Calcium-dependent release specificities of various cell lines loaded with different transmitters.

By loading cells in culture with acetylcholine (ACh) we have characterized a calcium-dependent release mechanism and shown that it was expressed independently of synthesis or storage of ACh. (Israël et al., 1994, Neurochemistry International 37, 1475-1483; Falk-Vairant et al., 1996a, Proc. Natl. Acad. Sci. U.S.A. 93, 5203-5207; Falk-Vairant et al., 1996b, Neuroscience 75, 353-360; Falk-Vairant et al., 1996c, Journal of Neuroscience Research 45, 195-201). The transmitter loading procedure was applied to two other transmitters, gamma-aminobutyric acid (GABA) and glutamate (Glu). We could then study the specificity of the release mechanism for the three transmitters in a variety of cell lines, including neural-derived cells. Four different calcium-dependent release phenotypes were identified: two were specific for ACh or GABA, and two co-released two transmitters ACh and GABA but not Glu, or ACh and Glu but not GABA. We conclude that release mechanisms having different specificities are expressed by the cell lines studied, they become functional after loading the cells with the relevant transmitters. These observations will help the identification of proteins controlling the specificity of release, and provide an interesting model for pharmacological studies.

TNF alpha gene expression is induced in neurones after a hippocampal lesion.

The tumour necrosis factor alpha (TNF alpha) protein is normally absent in the brain. Its production in the nervous tissue during pathological processes is commonly attributed to cells of the macrophage or astroglial lineages. However, an immunoreactivity for TNF alpha has been observed recently in adult mouse brain after a lesion to the hippocampus. The identification, in the present study, of the cells responsible for this synthesis demonstrates a neuronal localization of the TNF alpha messenger RNA. We propose that neurone-produced TNF alpha acts as a modulatory effector in post-traumatic regenerative attempts of the brain.

PDGF-alpha receptor is expressed by mature neurones of the central nervous system.

The platelet-derived growth factors (PDGF) are constitutively expressed by neurones in the central nervous system (CNS). The synthesis of the PDGF-alpha-receptor (PDGF-alpha R) is commonly attributed to oligodendrocyte precursors during late embryonic and early postnatal development, suggesting communication between neurones and glia which orchestrates amplification and final targeting of the myelinating cells. However PDGF A production persists when central myelination is achieved, which suggests that PDGF-alpha R are present in the adult CNS. In this study, we demonstrate the production of PDGF-alpha R transcripts and protein by various neuronal populations of the adult CNS. We propose a developmental shift, where glial cells and neurones are consecutive targets of PDGF A and neuromodulatory effects of PDGF, exerted on mature neurones via the expression of the PDGF-alpha R.

Transplantation of oligodendrocyte precursors in the adult demyelinated spinal cord: migration and remyelination.

A demyelinating lesion induced by an injection of lysolecithin into the spinal cord can be partly repaired by oligodendrocyte precursors transplanted at a distance of 6-8 mm from the lesion. Using a non-toxic fluorescent dye (Hoechst 33342) as a cell marker, we demonstrate that transplanted oligodendrocyte precursors from different origins (periventricular zone fragments from newborn mouse and cultured rat oligodendrocyte progenitor cells) can migrate along specific pathways (i.e. white matter fasciculi, ependymal wall, meninges and blood vessels). These cells can be attracted when passing at the vicinity of the lesion as well as differentiate and remyelinate axons with the lesion. Myelin repair thus appears to be the result of distinct successive events: migration, specific attraction, differentiation and myelination. This can occur in both shiverer and normal adult hosts.

Developmental expression of platelet-derived growth factor alpha-receptor in neurons and glial cells of the mouse CNS.

The synthesis of platelet-derived growth factor-alpha receptor (PDGF-alphaR) is commonly attributed to oligodendrocyte progenitors during late embryonic and postnatal development. However, we recently demonstrated that mature neurons could also synthesize PDGF-alphaR, emphasizing a larger role for this receptor than previously described. In the present study, to analyze the pattern of PDGF-alphaR expression during postnatal development of the mouse CNS, we used in situ hybridization and immunohistochemistry on brain and spinal cord tissue sections. We found that, in addition to immature cells of the oligodendrocyte lineage, neurons of various CNS regions express PDGF-alphaR transcripts and protein as early as postnatal day 1 (P1). Whereas neuronal expression was maintained at all ages, the oligodendroglial expression strongly decreased after P21. In the adult, PDGF-alphaR was detected in very few oligodendrocyte progenitors scattered in the cerebral cortex or in white matter tracts, thus suggesting the presence of PDGF-alphaR on O-2Aadult progenitors. In the mature CNS, PDGF-alphaR transcripts and protein were mainly localized in neurons of numerous structures, such as the olfactory bulb, cerebral cortex, hippocampus, and brainstem nuclei and in motor neurons of the ventral horn of the spinal cord. The differential expression of PDGF-alphaR in oligodendroglia and neurons argues in favor of several roles of PDGF during development.

