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

Immortalized hybrid cells were generated by somatic cell fusion of 18-d-old embryonic corpus striatum of the mouse strain C57BL/6J with the N18TG2 neuroblastoma. One of the cell populations obtained was treated with a combination of 1 mM n-butyric acid and 10 microM SKF 38393 (a specific D1 agonist), and a surviving cell population (E1X) was subcloned. Twenty-seven monoclonal cell lines were obtained and screened for the expression of striatal-specific characteristics including gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), acetylcholine (ACh), mRNA for specific dopamine receptors, and dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein, M(r) 32,000 (DARPP-32), and functional D1 and D2 dopamine receptors. Neither the parent hybrid cell population (E1X) nor any of the monoclonal cell lines examined expressed GABA levels significantly different than that of the N18TG2 parent neuroblastoma cells (1.36 +/- 0.07 micrograms/mg protein). The range of ChAT activity in the monoclonal hybrid cell lines was 5.5 +/- 0.3 to 921.3 +/- 97.4 pmol/min/mg protein. Two of the cell lines expressing ChAT activity (X52 and X58) contained ACh (49.64 +/- 4.23 and 1.78 +/- 0.07 ng/mg protein, respectively). The neuronal origin of four of the monoclonal hybrid lines was shown by their immunoreactivity, following differentiation with 10 microM forskolin, to neurofilament protein, a neuron-specific marker. The monoclonal hybrid cell lines, but not the N18TG2 neuroblastoma, were shown to express an array of D1, D2, and D5 receptor mRNA as well as DARPP-32 mRNA. Two monoclonal cell lines expressed D1 receptor binding sites (X57, 29.2 +/- 4.5 fmol/mg protein and X62, 43.8 +/- 6.8 fmol/mg protein) which mediated the stimulation of adenylate cyclase activity. One cell line, X58, expressed only D2 dopamine receptors (80.9 +/- 9.8 fmol/mg protein) which were negatively coupled to adenylate cyclase activity. These findings suggest that the immortalized monoclonal hybrid cell lines are of neuronal origin and have incorporated elements of the medium spiny and cholinergic neurons of the developing striatum.

Survival of developing dopaminergic neurons in reaggregate tissue culture following treatment with methamphetamine.

Developing central dopamine (DA)-containing neurons in rotation-mediated reaggregate tissue culture exposed to 10(-4) M methamphetamine for 1 week (at a time corresponding to the 2nd week of postnatal development) exhibit decreased DA levels. DA neurons were visualized either by a histofluorescent method after 'loading' with exogenous DA, or by tyrosine hydroxylase (TH) immunocytochemistry. An apparent loss of histofluorescent DA cell bodies was observed due to a methamphetamine-induced reduction in exogenous DA accumulation. No decrease in the number of TH-immunoreactive neurons was observed. Thus, developing DA neurons when visualized by TH immunocytochemistry survive despite methamphetamine-induced reductions in DA levels.

Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion.

To facilitate the study of trophic interactions between mesencephalic dopaminergic neurons and their target cells, clonal hybrid cell lines have been developed from rostral mesencephalic tegmentum (RMT) of the 14-day-old embryonic mouse employing somatic cell fusion techniques. Among the hybrid cell lines obtained, one contains a high level of dopamine (DA), another predominantly 3,4-dihydroxyphenylalanine (DOPA), and a third no detectable catecholamines. The hybrid nature of the cell lines is supported by karyotype analysis and by the expression of adhesion molecules as assessed by aggregation in rotation-mediated cell culture. The DA cell line shows neuronal properties including catecholamine-specific histofluorescence, neurite formation with immunoreactivity to neurofilament proteins, and large voltage-sensitive sodium currents with the generation of action potentials. In contrast to the pheochromocytoma cell line (PC12), the dopamine content of the DA hybrid cell line is depleted by low concentrations of N-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).