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 mu M SKF 38393 (a sp ecific D-1 agonist), and a surviving cell population (E1X) was subclon ed. Twenty-seven monoclonal cell lines were obtained and screened for the expression of striatal-specific characteristics including gamma-am inobutyric acid (GABA), choline acetyltransferase (ChAT), acetylcholin e (ACh), mRNA for specific dopamine receptors, and dopamine- and adeno sine 3':5'-monophosphate-regulated phosphoprotein, M(r) 32,000 (DARPP- 32), and functional D-1 and D-2 dopamine receptors. Neither the parent hybrid cell population (E1X) nor any of the monoclonal cell lines exa mined expressed GABA levels significantly different than that of the N 18TG2 parent neuroblastoma cells (1.36 +/- 0.07 mu g/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 expr essing 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, f ollowing differentiation with 10 mu M forskolin, to neurofilament prot ein, a neuron-specific marker. The monoclonal hybrid cell lines, but n ot the N18TG2 neuroblastoma, were shown to express an array of D-1, D- 2, and D-5 receptor mRNA as well as DARPP-32 mRNA. Two monoclonal cell lines expressed D-1 receptor binding sites (X57, 29.2 +/- 4.5 fmol/mg protein and X62, 43.8 +/- 6.8 fmol/mg protein) which mediated the sti mulation of adenylate cyclase activity. One cell line, X58, expressed only D-2 dopamine receptors (80.9 +/- 9.8 fmol/mg protein) which were negatively coupled to adenylate cyclase activity. These findings sugge st that the immortalized monoclonal hybrid cell lines are of neuronal origin and have incorporated elements of the medium spiny and choliner gic neurons of the developing striatum.