El agua, principal materia prima para la explotación minera

General anaesthesia is a drug-induced state, producing a controllable and reversible loss of consciousness and an absence of motor response to noxious stimuli. Depths of anaesthesia appropriate for the conduct of surgical and experimental procedures can be achieved with a wide variety of drugs, either alone, or in combinations (Flecknell, 1987). In this study, anaesthesia was produced with halothane, a potent inhalation anaesthetic which allows rapid induction and recovery (1-3 min). Like many anaesthetic agents, halothane causes depression of the cardiovascular system and moderate hypotension at surgical levels of anaesthesia as a result of a reduction in cardiac output and peripheral vasodilation. The electrical activity of the brain shows progressive replacement of fast, low-voltage activity by slow waves of greater amplitude as halothane anaesthesia is deepened (Marshall and Longnecker, 1996). Dilation of cerebral vessels may occur causing an increase in cerebral blood flow. There is no indication that halothane interferes with energy metabolism in the brain unless excessive doses are used (Marshall and Longnecker, 1996). The undesirable effects of halothane are not severe in comparison to the profound effects of other drugs, such as barbiturates and ketamine, and therefore halothane was considered to be a good agent for induction and maintenance of anaesthesia in the rat.

Halothane was administered in a 1:1 mixture of oxygen and nitrous oxide. The latter on its own has some anaesthetic potency and causes minimal cardiovascular and

respiratory system depression which may reduce the overall degree of depression of blood pressure or respiration at a particular depth of anaesthesia. In order to minimise a possible interference of halothane anaesthesia with the processes under investigation, once the surgical procedure had been completed, the depth of anaesthesia was carefully controlled by monitoring EEG and mean arterial blood pressure (MABP).

In most experiments, especially those that required minimal surgery, animals were allowed to breath spontaneously with close monitoring of their physiological variables. A tendency towards hypoventilation was observed in halothane anaesthetised rats breathing spontaneously, but this was unlikely to have altered the changes under study and their interpretation (Kraig and Cooper, 1987). In some experiments, animals were artificially ventilated. This allowed the precise control of the duration of inspiration and expiration, the volume of gas delivered to the lungs, and of blood gas parameters. As spontaneous respiratory movements may interfere with ventilation, artificially ventilated animals were relaxed with tubocurarine, a competitive blocking agent at the neuromuscular junction (Flecknell, 1987). Since administration of tubocurarine prevented all movements in response to pain, changes in mean arterial blood pressure, heart rate and EEG were constantly monitored to ensure anaesthesia adequacy.

5 .2 .2 Surgical Preparation

The initial preparation of animals was the same for all experiments. The different surgical procedures that were performed before implantation of an ISM and/or a microdialysis probe depended on the condition under investigation and the variables being measured.

5.2.2.1 Common Surgical Preparation

Experiments were performed on adult male Sprague-Dawley or Wistar rats (Bantin and Kingman, Grimston, U.K.) with food and water freely available. All animal procedures used were in strict accordance with the Home Office guidelines and specifically licensed under the Animals (Scientific Procedures) Act, 1986. Premedication with atropine sulphate (15-30 ^g.kg ‘ i.m .) reduced bronchial and salivary secretions which may partially occlude the airways. Atropine also increases heart rate and protects the heart from vagal inhibition which can occur during endotracheal intubation or surgical procedures (Flecknell, 1987). Anaesthesia was induced and maintained with halothane (2

% and 1.0-1.5 %, respectively) in O2/N2O (1:1). A femoral artery was cannulated for

continuous monitoring of MABP and determination of arterial blood gases, pH and glycaemia. A femoral vein was cannulated for drug administration. Body temperature was maintained at 37.5-38°C throughout the experiments using a homeothermic pad.

