Specific modulation of dopamine expression in neuronal hybrid cells by primary cells from different brain regions.

Abstract

MN9D is an immortalized dopaminecontaining neuronal hybrid cell line. When MN9D cells were coaggregated with primary embryonic cells of optic tectum, a brain region that does not receive a dopaminergic innervation, there was a marked reduction in their dopamine content, tyrosine hydroxylase immunoreactivity, and tyrosine hydroxylase mRNA. Similar reductions in dopamine content were produced by coaggregation with cells from embryonic thalamus, another brain region devoid ofdopaminergic innervation.Coaggregation of MN9D cells with dopaminoceptive cells from the corpus striatum or the cortex did not have a demonstrable stimulator effect on the dopamine content of MN9D cells. The decrease in MN9D dopamine content produced by optic tectum cells was not reversed by addition of corpus striatum cells.Thus, the MN9D hybrid cells are able to respond to an inhibitory factor(s) from cells derived from brain areas that are not targets for dopaminergic neurons. Catecholamineproducing PC12 cells did not respond in a similar manner, suggesting that the response ofMN9D cells is a function of their mesencephalic origin. Given the selective response of MN9D cells to different brain cell populations, this hybrid cell line should facilitate investigations of cell-cell interactions in the central nervous system that may be involved in the expression of neurotransmitter phenotype and establishment of specific neuronal connections.Examination of the molecular mechanisms involved in cellcell interactions and establishment of specific neuronal connections in the brain is difficult because of the complexity of the neuronal population. There is, however, considerable evidence for the participation of chemotrophic mechanisms involving soluble molecules in axonal guidance (1) and the direct contact of growth cones with cellular membranes and extracellular matrix (2). In addition to such positive signals, nontarget cells may play a role in inhibition of growth cone extension (3, 4). For dopaminergic neurons, primary tissue cultures have proven useful in approaching some of these issues of neuronal connectivity (5). In particular, the cell aggregation culture system developed by Moscona (6) for examining cell-cell interactions and applied to brain tissue by Garber and Moscona (7) Although the use of cell aggregation techniques simplifies the examination of neuronal interactions, this approach has limitations with respect to identification of the molecules involved because of cell heterogeneity in any brain area and the limited amounts ofmaterial obtainable from a specific cell type for isolation ofspecific molecules. For these reasons, we have developed a series of clonal cell lines using somatic cell fusion (11) that provides large quantities of cells for studies of cell-cell interactions and isolation of specific neurotrophic molecules. Among these cell lines is a clonal catecholamineproducing hybrid cell line designated as MN9D. It was derived from cells of the mesencephalon and contains substantial quantities of dopamine (12). Bri...

Immortalized murine striatal neuronal cell lines expressing dopamine receptors and cholinergic properties

“…Second, in a comparative study of D,-like receptor pharmacology and function, transfected D, receptors have been shown to be linked to the modulation of adenylate cyclase activity in the hybrid MN9D cells, but not in the fibroblast cell line CCL1.3 (Tang et al, 1994). Third, the MN9D cells, in contrast to PC12 cells, have been shown in reaggregate cultures with primary cells to be capable of responding in a differential fashion to dopaminergic target and nontarget cells (Choi et al, 1992). The MN9D cell line shows a marked reduction in its dopaminergic phenotype when coaggregated with nontarget cells such as optic tectum or thalamus.…”

“…It is unlikely that the high levels of ChAT activity and the ability to synthesize ACh are derived from genes that are silent in the N 18TG2 cell line (Mevel-Minio and Weiss, 1981;Killary and Foumier, 1984). No ChAT activity was detected in a mesencephalic dopaminergic cell line (MN9D) produced with N 18TG2 as the fusion partner (Choi et al, 1992). On the other hand, the fusion of N 18TG2 cells with primary septal cholinergic cells resulted in cell lines which do express ChAT activity (Hammond et al, 1986(Hammond et al, , 1990Lee et al, 1990a), suggesting that the expression of a cholinergic phenotype in cell lines produced by the somatic cell fusion method is most likely a function of the lineage of the primary cells participating in the fusion process.…”

“…The clonal hybrid cell lines X57, X58, and X62 and the parent neuroblastoma line N18TG2 were plated at a density of 5-20 x lo4 cells per 60 mm culture dish. The cells were maintained in culture for up to 5 d in the presence of 1 mM n-butyric acid, which causes some cultured cells to differentiate (Prasad and Sinha, 1976;Choi et al, 199 l), or 10 PM forskolin, which maximally stimulates adenylate cyclase activity, or vehicle (0.1% dimethylsulfoxide). Cultures were examined for the expression of the neuronal marker neurofilament protein (NFP) and a glial marker, glial fibrillary acidic protein (GFAP).…”

“…Furthermore, cell lines produced in this manner may be used as models for the examination of the molecular mechanisms governing the differential expression of specific neurochemical phenotypes (Choi et al, 1992). Somatic cell fusion has been used for the generation of adrenergic cell lines from the peripheral nervous system (Greene et al, 1975) as well as those of central nervous system lineage including septal, hippocampal (Hammond et al, 1986(Hammond et al, , 1990Lee et al, 1990a,b), and ventral spinal cord (Cashman, 1991) cell lines.…”

MPP+ Downregulates Mitochondrially Encoded Gene Transcripts and Their Activities in Dopaminergic Neuronal Cells: Protective Role of Bcl-2

“…Using a dopaminergic neuronal cell line, MN9D, derived from murine mesencephalon (21,22), several cDNA fragments that corresponded to putatively upregulated or downregulated transcripts were isolated following 200 M MPP ϩ treatment. Interestingly, sequence analysis revealed that two of the cDNA species matched the sequences of mitochondrially encoded cytochrome c oxidase subunit 3 (COX III) and ATPase subunit 6 (ATPase 6) which are catalytic components of multimeric cytochrome c oxidase and ATP synthase complex, respectively (23,24).…”