SEGUNDA PARTE DE LA LEY: MODIFICACIÓN DEL ARTÍCULO 2014 DEL CÓDIGO CIVIL

The subject was first trained to climb voluntarily into a primate chair. The monkey was made familiar with being situated in the chair for an initial period of 30- 60 minutes, which later was extended up to three hours, due to an increasing complexity of the training session and tasks to be learned by the monkey. This was achieved by restricting and closely controlling the amount of water and fruit available in the home cage (deprivation up to 12 hrs prior to training/recording session) and rewarding the monkey in the primate chair with liquids and fruits. One of the principal aims was to train the monkey on a colour discrimination task where a green LED light signalled reward and a red LED light instructed the monkey to withhold behavioural responses (see below).

To familiarise the monkey with the discrimination task, several basic applications were employed. The monkey was first trained to feed himself from a small plastic container held by the experimenter. It was not able to see the content of the box and was therefore forced to reach out and tap the box, resulting in the experimenter tilting it to enable the monkey to see the content and reach for the reward. Once the

monkey was able to feed himself, two coloured boxes were introduced. A red container with no food reward and a green container with a small piece of favourable food. The subject was to learn the association of the green coloured box with food reward. When the 90% correct criterion was reached, the coloured boxes were replaced with one white box having a LED (red or green light) attached to it. Now the monkey had to discriminate the colour of the LED before knowing whether the box would contained a food reward or not. Eventually, the floating LED light was distanced further and further away from the box and thereby from the monkey. Once the monkey was able to carry out the discrimination task with the LED placed at a distance of 4 m (the ultimate testing distance), the food reward was changed to liquid reward by introducing lick- tubes. The lick-tubes were attached in front of the monkey's mouth. As soon as the monkey touched the end of the lick-tubes with its tongue, the circuit between the chair and the lick-tubes closed, activating the solenoid driven pump system and resulting in liquid administration through the lick-tubes to the monkey. For this pump system to work, a very tight and secure contact between the monkey's tongue and the lick-tubes was required. Even with this occurring, the solenoid pumps were found to be relatively unreliable in delivering liquid. Since especially during the training phase, the delivery of fruit juice reward is cmcial, a new Infusion pump system (Hamilton MicroLab 941, Dundee, Scotland) was introduced. This system was found to be highly reliable in delivering the required quantity of liquid at any given time. In addition, the amount of liquid delivered could be closely controlled. And hence, during the training period, larger quantities of liquid could be delivered decreasing the amount as the monkey became familiar with the task. At first, longer periods of the LED stimulus were presented in order for the subject to learn the task. Subsequently, however, the presentation time was progressively reduced down to one second during which discrimination had to occur for possible reward. The LED stimuli were first presented in an experimenter controlled fashion, having a greater number of green stimuli than red stimuli. Once the monkey correctly carried out the discrimination task, the presentation pattern was gradually changed to a computer controlled pseudo-random order. It was also possible to manually change the LED position on the screen to five different locations (+/- 10° above or below, or +/- 15° to the left or the right of the central LED position).

General Methods 51 The final result of the training period was as follows: A short 500 ms signal tone, to indicate the start of a trial, obtained the monkey's attention. Once fixation occurred on the LED light presented on a white wall (at eye level) at a distance of four metres, the monkey's task was to discriminate the colour of the LED. Licking, while the LED light was green, resulted in fruit juice reward. The monkey was to withhold licking in order to avoid delivery of a weak saline solution while the LED was red. Towards the end of the training period the LED colour discrimination task was carried out at a high level of accuracy with fast responses (300-500 ms). This was achieved regardless of whether objects or images of objects were presented simultaneously with the LED light. At this stage, operation procedures for neurophysiological single unit experiments were prepared.

