Evidence for sustained elevation of IL-6 in the CNS as a key contributor of depressive-like phenotypes methods
Aim. Evidence-backed execution summary for Evidence for sustained elevation of IL-6 in the CNS as a key contributor of depressive-like phenotypes methods from Evidence for sustained elevation of IL-6 in the CNS as a key contributor of depressive-like phenotypes.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Central administration of IL-1β and IL-6
reagent used in the protocol.
- Use
- Recombinant mouse IL-1β (R&D Systems, Minneapolis, MN, USA) and recombinant mouse IL-6 (EBiosciences, San Diego, CA, USA) were diluted in artificial CSF containing 0.1% bovine serum albumin (BSA) as carrier protein. Proteins were injected directly into the brain (2-3 µl; intracerebroventricular...
Social interaction paradigm
reagent used in the protocol.
- Use
- Brain tissue was collected following decapitation of anesthetized animals under isoflurane anesthesia, dissected in cold PBS and stored at -80 °C. Frozen tissues were thawed in cold lysis buffer containing the following in PBS: 3% of 5 ℳ NaCl, 2% of 1 ℳ Tris-buffered saline,...
Multiplex cytokine ELISA
reagent used in the protocol.
- Use
- Custom multiplex ELISA kits were purchased from Meso-Scale Devices (Gaithersburg, MD, USA). The standard manufacturer's protocol was modified to optimize for the detection of cytokines in the brain tissue by the addition of fresh protease inhibitors to thawing homogenates, and increasing the sample aliquot to 50R...
SDS-PAGE and western blotting of IL-6
reagent used in the protocol.
- Use
- Reducing agents (NuPAGE, no. NP0004, Invitrogen, Grand Island, NY, USA) and loading buffer were added to samples and heat treated for 3 min (90 °C) before loading. A total of 30-50 µg of total protein were loaded in each lane (5 µg purified IL-6 was used as loading contro...
Blockade of IL-6-induced increases in protein levels and depressive-like behavior by central administration of anti-I...
reagent used in the protocol.
- Use
- To demonstrate a direct and specific interaction of IL-6 in mediating depressive-like behavior, the effects of central coadministration of anti-IL-6 antibody (1 µg) with IL-6 (1 µg) were evaluated. IL-6 administration produced the expected increase in immobility time relative to vehicle/vehicle...
Social competition paradigm of dominant-submissive behavior
The social competition paradigm is similar to the procedure we reported previously, which takes advantage of the natural dominant-submissive relationship formed between a pair of male conspecifics. Briefly, pair-housed subjects (non food-restricted) were trained individually in operant chambers (Med Associates...
- Use
- The social competition paradigm is similar to the procedure we reported previously, which takes advantage of the natural dominant-submissive relationship formed between a pair of male conspecifics. Briefly, pair-housed subjects (non food-restricted) were trained individually in operant chambers (Med Associates...
Tail suspension test
The tail suspension test used is a variant of a procedure described previously. Briefly, subjects were suspended by their tails with tape to a flat bar for 6 min in duration (Med Associates). Immobility time was automatically recorded (Med PC software, St Albans, VT, USA).
- Use
- The tail suspension test used is a variant of a procedure described previously. Briefly, subjects were suspended by their tails with tape to a flat bar for 6 min in duration (Med Associates). Immobility time was automatically recorded (Med PC software, St Albans, VT, USA).
Forced swim test
Subjects were placed into individual tanks (33 cm height × 10 cm diameter; 25±0.5 °C water temperature) for a 6-min test period. Immobility time (floating) was recorded via automated cameras connected to an interface and computer and analyzed using BiObserve software (St Augustin, Ge...
- Use
- Subjects were placed into individual tanks (33 cm height × 10 cm diameter; 25±0.5 °C water temperature) for a 6-min test period. Immobility time (floating) was recorded via automated cameras connected to an interface and computer and analyzed using BiObserve software (St Augustin, Ge...
