Dopamine neurons modulate neural encoding and expression of depression-related behaviour methods

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    "name": "Dopamine neurons modulate neural encoding and expression of depression-related behaviour methods",
    "description": "Evidence-backed execution summary for Dopamine neurons modulate neural encoding and expression of depression-related behaviour methods from Dopamine neurons modulate neural encoding and expression of depression-related behaviour.",
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        "position": 1,
        "name": "METHODS",
        "text": "TH::IRES-Cre homozygous male mice ( and ) obtained from the European Mouse Mutant Archive (mouse line EM:00254) were bred with wild-type C57BL/7 females to produce subjects. Only heterozygous TH::IRES-Cre male offspring that had been back-crossed for at least five generations were used for experiments. Animals were age matched, underwent surgery and were divided into two groups: the chronic mild stress (CMS) group and the control non-CMS group. Non-CMS animals were housed in a quiet room with a reverse 12 h light-dark cycle and given food and water ad libitum. CMS animals were housed separately and were subjected to standard CMS protocols; two stressors per day for 8 to 12 weeks before behavioural testing. Mice in this group experienced one stressor during the day and a different stressor during the night. Well-validated and approved standard stressors were randomly chosen from..."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "METHODS",
        "text": "TH::Cre BAC transgenic rats ( ) were bred by mating Cre-positive founders to wild-type rats to produce TH::Cre heterozygous transgenic rats on a Long-Evans background. Only male rats aged 5 to 7 months were used for experiments. All rats underwent a 5- to 7-week chronic mild stress paradigm before behavioural testing and electrophysiological recording. Animals were subjected to the same twice-daily stressors on the same time course, except crowded housing and individual housing were both replaced with a standard restraint tube stressor in which the animal is immobilized in a cylindrical plastic tube (Braintree Scientific) for 30 to 45 min. All rats were singly housed with a 12 h standard light-dark cycle and when not undergoing water deprivation, food deprivation or restraint stress, water and food were available ad libitum. Experimental protocols were approved by Stanford Univ..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Stereotactic injection into VTA, and cannula and fibre implantation",
        "text": "Mice in and were aged 2 to 4 months. These mice were deeply anaesthetized with isofluourane and placed into a stereotactic apparatus. The head was levelled using Bregma and Lambda reference points and a craniotomy was preformed. Virus was injected at 2 sites in the VTA of the right hemisphere using a 33-gauge metal needle and a 10-µl syringe controlled by an injection pump at 0.1 µl min -1 for 2 µl: (1 µl at anterior-posterior, -3.3; lateral-medial, 0.5; dorsal-ventral, -4.1; and 1 µl at dorsal-ventral, -4.6). The needle was left in place after injection for 10 min before slowly being withdrawn. For all mice, an implantable fibre-optic light guide (IFL), consisting of a metal ferrule, 2.5 mm in diameter with a 200-mm thick, 5-mm long cleaved bare optic fibre (Doric Lenses) was implanted at anterior-posterio..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Blue and yellow light delivery and protocols",
        "text": "A 200-µm patch cord was connected to the external portion of the chronically implantable optical fibre with a zirconia sleeve. Optic fibres were attached through an FC/PC adaptor to a 473-nm blue laser diode (no. BCL-473-050-M), and light pulses were generated through a stimulator (no. 33220A). For rats and mice expressing ChR2 and their eYFP controls, the light paradigm was 8 light pulses at 30 Hz every 5 s. Light-on epochs were 3 min for all assays (TST, OFT and FST) other than for anhedonia, for which the light epoch was 30 min. Optical-fibre light power from the patch cord was measured using a light sensor (S130A) and intensity calculated using a model based on empirical measurements from mammalian brain tissue for predicting irradiance values ( http://www.stanford.edu/group/dlab/cgi-bin/graph/chart.php ). For ChR2-transduced mice and controls, estimated light intensity at 0...."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Pharmacological infusion of glutamate and dopamine receptor antagonists",
        "text": "For pharmacological experiments, drug or saline were infused in a volume of 0.4 µl through a 26-gauge stainless steel double internal cannula (PlasticsOne) that was 0.5 mm longer than the guide cannula. Each graph in depicts a within-subjects matched comparison, counter-balanced for treatment order. The internal cannula was connected to a microsyringe pump by a PE20 tube. Solutions were administered at a constant rate of 100 nl min -1, and the injection cannula was removed 2 min after the termination of the injection; TST was performed 10 min post infusion. For dopamine receptor antagonist experiments, 800 ng of SCH23390 per 0.4 µl per side at a concentration of 6.16 mM SCH23390 was infused for antagonism of D1-like receptors and 400 ng per 0.4 µl per side at a concentration of 2.89 mM raclopride was infused for D2-like receptor antagonism. For glutamate receptor..."
