Volume 92, Issue 1, Pages 114-125 (October 2016) Dysfunction of Somatostatin-Positive Interneurons Associated with Memory Deficits in an Alzheimer’s Disease Model  Lena C. Schmid, Manuel Mittag, Stefanie Poll, Julia Steffen, Jens Wagner, Hans-Rüdiger Geis, Inna Schwarz, Boris Schmidt, Martin K. Schwarz, Stefan Remy, Martin Fuhrmann  Neuron  Volume 92, Issue 1, Pages 114-125 (October 2016) DOI: 10.1016/j.neuron.2016.08.034 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 In Vivo Imaging of Synapses on O-LM Interneurons (A) Schematic illustration of the experimental timeline. (B) Representation of an O-LM interneuron and its processes in hippocampal layers stratum oriens (SO), stratum pyramidale (SP), stratum radiatum (SR), and stratum lacunosum-moleculare (SLM). (C) Maximum-intensity side projection of a z stack acquired in the hippocampus of a Gad1-eGFP mouse showing putative O-LM interneurons (see also Figures S1A and S1B). (D) Time series of O-LM interneurons (indicated by star), dendrites in SO (upper panel), and axons in SLM (lower panel) imaged over a time period of 7 months in APP/PS1 mice. Aβ plaques indicated by yellow arrows were stained with MethoxyX04 (MeX04, blue) and emerged in SO and SLM. (E) Percent plaque volume per imaging volume covered with Aβ plaques in SLM and SO over time in APP/PS1 mice. n = 6 and 7 APP/PS1 mice in SLM and SO, respectively. Data are represented as mean ± SEM. Scale bars, 50 (C) and 20 μm (D). Neuron 2016 92, 114-125DOI: (10.1016/j.neuron.2016.08.034) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Axon Loss in SLM of APP/PS1 Mice (A) Overview images of SLM showing axons with boutons and Aβ plaques (blue) stained with MethoxyX04 (MeX04) at 4 and 11 months of age in an APP/PS1 mouse. (B) Magnification of the boxed region in (A) illustrating an axon loss (red arrows) between 4 and 5 months of age. Yellow arrows indicate persistent boutons. See also Figure S2. (C) Time series of axonal boutons in SLM. Yellow arrows indicate stable boutons; gained and lost boutons are labeled with turquoise and red, respectively. Note the axon loss between 10 and 11 months. (D and E) Axon survival over time from 4 to 11 months of age (D) and at beginning and end (E) comparing wild-type and APP/PS1 mice. (F) Percentage of axons lost below and above a distance of 50 μm to the next Aβ plaque at the time point prior to its loss. (G) Density of boutons on persistent axons in wild-type and APP/PS1 mice. (H) Bouton density of lost axons aligned to the time point of loss. (I) Average of bouton density in wild-type and APP/PS1 mice split into stable axons as well as axons pre- and post-loss. n = 5 wild-type and 6 APP/PS1 mice (D, E, and G), n = 19 axons in 6 APP/PS1 mice (F), n = 5 APP/PS1 animals (H), and n = 5 wild-type, 6 APP/PS1 mice for stable axons, and 5 APP/PS1 mice for pre- and post-loss axons (I). All data are represented as mean ± SEM, n.s. (not significant). p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Repeated-measures two-way ANOVA (D and G) and one-way ANOVA with Sidak’s multiple comparisons test (E and I). Scale bars, 20 (A) and 10 μm (B and C). Neuron 2016 92, 114-125DOI: (10.1016/j.neuron.2016.08.034) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Structural Plasticity Impairment of Dendritic Spines on O-LM Interneurons (A) Time series from 4 to 11 months of the same O-LM interneuron in SO and emerging Aβ plaques (blue) in an APP/PS1 mouse. (B) Magnification of the boxed region in (A) showing a dendritic element with spines. Arrows indicate spines that were stable (yellow), gained (turquoise), or lost (red) between subsequent imaging time