Astrocyte–neuron co-culture on microchips based on the model of SOD mutation to mimic ALS

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. ALS is believed to be a non-cell autonomous condition, as other cell types, including astrocytes, have been implicated in disease pathogenesis. Hence, to facilitate the development of therapeutics against ALS, it is crucial to better understand the interactions between astrocytes and neural cells. Furthermore, cell culture assays are needed that mimic the complexity of cell to cell communication at the same time as they provide control over the different microenvironmental parameters. Here, we aim to validate a previously developed microfluidic system for an astrocyte-neuron cell culture platform, in which astrocytes have been genetically modified to overexpress either a human wild-type (WT) or a mutated form of the super oxide dismutase enzyme 1 (SOD1). Cortical neural cells were co-cultured with infected astrocytes and studied for up to two weeks. Using our microfluidic device that prevents direct cell to cell contact, we could evaluate neural cell response in the vicinity of astrocytes. We showed that neuronal cell density was reduced by about 45% when neurons were co-cultured with SOD-mutant astrocytes. Moreover, we demonstrated that SOD-WT overexpressing astrocytes reduced oxidative stress on cortical neurons that were in close metabolic contact. In contrast, cortical neurons in metabolic contact with SOD-mutant astrocytes lost their synapsin protein expression after severe glutamate treatment, an indication of the toxicity potentiating effect of the SOD-mutant enzyme.

Molecular heterogeneity of the ganglionic eminence and its relationship to the birth of neocortical interneurons

Susan Willi Monnerat

Neocortical function and malfunction depends critically on a spectrum of inhibitory interneurons. To gain novel insight in normal brain function and mechanisms leading to diseases, such as epilepsy or schizophrenia, the knowledge about the specific functions of each interneuron type appears crucial. To date, the mystery about the diversity of neocortical interneurons remains still unresolved, even though much effort was devoted to the characterization of mature interneurons in the neocortex. Ten years ago, the discovery of the ganglionic eminence (GE) as origin of neocortical interneurons allowed a novel approach to assess their various identities, and thus prompted researchers to follow the fate of cells accommodated in this region. Despite the knowledge of the source location and its subdivision into three GEs, their molecular identity remained unknown apart from few genes, and consequently unabled the specific isolation of precursor cells. To this purpose, we performed extensive microarray analysis of the three GEs at various developmental timepoints to reveal their molecular heterogeneity and identify novel precursor cell surface markers that would enable their prospective isolation by flow cytometry and subsequent fate assessment in vitro and in vivo. Surprisingly, molecular heterogeneity of the three GEs was restricted to few differentially expressed genes. However, the differences in gene expression became more apparent at late developmental stage that may reflect the increased specification during development. Among the three GEs, the LGE and CGE resembled molecularly each other most, while the MGE and CGE were most distantly related to each other. Furthermore, our genome-wide expression study suggested that the CGE, a structure of so far controversial nature, was rather a unique structure than a fusion of the MGE and LGE. Analysis across developmental timepoints revealed that the MGE underwent tremendous changes in gene expression when compared to the differential expression existing between the homochronic MGE and cerebral cortex. In our microarray screen, we could identify Boc as a novel marker of LGE/CGE-resident precursor cells. Taking advantage of Boc that encodes a cell surface molecule, we could isolate the Boc+ cells from the GE by flow cytometry, and demonstrate that in contrast to Boc– cells, they self-renewed and differentiated into oligodendrocytes, astrocytes, and neurons, and thus identify them as progenitor cells. Furthermore, immunohistochemical studies suggested that Boc+ GE cells may correspond to radial glia, the embryonic neural stem cells. The specific expression of Boc in the LGE and CGE, but not in the MGE during their entire lifetime suggested that Boc is a reliable marker for LGE/CGE-resident precursor cells. To assess the fate of Boc+ GE cells, we performed homotypic transplantations of Boc+ and Boc– cells onto organotypic slice cultures, and observed that in contrast to Boc– cells, Boc+ cells did not migrate from the GE to the neocortex suggesting that Boc+ precursor cells of the GE do not generate neocortical interneurons. In future, additional fate-mapping studies of Boc+ GE cells, such as in vivo transplantation, should shed more light on the ability of Boc+ LGE/CGE precursor cells to generate neocortical interneurons, and dependent on the outcome serve to construct a cell-lineage tree of neocortical interneurons. Thus, Boc will remain a useful tool to investigate developmental processes underlying the generation of neocortical and/or other neurons, such as striatal neurons.

