The role of dopaminergic transmission on striatal mRNA levels of regulators of G protein signalli... more The role of dopaminergic transmission on striatal mRNA levels of regulators of G protein signalling proteins RGS 2-12 was evaluated by quantitative in situ hybridisation. A single injection of L-dopa (50 mg/kg i.p.) significantly increased the RGS2 mRNA level (by 25%), an effect that was specifically abolished by the D1 dopamine receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (R(+)-SCH23390), but not changed by S(-)-eticlopride. Interestingly, the administration of this D2 dopamine receptor antagonist alone markedly enhanced the expression of RGS2 (by 71%), which suggests a constitutive inhibition of RGS2 expression by D2 dopamine receptors. Opposite results were obtained concerning the regulation of RGS4 since L-dopa alone was without effect whereas co-administration of L-dopa and R(+)-SCH23390 significantly enhanced the RGS4 mRNA levels (by 38%). In conclusion, D1 and D2 dopamine receptors appear to mediate opposite r...
The International Journal of Neuropsychopharmacology, 1999
Family, twin and adoption studies suggest that genetic factors play an important role in the aeti... more Family, twin and adoption studies suggest that genetic factors play an important role in the aetiology of schizophrenia. The mode of inheritance, however, is complex and non-Mendelian. Although the aetiology of schizophrenia is unknown, it has been hypothesized that the necessary conditions for developing the disease are environmental stress and a vulnerability to psychosis. The implication of dopamine receptors to schizophrenia has been greatly studied. Several linkage and association studies have been performed in an attempt to establish the involvement of dopamine receptors in schizophrenia. However, although no conclusive evidence of linkage or association to any gene has been established, some results, suggestive of linkage for chromosomes 6, 22 and 13, await confirmation from other studies. Concerning association studies, it is also of interest that some studies support an association between schizophrenia and homozygosity at D(3). More work in larger samples is required before conclusive linkage hypothesis or association to a dopamine receptor may be established. Schizophrenic patients have been shown to have significant deficits in a wide range of cognitive processes, including memory, attention, reasoning ability and language. Since cognitive deficits are significant symptoms of schizophrenia which require effective treatment, their assessment in schizophrenic patients and during clinical trials of new potential antipsychotics is highlighted. Cognitive impairment in schizophrenia impedes psychosocial performance and is therefore an especially relevant target variable in the development of new therapeutic approaches. It is most prominent in tasks involving attention, memory and executive functions which are thought to reflect involvement of prefrontal and left-temporal brain areas. Semantic networks in schizophrenic patients with a younger age of onset are observed to be more disorganized and differ significantly to those of control subjects. The need to use broader approaches such as neuropsychological-related measures to identify pertinent phenotypes in non-affected subjects carrying vulnerability genes is also emphasized. Since dopamine receptors are the primary targets in the treatment of schizophrenia, improved therapy may be obtained by drugs that selectively target a particular subtype of dopamine receptor. In the development of novel antipsychotics, D(3) and D(4) receptors have received much attention and this is partly related to the fact that these receptors have a high abundance in brain areas associated with cognitive and emotional functions, such as parts of the limbic system and cortex. Recent studies suggest that atypical neuroleptics may significantly improve the cognitive deficits observed in schizophrenic patients and that atypical neuroleptics such as risperidone appear to improve memory and alertness suggesting that further clinical studies are needed to determine the precise influence of antipsychotics on the cognitive system of schizophrenic patients. Such studies could lead to useful insights as to the potential advantages of the newer antipsychotics which appear to have a sparing or beneficial effect on various components of cognitive function. However, the observation that cortical D(2) receptors are important sites of action for antipsychotics, that the cerebral cortex may harbour the common sites of actions of antipsychotics and that the balancing of the opposing actions of D(1) and D(2) receptor regulation may be an appropriate drug treatment suggests that the adjustment of D(1) receptor levels in the cortex may become an important goal of future antipsychotic generation. Such antipsychotics will be able to treat the positive, negative and cognitive deficits of schizophrenia.
