Isothesia

Sixteen adult male Sprague-Dawley rats were obtained from Charles River Labs (Wilmington, MA) weighing 250–300 g upon arrival. Rats were housed individually in plastic tub cages with free access to food and water and a short length of autoclaved manzanita wood for enrichment in the Boston College Animal Care Facility. The vivarium maintained a 12 h light/dark cycle. All rats were allowed to acclimate to the colony housing for 7 days prior to surgery. All experimental protocols were reviewed and approved by the Boston College Institutional Animal Care and Use Committee.
Under isoflurane anesthesia (3% in O², Isothesia, Henry Schein, Dublin, OH) in a stereotaxic frame, as previously (3 (link)), stainless steel guide cannulae (22 g; Plastics One, Roanoke, VA) were implanted to target the VH as in (13 (link)) at −5.8 mm posterior to bregma, ±5.2 mm from midline, and −7.0 mm ventral to the surface of the skull. A stylet was placed in each cannula, which extended 1 mm below the tip of the guide. Immediately after surgery, each rat received loxicom (1mg/kg, Eloxiject, Henry Schein) and penicillin G procaine (15,000 Units, Combi-Pen-48, Henry Schein). Behavioral testing began 7–10 days after surgery.
Microinjections were made by gently restraining the rat in a cloth towel. Stylets were removed and replaced with a microinjector that extended 1 mm beyond cannula tip (33g; Plastics One). Each rat was injected bilaterally with 1 μL of either muscimol (Sigma, St. Louis, MO) in saline (1 μg per side) or saline alone at a rate of 1 μL/min. Injectors were left in place for an additional minute to allow for diffusion. The concentration was equal to that used by Hobin et al. (13 (link)). Injections were made 1 h prior to behavioral treatments as previously (16 (link)).
Fear conditioning occurred in chambers made of black plastic with wire mesh lids 10 x 11 x 6-in (L x W x H) with a stainless steel shock grid (Model H10-11R-TC-SF, Coulbourn Instruments, Whitehall, PA) enclosed within a 15 x 12 x 27-in (L x W x H) ventilated light and sound-attenuating chamber. Two infrared LED arrays (CMVision Model IR30) illuminated the chamber, and overhead cameras (Model VX-5000, Microsoft, Redmond, WA) with the manufacture’s infrared blocking filters replaced with infrared passing filters allowed for automated freezing detection with ANY-Maze software (version 4.99, Stoelting, Wood Dale, IL) as previously (17 (link)). Stimuli were delivered by a white LED array (Model LPL620WTHD, Hampton Bay) and speaker mounted at the top of the enclosure; a ventilation fan provided masking noise at ~55dB. The conditioning stimuli were a flickering white LED light (264.0 Lux, 20ms on/off) and a white noise pip (pip duration = 10ms, interval = 3 Hz, 75dB). Assignment of light or pip to CS+ or CS− was counterbalanced. No effect of the cue stimulus was evident as both the light and pip produced equivalent fear conditioning and discrimination behavior (data not shown).
To begin conditioning, rats were transferred from the vivarium, placed in the apparatus, and conditioning trials began immediately. Conditioning trials lasted 90 seconds beginning with a 70 s blackout (inter-trial-interval) after which a 5 s, 1 kHz tone (75dB) signaled the upcoming CS presentation. Then, either the CS+ cue or the CS− cue was administered for 15 seconds. CS+ trials concluded with a 500 ms, 1 mA shock (Model H13-15, Coulbourn Instruments). Training consisted of 15 trials of each cue presented in quasi-randomized order so that one trial type never occurred more than 2 times in series. Recall tests began by placing the rat in the conditioning apparatus. After 2 minutes rats received 6, 1 min presentations of each the CS+, the CS− or the context alone in a quasi-random order.
A schematic diagram of the procedures is provided in Figure 1. On day 1, rats were randomly assigned to either muscimol or vehicle conditions, injected and returned to the homecage. 1 h later all rats received CS+/CS− conditioning. Fear recall and discrimination were assessed on Days 2 and 3 in identical tests. To test the role of VH in fear discrimination recall, all rats received additional conditioning and testing until both the vehicle and muscimol treated rats exhibited equal fear and discrimination. This required two additional drug-free CS+/CS− training sessions, which began in the afternoon on Day 3 and again on Day 4. Recall tests were given on the morning of Day 4 and Day 5 at which point all rats exhibited equal freezing and discrimination, regardless of past drug treatment. Rats were then assigned to new muscimol and vehicle groups each consisting of 4 rats from the previous muscimol group and 4 rats from the previous vehicle group. To test the role of VH in fear discrimination recall, on day 8 rats received either muscimol or vehicle, according to their new groups and 1 h later given a final recall test.
