Na(+)-K(+)-ATPase gene expression during in vitro development of rat fetal forebrain.

The existence of at least three isoforms of Na(+)-K(+)-ATPase in adult brain tissues [alpha 1, kidney type; alpha 2 [or alpha(+)]; alpha 3] suggests that these genes might be regulated in a cell-specific and time-dependent manner during development. We have studied this question in serum-free aggregating cell cultures of mechanically dissociated rat fetal telencephalon. At the protein level, the relative rate of synthesis of the pool of alpha 1-, alpha 2-, and alpha 3-subunits increased approximately twofold over 15 days of culture, leading to a marked increase in the immunochemical pool of alpha-subunits as measured by a panspecific polyclonal antibody. Concomitantly, Na(+)-K(+)-ATPase enzyme-specific activity increased three- (lower forebrain) to sixfold (upper forebrain). The transcripts of all three alpha-isoforms and beta-subunit were detected in vitro in similar proportion to the level observed in vivo. alpha 3-mRNA (3.7 kb) was more abundant than alpha 1 (3.7 kb) or alpha 2 (5.3 and 3.4 kb). Cytosine arabinoside (0.4 microM) and cholera toxin (0.1 microM) were used to selectively eliminate glial cells or neurons, respectively. It was found that alpha 2-mRNA is predominantly transcribed in glial cell cultures, whereas alpha 3- and beta 1-mRNA (2.7, 2.3, and 1.8 kb) are predominant in neuronal cultures.

Evaluation of the toxicity of different metal compounds in the developing brain using aggregating cell cultures as a model.

To evaluate their toxicity in the developing brain, eight metal compounds, [bismuth sodium tartrate (BiNA-tartrate), CdCl(2), CoCl(2), HgCl(2), dimethyl mercury, NiCl(2), TlCl and triethyltin chloride (TET)] were tested in aggregating cell cultures of foetal rat telencephalon. The compounds were applied to the cultures continuously, either during an early developmental stage (between days 5 and 14) or during and advanced stage of maturation (between days 24 and 34). Changes in the activities of cell type-specific enzymes were used as a criterion for toxicity. A general cytotoxic effect was observed after treatment with either CdCl(2), HgCl(2) or TET at 10(-6)m, and with TlCl at 10(-5)m. Selective effects were found with BiNa-tartrate and dimethylmercury. CoCl(2) did not modify the parameters tested, whereas a stimulant effect was found with NiCl(2). The effects of several compounds were development dependent: HgCl(2), TET and TlCl were more toxic in immature cultures, whereas BiNa-tartrate, dimethylmercury and NiCl(2) were more effective in differentiated cultures.

Induction of apoptosis by mercury compounds depends on maturation and is not associated with microglial activation

The earliest sign of neurotoxicity observed after exposure of three-dimensional brain cell cultures to low concentrations of mercury compounds is a microglial reaction. We hypothesized that an induction of apoptosis by mercury compounds could be an activating signal of the microglial reaction. Aggregating brain cell cultures of fetal rat telencephalon were treated for 10 days with either mercury chloride or monomethylmercury chloride at noncytotoxic concentrations during two developmental periods: from day 5 to 15, corresponding to an immature stage, and from day 25 to 35 corresponding to a mature stage. Apoptosis was evaluated by the TUNEL technique. It was found that both mercury compounds caused a significant increase in the number of apoptotic cells, but exclusively in immature cultures exhibiting also spontaneous apoptosis. Double staining by the TUNEL technique combined with either neuronal or astroglial markers revealed that the proportion of cells undergoing apoptosis was highest for astrocytes. Furthermore neither an association nor a colocalization was found between apoptotic cells and microglial cells. In conclusion, it appears that the induction of apoptosis by mercury compounds in immature cells is only an acceleration of a spontaneously occurring process, and that it is not a directly related to the early microglial reaction.

Differential regulation of Na-K-ATPase isoform gene expression by T3 during rat brain development.

