Table 2.
Protocols for receptor autoradiography. Substances listed between square brackets were only included in the buffer solution during the main incubation
| Transmitter | Receptor | Ligand | Displacer | Incubation buffer | Preincubation | Main incubation | Final rinsing |
|---|---|---|---|---|---|---|---|
| Glutamate | AMPA | [3H] AMPA (10 nM) | Quisqualat (10 µM) | 50 mM Tris–acetate (pH 7.2) [+ 100 mM KSCN] | 3 × 10 min, 4 °C | 45 min, 4 °C |
(1) 4 × 4 sec, 4 °C (2) Acetone-glutaraldehyde (100 ml + 2.5 ml) 2 × 2 s, 22 °C |
| Kainate | [3H] kainite (9.4 nM) | SYM 2081 (100 µM) | 50 mM Tris–acetate (pH 7.1) [+ 10 mM Ca-acetate] | 3 × 10 min, 4 °C | 45 min, 4 °C |
(1) 3 × 4 s, 4 °C (2) Acetone-glutaraldehyde (100 ml + 2.5 ml), 2 × 2 s, 22 °C |
|
| NMDA | [3H] MK-801 (3.3 nM) | ( +)MK-801 (100 µM) |
50 mM Tris–acetate (pH 7.2) + 50 µM glutamate [+ 30 µM glycine + 50 µM spermidine] |
15 min, 4 °C | 60 min, 22 °C |
(1) 2 × 5 min, 4 °C (2) Dip in distilled water, 22 °C |
|
| GABA | GABAA | [3H] Muscimol (7.7 nM) | GABA (10 µM) | 50 mM Tris–citrate (pH 7.0) | 3 × 5 min, 4 °C | 40 min, 4 °C |
(1) 3 × 3 s, 4 °C (2) Dip in distilled water, 22 °C |
| GABAB | [3H] CGP 54626 (2 nM) | CGP 55845 (100 µM) | 50 mM Tris–HCl (pH 7.2) + 2.5 mM CaCl2 | 3 × 5 min, 4 °C | 60 min, 4 °C |
(1) 3 × 2 s, 4 °C (2) Dip in distilled water, 22 °C |
|
| GABAA/BZ | [3H] Flumazenil (1 nM) | Clonazepam (2 µM) | 170 mM Tris–HCl (pH 7.4) | 15 min, 4 °C | 60 min, 4 °C |
(1) 2 × 1 min, 4 °C (2) Dip in distilled water, 22 °C |
|
| Acetylcholine | M1 | [3H] Pirenzepine (1 nM) | Pirenzepine (2 µM) | Modified Kreb’s buffer (pH 7.4) | 15 min, 4 °C | 60 min, 4 °C |
(1) 2 × 1 min, 4 °C (2) Dip in distilled water, 22 °C |
| M2 | [3H] Oxotremorine-M (1.7 nM) | Carbachol (10 µM) | 20 mM HEPES-Tris (pH 7.5) + 10 mM MgCl2 + 300 nM pirenzepine | 20 min, 22 °C | 60 min, 22 °C |
(1) 2 × 2 min, 4 °C (2) Dip in distilled water, 22 °C |
|
| M3 | [3H] 4-DAMP (1 nM) | Atropine sulfate (10 µM) | 50 mM Tris–HCl (pH 7.4) + 0.1 mM PSMF + 1 mM EDTA | 15 min, 22 °C | 45 min, 22 °C |
(1) 2 × 5 min, 4 °C (2) Dip in distilled water, 22 °C |
|
| Nic α4/β2 | [3H] Epibatidine (0.5 nM) | Nicotine (100 µM) | 15 mM HEPES (pH 7.5) + 120 mM NaCl + 5.4 mM KCl + 0.8 mM MgCl2 + 1.8 mM CaCl2 | 20 min, 22 °C | 90 min, 22 °C |
(1) 5 min, 4 °C (2) Dip in distilled water, 22 °C |
|
| Serotonin | 5-HT1A | [3H] 8-OH-DPAT (1 nM) |
5-Hydroxy-tryptamine (1 µM) |
170 mM Tris–HCl (pH 7.4) [+ 4 mM CaCl2 + 0.01% ascorbate] | 30 min, 22 °C | 60 min, 22 °C |
(1) 5 min, 4 °C (2) Dip in distilled water, 22 °C |
| 5-HT2 | [3H] Ketanserine (1.14 nM) | Mianserin (10 µM) | 170 mM Tris–HCl (pH 7.7) | 30 min, 22 °C | 120 min, 22 °C |
(1) 2 × 10 min, 4 °C (2) Dip in distilled water, 22 °C |
|
| Norepinephrine | α1 | [3H] Prazosin (0.2 nM) | Phentolamine mesylate (10 µM) | 50 mM Na/K-phosphate buffer (pH 7.4) | 15 min, 22 °C | 60 min, 22 °C |
(1) 2 × 5 min, 4 °C (2) Dip in distilled water, 22 °C |
| α2 | [3H] RX 821002 (1.4 nM) | Phentolamine mesylate (10 µM) | 50 mM Tris–HCl (pH 7.7) + 100 µM MnCl2 | 15 min, 22 °C | 90 min, 22 °C |
(1) 5 min, 4 °C (2) Dip in distilled water, 22 °C |
|
| Dopamine | D1 | [3H] SCH 23390 (1.67 nM) | SKF 83566 (1 µM) | 50 mM Tris–HCl (pH 7.4) + 120 mM NaCl + 5 mM KCl + 2 mM CaCl2 + 1 mM MgCl2 | 20 min, 22 °C | 90 min, 22 °C |
(1) 2 × 20 min, 4 °C (2) Dip in distilled water, 22 °C |
GABAA/BZ GABAA associated benzodiazepine binding sites