A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons methods
Aim. Evidence-backed execution summary for A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons methods from A critical role for Piezo2 channels in the mechanotransduction of mouse proprioceptive neurons.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Whole cell recordings
reagent used in the protocol.
- Use
- 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 we...
Virus production and in vivo injection
reagent used in the protocol.
- Use
- 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...
Immunohistochemistry
reagent used in the protocol.
- Use
- 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 se...
Statistical analysis of the data
The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA) and Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA). Statistical analyses were performed with SigmaStat (Systat Software Inc.) and the data are reported as the mean ± SEM: * P <...
- Use
- The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA) and Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA). Statistical analyses were performed with SigmaStat (Systat Software Inc.) and the data are reported as the mean ± SEM: * P <...
Whole cell recordings
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 we...
- Use
- 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 we...
Behavioral tests
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 exami...
- Use
- 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 exami...
Behavioral tests
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 T...
- Use
- 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 T...
Statistical analysis of the data
Software used for acquisition, scoring, statistics, or reporting.
- Use
- The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA) and Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA). Statistical analyses were performed with SigmaStat (Systat Software Inc.) and the data are reported as the mean ± SEM: * P <...
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Slice preparation
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.
Whole cell recordings
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...
Virus production and in vivo injection
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...
Immunohistochemistry
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,...
Behavioral tests
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.
Behavioral tests
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...
Measurement outputs
What raw and processed outputs should exist?
The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA) and Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA). Statistical an...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
A set of 4 shRNAs were designed against the Piezo2 coding region (Cat. No. TR509519: Origene, USA), and each shRNA sequence was cloned by standard methods into the adeno-associa...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
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 we...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
For two limb hanging we used a 2 mm diameter metal bar mounted between two ring stands 37 cm above a cotton pad that was situated under the wire in between the stand...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA) and Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA).
from paperScoring or quantification
Quantify the primary readouts for this experiment: The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA) and Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA). Statistical an...; A set of 4 shRNAs were designed against the Piezo2 coding region (Cat. No. TR509519: Origene, USA), and each shRNA sequence was cloned by standard methods into the adeno-associa...; 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 we...; For two limb hanging we used a 2 mm diameter metal bar mounted between two ring stands 37 cm above a cotton pad that was situated under the wire in between the stand....
from paperStatistical comparison
The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA) and Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA). Statistical an...
from paperReporting output
Report representative outputs alongside summary comparisons for The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA) and Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA). Statistical an..., A set of 4 shRNAs were designed against the Piezo2 coding region (Cat. No. TR509519: Origene, USA), and each shRNA sequence was cloned by standard methods into the adeno-associa..., 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 we..., For two limb hanging we used a 2 mm diameter metal bar mounted between two ring stands 37 cm above a cotton pad that was situated under the wire in between the stand....
inferred from protocolStructured statistical methods
The data were analyzed with the Clampex 10.1 software (Molecular Devices Corp., Sunnyvale, CA, USA) and Origin 7.5 (Origin Lab Corporation, Northampton, MA, USA). Statistical an...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
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.
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 fluorescence microscopy (Olympus BX50WI) coupled to an imaging system that included polychrome V, an Imago camera (both Till photonics GmbH, Germany) and the Imaging Workbench 6.0 software (INDEC BioSystems). As most sensory neurons are oval, their two diameters were measured and the area was deriv...
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 Buprenorphine as an analgesic and they remained on a heated pad until they recovered from the anesthesia. The mice were studied in the two weeks following surgery.
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, Littleton, USA); chicken anti-GFP (1:1000; AB13970, Abcam, Cambridge, UK), goat-anti rabbit AlexaFluor-594 (1:1000; A11012) and goat-anti chicken AlexaFluor-488 (1:2000; A11039, Life Technology).
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.
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 healthy, fully coordinated animals this value is 100%.
Machine-readable layer
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