Pregabalin effects on transmitter 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

Bahía Blanca, Buenos Aires, Argentina

Workshop; Workshop "Neuronal Communication: From structure to physiology; 2008

Sociedad Argentina de Investigación en Neurociencias (SAN)

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Arial; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:8.5in 11.0in; margin:70.85pt 85.05pt 70.85pt 85.05pt; mso-header-margin:.5in; mso-footer-margin:.5in; 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 hope of modulating brain GABA receptors, resulting ineffective. The mechanism of action of pregabalin (PBG) has been only partially characterized; the cellular and molecular basis of its inhibitory action neurotransmitter release is unknown. The primary high-affinity binding site for PBG is the α2-δ type 1 auxiliary subunit of voltage-gated calcium channels (Gee et al., 1996). The exact action of PBG on calcium channel function is still a matter of controversy. It is generally accepted that PBG subtly reduce calcium-dependent overflow of neurotransmitters from several neuronal tissues, thus reducing synaptic responses. Here, we studied the effect of PBG on auditory brainstem slices containing the Medial Nucleus of the Trapezoid Body (MNTB), whose principal neurons receive a giant synapse (Calyx of Held). Whole cell patch clamp technique recordings of excitatory postsynaptic currents (EPSCs) at the soma of the MNTB neurons during low and high frequency stimulation. Also, presynaptic calcium currents were recorded form the Calyx of Held. The efficacy of synaptic transmission on MNTB was assayed. The amplitude of the EPSCs didn’t show a significant difference. At 100 Hz PBG (100 μM) produced a significant increment on the recovery’s fraction (Before PBG=2.28±0.09s; after PBG=1.29±0.5s; Student’s t-Test, p=0.014) while at 300Hz it induced a decrement (Before PBG:tfast=180±14ms, tslow=3.9±0.2s; after PBG: t=2.74±0.8s). The P/Q-type calcium channels mediated currents decreased in presence of PBG after a short stimuli ramp like, action potential (AP) or high frequency trains of AP (100-300Hz). However, an increase of the calcium current and a lower facilitation was observed with PBG after high frequency trains of square pulses (100-300Hz). Thus, suggesting that PBG might interact with P/Q-type channels in their closed state, increasing the number of available channels to be open. On the other hand, we studied mouse lavator auris longus nerve terminals staining and distaining using fluorescence microscopy of FM2-10 dye. This technique allowed us to study neuromuscular junction (NMJ) synaptic vesicles recycling. The bi-exponential kinetic decay of FM2-10 distaining at 50HZ on the NMJ (tfast=32.25±3.85s (32.21%), tslow=172.09±19.87 (33.97%)) were significantly reduce (ptfast=0.011, ptslow=0.006) in the presence of PBG (tfast=74.59±8.88s (58.08%), tslow=652.39±137.88 (22.22%)), however the fast component weight was increased while the slow component was diminished. In addition, a 30% blockage of P/Q calcium channels with ω-agatoxin (10 nM) produced a PBG like effect on dye release. These results suggest an effect of Pregabalin on vesicle recycling at NMJ.