Quantitative in situ hybridization was used to investigate the effect of prolonged striatal dopam... more Quantitative in situ hybridization was used to investigate the effect of prolonged striatal dopamine or monoamine depletion on the mRNA density of regulators of G-protein signaling (RGS) 2–12 proteins. Two types of treatments were studied: a 6-hydroxydopamine-induced unilateral lesion of the nigrostriatal pathway and a 5-day reserpine treatment. The results clearly show a selective increase in the mRNA levels of
There are two families of dopamine (DA) receptors, called D1 and D2, respectively. The D1 family ... more There are two families of dopamine (DA) receptors, called D1 and D2, respectively. The D1 family consists of D1- and D5-receptor subtypes and the D2 family consists of D2-, D3-, and D4-receptor subtypes. The amino acid sequences of these receptors show that they all belong to a large superfamily of receptors with seven transmembrane domains, which are coupled to their
Regional brain glucose utilization was investigated with positron emission tomography and fluorod... more Regional brain glucose utilization was investigated with positron emission tomography and fluorodeoxyglucose in 2 patients with a seizure disorder associated with diffuse band heterotopia, a condition known as "double cortex." Although 1 patient was examined shortly after the onset of the first seizures, the other had a long history of intractable epilepsy before examination. Magnetic resonance imaging revealed a symmetric and generalized band of ectopic gray matter and an overlying normal-looking cortex, without focal abnormality. Metabolic studies yielded comparable results in both patients, with similar and even higher glucose uptake in the layer of gray matter heterotopia compared to the normal cortex. These data suggest the persistence of some synaptic activity in the heterotopic neurons, which seems unaffected by age or by the time-course of epilepsy.
Striatal glutamatergic inputs are known to participate in the modulation of dopaminergic transmis... more Striatal glutamatergic inputs are known to participate in the modulation of dopaminergic transmission. Accordingly, the non-competitive N-methyl-D-aspartate receptor antagonists memantine and amantadine increase striatal dopamine levels, the latter being widely used in Parkinson's disease therapy. Based on our previous work revealing increased function of dopamine receptors and dopamine transporter after amantadine treatment, we studied the effects of repeated memantine administration on dopaminergic neurotransmission. On rat striatal membranes, dopamine-stimulated [(35)S]GTPgammaS binding was significantly reduced (20%) after 2 days injection with memantine (20 mg/kg per day, i.p.) but not after longer treatments (4 or 7 days). Evaluation of [(3)H]SCH 23390 and [(3)H]spiperone specific bindings only revealed a significant increase in D1 receptor density after 4 or 7 days treatment. Finally, none of these treatments were found to change the activity of the neuronal dopamine transporter in striatal synaptosomes. This shows that amantadine and memantine differentially affect striatal dopaminergic transmission, which could indicate that these two related aminoadamantanes display distinct pharmacodynamic properties.
The possibility to isolate stem cells from the adult central nervous system and to maintain and p... more The possibility to isolate stem cells from the adult central nervous system and to maintain and propagate these cells in vitro has raised a general interest with regards to their use in cell replacement therapy for degenerative brain diseases. Considering the critical role played by astrocytes in the control of glutamate homeostasis, we have characterised the expression of functional glutamate transporters in neural stem cells exposed to selected culture conditions favouring their differentiation into astrocytes. Commonly, neural stem cells proliferate in suspension as neurospheres in serum-free medium. The addition of serum or a supplement of growth factors (G5) to the culture medium was found to trigger cell adhesion on coated surfaces and to favour their differentiation. Indeed, after 7 days in these conditions, the vast majority of the cells adopted markedly distinct morphologies corresponding to protoplasmic (with serum) or fibrous (with G5 supplement) astrocytes and approximately 35-40% acquired the expression of the glial fibrillary acidic protein (GFAP). Immunocytochemical analysis also revealed that the treatments with serum or with the G5 supplement triggered the expression of the glial glutamate transporters GLT-1 (35 and 21%, respectively) and GLAST (29 and 69%, respectively). This effect was correlated with a robust increase in the Na+ -dependent [3H]-d-aspartate uptake, which was partially inhibited by dihydrokainate, a selective blocker of GLT-1. Together, these results indicate that in vitro differentiation of cultured neural stem cells can give rise to distinct populations of astrocytes expressing functional glutamate transporters.
In rat brain, [3H]glibenclamide binds with high affinity to sulfonylurea receptors associated wit... more In rat brain, [3H]glibenclamide binds with high affinity to sulfonylurea receptors associated with ATP-sensitive potassium (KATP) channels. KATP channels may play a modulatory role in neurotransmitter release and are involved in acute pathological events occurring in the brain. Fluorescein derivatives, which are suitable tools for the labelling of nucleotide binding sites, influence KATP channels and sulfonylurea receptors properties in insulinoma and cardiac cells. In this study, a negative allosteric action of fluorescein derivatives on glibenclamide binding sites has been shown in rat cortical neurons. This supports the hypothesis of interactions between nucleotide- and sulfonylurea-binding sites within the sulfonylurea receptor.
