EPISODIC GONADOTROPIN-RELEASING-HORMONE GENE-EXPRESSION REVEALED BY DYNAMIC MONITORING OF LUCIFERASE REPORTER ACTIVITY IN SINGLE, LIVING NEURONS

The existence of an intrinsic oscillator for pulsatile gonadotropin-re leasing hormone (GnRH) secretion in normal and transformed GnRH neuron s raises the question of whether the corresponding gene also is expres sed in an episodic manner. To resolve this question, me used a modific ation of conventional luciferase technology, which enabled continuous monitoring of GnRH gene activity in single, living neurons, With this method, the relative rate of endogenous gene expression is estimated b y quantification of photons emitted by individual neurons microinjecte d with a GnRH promoter-driven luciferase reporter construct, Immortali zed GT1-1 neurons, which secrete the decapeptide GnRH in a pulsatile m anner conceptually identical to that of their non-transformed counterp arts irt vivo, were chosen as the model for these studies. First, we i njected individual cells with purified luciferase protein and establis hed that the reporter half-life was sufficiently short (50 min) to ena ble detection of transient changes in gene expression, Next, we subjec ted transfected GT1-1 cells to continuous monitoring of reporter activ ity for 16 h and found that the majority of them exhibited spontaneous fluctuations of photonic activity over time. Finally, we established that photonic activity accurately reflected endogenous GnRH gene expre ssion by treating transfected GT1-1 cells with phorbol 12-myristate 13 acetate (a consensus inhibitor of GnRH gene expression) and observing a dramatic suppression of photonic emissions from continuously monito red cells. Taken together, these results demonstrate the validity of o ur ''real-time'' strategy for dynamically monitoring GnRH gene activit y in living neurons, Moreover, our findings indicate that GnRH gene ex pression as well as neuropeptide release can occur in an intermittent manner.