Influence on 5-[3H]Hydroxytryptamine Binding Site Development in Chick Embryo by Serotonergic Compounds

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Abstract
Saturable and specific binding sites for 5-[3H]hydroxytryptamine (5-HT, serotonin) characterized by a Kd of 3.5-4.5 nM were detected in the chick embryo brain and developed linearly as a function of age, weight and protein content. Saturation and displacement studies using unlabeled 5-HT as the displacing ligand suggested a single population of binding sites. Displacement studies using 5-methoxytryptamine, lysergic acid diethylamide (LSD), 2-bromo-lysergic acid diethylamide (BOL), methysergide and spiperone as competing ligands suggested the existence of subclasses of [3H]5-HT binding sites because the Hill coefficients were less than unity. When compared with the reported [3H]5-HT binding sites (5-HT1) in the rat forebrain, the IC50 [mean inhibitory concentration] values of the competing ligands were similar. The Hill coefficients for LSD and methysergide were less than unity which suggested that the [3H]5-HT binding sites in the chick embryo brain may be more similar to those found in rat spinal cord than rat forebrain. To study [3H]5-HT binding site regulation and development, various serotonergic compounds were injected into the chorioallantoic fluid of the eggs at different times during embryonic development. Multiple pretreatments with d,l-5-hydroxytryptophan, 5-HT or BOL had no significant effects on either the affinity (Kd) or number (Bmax [maximum binding capacity]) of specific [3H]5-HT binding sites. Multiple pretreatments with p-chlorophenylalanine were found to increase the Bmax of specific [3H]5-HT binding by 23% (P < 0.01) whereas multiple pretreatments with LSD were found to decrease the Bmax of specific binding by 45% (P < 0.01). Neither of these pretreatments affected Kd values which indicated that only the number of specific [3H]5-HT binding sites was altered. These effects were probably not due to excess endogenous 5-HT or LSD remaining in the tissue preparation. The chick embryo brain may have a functioning serotonergic system and the chick embryo may be an ideal system for the study of [3H]5-HT binding site regulation and development.