Transgenic rats expressing a mutated form of the human Cu/Zn superoxide dismutase (hSOD1(G93A)) d... more Transgenic rats expressing a mutated form of the human Cu/Zn superoxide dismutase (hSOD1(G93A)) develop an amyotrophic lateral sclerosis (ALS)-like phenotype, including motor neurone degeneration and reactive gliosis in the spinal cord. This study aimed at examining the presence of endogenous neural progenitors in the lumbar spinal cord of these rats at the end-stage of the disease. Immunohistochemical data clearly demonstrated the induced expression of the stem cell factor reported as a chemoattractant and survival factor for neural stem cells as well as nestin (neuro-epithelial stem cell intermediate filament) in the spinal cord sections. While the stem cell factor immunolabelling appeared diffuse throughout the gray matter, nestin labelling was restricted to clusters within the ventral horn. Interestingly, as paralysis regularly develops asymmetrically, induction of nestin was only detected on the ipsilateral side of the predominant symptoms. Finally, immunohistochemical detection of the stem cell factor receptor (c-Kit) revealed its specific induction which coincided with nestin immunolabelling. Together, these results are indicative of endogenous recruitment of neural progenitors within lesioned tissues and could support the development of treatments involving endogenous or exogenous stem cells.
Quantitative in situ hybridization on rat coronal brain sections with radiolabelled oligonucleoti... more Quantitative in situ hybridization on rat coronal brain sections with radiolabelled oligonucleotide probes was performed to investigate the effects of antipsychotic drugs on mRNA levels of regulator of G-protein signalling (RGS) 2 and c-fos. This study demonstrated a similar increase of RGS2 mRNA level in the striatum upon both a single and a 21-day treatment with either haloperidol (2 mg/kg) or risperidone (7.5 mg/kg) in contrast to clozapine (20 mg/kg). Otherwise, the acute c-fos mRNA induction in the striatum was abolished by 74 to 89% upon chronic treatment with either haloperidol or risperidone. In conclusion, the induction of RGS2 mRNA in the striatum, in contrast to the immediate early gene c-fos mRNA, is preserved upon chronic treatment with haloperidol and risperidone.
When rat cultured neurons were incubated with unlabelled neurotensin (3nM) for 1 or 24h at 37°C, ... more When rat cultured neurons were incubated with unlabelled neurotensin (3nM) for 1 or 24h at 37°C, the [3H]-neurotensin specific binding measured in cell homogenates was decreased to about 35 and 65% of control values, respectively. In these experiments, the decreases in binding corresponded to reductions of Bmax values without changes in the affinity. The slow neurotensin-induced receptor down-regulation is thought
In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues an... more In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues and several methods have been reported for promoting their maturation into differentiated astrocytes. Hence, a combination of several nutriments/growth factors – the G5 supplement (insulin, transferrin, selenite, biotin, hydrocortisone, fibroblast growth factor and epidermal growth factor) – is widely used as a culture additive favouring the growth, differentiation and maturation of primary cultured astrocytes. Considering the key role played by glial cells in the clearance of glutamate in the synapses, cultured astrocytes are frequently used as a model for the study of glutamate transporters. Indeed, it has been shown that when tested separately, growth factors influence the expression and activity of the GLAST and GLT-1. The present study aimed at characterising the functional expression of these transporters during the time course of differentiation of cultured cortical astrocytes exposed to the supplement G5. After a few days, the vast majority of cells exposed to this supplement adopted a typical stellate morphology (fibrous or type II astrocytes) and showed intense expression of the glial fibrillary acidic protein. Both RT-PCR and immunoblotting studies revealed that the expression of both GLAST and GLT-1 rapidly increased in these cells. While this was correlated with a significant increase in specific uptake of radiolabelled aspartate, fluorescence monitoring of the Na+ influx associated with glutamate transporters activity revealed that the exposure to the G5 supplement considerably increased the percentage of cells participating in the uptake. Biochemical and pharmacological studies revealed that this activity did not involve GLT-1 but most likely reflected an increase in GLAST-mediated uptake. Together, these data indicate that the addition of this classical combination of growth factors and nutriments drives the rapid differentiation toward a homogenous culture of fibrous astrocytes expressing functional glutamate transporters.
Magma: Magnetic Resonance Materials in Physics, Biology, and Medicine, 1997
The aim of this study was to investigate the effects of riluzole on the lesion induced by a perma... more The aim of this study was to investigate the effects of riluzole on the lesion induced by a permanent middle cerebral artery occlusion (MCAO) in rats. Riluzole at 4 or 8 mg/kg i.v. significantly reduced the cortical ischemic brain damage. With the most effective dose of 8 mg/kg, the time evolution of the lesion was assessed by T2-weighted magnetic resonance imaging (MRI) repeated on the same animals after MCAO. MRI obtained at 24, 48, and 72 hours after MCAO showed a progressive increase of the ischemic lesion, except in the cortex of the riluzole-treated rats (8 mg/kg i.v.). Furthermore, there was no difference between lesion volumes as measured by MRI or by histology. This study indicates that MRI may be a valuable method to quantify in vivo the neuroprotective profile of a drug.
