CONSIDERACIONES GENERALES DE LA METODOLOGÍA

2.1.1 Drugs and Reagents.

All drugs were obtained from Sigma Chemical Company, Poole, Dorset, except for 5,7-dichlorokynurenic acid which was a gift from Pfizer Central Research, Sandwich, Kent. Carbetapentane and the DM and carbetapentane analogues were synthesised and donated by Dr. A H Newman, National Institute for Drug Abuse Addiction Research Centre, Baltimore, Maryland, USA. All other chemicals and reagents were obtained from commercial sources (Sigma or BDH Ltd, Poole, Dorset). Radiolabelled ligands, [^H]dextromethorphan ([^H]DM, 80-88Ci/mmole), [^H]-l-[l-(2-thienyl)cyclohexyl]- piperidine ([^H]TCP, 40.8Ci/ mmole) [^H]glycine (43Ci/mmole) l,3-di-(2-[5- 3H]tolyl)-guanidine ([^HJDTG, 39.4Ci/mmole) and [3h]PK-11195 ([3h]PK-11195,

8 6Ci/mmole) were obtained from New England Nuclear Products (NEN, Boston, MA, USA).

The compounds investigated were dissolved initially in distilled water at a concentration of 1 millimolar (ImM) and diluted with the appropriate incubation buffer for each assay, with the following exceptions. Haloperidol was dissolved in ethanol acidified with lactic acid (final concentration of less than 0.04% ethanol and 0.07%

lactic acid at lOpM haloperidol), 1,3-di-o-tolylguanidine (DTG) was dissolved in methanol (<0.05%) and 5,7-dichlorokynurenic acid (DCK) in dimethylsulphoxide (DMSO) and sodium hydroxide (less than 0.05% and 0.002% at lOpM DCK, respectively). The synthetic analogues of DM and carbetapentane were dissolved in distilled water at an initial concentration of ImM, except compound 3 which was dissolved in DMSO and lactic acid (<0.1% at ImM), and diluted with the appropriate assay buffer. The solvents were also tested in each assay at the highest concentration used. All radiolabelled ligands were diluted with the appropriate assay buffer.

2.1.2 Animals and Tissues.

For binding experiments, rat frozen brains (male, Sprague-Dawley, 250-3 5Og), were obtained direct from Charles River (Manston, Kent), supplied in dry ice, and were

stored immediately on arrival at -80°C. For autoradiography and rat middle cerebral artery experiments, Sprague-Dawley rats (male, weight 200-3OOg) were obtained from

Charles River, and housed with free access to food and water in the School of Pharmacy Animal Unit for not less than one and not more than three weeks prior to use.

2.2 PREPARATION OF RAT AND GUINEA PIG BRAIN MEMBRANE AND

CELL FRACTIONS

2.2.1 Tissue Preparation Buffers.

[^H]DM, [^H]DTG and [^H]PK-11195 Assays: SOmillimolar (mM) Tris

([hydroxymethyl]aminomethane, reagent grade. Sigma), buffered to pH 7.5 at 4°C with concentrated hydrochloric acid. [^H]TCP Assay: 5mM Tris, buffered to pH 7.7 at 4°C

with concentrated hydrochloric acid. [^H]Glycine Assay: 50mM Tris, buffered to pH 7.7 at 4°C with glacial acetic acid. For all the following methods described, the buffer referred to in each case was the tissue preparation or assay buffer appropriate to the assay for which the tissue was being prepared.

2.2.2 Rat and Guinea Pig Brain Crude Homogenate.

This preparation was the one used for [^H]DM displacement studies, and also for DM, DTG and PK-11195 saturation experiments. Frozen rat or guinea pig brains were

using either a Polytron or Ultra-Turrax homogeniser (low setting). The suspension was centrifuged at lOOOg (average centrifugal force, G^y), for 20 minutes and the pellet (nuclear fraction, P%) discarded. The supernatant was then centrifuged at 100,000g for 1 hour and the resultant pellet suspended in 2.5 volumes of the appropriate assay buffer (see Sections 2.3, 2.6 and 2.7) and frozen at -80°C.

