Opposite Effects of mGluR1a and mGluR5 Activation on Nucleus Accumbens Medium Spiny Neuron Dendritic Spine Density methods
Aim. Evidence-backed execution summary for Opposite Effects of mGluR1a and mGluR5 Activation on Nucleus Accumbens Medium Spiny Neuron Dendritic Spine Density methods from Opposite Effects of mGluR1a and mGluR5 Activation on Nucleus Accumbens Medium Spiny Neuron Dendritic Spine Density.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Drugs
reagent used in the protocol.
- Use
- 3-Cyano- N -(1,3-diphenyl-1 H -pyrazol-5-yl)benzamide (CDPPB) and ( RS )-2-Chloro-5-hydroxyphenylglycine sodium salt (CHPG) were obtained from Tocris Bioscience (Minneapolis, MN). CDPPB was suspended in 0.5% methyl cellulose (w/v in deionized water, Sigma-Aldrich, St. Louis, MO). CHPG was dissolved in sterile saline...
Surgical microinjection procedure
reagent used in the protocol.
- Use
- Animals were anesthetized with a 2.5-4% isoflurane (Piramal Critical Care, Bethlehem, PA)/oxygen mixture and placed in a stereotaxic apparatus. Drug or vehicle was injected bilaterally via a Hamilton microinjection syringe at the following coordinates targeting the core-shell border: AP: +1.80 mm from bregma,...
Tissue preparation
reagent used in the protocol.
- Use
- Tissue was prepared and ballistically labeled with DiI following protocols previously established in our lab [, ]. Twenty-four hours after drug treatment, animals were overdosed with Beuthanasia-D (0.3 ml i.p.; Schering, Union, NJ) and transcardially perfused with 25 mM phosphate buffered saline (PBS, pH = 7.2) for...
DiI labeling
reagent used in the protocol.
- Use
- DiI "bullets" were made by dissolving 2 mg DiI (Molecular Probes, Carlsbad, CA) in 100 µl dichloromethane and applying the solution to 90 mg of 1.3 µm tungsten particles (Bio-Rad, Hercules, CA). Particles were suspended in 10 ml of 15 mg/ml polyvinylpyrrolidone (PVP) and sonicated for 10 minute...
Confocal imaging
reagent used in the protocol.
- Use
- Cells were imaged with a Leica TCS SPE confocal microscope (Leica, Manheim, Germany). All images were taken at a xy pixel distribution of 512 x 512 and a frequency of 400 Hz. Whole medium spiny neurons were imaged at 20X magnification with a z-step size of 1 µm and reconstructed using the Leica LAS AF software...
Quantitation
reagent used in the protocol.
- Use
- Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the Surpass module of Imaris software (Bitplane, Inc., St. Paul, Minnesota). Dendrites were manually traced in the xy plane using the Filament t...
Data Analysis
reagent used in the protocol.
- Use
- Data were examined for both univariate and multivariate outliers in SPSS (version 20, SPSS, Inc. Chicago, IL). After outliers were removed ( n = 1 animal per experiment), means were compared using a Student's t -test for spine density analysis. For spine morphology analyses, groups were compared using a two-wa...
Surgical microinjection procedure
Animals were anesthetized with a 2.5-4% isoflurane (Piramal Critical Care, Bethlehem, PA)/oxygen mixture and placed in a stereotaxic apparatus. Drug or vehicle was injected bilaterally via a Hamilton microinjection syringe at the following coordinates targeting the core-shell border: AP: +1.80 mm from bregma,...
- Use
- Animals were anesthetized with a 2.5-4% isoflurane (Piramal Critical Care, Bethlehem, PA)/oxygen mixture and placed in a stereotaxic apparatus. Drug or vehicle was injected bilaterally via a Hamilton microinjection syringe at the following coordinates targeting the core-shell border: AP: +1.80 mm from bregma,...
Confocal imaging
Cells were imaged with a Leica TCS SPE confocal microscope (Leica, Manheim, Germany). All images were taken at a xy pixel distribution of 512 x 512 and a frequency of 400 Hz. Whole medium spiny neurons were imaged at 20X magnification with a z-step size of 1 µm and reconstructed using the Leica LAS AF software...
- Use
- Cells were imaged with a Leica TCS SPE confocal microscope (Leica, Manheim, Germany). All images were taken at a xy pixel distribution of 512 x 512 and a frequency of 400 Hz. Whole medium spiny neurons were imaged at 20X magnification with a z-step size of 1 µm and reconstructed using the Leica LAS AF software...