The dopamine receptor-mediated modulation of guanosine 5'-O-(gamma-[35S]thio)triphosphate... more The dopamine receptor-mediated modulation of guanosine 5'-O-(gamma-[35S]thio)triphosphate ([35S]GTP gamma S) binding has been characterized in rat striatal membranes. In optimized experimental conditions, the potency of dopamine was 4.47 microM [3.02-6.61 microM] and a maximal response representing 54.8 +/- 4.5% increase above basal level was observed. Data obtained with different agonists and antagonists clearly revealed that the most important fraction of this response was reflecting D2 receptor activation. Further analysis with specific antagonists also supported evidence for the involvement of D1 dopamine receptors. The potencies of compounds interacting with D1 and D2 receptors were deduced from [35S]GTP gamma S binding experiments and compared with their binding affinities for these receptors measured in similar experimental conditions. A good correlation between these parameters was observed, supporting the applicability of this technique for the study of dopamine receptors in the central nervous system.
Adult bone marrow mesenchymal stem cells are multipotent cells that can differentiate into a vari... more Adult bone marrow mesenchymal stem cells are multipotent cells that can differentiate into a variety of mesodermal tissues. Recent studies have reported on their ability to also evolve into non-mesodermal cells, especially neural cells. While most of these studies revealed that manipulating these cells triggers the expression of typical neurone markers, less is known about the induction of neuronal- or glial-related physiological properties. The present study focused on the characterisation of glutamate transporters expression and activity in rat mesenchymal stem cells grown in culture conditions favouring their differentiation into astroglial cells. Ten days exposure of the cells to the culture supplement G5 was found to increase the expression of nestin (neuro-epithelial stem cell intermediate filament), an intermediate filament protein expressed by neural stem cells. Simultaneously, a robust induction of the high-affinity glutamate transporter GLT-1 (and GLAST) expression was detected by RT-PCR and immunocytochemistry. This expression was correlated with a highly significant increase in the Na+-dependent [3H]D-aspartate uptake. Finally, while glial fibrillary acidic protein immunoreactivity could not be detected, the induced expression of the astrocytic enzyme glutamine synthetase was demonstrated. These results indicate that in vitro differentiation of adult mesenchymal stem cells in neural precursors coincides with the induction of functional glutamate transport systems. Although the astrocytic nature of these cells remains to be confirmed, this observation gives support to the study of mesenchymal stem cells as a promising tool for the treatment of neurological diseases involving glutamate excitoxicity.
Serotonin S2, benzodiazepine, and muscarinic receptors showed different regional distributions in... more Serotonin S2, benzodiazepine, and muscarinic receptors showed different regional distributions in the human brain but were present in all cortical areas. The laminar distributions of [3H]ketanserin, [3H]diazepam, and [3H]quinuclidinylbenzilate were investigated in the temporal cortex and revealed a high density in the IIIrd and IVth layers. Dopamine D2 receptors were not detected in the cortex.
Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization... more Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization of the neurotensin receptor at the second messenger level. Stimulation with nanomolar concentrations of neurotensin elicited rapid rises in the cytosolic calcium concentration ([Ca2+]i), which remained elevated throughout the peptide application. A significant response was already detected with neurotensin concentrations as low as 0.01 nM. This high efficiency of neurotensin in mediating this calcium response contrasts with the nanomolar affinity of the peptide for its receptor measured in binding experiments. Evidence indicated that the initial elevation of the [Ca2+]i resulted from release of Ca2+ from intracellular stores, whereas the sustained response involved an influx of extracellular origin. Return to the basal level was only reached after extensive washing of the peptide or its displacement with the neurotensin receptor antagonist SR48692. After washing, further stimulations were still able to mediate an increase in the [Ca2+]i, indicating an apparent absence of rapid desensitization of the intracellular signaling pathway that mediates calcium mobilization. In contrast with this absence of response desensitization, the neurotensin receptors were found to internalize after stimulation with the peptide. This internalization was maximal after 30 min and accounted for approximately 70% of the number of neurotensin binding sites located at the cell surface. These results indicate that despite the functional properties of the rat neurotensin receptor present in Chinese hamster ovary cells after transfection, the intracellular signaling pathway triggered by stimulation with neurotensin seems to be resistant to desensitization. This might be related to the high efficiency of the intracellular signaling pathway coupled to the neurotensin receptor observed in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective l... more Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.
