The impact of anterior thalamic lesions on active and passive spatial learning in stimulus controlled environments: geometric cues and pattern arrangement.

General Method

The study involved three experiments, each with a different cohort of rats. The first experiment examined passive location learning in a rectangular pool. The next two experiments both compared passive with active learning in a square pool where the appearance of the walls signified the correct location. In all cases, the test pool (rectangular or square) was set within a larger circular pool. The test pool was rotated within the circular pool after every trial to ensure that the rats did not use cues beyond the arena to solve the tasks.

The three experiments involved separate cohorts of adult male Lister Hooded rats (Cohort 1, Harlan, Bicester, U.K.; Cohort 2 and Cohort 3, Charles River, Kent, U.K.). The total numbers of rats were as follows; Experiment 1, n = 25; Experiment 2, n = 27; Experiment 3, n = 25. The rats weighed 270 to 320 g (Exp. 1–3) at the beginning of the experiment and were housed in pairs under a 12-hr light/dark cycle. The animals were given free access to food and water for the duration of the experiments. The rats either sustained bilateral lesions of the anterior thalamic nuclei (ATNx1 = 15; ATNx2 = 14; ATNx3 = 10) or sham surgeries (Sham1 = 10; Sham2 = 13; Sham3 = 15). All animals were habituated to handling before the start of the first experiment. All experiments were performed in accordance with the U.K. Animals (Scientific Procedures) Act (1986) and associated guidelines, as well as EU directive 2010/63/EU. The study was also been approved by local ethical review committees at Cardiff University.

Surgery

For Cohorts 1 and 2 the surgeries were performed under pentobarbitone sodium anesthesia (60 mg/kg i.p., Sigma-Aldrich Company Ltd, Dorset, U.K.). Once anesthetized, the animal was placed in the head-holder of the stereotaxic apparatus (Kopf Instruments, CA) with the incisor bar adjusted to +5.0 relative to the horizontal plane. Following an incision, the scalp was retracted to expose the skull. A craniotomy was made and the dura cut exposing the cortex above the target location. Lesions to the anterior thalamic nuclei were made by injecting 0.12M N-methyl-D-aspartic acid (NMDA; Sigma Chemicals U.K.) dissolved in sterile phosphate buffer (ph 7.4) over two separate sites within one hemisphere with the use of a 1-μl Hamilton syringe (Hamilton, Switzerland) that was attached to a moveable arm mounted on the stereotaxic frame. The lateral and medial sites were infused with 0.22 μl or 0.24 μl of NMDA over a period of five minutes, respectively. The syringe was left in situ for an additional four minutes before being retracted. The lesion coordinates for the ATNx1 group relative to bregma were anteroposterior (AP) −0.6; mediolateral (ML) ± 0.9 and ± 1.8 from the midline; dorso-ventral (DV) −7.0 and −6.3 from bregma for the medial site and the lateral site, respectively (these depth coordinates were changed to −7.1 and −6.4 for the ATNx2 group). For the sham surgeries, the syringe was lowered to + 0.2 above the target site for a few seconds, and then removed. No NMDA was injected in these rats.

Minor refinements were made to the surgical procedures for Cohort 3. For 21 of the 25 rats the surgery was performed under an isoflurane-oxygen mixture (1.5–2.5% isoflurane) with a reduced dose of sodium pentobarbital (14 mg/kg, i.p) when the surgery was nearly completed. For the remaining four rats the surgery was performed entirely under sodium pentobarbital anesthesia (i.e., like Cohorts 1 and 2). The injection site coordinates for Cohort 3 were as follows: medial injections, AP −0.1, ML ± 0.8, DV −6.8; lateral injections, AP −0.4, ML ± 1.5, DV −6.2. Each of the medial injections consisted of 0.20 μl of 0.12M NMDA while the more lateral injections consisted of 0.18 μl of 0.12M NMDA.

After removal of the Hamilton syringe, the incision was cleaned and sutured. A topical antibiotic powder (Aureomycin, Fort Dodge, Animal Health, Southampton, U.K.) was applied. The rats received glucose-saline (5 ml s.c.) for fluid replacement and were then placed in a recovery chamber until they regained consciousness. Rats were given the analgesic Metacam (0.06 ml s.c.; 5 mg/ml meloxicam; Boehringer Ingelheim Vetmedica, Germany). A respiratory stimulant millophylline (0.1 ml s.c., Arnolds Veterinary Products, Shropshire, U.K.), an antimicrobial Baytril in their water (2.5%; Bayer Ltd, Animal Health Division, Ireland), and low dose of diazepam (0.07 ml s.c., 5 mg/ml; CP Pharmaceuticals Ltd, U.K.) was administered to facilitate postoperative recovery as advised. All animals were monitored carefully until they had fully recovered.

