1. Adrenergic Gate Release for Spike Timing-Dependent Synaptic Potentiation 
Yanling Liu, Lei Cui, Martin K. Schwarz, Yan Dong, Oliver M. Schlüter 
Neuron 
Volume 93, Issue 2, Pages (January 2017)
DOI: /j.neuron
Copyright © 2017 Elsevier Inc. Terms and Conditions

2. Figure 1 NE Modulation of Kv1.1-Mediated Latency of APs
(A–C, E, and F) Repeated rheobase current injections elicited single APs in CA1 pyramidal neurons (second AP marked with “#”). Sample recordings before (gray, left traces) and after different treatments (color, right traces). A total of 100 nM dendrotoxin-K (+ DTX-K) (A) to block Kv1.1 or 20 μM NE (+ NE) (B and C) to activate adrenoceptors was applied for 10 min as treatment. Laser light pulses (473 nm) were applied onto the stratum radiatum to trigger endogenous NE release in DBH-Cre × iChR2 mice (E) or no release in iChR2 control mice (F). A total of 1 μM ICI blocked NE modulation (C).
(D) Coronal slices of the brain stem (brain scheme with position of slices, upper panel) were cut from DBH-Cre × iChR2 (left) and iChR2 (right) mice and ChR2Y expression visualized under fluorescent light.
(G) Summary graph shows normalized AP latency modulation after different triggers as in (A)–(C), (E), and (F) normalized to latency in control.
(H) AP latency jitter of control is illustrated in gray and after different triggers in color as in (A)–(C), (E), and (F). Scale bar, 20 mV and 250 ms.
Error bars are SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 2
The SAP97-Kv1.1 Interaction Is Required for NE Modulation of AP Latency
(A) Kv1.1 was immunoprecipitated from mouse hippocampal extracts from floxed SAP97 (97flx) mice and SAP97-Nex (97KONex) KO mice, which lack SAP97 in forebrain glutamatergic neurons. The western blot was decorated with a Kv1.1 antibody or SAP97 antibody to test the complex formation with SAP97. Lane “w/o ab” presents immunoprecipitation without primary antibody as control and “input” presents the hippocampal extract used for immunoprecipitation.
(B) Analysis of SAP97 KO in SAP97-Nex hippocampal P2 fractions.
(C) Scheme of Kv1.1 and SAP97 interaction for full-length protein (left) and Kv1.1Δ4 (right).
(D) Kv1.1 was immunoprecipitated from HEK293 cell extracts and transfected with SAP97 and Kv1.1 or Kv1.1Δ4. Samples with cell extract (input) and without primary antibody (w/o ab) were used as controls for protein expression and non-specific bead interactions.
(E, F, I, and J) Repeated rheobase current injections elicited single APs in CA1 pyramidal neurons. Sample recordings before trigger stimulus are presented in black (E and F) or green (I and J) (left traces) and after different stimuli in orange (E) or red (F, I, and J) (right traces). A total of 100 nM DTX-K (+ DTX-K) to block Kv1.1 (E) or 20 μM NE (+NE) to activate adrenoceptors (F, I, and J) was applied for 10 min as a trigger.CA1 pyramidal neurons from SAP97-Nex knockout mice were analyzed (E and F). Modulation of AP latency was analyzed in transduced CA1 pyramidal neurons expressing GFP-tagged Kv1.1 (I) or GFP-tagged Kv1.1Δ4 (J).
(G) Scheme of AAV molecular replacement vector to express Kv1.1 or Kv1.1Δ4 in CA1 pyramidal neurons. shRNA targeting endogenous Kv1.1 is driven by a human U6 promoter and recombinant Kv1.1 with silent mutations (∗∗∗∗∗) in the shRNA target sequence from the CAG promoter.
(H) Efficiency of the Kv1.1 shRNA was tested in dissociated hippocampal cultures, which were transduced at 5 days in vitro (DIV 5) and harvested at DIV 14.
(K) Summary of normalized AP latency modulation after different triggers as in (E), (F), (I), and (J) normalized to latency in control.
(L) AP latency jitter of control in black and green and after different triggers in orange and red as in (E), (F), (I), and (J). Scale bar, 20 mV and 250 ms.
Error bars are SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

4. Figure 3 NE Gates STD-LTP
(A and C) Schematic presentation of EPSP followed by single backpropagating AP (A) or AP burst (C) conditioning stimulus with time course of events (left) and example trace (right). Scale bar, 10 ms (left) and 20 mV and 50 ms (right).
(B, D, E, G, and I) EPSP amplitude versus time of sample recordings with 100 conditioning stimuli at 5 Hz with one AP (B, E, G, and I) or one burst (D), applied at vertical line. LTP induction includes conditioning with one AP pair (B), with burst pair (D), with one AP pair and 20 μM NE application (during horizontal line; E), with additional application of 100 μM APV (G), or with 1 μM ICI (I). EPSP (average of 3 min) before conditioning at position 1 and 25 min after conditioning at position 2 is presented in inset. Scale bar, 5 mV and 50 ms.
(F, H, and J) Summary graphs for conditions as in (B), (D), (E), (G), and (I), and indicated in inset. NE-LTP control summary graph (one AP pair with NE) from (F) is illustrated in copy as a red line (mean) with gray box (SEM) in (H) and (J).
Error bars are SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