The role of dopaminergic transmission on striatal mRNA levels of regulators of G protein signalli... more The role of dopaminergic transmission on striatal mRNA levels of regulators of G protein signalling proteins RGS 2-12 was evaluated by quantitative in situ hybridisation. A single injection of L-dopa (50 mg/kg i.p.) significantly increased the RGS2 mRNA level (by 25%), an effect that was specifically abolished by the D1 dopamine receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (R(+)-SCH23390), but not changed by S(-)-eticlopride. Interestingly, the administration of this D2 dopamine receptor antagonist alone markedly enhanced the expression of RGS2 (by 71%), which suggests a constitutive inhibition of RGS2 expression by D2 dopamine receptors. Opposite results were obtained concerning the regulation of RGS4 since L-dopa alone was without effect whereas co-administration of L-dopa and R(+)-SCH23390 significantly enhanced the RGS4 mRNA levels (by 38%). In conclusion, D1 and D2 dopamine receptors appear to mediate opposite r...
The International Journal of Neuropsychopharmacology, 1999
Family, twin and adoption studies suggest that genetic factors play an important role in the aeti... more Family, twin and adoption studies suggest that genetic factors play an important role in the aetiology of schizophrenia. The mode of inheritance, however, is complex and non-Mendelian. Although the aetiology of schizophrenia is unknown, it has been hypothesized that the necessary conditions for developing the disease are environmental stress and a vulnerability to psychosis. The implication of dopamine receptors to schizophrenia has been greatly studied. Several linkage and association studies have been performed in an attempt to establish the involvement of dopamine receptors in schizophrenia. However, although no conclusive evidence of linkage or association to any gene has been established, some results, suggestive of linkage for chromosomes 6, 22 and 13, await confirmation from other studies. Concerning association studies, it is also of interest that some studies support an association between schizophrenia and homozygosity at D(3). More work in larger samples is required before conclusive linkage hypothesis or association to a dopamine receptor may be established. Schizophrenic patients have been shown to have significant deficits in a wide range of cognitive processes, including memory, attention, reasoning ability and language. Since cognitive deficits are significant symptoms of schizophrenia which require effective treatment, their assessment in schizophrenic patients and during clinical trials of new potential antipsychotics is highlighted. Cognitive impairment in schizophrenia impedes psychosocial performance and is therefore an especially relevant target variable in the development of new therapeutic approaches. It is most prominent in tasks involving attention, memory and executive functions which are thought to reflect involvement of prefrontal and left-temporal brain areas. Semantic networks in schizophrenic patients with a younger age of onset are observed to be more disorganized and differ significantly to those of control subjects. The need to use broader approaches such as neuropsychological-related measures to identify pertinent phenotypes in non-affected subjects carrying vulnerability genes is also emphasized. Since dopamine receptors are the primary targets in the treatment of schizophrenia, improved therapy may be obtained by drugs that selectively target a particular subtype of dopamine receptor. In the development of novel antipsychotics, D(3) and D(4) receptors have received much attention and this is partly related to the fact that these receptors have a high abundance in brain areas associated with cognitive and emotional functions, such as parts of the limbic system and cortex. Recent studies suggest that atypical neuroleptics may significantly improve the cognitive deficits observed in schizophrenic patients and that atypical neuroleptics such as risperidone appear to improve memory and alertness suggesting that further clinical studies are needed to determine the precise influence of antipsychotics on the cognitive system of schizophrenic patients. Such studies could lead to useful insights as to the potential advantages of the newer antipsychotics which appear to have a sparing or beneficial effect on various components of cognitive function. However, the observation that cortical D(2) receptors are important sites of action for antipsychotics, that the cerebral cortex may harbour the common sites of actions of antipsychotics and that the balancing of the opposing actions of D(1) and D(2) receptor regulation may be an appropriate drug treatment suggests that the adjustment of D(1) receptor levels in the cortex may become an important goal of future antipsychotic generation. Such antipsychotics will be able to treat the positive, negative and cognitive deficits of schizophrenia.
