DEXTROMETHORPHAN SITE BINDING.

2.3.1 Buffers.

Incubation buffer; 50mM Tris, buffered to pH 7.5 at 23°C with concentrated hydrochloric acid. Filter pre-soak and wash buffers: 50mM Tris-HCl containing

lOOmM of choline chloride and 0.01% Triton X-100 (to reduce non-specific binding of the ligand to the filters), buffered to pH 7.5 at 4°C.

2.3.2 [^H]DM Binding Assay I: Binding Characteristics and Subcellular Distribution of [^H]DM Sites.

The method used was developed from that described by Craviso and Musacchio (1983a). For total binding of DM, 50pl of [^H]DM at a final concentration of approximately 5nM, was added to 50pl of assay buffer and 400pl of either rat or guinea pig brain (frozen stock solutions of either crude homogenate or the appropriate cell fraction, thawed and diluted with assay buffer to give approximately 250pg of protein in the assay) in a final assay volume of 0.5ml. Non-specific binding of [^H]DM was determined by substituting for buffer 50pl of ImM unlabelled DM to give a final concentration of lOOpM in the assay, and was defined as that which could not be displaced by excess unlabelled DM. The reaction was initiated with the addition of protein and allowed to incubate for 30 minutes at 23 °C. The ability of other pharmacological agents to displace [^H]DM binding was investigated by incubating

50pl of each compound at concentrations ranging from InM to lOOpM with [^H]DM and protein as described above. All points were determined in triplicate. In all experiments, DM (InM to lOOpM) was included as a standard.

The reaction was terminated by dilution with 3ml of ice-cold DM wash buffer, followed by rapid filtration through Whatman GF/B glass-fibre filters (SEMAT), which

had been pre-soaked for at least 2 hours in wash buffer. Filtering was performed using a Brandel M-48R (SEMAT). The filters were then washed 4 times with 3ml of wash buffer. Each filter sample was punched into individual vials to which was added 5ml of 'Optiphase Safe' scintillation fluid (LKB). The samples were allowed to stand for at least 12 hours to allow for maximum extraction of the radioactivity, and counted in an LKB 1219 Rackbeta liquid scintillation counter, with automatic corrections for quenching and efficiency.

For each experiment, a sample of radioactive [^H]DM stock solution was added to 5ml of scintillation fluid and counted as described above to determine the free concentration of [^H]DM in the assay. A sample of the tissue was analysed for the protein content (as described in Section 2.2.7), for determination of bound [^H]DM in moles per unit protein.

2.3.3 [^H]DM Binding Assay II; Competition Experiments with DM and Carbetapentane Analogues.

The method was essentially as described above (Section 2.3.2), with the following modifications. To measure the ability of DM and carbetapentane analogues to inhibit binding of [^H]DM, each compound at concentrations from InM to lOpM were incubated as described above (2.3.2) with [^H]DM and protein (approximately 200pg

of rat or guinea pig brain P2/P3 or crude P2 fraction, or 300pg of guinea pig brain P3). Reagents were diluted and dispensed into 1ml polystyrene minitubes, in an incubation volume of 0.5ml. All points were determined in duplicate. Standard agents for each assay (DM, chlorpromazine, MK-801 and 5,7-dichlorokynurenic acid) were included in each experiment. The reaction was terminated by dilution with 0.5ml of ice-cold wash buffer, followed by rapid filtration through pre-soaked GF/B filters, and the filters were

washed with 4 x 1ml of wash buffer. The filters were added to 5ml of 'Ecoscint A' scintillation fluid, and counted in a Beckman LSI701 liquid scintillation counter, with

automatic corrections for quenching and efficiency. Free ligand and protein concentrations were determined as described previously.

2.3.4 [^H]DM Binding Assay III: Saturation Experiments.

Binding of [^H]DM was measured at concentrations of approximately 0.1 nM to 2pM achieved by diluting the specific activity of [^H]DM 10-fold with unlabelled DM, to give a stock concentrations of 20pM. This solution was then diluted 2-fold sequentially, giving 12 concentrations from 20 pM to InM, to give approximate final assay concentrations of 2pM to O.lnM. These solutions were then incubated with approximately 250pg of rat or guinea pig brain homogenate as described for the DM binding assay (Section 2.3.2), with either buffer (for total binding) or lOOpM unlabelled DM (for non-specific binding), in a final volume of 300pl. The reaction was terminated, and the filters counted in 5ml of Ecoscint A as described in Section 2.3.3. Total and non-specific binding were determined in duplicate for each experiment. Absolute free ligand concentrations were measured by counting aliquots of the stock solutions to obtain the concentration of [^H]DM, and multiplying by the dilution factor.

