Objective
Serotonin (5-hydroxytryptamine, 5-HT) is a major neurotransmitter in the central nervous system whose actions are mediated by at least 15 different types of receptors that belong to the ligand-gated ion channel superfamily and the G-protein-coupled superfamily. Within the second group, the 5-HT receptor types deserve special attention because their selective ligands are of great potential interest in the treatment of several diseases which concern neuro-vascular interactions (such as migraine), feeding and drinking behavior, nociception, cognitive functions, mood and hedonia, etc. 5-HT1B/1D receptors are coded by two different genes, 5-ht1a and 5-ht1b, the products of which have species-dependent pharmacological profiles. These species-related differences explain why pharmacologists named the same receptor protein, encoded by the 5-ht1b gene, the 5-HT1B receptor in rodents and the 5-HT1Db receptor in other mammalian species including the human. Our research programme aimed at the detailed definition of the respective properties (molecular, pharmacological and functional characteristics, and distributions in brain and spinal cord in several species) of 5-HT1B/1D receptors as a prior obligatory step toward the development of novel selective ligands with clearcut therapeutic indications and/or major interest as tools for pharmacological and physiological investigations.
At the molecular level, site-directed mutagenesis focused on hydroxylated aminoacid residues possibly involved in hydrogen bonding between the rat 5-HT1B receptor and the hydroxyl and/or the N atom in the indole ring of serotonin. These studies demonstrated that Thr209 within the 5th putative transmembrane domain of the 5-HT1B raceptor is obligatory for the recognition of 5-HT, and that the adjacent Ser208 is necessary for the functional (negative) coupling of the activated receptor with adenylate cyclase.
Polyclonal antibodies were raised against a synthetic peptide corresponding to a highly selective portion (Val263-Lys287) of the putative third intracellular loop of the rat 5-HT1B receptor, which allowed the labeling of this receptor at the regional, cellular and subcellular levels in the central nervous system. In agreement with thorough studies achieved in parallel with the selective 5-HT1B/1D radioligand, S-CM-G[125I]TNH2 (or [125I]GTI), immunohistochemical investigations demonstrated that 5-HT1B receptors in rats and mice and the homologous 5-HT1Db receptors in guinea-pigs and human are especially abundant in the substantia nigra, the globus pallidus, the dorsal subiculum and the deep cerebellar nuclei. In contrast, in situ hybridization with a specific [35S]cRNA probe showed that these areas do not express the 5-HT1B receptor mRNA in the rat brain. Indeed, this transcript is present in the caudate putamen, the CA1 area of the hippocampus, the anterior raphe nuclei and Purkinje cell layer in the cerebellum, which all contain the cell bodies of neurons projecting to the central areas where antibodies allowed the visualization of the 5-HT1B receptor protein. These data suggested that this receptor is addressed exclusively to axon terminals, a hypothesis which could be directly verified by electron microscope studies at the level of the dorsal subiculum, the superior colliculi and the substantia nigra.
Studies of the mechanisms responsible for the addressing of 5-HT1B receptor at the axon terminals have been performed in LLC-PK1 epithelial cells (from pig kidney) used as a model of polarized cells. Data in the literature have shown that the baso-lateral domain of the plasmic membrane of LLC-PK1 cells is homologous to the somato-dendritic compartment of the plasmic membrane of neurons whereas the apical domain of epithelial cells corresponds to the terminal compartment of neurons. As expected of such a homology, the 5-HT1A receptor, which has an exclusive somato-dendritic location in central neurons, was found to be expressed at the baso-lateral pole of LLC-PK1 cells transfected with the corresponding plasmid. A differential addressing was observed in LLC-PK1 cells transfected with a plasmid encoding the rat 5-HT1B receptor as the corresponding receptor protein could be immunolabeled both within the cytoplasm and at the apical pole of the plasmic membrane. Construction of six 5-HT1A/5-HT1B receptor chimeras allowed the demonstration that the third intracellular loop of these receptors plays a key role in their respective addressing.
Major advances in the knowledge of the functional properties of 5-HT1B/1D receptors were achieved thanks to the knock-out of the 5-HT1B receptor gene (by homologous recombination of embryonic stem cells) in mice, the use of stereotaxic microinfusion of 5-HT1B/1D selective ligands in selected brain areas, and in vitro investigations using peripheral and central tissues. These studies clearly indicated that 5-HT1B receptors mediate an anti-aggressive action of endogenous 5-HT in mice, and participate in the efficiency of spatial memory mechanisms at the hippocampal level in rats. In line with the location of 5-HT1B/1D receptors on axon terminals, in vitro studies demonstrated that these receptors mediate a local negative control of the release of various neurotransmitters. Thus, 5-HT1B receptors are responsible for the inhibitory effect of 5-HT and related agonists (notably the anti-migraine drug sumatriptan) on the electrically-evoked release of pain-mediating neurotransmitters such as calcitonin gene-related peptide (CGRP) and substance P from primary afferent fibres within the dorsal horn of the spinal cord. In addition, in human, 5-HT1Da receptors mediate the 5-HT inhibition of noradrenaline release from cardiac sympathetic nerves, and 5-HT1Db receptors the negative control of the indoleamine on its own release in the cerebral cortex. Finally, 5-HT1D receptors have been found in rabbit eye tissues where they participate in the control of intraocular pressure. All these data support the idea that selective 5-HT1Da or 5-HT1Db receptor ligands are of great potential interest in the treatment of both central and peripheral diseases.
MAJOR SCIENTIFIC BREAKTHROUGHS:
The generation of mice that do not express the 5-HT1B receptors (by homologous recombination of the corresponding gene) allowed the clearcut demonstration of the key role of these receptors in the control of impulsivity and aggressiveness, at least in this species. Furthermore, the involvement of hippocampal 5-HT1B receptors in 5-HT modulation of cognitive functions has also been clearly demonstrated. These data suggest that 5-HT1Db ligands should be developed for the treatment of some personality disorders related to impulsivity (including obsessive compulsive disorders) and cognitive deficits (associated with aging or neurodegenerative disorders).
The production of antibodies which recognize selectively the 5-HT1B/1Db receptors allowed the immunohistochemical mapping of these receptors in the central nervous system of several species including the human. In addition, these antibodies were especially helpful to demonstrate the key role of the (putative) third intracellular loop of the receptor protein for the exclusive addressing of the 5-HT1B receptor to the plasma membrane of axon terminals.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- medical and health sciences clinical medicine psychiatry obsessive-compulsive disorder
- natural sciences biological sciences neurobiology cognitive neuroscience
- natural sciences biological sciences biochemistry biomolecules proteins
- natural sciences physical sciences optics microscopy
- medical and health sciences medical biotechnology cells technologies stem cells
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Coordinator
75634 Paris
France
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