SEGUIMIENTO Y MANEJO DE LAS POBLACIONES DE CONEJO

The nine-hole operant boxes were manufactured by Paul Fray Ltd (Cambridge, UK). All boxes were fitted with a house light in the ceiling, and nine-holes into which the rats could poke. Sucrose pellets were delivered into a food hopper which was covered by a hinged Plexiglas panel and positioned on the wall opposite to the nine-holes. Behind the panel was a light which could be switched on to indicate the presence of a sucrose pellet (Figure 2.5). The holes that were not used were covered by plastic dummies.

The inside length of the boxes was 26 cm. The inside width of the boxes was 25 cm in the middle of the box, and 22.5 cm on either short side of the side of the box. The inside height of the boxes was 26 cm. The stimulus holes measured 2.3x2.3 cm, and were 3 cm deep. All stimulus holes were positioned 3 cm apart. The sensors detecting nose pokes into the stimulus holes were positioned 0.5 cm into the holes. The panel measured 8x8 cm, and the food hopper located behind the panel was 3.5 cm deep with a slight indentation in the middle that caused the sucrose pellets (AIN-76A Rodent Tablet, TestDiet, UK) to naturally roll into the middle of the food hopper. The floor consisted of a metal grid which was located above a litter tray. The grids were placed 1 cm apart from each other.

61 All hardware and software controlling the boxes was manufactured by Cambridge Cognition (Cambridge, UK).

Figure 2.5. A photograph of a nine-hole operant box, with arrows indicating the location of the stimulus holes in which rats responded during testing, the food hopper from which sucrose pellets were delivered following a correct trial, and the sucrose pellet dispenser. Stimulus lights were located in the end of each stimulus hole and at the back of the Plexiglas panel covering the front of the food hopper (not visible in photo). The picture is modified from Camden Instruments‟ website.

2.7.2.1. Lateralised Choice Reaction Time task

Task overview

During LCRT task testing, only three of the stimulus holes in the nine-hole boxes were exposed and the house light was by default turned on. Each trial began with the automatic illumination of the central stimulus light. The rats were required to maintain a nose poke into the illuminated hole for a variable length of time (200, 400, 600, or 800 ms) until either of the two lateral stimulus holes lit up for 50 ms. If the rat responded into the recently illuminated stimulus hole, the panel light was turned on and a sucrose pellet was delivered. If the rat terminated the nose poke before the end of the hold, responded into the incorrect hole, or neglected to make a response within 10 s, a “time-out” occurred during which the house light was turned off for 2 s. Following both time-outs and correct responses, a 2 s long intertrial period commenced after which another trial was automatically generated. Each session lasted for 30 min. There was no limit to the number of trials a rat could complete in one session.

A usable trial was defined as a trial where the rats maintained the hold into the central stimulus hole for the required length. Reaction time was defined as the time elapsed between the illumination of the lateral stimulus light and the discontinuation of the central nose poke.

62 Movement time was defined as the time elapsed between the discontinuation of a nose poke in the central hole and execution of a nose poke into a lateral stimulus hole. Only reaction and movement times following correct responses were reported.

2.7.2.2.4. Pre-lesion training

Prior to training, rats were food restricted to between 85% and 90% of their baseline weights. In instances where rats had not reached adulthood, their target weights were adjusted as previously described (2.1.3). To avoid neophobic reactions, rats were exposed to sucrose pellets twice in their home cages prior to pre-lesion training. Sucrose pellets were placed on the floor of their home cages in the evening and the cages were checked the following morning to confirm that the sucrose pellets had been consumed. During their first exposure to the operant boxes, rats were allowed one 15 min habituation session in the boxes. During this session, sucrose pellets were left in the three stimulus holes that were used in the LCRT task to encourage explorative behaviour, while the other holes were covered by plastic dummies.

During their second exposure to the boxes the rats completed a magazine training session, during which a sucrose pellet was automatically dropped into the illuminated food hopper in the beginning of the session and another pellet was dropped following each panel entry. During magazine training, the house light was kept on but all stimulus lights were off. Magazine training sessions were 15 min long.

After rats had learned to enter the magazine for sucrose pellets, central nose poke training commenced. During such training, the central stimulus hole was automatically illuminated in the beginning of each trial. Following a nose poke into the lit hole, the stimulus light was switched off, a sucrose pellet dropped into the panel and the panel light illuminated. A new trial was automatically generated when the rats‟ entered the food hopper. Central nose poke training sessions were 30 min long and followed by general nose poke training, where rats were trained to respond to any illuminated hole regardless of its location. General nose poke training sessions followed the same format as central nose poke training, but also included the use of the two lateral stimulus lights. At the beginning of a trial, one of the three stimulus lights was chosen by the software in a pseudo-randomised order, and automatically illuminated. Responding into the illuminated hole resulted in the delivery of a sucrose pellet as described previously.

Once the nose poke training was completed the rats were trained on the LCRT task. Initially, there was no minimum time the rats‟ had to maintain their response to the central stimulus light and the lateral lights were illuminated for 2 s. As the rats learned the task, the time which they had to maintain the central hold prior to onset of a lateral light cue was

63 gradually increased, and the length of time the lateral stimulus lights were switched on for was decreased. At the end of training, rats had to maintain their central nose pokes for 200, 400, 600, or 800 ms, and the lateral lights were only illuminated for 50 ms. The length of time rats had to maintain their central nose poke was determined by the software in a pseudo- randomised order, to ensure that lateralised nose pokes were made in response to the presentation of a visual cue and not merely executed following a pre-learned time interval.