Inflammasome signaling affects anxiety- and depressive-like behavior and gut microbiome composition methods
Aim. Evidence-backed execution summary for Inflammasome signaling affects anxiety- and depressive-like behavior and gut microbiome composition methods from Inflammasome signaling affects anxiety- and depressive-like behavior and gut microbiome composition.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Statistical analysis
reagent used in the protocol.
- Use
- Statistical analyses were performed using the Statistical Package for the Social Sciences version 22.0 for Windows (SPSS, Chicago, IL, USA) using a general linear model for repeated measures. The effects of genotype, stress, treatment and their interaction were explored and the significance set at P <0.05. Sphericit...
Materials and methods
Procedures were approved by the Animal Ethics Committees of the Australian National University and the South Australian Health and Medical Research and are in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). Male mice (C57BL/6J background, wt, n =81; ca...
- Use
- Procedures were approved by the Animal Ethics Committees of the Australian National University and the South Australian Health and Medical Research and are in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). Male mice (C57BL/6J background, wt, n =81; ca...
Respirometry
Minocycline- or saline-treated restrained animals were individually housed in the Promethion Metabolic Monitoring System (Sable Systems International, Las Vegas, NV, USA) for 48 h to assess the effects of minocycline on exploratory behavior, food intake, energy expenditure and volume of oxygen inhaled and of c...
- Use
- Minocycline- or saline-treated restrained animals were individually housed in the Promethion Metabolic Monitoring System (Sable Systems International, Las Vegas, NV, USA) for 48 h to assess the effects of minocycline on exploratory behavior, food intake, energy expenditure and volume of oxygen inhaled and of c...
16S rRNA analysis
Please see for a detailed explanation of the methods used for the 16S rRNA analysis. Briefly, fecal pellets were collected with autoclaved toothpicks, placed in 1.5 ml tubes, snap-frozen on dry ice and stored at -80 °C. Following DNA extraction, fecal microbiota profiling was performed by pair...
- Use
- Please see for a detailed explanation of the methods used for the 16S rRNA analysis. Briefly, fecal pellets were collected with autoclaved toothpicks, placed in 1.5 ml tubes, snap-frozen on dry ice and stored at -80 °C. Following DNA extraction, fecal microbiota profiling was performed by pair...
Caspase-1 deficiency affects chronic restraint stress response
Our results suggest that casp-1 -/- mice had an attenuated response to chronic stress. We found a significant (genotype × stress) interaction for swimming and climbing time in the forced swim test (respectively F 1, 45 =7.02, P =0.011, and F 1, 45 =8.60, P =0.005). The wt mice showed a greater decre...
- Use
- Our results suggest that casp-1 -/- mice had an attenuated response to chronic stress. We found a significant (genotype × stress) interaction for swimming and climbing time in the forced swim test (respectively F 1, 45 =7.02, P =0.011, and F 1, 45 =8.60, P =0.005). The wt mice showed a greater decre...
Caspase-1 deficiency increases locomotion and locomotor skills
We found that caspase-1 deficiency increases locomotor activity in the open field test (F 1, 45 =10.54, P =0.002, ). Moreover, casp1 -/- mice acquired skills more quickly than wt mice to perform in the accelerating rotarod test (F 1, 45 =15.35, P <0.0001, and ).
- Use
- We found that caspase-1 deficiency increases locomotor activity in the open field test (F 1, 45 =10.54, P =0.002, ). Moreover, casp1 -/- mice acquired skills more quickly than wt mice to perform in the accelerating rotarod test (F 1, 45 =15.35, P <0.0001, and ).
Chronic restraint stress increases anxiety-like and depressive-like behaviors
Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming (F 1, 45 =37.80, P <0.0001) and climbing behavior (F 1, 45 =109.52, P <0.0001). It also increased anxiety-like behavior in the elevated plu...