A fetal rat telencephalon organotypic cell culture system was found to reproduce the developmental pattern of Na-K-adenosinetriphosphatase (ATPase) gene expression observed in vivo [Am. J. Physiol. 258 (Cell Physiol. 27): C1062-C1069, 1990]. We have used this culture system to study the effects of triiodothyronine (T3; 0.003-30 nM) on mRNA abundance and basal transcription rates of Na-K-ATPase isoforms. Steady-state mRNA levels were low at culture day 6 (corresponding to the day of birth) but distinct for each isoform alpha 3 much greater than beta 1 = beta 2 greater than alpha 2 greater than alpha 1. At culture day 6, T3 did not modify mRNA abundance of any isoform. At culture day 12 (corresponding to day 7 postnatal), T3 increased the mRNA level of alpha 2 (4- to 7-fold), beta 2 (4- to 5-fold), alpha 1 (3- to 6-fold), and beta 1 (1.5-fold), whereas alpha 3 mRNA levels remained unchanged. Interestingly, the basal transcription rate for each isoform differed strikingly (alpha 2 greater than alpha 1 much greater than beta 1 = beta 2 greater than alpha 3) but remained stable throughout 12 days of culture and was not regulated by T3. Thus we observed an inverse relationship between rate of transcription and rate of mRNA accumulation for each alpha-isoform, suggesting that alpha 1- and alpha 2-mRNA are turning over rapidly whereas alpha 3-mRNA is turning over slowly. Our data indicate that one of the mechanisms by which T3 selectively controls Na-K-ATPase gene expression during brain development in vitro occurs at the posttranscriptional level.

Differential expression of 240 kDa ConA-binding glycoprotein in vitro and in vivo detected by immunochemical methods.

The expression of the 240 ConA-binding glycoprotein (240 kDa), a marker of synaptic junctions isolated from the rat cerebellum, was studied by immunocytochemical techniques in forebrain and cerebellum from rat and chicken, and in chick dorsal root ganglia. Parallel studies were carried out either on tissue sections or in dissociated cell cultures. In all cases non neuronal cells were not immunostained. The tissue sections of cerebellum from rat and chick exhibited 240 kDa glycoprotein immunoreactivity, especially in the molecular layer, while the forebrain sections from rat and chick did not show any significant immunostaining. In contrast, in dissociated forebrain cell cultures, all neuronal cells expressed 240 kDa glycoprotein immunoreactivity, while glial cells remained totally unlabelled. In tissue sections of dorsal root ganglion (DRG), sensory neurons expressed the 240 kDa only after the embryonic day (E 10). A large number of small neurons in the dorsomedial part of DRG were immunostained with 240 kDa glycoprotein antiserum, whereas only a small number of neurons in the ventrolateral part of the ganglia displayed 240 kDa immunoreactivity. In dissociated DRG cells cultures (mixed or neuron-enriched DRG cell cultures) all the neuronal perikarya but not their processes were stained. These studies indicate that 240 kDa glycoprotein expression is completely modified in cultures of neurons of CNS or PNS since the antigen becomes synthetized in high amount by all cells independent of synapse formation. This demonstrates that the expression of 240 kDa is controlled by the cell environment.

Myelin basic protein content of aggregating rat brain cell cultures treated with cytokines and/or demyelinating antibody: effects of macrophage enrichment.

The demyelinative potential of the cytokines interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) has been investigated in myelinating aggregate brain cell cultures. Treatment of myelinated cultures with these cytokines resulted in a reduction in myelin basic protein (MBP) content. This effect was additively increased by anti-myelin/oligodendrocyte glycoprotein (alpha-MOG) in the presence of complement. Qualitative immunocytochemistry demonstrated that peritoneal macrophages, added to the fetal telencephalon cell suspensions at the start of the culture period, successfully integrated into aggregate cultures. Supplementing the macrophage component of the cultures in this fashion resulted in increased accumulation of MBP. The effect of IFN-gamma on MBP content of cultures was not affected by the presence of macrophages in increased numbers.

Calmodulin kinase IV: expression and function during rat brain development.

The expression of calmodulin kinase IV (CaMKIV) can be induced by the thyroid hormone T3 in a time- and concentration-dependent manner at a very early stage of brain differentiation using a fetal rat telencephalon primary cell culture system which can grow and differentiate under chemically defined conditions (Krebs et al. (1996) J. Biol. Chem. 271, 11055-11058). After the induction of CaMKIV by T3 we examined the influence of prolonged absence of T3 from the culture medium on the expression of CaMKIV. We could demonstrate that after the T3-dependent induction of CaMKIV, omission of the hormone, even for 8 days, from the medium did not downregulate the expression of CaMKIV indicating that different regulatory mechanisms became important for the expression of the enzyme. We further showed that CaMKIV could be involved in the Ca(2+) -dependent expression of the immediate early gene c-fos, probably via phosphorylation of the transcription factor CREB. Convergence of signal transduction pathways on this transcription factor by using different protein kinases may explain the importance of CREB for the regulation of different cellular processes.

Effects of trimethyltin (TMT) on glial and neuronal cells in aggregate cultures: dependence on the developmental stage.