Quantitative in situ hybridization was used to investigate the effect of prolonged striatal dopam... more Quantitative in situ hybridization was used to investigate the effect of prolonged striatal dopamine or monoamine depletion on the mRNA density of regulators of G-protein signaling (RGS) 2–12 proteins. Two types of treatments were studied: a 6-hydroxydopamine-induced unilateral lesion of the nigrostriatal pathway and a 5-day reserpine treatment. The results clearly show a selective increase in the mRNA levels of
There are two families of dopamine (DA) receptors, called D1 and D2, respectively. The D1 family ... more There are two families of dopamine (DA) receptors, called D1 and D2, respectively. The D1 family consists of D1- and D5-receptor subtypes and the D2 family consists of D2-, D3-, and D4-receptor subtypes. The amino acid sequences of these receptors show that they all belong to a large superfamily of receptors with seven transmembrane domains, which are coupled to their
Regional brain glucose utilization was investigated with positron emission tomography and fluorod... more Regional brain glucose utilization was investigated with positron emission tomography and fluorodeoxyglucose in 2 patients with a seizure disorder associated with diffuse band heterotopia, a condition known as "double cortex." Although 1 patient was examined shortly after the onset of the first seizures, the other had a long history of intractable epilepsy before examination. Magnetic resonance imaging revealed a symmetric and generalized band of ectopic gray matter and an overlying normal-looking cortex, without focal abnormality. Metabolic studies yielded comparable results in both patients, with similar and even higher glucose uptake in the layer of gray matter heterotopia compared to the normal cortex. These data suggest the persistence of some synaptic activity in the heterotopic neurons, which seems unaffected by age or by the time-course of epilepsy.
Striatal glutamatergic inputs are known to participate in the modulation of dopaminergic transmis... more Striatal glutamatergic inputs are known to participate in the modulation of dopaminergic transmission. Accordingly, the non-competitive N-methyl-D-aspartate receptor antagonists memantine and amantadine increase striatal dopamine levels, the latter being widely used in Parkinson's disease therapy. Based on our previous work revealing increased function of dopamine receptors and dopamine transporter after amantadine treatment, we studied the effects of repeated memantine administration on dopaminergic neurotransmission. On rat striatal membranes, dopamine-stimulated [(35)S]GTPgammaS binding was significantly reduced (20%) after 2 days injection with memantine (20 mg/kg per day, i.p.) but not after longer treatments (4 or 7 days). Evaluation of [(3)H]SCH 23390 and [(3)H]spiperone specific bindings only revealed a significant increase in D1 receptor density after 4 or 7 days treatment. Finally, none of these treatments were found to change the activity of the neuronal dopamine transporter in striatal synaptosomes. This shows that amantadine and memantine differentially affect striatal dopaminergic transmission, which could indicate that these two related aminoadamantanes display distinct pharmacodynamic properties.
The possibility to isolate stem cells from the adult central nervous system and to maintain and p... more The possibility to isolate stem cells from the adult central nervous system and to maintain and propagate these cells in vitro has raised a general interest with regards to their use in cell replacement therapy for degenerative brain diseases. Considering the critical role played by astrocytes in the control of glutamate homeostasis, we have characterised the expression of functional glutamate transporters in neural stem cells exposed to selected culture conditions favouring their differentiation into astrocytes. Commonly, neural stem cells proliferate in suspension as neurospheres in serum-free medium. The addition of serum or a supplement of growth factors (G5) to the culture medium was found to trigger cell adhesion on coated surfaces and to favour their differentiation. Indeed, after 7 days in these conditions, the vast majority of the cells adopted markedly distinct morphologies corresponding to protoplasmic (with serum) or fibrous (with G5 supplement) astrocytes and approximately 35-40% acquired the expression of the glial fibrillary acidic protein (GFAP). Immunocytochemical analysis also revealed that the treatments with serum or with the G5 supplement triggered the expression of the glial glutamate transporters GLT-1 (35 and 21%, respectively) and GLAST (29 and 69%, respectively). This effect was correlated with a robust increase in the Na+ -dependent [3H]-d-aspartate uptake, which was partially inhibited by dihydrokainate, a selective blocker of GLT-1. Together, these results indicate that in vitro differentiation of cultured neural stem cells can give rise to distinct populations of astrocytes expressing functional glutamate transporters.
In rat brain, [3H]glibenclamide binds with high affinity to sulfonylurea receptors associated wit... more In rat brain, [3H]glibenclamide binds with high affinity to sulfonylurea receptors associated with ATP-sensitive potassium (KATP) channels. KATP channels may play a modulatory role in neurotransmitter release and are involved in acute pathological events occurring in the brain. Fluorescein derivatives, which are suitable tools for the labelling of nucleotide binding sites, influence KATP channels and sulfonylurea receptors properties in insulinoma and cardiac cells. In this study, a negative allosteric action of fluorescein derivatives on glibenclamide binding sites has been shown in rat cortical neurons. This supports the hypothesis of interactions between nucleotide- and sulfonylurea-binding sites within the sulfonylurea receptor.
