Table 1.
Ligands and binding protocols used for receptor autoradiography
| Transmitter | Receptor | [3H]-Ligand | Displacer | Incubation buffer | Preincubation | Main incubation | Final rinsea |
|---|---|---|---|---|---|---|---|
| Glutamate | AMPA | AMPA [10 nM] |
Quisqualate [10 μM] |
50 mM Tris-acetate (pH 7.2) +100 mM KSCNb |
3 × 10 minutes, 4 °C | 45 minutes, 4 °C | 1. 4 × 4 seconds 2. Acetone/glutaraldehyde (100 ml/2.5 ml), 2 × 2 seconds, 4 °C |
| Kainate | Kainate [9.4 nM] |
SYM 2081 [100 μM] |
50 mM Tris-acetate (pH 7.1) + 10 mM Ca2+-acetate |
3 × 10 minutes, 4 °C | 45 minutes, 4 °C | 1. 4 × 4 seconds, 4 °C 2. Acetone/glutaraldehyde (100 ml/2.5 ml), 2 × 2 seconds, 4 °C |
|
| NMDA | MK-801 [3.3 nM] |
(+)MK-801 [100 μM] |
50 mM Tris-acetate (pH 7.2) + 50 μM glutamate + 30 μM glycineb + 50 μM spermidineb |
15 minutes, 4 °C | 60 minutes, 22 °C | 2 × 5 minutes | |
| mGlu2/3 |
LY341495 [1 nM] |
l-Glutamate [1 mM] |
10 mM phosphate buffer (pH 7.6) + 100 mM KBrb |
2 × 5 minutes, 22 °C | 60 minutes, 4 °C | 2 × 5 minutes | |
| GABA | GABAA | Muscimol [7.7nM] |
GABA [10 μM] |
50 mM Tris-citrate (pH 7.0) | 3 × 5 minutes, 4 °C | 40 minutes, 4 °C | 3 × 3 seconds |
| GABAB | CGP 54626 [2 nM] |
CGP 55845 [100 μM] |
50 mM Tris-HCl (pH 7.2) + 2.5 mM CaCl2 |
3 × 5 minutes, 4 °C | 60 minutes, 4 °C | 3 × 2 seconds | |
| BZ | Flumazenil [1 nM] |
Clonazepam [2 μM] |
170 mM Tris-HCl (pH 7.4) | 15 minutes, 4 °C | 60 minutes, 22 °C | 2 × 1 seconds | |
| pBZ | PK 11195 [0.1 nM] |
PK 11195 [10 μM] |
50 mM Tris-HCl (pH 7.4) | 15 minutes, 22 °C | 60 minutes, 22 °C | 3 × 5 minutes | |
| Acetylcholine | M1 | Pirenzepine [1 nM] |
Pirenzepine [2 μM] |
Krebs buffer (pH 7.4) + 4 mM KCl + 120 mM NaCl |
15 minutes, 22 °C | 60 minutes, 22 °C | 3 × 4 minutes |
| M2 | Oxotremorine-M [1.7 nM] |
Carbachol [10 μM] |
20 mM HEPES-Tris (pH 7.5) + 10 mM MgCl2 + 300 nM pirenzepine |
20 minutes, 22 °C | 60 minutes, 22 °C | 2 × 2 minutes | |
| M3 | 4-DAMP [1 nM] |
Atropine sulfate [10 μM] |
50 mM Tris-HCl (pH 7.4) + 0.1 mM PMSF + 1 mM EDTA |
15 minutes, 22 °C | 45 minutes, 22 °C | 2 × 5 minutes | |
| nACh | Epibatidine [0.5 nM] |
Nicotine [100 μM] |
1 mM HEPES (pH 7.5) + 120 mM NaCl + 5.4 mM KCl + 0.8 mM MgCl2 + 1.8 mM CaCl2 |
20 minutes, 22 °C | 90 minutes, 22 °C | 5 minutes | |
| Noradrenaline | α1 | Prazosin [0.2 nM] |
Phentolamine mesylate [10 μM] |
50 mM Na+/K+-phosphate buffer (pH 7.4) | 15 minutes, 22 °C | 60 minutes, 22 °C | 2 × 5 minutes |
| α2 | UK 14,304 [0.64 nM] |
Phentolamine mesylate [10 μM] |
50 mM Tris-HCl (pH 7.7) + 100 μM MnCl2 |
15 minutes, 22 °C | 90 minutes, 22 °C | 5 minutes | |
| Serotonin | 5-HT1A | 8-OH-DPAT [1 nM] |
5-Hydroxytryptamine [1 μM] |
170 mM Tris-HCl (pH 7.4) + 4 mM CaCl2 b + 0.01% ascorbateb |
30 minutes, 22 °C | 60 minutes, 22 °C | 5 minutes |
| 5-HT2 | Ketanserin [1.14 nM] |
Mianserin [10 μM] |
170 mM Tris-HCl (pH 7.7) | 30 minutes, 22 °C | 120 minutes, 22 °C | 2 × 10 minutes | |
| Dopamine | D1 | 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 minutes, 22 °C | 90 minutes, 22 °C | 2 × 10 minutes |
| D2 | Raclopride [0.55 nM] |
Butaclamol [1 μM] |
50 mM Tris-HCl (pH 7.4) + 0.1% ascorbate + 150 mM NaCl |
20 minutes, 22 °C | 45 minutes, 22 °C | 6 × 1 minutes | |
| Adenosine | A1 | DPCPX [1 nM] |
R-PIA [100 μM] |
170 mM Tris-HCl (pH 7.4) + 2 U/L adenosine deaminase + 100 μM Gpp(NH)pb |
15 minutes, 4 °C | 120 minutes, 22 °C | 2 × 5 minutes |
| A2A | ZM 241385 [0.42 nM] |
2-Chloroadenosine [20 μM] |
170 mM Tris-HCl (pH 7,4) + 2 U/L adenosine deaminase + 10 mM MgCl2 |
2 × 10 minutes, 22 °C | 120 minutes, 22 °C | 2 × 5 minutes |
Abbreviations: A 1 Adenosine receptor type 1, A 2A Adenosine receptor type 2A, α 1 Adrenoceptor type 1, α 2 Adrenoceptor type 2, AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, BZ γ-Aminobutyric acid type A-associated benzodiazepine-binding site, D1 Dopamine receptor type 1, D2 Dopamine receptor type 2, 4-DAMP 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide, EDTA Ethylenediaminetetraacetic acid, GABA A γ-Aminobutyric acid receptor type A, GABA B γ-Aminobutyric acid receptor type B, Gpp(NH)p 5′-guanylimidodiphosphate, 5-HT 1A Serotonin receptor type 1A, 5-HT 2 Serotonin receptor type 2, KBr Potassium bromide, KSCN Potassium thiocyanate, M 1, M 2 , and M 3 Muscarinic cholinergic receptor types 1, 2, and 3, mGlu2/3 Metabotropic glutamate receptor type 2/3, MK-801 Dizocilpine, nACh Nicotinic cholinergic receptor of the α4/β2 type, NMDA N-methyl-d-aspartate, 8-OH-DPAT 8-Hydroxy-2-(dipropylamino)tetralin, pBZ Peripheral benzodiazepine receptor, PMSF Phenylmethylsulfonyl fluoride, R-PIA N 6-R-phenylisopropyladenosine, SYM 2081 (2S,4R)-4-Methylglutamic acid
a Final rinsing was carried out in incubation buffer at 4 °C, followed by one to three dips in distilled water at room temperature, unless otherwise specified
b Substance included only in the main incubation buffer solution