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Research ArticleNew Research, Cognition and Behavior

Activation of a Habenulo–Raphe Circuit Is Critical for the Behavioral and Neurochemical Consequences of Uncontrollable Stress in the Male Rat

Samuel D. Dolzani, Michael V. Baratta, Jose Amat, Kara L. Agster, Michael P. Saddoris, Linda R. Watkins and Steven F. Maier
eNeuro 6 October 2016, 3 (5) ENEURO.0229-16.2016; DOI: https://doi.org/10.1523/ENEURO.0229-16.2016
Samuel D. Dolzani
1Department of Psychology and Neuroscience and the Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado 80309
2Institute for Behavioral Genetics, University of Colorado Boulder, Boulder, Colorado 80309
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Michael V. Baratta
1Department of Psychology and Neuroscience and the Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado 80309
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Jose Amat
1Department of Psychology and Neuroscience and the Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado 80309
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Kara L. Agster
1Department of Psychology and Neuroscience and the Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado 80309
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Michael P. Saddoris
1Department of Psychology and Neuroscience and the Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado 80309
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Linda R. Watkins
1Department of Psychology and Neuroscience and the Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado 80309
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Steven F. Maier
1Department of Psychology and Neuroscience and the Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado 80309
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Article Figures & Data

Figures

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  • Figure 1.
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    Figure 1.

    Optical inhibition of the LHb during IS prevents later anxiety-like behavior. A, Top left, Changes in LHb firing rate in response to continuous (10 s) green light illumination of LHb in rats expressing NpHR. Middle, Raster plot and perievent histogram showing the firing rate of a representative LHb neuron in a rat expressing NpHR in the LHb (bin size, 100 ms). Green bar represents continuous laser illumination. Bottom, The z-normalized firing rate of all valid neurons recorded during green light illumination of LHb (n = 36), B, Coronal micrographs demonstrating NpHR expression in the LHb in a rat that received IS plus optical silencing (IS+NpHR). Top left, Bilateral NpHR 3.0-eYFP expression (4× mosaic; scale bar, 1000 μm) and (top right) unilateral NpHR3.0-eYFP expression (inverted arrow indicates tissue damage from optical fiber implant (10×; scale bar, 200 μm). Middle, JSI data expressed as the percentage of baseline for rats previously injected with either eYFP or NpHR and later exposed to IS or HC treatment with green light illumination of the LHb (n = 10-11/group). Bottom, Diagram indicating the location of optical fiber tips in the LHb of rats that received IS plus NpHR 3.0 silencing (green dots) or IS plus eYFP (black dots). Position relative to bregma is denoted above individual images. Hipp, Hippocampus.

  • Figure 2.
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    Figure 2.

    Optical inhibition of the LHb during IS prevents the neurochemical consequences of the stressor. A, Rats were injected with NpHR or eYFP, and implanted with LHb optical fibers and BLA microdialysis probes 3 weeks later. B, Left, BLA 5-HT levels measured during IS in rats that received IS plus NpHR or IS plus eYFP with green light delivery during tailshocks. Right, Area under the curve for BLA 5-HT levels measured during IS (arbitrary units, relative to average of baseline BLA 5-HT samples). C, Left, BLA 5-HT measured during JSI in rats that previously received IS plus NpHR or IS plus eYFP with green light delivery during tailshocks. Right, Area under the curve for BLA 5-HT measured during JSI (arbitrary units, relative to average of baseline BLA 5-HT samples). D, Left, JSI data for all rats that received IS plus NpHR or IS plus eYFP (n = 7-8/group). Middle, Correlation between BLA 5-HT levels measured during JSI and social investigation. Right, Schematic of BLA microdialysis probes for all rats included in statistical analysis. Position relative to bregma is denoted.

  • Figure 3.
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    Figure 3.

