[Show abstract][Hide abstract] ABSTRACT: We have studied the consequences of the constitutive acetylcholinesterase (AChE) deficiency in knockout mice for the AChE gene on the subcellular localization of the m2 receptor (m2R) and the regulation of its intraneuronal compartmentalization by the cholinergic environment, using immunohistochemistry at light and electron microscopic levels. (1) In AChE +/+ mice in vivo, m2R is mainly located at the neuronal membrane in striatum, hippocampus, and cortex. In AChE -/- mice, m2R is almost absent at the membrane but is accumulated in the endoplasmic reticulum and Golgi complex. (2) In vivo and in vitro (organotypic culture) dynamic studies demonstrate that the balance between membrane and intracytoplasmic m2R can be regulated by the cholinergic influence: In AChE -/- mice, m2R is translocated from the cytoplasm to the cell surface after (1) blockade of muscarinic receptors by atropine, (2) supplementation of AChE -/- neurons with AChE in vitro, and (3) disruption of the cortical and hippocampal cholinergic afferents in vitro. Our results suggest that the neurochemical environment may contribute to the control of the abundance and availability of cell surface receptors, and consequently to the control of neuronal sensitivity to neurotransmitters or drugs, by regulating their delivery from the endoplasmic reticulum and Golgi complex.
Molecular and Cellular Neuroscience 06/2003; 23(1):121-33. · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A bilamination involving the whole dentate stratum granulosum associated with a hippocampal sclerosis is reported. This morphological abnormality could be an unusual aspect of granule cell dispersion, plastic change induced by an early post-natal injury, or the the result from a neuronal migration disorder during the embryonic period. Whatever its origin, this bilamination is an abnormality of the hippocampal development which continues during the first years of life.
[Show abstract][Hide abstract] ABSTRACT: Many experimental studies suggest that NFkappaB, a transcription factor involved in acute inflammation, and cytokines participate in neuronal excitability and/or glial scar formation in epilepsy. In this report, we looked for the expression of NFkappaB in hippocampi surgically removed in patients with medial temporal lobe epilepsy (MTLE) and hippocampal sclerosis (HS) who had an history of febrile convulsions. We analyzed 18 hippocampi from epileptic patients with MTLE and HS, and we used as control specimens three hippocampi from non-epileptic patients and four hippocampi from patients with cryptogenic MTLE without HS. We used antibodies raised against the NFkappaB-p65 subunit and we identified glial cells with specific antibodies. Hippocampi from patients with MTLE and HS displayed severe neuronal loss surrounded by gliosis in CA1 area and more or less in CA3/CA4 areas. Double immunolabeling showed that reactive astrocytes of lesioned areas over-expressed NFkappaB-p65 (significantly when compared to control specimens). Moreover, some surviving pyramidal neurons in these areas and numerous dentate granule cells were strongly positive for NFkappaB-p65 in cytoplasm and nucleus, whereas control hippocampi showed a faint basal cytoplasmic staining in neurons. These results suggest that in epileptic hippocampi with typical sclerosis, inflammatory processes are chronically active or transiently re-induced by recurrent seizures. Whether NFkappaB over-expression reflects protective or deleterious mechanisms in the epileptic focus remains to be elucidated.
Brain Research 11/2002; 952(2):159-69. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The fluorescent exclusion dye propidium iodide (PI) is widely used as a vital dye in tissue culture systems and labels the nucleus in dying cells which lack an intact plasma membrane. We have developed a method, which allows the preservation of the PI signal in paraformaldehyde-fixed tissue, enabling subsequent immunohistochemical characterisation of labelled cells. We have tested this method in a model of ischemia based on oxygen and glucose deprivation in organotypic hippocampal slice cultures, in combination with immunocytochemical detection of calpain-I mediated spectrin breakdown products (BDPs). Using confocal laser microscopy it was possible to correlate at the single cell level which cells were PI positive and which cells expressed BDPs. This method can also be used with other immunocytochemical markers to determine the phenotype of cells, which accumulate PI in vitro. By fixing tissue at different times after insults, it is possible to obtain a 'snapshot' of viability at any time during the experimental protocol and subsequently characterise those cells which had accumulated PI at the time of fixation. The technique may also prove useful in characterising cell death in other in vitro and in vivo systems.