2.4 PCP SITE BINDING.

2.4.1 Buffers.

Incubation buffer: 5mM Tris-HCl, buffered to pH 7.7 at 23°C. Filter pre-soak buffer: 5mM Tris-HCl containing 0.5% polyethyleneimine (PEI, Sigma), pH 7.7 at 4°C. Wash buffer: 5mM Tris-HCl, pH 7.7 at 4°C.

2.4.2 [^H]TCP Binding Assay.

This method was based on one described by Monahan et al (1989). For total binding, SOpl of [^H]thienylcyclohexylpiperidine ([^H]TCP) at a final concentration of 5nM,

was incubated for 60 minutes at 23°C, with 50pl of assay buffer and 400pl of rat brain crude SPM (frozen stock diluted with assay buffer to give approximately 200pg of protein). For non-specific binding of [^H]TCP, the buffer was substituted with of 50pl

of phencyclidine (PCP) at a final concentration of lOOpM. Competition experiments were performed as described for the DM assay (Section 2.3.3). Phencyclidine (InM to lOOpM) was included as a standard in all experiments. The reaction was terminated by dilution with ice-cold wash buffer, followed by rapid filtration through GF/B filters pre-soaked at least 2 hours in filter pre-soak buffer. The filters were then washed 4 times with 1ml of wash buffer, and counted in 5ml of Ecoscint A as described for the DM assay.

2.5 GLYCINE SITE BINDING

2.5.1 Buffers.

Incubation buffer; 50mM Tris, buffered to pH 7.7 at 4°C with glacial acetic acid. Filter pre-soak buffer: 50mM Tris-acetate containing 0.1% PEI, and ImM glycine (to reduce

non-specific binding to the filter), pH 7.7 at 4°C. Wash buffer: 50mM Tris-acetate containing lOmM magnesium chloride, pH 7.7 at 4°C.

2.5.2 [^HJGlycine Binding Assay.

The method was a modification of those employed by Bristow et al (1986) and Sacaan and Johnson (1989). For total binding, 50pl of buffer was incubated for 20 minutes at

4°C with 50^1 of [^H]glycine (final concentration approximately 20nM) and 400pl of rat brain synaptic plasma membranes (approximately 200pg of protein). Non-specific binding was defined with ImM D-serine, and all points were determined in triplicate. Competition experiments were performed as described for the DM assay (Section 2.3.3), and glycine (lOnM to lOOpM) was included in each assay as a standard. The reaction was terminated by dilution with 1ml of ice-cold wash buffer (which contained magnesium chloride to reduce dissociation of the ligand-receptor complex), followed by rapid filtration through GF/B filters pre-soaked at least 2 hours in filter pre-soak buffer. The filters were then washed 3 times with 1ml of ice-cold wash buffer. The duration of the separation procedure was kept to less than 6 seconds and the reaction mixture and reagents were kept below 5°C to minimise dissociation of the relatively low affinity [^HJglycine from its binding site. The filters were counted as described for the DM assay.

2.6 SIGMA SITE BINDING

2.6.1 Buffers.

Incubation Buffer; 50mM Tris-HCl, pH 7.5 at 23°C. Filter pre-soak buffer: 50mM Tris-HCl containing 0.5% PEI, pH 7.5 at 4°C. Wash Buffer: 50mM Tris-HCl, pH 7.5

at 4°C.

2.6.2 [^H]DTG Binding Assay I: Binding Characteristics.

This assay was adapted from those described by Bowen et al (1989) and Karbon et al

(1991). Experiments were performed in 1ml minitubes. l,3-di-(2-[5-^H]tolyl)- guanidine ([^H]DTG), at a final concentration of approximately 3nM, was incubated for 60 minutes at 23°C with approximately 2 0 0pg of rat brain crude homogenate, and

either buffer (for total binding) or lOgM haloperidol (non-specific binding), in a final assay volume of 0.3ml. For displacement experiments, buffer was substituted with 30pl of cold compound at final concentrations from InM to lOOpM. All points were determined in triplicate. The reaction was terminated by dilution with ice-cold wash buffer, followed by rapid filtration through GF/B filters pre-soaked for at least 2 hours in filter pre-soak buffer. The filters were then washed 4 times with 1ml of wash buffer, and counted in 5ml of Ecoscint A.