    Stress, per se, increases Fos-IR in the LHbM. A, Photomicrographs of Fos-IR in the habenula from rats that were killed 2 h after receiving HC (top), IS (middle), or ES (bottom). Images are shown at 10× (left), 20× (middle), and 40× (right) magnification. Scale bars: left, 200 μm; middle, 100 μm; and right, 50 μm. B, Fos-IR was examined in the habenula at 0, 2, and 4 h following HC, IS, or ES (left to right). n = 8/group, two habenula sections/rat at all time points.

  • Figure 4.
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    Figure 4.

    Stress, per se, increases Fos-IR in the LHb–DRN pathway. A, Schematic showing FG injected directly into the DRN prior HC, IS, or ES (left). Photomicrographs show the site of injection at 4× (top) and 10× (bottom) magnification. Scale bars, 200 μm. Fluoro-Gold traveled retrogradely to the LHb prior to rats receiving HC, IS, or ES (right). B, Two weeks after Fluoro-Gold injection, rats received HC, IS, or ES and were killed 2 h later. Total FG (left), Fos-IR (middle), and the percentage of FG cells also expressing Fos (FG+Fos; right) were quantified in the MHb, LHbM, and LHbL (top). The total number of FG-labeled cells in each habenula subregion (FG+Fos) did not differ across treatment groups (left). Total Fos-IR was increased in the LHbM and LHbL of rats that received either IS or ES (middle). Similarly, activation of the LHb–DRN pathway (% FG+Fos) was increased in rats that received either IS or ES (right). Photomicrographs (bottom) of representative LHbM tissue samples are shown for rats that received HC (top), IS (middle), or ES (bottom). Images in the first three panels are taken at 20× (scale bar, 50 um) and magnified to 40× at the far right (scale bar, 50 μm). FG reliably labeled cell bodies in the LHbM 2 weeks after injection (left). Increased Fos-IR is evident in rats that received IS or ES, relative to HC (middle). IS and ES also increased LHb–DRN pathway activation relative to HC, as measured by the colocalization of FG and Fos-IR (right). Arrows indicate colocalized FG and Fos-IR (magenta spot). N = 12/group, two habenula sections/rat.

Tables

  • Figures
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    Table 1.