Brain Research Protocols 11/2002; 10(2):109-14. · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used in situ hybridization techniques to study the distribution of neurones synthesizing somatostatin mRNA and neuropeptide Y mRNA in the hilar region of the hippocampal formation of patients with temporal lobe epilepsy. In the dentate gyrus, somatostatin mRNA- and neuropeptide Y mRNA-synthesizing neurones were found to be exclusively located within the hilar region. Unlike animal models, no ectopic expression of either peptide was found in principal cells. The numbers of hilar interneurones expressing somatostatin mRNA and neuropeptide Y mRNA were compared with the degree of hilar cell loss determined by immunohistochemistry against neuronal nuclear antigen. The numbers of somatostatin and neuropeptide Y mRNA-synthesizing neurones varied considerably between patients, but both were found to be highly correlated to the total number of neuronal nuclear antigen-immunoreactive hilar neurones. These results suggest that loss of somatostatin and neuropeptide Y interneurones occurs in proportion to overall hilar cell loss, and therefore the hypothesis of a selective loss of these interneurones in temporal lobe epilepsy seems unlikely.
[Show abstract][Hide abstract] ABSTRACT: Prior epileptic episodes have been shown to decrease markedly the neuronal damage induced by a second epileptic episode, similar to the tolerance following an episode of mild ischemia. Endogenous neuroprotective effects mediated by various mechanisms have been put forward. This study investigated whether neuroprotection against the excitotoxic damage induced by re-exposure to an epileptic challenge can reflect a change in epileptic susceptibility. Tolerance was elicited in rats by a preconditioning session using intrahippocampal kainic acid (KA) administration followed at 1, 7 and 15-day intervals by a subsequent intraventricular KA injection. The degree of pyramidal cell loss in the vulnerable CA3 subfield contralateral to the KA-injected hippocampus was extensively reduced in animals experiencing KA ventricular administration. This neuroprotection was highly significant 1 and 7 days after injection, but not 15 days after injection. In preconditioned animals, the after-discharge threshold was assessed as an index of epileptic susceptibility. It increased significantly from 1 to 15 days after intrahippocampal KA administration. Finally, an enhancement of neuropeptide Y expression in both non-principal cells and mossy fibers was detected, occurring at the same time as the decrease in epileptic susceptibility. These results provide further evidence of an 'epileptic tolerance' as shown by the substantial neuroprotective effect of a prior episode of epileptic activity upon subsequent epileptic insult and suggest that the prevention of excitotoxic damage after preconditioning results from an endogenous neuroprotective mechanism against hyperexcitability and seizures.
Brain Research 04/2001; 894(2):209-17. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have developed an in vitro system that allows the study of the effects of factors released from macrophages on neuronal and glial survival in cultured hippocampal slices. Organotypic hippocampal slice cultures are grown on semi-permeable membranes in stationary co-culture with a murine macrophage cell line (RAW 264.7). The two culture systems are separated by a semi-permeable membrane specifically allowing the study of diffusable factors between the two culture systems. The use of the fluorescent exclusion dye propidium iodide as an in vitro marker of cell viability allows the study of progressive toxicity as it evolves in the slice cultures. We demonstrate that the HIV-1 derived nuclear regulatory protein Tat induces toxicity in slice cultures via the production of soluble mediators. The advantages of organotypic cultures over other in vitro systems is discussed as well as the general applicability of this method to the study of other brain pathologies, where macrophage derived factors are thought to play a role in neuronal survival.
Journal of Neuroscience Methods 09/1999; 90(1):7-11. · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is now a strong consensus that the neurotoxic properties of HIV-1 are likely to be mediated by an indirect mechanism in which neurones are damaged by infected mononuclear cells. The aim of this study was to determine the ability of HIV-1 Tat to induce neurotoxic properties in a murine macrophage cell line RAW264.7.