SDS-PAGE and western blotting of IL-6
Reducing agents (NuPAGE, no. NP0004, Invitrogen, Grand Island, NY, USA) and loading buffer were added to samples and heat treated for 3 min (90 °C) before loading. A total of 30-50 µg of total protein were loaded in each lane (5 µg purified IL-6 was used as loading contro...
- Use
- Reducing agents (NuPAGE, no. NP0004, Invitrogen, Grand Island, NY, USA) and loading buffer were added to samples and heat treated for 3 min (90 °C) before loading. A total of 30-50 µg of total protein were loaded in each lane (5 µg purified IL-6 was used as loading contro...
Central administration of IL-6 induces depressive-like behavior in the absence of sickness or elevations in IL-1β
To understand the role of IL-6 in the depressive and sickness behavior observed after administration of IL-1β, we evaluated the direct central effects of IL-6. Central administration of IL-6 (500-1000 ng, i.c.v.; 2 h pretreatment) to naïve mice produced dose-dependent increases in immobil...
- Use
- To understand the role of IL-6 in the depressive and sickness behavior observed after administration of IL-1β, we evaluated the direct central effects of IL-6. Central administration of IL-6 (500-1000 ng, i.c.v.; 2 h pretreatment) to naïve mice produced dose-dependent increases in immobil...
Depressive-like phenotype and reduced antidepressant-like efficacy in a mouse model with endogenous overexpression of...
To further test the hypothesis that central elevation of IL-6 protein is a relevant mechanism of depressive-like behavior, we employed LPR mice that have previously been reported to have increased IL-6 mRNA expression in brain regions, and a depressive-like phenotype relative to control MRL WT mice.,,, In the tai...
- Use
- To further test the hypothesis that central elevation of IL-6 protein is a relevant mechanism of depressive-like behavior, we employed LPR mice that have previously been reported to have increased IL-6 mRNA expression in brain regions, and a depressive-like phenotype relative to control MRL WT mice.,,, In the tai...
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Materials and methods
All experiments were performed in accordance with the specifications of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Pub. 85-23, rev 1996) and under the approval of the Institutional Animal Care and Use Committee (IACUC). Experiments were conducted in an AAALAC-accredited facility maintained on a 12-h light/dark cycle (lights on at 0600 h) with food and water provided ad libitum. Behavioral experiments were conducted during the light cycle between the hours of 0800-1700. Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were used for studies investigating stress-induced behavioral and cytokine responses. Adult male Swiss Webster mice (Charles River Laboratories) were used for studies evaluating exogenous administration of IL-1β or IL-6. For studies evaluating effects of endogenous overexpress...
Learned helplessness
Learned helplessness was assessed in Sprague-Dawley rats in an escape-avoidance paradigm following exposure to inescapable footshock. Briefly, on day 1 subjects were sequestered to one side of a shuttle box (Med Associates, St Albans, VT, USA) and were administered a series of inescapable footshocks (60 shocks, 0.8 mA, 15 s duration, 2-s intertrial interval) paired with a light cue and audible tone. Sham controls were exposed to the chambers but did not receive any footshocks or cues. On day 2, all subjects were assessed for helpless behavior in a 30-trial active-avoidance task (see Supplementary Information for detailed methods).
Tail suspension test
The tail suspension test used is a variant of a procedure described previously. Briefly, subjects were suspended by their tails with tape to a flat bar for 6 min in duration (Med Associates). Immobility time was automatically recorded (Med PC software, St Albans, VT, USA).
Forced swim test
Subjects were placed into individual tanks (33 cm height × 10 cm diameter; 25±0.5 °C water temperature) for a 6-min test period. Immobility time (floating) was recorded via automated cameras connected to an interface and computer and analyzed using BiObserve software (St Augustin, Germany).
SDS-PAGE and western blotting of IL-6
Reducing agents (NuPAGE, no. NP0004, Invitrogen, Grand Island, NY, USA) and loading buffer were added to samples and heat treated for 3 min (90 °C) before loading. A total of 30-50 µg of total protein were loaded in each lane (5 µg purified IL-6 was used as loading control; EBiosciences, no. 34-8061). Protein samples were separated by SDS-PAGE gel electrophoresis (Invitrogen; 4-12% Bis-Tris) in running buffer, washed in Tris-buffered saline with Tween-20 and transferred to a nitrocellulose membrane. Following successful transfer, membranes were cut at the ∼28 kDa band, which allowed us to simultaneously probe for the protein of interest (IL-6 at ∼21 kDa) and the control protein (actin at ∼45 kDa) on the same gel (within sample control). Membranes were blocked with blocking buffer (Pierce SuperBlock,...