    Statistical analyses performed in all experiments

    FigureData structureType of test/comparisonsp Value
    1ASee below
    1BNormal distributionTwo-way ANOVA (stress treatment × virus type)Main effect of virus type: p = 0.0044
    Main effect of stress treatment: p < 0.0001
    Sidak’s multiple-comparisons testInteraction: p < 0.0001
    Home cage–inescapable Stress95% CI:
    eYFP30.24–58.97
    NpHR−7.976 to 20.10
    2BNormal distributionTwo-way repeated-measures ANOVAMain effect virus type: p = 0.0007
    Sidak’s multiple comparisons testMain effect time: p < 0.0001
    eYFP – NpHRInteraction: p = 0.0002
    BL195% CI:
    BL2−43.11 to 65.40
    BL3−59.67 to 48.84
    BL4−47.73 to 60.78
    S1−66.85 to 41.66
    S233.82–142.3
    S3−9.440 to 99.07
    S47.267–115.8
    S51.613–110.1
    PS10.8602–109.4
    PS2−5.678 to 102.8
    PS313.43–121.9
    PS42.628–111.1
    Independent samples t test (AUC)0.5271–109.0
    T, dfp = 0.0023
    t = 3.859 df = 12.00
    2CNormal distributionIndependent-samples t test for baseline JSI 5-HT valuesMaine effect virus type: p = 0.0029
    Two-way repeated-measures ANOVAMain effect time: p < 0.0001
    Sidak’s multiple-comparisons testInteraction: p < 0.0001
    eYFP – NpHR95% CI:
    BL1−31.60 to 60.99
    BL2−32.35 to 60.23
    BL3−67.83 to 24.76
    BL4−41.90 to 50.69
    JSI78.49–171.1
    PI1−9.353 to 83.23
    PI2−34.55 to 58.03
    PI3−25.61 to 66.98
    Independent-samples t test (AUC)p = 0.0102
    T, dft = 3.580 df = 6.479
    2DNormal distributionIndependent-samples t testp < 0.0001
    T, dft = 6.344 df = 9.716
    2DNormal distributionLinear regressionp = 0.0125
    R2 = 0.3921
    R = −0.6257
    T, dft = 6.344 df = 9.716
    3Normal distributionOrdinary one-way ANOVAp = 0.0065
    Tukey’s post hoc test95% CI
    MHb Fos 0 h0.03556 to 2.589
    HC vs ES0.4731 to 3.027
    HC vs IS-0.8394 to 1.714
    ES vs ISp < 0.0001
    LHbM Fos 0 h−37.59 to −19.54
    HC vs ES−34.15 to −16.10
    HC vs IS−5.587 to 12.46
    ES vs ISp = 0.1416
    LHbL Fos 0 hp = 0.3300
    MHb Fos 2 hp = 0.0013
    LHbM Fos 2 h95% CI:
    HC vs ES−38.21 to −8.789
    HC vs IS−34.40 to −4.977
    ES vs IS−10.90 to 18.52
    LHbL Fos 2 hp = 0.1888
    MHb Fos 4 hp = 0.8780
    LHbM Fos 4 hp = 0.8652
    LHbL Fos 4 hp = 0.0348
    HC vs ES95% CI:
    HC vs IS−3.706 to 0.4562
    ES vs IS−4.331 to −0.1688
    −2.706 to 1.456
    4Normal distributionOrdinary one-way ANOVA95% CI:
    Tukey’s post hoc testp = 0.6651
    Total FG MHbp = 0.7163
    Total FG LHbMp = 0.5220
    Total FG LHbLp = 0.9074
    Total Fos-IR MHbp < 0.0001
    Total Fos-IR LHbM95% CI:
    HC vs ES−16.94 to −6.726
    HC vs IS−18.73 to −8.517
    ES vs IS−6.899 to 3.316
    Total Fos-IR LHbLp = 0.0109
    HC vs ES95% CI:
    HC vs IS−8.395 to −1.022
    ES vs IS−7.020 to 0.3534
    Total % double-label MHb−2.312 to 5.062
    Total % double-label LHbMp = 0.4152
    HC vs ESp < 0.0001
    HC vs IS95% CI:
    ES vs IS−21.49 to −7.661
    Total % double-label LHbL−18.44 to −4.614
    −3.866 to 9.960
    p = 0.0764
    • View popup
    Table 2.

    Electrophysiology statistics for all recorded neurons

    Cell firing rate (Hz)ANOVABL vs laserBL vs postLaser vs post
    31.050.68961273n.s.n.s.n.s.
    2.010.1004690150.8917544750.1031188660.239922898
    1.117.62002E-100.573159780.0001240210.000124007
    4.234.98676E-050.0001716310.0170464950.023234465
    22.987.71135E-080.0001494370.1836157440.000150392
    19.4451.55431E-150.0001240060.0001874330.000124006
    18.463.07286E-090.0001240070.0144282780.000139218
    19.128.43769E-150.0001240060.0003558660.000124006
    1.634.95913E-110.0001240140.1062837360.000124006
    6.03500.0001240060.36400620.000124006
    8.49500.0001240060.0001240060.000124006
    7.312.77556E-1400.0001240070
    2.3159.95919E-080.0007599050.0121109370.000124017
    2.0051.39015E-050.0001336810.2984437360.001375559
    1.9750.0001559280.0003591920.7682121690.002040156
    6.20500.0001243310.5864627260.308035137
    1.4053.94043E-090.0007919130.0006309680.000124007
    3.65500.0001240060.2170181080.000124006
    8.67500.0001240060.3154967190.000124006
    4.7351.11022E-160.0001240060.0002021960.000124006
    2.05789473700.0001267570.7225589180.000126757
    4.1631578951.0224E-080.000142950.0341343560.000126757
    9.1473684212.22045E-160.0001267570.000126860.000126757
    2.191.82944E-080.0001241830.8973613660.000124035
    3.5852.02568E-100.0001241830.8973613660.000135351
    2.3400.0001240060.0001240060.000124006
    2.437.2915E-120.0001240070.0001240060.107882478
    16.191.08128E-050.0001551380.2002738490.000538695
    8.6456.02274E-120.0001240060.0043294250.000124034
    24.947.95227E-080.000124060.5121408550.000127955
    20.8951.20682E-100.0001240060.0003224190.000161763
    1.230.0004373880.0019321980.9904240630.001355133
    2.785.77942E-060.001763290.1357446770.00012667
    7.1100.0001240060.685207930.000124006
    1.1852.26193E-100.1675937620.0001240350.000124006
    2.3421052637.6863E-100.0001269570.1040592730.000126757
    • View popup
    Table 3.