EPFL2007

VIP receptor subtypes in mouse cerebral cortex: evidence for a differential localization in astrocytes, microvessels and synaptosomal membranes

Pierre Magistretti, Jean-Luc Martin

The binding characteristics of a monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) to the membranes of astrocytes, intraparenchymal microvessels and synaptosomes were analyzed in mouse cerebral cortex. Binding to astrocytes, studied in primary cultures, indicates the presence of a single class of high affinity binding sites with a Kd of 3.3 nM and a Bmax of 565 fmol/mg protein. The structurally related peptide secretin does not compete for sites labeled by M-[125I]VIP. In cultured astrocytes, VIP has been previously shown to promote glycogenolysis. Secretin, despite its lack of interaction with sites labeled by M-[125I]VIP, stimulates glycogenolysis with an EC50 of 0.5 nM, thus demonstrating the presence in astrocytes of functional secretin receptors independent from those for VIP. Trypsinization of the primary astrocyte cultures followed by replating as secondary cultures, reveals a second class of low affinity binding sites, with a Kd of 41.3 nM and a Bmax of 881 fmol/mg protein. Secretin does not compete for this class of low affinity binding sites either. Binding of M-[125I]VIP to intraparenchymal microvessels reveals the presence of two classes of binding sites with Kd of 1.4 and 30.3 nM, and Bmax of 7.1 and 73.8 pmol/mg protein, respectively. Similar to what is observed in primary or secondary astrocyte cultures, secretin does not interact with these sites. In this cell type VIP stimulates cAMP formation with an EC50 of 18 nM, while secretin is ineffective. Finally, in agreement with previous reports in rat and guinea pig cerebral cortex, two classes of binding sites are observed in synaptosomal membranes: a high affinity class with a Kd of 4.9 nM and a Bmax of 316 fmol/mg protein, and a low affinity class with a Kd of 42.8 nM and a Bmax of 1578 fmol/mg protein. In contrast to what is observed in non-neuronal membranes, in synaptosomal membranes, secretin effectively competes for sites labeled by M-[125I]VIP with an EC50 of approximately 150 nM. These results indicate that secretin may represent a useful tool to discriminate between neuronal and non-neuronal VIP binding sites, since it competes with M-[125I]VIP exclusively for the neuronal class of binding sites.

1992

Regulation of glycogenolysis by neurotransmitters in the central nervous system

Pierre Magistretti

Neurotransmitters are the molecules that neurons use to communicate with each other and with the other cell types of the nervous system, such as glial cells and cells of the vasculature. The best characterized actions of neurotransmitters are the alterations in excitability that they elicit in other neurons. These changes in neuronal firing rate are due to the opening or closing of transmembrane channels selectively permeable to given ionic species. We have however recently demonstrated that certain neurotransmitters can regulate energy metabolism within discrete regions of the central nervous system. In particular we have observed that Vasoactive Intestinal Peptide stimulates glycogenolysis in the cerebral cortex. This action is also exerted by the monoamines noradrenaline, serotonin and histamine. Studies in primary cultures indicate that the glycogenolysis elicited by neurotransmitters may take place in astrocytes, which are glial cells and where glycogen is predominantly stored in the nervous system. These observations suggest that the primary function of certain neuronal circuits may be to regulate the availability of energy substrates within discrete neuronal ensembles.