- Email: [email protected]
Serotonin receptors in brain revisited
BRES : 44633
Model7
pp:024ðcol:fig: : NILÞ brain research ] (]]]]) ]]]–]]]
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Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Review Q2
Serotonin receptors in brain revisited
Q1
Jose´ M. Palacios Frontera Biotechnology S.L., Barcelona 08013, Spain
art i cle i nfo
ab st rac t
Article history:
In the early 1980’s, the dispute on the existence of multiplicity of receptor for neuro-
Accepted 19 December 2015
transmitter was at its height. Several subtypes of serotonin (5-HT) receptors were proposed on the basis of radioligand binding assays. In order to provide further support to the
Keywords:
existence of these receptors we performed quantitative autoradiographic mapping of the
Receptor multiplicity
binding of several ligands for the 5-HT1 receptor labeling the subtypes 5-HT1A, 5-HT1B and
Serotonin (5-HT)
5-HT1C, and characterize pharmacologically these different receptors. The results demon-
Receptor autoradiography
strated differential localization of the subtypes of 5-HT1 receptor indicating that they were
Molecular neuroanatomy
expressed by different cell populations, probably neurons, in the brain and further
Human brain
supporting their reality. Shortly afterwards, the cloning of the genes coding for these 5HT receptors, and many others, ended the dispute by demonstrating that they were different proteins. The advent of Molecular Biology provided new methodologies for the study the chemical and molecular anatomy of 5-HT receptors in brain, by visualizing cells expressing their mRNA by in situ hybridization and showed that the family of mammalian 5-HT receptors has 14 members, a figure much larger than ever suspected. Original article abstract: Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors: The distribution of serotonin-1 (5-HT1) receptors in the rat brain was studied by light microscopic quantitative autoradiography. Receptors were labeled with [3H]serotonin (5-[3H]HT), 8-hydroxy-2-[H-dipropylamino-3H]tetralin (8-OH-[3H] DPAT), [3H]LSD and [3H]mesulergine, and the densities quantified by microdensitometry with the aid of a computer-assisted image-analysis system. Competition experiments for 5-[3H]HT binding by several serotonin-1 agonizts led to the identification of brain areas enriched in each one of the three subtypes of 5-HT1 recognition sites already described (5HT1A, 5-HT1B, 5-HT1C). The existence of these'selective' areas allowed a detailed pharmacological characterization of these sites to be made in a more precise manner than has been attained in membrane-binding studies. While 5-[3H]HT labeled with nanomolar affinity all the 5-HT1 subtypes, the other 3H-labeled ligands labeled selectively 5-HT1A (8OH-[3H]DPAT), 5-HT1C ([3H]mesulergine) and both of them ([3H]LSD). Very high concentrations of 5-HT1 receptors were localized in the choroid plexus, lateroseptal nucleus, globus pallidus and ventral pallidum, dentate gyrus, dorsal subiculum, olivary pretectal nucleus, substantia nigra, reticular and external layer of the entorhinal cortex. The different fields of the hippocampus (CA1–CA4), some nuclei of the amygdaloid complex, the hypothalamic nuclei and the dorsal raphé, among others, also presented high concentrations of sites. Areas containing intermediate densities of 5-HT1 receptors included the claustrum, olfactory tubercle, accumbens, central gray and lateral cerebellar nucleus. The nucleus
E-mail address: [email protected] http://dx.doi.org/10.1016/j.brainres.2015.12.042 0006-8993/& 2016 Published by Elsevier B.V.
Please cite this article as: Palacios, J.M., Serotonin receptors in brain revisited. Brain Research (2016), http://dx.doi.org/10.1016/ j.brainres.2015.12.042
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caudate-putamen and the cortex, at the different levels studied, presented receptor densities ranging from intermediate to low. Finally, in other brain areas-pons, medulla, and spinal cord-only low or very low concentrations of 5-HT1 receptors were found. From the areas strongly enriched in 5-HT1 sites, dentate gyrus and septal nucleus contained 5HT1A sites, while globus pallidus, dorsal subiculum, substantia nigra and olivary pretectal nucleus were enriched in 5-HT1B. The sites in the choroid plexus, which presented the highest density of receptors in the rat brain, were of the 5-HT1C subtype. The distribution of 5-HT1 receptors reported here is discussed in correlation with the distribution of serotoninergic neurons and fibers, the related anatomical pathways and the effects which appear to be mediated by these sites. © 1985. This article is part of a Special Issue entitled SI:50th Anniversary Issue. & 2016 Published by Elsevier B.V.