    The z-normalized firing rate for all recorded neurons

    BLLaserRecovery
    −9.21485E-17−0.068442491−0.034221245
    −9.10383E-170.0195841640.090087152
    −1.04812E-16−0.0387792310.268952733
    −9.99201E-18−0.455220728−0.233230373
    −5.96745E-17−1.024094554−0.207156056
    −0.132067429−0.919189306−0.410609643
    −0.102381307−0.789798657−0.357359516
    −0.100727222−0.850484148−0.342746023
    −0.015070323−0.2624747970.04521097
    −0.047670764−0.632476897−0.09332727
    −0.0342648110.0272956970.554625337
    −0.077528279−0.439972986−0.200927458
    −0.009444506−0.119630410.073457269
    −0.019688117−0.189932426−0.068329348
    −0.015790119−0.205271549−0.078950596
    −3.94129E-17−0.6150021660.245870431
    1.02141E-16−0.1905539670.194579755
    3.33067E-17−0.5448640810.05771146
    −6.55032E-17−0.690738882−0.054442473
    −1.73195E-16−0.3951966010.187793805
    −0.017634339−0.285847671−0.003923753
    −0.032238634−0.2328028810.067656299
    −0.051458356−0.3670478920.193812657
    1.11022E−17−0.2012575410.013916745
    −7.54952E-17−0.1448959390.198278654
    −0.0315278330.631607590.265884726
    4.55191E-170.3415493350.432560984
    −1.17684E-16−0.588112809−0.160488531
    −0.073112074−0.45375906−0.18858273
    −0.067924124−0.424629951−0.145849345
    −0.094099006−0.442713419−0.259444403
    −0.021839826−0.123759015−0.004367965
    1.29341E-16−0.1376448870.072012888
    8.46545E-17−0.3915547280.022322843
    −1.9984E-17−0.0520935190.193324838
    −0.024702377−0.2451363950.047017782
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Activation of a Habenulo–Raphe Circuit Is Critical for the Behavioral and Neurochemical Consequences of Uncontrollable Stress in the Male Rat
Samuel D. Dolzani, Michael V. Baratta, Jose Amat, Kara L. Agster, Michael P. Saddoris, Linda R. Watkins, Steven F. Maier
eNeuro 6 October 2016, 3 (5) ENEURO.0229-16.2016; DOI: 10.1523/ENEURO.0229-16.2016

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Activation of a Habenulo–Raphe Circuit Is Critical for the Behavioral and Neurochemical Consequences of Uncontrollable Stress in the Male Rat
Samuel D. Dolzani, Michael V. Baratta, Jose Amat, Kara L. Agster, Michael P. Saddoris, Linda R. Watkins, Steven F. Maier
eNeuro 6 October 2016, 3 (5) ENEURO.0229-16.2016; DOI: 10.1523/ENEURO.0229-16.2016
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Keywords

  • amygdala
  • habenula
  • optogenetics
  • raphe
  • serotonin
  • stress

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