Quantitation
Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the Surpass module of Imaris software (Bitplane, Inc., St. Paul, Minnesota). Dendrites were manually traced in the xy plane using the Filament t...
- Use
- Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the Surpass module of Imaris software (Bitplane, Inc., St. Paul, Minnesota). Dendrites were manually traced in the xy plane using the Filament t...
Quantitation
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the Surpass module of Imaris software (Bitplane, Inc., St. Paul, Minnesota). Dendrites were manually traced in the xy plane using the Filament t...
Data Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data were examined for both univariate and multivariate outliers in SPSS (version 20, SPSS, Inc. Chicago, IL). After outliers were removed ( n = 1 animal per experiment), means were compared using a Student's t -test for spine density analysis. For spine morphology analyses, groups were compared using a two-wa...
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Drugs
3-Cyano- N -(1,3-diphenyl-1 H -pyrazol-5-yl)benzamide (CDPPB) and ( RS )-2-Chloro-5-hydroxyphenylglycine sodium salt (CHPG) were obtained from Tocris Bioscience (Minneapolis, MN). CDPPB was suspended in 0.5% methyl cellulose (w/v in deionized water, Sigma-Aldrich, St. Louis, MO). CHPG was dissolved in sterile saline. 9H-Xanthene-9-carboxylic acid (4-trifluoromethyl-oxazol-2-yl)-amide (SYN119) was synthesized by EAG Laboratories (Sunnyvale, CA) and was suspended in 20% 2-hydroxypropyl-β-cyclodextrin (w/v in deionized water, Sigma-Aldrich). For systemic treatments, CDPPB (5-10 mg/kg), SYN119 (10 mg/kg), or their corresponding vehicles were administered intraperitoneally (i.p.) at a volume of 2 ml/kg. For site-specific drug administration, CHPG (10 µg/0.5 µl/side) or an equivalent volume of vehicle was infused directly into the NAc. For all experiments, animals were...
Surgical microinjection procedure
Animals were anesthetized with a 2.5-4% isoflurane (Piramal Critical Care, Bethlehem, PA)/oxygen mixture and placed in a stereotaxic apparatus. Drug or vehicle was injected bilaterally via a Hamilton microinjection syringe at the following coordinates targeting the core-shell border: AP: +1.80 mm from bregma, ML: ±1.50 mm from bregma, DV: -6.20 mm from dura. Infusions of 0.5 µl were given manually over the course of 2.5 minutes. The injection needle was then left in place for an additional 2.5 minutes to allow for diffusion of the drug away from the needle tip. Animals were given a subcutaneous injection of 2.5 mg/kg ketoprofen 20 minutes prior to surgery and every 12 hours after surgery to induce analgesia and a subcutaneous injection of 10 mg/kg Baytril at the time of surgery to prevent infection. Immediately following the surgery, animals were monitored until they r...
Tissue preparation
Tissue was prepared and ballistically labeled with DiI following protocols previously established in our lab [, ]. Twenty-four hours after drug treatment, animals were overdosed with Beuthanasia-D (0.3 ml i.p.; Schering, Union, NJ) and transcardially perfused with 25 mM phosphate buffered saline (PBS, pH = 7.2) for 3 minutes followed by 1.5% paraformaldehyde in PBS for 20 minutes. Brains were removed, coronally blocked, and then post-fixed in 1.5% paraformaldehyde for 1 hour before being stored in PBS. Brains were sliced into 200-300 µm sections through the striatum using a Vibratome (Leica VT1000 S, Buffalo Grove, IL). Sections were stored in PBS until DiI labeling.
DiI labeling
DiI "bullets" were made by dissolving 2 mg DiI (Molecular Probes, Carlsbad, CA) in 100 µl dichloromethane and applying the solution to 90 mg of 1.3 µm tungsten particles (Bio-Rad, Hercules, CA). Particles were suspended in 10 ml of 15 mg/ml polyvinylpyrrolidone (PVP) and sonicated for 10 minutes with intermittent vortexing. The suspension was quickly pulled through a length of Teftzel tubing (Bio-Rad) pre-treated with PVP and allowed to settle before the remaining PVP was expelled. The tubing was dried for 20 minutes with nitrogen gas flow and then cut into 1.3 cm bullets. To deliver the DiI-coated tungsten particles, bullets were loaded into a Helios Gene Gun (Bio-Rad) with a modified barrel, 40 mm spacer, and 70 µm mesh filter. PBS was removed from wells containing brain sections, and one bullet was shot per section using helium gas at 100 PSI. After shoot...
