1. A Wnt/Calcium Signaling Cascade Regulates Neuronal Excitability and Trafficking of NMDARs 
Andrea McQuate, Elena Latorre-Esteves, Andres Barria 
Cell Reports 
Volume 21, Issue 1, Pages (October 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 21, 60-69DOI: (10.1016/j.celrep.2017.09.023)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 Wnt5a Mobilizes Intracellular Ca2+
(A) Dendrites of a dissociated CA1 neuron loaded with 3 μM Asante Calcium Red (ACR) at baseline and 1 and 10 min after adding Wnt5a to the bath. Scale bar, 10 μm.
(B) Change in ACR fluorescence normalized to baseline. Either Wnt5a (black circles; n = 18), Wnt7a (white squares; n = 9), or control medium (gray circles; n = 11) was added to the bath after a 3-min baseline. ACSF containing 50 mM KCl was added to the bath as a positive control. Asterisk indicates significance measured at a time window between 5.5 and 6.5 min (p < 0.01).
(C) Change in ACR fluorescence as in (A) induced by Wnt5a in neurons expressing shRNA targeting the RoR2 receptor (n = 10).
(D–G) Change in ACR fluorescence as in (A) induced by Wnt5a in neurons bathed in nominally Ca2+ free ACSF (D; n = 8), pretreated with 30 μM CPA (E; n = 8), in the presence of 10 μM nifedipine and 1 μM ω-conotoxin MVIIC (F; n = 6), or pretreated with 5 μM U73122 (G; n = 8).
Error bars indicate ± SEM.
Cell Reports  , 60-69DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

4. Figure 2 Wnt5a Depolarizes Neurons in Hippocampal Slices
(A) Sample trace of resting membrane potential of a CA1 neuron before and after bath application of Wnt5a.
(B) Average change in resting membrane potential normalized to baseline over the course of 20 min after bath application of Wnt5a (n = 18), Wnt7a (n = 9), or control conditioned media containing no Wnt ligand (n = 11) as indicated. Data are expressed as 1 normalized membrane potential (MP) for directionality.
(C) Average change in resting membrane potential normalized to baseline over the course of 20 min after bath application of Wnt5a to neurons recorded with 10 mM BAPTA in the patch pipette (gray circles; n = 11), in the presence of 10 μM nifedipine (black squares; n = 8), or pretreated with 5 μM U73122 (white triangles; n = 16).
(D) Absolute change in resting membrane potential from experiments in (B) and (C) 20 min after bath application of control medium, Wnt5a, Wnt7a (black bars; n = 11, 38, 9, respectively), or Wnt5a in the presence of PLC inhibitor U73122 (n = 16), its inactive analog U73343 (n = 4), BAPTA (n = 11), or nifedipine (n = 8). ∗p < 0.05 compared to control.
(E) Absolute change in resting membrane potential 20 min after bath application of Wnt5a (same as in D) for neurons treated with G protein inhibitors (gray bars) suramin 50 μM (n = 7), PTX 100 ng/mL (n = 9), YM  μM (n = 8), or with retigabine 10 μM (stripped bar; n = 7). ∗p ≤ 0.05 compared to Wnt5a.
Error bars indicate ± SEM.
Cell Reports  , 60-69DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

5. Figure 3
Wnt5a Increases Neuronal Excitability and Decreases High-Voltage-Activated K+ Channels
(A and B) Input-output function for neurons before and 20 min after bath application of Wnt5a (A; n = 6) or Wnt7a (B; n = 6). Current steps were 300 ms in duration. ∗p < 0.01.
(C) Sample traces of voltage response to a 0.06-nA, 300-ms current injection before and after treatment with either Wnt5a (top) or Wnt7a (bottom).
(D) Number of action potentials per 300 ms, 0.06 nA current injection over the course of 20 min during treatment with either Wnt5a (black circles; n = 6) or Wnt7a (white squares; n = 6). ∗p < 0.01 compared to baseline.
(E) Example traces of potassium currents evoked by a step to +20 mV before and after 20 min of treatment with either Wnt5a (top) or Wnt7a (bottom). Steady-state current was measured at a window indicated by arrowheads.
(F) K+ current measured as indicated in (E) before and after bath application of Wnt5a (n = 7), Wnt7a (n = 6), or Wnt5a in the presence 5 μM PLC inhibitor U73122 (n = 9). ∗p < 0.01.
(G–I) Current-voltage relationship for K+ currents acquired in the presence of 2 μM NBQX and 1 μM TTX before and 20 min after bath application of Wnt5a (G; n = 7), Wnt7a (H; n = 6), or Wnt5a in the presence 5 μM PLC inhibitor U73122 (I; n = 9).
Error bars indicate ± SEM.
Cell Reports  , 60-69DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions

6. Figure 4
Intracellular Ca2+ Release from Stores Is Necessary for Wnt5a to Upregulate NMDAR Currents
(A) Sample traces of isolated NMDAR-mediated EPSCs recorded at +40 mV in CA1 neurons during baseline, 20, and 50 min after bath application of Wnt5a from control hippocampal slices or slices pretreated with 30 μM CPA.
(B) Normalized peak amplitude of NMDAR-mediated EPSCs from control neurons (n = 10), neurons pretreated with 30 μM CPA (n = 9), neurons pretreated with 5 μM U73122 (n = 13), or in the presence of 10 μM nifedipine (n = 9) as indicated. Schafer collaterals where stimulated at 0.1 Hz to evoke postsynaptic responses.
(C) Normalized peak amplitude of isolated NMDAR-mediated EPSCs recorded at +40 mV from control CA1 neurons (black circles; n = 5) and neurons recorded with 10 μM Snap25 peptide in the patching pipette (white circles; n = 5). Schafer collaterals where stimulated at 0.1 Hz to evoke postsynaptic responses.
Error bars indicate ± SEM.
Cell Reports  , 60-69DOI: ( /j.celrep )
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7. Figure 5 Wnt5a Promotes Trafficking of GluN2B-Containing NMDARs
(A) Sample images of dendrites expressing SEP-tagged GluN2B containing NMDARs before or 60 min after bath application of Wnt5a (top) or Wnt7a (bottom). Scale bar, 5 μm.
(B) Surface expression of optically tagged GluN2B in dendrites of dissociated neurons. Normalized fluorescence intensity in dendrites of neurons expressing SEP-tagged GluN2B receptors before and after bath application of Wnt5a (black circles; n = 8), Wnt7a (white squares; n = 5), or control conditioned medium (gray triangles; n = 3).
(C) Surface expression of SEP-GluN2B (as in B) in dendrites expressing shRNA targeting the RoR2 receptor (n = 5).
(D) Surface expression of SEP-tagged GluN2B (as in B) in dendrites pretreated with 30 μM CPA (n = 5). CPA was also maintained in the bath during the experiment.
(E) Quantification of SEP-fluorescence for a window between 30 and 40 min after addition of Wnt ligand. ∗p < 0.01 compared to baseline.
(F) Biotinylation of surface endogenous GluN2B. Top, sample immunoblot of total and surface GluN2B from control neurons or neurons treated for 1 hr with Wnt5a. Bottom, quantification of immunoblots intensity (n = 8). ∗p < 0.01.
Error bars indicate ± SEM.
Cell Reports  , 60-69DOI: ( /j.celrep )
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8. Figure 6 Wnt/Ca2+ Signaling Cascade in Neurons
Wnt5a, but not Wnt7a, increases dendritic Ca2+ in a process that requires RoR2, Gq protein, and PLC. This signaling cascade leads to a decrease in potassium currents, a subsequent increase neuronal excitability, and VGCC-dependent mobilization of Ca2+ from intracellular stores. As a consequence of this signaling cascade, the trafficking of NMDARs into synapses via SNARE-dependent mechanism is increased.
Cell Reports  , 60-69DOI: ( /j.celrep )
Copyright © 2017 The Author(s) Terms and Conditions