Influence of triiodothyronine (L-T3) on the morphological and biochemical development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium

In cerebral hemisphere cultures initiated from 15-day-old rat embryos, the number of acetylcholinesterase-positive (AChE+) cells increased from 6.8 +/- 1.6 cells/well on day 3 to 112 +/- 16 cells/well on day 15. With time in culture, AChE+ cells increased both in size of the perikarya and neurite length. The addition of L-triiodothyronine (L-T3) at a concentration of 3 x 10(-8) M at the initiation of the culture had no effect on the number of AChE+ cells but significantly increased the size and neurite length of AChE+ neurons after 5 days in vitro. These morphological effects are associated with biochemical effects. L-T3 increased AChE activity in both a dose- and time-dependent manner (the stimulatory effect of L-T3 becomes significant between day 8 and day 15). Since a major part of AChE+ cells may be cholinergic neurons, we have also measured the effect of L-T3 on ChAT activity. L-T3 also increased ChAT activity in a dose and time dependent manner. Furthermore, treatment of cultures with L-T3 at different times in culture demonstrated the presence of a critical period which occurs in vitro around day 20, since the stimulatory effect of L-T3 on ChAT activity is lost after 20 days in vitro. Studies of the time necessary for L-T3 to increase both ChAT and AChE activities show that 2 days and 15 days, respectively, are required for L-T3 to significantly stimulate both enzyme activities. This in vitro analysis demonstrated the morphological effect of L-T3 on the size and the neurite length of AChE+ cells. These effects are associated with biochemical effects on ChAT and AChE activities. Thus, it appears that thyroid hormones regulate several steps of neuronal maturation.     

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.     

Enhanced survival of cultured dopamine neurons by treatment with soluble extracts from chemically deafferentiated striatum of adult rat brain

A soluble fraction was extracted from a chemically deafferentiated striatum of adult Wistar rats after unilateral lesioning of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) injection. The soluble extract from the lesioned side enhanced the survival of cultured mesencephalic dopamine (DA) neurons of 14-day-old rat embryos as evidenced by quantitative counting of tyrosine hydroxylase-like immunoreactive cells. The neurotrophic activity of this striatal extract for DA neurons was highest 14 days after 6-OHDA injection and became negligible in 28 days. The extract showed no promoting effects on cultured γ-aminobutyric acid (GABA)-containing mesencephalic neurons. These observations indicate that the striatum of adult rats may initiate de novo synthesis of trophic substance(s) for DA neurons but not for GABA neurons when subjected to nigral dopaminergic deafferentiation.     

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.     

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.     

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a serum-free chemically defined medium: effects on acute and chronic opiate regulation of adenylate cyclase activity

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a chemically defined medium within 3 cell passages, exhibited viability, growth rate and morphology similar to those of cells grown in medium supplemented with 5% fetal calf serum. Hybrid cells cultured in the chemically defined medium within these periods of time also did not exhibit a difference in basal adenylate cyclase activity nor in the enzymatic activities stimulated by adenosine, forskolin, NaF, GppNHp or Mn²⁺. Furthermore, opiate receptor density in chemically defined medium cultured cells remained identical to that in cells cultured in 5% fetal calf serum. The acute and chronic effects of opiates on adenylate cyclase were similar for cells grown under either set of conditions.     

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.     

Time course of in vitro expression of NADPH-diaphorase in cultured rat brain neurons: comparison with in vivo expression

The time course of NADPH-diaphorase expression was examined in primary cultures of rat central nervous system and in embryonic or neonatal rat brains using a histochemical method. In cerebral and brainstem cultures from 17-day-old embryonic rats, neuronal cells moderately expressing NADPH-diaphorase were first detected on about the 5th to 7th day of culturing. Both the density of positive cells and the staining intensity increased with age of cultures. The density of positive cells, calculated as a percent of the total number of cells, increased up to day 21 in cultures from both the cerebrum and the brainstem, indicating that NADPH-diaphorase is preferentially expressed in neurons with longer viability. On the other hand, virtually no intensely positive cells were detectable in cerebellar cultures at any period examined up to 21 days. In the in vivo study, moderately stained NADPH-diaphorase-positive neurons were first detected, mainly in the laterodorsal-pedunculopontine tegmental nuclei complex and partly in the striatum, in 16-day-old embryonic rat brain. At 2 days postnatal, intensely stained neurons were detectable in the cerebral cortex as well as in the tegmental nuclei complex and the striatum, indicating some delay in the in vitro, as compared to the in vivo, expression of neuronal NADPH-diaphorase.     

