1. Synaptic Plasticity onto Dopamine Neurons Shapes Fear Learning
Marco Pignatelli, George Kwabena Essien Umanah, Sissi Palma Ribeiro, Rong Chen, Senthilkumar Senthil Karuppagounder, Hau-Jie Yau, Stephen Eacker, Valina Lynn Dawson, Ted Murray Dawson, Antonello Bonci 
Neuron 
Volume 93, Issue 2, Pages (January 2017)
DOI: /j.neuron
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2. Figure 1
Stress-Induced Synaptic Plasticity in the VTA Affects the Associative Strength of a Subsequent Fear Conditioning
(A) Experimental timeline of the stress-enhanced fear learning.
(B) Percentage of freezing during fear conditioning training (day 2) during the first 2 min of the acquisition before delivery of the auditory cue, 30 s cue presentation, 2 s shock presentation, and 45 s of post-shock reactivity.
(C) Percentage of freezing during 5 min of context-induced fear conditioning (day 3). Two-tailed Student’s t test: ∗p < 0.05.
(D) Percentage of freezing during the first 2 min of acclimation period (baseline) in context B and during 3 min of the auditory cue presentation (day 4). Two-way ANOVA: ∗p < (In B–D: n = 8–10 mice per group.)
(E) Percentage of freezing during fear conditioning training (day 2) during the first 2 min of the acquisition before delivery of the auditory cue, 30 s cue presentation, 2 s shock presentation, and 45 s of post-shock reactivity.
(F) Percentage of freezing during 5 min context-induced fear conditioning (day 3). One-way ANOVA: ∗p < 0.05.
(G) Percentage of freezing during the first 2 min of acclimation period (baseline) in context B and during 3 min of the auditory cue presentation (day 4). Two-way ANOVA: ∗p < Blue shading indicates the period of cue presentation, and red vertical bar indicates the period of foot shock presentation.
(H) Left: average AMPAR/NMDAR ratio 24 hr after intra-VTA infusions of either saline (n = 6 cells; 5 animals) or APV (n = 7 cells; 4 animals). Two-tailed Student’s t test: ∗p < Right: representative traces of AMPAR/NMDAR ratio. Values represent the mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2
Conditional Deletion of Thorase from DAT+ Neurons Does Not Affect DA Neuron Cell Density or the Relative Metabolic State
(A) Simplified breeding scheme of the conditional DATIREScre/wt;Thoraseflox/+ (cHet or control group) or DATIREScre/wt;Thoraseflox/flox (cKO or experimental group).
(B) Representative images of double immunofluorescence staining for tyrosine hydroxylase (TH, green) and Thorase (red). Cell nuclei are stained with DAPI (blue). The images of SNpc and VTA are shown for both cHet and cKO mice.
(C) Quantification of TH cells expressing Thorase. Percentage of TH cells that co-localized with Thorase was assessed in the SNpc and VTA. The values represent the mean ± SEM (n = 3, ∗∗∗p < 0.001, two-way ANOVA, Holm-Sidak multiple comparisons tests).
(D) TH-positive neurons were counted in the SNpc of Thorase cHet and cKO mice by unbiased stereological counting.
(E–G) Representative images of TH immunohistochemistry from cHet and cKO mice. Stereological quantitation of TH- and Nissl-positive neurons (E) in the SNpc (n = 4 mice per group). High-performance liquid chromatography-electrochemical quantitation of striatal DA, 5HT, and NE (F) and dopamine metabolites dihydroxyphenylacetic acid (DOPAC), Homovanillic acid (HVA), and 5-HIAA levels (G) in Thorase cHet or cKO mice. Error bars represent the mean ± SEM, n = 4 mice per group. (One-way ANOVA, p > 0.05.)
Neuron  , DOI: ( /j.neuron )
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4. Figure 3
Increased AMPAR Surface Expression in Mice with Conditional Deletion of Thorase from DA Neurons
(A) Representative image of TH-labeled and DAB-stained ventral mesencephalon of mouse brain taken with laser capture microdissection (LCM).
(B) Left: representative images of a TH-stained single neuron before LCM and neurons in SNpc and VTA. Cells ready for excision and capture are outlined in yellow. Right: the same sections as the left panel after cells were dissected out with the laser.
(C) Representative images of immunoblots of LCM samples after the BS3-crosslinking assay to assess surface and total protein expression in cHet and cKO brain sections. Actin, TH, and DAT serve as loading controls.
(D) Quantification of relative proteins levels in samples not treated with BS3. Signal intensities normalized to actin signal.
(E) Quantification of percentage surface protein (indicated by “s-”) levels in samples treated with BS3.
(F) Quantification of AMPAR subunit composition in cHet versus cKO mice.
(G) Quantification of relative phosphorylation state of GluR2. The experiments were repeated three times with similar results. The optical densitometry quantification values represent the mean ± SEM (n = 3, ∗∗p < 0.005, ∗p < 0.05, n.s. p > 0.05, two-way ANOVA, Holm-Sidak multiple comparisons tests).
