Mecanismos biofísicos do efeito excitatório de receptores metabotrópicos de glutamato do grupo I em neurônios do Locus Coeruleus

Abstract

Metabotropic glutamate receptors (mGluR) mediate important roles in cellular excitability and plasticity in several regions of the nervous system. The activation of these receptors may involve ion channels and membrane transporters however the protein profile behind these changes are diverse and remain incompletely understood. In Locus Coeruleus neurons (LC), previous studies have shown the impact of group II and III mGluR activation, but the impact of activation of group I remains unclear. Recently, we identified that activation of group I metabolic glutamate receptors (mGluR-I) in Locus Coeruleus neurons causes an inward current and increases firing rate of pre-existing spontaneous action potentials. The present study investigates the membrane transport mechanisms behind these changes in excitability. Experiments under whole-cell voltage clamp (Vm = -60 mV) were conducted in horizontal slices (200 μM) of brainstem from Wistar rats. The activation of mGluR-I with the selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) by local and brief (10 ms) pulsatile application generated transient inward currents of -24.0 ± 7.6 pA (n=39). This current was not inhibited by the combined presence of inhibitors for AMPA (CNQX, 10 μM), NMDA (MK801, 10 μM), GABAergic (picrotoxin, 25 μM) and glycinergic (strychnine, 10 μM) receptors, nor by the neuronal NaV blocker, tetrodotoxin (1 μM). We investigated the ionic basis of the mGluR-I current through ramp protocols under voltage clamp and calculated the mGluR-I current from the difference (DHPG - Control). The I-V relation for this current exhibited little voltage dependence in the range -100 mV to -50 mV, with no decrease as Vm approached the K+ equilibrium potential. In related experiments, we used Cs+ as the principal cation in the pipette solution instead of K+ and we did not observe a significant difference in the amplitude of the current evoked by DHPG. The participation of TRPC channels was tested using the selective antagonist SKF96365 (50 µM), which did not significantly reduce the mGluR-I current. The involvement of the Na+ /Ca2+ exchanger and, LVA and HVA Ca2+ channels were tested by observing the reduction of DHPG current in the presence of different concentrations of Ni2+. The inhibition of DHPG current by Ni2+ was concentration dependent with IC50= 0.9 ± 0.09 mM. Lower concentrations of Ni2+, which selectively inhibit LVA Ca2+ channels, exhibited little or no effect. In contrast, higher concentrations of Ni2+, which additionally inhibit HVA Ca2+ channels and the Na+ /Ca2+ exchanger, blocked almost all of the DHPG current. The involvement of HVA Ca2+ channels and specifically L-type Ca2+ channels were tested with Cd2+ (100 µM) and dyhidropirydines (nifedipine, 100 µM and nimodipine, 5 µM), respectively. With these blockers the mGluR-I current was partially inhibited. The Na+ /Ca2+ exchanger was tested replacing extracellular Na+ by choline. The changes in gradient reduced the amplitude of DHPG current. Taking together, these data indicate: i)The intensity of the current observed with local application of DHPG, matches the depolarization effects of these receptors reported in our previous study, considering the current amplitude; ii) Activation of group I mGluR receptors occurs directly on LC neurons, generating an inward current at the resting potential; iii) Unlike other neuronal systems in which Group I mGluR may act through KIR, BK or SK channels, in the LC activation with DHPG did not require changes in K+ conductance; iv) The biophysical mechanism probably involved the combination of L-Type Ca2+ channels and the Na+ /Ca2+ exchanger. Our findings extend the knowledge about mGluR-I action on LC neurons complementing the existing literature and shows that activation of mGluR-I in the LC acts distinctly compared to other neurons in the brainstem.