Implant construction

An implant frame was constructed from two stainless steel recording rings (internal diameter of 16 mm, outer diameter of 19 mm, 10 mm deep) fitting a David Kopf hydraulic micro-drive, and two plastic tubes (5 mm internal diameter) used for the restraining of the monkey’s head during recording sessions. These tubes were made of either PTFE or Tuffnal, since experience showed that perspex tubes are prone to cracking and breaking. Tuffnal was found to be the strongest and most lasting type of substance especially with the use of ethanol for cleaning purposes. A proportional plan (1:1) of the implant was drawn onto graph paper which was then placed underneath a glass plate on which construction of the implant occurred. On the graph, the orthogonal axes represented the saggital and interaural planes of the monkey's head. Therefore, precise positioning of the recording rings was possible. The rings were centred on the glass plate with the stereotaxic co-ordinates between 12 and 15 mm to the left and the right of the implant mid-line and between 11 and 15 mm anterior to the interaural plane. The two plastic tubes (5 mm diameter) were positioned perpendicular to the mid-line (corresponding to the interaurial plane in the monkey) in front and behind the wells with a distance of approximately 65 mm between them. This layout of the two wells and two tubes was then fixed to hold in position by cementing them down to the glass plate with dental acrylate (Autenal Dental Products Ltd., Harrow, England). Small amounts of liquid dental acrylic was applied to the wells and the glass, making

sure that the acrylic was not placed higher than 2 mm on the walls of the rings allowing good grip for the micro-drive at a later stage. The plastic tubes were fixed in position in a similar way to the glass. By joining up the dental acrylic between all four parts in a cross-like fashion, the base of the implant was formed.

Once the dental acrylic hardened, small quantities of water were used to 'float- off the implant from the glass. The implant was now readily prepared for the operation and therefore placed with the estimated anterior and posterior co-ordinates in a sterotaxic holder (David Kopf Instruments, California).

Sureerv

Twenty-four hours prior to the surgical operation, the subject was taken off liquid and food. On the day of the operation, the monkey was given an intra-muscular injection of 1 ml of atrophine to reduce mucous secretion, 1 ml dupocilin to reduce bacterial infection and sedated with a weight dependent dose of ketamine (Vetalar, 10 mg/kg. Park Davis and Co., Gwent). Once sedated, the head was closely shaven and swabbed with alcohol and iodine. In addition, a drop of paraffin oil (Atropine) was placed in each eye of the monkey to protect and avoid the drying of the eyes. Finally, a barbituate anaesthetic (Sagatal, 60 mg/ml. May and Barker Ltd., Dagenham) was administered intravenously. The usage of a Butterfly vein set (JERUM, Surflo IV catheter; obtained through the district of St. Andrews vet Dr. Wilson) connected by a infusion set (Bioset) to a Hartmann's solution drip (compound sodium lactate, Baxter) was introduced to aid the administration of anaesthetic drugs during the operation. Once the cessation of the gabella reflex took place, the monkey's head was carefully positioned in the David Kopf stereotaxic frame. Under full sterile conditions the operation proceeded. To monitor and regulate the monkey’s body state, a breathing counter and a rectal thermometer linked to a heating plate were set up. The depth of anaesthetic level were closely monitored by regularly checking the breathing rate (30- 40 per min) and stretch reflexes.

By making an anterior-posterior incision, the skull was exposed and the skin pulled back. Connective tissue covering the area of interest of the skull (equivalent to the size of the implant) was reflected and the skull cleaned. The already fixed implant was now lowered down onto the skull using the David Kopf stereotactic instrument

General Methods 53 set-up with the predetermined co-ordinates (which are to be such that the implant is 0° lateral/medial and 0° anterior/posterior). The relative size and position of the recording rings were marked on the skull and the implant was temporarily raised. Using an electric drill (constantly applying distilled water to prevent an increase in bone temperature), the bone underlying the two ring marks were removed, leaving the dura intact and thereby establishing a cranial defect. The implant was once again lowered down onto the exposed skull by the stereotaxic apparatus. To be able to fix the implant securely in position onto the skull, several small elongated holes were drilled into the skull for the insertion of 6-8 small (1.0 cm in length, with two cross-bars of approximately 1.0 cm in length and 0.3 cm wide) stainless steel H-shaped pieces which were subsequently locked into position. In addition, 0.5 cm long stainless steel screws fixed to the skull and stainless steel wire were added around the implant. Finally, a larger mass of liquid dental acrylic was once more applied to the implant frame, the screws, the wires and to the skull to securely fixate the implant.

A small amount of topical antibiotic (PEP, 3% powder) (Intervet Laboratories Ltd., England) was placed onto the exposed dura in the wells and around the implant. The well rings were then covered with plastic aerated caps.

The monkey was returned to its home cage where it regained consciousness after approximately two hours and was allowed to recover from surgery. Re-training sessions started after one to two weeks after the operation until pre-surgical performance in the colour discrimination task was achieved. Restraining of the head was introduced gradually by inserting two metal rods through the plastic tubes of the implant and fixing them to the primate chair.