Simple culture systems using dissociated neurones may not provide the appropriate microenvironment in which to observe the complex cell-cell interactions that occur in the brain. We have therefore developed a more physiological model in which rat organotypic hippocampal slices are co-cultured with the murine macrophage cell line RAW264.7. Effects of Tat were studied by using a stable Tat expressing RAW264.7 cell line or by addition of recombinant Tat protein to co-cultures.
Organotypic hippocampal slices prepared from 8-10 day rat pups were grown on membrane inserts that were placed into six-well plates on which RAW264.7 cells were growing as an adherent monolayer. Cell death in the slices was assessed using propidium iodide. Specific astrocytic (glial fibrillary acidophilic protein; GFAP) and neuronal (microtubule-associated protein; MAP2) markers were visualized by immunocytochemistry.
RAW264.7 cells that either expressed or were exposed to HIV-1 Tat protein, produced a soluble factor that caused profound degeneration in brain slice cultures involving loss of both glial cells and neurones. By contrast treatment of slice cultures with Tat in the absence of RAW264.7 cells was not neurotoxic.
The neurotoxic properties previously attributed to HIV-1 Tat are likely to be mediated via induction of macrophage derived soluble factor(s).
AIDS 09/1999; 13(12):1443-52. · 6.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been suggested that, after ischaemia, activation of proteases such as calpains could be involved in cytoskeletal degradation leading to neuronal cell death. In vivo, calpain inhibitors at high doses have been shown to reduce ischaemic damage and traumatic brain injury, however, the relationship between calpain activation and cell death remains unclear. We have investigated the role of calpain activation in a model of ischaemia based on organotypic hippocampal slice cultures using the appearance of spectrin breakdown products (BDPs) as a measure of calpain I activation. Calpain I activity was detected on Western blot immediately after a 1-h exposure to ischaemia. Up to 4 h post ischaemia, BDPs were found mainly in the CA1 region and appeared before uptake of the vital dye propidium iodide (PI). 24 h after the insult, BDPs were detected extensively in CA1 and CA3 pyramidal cells, all of which was PI-positive. However, there were many more PI-positive cells that did not have BDPs, indicating that the appearance of BDPs does not necessarily accompany ischaemic cell death. Inhibition of BDP formation by the broad-spectrum protease inhibitor leupeptin was not accompanied by any neuroprotective effects. The more specific and more cell-permeant calpain inhibitor MDL 28170 had a clear neuroprotective effect when added after the ischaemic insult. In contrast, when MDL 28170 was present throughout the entire pre- and post-incubation phases, PI labelling actually increased, indicating a toxic effect. These results suggest that calpain activation is not always associated with cell death and that, while inhibition of calpains can be neuroprotective under some conditions, it may not always lead to beneficial outcomes in ischaemia.
European Journal of Neuroscience 08/1999; 11(7):2375-84. · 3.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: D2 dopamine receptor (D2R) gene expression was analyzed by in situ hybridization and D2R ligand autoradiography in the human striatum during ontogeny. D2R mRNA and ([3H]YM-09151-2)-binding sites were detected in the striatum from week 12 of fetal life. At this time, D2R mRNA and binding sites were predominant in the putamen and occurred in a pattern of clusters. D2R-binding sites displayed a similar pattern. The signal in the caudate nucleus was weak from weeks 12 to 16. From week 20 of fetal life, D2R mRNA and D2R-binding sites signals became intense in the ventral striatum. At birth, D2R mRNA became homogeneously distributed while D2R-binding sites kept an heterogeneously distribution. Comparative topological and temporal analysis of the D2R, enkephalin and D1 dopamine receptor (D1R) mRNAs showed a distinct developmental pattern for each mRNA. Before birth, the neurons expressing enkephalin and D1R mRNAs were preferentially distributed in the matrix and in the striosomes, respectively, while the neurons expressing D2R mRNA did not display a preferential localization. At birth, high levels of enkephalin mRNA were restricted to the matrix; D1R mRNA level was homogeneous throughout the striatum. D2R mRNA was heterogeneously distributed in the whole striatum with high signals located both in the striosomes and the matrix. These results demonstrate that functional D2R are expressed as early as week 12 in the striatum with a heterogeneous distribution. Our findings also demonstrate that, in contrast to what was expected from similar studies in rodents, D2R mRNA and enkephalin mRNA do not display identical, overlapping expression patterns in striatal neurons during human ontogeny.