Histology

After behavioral testing, the animals were administered with an intraperitoneal injection of a lethal overdose of Euthatal (200 mg/ml sodium pentobarbital, Marial Animal Health Ltd., Harlow, Essex, U.K.) and perfused intracardially with 0.1M phosphate buffer saline (PBS) followed by 4% paraformaldehyde in 0.1M PBS (PFA). The brains were extracted from the skull and placed on a stirrer to postfix in PFA for four hours, after which the brains were placed in 25% sucrose overnight. The brains were frozen on a microtome (Leica, U.K.) and sectioned at 40 μm in the coronal plane. One-in-five sections were mounted and stained with cresyl violet, a Nissl stain.

Volumetric Analysis

The extent of the lesions in the anterior thalamic nuclei was first drawn by hand onto five equidistant coronal sections ( Paxinos & Watson, 2005 ). Any unintended hippocampal damage was also plotted onto the appropriate subset of sections from a series of 20 equidistant coronal plates ( Paxinos & Watson, 2005 ). These images were scanned, and the area of damage was quantified using the program analySIS^D (Soft-Imaging Systems, Olympus). The percent damage to the anterior thalamic nuclei and to the hippocampus was quantified by taking the area of damage within the region of interest and dividing it by the total area of that region summed across each drawing.

Behavioral Testing

Both Cohort 1 and Cohort 2 received other spatial and nonspatial behavioral testing, but none was in a swim pool. Previous tasks given to Cohort 1 involved the following: object recognition memory and object recency memory, T maze alternation, and a contextual biconditional discrimination task ( Dumont & Aggleton, 2013 ; Dumont et al., 2014 ). Cohort 2 was previously trained on a spatial go/no-go discrimination that involved digging for food rewards in different locations and had also learnt a spatial biconditional problem in a separate room from that used in the current study ( Dumont et al., 2014 ). Cohort 3 had learnt a series of force-choice nonspatial discriminations involving different odors and digging media, along with an automated task based on the Stroop test ( Haddon & Killcross, 2005 , 2006 ). The rats were approximately 12 to 13 months (Cohort 1), 10 months (Cohort 2), and 7 to 8 months (Cohort 3) old at the start of the experiments reported below.

Procedure

The ATNx1 and Sham1 rats completed one session of four training trials each day. For each session they were carried into a room adjacent to the test room in groups of five in a light-tight aluminum carrying box and remained in this box between trials. For each trial, the rat was carried from the box to the pool and placed on the platform. The rat was allowed to stay on the platform for 30 s, undisturbed, before being removed, dried and returned to the holding box.

Pretraining

Pretraining (three sessions) was designed to discourage the rats from stepping off the platform during the placement sessions. For these sessions the escape platform was placed in a quadrant (NE, NW, SW, or SE) in the circular pool, that is, not the rectangular pool used in the experiment proper. Each location was used once in a session. The platform was randomly positioned either 25 cm or 50 cm from the edge of the pool, each for two trials per session. The rats were placed on the platform for 30 s. If a rat stepped off the platform and did not immediately climb back unto the platform, the experimenter indicated the location of the platform by tapping on the escape platform. If the rat still failed to return to the platform, the experimenter would guide the rat (they would follow the experimenter’s hand through the water) back to the platform, where the rat remained for 30 s.

Training

The rats next received 12 sessions of training in the rectangular pool ( Figure 1A ). The platform was positioned 25 cm from a corner on an imaginary line that bisected the corner. The position of the platform was counterbalanced, so that half of the rats from each group had the platform placed in a corner where the short wall was to the right of the long walls and the other half received the platform in the corner where the short wall was to the left of the long wall (see Figure 1A ). Between each trial, the rectangular pool was randomly rotated 90°, 180°, or 270° clockwise. Four possible orientations were used (North, South, East, or West) with each orientation being used once for any given session (see Figure 1A ). Similar to pretraining, the rats were placed on an escape platform. If any rat fell into the pool and failed to climb back onto the platform immediately, the experimenter would remove the rat from the pool and return it to the platform.

The first three trials of the final session, Session 12, were conducted in the same manner as previous trials. The fourth trial consisted of a Probe Test. The platform was removed and the rats were released into the water in the center of the rectangle, facing away from the experimenter. Each rat was allowed to swim for 60 s.