Contents Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
The work reported in our paper was conducted in Basle (Switzerland) at Sandoz, now Novartis, in a moment of tremendous change in the study of receptors for neurotransmitters. In the late 1970's and early 1980's the dispute between the supporters of a limited number of receptor for a neurotransmitter (one, at the most two), the classical isolated organ, functional pharmacologists and those, the new biochemical “radioactive” pharmacologists, postulating multiplicity of receptors for a neurotransmitter, was at its height (Palacios et al., 2010). This was driven by, in one side, the development of new relatively simple methods, such as radioligand binding techniques, Robert Lefkowitz has written: “if a single technical advance can be said to have opened the door to the molecular era of receptors, it was the development of radioligand binding methods during the 1970's” (Lefkowitz, 2004). On the other hand, the pharmaceutical industry was committed to the discover y and development of new molecules with significant potential for the treatment of diseases of the CNS, and significant economic interest. I joined Sandoz in August 1981, following a 3 years postdoctoral stay at the Department of Neuroscience, Medical School, Johns Hopkins University, working with Michael Kuhar (Kuhar, 1981), and in the exciting environment created by Sol Snyder. There I took part in the development of the technique of receptor autoradiography, a daughter of the “grind and bind” assays. This relatively simple procedure consisted, basically in labeling receptors in microtome sections of brain tissues and generating autoradiographic images of the radiolabeled sites by apposing it to photographic emulsions. The visualization of the binding sites for receptor ligands at the light microscopic level and recently developed digital computerized image analysis systems, allowing the mapping of the brain areas, nuclei and cell layers where the receptors were located and its ligands would exert their actions (Palacios et al., 1981). The methods were quantitative and made it possible to characterize pharmacologically the
sites being visualized. Together with the development in the preceding decades of histochemical and later immunohistochemically, methods for the transmitter and related proteins, our knowledge of the anatomical and cellular geography of neurotransmission in the mammalian brain made a significant leap forward. The field of serotonin receptors (5-HTRs) was a paradigmatic example of receptor complexities and promises. The existence of multiple 5-HTRs had been postulated already on the 1950's, on the basis of classical pharmacology. Peroutka and Snyder (1979) presented, in 1979, the first evidences for 5HTRs subtypes from radioligand studies.Their results showed that 5-HTRs could be classified into two classes, 5 HT1 and 5 HT2 as differentially labeled by [3H]-5-HT, [3H]-spiperone and [3H]-LSD. Further subdivisions were proposed, based in the properties of new ligand and selective compounds, subdividing 5-HT1 into 5-HT1A and 5-HT1B (Pedigo et al., 1981). We had proposed 5 HT1C based on the localization and characteristics of the sites labeled by a Sandoz compound mesulergine (Palacios et al., 2010; Pazos et al., 1984). Obviously not everybody in the neuropharmacological community was happy with the uncontrolled proliferation of 5 HTRs and the polemic took different aspects see (Palacios et al., 2010). The question Angel Pazos and I put was that if these different proposed 5-HTRs (or “sites”, as we were required to call them at the time) were really different molecular entities, they would probably be expressed by different cell populations and show different regional distributions in the brain. To do that properly we should first look at the pharmacological characteristics of the binding sites of different radioligands, using as many unlabeled displacers as necessary and construct saturation and displacement curves, all that at the microscopic level, and then analyze the differential anatomical localization of these sites throughout the brain. We generated an atlas of the rat brain showing the detailed distribution, density and molecular pharmacological
Please cite this article as: Palacios, J.M., Serotonin receptors in brain revisited. Brain Research (2016), http://dx.doi.org/10.1016/ j.brainres.2015.12.042
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characteristics of 5-HT1A, 5-HT1B and 5-HT1C (later to become 5-HT2C) in the rat brain (Pazos and Palacios, 1985). A companion paper (Pazos et al., 1985) presented similar data for 5HT2, then still considered a single population. The results showed a clear difference in the brain areas labeled or enriched in one or the other population. These differences were not random distributions but rather showed association of receptor subtypes with well-defined anatomical and functional brain areas. For example, we found 5-HT1A enriched in the components of the limbic system, while 5HT1B where predominant in the basal ganglia and the striato nigral pathway. This suggested different cellular populations expressing these receptors, its involvement in the functions of different brain functions thus adding further support to the actual existence of these receptors, and their interest as new targets for drug development. The concept of target identification did not existed at the time. Because of the limited cellular resolution of the technique, it was not possible to assign receptors to specific neuronal or glial populations. A way to overcome this limitation was to study the effects of selective lesions in the brain of the experimental animal and examine changes in receptor densities and localizations, and later studying human neuropathologies. Thanks to our collaboration with Alphonse