- Use
- Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming (F 1, 45 =37.80, P <0.0001) and climbing behavior (F 1, 45 =109.52, P <0.0001). It also increased anxiety-like behavior in the elevated plu...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analyses were performed using the Statistical Package for the Social Sciences version 22.0 for Windows (SPSS, Chicago, IL, USA) using a general linear model for repeated measures. The effects of genotype, stress, treatment and their interaction were explored and the significance set at P <0.05. Sphericit...
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Materials and methods
Procedures were approved by the Animal Ethics Committees of the Australian National University and the South Australian Health and Medical Research and are in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). Male mice (C57BL/6J background, wt, n =81; casp1 -/-, n =20) aged 60-90 days were obtained from the Australian Phenomics (Canberra, ACT, Australia) or the Bioresources Facilities (Adelaide, SA, Australia). Genetic caspase-1 deficiency was confirmed by genotyping in experimental mice ( ). Littermates were group housed (Green Line IVC Sealsafe PLUS mouse, Tecniplast, Varese, Italy) in a temperature-specific (22C±1 °C) and light-specific (12 h cycles, lights on at 0700 h) pathogen-free room with water and standard regular chow ad libitum. Animals were assigned and randomize...
Materials and methods
After baseline behavioral testing, animals were submitted to restraint stress for 21 days. Every day, mice were placed in a horizontal resting position inside a well-ventilated (12 holes, 0.5 mm diameter) 50 ml falcon tube at 1000 h and after 4-6 h they were unrestrained.
Pharmacological caspase-1 inhibition with minocycline
The wt animals were treated with either saline (0.2 ml, intraperitoneally, n =27) or minocycline (LKT Laboratories, St Paul, MN, USA; 5 mg kg -1 per day in 10 ml kg -1 saline, intraperitoneally, n =27). Treatment lasted for the same duration of the restraint procedure (21 days).
Chronic restraint stress increases anxiety-like and depressive-like behaviors
Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming (F 1, 45 =37.80, P <0.0001) and climbing behavior (F 1, 45 =109.52, P <0.0001). It also increased anxiety-like behavior in the elevated plus maze test, decreasing the time spent in the open arms (F 1, 45 =5.65, P =0.022) and the open to closed arms time ratio (F 1, 45 =4.55, P =0.038, ), as well as in the open field test, increasing the number of defecations (F 1, 45 =12.74, P =0.001, ). Furthermore, restraint decreased body weight gain (F 1, 45 =6.06, P =0.018) and food intake (F 1, 43 =5.75, P =0.021). Nevertheless, restrained mice showed an increase in ratio quotient (F 1, 28 =4.79, P =0.037). Following restraint, no changes were observed in the sucrose preference test (F 1, 33 =0.05, P =0.817, ) or in loco...
Chronic restraint stress affects the gut microbiome
Chronic restraint stress (4-6 h per day for 21 days) affected the gut microbiome compared with nonstressed animals (PERMANOVA P =0.0027, t =2.3492). Although the shallowest level of classification (that is, phylum level) only revealed a nonsignificant trend toward an increased ratio of Firmicutes to Bacteroidetes ( ), the deeper analysis did identify clear differences between the animal groups. In particular, the relative abundances of the genera Allobaculum (difference in median relative abundance -7.8%, P <0.0001 Mann-Whitney U- test), Bifidobacterium (-4.6%, P =0.0002), Turicibacter (-3.4%, P <0.0007), Clostridium (-0.7%, P <0.0001) and the family S24-7 (-5.8%, P =0.0021) were all reduced in restrained animals, and the relative abundance of the family Lachnospiraceae was increased (+0.3%, P =0.0244). Variance in the relative abundance...