Confocal imaging
Cells were imaged with a Leica TCS SPE confocal microscope (Leica, Manheim, Germany). All images were taken at a xy pixel distribution of 512 x 512 and a frequency of 400 Hz. Whole medium spiny neurons were imaged at 20X magnification with a z-step size of 1 µm and reconstructed using the Leica LAS AF software to measure the distance from the soma to each dendritic segment. Cells were imaged from the NAcC and NAcSh in all experiments, and cells were also imaged in the dorsolateral caudate for experiments using systemic drug manipulations. Data from a minimum of 7 animals per treatment group were collected for each experiment (see figure legends for specific group sample sizes). For each animal, each brain region had a minimum of six dendritic segments imaged for analysis, with two or three distal dendritic segments (distance of 70-200 µm from the soma) taken from each...
Measurement outputs
What raw and processed outputs should exist?
3-Cyano- N -(1,3-diphenyl-1 H -pyrazol-5-yl)benzamide (CDPPB) and ( RS )-2-Chloro-5-hydroxyphenylglycine sodium salt (CHPG) were obtained from Tocris Bioscience (Minneapolis, MN...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Cells were imaged with a Leica TCS SPE confocal microscope (Leica, Manheim, Germany). All images were taken at a xy pixel distribution of 512 x 512 and a frequency of 400 Hz. Wh...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Data were examined for both univariate and multivariate outliers in SPSS (version 20, SPSS, Inc. Chicago, IL). After outliers were removed ( n = 1 animal per experiment), means...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the Surpass module of Imaris software (Bitplane, Inc., St.
from paperScoring or quantification
Quantify the primary readouts for this experiment: 3-Cyano- N -(1,3-diphenyl-1 H -pyrazol-5-yl)benzamide (CDPPB) and ( RS )-2-Chloro-5-hydroxyphenylglycine sodium salt (CHPG) were obtained from Tocris Bioscience (Minneapolis, MN...; Cells were imaged with a Leica TCS SPE confocal microscope (Leica, Manheim, Germany). All images were taken at a xy pixel distribution of 512 x 512 and a frequency of 400 Hz. Wh...; Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the...; Data were examined for both univariate and multivariate outliers in SPSS (version 20, SPSS, Inc. Chicago, IL). After outliers were removed ( n = 1 animal per experiment), means....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the...; Data were examined for both univariate and multivariate outliers in SPSS (version 20, SPSS, Inc. Chicago, IL). After outliers were removed ( n = 1 animal per experiment), means...
from paperReporting output
Report representative outputs alongside summary comparisons for 3-Cyano- N -(1,3-diphenyl-1 H -pyrazol-5-yl)benzamide (CDPPB) and ( RS )-2-Chloro-5-hydroxyphenylglycine sodium salt (CHPG) were obtained from Tocris Bioscience (Minneapolis, MN..., Cells were imaged with a Leica TCS SPE confocal microscope (Leica, Manheim, Germany). All images were taken at a xy pixel distribution of 512 x 512 and a frequency of 400 Hz. Wh..., Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the..., Data were examined for both univariate and multivariate outliers in SPSS (version 20, SPSS, Inc. Chicago, IL). After outliers were removed ( n = 1 animal per experiment), means....
inferred from protocolStructured statistical methods
Confocal images were processed through 3D deconvolution using Autoquant X3 AutoDeblur software (Media Cybernetics, Bethesda, MD) and reconstructed z-stacks were rendered by the...; Data were examined for both univariate and multivariate outliers in SPSS (version 20, SPSS, Inc. Chicago, IL). After outliers were removed ( n = 1 animal per experiment), means...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
3-Cyano- N -(1,3-diphenyl-1 H -pyrazol-5-yl)benzamide (CDPPB) and ( RS )-2-Chloro-5-hydroxyphenylglycine sodium salt (CHPG) were obtained from Tocris Bioscience (Minneapolis, MN). CDPPB was suspended in 0.5% methyl cellulose (w/v in deionized water, Sigma-Aldrich, St. Louis, MO). CHPG was dissolved in sterile saline. 9H-Xanthene-9-carboxylic acid (4-trifluoromethyl-oxazol-2-yl)-amide (SYN119) was synthesized by EAG Laboratories (Sunnyvale, CA) and was suspended in 20% 2-hydroxypropyl-β-cyclodextrin (w/v in deionized water, Sigma-Aldrich). For systemic treatments, CDPPB (5-10 mg/kg), SYN119 (10 mg/kg), or their corresponding vehicles were administered intraperitoneally (i.p.) at a volume of 2 ml/kg. For site-specific drug administration, CHPG (10 µg/0.5 µl/side) or an equivalent volume of vehicle was infused directly into the NAc. For all experiments, animals were sacrificed 24 hours after drug administration and tissue was processed for dendritic spine analysis.