Neuron  , DOI: ( /j.neuron )
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5. Figure 4
Selective Increase in AMPAR-Mediated Synaptic Transmission in Mice with Conditional Deletion of Thorase from DA Neurons
(A and B) Left: cumulative probability plots of the amplitudes (A) or frequencies (B) of mEPSCs recorded from putative VTA dopamine neurons in brain slices from cHet (n = 7 cells; 7 animals) and cKO (n = 8 cells; 8 animals) mice. Right: histograms of the means obtained from mEPSC amplitude (A) or frequency (B). Kolmogorov-Smirnov test: p < 0.01; two-tailed Student’s t test: ∗p < 0.05.
(C and D) Mean mEPSC parameters: rise (C) and decay (D) times.
(E and F) Left: cumulative probability plots of the amplitudes (E) or frequencies (F) of mIPSCs recorded from putative VTA dopamine neurons in brain slices from cHet (n = 6 cells; 6 animals) and cKO (n = 5 cells; 5 animals) mice. Right: histograms of the means obtained from mIPSC amplitude (E) and frequency (F). Kolmogorov-Smirnov test: p > 0.05; two-tailed Student’s t test: p > 0.05.
(G and H) Mean mIPSC parameters: rise (G) and decay (H) times.
(I and J) Left: paired-pulse ratios obtained at various inter-pulse intervals (20–200 ms) of electrically evoked EPSCs (Vh = −70 mV) (I) and IPSCs (Vh = 0 mV) (J) (n = 6–7 per group; 5–6 animals per group). Right: representative traces of electrically evoked EPSCs (I) and IPSCs (J) evoked by paired-pulse stimulation.
(K) Schematic of the protocol used to measure excitation-inhibition ratio.
(L) Left: histogram of the means of excitation-inhibition ratio from cHet (n = 9 cells; 6 animals) and cKO (n = 11 cells; 9 animals) mice. Right: representative traces of electrically evoked AMPAR- and GABAR-mediated currents. Two-tailed Student’s t test: ∗∗p < Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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6. Figure 5
Thorase Deficiency Increases AMPAR/NMDAR Ratio by Enhancing AMPAR- over NMDA-Mediated Currents
(A) Left: input-output curves from stimulus-evoked AMPAR EPSCs expressed as a fixed increase over threshold from cHet (n = 9 cells; 8 animals) and cKO (n = 11 cells; 11 animals) mice. Two-way ANOVA: p < 0.01; post hoc tests: ∗∗p < Right: representative traces of input-output curves.
(B) Left: average AMPAR/NMDAR ratio from cHet (n = 6 cells; 5 animals) and cKO (n = 8 cells; 6 animals) mice. Two-tailed Student’s t test: ∗∗∗p < Right: representative traces of AMPAR/NMDAR ratio.
(C) AMPAR rectification indices from cHet (n = 5 cells; 4 animals) and cKO (n = 6 cells; 4 animals) mice. Two-tailed Student’s t test: p > 0.05.
(D and E) Left: average I-V plots of AMPAR-mediated (D) and NMDAR-mediated (E) currents from cHet (n = 6–8 cells; 5 animals) and cKO (n = 6–7 cells; 5 animals) mice. Right: representative traces of I-V plot of AMPA (D) and NMDA (E) EPSCs. Two-way ANOVA: p > 0.05.
(F) Decay time for NMDAR EPSCs from cHet (n = 11 cells; 7 animals) and cKO (n = 8 cells; 6 animals) mice. Two-tailed Student’s t test: p > Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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7. Figure 6
Impaired LTD and LTP at Glutamatergic Synapses onto DA Neurons in Mice with Conditional Deletion of Thorase
(A) Left: LTD induced with three consecutive low-frequency stimulation (LFS) pairing protocols in putative dopamine neurons from cHet (n = 7 cells; 6 animals) and cKO (n = 6 cells; 6 animals) mice. Right: representative traces of LTD experiments.
(B) Left: LTP-induced by spike-timing-dependent plasticity (STDP) in putative dopamine neurons from cHet (n = 6 cells; 6 animals) and cKO (n = 8 cells; 7 animals) mice. Right: representative traces of LTP experiments.
(C) Left: time course of EPSC amplitudes showing the effect of NASPM (100 μM) for 10 min from cHet (n = 5 cells; 5 animals) and cKO (n = 7 cells; 5 animals) mice. Right: representative traces of NASPM experiments.
(D) Left: effect of NASPM on expression of LTP in cHet (n = 6 cells; 6 animals) and cKO (n = 5 cells; 5 animals). Right: representative traces of LTP-NASPM experiments.
(E) Summary of magnitude of LTD and LTP during the last 5 min of recordings of experiments shown in (A) and (B). Two-tailed Student’s t test: ∗∗∗p < for LTP; ∗∗p < 0.01 for LTD.
(F) Scheme of Thorase-mediated AMPAR trafficking in the context of LTP and LTD in putative dopamine neurons (see text for further details). Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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8. Figure 7
Conditional Deletion of Thorase from DA Neurons Affects Associative Fear Learning
(A) Top: schematic drawing of fear conditioning training (day 1). Bottom: percentage of freezing during fear conditioning training (day 1). Blue shading indicates the period of cue presentation, and red vertical bar indicates the period of foot shock presentation.
