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NMDA receptor–BK channel coupling regulates synaptic plasticity in the barrel cortex

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Summary

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BK channel produces supression of NMDARs activity in about 40% of the basal dendrites of BC-L5 PYRs

Introduction

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  • NMDA characteristics:
    • High sensitivity to glutamate
    • Voltage-dependent block by Mg2+
    • Slow gating kinetics
    • High permeability to Ca2+
  • BK micromolar range of activation is restricted to the vecinity of VGCCs and RyRs
  • BK channel blunts NMDAR-mediated excitatory responses
  • BC proccess information relayed from different nuclei of thalamus.
  • VPM, VB and PM nuclei from thalamus directly innervate BC-L5 pyramidal neurons
  • Inputs to basal dendrites of BC-L5 PYRs produce long lasting Ca2+ transients and long-lasting depolarizations
  • BK channels asociated with NMDAR would thus attenuate postsynaptic responses, working as negative feedback

Results

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NMDAR Activation Opens BK Channels in BC-L5PN

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  • Methods: Whole cell voltage-clamp recordings on BC-L5 PYRs
  • Bath contained a Mg-free ACSF solution, glycine and TTX.
  • When clamping the cell to >= 40mV, 40% of the cells developed a slower outward current, dependent on membrane voltage.
  • These neurons were classified as type B neurons in contrast to type A that didn't have this outward current.
  • Outward currents of type B were proportional to clamped voltage and decreased the inward current.
  • In this case NMDA pippete was pointed in direction to dendrites of PYRs in L5 (just behind L4). Then additionally the proccedure was tried with the pipette pointing towards L4 and in this case only the inward current was observed, so it was determined that the source of these currents was on the dendrites but not in the soma.
  • Outward current was pharmacollogically characterized:
    • AP5/D-AP5 abolished the current, confirmed NDMA dependency
    • ZnCl2/IFEN suppresssed partially the outward current, evidence mediation of GluN2A/GluN2B containing NMDARs
    • Paxiline supressed completelly the outward current. Evidence of BK channel being the source of the outward current.

NMDARs and BK Channels Are Within Functional Proximity in B-type BC-L5PNs

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  • Methods: Whole cell voltage-clamp recordings on BC-L5 PYRs in presence of chelators BAPTA and EGTA
  • BAPTA and EGTA quelators were used before to estimate the lineal physical distance between channels in BK-VGCC macrocomplexes
  • BAPTA and EGTA have similar affinities for calcium, but BAPTA has a faster association rate than EGTA.
  • Type 2 outward currents were abolished at EGTA 15 mM in contrast to BAPTA 15mM where the outward current was largely preserved. Also it was preserved lowering EGTA concentration to 1mM.
  • Using diffusion data in the presence of BAPTA and EGTA, authors calculated that the NMDAR and BK channels are located within 15 to 60 nm of each other.

Both GluN2A- and GluN2B-Containing NMDARs Can Functionally Couple to BK Channels

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  • Methods: Proximity ligation assay (PLA); HEK293T heterlogous expression system; whole cell patch clamp recording
  • Positive PLA signals were observed for HEK293T containing GluN1/GluN2A or GluN1/GluN2B subunits (no preference between GluN2A and GluN2B subunits).
  • NMDARs were activated by 200uM NMDA / 10 uM glycine