5. Figure 4
NE-Facilitated STD-LTP Requires SAP97 and Kv1.1 C-Terminal SAP97 Interaction Motif
(A, B, E, G, and H) EPSP amplitude versus time of sample recordings with 20 μM NE application during baseline (horizontal line) and 100 conditioning stimuli with one AP at 5 Hz, applied at vertical line for different conditions. Conditions include floxed SAP97 mice (97flx; A), SAP97-Nex KO mice (97KONex; B), single-cell SAP97 KO mice (97KOAAV), AAV-mediated Kv1.1 wild-type replacement (Kv1.1), and KvΔ4 replacement (H and I). EPSP (average of 3 min) before conditioning at position 1 and 25 min after conditioning at position 2 presented in inset. Scale bar, 5 mV and 50 ms.
(C, F, and I) Summary graphs for different conditions as in (A), (B), (E), (G), and (H) and indicated in inset. Copy of control NE-LTP from Figure 1F (F).
(D) Schematic presentation of recording configuration and sample fluorescent image of AAV-Cre-transduced CA1 pyramidal neurons to generate single-cell SAP97 KOs.
Error bars are SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

6. Figure 5 β2-Adrenoceptor-Dependent LTP of Dentate Gyrus Requires SAP97
(A–C) Field EPSP slope versus time of sample recordings in dentate gyrus with MPP stimulation. LTP was triggered with three conditioning stimuli, each 5 min apart and consisting of a 100 Hz for 1 s stimulus. Conditions included floxed SAP97, control slices (A), β2-adrenoceptor blockade with 1 μM ICI (B), and in conditional Dlg1/SAP97 KO mice with CaMKIIα-Cre driver (97KOCaMKII; C). Average (3 min) field EPSP before conditioning at position 1 and 60 min after conditioning at position 2 presented in inset. Scale bar, 0.5 mV and 10 ms.
(D) Summary graphs for different conditions as in (A)–(C).
Error bars are SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

7. Figure 6 NE-Triggered Reduction in Surface Kv1.1 Requires SAP97
(A and B) Dissociated hippocampal cultures were fixed, permeabilized, and decorated with antibodies directed against SMI-312 and Kv1.1 (A) or MAP-2 and Kv1.1 (B). Fluorescence images were acquired with a confocal microscope. Arrows mark dendrites, while arrowheads mark axons.
(C) Neurons were fixed and decorated with a Kv1.1 antibody recognizing an extracellular epitope in non-permeabilizing conditions. Three primary dendrites were analyzed as the exemplified one with the boxed dendrite, magnified at bottom. Reduction of Kv1.1 surface expression was induced with 20 μM NE and was blocked with 1 μM ICI
(D) Summary graph of normalized surface fluorescence relative to control for different conditions as in (C).
(E) Analysis of AAV-Cre-transduced cultures of floxed SAP97 KO mice. Single-cell KOs (sKOs) were identified by their green fluorescence. Surface expression of Kv1.1 was analyzed in untreated control cultures and NE-treated sister coverslips.
(F) Summary graph of surface fluorescence for the conditions in (E) normalized to control condition.
Error bars are SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

8. Figure 7
β-Adrenoceptor-Facilitated Increase of Dendritic Excitability Requires SAP97
(A) AAV-ChR2Y was stereotactically injected into the CA1 area of SAP97-Nex (97KONex) KO mice or littermate floxed SAP97 (97flx) controls. ChR2Y expression was visualized in hippocampal slices with fluorescent illumination.
(B) Schematic representation of recording configuration with somatic patch-clamp recording from CA1 pyramidal neurons and 473 nm laser light stimulation in a 40 μm diameter spot in stratum radiatum (blue).
(C) Sample traces of triggered APs in 97flx mice with 2 ms current injection (1 nA; left) or 1–2 ms 473 nm laser light stimulation (blue mark) on soma (middle) or dendrite (right). Scale bar, 20 mV and 50 ms.
(D) Summary graph of AP threshold of somatic current injections (ΔIsoma) and somatic (hνsoma) or dendritic (hνdendrite) ChR2Y activation in 97flx control or 97KONex mice. Statistical analysis was performed with two-factor ANOVA and Sidak post hoc analysis.
(E and F) Modulation of AP threshold and latency with 10 min β-adrenoceptor activation in 97flx (E) and 97KONex (F) mice. Sample traces with AP before 10 μM isoproterenol (ISO) perfusion (control) and after 5 min ISO perfusion. Magnification of traces on right. Scale bar, 20 mV and 5 ms.
(G and H) Summary graph of AP latency (G), calculated relative to control condition and change in AP threshold (H) after ISO perfusion.
(I) Schematic recording configuration of dendritic spikes with dendritic patch-clamp electrophysiology of apical CA1 pyramidal neuron dendrites and electrical stimulation in the alveus to trigger backpropagating APs.
(J–L) Dendritic spikes before and after ISO (2 μM; J and K) or DTX-K (L) perfusion of 97flx (J and L) and 97KONex (K) mice. Scale bar, 10 mV and 5 ms.
(M and N) Summary graph of time course of change (M) and relative change after 10 min (N) in dendritic spike amplitude after drug perfusion.
(O and P) Light (blue bar) pulses (0.5 ms) triggered somatic subthreshold depolarization in 97flx neurons (O) or 97KONex neurons (P). Scale bar, 10 mV and 25 ms. After 10 min stimulation of β-adrenoceptors, depolarizations with same light pulse intensities became superthreshold in 97flx (O), but not in 97KONex (P).
(Q) Summary graph of average subthreshold depolarizations before ISO application.
(R) Summary graph of fraction of APs triggered before ISO (−ISO) and after ISO (+ISO) perfusion.
Error bars are SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions