Materials and Methods
Animals and Housing
24 male C57Bl/6 mice were obtained from Animal Resources Centre (Murdoch, Western Australia) after weaning at 4 weeks of age. Mice were randomly assigned to one of two conditions with water ad libitum: standard-housed (SH) or environmentally enriched (EE). SH mice were housed in open top standard mouse cages (34 x 16 x 16 cm) with basic nesting materials, with 3 mice per box. EE mice were housed in larger cages (40 x 28 x 18 cm) with nesting material and six objects (climbing apparatus, floor gratings, tunnels, shelters, hanging toys and random objects) changed once per week. Animals were not re-exposed to the same objects until 20 weeks of the cycle had elapsed. No running wheels were used in this paradigm. Four weeks of post-weaning EE were completed before behavioural testing began. The holding room was maintained on a 12:12h reversed light/dark cycle at 20±1 °C. From XXX weeks animals were weighed daily for five days and subsequently maintained at 85% of their free-feeding weight (FFW) with 1 g per week added until mice reached 13 weeks of age to prevent stunting. All procedures were approved by The Florey Institute of Mental Health and Neuroscience Animal Ethics Committee.
All animals were tested in automated touchscreen-based operant systems (Campden Instruments Ltd), with instructions and event recordings managed through the software Whisker Server and ABET II. [Expand]
VD was conducted as described previously. I brief, VD consisted of pairwise discrimination between a rewarded (S+) and unrewarded (S-) stimulus. The location of the S+ was pseudorandomised between trials, with no location appearing more than 3 times in a row. Designation of S+ and S- was pseudorandomised and counterbalanced between our housing groups. Touching the S+ initiated a reward sequence; whereas S- touches triggered a 5 second timeout, no reward provision and started a correction trial. Correction trials consisted of representation of the previous trial until a correct response is made and are not counted towards the trial limit or number of correct/incorrect responses. Each session ended once a mouse finished 30 trials or reached a 60 minute time limit (whichever came first). Criterion for the VD task was 80% correct responses for two consecutive days.
Once all mice had acquired VD, they were collectively given two further sessions of VD to ensure performance remained at criterion level. Reversal commenced the following day with an inversion of the S+ and S- designation. All other aspects of the test were identical to VD.
Trial-Unique Delayed Non-Matching to Location
After all mice had completed RL, the mice were given XX sessions of five-location Punish Incorrect trials to adjust the mice to the TUNL setup. The TUNL was then performed as described in Kim 2015
with some modifications. Each trial commenced with a sample phase, in which initiation triggered the display of a white square in one of five possible locations which disappeared upon nose-poke. After a delay a second initiation procedure was triggered by breaking the back IR beam. This commenced the choice phase, in which the two stimuli were presented: one in the old (sample, incorrect) location and the other in the new (correct) location. Touching the correct location resulted in reward delivery whereas a touch to the incorrect location resulted in a 5s timeout and commencement of a correction trial.
Mice were initially trained to criterion on the maximal spatial separation level, consisting of three blank locations (S3) between the correct and incorrect stimulus. Once an individual mouse reached 70% performance for two sessions, the level was reduced S2 and then S1 excluding . Task parameters consisted of a 2s delay, 15s ITI, 5s correction trial ITI, 5s timeout and the session finishing after 45 min or completion of 36 trials.
During Stage 2, mice continued to be trained on S1 but with the centre location added as a sample location. Mice were trained on S1 until group performance became stable and then moved collectively onto S0. When group performance stabilised the mice were moved onto probe trials. Task parameters were identical to those in Stage 1 with the exception of baseline training at a 0s delay and the session finishing after 60 minutes or completion of 48 trials.
For the pattern separation (PS) probe, S0 and S1 trials were mixed within-session until performance stabilised. For the working memory (WM) probe, mice were exposed to S1 trials with mixed sample-choice delays of 0, 3 or 6 s within sessions until performance stabilised.
-Injection stress probe
The MTEP probe was similar to that of the WM probe, except that mice were injected with 20mg/kg MTEP 10 minutes prior to the commencement of the session. Mice had alternating vehicle and MTEP injection sessions for 10 days (5 days MTEP, 5 days vehicle), counterbalanced across the housing groups.
While all mice completed the VD and RL tasks, four mice were excluded from TUNL. Two mice (one SH, one EE) failed to acquire Stage 1 criterion performance. One mouse (EE) was in a chamber with an equipment failure that prevented its progression. One mouse (EE) died due to a traumatic interaction with toys in its enrichment cage.
The effects of housing, stimulus type, location, separation level and delay on our dependent variables were examined as appropriate. Generalised linear and latent mixed models (GLLAMM) were used to calculate the odds ratio of correct selection. Median regressions were performed on the number of back beam breaks per session. All GLLAMMs and median regressions were clustered by individual animal. For VD and RL, trials to criterion and correction trials were also compared using 'traditional' statistical tests, two-tailed Mann Whitney U-tests. All statistical analyses were conducted using StataMP version 13.