Figure 3.
Synaptic zinc release impacts dendritic non-linearities in basal dendrites of L2/3 PNs
(A) Two-photon laser scanning microscopy (2PLSM) image (maximum-intensity projection, MIP) of L2/3 PN. White dotted line: the location of the theta glass pipette used for focal dendritic stimulation.
(B) Representative traces of recorded EPSPs for increasing (left, average of 6) intensity of stimulation in control (black), with ZX1 (100 μM) and after addition of the NMDAR antagonist D-AP5 (50 μM).
(C) Plot of EPSP integral (third pulse) in function of stimulus intensity obtained from the experiment illustrated in (B). Dotted lines represent linear regression to the stimulation intensity values before occurrence of non-linear EPSPs.
(D) Recorded EPSPs (six individual sweeps) for conditions listed in (B) with constant stimulation intensity.
(E) Left: summary plot of integral of synaptically evoked EPSPs (third pulse) at the stimulus intensity defined as the minimum value needed to induce non-linear dendritic behavior (in zinc chelated) in control conditions and in the presence of ZX1. ∗p < 0.05. Right: the first peak amplitude is not affected by the application of the zinc chelator (ctrl: 7.82 ± 0.83 mV; ZX1: 7.97 ± 0.70 mV; n = 11 p = 0.68, Wilcoxon matched-pairs signed rank test).
(F) Chelating extracellular zinc induces a shift to the left of the stimulus current-voltage relationship. Stimulation intensity is shown relative to the maximum value used in control for each cell defined as 1 (n = 11, ∗p < 0.01, Wilcoxon matched-pairs signed rank test).
(G) 2PLSM image of a basal dendrite from an L2/3 PN with nine selected glutamate uncaging locations (orange).
(H) Photolysis-evoked EPSPs (pEPSPs) in response to increasing number of laser spot locations in control conditions and in the presence of the zinc chelator ZX1. Right: algebraic sum of individual pEPSPs.
(I) Subthreshold input-output relationship of pEPSPs obtained in control conditions (black) and in the presence of ZX1 (orange) for dendrite illustrated in (G) and (H) (upper left).
(J) Summary plot of supralinearity for control conditions and after ZX1 application.
(K) Biophysical model of NMDA zinc modulation at single synapse (see STAR Methods). Zinc binding (bZn) is modeled by an increment-and-decay dynamics following glutamatergic events. At a given synaptic event, the current level of zinc binding (bZn, black curve) sets the zinc modulation level (mZn, piecewise function in orange) that reduces NMDA conductance according to the factor (1–αZn·mZn), where αZn accounts for the efficacy of zinc inhibition at full binding (see STAR Methods).
(L) Morphological reconstruction of layer 2/3 PN used for the model (Jiang et al., 2015). The orange dot indicates the location of the synaptic stimulation used to obtain the traces in (M).
(M) Membrane potential (top) and conductance (bottom) traces obtained following the activation of an increasing number of recruited synapses (Nsyn) for free-zinc (black), chelated-zinc (green), and AMPA-only (blue) conditions.
(N) Summary plot of the integral of the third EPSP in the traces obtained during simulations shown in (M).
(O) Left: considering multiple locations of synaptic stimulation over the basal dendrite dendritic tree (n = 25 locations, color-coded). Right: half-activation level (Nsyn1/2) and respective third pulse integral measured in zinc-free (black) or in zinc-chelated (green) conditions obtained for the point of stimulation reported (left). ∗∗∗p < 0.0001, Wilcoxon matched-pairs signed rank test.