Statistical analysis
Behavioral data are expressed as mean±s.e.m. Data were analyzed using a one-way analysis of variance (ANOVA) (treatment) or two-way ANOVA (treatment × pretreatment), (treatment × strain) or (treatment × time) depending upon the experimental design. A two-way ANOVA with repeated measures was used to analyze data of which time was a factor. ANOVA was followed by planned post hoc comparisons using Bonferroni's post hoc test. Significance was achieved at P <0.05.
Central administration of IL-1β induces sickness and depressive-like behavior and elevations of IL-6
It has been previously reported that IL-1β induces depressive-like behaviors with accompanying increases in 'sickness behavior' and elevations of IL-6 in rodents., Reassuringly, we were able to confirm and extend these observations. IL-1β (50-200 ng, 2 h pretreatment, i.c.v.) produced dose-dependent increases in immobility time as analyzed by one-way ANOVA with Dunnett's post hoc comparisons in the tail suspension test (F(3,82)=4.864; P <0.01 vs vehicle; ) and forced swim test (F(3,68)=3.251; P <0.05 vs vehicle; ), an effect suggestive of a prodepressive profile. IL-1β-treated mice also demonstrated dose-related increases in sickness behaviors (F(3,59)=41.54; P <0.0001 vs vehicle; ). Cortex homogenates of mice administered IL-1β (100 ng, i.c.v.) revealed significant increases in IL-1β protein levels relative to vehicle-infused co...
Central administration of IL-6 induces depressive-like behavior in the absence of sickness or elevations in IL-1β
To understand the role of IL-6 in the depressive and sickness behavior observed after administration of IL-1β, we evaluated the direct central effects of IL-6. Central administration of IL-6 (500-1000 ng, i.c.v.; 2 h pretreatment) to naïve mice produced dose-dependent increases in immobility time as analyzed by one-way ANOVA with Dunnett's post hoc comparisons in the tail suspension (F(3,37)=2.998; P <0.05 vs vehicle; ) and forced swim tests (F(3,48)=2.272; P <0.05 vs vehicle; ), indicative of depressive-like activity of IL-6. Central administration of IL-6 at doses that increased immobility did not produce significant increases in sickness behaviors (F(3,37)=1.342; P >0.05 vs vehicle; ) or alterations in general exploratory behavior in an open field ( ). Importantly, as measured by multiplex ELISA, cortex homogenates revealed the expected increase in IL-6...
Measurement outputs
What raw and processed outputs should exist?
All experiments were performed in accordance with the specifications of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Pub. 85-23, rev 1996)...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Learned helplessness was assessed in Sprague-Dawley rats in an escape-avoidance paradigm following exposure to inescapable footshock. Briefly, on day 1 subjects were seque...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
The social competition paradigm is similar to the procedure we reported previously, which takes advantage of the natural dominant-submissive relationship formed between a...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
A trained observer blind to the treatment conditions observed and scored the absence or presence of the following four behaviors in individually housed mice: hypoactivity, ptosi...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Behavioral data are expressed as mean±s.e.m.
from paperScoring or quantification
Quantify the primary readouts for this experiment: All experiments were performed in accordance with the specifications of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Pub. 85-23, rev 1996)...; Learned helplessness was assessed in Sprague-Dawley rats in an escape-avoidance paradigm following exposure to inescapable footshock. Briefly, on day 1 subjects were seque...; The social competition paradigm is similar to the procedure we reported previously, which takes advantage of the natural dominant-submissive relationship formed between a...; A trained observer blind to the treatment conditions observed and scored the absence or presence of the following four behaviors in individually housed mice: hypoactivity, ptosi....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Behavioral data are expressed as mean±s.e.m. Data were analyzed using a one-way analysis of variance (ANOVA) (treatment) or two-way ANOVA (treatment × pretreatment), (...; It has been previously reported that IL-1β induces depressive-like behaviors with accompanying increases in 'sickness behavior' and elevations of IL-6 in rodents., R...; To understand the role of IL-6 in the depressive and sickness behavior observed after administration of IL-1β, we evaluated the direct central effects of IL-6. Central admi...; To further test the hypothesis that central elevation of IL-6 protein is a relevant mechanism of depressive-like behavior, we employed LPR mice that have previously been reporte...