Animals were anesthetized with a 2.5-4% isoflurane (Piramal Critical Care, Bethlehem, PA)/oxygen mixture and placed in a stereotaxic apparatus. Drug or vehicle was injected bilaterally via a Hamilton microinjection syringe at the following coordinates targeting the core-shell border: AP: +1.80 mm from bregma, ML: ±1.50 mm from bregma, DV: -6.20 mm from dura. Infusions of 0.5 µl were given manually over the course of 2.5 minutes. The injection needle was then left in place for an additional 2.5 minutes to allow for diffusion of the drug away from the needle tip. Animals were given a subcutaneous injection of 2.5 mg/kg ketoprofen 20 minutes prior to surgery and every 12 hours after surgery to induce analgesia and a subcutaneous injection of 10 mg/kg Baytril at the time of surgery to prevent infection. Immediately following the surgery, animals were monitored until they recovered ambulatory posture. Afterwards, animals were observed for eating, drinking, and or the display of any discomfort. None of the animals exhibited any symptoms of problems. Location of the injection site was verified following euthanasia.
Tissue was prepared and ballistically labeled with DiI following protocols previously established in our lab [, ]. Twenty-four hours after drug treatment, animals were overdosed with Beuthanasia-D (0.3 ml i.p.; Schering, Union, NJ) and transcardially perfused with 25 mM phosphate buffered saline (PBS, pH = 7.2) for 3 minutes followed by 1.5% paraformaldehyde in PBS for 20 minutes. Brains were removed, coronally blocked, and then post-fixed in 1.5% paraformaldehyde for 1 hour before being stored in PBS. Brains were sliced into 200-300 µm sections through the striatum using a Vibratome (Leica VT1000 S, Buffalo Grove, IL). Sections were stored in PBS until DiI labeling.
DiI "bullets" were made by dissolving 2 mg DiI (Molecular Probes, Carlsbad, CA) in 100 µl dichloromethane and applying the solution to 90 mg of 1.3 µm tungsten particles (Bio-Rad, Hercules, CA). Particles were suspended in 10 ml of 15 mg/ml polyvinylpyrrolidone (PVP) and sonicated for 10 minutes with intermittent vortexing. The suspension was quickly pulled through a length of Teftzel tubing (Bio-Rad) pre-treated with PVP and allowed to settle before the remaining PVP was expelled. The tubing was dried for 20 minutes with nitrogen gas flow and then cut into 1.3 cm bullets. To deliver the DiI-coated tungsten particles, bullets were loaded into a Helios Gene Gun (Bio-Rad) with a modified barrel, 40 mm spacer, and 70 µm mesh filter. PBS was removed from wells containing brain sections, and one bullet was shot per section using helium gas at 100 PSI. After shooting, sections were stored overnight in the dark in PBS and then post-fixed in 4% paraformaldehyde for 1 hour the next day. Sections were mounted on slides and coverslipped with FluorGlo mounting media for lipophilic dyes (Spectra Services, Ontario, NY).
Cells were imaged with a Leica TCS SPE confocal microscope (Leica, Manheim, Germany). All images were taken at a xy pixel distribution of 512 x 512 and a frequency of 400 Hz. Whole medium spiny neurons were imaged at 20X magnification with a z-step size of 1 µm and reconstructed using the Leica LAS AF software to measure the distance from the soma to each dendritic segment. Cells were imaged from the NAcC and NAcSh in all experiments, and cells were also imaged in the dorsolateral caudate for experiments using systemic drug manipulations. Data from a minimum of 7 animals per treatment group were collected for each experiment (see figure legends for specific group sample sizes). For each animal, each brain region had a minimum of six dendritic segments imaged for analysis, with two or three distal dendritic segments (distance of 70-200 µm from the soma) taken from each of two or three cells per brain region. Distal dendritic segments were chosen for analysis as this dendritic region of medium spiny neurons receives the majority of inputs from glutamatergic projections to the NAc [ ]. Images of individual dendrites were taken with a 63X oil immersion lens and 5.6 o...
Machine-readable layer
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