5.2.2.2 Surgery for Artificial Ventilation

The animal was placed on it’s back and the neck extended. The skin above the trachea was incised, the underlying muscles separated, and the trachea exposed and isolated. A small incision was made across the trachea between two of the rings of cartilage and a specially prepared tube inserted and secured with 2 ligatures. In animals being prepared for ischaemia, a thread was placed dorsal to the trachea and the two ends secured loosely around the rat’s neck to form a tourniquet if required. The animal was then relaxed with tubocurarine ( 1 mg.kg ' i.v., repeated every hour) and ventilated

mechanically (75 cycles.m in'; Rodent Ventilator, Harvard Apparatus Co. Edenbridge, U.K.) with an appropriate stroke volume to maintain normocapnia.

5.2.2.3 Surgery for Transient Forebrain Ischaemia

Ischaemia was produced by occlusion of the four major cerebral vessels (Pulsinelli and Brierley, 1979). After a tracheotomy, the common carotid arteries were isolated and encircled with inflatable vascular occluders (Type OC2A, In Vivo Metric, Healdsburg, CA. U .S.A.). The tubing from the occluders was brought out of the wound and connected to a compressed air cylinder to allow remote induction of ischaemia. After closing the wound, animals were placed in the prone position with the head secured in a stereotactic frame. A dorsal cervical incision was made and the cervical muscles divided down to the first cervical vertebra. A monopolar electrode was passed down through the alar foramen on one side, and an intermittent current was applied to electrocoagulate the underlying vertebral artery (Pulsinelli and Buchan, 1988). This was repeated for the other side, and the wound closed. The skull was then exposed in preparation for a craniectomy, the position of which was determined by the region of the brain under investigation. Once surgical procedures were completed the concentration of halothane in the breathing mixture was reduced to 0 .8-1 . 2 %.

5.2.2.4 Ion-Selective Microelectrode Implantation

Pre-experimental in vitro calibrations were carried out as described in Section 3.4. A 1-2 mm craniectomy was performed 3 mm posterior to the bregma and 1.5 mm left to the sagittal suture (Paxinos and Watson, 1986). The dura was incised to avoid damage to the electrode tip upon implantation. The amplifier holding the electrode was secured to the stereotactic frame, and the electrode was positioned and lowered until its tip touched the brain surface. The electrode was implanted 1 mm deep into the cortex. The exposed cortical surface was prevented from drying out by covering it with warm paraffin oil. In these experiments, animals were kept in the stereotactic frame throughout.

At the end of the experiment before the ISM was withdrawn, the brain surface was washed repeatedly to remove the paraffin as this can enter the barrels rendering the electrode inactive. A post-experimental calibration in vitro was performed to ensure the ISM had not been altered with implantation. If there was a marked difference in the pre- and post-calibrations the experiment was excluded.

5.2.2.5 Microdialysis Probe Implantation

For recordings in the dorsolateral striatum, a 2-3 mm craniectomy was performed 0.7 mm anterior to the bregma and 3 mm left to the sagittal suture (Paxinos and Watson, 1986). The dura was incised to avoid damage to the dialysis membrane on implantation. Probes (4 mm in length) were tested with a syringe containing normal ACSF to ensure there were no leaks or abnormal resistance to flow, and then secured into the electrode holder of the stereotactic frame. The probe was lowered until it touched the brain surface, and was then inserted 7 mm deep into the brain. In some experiments a 2 mm probe was implanted laterally into the cortex, 1. 6 mm under the upper brain surface. In these cases,

the craniectomy was performed 3 mm posterior to the bregma and 5.6 mm lateral to the sagittal suture. The implanted probe was secured to the skull with surgical bone cement (Surgical Simplex P, Howemedica Ltd, London, U.K.).

With experiments involving microdialysis alone, when the bone cement was dry, the animal was removed from the stereotactic frame and laid on the homeothermic pad. This provided greater flexibility when setting up the equipment around the animal. The implanted probe was perfused with ACSF (see Section 4.3.2) and the outlet tube was connected to the reaction loop (see Section 4.5).