Pregabalin effects on neurotransmitter release at the mouse Calyx of Held and Neuromuscular Junction

MARIANO N. DI GUILMI, M. EUGENIA LOPEZ, FRANCISCO J. URBANO, CARLOTA GONZALEZ INCHAUSPE AND OSVALDO D. UCHITEL.

Montevideo, Uruguay

Otro; XIV Escuela Latinoamericana de Neurociencias; 2009

Instituto de Investigaciones Biológicas Clemente Estable - IBRO LARC

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} p.Left, li.Left, div.Left {mso-style-name:Left; mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:none; mso-layout-grid-align:none; text-autospace:none; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Pregabalin [S-[+]-3-isobutylGABA, Lyrica] is an anticonvulsant and analgesic medication which was originally synthesized with the intention of modulating brain GABA receptors, resulting ineffective. The mechanism of action of pregabalin (PBG) has been only partially characterized; it is generally accepted that PBG subtly reduce calcium-dependent overflow of neurotransmitters in several tissues. However, the cellular and molecular basis of its inhibitory action on neurotransmitter release is unknown. The α2-δ type 1 auxiliary subunit of voltage-gated calcium channels is the primary high-affinity binding site for PBG (Gee et al., 1996). The exact action of PBG on calcium channels function is still a matter of controversy.   Here, we studied the effect of PBG on transmitter release at the mouse neuromuscular junction (lavator auris longus) using intracellular recordings and at the Calyx of Held-Medial Nucleus of the Trapezoid Body (MNTB) synapse in brainstem slices using whole cell patch clamp. Excitatory postsynaptic currents (EPSCs) and presynaptic calcium currents were recorded at Calyx of Held-MNTB complex at both low and high frequency stimulation.   The P/Q-type calcium channels mediated currents decreased in presence of PBG (100 μM) after short stimuli ramp like, action potential (AP) or high frequency trains of AP (100-300Hz). The calcium current was partially recovered by isoleucine (500 μM). Calcium currents activation curves, obtained from plotting tail currents versus command voltage showed no differences. However, two pulses inactivation protocol shows a larger rescue of the inactivation.  Although the amplitude of EPSCs at the Calyx – MNTB was not affected, differences in the recovery from high frequency depression were observed. Similar effects after high frequency stimulation were observed at the NMJ showing principally an enhancement of the initial facilitation and a recovery of depression at the last part of the train. This pool of data suggests that PBG might interact with P/Q-type channels, increasing the number of available closed channels to be open. Additionally, we studied the NMJ FM2-10 dye staining and distaining using fluorescence microscopy. The bi-exponential kinetic decay of FM2-10 distaining at 50Hz on the NMJ was significantly reduced in the presence of PBG.   Taken together, these results suggest that PBG might accelerate the recovery of the calcium channel from inactivated states interacting with P/Q-type and/or with vesicle recycling.