points. (C and D) Spine density over time in wild-type (C) and APP/PS1 (D) mice. Additionally, the densities of lost (red) and gained spines (turquoise) are depicted on the secondary y axis for both genotypes. (E) Average spine density in old (7–11 months) wild-type and APP/PS1 mice, as well as spine density in the vicinity of (<50 μm) or at a distance (>50 μm) from Aβ plaques in old APP/PS1 animals. (F) Monthly turnover rate of dendritic spines of wild-type and APP/PS1 mice. (G) Average turnover rate for young (5–6 months) and old (7–11 months) wild-type and APP/PS1 mice, as well as the turnover rate in old APP/PS1 animals at a distance below and above 50 μm proximity to Aβ plaques. n = 5 wild-type and 7 APP/PS1 mice (C, D, and F), n = 125 dendrites in wild-type and 175 dendrites in APP/PS1 (E), and n = 50 and 125 dendrites in wild-type and 70 and 175 dendrites in APP/PS1 (G). All data are represented as mean ± SEM, n.s. p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Repeated-measures one-way ANOVA with post-test for linear trend (C and D), one-way ANOVA with Sidak’s multiple comparisons test (E), repeated-measures two-way ANOVA (F), and Kruskal-Wallis test with Dunn’s multiple comparisons test (G). Scale bars, 20 (A) and 2 μm (B). Neuron 2016 92, 114-125DOI: (10.1016/j.neuron.2016.08.034) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Learning-Dependent Spine Gain on GABAergic Interneurons (A) Schematic illustration of the experimental outline. (B) Freezing rates of wild-type and APP/PS1 mice before fear conditioning (FC) and during memory retrieval (RE). (C) Exemplary pictures of a putative O-LM interneuron dendrite with spines from a wild-type mouse during BL and L&M. Arrows indicate stable (yellow), gained (turquoise), and lost (red) spines over a 4-day interval. Note the emergence of new spines on day 4 during L&M. (D) Normalized density of gained spines comparing BL and FC in wild-type and APP/PS1 mice. (E) Left panel: schematic illustrating the stabilization of newly gained spines after FC. Newly gained spines can either become stabilized (yellow) or removed (red). Right panel: normalized density of stabilized spines comparing wild-type and APP/PS1 mice. (F) Left panel: schematic illustration of clustered and non-clustered spine gain. Right panel: fraction of spines emerging clustered after FC comparing wild-type with APP/PS1 mice. n = 21 wild-type and 11 APP/PS1 mice (B), and n = 6 wild-type and 8 APP/PS1 animals (D–F). All data are represented as mean ± SEM, n.s. p > 0.05, ∗p < 0.05, ∗∗p < 0.01. Unpaired t test (B) and one-way ANOVA with Sidak’s multiple comparisons test (D and E). Age of animals at start of experiment was as follows: 9–14 (B) and 12–14 months (D and F). Scale bar, 1 μm (C). Neuron 2016 92, 114-125DOI: (10.1016/j.neuron.2016.08.034) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Decreased Ca2+ Response of O-LM Interneurons to Aversive Stimulation (A) Schematic of juxtacellular recordings of SO neurons in awake mice during airpuff stimulation. (B) Action potential firing in response to airpuff stimulation. Bin size 30 ms. (C) SO neurons increase firing frequency following airpuff stimulation (100 ms bin size, 16 trials). (D) Schematic illustration of two-photon Ca2+ imaging and pharmacological block of subcortical cholinergic input to O-LM interneurons in awake mice. (E) Putative O-LM interneurons expressing GCaMP6m in SO of the hippocampus. (F) Change in somatic GCaMP6m fluorescence in response to airpuff with and without the cholinergic blocker Pirenzipine. (G) Normalized average Ca2+ transients of putative O-LM interneurons responding to