A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons methods

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    "name": "A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons methods",
    "description": "Protocol for A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons methods",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "Slice preparation",
        "text": "Coronal slices from adult mice were prepared 4-7 days after tracer injections as described previously. The animals were sacrificed by cervical dislocation, decapitated, and their brain was removed rapidly and immersed in ice-cold, oxygenated (95% O 2 -5% CO 2 ) artificial cerebrospinal fluid (ACSF) composed of (in mM): 120 NaCl, 2.5 KCl, 1 NaH 2 PO 4, 26 NaHCO 3, 2.5 CaCl 2, 1.2 MgCl 2 and 11 glucose. The brainstem was attached at its rostral end to the platform of the vibrating slicer (Pelco, TPI, series 1000, St. Louis, USA) and covered with ice-cold ACSF. Slices (200 µm thick, -4.16 to -5.80 mm posterior to bregma) were obtained in ACSF at 4 °C and then maintained at 34 °C."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "Whole cell recordings",
        "text": "Patch clamp recordings were performed in the whole cell configuration using a Multiclamp 700 amplifier, and the data were acquired using pCLAMP 10 software and a Digidata 1440 (Molecular Devices, Sunnyvale, USA), sampled at a frequency of 20 KHz and filtered at 10 KHz. Patch electrodes of 4-7 MΩ were pulled from borosilicate glass capillaries and 70% series resistance compensation was used. The pipettes were filled with an internal solution containing (in mM): 115 K-gluconate, 25 KCl, 9 NaCl, 10 HEPES, 0.2 EGTA, 1 MgCl 2, 3 K 2 -ATP and 1 Na-GTP, pH 7.2 adjusted with KOH and 280-290 mosmol Kg -1. The control external solution consisted of (in mM): 140 NaCl, 3 KCl, 1 CaCl 2 2 MgCl 2, 10 glucose and 10 HEPES (pH 7.2). Slices were transferred to a recording chamber and perfused with oxygenated external control solution. MTN neurons were identified under a..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Virus production and in vivo injection",
        "text": "Mice anaesthetized under isoflurane (2.5%) underwent surgical procedures in which the area of the skull over the midbrain was thinned and a ~2 mm 2 craniotomy was performed. The virus was delivered using a glass micropipette controlled by a nanoliter injector microprocessor (Nanoliter2010, WPI). The needle was held for 1 minute prior to the injection and the viral solution was injected in a total volume of 700 nl and at a rate of 46 nl/s. Injections were performed at the location 5.4 mm anteroposterior to Bregma, ±1.2 mm mediolateral, 2.2 mm dorsoventral. After the full volume of virus was injected, the needle was held in place for a further minute prior to withdrawal and a layer of adhesive cement (bonewax) was applied around the hole. We then sutured the skin with a tissue adhesive (cyanocrylate). The mice were given 0.1 mg/Kg Bup..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Immunohistochemistry",
        "text": "Mice were anesthetized with ketamine (200 mg/ml) and xylacine (50 mg/ml), and they were perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). The brain was then removed from each mouse and after post-fixing by immersion in 4% PFA (24 h, 4 °C), coronal vibratome sections (50 µm; VT 1000S, Leica) were obtained and then incubated for 30 min in blocking solution at room temperature (3% goat serum in TTBS). The sections were immunostained overnight at 4 °C with the primary antibodies and after washing three times in PBS (5 min) the secondary antibodies were applied for 2 h at room temperature. After three 5 min rinses with PBS, the nuclei were stained with DAPI (2 µM; Life Technology, Carlsbad, USA). The antibodies used were: rabbit anti-Piezo2 (1:100; NBP1-78624, Novus Biological,..."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Behavioral tests",
        "text": "In this study we tested a cohort of 6-9 week-old female Pvalb-Cre:RCE:Piezo2 cKO mice (n = 7) and their control littermates (n = 7). The animals were housed on a 12 h light/dark cycle at 21 °C with food and water available ad libitum. Balance and coordination was examined using four tests: the balance beam, two limb hanging, rotarod, and walking track. Balance was tested on a 1 m long, 2 cm wide beam suspended on two poles 50 cm above a table top. Food was placed in a box at the finish point to attract the mouse and a lamp above the start point served as an aversive stimulus. A video camera on a tripod was used to record the time required to cross the beam. The balance beam is a useful measure of fine coordination and balance."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "Behavioral tests",
        "text": "In the rotarod test (Ugo Basile), the mice were placed on a 5 cm diameter rotating cylinder along which they must walk in order not to fall off. The initial speed of rotation was 5 rpm and it accelerated to 20 rpm. The animal's gait was tested with the CatWalk system 7.1 (Noldus Information Technology), where the mice were placed in a glass chamber (85 cm long and 8.5 cm wide) and they were allowed to move freely across the walkway. Fluorescent light is emitted inside the apparatus and reflected internally. The contact of the paws with the glass reflects the light, allowing the paw prints to be captured by a video camera and analyzed with the CatWalk software. We analyzed the regularity index (%) that expresses the number of normal step sequence patterns relative to the total number of paw placements, a fractional measure of inter-paw coordination. In hea..."