Interleukin-2 as a neurotrophic factor for supporting the survival of neurons cultured from various regions of fetal rat brain

Interleukin(IL)-2 supported the survival and enhanced neurite extension of cultured hippocampal neurons prepared from embryonic 18-day-old rats. This neurotrophic effect was observed at concentrations of 2 to 200 U/ml, and almost all the neurons could survive for more than 2 days in the presence of 200 U/ml of IL-2. This viability-promoting effect of IL-2 on the neurons was completely blocked with anti-IL-2 antibodies. IL-2 also supported the survival of cultured cortical, striatal, and septal neurons. These results indicate that IL-2 has a survival-promoting effect on a wide variety of neurons. On the other hand, IL-2 did not affect the choline acetyltransferase (ChAT) activity of striatal neurons, suggesting that this cytokine does not act as a differentiation factor for striatal cholinergic neurons. © 1993 Wiley-Liss, Inc.     

Etazolate (SQ20009): electrophysiology and effects on [3H]flunitrazepam binding in cultured cortical neurons

The actions of etazolate (SQ20009) on cultured cortical neurons have been examined in electrophysiological experiments and in receptor binding studies. Etazolate (0.3 to 100 microM) prolongs the duration of spontaneously occurring IPSPs. Higher concentrations of etazolate produce an increase in membrane chloride conductance, an effect which is picrotoxinin and bicuculline sensitive. Etazolate potentiates the response to exogenously applied GABA and acts synergistically with diazepam to enhance GABA-mediated conductance. Etazolate does not increase glycine-mediated conductance changes. Etazolate increases [3H]flunitrazepam binding and stimulates the GABA enhancement of [3H]flunitrazepam binding. In addition, GABA stimulates the etazolate enhancement of [3H]flunitrazepam binding. The etazolate-mediated increases in binding are Cl- dependent and picrotoxinin and bicuculline sensitive. The dose response relationships for etazolate-mediated effects are similar in physiological experiments and in binding studies. These data suggest that etazolate interacts with the postsynaptic GABA receptor complex at a site distinct from either the GABA recognition site or the benzodiazepine binding site to enhance GABA-mediated inhibition and to increase benzodiazepine binding.     

Chronic treatment with sabeluzole protects cultured rat brain neurons from the neurotoxic effects of excitatory amino acids.

The neuroprotective properties of the cognitive enhancer sabeluzole were investigated in rat brain neuronal cultures derived from the hippocampal formation of 17-day-old rat embryos. Measurement of the neuronal cytoskeletal microtubule-associated protein, MAP2, was used to assess survival of neurons after exposure of neuronal cultures to glutamate. MAP2 was quantified in neuronal cell homogenates by means of an enzyme-linked immunosorbent assay (ELISA) using a mouse monoclonal MAP2 antibody, peroxidase-labeled goat anti-mouse Ig antiserum, and 2,2'-azido-di-[3-ethylbenz-thiazoline] sulphonate (ABTS) as substrate. Exposure of 7-day-old neuronal cultures to 1 mM glutamate for 16 hours led to a three-fold increase in released lactate dehydrogenase (LDH) and a 40% decrease in cellular MAP2 content. Acute treatment of neuronal cultures with 10 microM sabeluzole yielded a 40% drop in released LDH induced by glutamate. Cultures treated chronically with 0.1 microM sabeluzole on days 1 and 4 in culture showed, after 1 week in culture, a MAP2 content and total LDH activity that was not different from control cultures. A 16-hour exposure to 1 mM glutamate did not induce LDH release or changes in MAP2 levels in sabeluzole-treated cultures. A single treatment with 0.1 microM sabeluzole between day 1 to 5 induced a 70-80% drop in glutamate-induced released LDH in 7-day-old neuronal cultures. Full and partial neuronal protection after chronic sabeluzole treatment at 0.1 microM was also observed for neurotoxicity induced by 5 mM N-methyl-D-aspartate (NMDA) and 1 mM kainic acid or 30 microM veratridine, respectively. Within a series of compounds such as Ca++ and Na+ channel antagonists, glutamate receptor antagonists and various neurotransmitter receptor antagonists, sabeluzole, chronically given, were the most potent for inhibition of released LDH induced by 1 mM glutamate (IC50-value: 34 +/- 13 nM). In conclusion, chronic sabeluzole treatment protects cultured rat brain neurons from excitotoxic aggression.     

Triiodothyronine stimulates the production of insulin-like growth factor (IGF) by fetal hypothalamus cells cultured in serum-free medium

Grown in serum-free medium, dissociated cells from fetal mouse hypothalami release insulin-like growth factors (IGFs) and their binding proteins (IGF BPs) into the culture medium. Addition of triiodothyronine (10-12-10-8 M), which enhances neuron maturation, resulted in a significant increase in IGF concentration. By contrast, there was no significant effect on IGF BP. These results suggest a role for thyroid hormone in the control of IGF biosynthesis in nerve cells.     

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

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 (≈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 (≈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 (≈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. J. Neurosci. Res. 50:605–617, 1997. © 1997 Wiley-Liss, Inc.     

Expression of a marrow stroma and thymus-associated antigen (ST3) in the rat brain: comparison with Thy-1

Cells of the immunohemopoietic and nervous systems express certain molecules that generally are not found in other tissues. One example is the ‘ST3’ antigen, which is present on the major population of fibroblastoid cells grown from rat bone marrow, but is not detected on adherent cells from most peripheral organs (e.g. lung). An immunohistological survey revealed ST3 also in the thymic cortex, the glomerular mesangial area, and the brain. Because this pattern of distribution is similar to that described for Thy-1, we compared the localization of the two antigens in the adult rat brain and found that there were areas where it was congruent and others where it was distinct. Staining for ST3 was absent from the white matter, but was especially notable in discrete layers of the frontal, orbital, parietal, and cingulate cortices, the substantia nigra, the inferior olivary nuclei, and the deep molecular layer of the cerebellum, as well as other scattered regions in the gray matter. This is in contrast to Thy-1, which stained more diffusely throughout the gray zones. In further experiments using primary brain cell cultures. ST3 was demonstrated on neurons, but not on oligodendrocytes or astrocytes. Similarly, it was found on the surface of cells of the PC12 neuronal line, but not on the C6 astrocytoma. This restricted distribution on a subpopulation of neurons raises the possibility that the ST3 epitope might be part of a cell interaction molecule of the marrow stroma, thymus, and brain.     

The noradrenergic system in cultured aggregates of fetal rat brain cells: Morphology of the aggregates and pharmacological indices of noradrenergic neurons

Spherical aggregates formed rapidly in culture by re-aggregation of trypsin-dissociated brain cells from the 17-day-old fetal rat. Over days 10 days an initially random distribution of cells evolved into a 30layered arrangement; cells with characteristics of neurons were found largely in the intermediate layer. The survival of neuronal and glial cell types was evaluated histologically and verified by electron microscopy, which revealed synaptic and myelin structures that rapidly increased in number after 18 days in culture. Levels of norepinephrine (NE) and dopamine (DA) reached peaks of 9.5 and 4.4 ng/mg protein, respectively, at culture day 21. Uptake of [³H]NE paralleled these amine levels and was blocked by desipramine or pretreatment with either reserpine or 6-OH-DA. Autoradiographs of aggregates labeled with [³H]NE showed a high density of silver grains over cells, apparently neurons, with branching processes traced for 120 μm. Previously accumulated [³H]NE was released under depolarizing conditions (high [K⁺] or vertridine) only in the presence of Ca²⁺. Release was induced to a lesser extent by kainic > glutamic acid. Thus, such aggregates appear to contain catecholaminergic neurons capable of synthesis, uptake and release of NE. The time course of development of these functions supports suggestions that aggregate preparations might be useful in studying neurochemical or morphological aspects of brain development and function in vitro.     

The fetal development of the luteinizing hormone-releasing hormone (LHRH) neuronal systems of the guinea pig brain

We have studied the distribution of LHRH-like immunoreactive material in fetal guinea pig brains beginning at day 25 of gestation. Cells and processes were first detected throughout the peripheral, intracranial and central course of the nervus terminalis at 28 (but not 25) days of gestation. The localization of LHRH in this structure preceded its appearance in the hypothalamus and coincided with the initial detection of immunoreactive LH in the pituitary gland. The possible role of the LHRH neuronal network within the nervus terminalis in the development of reproductive function is discussed. Comparisons between the brains of littermates of both sexes were made at each age (days 28 through 60 of gestation) to determine possible differences between the sexes in the development of the LHRH neurosecretory systems. No sexually dimorphic features were evident in these systems throughout the prenatal period except at days 40 and 45. At these ages, differences in the number of LHRH neurons in the arcuate nucleus were found between the sexes in some but not all of the brains examined. These differences in LHRH concentrations may reflect the onset of testicular activity as indicated by an increase in serum testosterone levels. Increased serum testosterone concentrations were observed in the male fetuses beginning at 45 days of gestation. However, cell counts made within this nucleus from days 40 through 60 of gestation indicated no comparable sexual dimorphism in the total neuronal population which appeared to be relatively stable throughout this period of brain growth. The number of immunoreactive LHRH neurons visible throughout the brain increased from days 30 through 45 and fewer LHRH cells were seen on days 50 and 60 of gestation, particularly in the arcuate nucleus. The apparent decrease in visible LHRH neurons was concomitant with an increase in number and more extensive distribution of immunoreactive processes throughout the hypothalamus and in certain extrahypothalamic areas of the brain.