Long-term effects of trimethyltin (TMT) applied at concentrations below the cytotoxic level were examined in three-dimensional cell cultures of fetal rat telencephalon using biochemical, immunochemical and morphological criteria. It was found that in immature cultures low concentrations of TMT (10(-8) M) specifically induced a gliotic response in astrocytes, with increased immunoreactivity for glial fibrillary acidic protein, and a greater number of astrocytic processes. Significant changes in oligodendrocytic and neuronal parameters were found only at 10(-6) M of TMT. In differentiated cultures, distinct changes in cell type-specific parameters occurred at 10(-6) M of TMT (the lowest effective concentration). In addition, different patterns of responses were found for astrocytes and oligodendrocytes, as compared to immature cultures. These results suggest that among neural cells, astroblasts are most sensitive to TMT, and that the glial responses to this neurotoxicant are development-dependent.

Hypocretins regulate the anxiogenic-like effects of nicotine and induce reinstatement of nicotine-seeking behavior

Emerging evidence suggests that the hypocretinergic system is involved in addictive behavior. In this study, we investigated the role of these hypothalamic neuropeptides in anxiety-like responses of nicotine and stress-induced reinstatement of nicotine-seeking behavior. Acute nicotine (0.8 mg/kg, s.c.) induced anxiogenic-like effects in the elevated plus-maze and activated the paraventricular nucleus of thehypothalamus (PVN) as revealed by c-Fos expression. Pretreatment with the hypocretin receptor 1 (Hcrtr-1) antagonist SB334867 orpreprohypocretin gene deletion blocked both nicotine effects. In the PVN, SB334867 also prevented the activation of corticotrophinreleasing factor (CRF) and arginine-vasopressin (AVP) neurons, which expressed Hcrtr-1. In addition, an increase of the percentage of c-Fos-positive hypocretin cells in the perifornical and dorsomedial hypothalamic (PFA/DMH) areas was found after nicotine (0.8 mg/kg,s.c.) administration. Intracerebroventricular infusion of hypocretin-1 (Hcrt-1) (0.75 nmol/1 l) or footshock stress reinstated a previouslyextinguished nicotine-seeking behavior. The effects of Hcrt-1 were blocked by SB334867, but not by the CRF1 receptor antagonistantalarmin. Moreover, SB334867 did not block CRF-dependent footshock-induced reinstatement of nicotine-seeking while antalarmin was effective in preventing this nicotine motivational response. Therefore, the Hcrt system interacts with CRF and AVP neurons in the PVN and modulates the anxiogenic-like effects of nicotine whereas Hcrt and CRF play a different role in the reinstatement of nicotineseeking.Indeed, Hcrt-1 reinstates nicotine-seeking through a mechanism independent of CRF activation whereas CRF mediates the reinstatement induced by stress.

Weed or wheel! FMRI, behavioural, and toxicological investigations of how cannabis smoking affects skills necessary for driving.

Marijuana is the most widely used illicit drug, however its effects on cognitive functions underling safe driving remain mostly unexplored. Our goal was to evaluate the impact of cannabis on the driving ability of occasional smokers, by investigating changes in the brain network involved in a tracking task. The subject characteristics, the percentage of Δ(9)-Tetrahydrocannabinol in the joint, and the inhaled dose were in accordance with real-life conditions. Thirty-one male volunteers were enrolled in this study that includes clinical and toxicological aspects together with functional magnetic resonance imaging of the brain and measurements of psychomotor skills. The fMRI paradigm was based on a visuo-motor tracking task, alternating active tracking blocks with passive tracking viewing and rest condition. We show that cannabis smoking, even at low Δ(9)-Tetrahydrocannabinol blood concentrations, decreases psychomotor skills and alters the activity of the brain networks involved in cognition. The relative decrease of Blood Oxygen Level Dependent response (BOLD) after cannabis smoking in the anterior insula, dorsomedial thalamus, and striatum compared to placebo smoking suggests an alteration of the network involved in saliency detection. In addition, the decrease of BOLD response in the right superior parietal cortex and in the dorsolateral prefrontal cortex indicates the involvement of the Control Executive network known to operate once the saliencies are identified. Furthermore, cannabis increases activity in the rostral anterior cingulate cortex and ventromedial prefrontal cortices, suggesting an increase in self-oriented mental activity. Subjects are more attracted by intrapersonal stimuli ("self") and fail to attend to task performance, leading to an insufficient allocation of task-oriented resources and to sub-optimal performance. These effects correlate with the subjective feeling of confusion rather than with the blood level of Δ(9)-Tetrahydrocannabinol. These findings bolster the zero-tolerance policy adopted in several countries that prohibits the presence of any amount of drugs in blood while driving.