(B) Top: schematic drawing of context-induced fear conditioning (day 2). Bottom: percentage of freezing during context-induced fear conditioning (day 2). Two-tailed Student’s t test: ∗p < 0.05.
(C) Top: schematic drawing of cued-induced fear conditioning (days 3–15). Bottom: percentage of freezing during the first 2 min of acclimation period (baseline) in context B and during presentation of the cue (days 3–15). Two-way ANOVA: p < 0.01; post hoc tests: ∗∗∗p < (In A–C: n = 12–13 mice per group.)
(D) Top: representative traces of AMPAR/NMDAR ratio. Bottom: average AMPAR/NMDAR ratio collected from cHet and cKO 24 hr after exposure to the cue (n = 6 cells; 5 animals) and from cHet and cKO exposed to cue-shock pairing (n = 7 cells; 5 animals). Two-way ANOVA: ∗p < 0.05.
(E and F) Top: schematic drawings of first-order fear conditioning training (E: day 1, F: day 2). Bottom: percentage of freezing during first-order fear conditioning training (E: day 1, F: day 2). Blue shadings indicate the period of cue presentation, and red vertical bars indicate the period of foot shock presentation.
(G) Top: schematic drawing of CS1-CS2 pairings of second-order fear conditioning (day 3). Bottom: percentage of freezing during CS1-CS2 pairings of second-order fear conditioning. Blue and yellow shadings indicate the period of CS1 (white noise) and CS2 (cue light) presentations, respectively.
(H) Top: schematic drawing of the test day for second-order fear conditioning (day 4). Bottom: percentage of freezing during test day. Two-tailed Student’s t test: ∗p < Yellow shading indicates the period of cue light presentation. (In E–H: n = 10–11 mice per group.) Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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9. Figure 8
Cell-type-Specific Restoration of Thorase Rescue Electrophysiological and Behavioral Phenotypes
(A) Schematic drawing of viral stereotaxic injection of AAV1-CAG-DIO-Thorase-eGFP or AAV1-CAG-DIO-eGFP into VTA/SNpc of ThoraseDAKO mice.
(B) Immunofluorescence for TH (white), eGFP (green), and DAPI (blue) in midbrain coronal sections from ThoraseDAKO mice that receive either AAV1-CAG-DIO-Thorase-eGFP or AAV1-CAG-DIO-eGFP into VTA/SNpc (scale bar, 100 μm).
(C) Fluorescence and differential interference contrast (DIC) images of eGFP+ cell selected for recording from ex vivo midbrain section of ThoraseDAKO with either AAV1-CAG-DIO-Thorase-eGFP or AAV1-CAG-DIO-eGFP (scale bar, 20 μm).
(D) Left: average AMPAR/NMDAR ratio from eGFP (n = 6 cells; 5 animals) and Thorase-eGFP (n = 7 cells; 5 animals). Two-tailed Student’s t test: ∗p < Right: representative traces of AMPAR/NMDAR ratio.
(E) Decay time for NMDAR-EPSCs from eGFP (n = 6 cells; 5 animals) and Thorase-eGFP (n = 7 cells; 5 animals). Two-tailed Student’s t test: p > 0.05.
(F) Left: paired-pulse ratios obtained at various inter-pulse intervals (20–200 ms) of electrically evoked EPSCs (Vh = −70 mV) (n = 6 per group; 5 animals per group). Right: representative traces of electrically evoked EPSCs.
(G and H) Cumulative probability plots of the amplitudes (G) or frequencies (H) of mEPSCs recorded from putative VTA dopamine neurons in brain slices from eGFP (n = 6 cells; 4 animals) and Thorase eGFP (n = 7 cells; 4 animals) mice. Inset: histograms of the means obtained from mEPSC amplitude (G) or frequency (H). Kolmogorov-Smirnov test: p < 0.01; two-tailed Student’s t test: ∗p < 0.05.
(I and J) mEPSC parameters. Mean mEPSC rise (I) and decay (J) times from eGFP (n = 6 cells; 4 animals) and Thorase-eGFP (n = 7 cells; 4 animals) Two-tailed Student’s t test: p > 0.05.
(K) Percentage of freezing during fear conditioning training (day 1) during the first 2 min of the acquisition before delivery of the auditory cue, 30 s cue presentation, 2 s shock presentation, and 45 s of post-shock reactivity. Blue shading indicates the period of cue presentation, and red vertical bar indicates the period of foot shock presentation.
(L) Percentage of freezing during 5 min of context-induced fear conditioning (day 2). Two-tailed Student’s t test: ∗p < 0.05.
(M) Percentage of freezing during the first 2 min of acclimation period in context B and during 3 min of the auditory cue presentation (day 3). Blue shading indicates the period of cue presentation. Two-way ANOVA: p < 0.01; post hoc tests: ∗∗p < (In K–M: n = 8–10 mice per group). Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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