airpuff. (H) Comparison of average Ca2+ response peak. (I) Left panel: schematic illustrating the injection of viral components in SO of the right dorsal hippocampus (HPC) in SST-Cre and APP/PS1::SST-Cre mice. RGTVA, rAAV1/2.EF1a.DIO.TVA.IRES.RG.pA; mCh, rAAV1/2.EF1a.DIO.mCherry.pA; eGFP, RABVΔG-EGFP(EnvA). Right panel: mono-transsynaptic retrograde tracing of O-LM inputs (green, eGFP) in the medial septum diagonal band (MSDB) of SST-Cre and APP/PS1 mice. Counterstaining with the nuclear marker DAPI (blue). (J and K) Density of mono-transsynaptically connected eGFP+ neurons (J) and fibers (K) in MSDB normalized to the number of O-LM source neurons after retrograde tracing in wild-type and APP/PS1 mice. n = 8 trials (B) and n = 16 trials in 2 neurons (C). n = 76 neurons before pharmacology (black) and 75 neurons after pharmacology (dark blue) in 3 wild-type mice; n = 46 neurons before pharmacology (gray) and 55 neurons after pharmacology (light blue) in 3 APP/PS1 mice (H). n = 3 mice per group and n = 5–7 slices per animal (J and K). All data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01. Unpaired t test (C, J, and K) and Kruskal-Wallis test with Dunn’s multiple comparisons test (H). Age of animals at start of experiment was as follows: 11–13 (D–H) and 8–10 months (I–K). Scale bars, 20 (E) and 200 μm (I). Neuron 2016 92, 114-125DOI: (10.1016/j.neuron.2016.08.034) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Spine Gain on O-LM Interneurons Requires Cholinergic Input (A) Schematic illustration of the experimental outline for (B)–(D). (B) Representation of an O-LM interneuron expressing tdTomato with application of Pirenzipine and NBQX through a small hole in the cranial window. (C) Exemplary pictures of a putative O-LM interneuron dendrite in a wild-type mouse during BL and L&M. Arrows indicate stable (yellow), gained (turquoise), and lost (red) spines over a 4-day interval. (D) Normalized density of gained spines comparing wild-type mice treated with vehicle, Pirenzepine, or NBQX. (E) Freezing response after bilateral infusion of vehicle, Pirenzepine, or NBQX prior to memory acquisition. (F) Bilateral expression of hM4D(Gi)-mCherry (DREADD) in SST-positive interneurons in SO of the dorsal hippocampus. (G) Schematic illustration of experimental outline for pharmacogenetic inhibition of O-LM interneurons in hippocampus. (H) Freezing rates of SST-Cre mice either untreated (control) or hM4D(Gi)-mCherry injected (DREADD) in SO of dorsal hippocampus inactivating O-LM interneurons with CNO during training. (I) Illustration of an implanted double guide cannula. (J) Experimental timeline for bilateral infusion of Cevimeline into the hippocampus prior to fear conditioning. (K) Freezing response during memory recall in wild-type mice treated with vehicle as well as APP/PS1 mice treated with vehicle or Cevimeline. n = 37 dendrites in 6 mice (vehicle), n = 25 dendrites in 4 mice (Pirenzepine), and n = 29 dendrites in 5 mice (NBQX) (D); n = 6 vehicle-, 7 Pirenzepine-, and 6 NBQX-treated mice (E); n = 4 control and 3 DREADD mice (H); and n = 4 wild-type vehicle-, 5 APP/PS1 vehicle-, and 7 APP/PS1 Cevimeline-treated mice (K). All data are represented as mean ± SEM, n.s. p > 0.05, ∗p < 0.05. Kruskal-Wallis test with Dunn’s multiple comparisons test (D), one-way ANOVA with Sidak’s multiple comparisons test (E and K), and unpaired t test (H). Age of mice at start of experiments was as follows: 9–14 (C and D), 5–7 (E), 2–5 (F–H), and 9–12 months (I–K). Scale bar, 2 (C), 500, and 100 μm (F). Neuron 2016 92, 114-125DOI: (10.1016/j.neuron.2016.08.034) Copyright © 2016 Elsevier Inc. Terms and Conditions