Molecular Brain Research 10/1997; 48(2):389-400. · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A series of 15 fetal and perinatal human brains (from week 12 of fetal life to day 2 after birth) was studied in order to describe the anatomical and molecular correlates of the substantia nigra ontogeny. In situ hybridization, immunohistochemistry and binding studies were used to detect D2 dopamine receptor (D2R) mRNA, D2R binding sites, dopamine membrane transporter (DAT) mRNA, tyrosine hydroxylase (TH) protein D1 dopamine receptor (D1R) protein and D1R binding sites. Dopaminergic (DA) neurons of the substantia nigra were detected through TH immunoreactivity from week 12. At week 16, the substantia nigra was clearly delineated as a compact group of intermingled neurons and fibers. From week 19, groups of DA neurons were segregated from the pars reticulata. These groups have been divided into the substantia nigra pars compacta, the ventral tegmental area and the retrorubral area. The DA neurons exhibited a gradual increase in size and branching development until birth. From week 12 onward they expressed several other markers of dopamine transmission, i.e., D2R mRNA, D2R binding sites and DAT mRNA. The ventral tegmental area expressed lower levels of mRNA for DAT and D2R than the pars compacta. From week 12, D1R immunoreactivity and D1R binding sites were also present in the substantia nigra pars reticulata. This suggests that projecting striatonigral neurons, known to express the D1R gene, have developed pathways connecting with the substantia nigra by week 12. Our results demonstrate that the developing substantia nigra in human displays early transcriptional and translational activity for the main constituents of dopaminergic transmission from week 12 and receives at this time dopaminoceptive inputs bearing D1 receptors from the striatum.
The Journal of Comparative Neurology 04/1997; 379(1):72-87. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The stratum granulosum (SG) of the fascia dentata from 17 human epileptic hippocampi was assessed in terms of width, volumetric cell density (VCD) and percentage of cell loss to study the granule cell dispersion (GCD) phenomenon described by Houser. GCD was considered when three conditions were observed, the SG was wider than 120 microns, granule cell (GC) somata did not remain in close apposition to one another the normal clear boundary between the molecular layer and the SG was not maintained. GCD involved a partial zone of the SG in six cases and the whole SG in two cases. Dynorphin mRNA in-situ hybridization was performed in two cases and allowed us to affirm that dispersed cells are actually GC. A close correlation linked GCD, GC loss and VCD decrease in diffuse CA4, laminated CA4, CA3, CA2 and CA1. The discussion is focused on the possible causes of dispersion. Some arguments did not suggest for a migration arrest during development. Nevertheless, in one case, a cluster of horizontal cells in the inner part of the molecular layer could evoke the persistence of normally transient cells during ontogenesis. A neo-migration due to permissive phenomenon induced by gliogenesis, mossy fibers sprouting in the supra-granular layer and over-expression of growth factors is suggested from experimental data. Nevertheless a straining due to the tissue shrinkage observed in severe hippocampal sclerosis (HS) could also be involved in the origin of GCD.