At the conclusion of the experiment rats were overdosed with tribromoethanol (Sigma), sacrificed and brains were flash frozen in 2-methylbutane on dry ice. Sections (40 μm) containing the ventral hippocampus were stained with cresyl violet and cannula placement was determined by comparison to the Rat Brain Atlas in Stereotaxic Coordinates (Paxinos and Watson, 2006). Only rats with cannula located in the ventral hippocampus were used in the statistical analysis (Figure 2).

Surgical procedures and cannula placements were conducted as previously described (Christianson et al., 2011 (link); Chen et al., 2015 (link)). Rats were anesthetized with isoflurane (3% in oxygen; Isothesia, Henry Schein, Dublin, OH) and placed in a stereotaxic apparatus. Stainless steel guide cannula (22 g; Plastics One, Roanoke, VA) were implanted bilaterally to target anterior (AP +2.7, ML ±3.9, DV −5.2), medial (AP +0.5, ML ±4.9, DV −6.2), or posterior (AP −1.8, ML ±6.5, DV − 6.2) insular cortex. All coordinate measures (mm) were taken from the skull surface at bregma. The IC can be subdivided into granular, dysgranular and agranular regions along its dorsal-ventral axis. Here, cannula were targeted for the central agranular region and the injection volume (0.5μL) was selected to permit diffusion throughout the three regions. Cannula tips found within any of the three subdivisions were included, thus conclusions from these were not intended to be specific to any of the IC subregions. Cannula were fixed to the skull with stainless steel screws and acrylic cement and a stylet extending 1mm below the tip of the guide was placed in the cannula. Rats were given 1 mg/kg Loxicom (Eloxiject, Henry Schein) and penicillin (15,000 Units, Combi-Pen-48, Henry Schein) after surgery. A minimum of 7 days of recovery were allotted before behavioral testing, during which time rats were periodically handled and stylets were checked to ensure the cannula remained unobstructed. Microinjections were made by gently restraining the rat in a cloth towel and replacing the stylet with a microinjector connected with PE-50 tubing to a 25 μL syringe (Hamilton, Reno, NV) in a micromanipulator (Model 5000, Kopf Instruments, Tujunga, CA). The injector protruded 1 mm beyond the cannula tip (33g; Plastics One, Roanoke, VA). NMDArs were blocked with receptor antagonist D-(−)-2-Amino-5-phosphonopentanoic acid (AP5). AP5 (Tocris, Minneapolis, MN) was dissolved in sterile saline at 6 μg/μL (as in (Bast et al., 2005 (link); Amat et al., 2014 (link); Christianson et al., 2014 (link)). The GABA_A agonist muscimol was used to temporarily inactivate IC. Muscimol (Sigma, St. Louis, MO) was dissolved in sterile saline at 100 ng/μL (Moscarello and LeDoux, 2013 (link)). Each drug was administered bilaterally at 0.5 μL per side at a rate of 1 μL/min, with an additional minute allowed for diffusion. Vehicle treated animals received saline injections at the same volume and rate as the drug infusions. AP5 injections were completed 15 minutes before conditioning (Bast et al., 2005 (link)) and muscimol injections were completed an hour before testing (Amat et al., 2005 (link)). At the end of each experiment, rats were overdosed with tribromoethanol (Sigma, St. Louis, MO). Brains were removed and flash-frozen in 2-methylbutane on dry ice, and stored at −80°C until they were sliced at 40 μm on a freezing cryostat (−20°C). Slices were stained with cresyl violet, coverslipped, and allowed to dry overnight before cannula placement was determined by comparison with the Rat Brain Atlas in Stereotaxic Coordinates (Paxinos and Watson, 2007 ). Data from rats for which cannulas were not found or were located outside of the targeted areas of IC were excluded in statistical analysis (see Figure 2).