Transgenic rats expressing a mutated form of the human Cu/Zn superoxide dismutase (hSOD1(G93A)) d... more Transgenic rats expressing a mutated form of the human Cu/Zn superoxide dismutase (hSOD1(G93A)) develop an amyotrophic lateral sclerosis (ALS)-like phenotype, including motor neurone degeneration and reactive gliosis in the spinal cord. This study aimed at examining the presence of endogenous neural progenitors in the lumbar spinal cord of these rats at the end-stage of the disease. Immunohistochemical data clearly demonstrated the induced expression of the stem cell factor reported as a chemoattractant and survival factor for neural stem cells as well as nestin (neuro-epithelial stem cell intermediate filament) in the spinal cord sections. While the stem cell factor immunolabelling appeared diffuse throughout the gray matter, nestin labelling was restricted to clusters within the ventral horn. Interestingly, as paralysis regularly develops asymmetrically, induction of nestin was only detected on the ipsilateral side of the predominant symptoms. Finally, immunohistochemical detection of the stem cell factor receptor (c-Kit) revealed its specific induction which coincided with nestin immunolabelling. Together, these results are indicative of endogenous recruitment of neural progenitors within lesioned tissues and could support the development of treatments involving endogenous or exogenous stem cells.
Quantitative in situ hybridization on rat coronal brain sections with radiolabelled oligonucleoti... more Quantitative in situ hybridization on rat coronal brain sections with radiolabelled oligonucleotide probes was performed to investigate the effects of antipsychotic drugs on mRNA levels of regulator of G-protein signalling (RGS) 2 and c-fos. This study demonstrated a similar increase of RGS2 mRNA level in the striatum upon both a single and a 21-day treatment with either haloperidol (2 mg/kg) or risperidone (7.5 mg/kg) in contrast to clozapine (20 mg/kg). Otherwise, the acute c-fos mRNA induction in the striatum was abolished by 74 to 89% upon chronic treatment with either haloperidol or risperidone. In conclusion, the induction of RGS2 mRNA in the striatum, in contrast to the immediate early gene c-fos mRNA, is preserved upon chronic treatment with haloperidol and risperidone.
When rat cultured neurons were incubated with unlabelled neurotensin (3nM) for 1 or 24h at 37°C, ... more When rat cultured neurons were incubated with unlabelled neurotensin (3nM) for 1 or 24h at 37°C, the [3H]-neurotensin specific binding measured in cell homogenates was decreased to about 35 and 65% of control values, respectively. In these experiments, the decreases in binding corresponded to reductions of Bmax values without changes in the affinity. The slow neurotensin-induced receptor down-regulation is thought
In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues an... more In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues and several methods have been reported for promoting their maturation into differentiated astrocytes. Hence, a combination of several nutriments/growth factors – the G5 supplement (insulin, transferrin, selenite, biotin, hydrocortisone, fibroblast growth factor and epidermal growth factor) – is widely used as a culture additive favouring the growth, differentiation and maturation of primary cultured astrocytes. Considering the key role played by glial cells in the clearance of glutamate in the synapses, cultured astrocytes are frequently used as a model for the study of glutamate transporters. Indeed, it has been shown that when tested separately, growth factors influence the expression and activity of the GLAST and GLT-1. The present study aimed at characterising the functional expression of these transporters during the time course of differentiation of cultured cortical astrocytes exposed to the supplement G5. After a few days, the vast majority of cells exposed to this supplement adopted a typical stellate morphology (fibrous or type II astrocytes) and showed intense expression of the glial fibrillary acidic protein. Both RT-PCR and immunoblotting studies revealed that the expression of both GLAST and GLT-1 rapidly increased in these cells. While this was correlated with a significant increase in specific uptake of radiolabelled aspartate, fluorescence monitoring of the Na+ influx associated with glutamate transporters activity revealed that the exposure to the G5 supplement considerably increased the percentage of cells participating in the uptake. Biochemical and pharmacological studies revealed that this activity did not involve GLT-1 but most likely reflected an increase in GLAST-mediated uptake. Together, these data indicate that the addition of this classical combination of growth factors and nutriments drives the rapid differentiation toward a homogenous culture of fibrous astrocytes expressing functional glutamate transporters.
Magma: Magnetic Resonance Materials in Physics, Biology, and Medicine, 1997
The aim of this study was to investigate the effects of riluzole on the lesion induced by a perma... more The aim of this study was to investigate the effects of riluzole on the lesion induced by a permanent middle cerebral artery occlusion (MCAO) in rats. Riluzole at 4 or 8 mg/kg i.v. significantly reduced the cortical ischemic brain damage. With the most effective dose of 8 mg/kg, the time evolution of the lesion was assessed by T2-weighted magnetic resonance imaging (MRI) repeated on the same animals after MCAO. MRI obtained at 24, 48, and 72 hours after MCAO showed a progressive increase of the ischemic lesion, except in the cortex of the riluzole-treated rats (8 mg/kg i.v.). Furthermore, there was no difference between lesion volumes as measured by MRI or by histology. This study indicates that MRI may be a valuable method to quantify in vivo the neuroprotective profile of a drug.
