Selective labeling of serotonin receptors by d-[3H]lysergic acid diethylamide in calf caudate.

Abstract

Since it was known that d-lysergic acid diethylamide (LSD) affected catecholaminergic as well as serotoninergic neurons, the objective in this study was d-Lysergic acid diethylamide (LSD) has been extensively studied since its discovery (1). The structural resemblance of serotonin (2) to LSD was subsequently noted, and evidence accumulated suggesting that LSD exerts its effects through the serotoninergic neurotransmitter system (3-11). Convincing evidence also indicates dopaminergic involvement in LSD activity (12)(13)(14)(15)(16)(17)(18)(19) clearance); this piston, rotating at 500 rpm, was passed up and down 10 times. The crude homogenate was incubated at 370C for 60 min and then stored in 5-ml aliquots at -20°C for future use. Before use, the samples were thawed, resuspended with an additional 5 ml of buffer, and centrifuged at 39,000 X g for 15 min at 4°C. The supernatant was discarded and the pellet was resuspended in 15 ml of buffer by using five up-and-down strokes of a ground-glass homogenizer. This suspension was then homogenized by a Polytron homogenizer (Brinkmann, Westbury, NY) at a setting of 7 (full range = 10) for 10 sec, using a PT-10 homogenizer probe and a 50-ml polycarbonate tube to contain the suspension. This resulted in a final protein concentration of 0.2 mg per tube. Because the membrane homogenate was made up immediately prior to use, it was not necessary to store it on ice.Serotonin-Specific [3HJLSD Binding Assays. The assays were done by using 12 X 75 mm glass test tubes in which the following aliquots were placed (Eppendorf Brinkmann pipettes with polypropylene tips) for control binding: 0.1 ml of nonradioactive LSD (final concentration, 200 nM) or 0.1 ml of buffer; 0.1 ml of buffer containing apomorphine, phentolamine, and spiperone (final concentration, 50 nM for each, referred to as the APS system); 0.2 ml of [3H]LSD (11.5 Ci/mmol; New England Nuclear; final concentration, 2.0 nM); and 0.2 ml of brain homogenate. For tubes with varying concentrations of drugs competing against [3H]LSD, the final contents of the tubes were the same as for control except that the 0.1 ml of buffer or nonradioactive LSD was replaced with 0.1 ml of drug solution.Abbreviations: LSD, d-lysergic acid diethylamide; IC50, concentration that inhibits [3HJLSD binding by 50%; APS system, mixture containing 50 nM each of apomorphine, phentolamine, and spiperone.

Resolution of dopamine and serotonin receptor components of [3H]spiperone binding to rat brain regions.

“…In the D2 binding system, which appeared to have a dopaminergic distribution in tissue areas (i.e., sites detectable only in regions with dopaminergic innervation), binding isotherms and Scatchard plots became monophasic and revealed similar low Kd values (0.05-0.1 nM) in areas where the D2 site was present. Although it may seem surprising that no D2 sites were detected in the frontal cortex, the present results are consistent with those of other workers, who have failed to detect dopaminergic [3H]spiperone binding in whole frontal cortex (6,9,10); when dopaminergic areas of the frontal cortex are specifically dissected out, however, D2-type binding is detected (17 (20 …”

Serotonin2 (5-HT2) receptor binding in the frontal cortex of schizophrenic patients

“…Recent studies indicate that 3H-5-HT and 3H-LSD are not specific ligands for 5-HT2 receptor binding (Peroutka and Snyder, 1979;Schmidt and Peroutka, 1989). 3H-LSD, in addition to binding to 5-HT2 receptors, labels dopamine2 (D2) receptors (Burt et al, 1976;Whitaker and Seeman, 1978). It is possible that studies which used 3H-5-HT and 3H-LSD as binding ligands did not accurately quantify 5-HT2 recpetors in the human frontal cortex.…”

Behavioral and cellular consequences of increasing serotonergic activity during brain development: a role in autism?

“…In ongoing studies using this agonist, pregnant rats are injected from gestational day 12 until birth and then the pups injected individually up until postnatal day 20 at a dose of 1 mg/kg. 5‐MT has relatively high potency at all tested serotonin receptors, approximately 1/10th the potency of serotonin itself (Whitaker and Seeman, 1978a, 1978b). Using this potency and known pharmacokinetic principles for calculating blood concentration based on the presumed volume of distribution, this dose would approximate the effects of the 50% increase in blood serotonin seen in autism.…”