2.2.3 Rat Brain Subcellular Fractions.

This preparation was used to investigate the subcellular distribution of DM binding sites, and was adapted from the method of De Robertis et al (1962). Frozen rat brains were suspended in 10 volumes of ice-cold 0.32M sucrose and homogenised as described above. The suspension was then centrifuged at lOOOg for 15 minutes, and the supernatant decanted off from the P% pellet. The pellet was washed once in DM tissue preparation buffer, re-centrifuged at lOOOg for 15 minutes and resuspended in 20 volumes of DM assay buffer, and frozen at -80°C. The supernatant, Sj, was then centrifuged at 20,000g for 20 minutes. The pellet from this spin was resuspended in buffer and centrifuged at ll,500g for 20 minutes. The resultant pellet, the crude mitochondrial fraction (P2), was washed once in buffer, re-centrifuged at 11,5 OOg for 20 minutes and suspended in 2.5 volumes of DM assay buffer and frozen at -80°C. The supernatant (S2) was centrifuged at 100,000g for 60 minutes. This pellet, the microsomal (P3) fraction, was resuspended in 2 volumes of DM assay buffer, and frozen at -80°C. The supernatant (cytosolic fraction, S3), was also frozen at -80°C.

2.2.4 Guinea Pig Brain Subcellular Fractions.

This preparation was used for displacement experiments with the DM and carbetapentane analogues. Based on the method described above, frozen guinea pig

brains were homogenised in 0.32M sucrose and centrifuged at lOOOg to separate the P% pellet. The supernatant from this spin was first centrifiiged at 20,000g for 20

minutes. The resultant pellet was resuspended in 10 volumes of ice-cold tissue

preparation buffer and centrifuged at ll,500g for 20 minutes. This pellet (P2) was washed once, re-centrifuged at 11,5OOg, and then resuspended in 2.5 volumes of DM assay buffer and frozen at -80°C. The supernatant from the 20,000g spin was

centrifuged at 100,0 0 0g for 60 minutes to give the microsomal pellet, which was then resuspended in 2 volumes of DM assay buffer and frozen at -80°C.

2.2.5 Rat Brain Crude Synaptic Plasma Membranes.

Rat brain crude synaptic plasma membranes (SPM), which had been extensively

washed to remove endogenous glutamate and glycine, were used for the [^H]TCP and [^H]glycine binding assays, and the method was modified from one developed by Bristow et al (1986). Frozen rat brains (minus the cerebellum) were thawed and suspended in 10 volumes of ice-cold 0.32M sucrose and homogenised as above. The homogenate was centrifuged at lOOOg for 20 minutes, the pellet (P%) discarded and the supernatant centrifuged at 20,000g for 20 minutes. The pellet was resuspended in 10 volumes of ice-cold distilled water, vortexed and allowed to stand for 10 minutes to allow lysis of the cells in the hypo-osmotic solution, before being centrifuged at 8,00 0g for 20 minutes. The supernatant was then used to wash the huffy uppercoat away from the brown mitochondrial-enriched pellet, and centrifuged at 40,000g for 20 minutes to obtain crude synaptic plasma membranes. These were washed twice in distilled water and the pellets frozen at -20°C for at least 18 hours. They were then thawed and resuspended in 10 volumes of either TCP or glycine tissue preparation buffer, vortexed and washed a further six times by centrifuging at 40,000g with a 10 minute incubation at room temperature between each spin. The final pellet was resuspended in 2.5 volumes of either TCP or glycine assay buffer and frozen at -80°C.

2.2.6 Rat Brain Sections.

Microscope slides (BDH) were washed in a solution of methanol acidified with 5% hydrochloric acid for approximately 25 minutes and dried under a stream of warm air. They were then immersed in a solution of 0.5% gelatine and 0.05% chromic potassium sulphate in distilled water for 5 minutes and dried as before.

Sprague-Dawley rats were sacrificed by cervical dislocation and decapitation. The brains were rapidly removed and mounted on cork. They were then fi'ozen by immersion in isopentane (2-methylbutane), cooled in liquid nitrogen, and stored at - 80°C. For the preparation of sections, the brains were removed from the fi^eezer, mounted onto a metal chuck and placed in a cryostat (2800 Frigocut, Reichert-Jung), to equilibrate at -20°C. The brain was then cut into coronal sections of 20 micron (pm) thickness at predetermined brain levels, measured using reference to a rat brain atlas (Paxinos & Watson, 1982). The co-ordinates used were the interaural co­ ordinates, and refer to the anteroposterior distance of the plane from the vertical line passing through the interaural line. The sections were thaw-dried onto the gelatine- coated microscope slides, either two or three sections per slide. They were then allowed to dry at room temperature and frozen at -20°C for not more than 3 weeks prior to use.

2.2.7 Protein Assay.

The protein concentration in all experiments was determined by the method of Bradford (1976). Briefly, 900pl of Bradford reagent was added to lOOpl of protein, and the fluorescence of the resulting solution was measured at 595nM using a CE505 Ultraviolet Spectrophotometer. These readings were then converted to protein concentration by reference to a series of standard protein solutions (0.05 to 0.5mg/ml solutions of bovine serum albumin (fraction V).