from paperReporting output
Report representative outputs alongside summary comparisons for All experiments were performed in accordance with the specifications of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Pub. 85-23, rev 1996)..., Learned helplessness was assessed in Sprague-Dawley rats in an escape-avoidance paradigm following exposure to inescapable footshock. Briefly, on day 1 subjects were seque..., The social competition paradigm is similar to the procedure we reported previously, which takes advantage of the natural dominant-submissive relationship formed between a..., A trained observer blind to the treatment conditions observed and scored the absence or presence of the following four behaviors in individually housed mice: hypoactivity, ptosi....
inferred from protocolStructured statistical methods
Behavioral data are expressed as mean±s.e.m. Data were analyzed using a one-way analysis of variance (ANOVA) (treatment) or two-way ANOVA (treatment × pretreatment), (...; It has been previously reported that IL-1β induces depressive-like behaviors with accompanying increases in 'sickness behavior' and elevations of IL-6 in rodents., R...; To understand the role of IL-6 in the depressive and sickness behavior observed after administration of IL-1β, we evaluated the direct central effects of IL-6. Central admi...; To further test the hypothesis that central elevation of IL-6 protein is a relevant mechanism of depressive-like behavior, we employed LPR mice that have previously been reporte...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
All experiments were performed in accordance with the specifications of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Pub. 85-23, rev 1996) and under the approval of the Institutional Animal Care and Use Committee (IACUC). Experiments were conducted in an AAALAC-accredited facility maintained on a 12-h light/dark cycle (lights on at 0600 h) with food and water provided ad libitum. Behavioral experiments were conducted during the light cycle between the hours of 0800-1700. Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were used for studies investigating stress-induced behavioral and cytokine responses. Adult male Swiss Webster mice (Charles River Laboratories) were used for studies evaluating exogenous administration of IL-1β or IL-6. For studies evaluating effects of endogenous overexpression of IL-6, male MRL/MpJ-Fas LPR/LPR (LPR) mice and MRL/MpJ +/+ (MRL) wild-type (WT) controls were used (Jackson Laboratories, Bar Harbor, ME, USA). All subjects were acclimated to the facility for a minimum of 1 week before experiments. Intact female C57Bl6/J mice (Jackson Laboratories) were maint...
Learned helplessness was assessed in Sprague-Dawley rats in an escape-avoidance paradigm following exposure to inescapable footshock. Briefly, on day 1 subjects were sequestered to one side of a shuttle box (Med Associates, St Albans, VT, USA) and were administered a series of inescapable footshocks (60 shocks, 0.8 mA, 15 s duration, 2-s intertrial interval) paired with a light cue and audible tone. Sham controls were exposed to the chambers but did not receive any footshocks or cues. On day 2, all subjects were assessed for helpless behavior in a 30-trial active-avoidance task (see Supplementary Information for detailed methods).
The tail suspension test used is a variant of a procedure described previously. Briefly, subjects were suspended by their tails with tape to a flat bar for 6 min in duration (Med Associates). Immobility time was automatically recorded (Med PC software, St Albans, VT, USA).
Subjects were placed into individual tanks (33 cm height × 10 cm diameter; 25±0.5 °C water temperature) for a 6-min test period. Immobility time (floating) was recorded via automated cameras connected to an interface and computer and analyzed using BiObserve software (St Augustin, Germany).
