Projection-Target-Defined Effects of Orexin and Dynorphin on VTA Dopamine Neurons  Corey Baimel, Benjamin K. Lau, Min Qiao, Stephanie L. Borgland  Cell Reports  Volume 18, Issue 6, Pages 1346-1355 (February 2017) DOI: 10.1016/j.celrep.2017.01.030 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 18, 1346-1355DOI: (10.1016/j.celrep.2017.01.030) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 DA-lAcbSh, DA-mAcbSh, and DA-BLA VTA Neurons Are Largely Non-overlapping Cell Populations (A) Schematic and examples of RetroBead injection sites in the lAcbSh (purple), mAcbSh (orange), and BLA (cyan) of adult C57BL/6J mice. (B) 20× images of VTA coronal sections with TH-positive neurons in gray and retrograde-labeled neurons from the lAcbSh and mAcbSh (i), the mAcbSh and BLA (ii), and the lAcbSh and BLA (iii). Scale bar, 100 μm. (C) Distribution of normalized retrograde fluorescence across the medial to lateral, dorsal to ventral, and rostral to caudal borders of the VTA. (D) The number of labeled and co-labeled neurons in the VTA from paired RetroBead injections. (E) Summary of the percentage of overlap with TH. Line and shading represents mean ± SEM. Error bars represent median and range with circles for each individual data point. Cell Reports 2017 18, 1346-1355DOI: (10.1016/j.celrep.2017.01.030) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 DA-lAcbSh, DA-mAcbSh, and DA-BLA VTA Neurons Have Different Intrinsic Electrophysiological Properties (A) 60× image of a biocytin-labeled (gray), GFP-positive (green), retrograde-labeled (purple) neuron from the lAcbSh. Scale bars, 10 μm. (B) Summary of capacitance and input resistance. (C) Sample traces (i) and current-voltage relationship (ii) showing increased activation of Ih in DA-lAcbSh neurons relative to DA-mAcbSh (orange) and DA-BLA (cyan) neurons. Symbols represent mean ± SEM. Transparent colored lines behind the averaged curves represent individual cells. Peak Ih current at a hyperpolarizing step to −130 mV (iii). Summary of leak conductance activation at −130 mV (iv). (D) Sample traces and summary data of spontaneous action potential firing. (E) Sample traces of action potentials evoked with 50, 100, and 150 pA (light to dark) depolarizing current steps and action potential number/current step plot demonstrating increased excitability of DA-mAcbSh and DA-BLA neurons compared to DA-lAcbSh neurons. Error bars represent median ± range. Symbols represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Cell Reports 2017 18, 1346-1355DOI: (10.1016/j.celrep.2017.01.030) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 OxA Preferentially Activates NAc-Projecting VTA Dopamine Neurons (A–C) Sample recordings of current-step-induced firing before and after application of 100 nM oxA (i). Time course of evoked firing with bath application of 100 nM oxA (ii). 100 nM oxA increased firing of DA-lAcbSh and DA-mAcbSh, but not DA-BLA, neurons (iii). Summary of the percentage of responding neurons in each population (iv). (D–F) Example traces of action potentials evoked with depolarizing current steps of 100, 200, and 300 pA (light to dark) for DA-lAcbSh or 25, 50, and 100 pA (light to dark) for DA-mAcbSh and DA-BLA neurons before and after application of 100 nM oxA (i). OxA increased the number of action potentials per current step in DA-lAcbSh and DA-mAcbSh, but not DA-BLA, neurons (ii). Error bars represent median ± range, symbols represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Cell Reports 2017 18, 1346-1355DOI: (10.1016/j.celrep.2017.01.030) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Dyn Inhibits Firing in Subpopulations of DA-lAcbSh, DA-mAcbSh, and DA-BLA Neurons (A–C) Sample recordings of current-step-induced firing before and after application of 200 nM dyn in responding and non-responding neurons (i). Time course of evoked firing with bath application of 200 nM dyn in all neurons (gray), responders (light red), and non-responders (dark red) (ii). 200 nM dyn decreased firing in a subset of DA-lAcbSh neurons and in the majority of DA-mAcbSh and DA-BLA neurons (iii). Summary of the percentage of responding neurons in each population (iv). (D–F) Example traces of action potentials evoked with depolarizing current steps of 100, 200, and 300 pA in DA-lAcbSh neurons and 25, 50, and 100 pA in DA-mAcbSh and DA-BLA neurons (i). Dyn decreased the number of action potentials per current step in DA-lAcbSh, DA-mAcbSh, and DA-BLA neurons (ii). Figure shows responders (light red) and non-responders (dark red). Error bars represent median ± range. Symbols represent mean ± SEM. ∗p < 0.05, ∗p < 0.01, ∗∗∗p < 0.001. Cell Reports 2017 18, 1346-1355DOI: (10.1016/j.celrep.2017.01.030) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 OxA and Dyn Have Opposing Actions on First Spike Latency and AHP Area Responders were identified as neurons that had a >15% change in firing rate to oxA or dyn. (A) OxA significantly decreased FSL in DA-lAcbSh (i) and DA-mAcbSh (ii) responding neurons, but not in DA-BLA non-responding neurons (iii). (B) Responders to dyn increased FSL in DA-lAcbSh (i), DA-mAcbSh (ii), and DA-BLA neurons (iii). FSLs in non-responders to dyn were not significantly different in any projection target. (C) OxA decreased AHP area in the responding population of DA-lAcbSh (i) and DA-mAcbSh (ii) neurons, with no change in the non-responders of DA-BLA neurons (iii). (D) AHP area of responders to dyn was increased in DA-mAcbSh neurons (ii), but not in DA-lAcbSh (i) or DA-BLA neurons (iii). In some responders, AHP area could not be determined, as there was only one or no action potential after dyn application. A Wilcoxon matched-pairs signed rank test was used to assess statistical differences in action potential characteristics between baseline (bl) and peptide application. We were unable to assess statistical significance in non-responders of Ai, Aii, Ci, and Cii and responders in Aiii and Ciii due to insufficient N. Error bars represent median ± range, and symbols represent individual data points. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001. Cell Reports 2017 18, 1346-1355DOI: (10.1016/j.celrep.2017.01.030) Copyright © 2017 The Author(s) Terms and Conditions