Animals
Forty adult male Sprague Dawley rats (Charles River, Quebec, Canada) weighing approximately 350-400 grams were used. Rats were double-housed in polypropylene cages until surgery after which they were housed singly. Both the housing room and experimental room were kept on a reverse 12-hour light/dark cycle. Due to the duration of the present study, rats were food restricted, receiving 15 g of 18% chow per day to maintain weights. They were handled for 5 minutes per day for five days before the beginning of the experiments. All the protocols were in accordance with the guidelines set out by the Animal Care Committee (ACC) at the University of Guelph.
Constructs
AAV8-hSYN1-GSK-3β(S9A)-HA-WPRE and AAV8-hSYN1-HA-WPRE viruses were generated by Vector Biolabs (Malvern, PA). Construct size restrictions for the AAV required the use of an HA tag with GSK-3β. The GSK-3β S9A pcDNA3 construct was a gift from Jim Woodgett (Addgene plasmid # 14754 ; http://n2t.net/addgene:14754 ; RRID:Addgene_14754) (Stambolic and Woodget, 1994). The constitutively active GSK-3β(S9A) was generated by a point mutation in serine 9 converting it to adenine, thus preventing Akt-mediated phosphorylation and inhibition of GSK-3β.
Surgery
Rats underwent stereotaxic surgery to introduce the AAV8-hSYN1-GSK-3β(S9A)-HA-WPRE or control virus bilaterally into the prelimbic region of PFC or ventral HIP. Isoflurane was used to anesthetize the rats at 5% induction and 2.5% maintenance and body temperature maintained at 37 οC using a thermostat regulated heating pad. Animals were injected subcutaneously with 0.9 % saline (3 mL) to keep them hydrated during surgeries, 5mg/ml carprofen (0.4 mL) as well as a lidocaine/bupivacaine injection at the incision site. Injection coordinates to the PFC (AP +3.24, ML ±0.6, DV 3.5) or ventral HIP (AP -5.5, ML ±5.1, DV 7.6 & 5.6) were obtained from Paxinos and Watson (2013). AAV was infused at a rate of 0.3μl/min and syringes were not removed for 5 minutes post-injection to avoid backflow of the virus to the surface of the skull. Four weeks following the AAV infusion surgery rats underwent a second surgery to implant electrodes bilaterally into the PFC and HIP at the same coordinates as the AAV infusion. Custom electrode microarrays were built using pre-fabricated Delrin templates and polyimide-insulated stainless-steel wires (A-M Systems: 791600, 0.008”). All arrays used had an electrode impedance of less than 2MΩ. Local field potential (LFP) recordings were collected and placements verified at the end of the study.
Electrophysiology
Four days post electrode implantation surgery animals underwent five minutes habituation in transparent plexiglass recording chambers (45cm x 45cm x 45cm) for four days. Following that, LFP recordings were taken from freely moving animals for 30 minutes using a wireless W2100 system (MultiChannel Systems) and were recorded at a sampling frequency of 1000 Hz. The spectral power of LFP oscillations in each region, coherence between regions, and cross correlation analysis was performed using routines from the Chronux software package for MATLAB (MathWorks). Recordings were downsampled, segmented, detrended and low-pass filtered to remove high frequencies greater than 100 Hz. Continuous multitaper spectral power and coherence (tapers = [5 9]) between regions was calculated for each segment in the following frequency bands: delta (1–4 Hz), theta (>4–12 Hz), beta (>12–32 Hz), slow gamma (>32–59 Hz), and fast gamma (>61–100 Hz). Electrode placements were verified post-mortem.
Behavioural Tests
Four days after the second surgery, rat groups underwent five minutes of habituation to an open field arena every day for four days. Animals then underwent three different tests in this arena that included the Novel Object Recognition task (NOR), Object Location (OL) and Object in Place test (OiP). Discrimination ratio was used as the output variable for these tests which was calculated as the difference between the time spent exploring the novel object and familiar object divided by the sum of both times. Animals also underwent tests for spatial memory and reversal learning in the T-maze.
