Episodic gonadotropin-releasing hormone gene expression revealed by dynamic monitoring of luciferase reporter activity in single, living neurons

摘要

The existence of an intrinsic oscillator for pulsatile gonadotropin-releasing hormone (GnRH) secretion in normal and transformed GnRH neurons raises the question of whether the corresponding gene also is expressed in an episodic manner. To resolve this question, we used a modification 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 by quantification of photons emitted by individual neurons microinjected with a GnRH promoter-driven luciferase reporter construct. Immortalized GT1–1 neurons, which secrete the decapeptide GnRH in a pulsatile manner conceptually identical to that of their nontransformed counterparts in vivo, were chosen as the model for these studies. First, we injected individual cells with purified luciferase protein and established that the reporter half-life was sufficiently short (50 min) to enable detection of transient changes in gene expression. Next, we subjected transfected GT1–1 cells to continuous monitoring of reporter activity 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 expression 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 monitored cells. Taken together, these results demonstrate the validity of our “real-time” strategy for dynamically monitoring GnRH gene activity in living neurons. Moreover, our findings indicate that GnRH gene expression as well as neuropeptide release can occur in an intermittent manner.