Twenty-four adult male Lister-hooded rats (Harlan, UK) approximately 3 months of age on starting the experiment were used in this study. During the training and testing periods, animals were kept under a restricted diet with access to 15-18g of standard laboratory chow per day in addition to sucrose pellets earned during the training sessions (~5g, per day). Water was
availablead libitum. Animals were kept in a 12 hours light/dark cycle with lights off at 7:00pm. They were pair-housed in 25x45x15cm solid-base plastic
cages with wire-mesh ceiling, in a temperature and humidity controlled room. All procedures used in this study were performed under licence in accordance with the UK Animals (Scientific Procedures) Act 1986.
3.3.2. Apparatus
Behavioural training and testing was carried out in eight standard 9-hole operant chambers (CeNes Ltd., Cambridge, U.K., see Figure 3.1). The chambers were situated within sound attenuated cubicles supplied with fans. A 9-hole array was situated at the rear wall of each chamber. For the purpose of the present experiment only the central 3 holes of the 9-hole array were used. A light bulb was situated deep inside each hole. Behaviour (nose pokes and withdrawals) was recorded through infra-red beams at the entrance of each hole. A food magazine, accessible via a plastic flap, was situated on the wall opposite the 9-hole array. The floor of the chamber was comprised of a metal grid. A speaker and a 6V, 3W, house light were situated on the ceiling of the chamber. An automatic pellet-dispenser delivered sucrose pellets (Noyes Precision Pellets, PJAI-0045, Formula A/I, Noyes, New Jersey) into the food magazine following correct responses. A PC486 computer equipped with a SPIDER extension for online control (Paul Fray Ltd, Cambridge, UK) controlled the equipment and recorded responses.
Figure 3.1. Photograph of the 9-hole box chamber
3.3.3. Behavioural Training Regimen
Rats had to go through several stages of training before the final testing protocol could commence. The first stage of training involved the habituation with the operant chambers. Rats were introduced to the chambers, the food trays of which were filled with 20-25 sucrose pellets, in order to accustom the animals with pushing the panel to gain access to the food. During the second stage of training, rats were trained to make sustained nose pokes for a
standard delay of 0.2 seconds in the central hole of the array when illuminated (the remaining 8 holes were covered). After each successful nose poke, the light in the central hole was switched off and a sucrose pellet was delivered in the, now illuminated, tray behind the animal. After the animal made a panel push to gain access to the pellet, the tray lamp was switched off and the one
in the central hole was switched on again, indicating the beginning of a new trial. Early withdrawals from the central hole (i.e. before the lapse of the 0.2 second foreperiod) were penalised with a 2-seconds timeout during which all lights in the chamber were kept switched off. When all animals demonstrated a reliable ability to sustain successful nose-pokes for more than 70% of the trials per training session, they were moved to the next training stage.
The third training stage involved a brightness discrimination task (see Figure 3.2A). After each successful central nose-poke, a pair of lights was presented for a 1.5-second period in the two (left and right) side holes, which were now open. On the basis of the brightness of these lights rats were required to make a response by poking either to the left or right hole. More specifically, a nose-poke to the right hole was required when the lights were bright, whereas a left nose-poke was required when the lights were dim. A sucrose pellet was delivered in the tray after the completion of a correct trial. Parallel*to the brightness discrimination task, all rats were also trained in a tone
discrimination task (see Figure 3.2B). For this task, rats were required to make a left nose poke when presented with a continuous tone and a right nose poke when presented with an intermitted tone (both presented for a 1.5- second period, following a successful central nose-poke). Initially, the visual and auditory discrimination tasks were carried out in separate,unimodal,
*
It has to be noted however that auditory discrimination training started 2 weeks after training for the visual discrimination task started. The reason we chose not to introduce the two tasks simultaneously was to avoid overwhelming the animals. The abrupt introduction of two difficult tasks could have resulted in a reluctance on the animals’ behalf in performing them. We chose to introduce the visual discrimination task first due to the fact that the visual stimuli were presented inside the response holes and would therefore establish a stimulus-response association more readily than the auditory stimuli. Once this association was established for
blocks of 120 trials. That meant that animals were trained in the visual and auditory tasks on alternating days (visual-auditory-visual-auditory, etc). When animals reached, on average, a stable performance of more than 70%
accuracy for light discrimination blocks and 60% for tone discrimination blocks, they moved to the final stage of training which involved blocks of randomly alternating visual and auditory trials (bimodalblocks). More
specifically, within a bimodal block the modality of a given trial was randomly determined and thus unpredictable. This final manipulation intended to “divide” rats’ attention between the two modalities in order to detect the stimulus as quickly and efficiently as possible. When performance was stable and accuracy was at least 70% for the visual and 60% for the auditory
discrimination trials, in both the unimodal and bimodal condition, rats
proceeded to surgery. For all training stages, a session was terminated after 120 successful trials or otherwise after 30 minutes. Training was carried out daily, during the light cycle. Training lasted 21 weeks in total.
Figure 3.2. Graphic representation of the visual (A) and auditory (B) discrimination tasks
3.3.4. Surgery
Surgery was performed under isoflurane anaesthesia (induction: 5%,
maintenance 2%). Rats were placed in a stereoscopic frame (Kopf, Tujunga, CA) with atraumatic ear bars. The incision bar was adjusted at 3.3 (flat skull). The head was shaved, loose hairs removed with duct tape, and the shaved area was cleaned with saline and 70% alcohol solution. Prior to any invasive procedures, a 0.05ml injection of the anti-inflammatory Carprofen (Rimadyl™) was given sub-cutaneously. A midline incision was made on the scalp to expose the skull. Using a dental drill (Volvere GX, NE22L, Tokyo, Japan) a 0.3 x 1 cm rectangular craniotomy perpendicular to the sagittal suture was made from about –1.5 to –4.5mm from bregma. The piece of skull within the craniotomy was removed and the area underneath was cleaned carefully with saline-soaked cotton buds and pieces of absorbable gelatine sponge
(Spongostan, Ferrosan, Denmark). In addition, the edges of the craniotomy
1. Animal nose-