Ruolo dei canali ionici TRPC nella modulazione delle proprietà elettrofisiologiche di neuroni GnRH immortalizzati

The study reported here has focused on the modulation of the intrinsic electrical properties of GT1-7 cells by the Ca2+-permeable TRPC ion channels, which are Ca2+-permeable, non-selective cationic channels, and their involvement in the GnRH-induced oscillations of the membrane potential. In particular, during my thesis project, I performed patch clamp experiments on GT1-7 cells using three experimental techniques: conventional whole-cell, cell-attached and perforated patch. The perforated patch technique, in particular, allowed us to record for relatively long times without heavily perturbing the intracellular milieu, and thus it was particularly appropriate for the study of relatively small ionic events modulated by intracellular messengers, such as those regulating the pacemaker-like activity in GT1-7 cells. In our hands, GT1-7 cells showed different patterns of activity, and the pooled percentages of spontaneously active versus silent cells are well in the range of those reported in the literature. The different patterns of activity have been related to the different degree of differentiation of GT1-7 cells. In our case, we recorded from morphologically differentiated GT1-7 cells after 3 to 7 days of culturing in defined medium in 0,5% of serum without observing significant differences. Furthermore, even synaptic and electrical coupling between cells have been taken into account to explain these different activities, however the isolated cells we investigated showed the same patterns as the clustered ones. Finally, as previously reported, the majority of silent GT1-7 cells could be induced to fire under GnRH stimulation. We made use SKF96365, one of the most used TRPC blockers, together with voltage-clamp protocols in order to check that the effect of this drug on the electrical activity was not due to the inhibition of voltage-dependent Ca2+ channels, that have been recognized as an essential component of the pacemaker activity in GT1-7 neurons. SKF96365 decreases, and sometimes completely abolishes both spontaneous and GnRH-induced action potentials either in the form of bursts or in the form of regular firing. Therefore, we can preliminary conclude that a SKF96365-sensitive effect, most probably mediated by a TRPC subfamily member, is involved in the spontaneous pacemaker activity of GT1-7 cells.