The dopamine receptor-mediated modulation of guanosine 5'-O-(gamma-[35S]thio)triphosphate... more The dopamine receptor-mediated modulation of guanosine 5'-O-(gamma-[35S]thio)triphosphate ([35S]GTP gamma S) binding has been characterized in rat striatal membranes. In optimized experimental conditions, the potency of dopamine was 4.47 microM [3.02-6.61 microM] and a maximal response representing 54.8 +/- 4.5% increase above basal level was observed. Data obtained with different agonists and antagonists clearly revealed that the most important fraction of this response was reflecting D2 receptor activation. Further analysis with specific antagonists also supported evidence for the involvement of D1 dopamine receptors. The potencies of compounds interacting with D1 and D2 receptors were deduced from [35S]GTP gamma S binding experiments and compared with their binding affinities for these receptors measured in similar experimental conditions. A good correlation between these parameters was observed, supporting the applicability of this technique for the study of dopamine receptors in the central nervous system.
Adult bone marrow mesenchymal stem cells are multipotent cells that can differentiate into a vari... more Adult bone marrow mesenchymal stem cells are multipotent cells that can differentiate into a variety of mesodermal tissues. Recent studies have reported on their ability to also evolve into non-mesodermal cells, especially neural cells. While most of these studies revealed that manipulating these cells triggers the expression of typical neurone markers, less is known about the induction of neuronal- or glial-related physiological properties. The present study focused on the characterisation of glutamate transporters expression and activity in rat mesenchymal stem cells grown in culture conditions favouring their differentiation into astroglial cells. Ten days exposure of the cells to the culture supplement G5 was found to increase the expression of nestin (neuro-epithelial stem cell intermediate filament), an intermediate filament protein expressed by neural stem cells. Simultaneously, a robust induction of the high-affinity glutamate transporter GLT-1 (and GLAST) expression was detected by RT-PCR and immunocytochemistry. This expression was correlated with a highly significant increase in the Na+-dependent [3H]D-aspartate uptake. Finally, while glial fibrillary acidic protein immunoreactivity could not be detected, the induced expression of the astrocytic enzyme glutamine synthetase was demonstrated. These results indicate that in vitro differentiation of adult mesenchymal stem cells in neural precursors coincides with the induction of functional glutamate transport systems. Although the astrocytic nature of these cells remains to be confirmed, this observation gives support to the study of mesenchymal stem cells as a promising tool for the treatment of neurological diseases involving glutamate excitoxicity.
Serotonin S2, benzodiazepine, and muscarinic receptors showed different regional distributions in... more Serotonin S2, benzodiazepine, and muscarinic receptors showed different regional distributions in the human brain but were present in all cortical areas. The laminar distributions of [3H]ketanserin, [3H]diazepam, and [3H]quinuclidinylbenzilate were investigated in the temporal cortex and revealed a high density in the IIIrd and IVth layers. Dopamine D2 receptors were not detected in the cortex.
Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization... more Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization of the neurotensin receptor at the second messenger level. Stimulation with nanomolar concentrations of neurotensin elicited rapid rises in the cytosolic calcium concentration ([Ca2+]i), which remained elevated throughout the peptide application. A significant response was already detected with neurotensin concentrations as low as 0.01 nM. This high efficiency of neurotensin in mediating this calcium response contrasts with the nanomolar affinity of the peptide for its receptor measured in binding experiments. Evidence indicated that the initial elevation of the [Ca2+]i resulted from release of Ca2+ from intracellular stores, whereas the sustained response involved an influx of extracellular origin. Return to the basal level was only reached after extensive washing of the peptide or its displacement with the neurotensin receptor antagonist SR48692. After washing, further stimulations were still able to mediate an increase in the [Ca2+]i, indicating an apparent absence of rapid desensitization of the intracellular signaling pathway that mediates calcium mobilization. In contrast with this absence of response desensitization, the neurotensin receptors were found to internalize after stimulation with the peptide. This internalization was maximal after 30 min and accounted for approximately 70% of the number of neurotensin binding sites located at the cell surface. These results indicate that despite the functional properties of the rat neurotensin receptor present in Chinese hamster ovary cells after transfection, the intracellular signaling pathway triggered by stimulation with neurotensin seems to be resistant to desensitization. This might be related to the high efficiency of the intracellular signaling pathway coupled to the neurotensin receptor observed in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective l... more Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.
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