Reducing agents (NuPAGE, no. NP0004, Invitrogen, Grand Island, NY, USA) and loading buffer were added to samples and heat treated for 3 min (90 °C) before loading. A total of 30-50 µg of total protein were loaded in each lane (5 µg purified IL-6 was used as loading control; EBiosciences, no. 34-8061). Protein samples were separated by SDS-PAGE gel electrophoresis (Invitrogen; 4-12% Bis-Tris) in running buffer, washed in Tris-buffered saline with Tween-20 and transferred to a nitrocellulose membrane. Following successful transfer, membranes were cut at the ∼28 kDa band, which allowed us to simultaneously probe for the protein of interest (IL-6 at ∼21 kDa) and the control protein (actin at ∼45 kDa) on the same gel (within sample control). Membranes were blocked with blocking buffer (Pierce SuperBlock, Thermo Scientific, Rockford, IL, USA no. 37517, +0.1% Tween-20) followed by incubation overnight at 4 °C with primary antibodies diluted in blocking buffer (IL-6 1:200 (BD Pharmingen), no. 554400; actin 1:10 000 (Sigma, St Louis, MO, USA), no. A2066). Membranes were then washed in...
Behavioral data are expressed as mean±s.e.m. Data were analyzed using a one-way analysis of variance (ANOVA) (treatment) or two-way ANOVA (treatment × pretreatment), (treatment × strain) or (treatment × time) depending upon the experimental design. A two-way ANOVA with repeated measures was used to analyze data of which time was a factor. ANOVA was followed by planned post hoc comparisons using Bonferroni's post hoc test. Significance was achieved at P <0.05.
It has been previously reported that IL-1β induces depressive-like behaviors with accompanying increases in 'sickness behavior' and elevations of IL-6 in rodents., Reassuringly, we were able to confirm and extend these observations. IL-1β (50-200 ng, 2 h pretreatment, i.c.v.) produced dose-dependent increases in immobility time as analyzed by one-way ANOVA with Dunnett's post hoc comparisons in the tail suspension test (F(3,82)=4.864; P <0.01 vs vehicle; ) and forced swim test (F(3,68)=3.251; P <0.05 vs vehicle; ), an effect suggestive of a prodepressive profile. IL-1β-treated mice also demonstrated dose-related increases in sickness behaviors (F(3,59)=41.54; P <0.0001 vs vehicle; ). Cortex homogenates of mice administered IL-1β (100 ng, i.c.v.) revealed significant increases in IL-1β protein levels relative to vehicle-infused controls ( t -test: P <0.001 vs vehicle; ), as well as a significant increases in IL-6 protein levels ( t -test: P <0.001 vs vehicle; ).
To understand the role of IL-6 in the depressive and sickness behavior observed after administration of IL-1β, we evaluated the direct central effects of IL-6. Central administration of IL-6 (500-1000 ng, i.c.v.; 2 h pretreatment) to naïve mice produced dose-dependent increases in immobility time as analyzed by one-way ANOVA with Dunnett's post hoc comparisons in the tail suspension (F(3,37)=2.998; P <0.05 vs vehicle; ) and forced swim tests (F(3,48)=2.272; P <0.05 vs vehicle; ), indicative of depressive-like activity of IL-6. Central administration of IL-6 at doses that increased immobility did not produce significant increases in sickness behaviors (F(3,37)=1.342; P >0.05 vs vehicle; ) or alterations in general exploratory behavior in an open field ( ). Importantly, as measured by multiplex ELISA, cortex homogenates revealed the expected increase in IL-6 protein levels in IL-6-treated mice (1 µg, i.c.v.) relative to vehicle controls ( P <0.05 vs vehicle; ), which were not accompanied by significant increases in IL-1β protein ( P >0.05 vs vehicle; ). The duration of depressive-like behavior in the tail suspension test following IL-6 a...
Machine-readable layer
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"item": "https://replicatescience.com/experiments/evidence-for-sustained-elevation-of-il-6-in-the-cns-as-a-key-contributor-of-depressive-like-phenotypes-methods-s-j-sukoff-rizzo-pmc3565187/evidence-for-sustained-elevation-of-il-6-in-the-cns-as-a-key-contributor-of-depressive-like-phenotyp-mlph391d"
}
]
}
]