Minocycline affects the gut microbiome
Minocycline treatment (5 mg kg -1 per day for 21 days) also affected microbiota composition compared with saline-treated controls (PERMANOVA P =0.0001, t =3.0947, ) and, interestingly, in a manner very similar to that observed for restrained animals. In particular, minocycline-treated animals were also found to possess lower relative abundances of the genera Allobaculum (difference in median relative abundance -7.8%, P <0.0001 Mann-Whitney U -test), Bifidobacterium (-5.8%, P <0.001), Turicibacter (-4.2%, P <0.0001), Clostridium (-0.7%, P <0.0001) and the family S24-7 (-7.4%, P =0.003), and significantly high relative abundances of the family Lachnospiraceae (+25.3%, P =0.005) and Ruminococcaceae incertae sedis (+2.4%, P =0.024). Variance in the relative abundance of these taxa accounted for >67% of intergroup variance.
Measurement outputs
What raw and processed outputs should exist?
Statistical analyses were performed using the Statistical Package for the Social Sciences version 22.0 for Windows (SPSS, Chicago, IL, USA) using a general linear model for repe...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Minocycline- or saline-treated restrained animals were individually housed in the Promethion Metabolic Monitoring System (Sable Systems International, Las Vegas, NV, USA) for 48...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Please see for a detailed explanation of the methods used for the 16S rRNA analysis. Briefly, fecal pellets were collected with autoclaved toothpicks, placed in 1.5 ml tub...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
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
Statistical analyses were performed using the Statistical Package for the Social Sciences version 22.0 for Windows (SPSS, Chicago, IL, USA) using a general linear model for repeated measures.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Statistical analyses were performed using the Statistical Package for the Social Sciences version 22.0 for Windows (SPSS, Chicago, IL, USA) using a general linear model for repe...; Minocycline- or saline-treated restrained animals were individually housed in the Promethion Metabolic Monitoring System (Sable Systems International, Las Vegas, NV, USA) for 48...; Please see for a detailed explanation of the methods used for the 16S rRNA analysis. Briefly, fecal pellets were collected with autoclaved toothpicks, placed in 1.5 ml tub...; Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming....
from paperStatistical comparison
Statistical analyses were performed using the Statistical Package for the Social Sciences version 22.0 for Windows (SPSS, Chicago, IL, USA) using a general linear model for repe...; Our results suggest that casp-1 -/- mice had an attenuated response to chronic stress. We found a significant (genotype × stress) interaction for swimming and c...; We found that caspase-1 deficiency increases locomotor activity in the open field test (F 1, 45 =10.54, P =0.002, ). Moreover, casp1 -/- mice acquired skills more qu...; Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming...
from paperReporting output
Report representative outputs alongside summary comparisons for Statistical analyses were performed using the Statistical Package for the Social Sciences version 22.0 for Windows (SPSS, Chicago, IL, USA) using a general linear model for repe..., Minocycline- or saline-treated restrained animals were individually housed in the Promethion Metabolic Monitoring System (Sable Systems International, Las Vegas, NV, USA) for 48..., Please see for a detailed explanation of the methods used for the 16S rRNA analysis. Briefly, fecal pellets were collected with autoclaved toothpicks, placed in 1.5 ml tub..., Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming....
inferred from protocolStructured statistical methods
Statistical analyses were performed using the Statistical Package for the Social Sciences version 22.0 for Windows (SPSS, Chicago, IL, USA) using a general linear model for repe...; Our results suggest that casp-1 -/- mice had an attenuated response to chronic stress. We found a significant (genotype × stress) interaction for swimming and c...; We found that caspase-1 deficiency increases locomotor activity in the open field test (F 1, 45 =10.54, P =0.002, ). Moreover, casp1 -/- mice acquired skills more qu...; Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
Procedures were approved by the Animal Ethics Committees of the Australian National University and the South Australian Health and Medical Research and are in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013). Male mice (C57BL/6J background, wt, n =81; casp1 -/-, n =20) aged 60-90 days were obtained from the Australian Phenomics (Canberra, ACT, Australia) or the Bioresources Facilities (Adelaide, SA, Australia). Genetic caspase-1 deficiency was confirmed by genotyping in experimental mice ( ). Littermates were group housed (Green Line IVC Sealsafe PLUS mouse, Tecniplast, Varese, Italy) in a temperature-specific (22C±1 °C) and light-specific (12 h cycles, lights on at 0700 h) pathogen-free room with water and standard regular chow ad libitum. Animals were assigned and randomized as described in the. The investigators were not blinded to group assignment. Behavioral phenotyping was performed between 0900 h and 1600 h. Animals were given 30 min of habituation to the behavioral testing room. Tests were performed from the least to the most invasive to minim...