      }
    ],
    "tool": [
      {
        "@type": "HowToTool",
        "name": "CatWalk system 7.1"
      },
      {
        "@type": "HowToTool",
        "name": "Digidata 1440"
      },
      {
        "@type": "HowToTool",
        "name": "fluorescence microscopy (Olympus BX50WI)"
      },
      {
        "@type": "HowToTool",
        "name": "Imago camera"
      },
      {
        "@type": "HowToTool",
        "name": "MM3A-LS piezoelectric micromanipulator"
      },
      {
        "@type": "HowToTool",
        "name": "Multiclamp 700 amplifier"
      },
      {
        "@type": "HowToTool",
        "name": "patch electrodes"
      },
      {
        "@type": "HowToTool",
        "name": "rotarod"
      },
      {
        "@type": "HowToTool",
        "name": "vibrating slicer"
      },
      {
        "@type": "HowToTool",
        "name": "balance beam"
      },
      {
        "@type": "HowToTool",
        "name": "cotton pad"
      },
      {
        "@type": "HowToTool",
        "name": "fluorescent light"
      },
      {
        "@type": "HowToTool",
        "name": "glass chamber"
      },
      {
        "@type": "HowToTool",
        "name": "glass micropipette"
      },
      {
        "@type": "HowToTool",
        "name": "heat-closed glass pipette"
      },
      {
        "@type": "HowToTool",
        "name": "lamp"
      },
      {
        "@type": "HowToTool",
        "name": "metal bar"
      },
      {
        "@type": "HowToTool",
        "name": "Nanoliter2010"
      },
      {
        "@type": "HowToTool",
        "name": "recording chamber"
      },
      {
        "@type": "HowToTool",
        "name": "ring stands"
      },
      {
        "@type": "HowToTool",
        "name": "rotating cylinder"
      },
      {
        "@type": "HowToTool",
        "name": "table top"
      },
      {
        "@type": "HowToTool",
        "name": "tripod"
      },
      {
        "@type": "HowToTool",
        "name": "video camera"
      }
    ],
    "supply": [
      {
        "@type": "HowToSupply",
        "name": "AAV-Piezo2-sh-1"
      },
      {
        "@type": "HowToSupply",
        "name": "artificial cerebrospinal fluid (ACSF)"
      },
      {
        "@type": "HowToSupply",
        "name": "blocking solution"
      },
      {
        "@type": "HowToSupply",
        "name": "borosilicate glass capillaries"
      },
      {
        "@type": "HowToSupply",
        "name": "Buprenorphine"
      },
      {
        "@type": "HowToSupply",
        "name": "chicken anti-GFP"
      },
      {
        "@type": "HowToSupply",
        "name": "cyanoacrylate"
      },
      {
        "@type": "HowToSupply",
        "name": "DAPI"
      },
      {
        "@type": "HowToSupply",
        "name": "DiI tracer"
      },
      {
        "@type": "HowToSupply",
        "name": "external control solution"
      },
      {
        "@type": "HowToSupply",
        "name": "goat serum"
      },
      {
        "@type": "HowToSupply",
        "name": "goat-anti chicken AlexaFluor-488"
      },
      {
        "@type": "HowToSupply",
        "name": "goat-anti rabbit AlexaFluor-594"
      },
      {
        "@type": "HowToSupply",
        "name": "internal solution"
      },
      {
        "@type": "HowToSupply",
        "name": "isoflurane"
      },
      {
        "@type": "HowToSupply",
        "name": "ketamine"
      },
      {
        "@type": "HowToSupply",
        "name": "paraformaldehyde (PFA)"
      },
      {
        "@type": "HowToSupply",
        "name": "PBS"
      },
      {
        "@type": "HowToSupply",
        "name": "phosphate buffer (PB)"
      },
      {
        "@type": "HowToSupply",
        "name": "rabbit anti-Piezo2"
      },
      {
        "@type": "HowToSupply",
        "name": "scrambled shRNA"
      },
      {
        "@type": "HowToSupply",
        "name": "TTBS"
      },
      {
        "@type": "HowToSupply",
        "name": "xylacine"
      },
      {
        "@type": "HowToSupply",
        "name": "AAV backbone of the PAM vector"
      },
      {
        "@type": "HowToSupply",
        "name": "artificial cerebrospinal fluid (ACSF)"
      },
      {
        "@type": "HowToSupply",
        "name": "bonewax"
      },
      {
        "@type": "HowToSupply",
        "name": "cyanocrylate"
      },
      {
        "@type": "HowToSupply",
        "name": "GFP reporter cassette"
      },
      {
        "@type": "HowToSupply",
        "name": "HEK-293 cells"
      },
      {
        "@type": "HowToSupply",
        "name": "rAAV serotype 2/8 particles"
      },
      {
        "@type": "HowToSupply",
        "name": "U6 promoter"
      }
    ],
    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons",
      "datePublished": "2016",
      "author": [
        {
          "@type": "Person",
          "name": "Danny Florez-Paz"
        },
        {
          "@type": "Person",
          "name": "Kiran Kumar Bali"
        },
        {
          "@type": "Person",
          "name": "Rohini Kuner"
        },
        {
          "@type": "Person",
          "name": "Ana Gomis"
        }
      ],
      "identifier": "10.1038/srep25923"
    }
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        "name": "A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons methods",
        "item": "https://replicatescience.com/experiments/a-critical-role-for-piezo2-channels-in-the-mechanotransduction-of-mouse-proprioceptive-neurons-methods-danny-florez-paz-pmc4869095/a-critical-role-for-piezo2-channels-in-the-mechanotransduction-of-mouse-proprioceptive-neurons-mlph8wb1"
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