Sukoff Rizzo et al. IL-6 mediates depressive behaviors in the CNS

Supplementary Information

SUPPLEMENTARY METHODS

Learned Helplessness

All testing was conducted in commercially available shuttle boxes enclosed in sound-attenuated chambers (Med Associates, Vermont, USA). The shuttle boxes were separated into two equal compartments with a retractable door and fitted with an electrified grid floor through which a scrambled electroshock was delivered. Med-PC software (Med Associates, Vermont, USA) was employed to detect the location of the subject within the shuttle box using infrared beams and to deliver the footshock per the software program. On day 1, subjects were sequestered to only one side of the shuttle box and were administered a series of inescapable footshocks (60 shocks, 0.8mA, 15 sec duration, 2 sec ITI) 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. During each trial, the presentation of footshock was preceded by light and tone cues for a 3 sec duration and the opening of the center door. Movement into the opposite side of the shuttle box during this 3 second cue period (avoidance) prevented the commencement of the footshock (0.6mA, 5 sec duration), triggered the closing of the center door, and initiated the ITI (30 sec). Crossing over to the opposite side during the 5 sec shock presentation (escape) terminated the shock, triggered the closing of the center door, and initiated the ITI. Failure to cross over either before or during the shock presentation was an indication of helplessness. Data were analyzed as the mean % failures per subject for the last 10 trials of the test day only (Day 2). Subjects with a failure rate of 50% were defined as helpless.

Social Competition Paradigm of Dominant/Submissive Behavior

Six week old male Sprague-Dawley rats were randomly pair-housed upon arrival to the facility. Pairs were left undisturbed with the exception of routine husbandry for a two week period through sexual maturation to allow for the natural homecage dominant-submissive relationship to form. At eight weeks of age pair-housed subjects (non-food restricted) were trained individually in operant chambers (Med Associates, Vermont USA) to anticipate the presentation of a palatable reward (sugar pellet; BioServ, USA) following a series of stimulus light cues. Each 15 minute training session consisted of 15 trials each initiated by a 50 sec ITI followed by illumination of a stimulus light on the rear chamber wall, then followed 5 sec later by illumination of a stimulus light on the opposite wall directly above the feeder. Five seconds after the second stimulus, the sugar pellet was presented and paired with an audio feedback click. Collection of the food pellet initiated the ITI for the next trial during which the chamber was dark and there were no programmed consequences. A priori criteria established for successful training before subjects were transitioned to the testing phase required a response time of <3 seconds from presentation of the reward to collection for 3 consecutive training days. The testing paradigm was identical to the training paradigm in which each trial consisted of two 5-sec presentations of the stimulus lights followed by the presentation of the sugar pellet paired with an audio feedback click. During the testing phase both cagemates were placed within the operant chamber and assessed for dominant/submissive behavior by a trained observer. The rat within the pair which obtained the food pellet was declared the winner and the other rat the loser. Dominance was expressed as the rat within each pair which consistently demonstrated the dominance of wins (>60%) in daily sessions over the course of a 6 week period.

Assessment of exploratory activity: VersaMax Animal Activity Monitoring Chambers (AccuScan Instruments, Columbus, OH, USA) were used to assess alterations in general exploratory and locomotor activity following central administration of IL-6 or vehicle control. Chambers were housed in a testing room with similar environmental conditions to the housing room (~400lux and 60-70dB background noise). Mice were habituated to the testing room for a 30 minutehabituation period prior to testing. Following habituation, mice were placed into individual open field chambers and behavior was recorded in the open field (40 x 40 x 40 cm) for a 60 min period.

Assessment of SERT levels and Receptor Occupancy: Adult male MRL or LPR mice (n=3 per treatment) were administered a single dose of either vehicle or fluoxetine (3 or 10 mg/kg, 60 min pre, i.p.). Subjects were euthanized and brain tissue (hippocampus and prefrontal cortex) were immediately dissected and stored at -80°C. The brain regions were subsequently homogenized in 40 volumes of assay buffer (50 mM Tris, 120 mM NaCl, and 5 mM KCl; pH 7.5) with a Polytron for 10 seconds. Ex-vivo [3H]-DASB binding studies were conducted in 96-well microtiter plates in a total volume of 250 μl containing 5 nM [3H]-DASB. Non-specific binding was defined with 10 μM fluoxetine. The binding assay was initiated by the addition of 50 μl of tissue homogenate (1.25 mg wet wt/well) and incubated at room temperature for one hour. The reaction was terminated by rapid vacuum filtration through pre-soaked (0.5% polyethylenimine) Whatman GF/B filter mats. Filters were washed with ice-cold 50 mM Tris, pH 7.5 and transferred to scintillation vials containing Ultima Gold LSC scintillation cocktail. Radioactivity was measured using the PerkinElmer TriCarb liquid scintillation counter. Absolute receptor level was calculated as the inhibition of specific [3H]-DASB binding (total binding – nonspecific binding), expressed as disintegrations per minute (dpm). Receptor occupancy was calculated as the percent inhibition of specific [3H]-DASB binding for fluoxetine treated subjects relative to vehicle treated controls as follows: % Receptor Occupancy = (1-(specific binding with fluoxetine) / (specific binding with vehicle))*100).

Pharmacokinetics: Whole blood was collected via cardiac puncture under isoflourane anesthesia into EDTA anti-coagulant coated tubes and centrifuged@ 4C for 20 min x 3500g. Brain tissue was dissected and rinsed in PBS, blotted dry, and stored at -80C. Plasma samples and standards were subjected to protein precipitation with acetonitrile containing an internal standard at 4X the sample volume. Samples were vortexed and centrifuged to obtain supernatant which was analyzed using LC-MS/MS. Brains were homogenized at 5X dilution with 60:40 Isopropyl: water and prepared using mixed matrix with blank plasma and subject to protein precipitation using the same conditions as above in the plasma prep. The resultant supernatant from the precipitation was analyzed via a reverse-phase HPLC system in combination with a mass spectrometric detection system (LC-MS/MS). Pharmacokinetic parameters were determined by non-compartmental analysis using WinNonlin (Pharsight Corp) pharmacokinetic software.

SUPPLEMENTARY TABLES

SUPPLEMENTARY FIGURE LEGENDS

SF1. No significant differences in exploratory activity in the open field in naïve Swiss Webster mice following central administration of recombinant mouse IL-6 (2 hr pre, i.c.v.). Data are presented as mean ± s.e.m.; n=10 per treatment group.

SF2. No significant differences in exploratory activity in the open field in naïve Swiss Webster mice following central administration of recombinant mouse IL-6 (24 hr pre, i.c.v.). Data are presented as mean ± s.e.m.; n=7 per treatment group.

SF3. Representative images of western blots (see statistical results in main figure 3G-3J. Twenty-four hour pretreatment of recombinant mouse IL-6 (1ug, i.c.v.) to Swiss Webster mice produces significant increases in IL-6 protein levels in hippocampus, hypothalamus, frontal cortex, and a non-significant increase in striatal IL-6 protein levels relative to vehicle infused controls. Membranes were cut at the ~28 kDA band which allowed simultaneous probing for the protein of interest (IL-6 @ ~ 21 kDA) and the control protein (actin @ ~45 kDA) on the same gel (within sample control; n=4 per treatment group).

1