Cloning and differentiation-induced expression of a murine serotonin1A receptor in a septal cell line

A neuronal cell model endogenously expressing the 5-HT1A receptor, in which to study the function and regulation of this gene, has yet to be identified. We examined murine SN-48 cells, a septum x neuroblastoma fusion cell line that proliferates in a nondifferentiated state but can be induced to differentiate into neurofilament-positive cells following 24-96 hr treatment with 10 microM retinoic acid in low serum. Northern blot analysis demonstrated the presence of a single 10.9 kilobase (kb) 5-HT1A receptor RNA species in differentiated SN-48 cells, which was not detected in undifferentiated SN-48 cells. The presence of receptor RNA in differentiated SN-48 cells correlated with the appearance of functional responses (i.e., pertussis toxin-sensitive inhibition of cAMP accumulation) to 5-HT1A agonists in differentiated but not in undifferentiated cells. In order to verify that the large 10.9 kb RNA species in SN-48 cells truly corresponded to the mouse 5-HT1A receptor RNA, a cDNA fragment from differentiated SN-48 cells was used to clone the corresponding mouse brain cDNA. The 2.4 kb cDNA contained a single open reading frame that displayed high (> 85% predicted amino acid identity) homology to the human and rat 5-HT1A receptor genes. When transfected into receptor-negative Ltk- cells, this cDNA was found to direct expression of a murine 5-HT1A receptor. Thus, we conclude that upon differentiation SN-48 cells express RNA encoding functional 5-HT1A receptors. The SN-48 septal cells will provide a useful cellular model system for investigating aspects of neuronal differentiation leading to the development of sensitivity to serotonergic input.