Novel Object Recognition
The NOR task was used to evaluate recognition memory and was conducted in a square opaque open field arena (1 m2). The NOR task was comprised of an acquisition phase and a test phase that were separated by a 2 hour delay. Rats were allowed explore two identical objects placed in two corners of the arena. During the delay time, objects were again cleaned and placed in the same position, however one of the objects was switched with a novel object. Object types were randomized, and positions counterbalanced between rats. The time spent exploring the novel object was be compared to the time spent exploring the familiar object.
Object Location
To evaluate spatial memory the OL task was used and was comprised of an acquisition phase and a test phase, three minutes in length each, that were separated by 5-minute delay. In the acquisition phase rats were allowed to explore two similar objects placed in the corners of the arena. During the delay period, the two objects were cleaned with 10% ethanol. For the testing phase, one object was relocated to opposite corner in which it was originally place. The position of the objects was counterbalanced between animals. In this test phase the time spent exploring the object in the changed position was compared to the time spent exploring the object in the original position.
Object in Place
The OiP task, used to test associative object recognition memory, requires coordinated PFC and HIP communication [45]. The task was comprised of two phases, an acquisition phase as well as a test phase separated by a 10 minute delay. During the acquisition phase animals were placed in the open field and allowed to explore four different objects for five minutes. In the test phase, two objects were switched positions and the other two remained unmoved. Rats were then allowed three minutes to investigate the objects. The time spent exploring the objects in the changed positions was compared to the time spent exploring the objects in the original positions. Object locations were counterbalanced between rats and cleaned between trials with 10% ethanol. All trials were recorded using AnyMaze software (Stoelting).
Reversal Learning
This test was comprised of a four-day training phase with a test of reversal learning on the fifth day. Prior to testing, animals were habituated to the T-maze for four days by raising all doors and baiting both food wells. In the training phase, only one of the two food wells were baited, and animals received four trials daily for four days with a minimum of 10 minutes between each trial. The time required to reach the baited food well was recorded and used to calculate the cut-off time for the reversal testing phase (3 seconds). In the reversal learning phase, the bait was placed in the opposing food well and the number of trials to needed reach three consecutive successes within the cut-off time recorded.
Immunohistochemistry
Fluorescence immunohistochemistry was performed as done previously [46] on PFA-fixed free-floating brain sections (30µm). Free-floating sections were first washed in TBS (60.5 mM Tris, 87.6 mM NaCl ph 7.6), then blocked for 2 hours (10% goat serum, 1% BSA, 0.2% Triton-X, 1X TBS) and incubated with rabbit anti-GSK3β, rabbit anti-HA and mouse anti-phospho tau (AT8) primary antibodies (source: Cell Signalling) for 60 hours at 4 ºC. Following the primary incubation, sections were washed in TBS, blocked (5% goat serum, 0.5% BSA, 0.01% Triton-X, 1X TBS), and incubated in anti-mouse-Alexa 488 and Alexa 594 anti-rabbit secondary antibody for 2 hours. Brain slices were then washed three times in TBS and mounted on slides using Prolong Gold (Thermo Fisher Scientific). Fluorescence microscopy (Etaluma Lumascope) was used to evaluate construct expression in the PFC and HIP. Control brains that received GFP injection were sectioned, mounted on slides and visualized directly under the fluorescence microscopy.
Data Analysis
For LFP data, power and coherence curves are presented as normalized data with jackknife estimates of SEM. Data may log-transformed to better exhibit group differences as indicated. Quantification of the LFP power and coherence data at each frequency measure is reported as means ± sem for between group comparisons, or as percent change from baseline for within group comparisons. LFP power data analysis were performed using independent samples t-test. Data analysis for the behaviours were performed using student’s t-test, with the exception of the T-maze training where a repeated measure ANOVA was used, followed by student’s t test for comparisons at each day of training. For the IHC data, baseline sampling was taken outside the regions of construct expression with paired t-tests used for analysis. Prior to all analyses, normality was assessed using the Shapiro-Wilk test and Levene’s test for equality of variance. Computations were performed using IBM SPSS 24 software.