20 pmoles) were injected into the right lateral cerebral ventricle (intracerebroventricularly). CoQ(10) was given intraperitoneally (i.p.) just before the operation (i.p. 10 mgkg b. wt.). More severe changes of investigated biochemical parameters were observed in the animals treated with ET-1 in comparison with ET-3. Recovery was noted earlier in the group subjected to ET-3 and CoQ(10) administration, than in the animals subjected to ET-1 and CoQ(10) treatment. Histopathological observations showed sparse foci of a neuronal loss in the cerebral cortex and in the hippocampus only in the ET-1 model of ischemia. Additionally more numerous dark neurons were present in above brain structures following ET-1 administration comparing with ET-3 one. Morphometrical studies demonstrated that CoQ(10) diminished neuronal injury in the hippocampal CA1, CA2 and CA3 zones. Above data indicate on neuroprotective effect of CoQ(10) as a potent antioxidant and oxygen derived free radicals scavenger in the cerebral ischemia.     

Effects of triiodothyronine (T3) on the development of rat cerebellar cells in culture

To determine whether or not the effects of thyroxine on the cells of the external granular layer of rat cerebellum are direct or indirect, Purkinje cell-free dissociated cell cultures from 5-day-old rat cerebellum in serum-free medium were treated with triiodothyronine (T3) at concentrations of 20–3000 ng/ml. Cultures were assayed for uptake and synthesis of γ-aminobutyric acid (GABA) after 3 weeks, and for thymidine incorporation at 1, 2, 3 and 4 days. Specific (per mg protein) activity of glutamic acid decarboxylase and of GABA uptake into neurons was reduced by the more physiologic (50 or 500 ng/ml) concentrations of T3, probably due to a non-neuronal increase in overall protein synthesis, since the total per culture values seem unaffected by hormone. Thymidine uptake was significantly increased only at the highest (unphysiologic) concentration of T3 on the second day after treatment. None of the label appeared to be in the nuclei of the neuronal cells at any of the times and concentrations tested.These results are consistent with the hypothesis that T3 does not have a direct effect on the proliferation of neurons derived from the external granular layer, although it does seem to affect some non-neuronal cells. T3 also does not seem to enhance differentiated GABA functions (uptake or synthesis) beyond the levels achieved using insulin, progesterone, putrescine, selenium and transferrin.     

Interleukin-2 promotes survival and neurite extension of cultured neurons from fetal rat brain

We investigated the effects of interleukin-2 (IL-2) on the survival and morphology of primary cultured neurons from fetal rat brain. Addition of recombinant human IL-2 significantly supported the survival of brain neurons in high cell density culture, but did not show any effect on the neuronal survival in low cell density culture. In addition, IL-2 significantly promoted the neurite elongation and branching of hippocampal neurons in low cell density culture. These results suggest that IL-2 supports neuronal survival indirectly and promotes neuritogenesis by directly acting on brain neurons.