After baseline behavioral testing, animals were submitted to restraint stress for 21 days. Every day, mice were placed in a horizontal resting position inside a well-ventilated (12 holes, 0.5 mm diameter) 50 ml falcon tube at 1000 h and after 4-6 h they were unrestrained.
The wt animals were treated with either saline (0.2 ml, intraperitoneally, n =27) or minocycline (LKT Laboratories, St Paul, MN, USA; 5 mg kg -1 per day in 10 ml kg -1 saline, intraperitoneally, n =27). Treatment lasted for the same duration of the restraint procedure (21 days).
Chronic restraint stress (4-6 h per day for 21 days) increased the floating time in the forced swim test (F 1, 45 =66.92, P <0.0001, ), whereas it decreased swimming (F 1, 45 =37.80, P <0.0001) and climbing behavior (F 1, 45 =109.52, P <0.0001). It also increased anxiety-like behavior in the elevated plus maze test, decreasing the time spent in the open arms (F 1, 45 =5.65, P =0.022) and the open to closed arms time ratio (F 1, 45 =4.55, P =0.038, ), as well as in the open field test, increasing the number of defecations (F 1, 45 =12.74, P =0.001, ). Furthermore, restraint decreased body weight gain (F 1, 45 =6.06, P =0.018) and food intake (F 1, 43 =5.75, P =0.021). Nevertheless, restrained mice showed an increase in ratio quotient (F 1, 28 =4.79, P =0.037). Following restraint, no changes were observed in the sucrose preference test (F 1, 33 =0.05, P =0.817, ) or in locomotor activity in the open field test (F 1, 45 =3.64, P =0.063, ).
Chronic restraint stress (4-6 h per day for 21 days) affected the gut microbiome compared with nonstressed animals (PERMANOVA P =0.0027, t =2.3492). Although the shallowest level of classification (that is, phylum level) only revealed a nonsignificant trend toward an increased ratio of Firmicutes to Bacteroidetes ( ), the deeper analysis did identify clear differences between the animal groups. In particular, the relative abundances of the genera Allobaculum (difference in median relative abundance -7.8%, P <0.0001 Mann-Whitney U- test), Bifidobacterium (-4.6%, P =0.0002), Turicibacter (-3.4%, P <0.0007), Clostridium (-0.7%, P <0.0001) and the family S24-7 (-5.8%, P =0.0021) were all reduced in restrained animals, and the relative abundance of the family Lachnospiraceae was increased (+0.3%, P =0.0244). Variance in the relative abundance of these taxa accounted for >40% of intergroup variance.
Minocycline treatment (5 mg kg -1 per day for 21 days) also affected microbiota composition compared with saline-treated controls (PERMANOVA P =0.0001, t =3.0947, ) and, interestingly, in a manner very similar to that observed for restrained animals. In particular, minocycline-treated animals were also found to possess lower relative abundances of the genera Allobaculum (difference in median relative abundance -7.8%, P <0.0001 Mann-Whitney U -test), Bifidobacterium (-5.8%, P <0.001), Turicibacter (-4.2%, P <0.0001), Clostridium (-0.7%, P <0.0001) and the family S24-7 (-7.4%, P =0.003), and significantly high relative abundances of the family Lachnospiraceae (+25.3%, P =0.005) and Ruminococcaceae incertae sedis (+2.4%, P =0.024). Variance in the relative abundance of these taxa accounted for >67% of intergroup variance.
Machine-readable layer
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