Epilepsy Research 02/1997; 26(2):351-61. · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied D1 dopamine receptor (D1R) gene expression in the human striatum during ontogeny by in situ hybridization, immunohistochemistry, and D1R ligand autoradiography. D1R mRNA, protein, and binding sites ([3H]SCH 23390) were detected in the striatum from week 12 of fetal life. At this time, D1R mRNA was predominant in the striosomal neurons; D1R immunoreactivity (D1R-IR) and D1R binding sites displayed a pattern similar to D1R mRNA. D1R-IR was essentially present in striosomal cell bodies and neuropil, whereas only a few cell bodies were detected in the matrix. From week 20 of fetal life, D1R gene expression developed in the matrix neurons as well, thus leading to an even D1R mRNA expression throughout striosomes and matrix compartments at birth. Comparative analysis of the expression of D1R and dynorphin mRNA show the same developmental patchy pattern up to week 26. Indeed, neurons expressing the D1R gene contain dynorphin mRNA; in contrast, they do not express the preproenkephalin A gene. At birth, the pattern of D1R mRNA expression level was sharply different from that of dynorphin (DYN) gene expression. High DYN mRNA expression was restricted to the striosomes, whereas high D1R mRNA expression was present in the whole striatum. These results demonstrate that, during human ontogeny, functional D1 receptors are expressed as early as week 12 in the striatum, developing initially in the striosomal neurons containing high dynorphin mRNA content. Toward the end of fetal life, there is a dissociation between D1R and DYN expression levels, suggesting that neuroanatomical or neurochemical modifications occur at this period, which may contribute to the regulation of the tone of the striatal D1R and DYN gene with topological specificity.
The Journal of Comparative Neurology 07/1996; 370(1):23-34. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for dopaminergic neurons. Since dopaminergic neurons degenerate in Parkinson's disease, this factor is a potential therapeutical tool that may save dopaminergic neurons during the pathological process. Moreover, a reduced GDNF expression may be involved in the pathophysiology of the disease. In this study, we tested whether altered GDNF production may participate in the mechanism of cell death in this disease. GDNF gene expression was analyzed by in situ hybridization using riboprobes corresponding to a sequence of the exon 2 human GDNF gene. Experiments were performed on tissue sections of the mesencephalon and the striatum from 8 patients with Parkinson's disease and 6 control subjects matched for age at death and for post mortem delay. No labelling was observed in either group of patients. This absence of detectable expression could not be attributed to methodological problems as a positive staining was observed using the same probes for sections of astroglioma biopsies from human adults and for sections of a newborn infant brain obtained at post-mortem. These data suggest that GDNF is probably expressed at a very low level in the adult human brain and its involvement in the pathophysiology of Parkinson's disease remains to be demonstrated. GDNF may represent a powerful new therapeutic agent for Parkinson's disease, however.
Journal of Neural Transmission 02/1996; 103(8-9):1043-52. · 3.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The distribution patterns of neurons expressing mRNAs for four neuropeptides in the human striatum were studied during ontogeny by the use of in situ hybridization. The results of our study demonstrate that somatostatin, enkephalin, dynorphin, and substance P mRNAs are present in striatal neuronal populations from week 12 of fetal life. Each neuronal population undergoes a specific differentiation. Neurons containing somatostatin mRNA are scattered throughout the caudate-putamen up until birth. Neurons containing enkephalin, dynorphin, or substance P mRNAs evolve throughout fetal life in relation to caudate-putamen and patch-matrix compartmentalization. Neurons containing enkephalin mRNA (distinct from those containing substance P or dynorphin mRNAs) are present in the matrix from week 12 of fetal life. These neurons are preferentially distributed in the matrix and, at birth, display higher enkephalin mRNA content in the matrix than in the patches. Dynorphin mRNA is found in the caudate and putamen, preferentially in the patch neurons; nevertheless, a low level of dynorphin mRNA is also present in neurons of the caudate matrix. Substance P mRNA is initially restricted to caudate neurons. At birth, both substance P and dynorphin mRNAs are expressed at high levels in the patches. These results demonstrate that each neuropeptide gene is expressed during human fetal life in neurons with a specific topology and pace of development in relation to caudate-putamen and patch-matrix differentiation. These results also contribute evidence that neurochemical evolution of the striatal neuronal populations is not complete at birth in humans.
The Journal of Comparative Neurology 10/1995; 360(3):488-505. · 3.66 Impact Factor