Cell-cell interactions during the migration of myelin-forming cells transplanted in the demyelinated spinal cord.

In the present paper, Dil-labeled myelin-forming cells were traced after their transplantation at a distance from a lysolecithin induced lesion in the adult wild-type and shiverer mouse spinal cord. Optical and ultrastructural observations indicate that after their transplantation, Dil-labeled Schwann cells and oligodendrocyte progenitors were found at the level of the graft as well as at the level of the lesion thus confirming that myelin-forming cells were able to migrate in the adult lesioned CNS (Gout et al., Neurosci Lett 87:195-199, 1988). Between the graft and the lesion, labeled Schwann cells and oligodendrocyte progenitors were absent in the gray matter, but were found as previously described, in specific locations (Baron-Van Evercooren et al., J Neurosci Res 35:428-438, 1993; Vignais et al., J Dev Neurosci 11:603-612, 1993). Both cell types were found along blood vessel walls and more precisely in the Virchow-Robin perivascular spaces. They were identified in the meninges among meningeal cells, collagen fibers, or occasionally in direct contact with the basement membrane forming the glia limitans. In addition to these findings, three major observations were made. In the ependymal region, myelin-forming cells were localized between or at the basal pole of ependymocytes. While Dil-labeled oligodendrocyte progenitors were noted to migrate along the outer surface of myelin sheats in CNS wild-type and shiverer white matter, Schwann cells were excluded from this structure in the wild-type mouse spinal cord. Moreover, in the shiverer mouse, migrating Schwann cells did not seem to interact directly with myelin sheats nor with mature oligodendrocytes. Finally, both cell types were seen to invade extensively the spinal peripheral roots. Our ultrastructural observations clearly suggest that multiple cell-cell and cell-substrate interactions rule the migration of myelin-forming cells in the adult CNS infering that multiple mechanisms are involved in this process.

Expression of the highly polysialylated neural cell adhesion molecule during postnatal myelination and following chemically induced demyelination of the adult mouse spinal cord.

We have investigated the expression of the highly polysialylated neural cell adhesion molecule in the mouse spinal cord during postnatal myelination and in the adult after chemically induced demyelination. By double immunohistochemistry, using a monoclonal antibody (anti-Men B) which specifically recognizes polysialic acid (PSA) units on neural cell adhesion molecule (N-CAM), and an anti-myelin basic protein, a caudorostral gradient of expression of PSA-NCAM was observed at postnatal day 1 (P1), which was inversely related to the gradient of myelination. At P7, PSA-NCAM labelling decreased relative to P1. In white matter, this decrease was correlated with the progression of myelination. PSA-NCAM immunoreactivity persisted in as yet unmyelinated structures, i.e. the corticospinal tract, the dorsomedial part of the ventral funiculus and the lateral funiculi, and decreased with the onset of myelination of these structures at P15. In the adult, PSA-NCAM expression remained in discrete structures, i.e. laminae I and II of the dorsal horn and lamina X around the central canal. The ependymal cells and the astrocyte endfeet under the meninges were also labelled. In addition, PSA-NCAM expression was reinduced on various cells and structures after lysolecithin-induced demyelination of the adult mouse spinal cord. At early times after demyelination, PSA-NCAM was expressed on glial cells around the lesion but also at a distance from this zone. Seven days after injection, cellular PSA-NCAM expression was found around but also within the lesion. This expression was totally abolished 15 days after injection. Double immunohistochemistry for PSA and cell-specific markers showed that the cells which expressed PSA-NCAM after demyelination were oligodendrocyte precursors, reactive astrocytes and Schwann cells. PSA-NCAM re-expression on all cell types was transient and ceased when myelin repair was accomplished. The spatial and temporal regulation of PSA-NCAM expression during development and after demyelination suggests a role for PSA-NCAM in glial plasticity during the myelination and remyelination processes.

Gene transfer to the central nervous system by transplantation of cerebral endothelial cells.

A cerebral endothelial immortalized cell line was used in transplantation experiments to deliver gene products to the adult rat brain. Survival of grafted cells was observed for at least 1 year, without any sign of tumor formation. When genetically modified to express bacterial beta-galactosidase and transplanted into the striatum, these cells were shown, by light and electron microscope analysis, to integrate into the host brain parenchyma and microvasculature. Following implantation into the striatum and nucleus basalis of adult rats, endothelial cells engineered to secrete mouse beta-nerve growth factor (NGF) induced the formation of a dense network of low-affinity NGF receptor-expressing fibers near the implantation sites. This biological response was observed from 3 to 8 weeks after engraftment. The present study establishes the cerebral endothelial cell as an efficient vector for gene transfer to the central nervous system.