      },
      {
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        "position": 6,
        "name": "Immunohistochemistry",
        "text": "To determine the specificity of eNpHR3.0-eYFP expression in dopamine neurons, TH::IRES-Cre mice transduced with the double-floxed AAV encoding eNpHR3.0-eYFP were anaesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA) dissolved in PBS. Brains were removed and post-fixed in PBS containing 4% PFA overnight at 4 °C, and subsequently immersed in a cryoprotectant consisting of PBS containing 30% sucrose until settling (~48 h at 4 °C). Coronal brain sections (40 µm) were collected and washed in PBS; blocking solution, primary and secondary solutions contained 0.3% Triton X-100 (PBST) and 3% normal donkey serum dissolved in PBS. Localization of dopamine cell bodies and fibres was confirmed by labelling with chicken anti-tyrosine hydroxylase antibody (1:300). Cell bodies were identified using the 4′,6-diamidino-2-phenylindole (..."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Behavioural testing",
        "text": "For animals undergoing CMS, behavioural testing occurred after 8 to 12 weeks of CMS for mice (or 8 to 12 weeks of no CMS for non-CMS controls) or 4 to 6 weeks of CMS for rats. For NpHR animals and eYFP controls, behavioural testing occurred at least 1 month post surgery. All behaviour was conducted during the dark cycle (07:00 to 19:00)."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "Open field test",
        "text": "The open-field test was conducted in an open plastic chamber (50 cm long, 50 cm wide and 40 cm deep). Mice were plugged into a patch cord connecting to the external portion of the chronically implanted optical fibre, individually placed in the centre of the chamber and allowed to freely explore for 12 min. Velocity of the animal in the field was measured using an automated video-tracking system (Viewer II, BiObserve). Measurement began immediately after placement in the chamber. Light stimulation occurred for minutes 3 to 6 and 9 to 12 only. Although no difference was detected, any trend towards subtly decreased locomotion in the eNpHR3.0 group after illumination in the OFT would still be consistent with a depression-related phenotype, as related motor changes are clinically observed as psychomotor retardation and/or reduced motivation to explore."
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        "@type": "HowToTool",
        "name": "Stereotactic injection into VTA, and cannula and fibre implantation"
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      {
        "@type": "HowToTool",
        "name": "Blue and yellow light delivery and protocols"
      },
      {
        "@type": "HowToTool",
        "name": "Pharmacological infusion of glutamate and dopamine receptor antagonists"
      },
      {
        "@type": "HowToTool",
        "name": "Immunohistochemistry"
      },
      {
        "@type": "HowToTool",
        "name": "Open field test"
      },
      {
        "@type": "HowToTool",
        "name": "Tail-suspension test"
      },
      {
        "@type": "HowToTool",
        "name": "Sucrose-preference test"
      },
      {
        "@type": "HowToTool",
        "name": "In vivo NAc electrophysiology"
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        "name": "Pharmacological infusion of glutamate and dopamine receptor antagonists"
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        "@type": "HowToSupply",
        "name": "Immunohistochemistry"
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      {
        "@type": "HowToSupply",
        "name": "Tail-suspension test"
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      {
        "@type": "HowToSupply",
        "name": "Sucrose-preference test"
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    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "Dopamine neurons modulate neural encoding and expression of depression-related behaviour",
      "datePublished": "2012",
      "author": [
        {
          "@type": "Person",
          "name": "Kay M. Tye"
        },
        {
          "@type": "Person",
          "name": "Julie J. Mirzabekov"
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        {
          "@type": "Person",
          "name": "Melissa R. Warden"
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        {
          "@type": "Person",
          "name": "Emily A. Ferenczi"
        },
        {
          "@type": "Person",
          "name": "Hsing-Chen Tsai"
        },
        {
          "@type": "Person",
          "name": "Joel Finkelstein"
        },
        {
          "@type": "Person",
          "name": "Sung-Yon Kim"
        },
        {
          "@type": "Person",
          "name": "Avishek Adhikari"
        },
        {
          "@type": "Person",
          "name": "Kimberly R. Thompson"
        },
        {
          "@type": "Person",
          "name": "Aaron S. Andalman"
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        {
          "@type": "Person",
          "name": "Lisa A. Gunaydin"
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          "@type": "Person",
          "name": "Ilana B. Witten"
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        {
          "@type": "Person",
          "name": "Karl Deisseroth"
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      "identifier": "10.1038/nature11740"
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