Use of aggregating brain cell cultures to study developmental effects of organophosphorus insecticides.

Aggregating brain cell cultures of fetal rat telencephalon can be grown in a chemically defined medium for extended periods of time. After a phase of intense mitotic activity, these three-dimensional cell cultures undergo extensive morphological differentiation, including synaptogenesis and myelination. To study the developmental toxicity of organophosphorus compounds (OP), aggregating brain cell cultures were treated with parathion. Protein content and cell type-specific enzyme activities were not affected up to a concentration of 10(5) M. Gliosis, characterized by an increased staining for glial fibrillary acidic protein (GFAP), was observed in immature and in differentiated cells. In contrast, uridine incorporation and myelin basic protein (MBP) immunoreactivity revealed strong differences in sensitivity between these two developmental stages. These results are in agreement with the view that in vivo the development-dependent toxicity is not only due to changes in hepatic detoxification, but also to age-related modifications in the susceptibility of the different populations of brain cells. Furthermore, they underline the usefulness of histotypic culture systems with a high developmental potential, such as aggregating brain cell cultures, and stress the importance of applying a large range of criteria for testing the developmental toxicity of potential neurotoxicants.

A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

Glial hyaluronate-binding protein expression in aggregating brain cell cultures.

We analyzed the expression of glial hyaluronate-binding protein (GHAP), an integral component of the extracellular matrix, in aggregating brain cell cultures of fetal rat telencephalon using immunofluorescence. GHAP immunoreactivity appeared after 1 week in culture, simultaneous with the first deposits of myelin basic protein, and showed a development-dependent increase. Comparison of glia-enriched and neuron-enriched cultures showed that only glial cells express GHAP. Three peptide growth factors, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor, which are known to stimulate the differentiation of glial cells, modulated the deposit of GHAP immunoreactivity. The 3-dimensional structure of aggregate cultures promoted GHAP deposition, suggesting that cell-cell interactions are required for extracellular matrix formation. Furthermore GHAP production seemed to depend on the developmental stage of the glial cells.

Ochratoxin A at nanomolar concentration perturbs the homeostasis of neural stem cells in highly differentiated but not in immature three-dimensional brain cell cultures.

Ochratoxin A (OTA), a fungal contaminant of basic food commodities, is known to be highly cytotoxic, but the pathways underlying adverse effects at subcytotoxic concentrations remain to be elucidated. Recent reports indicate that OTA affects cell cycle regulation. Therefore, 3D brain cell cultures were used to study OTA effects on mitotically active neural stem/progenitor cells, comparing highly differentiated cultures with their immature counterparts. Changes in the rate of DNA synthesis were related to early changes in the mRNA expression of neural stem/progenitor cell markers. OTA at 10nM, a concentration below the cytotoxic level, was ineffective in immature cultures, whereas in mature cultures it significantly decreased the rate of DNA synthesis together with the mRNA expression of key transcriptional regulators such as Sox2, Mash1, Hes5, and Gli1; the cell cycle activator cyclin D2; the phenotypic markers nestin, doublecortin, and PDGFRα. These effects were largely prevented by Sonic hedgehog (Shh) peptide (500ngml(-1)) administration, indicating that OTA impaired the Shh pathway and the Sox2 regulatory transcription factor critical for stem cell self-renewal. Similar adverse effects of OTA in vivo might perturb the regulation of stem cell proliferation in the adult brain and in other organs exhibiting homeostatic and/or regenerative cell proliferation.

Nkx2.1-derived astrocytes and neurons together with Slit2 are indispensable for anterior commissure formation.

Guidepost cells present at and surrounding the midline provide guidance cues that orient the growing axons through commissures. Here we show that the transcription factor Nkx2.1 known to control the specification of GABAergic interneurons also regulates the differentiation of astroglia and polydendrocytes within the mouse anterior commissure (AC). Nkx2.1-positive glia were found to originate from three germinal regions of the ventral telencephalon. Nkx2.1-derived glia were observed in and around the AC region by E14.5. Thereafter, a selective cell ablation strategy showed a synergistic role of Nkx2.1-derived cells, both GABAergic interneurons and astroglia, towards the proper formation of the AC. Finally, our results reveal that the Nkx2.1-regulated cells mediate AC axon guidance through the expression of the repellent cue, Slit2. These results bring forth interesting insights about the spatial and temporal origin of midline telencephalic glia, and highlight the importance of neurons and astroglia towards the formation of midline commissures.