2.6.3 [^H]DTG Binding Assay II: Saturation Experiments.

Concentrations of [^H]DTG ranging from 0.24nM to SOOnM were prepared in a similar manner to that described for the [^H]DM saturation assay, except that the specific activity of the [^H]DTG was diluted five-fold with unlabelled DTG to give a stock solution of 5pM. This solution was then diluted 2-fold sequentially, giving 12 concentrations from 5pM to 2.4nM, to obtain approximate final assay concentrations of SOOnM to 0.24nM. These solutions were then incubated with approximately 200pg of rat brain homogenate as described above, with either buffer (for total binding) or lOpM haloperidol (for non-specific binding). The reaction was terminated and the filters counted in 5ml of Ecoscint A. Total and non-specific binding were determined in duplicate for each experiment. Absolute free radiolabelled ligand concentrations were measured by counting aliquots of the stock solutions and multiplying by the dilution factor.

2.7 PERIPHERAL BENZODIAZEPINE SITE BINDING

2.7.1 Buffers.

Incubation, filter pre-soak and wash buffers: SOmM Tris-HCl adjusted to pH 7.5 at 4°C.

2.7.2 [^H]PK-11195 Binding Assay.

This assay was based on a method described by Benavides et al (1990). [^H]PK- 11195 (8 6Ci/mmol) was diluted in assay buffer to give a stock solution of 500nM.

This solution was then diluted in 2-fold steps to give approximate final assay concentrations ranging from 50nM to 2pM. The ligand was incubated for 60 minutes at 4°C with approximately 200pg of protein, and either buffer (total binding) or 1 pM unlabelled PK-11195 (non-specific binding) in 1ml minitubes in a final assay volume of 0.3ml. The reaction was terminated by dilution with ice-cold wash buffer, followed by rapid dilution through GF/B filters pre-soaked for at least 2 hours in filter pre-soak buffer. The filters were then washed with 4 x 1ml of wash buffer, and counted as described previously (Section 2.3.3). Total and non-specific binding were determined in duplicate. Protein concentration was measured as described, and free [^H]PK-

11195 concentrations were measured by counting an aliquot of each stock solution.

2.8 DM SITE AUTORADIOGRAPHY

2.8.1 Buffers.

The buffers used in these experiments were those described for the [^H]DM binding

assay.

2.8.2 [^H]DM Autoradiography Assay.

This method was modified from that used for the DM binding assay as described above (Section 2.3). The slide-mounted sections were immersed in DM assay buffer at room temperature for 30 minutes and then allowed to dry. Onto each slide was then pipetted 350pl (two sections per slide) or 500pl (three sections per slide) of DM incubation

buffer containing approximately 25nM of [^H]DM (83Ci/mmole), ensuring that the whole of each section was completely covered. For non-specific binding, the incubation buffer also contained unlabelled DM at a concentration of lOOpM. The sections were allowed to incubate for 30 minutes at room temperature, which was monitored and was between 21-23°C. The buffer was then aspirated off and the slides rinsed in ice-cold wash buffer. They were then washed three times for five minutes each wash in ice-cold wash buffer, and dried under a stream of cool air.

The slides were affixed to cardboard using double-sided adhesive tape, and placed in autoradiography film cases closely juxtaposed with tritium-sensitive film ([^H]- Hyperfilm, Amersham). The films were then left to develop for approximately 28 days. Extra sections in each experiment were included to determine total and non-specific binding by liquid scintillation counting. Two sections each of total and non-specific binding from two different brain regions were incubated with [^H]DM to determine total and non-specific binding as described above (Section 2.3.2), and counted in 10ml of Optiphase Safe scintillation fluid in an LKB liquid scintillation counter after a 12 hour extraction period. The level of [^H]DM bound was used to give an indication of the length of time required for exposure of each film.

The tritium-sensitive films were developed by immersion in D-19 developer (Kodak) for approximately 1 minute, followed by a 1 minute wash in water. They were then fixed for 1 minute, washed again for 30 minutes in water and allowed to dry. The films were analysed by image analysis to determine the amount of radiolabelled DM bound to each section as described in Section 2.10.3.