Probst of the Pathology Institute of the University of Basel, and to the fact that receptors could stand the conditions of human postmortem period (Palacios et al., 1986), we went ahead with the characterization of human 5-HTRs (Pazos et al., 1987a, 1987b). These studies provided important information for later imaging studies of 5-HT receptors in the living human brain (Paterson et al., 2013) It is worth mentioning that all these investigations were always complemented with extensive medicinal chemistry and molecular pharmacology, including detailed membrane binding assays as well as the study signal mechanisms and function of the sites. The expertize of Daniel Hoyer, and that of many other colleagues at Sandoz, was essential for the success of the project (Hoyer et al., 1986a, 1986b). Human studies revealed differences in pharmacology that we extended to other species. In a short time the complexity of the system grew incredibly. Soon important species differences were detected affecting for example drug targets such as the 5-HT1B, not detected in man. This led eventually to the discovery of a new subtype the 5-HT1D (Waeber et al., 1988). Shortly afterwards 5-HT4 and others will come (see Mengod et al., 2006). The advent of the molecular age of receptors will change things dramatically. In 1986 Lefkowitz and colleagues (Dixon et al., 1986) reported the cloning of the gene coding for the beta 2 adrenergic receptor, the first G-protein coupled receptor (GPCR) to be cloned. The following year the same group reported the cloning of a gen coding for a receptor very similar to the beta 2, G21 (Kobilka et al., 1987), which was identified as the 5-HT1A in 1988 (Fargin et al., 1988). In the meantime the 5-HT1C was also cloned (Julius et al., 1988; Lubbert et al., 1987) and from there the 5-HT2 (Julius et al., 1990) demonstrating that the 5-HT1C receptor was in fact a 5HT2C, showing the family of 5 HT2 receptors to be also a multiple one. The entry of the molecular biological techniques into the receptor field signaled the end of the dispute
3
“one transmitter one receptor, or several?” The binding sites were now gen products, proteins with well-defined structural characteristics and properties, looking for a function, selective ligands and a therapeutical utility. Through the use of molecular gene techniques more and more genes coding for new 5-HTRs were identified, while new ligand studies kept postulating new receptors, although not always simultaneous. Only in the 5-HT1 receptor family up to 6 different receptors were defined by 1993. The naming of receptors became terribly complex (Green, 1987), until Hoyer et al. (1994) decided to put some order. The 1994 classification paper became a classic. The “fishing” for new receptors continued. At the end, up to 14 different 5-HTRs were characterized, all of them GCPRs with the exception of the 5-HT3 receptors a ligand-gated ion channel receptor. Some of these receptors presented different molecular forms, reaching a number that we would have not dream of, even in our more lysergic dreams. For us, those interested in the molecular cartography of the brain, the advent of molecular biology into neuropharmacology represented a golden opportunity to expand our geographical activities to the mapping of the mRNAs coding for the different receptors using in situ hybridization, which Guadalupe Mengod brought to the team. In situ hybridization was a technique well fitted for receptor mapping, using similar tissue preparation, generating results in a format similar to receptor autoradiography and amenable to the same type of image analysis. Additionally it offered the opportunity for double or even triple labeling in the same section what aided to identify the type of cell expressing the mRNA for a receptor (Mengod et al., 2015). Now we could correlate the distribution of cells, mainly neurons, expressing the different mRNAs, and which we could phenotype, with that of the binding sites of ligands reputed as specific for a given receptor. The correlative studies of binding sites and mRNA expression beautifully confirmed some of the observations made in lesion studies and human brain lesions, and pointed to specific neuronal populations, including long projecting neurons as well as interneurons throughout the brain (Bruinvels et al., 1994; Pompeiano et al., 1992, 1994). The picture was that of a complex system, where several receptors populations are co-expressed by a single neuronal population and distributed in different cell layers or brain nuclei (for a review see Mengod et al. (2010). Thirty years later many of the main questions we put ourselves in the beginning and those raised by the new findings still remain to be answered. For example: Why so many different receptors for a single transmitter? How does it work serotonergic transmission, at the end of the day? Are then, selective compounds better than non-selective, clinically? How do we explain the different actions of 5-HTRs? Answering these questions will probably need at least 50 more years of Brain Research!
Acknowledgments In addition to those mentioned in the text, I would like to express my gratitude to all technicians, M. Rigo, R. Lenher, and K-H Wiederhold, graduate students, particularly Roser
Please cite this article as: Palacios, J.M., Serotonin receptors in brain revisited. Brain Research (2016), http://dx.doi.org/10.1016/ j.brainres.2015.12.042
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Cortés, Christian Waeber and Maria Pompeiano, postdocs, coworkers and collaborators that contributed so much to this field with their invaluable work in my laboratory in Basel.
references
Bruinvels, A.T., Landwehrmeyer, B., Gustafson, E.L., Durkin, M.M., Mengod, G., Branchek, T.A., Hoyer, D., Palacios, J.M., 1994. Localization of 5-HT1B, 5-HT1D alpha, 5-HT1E and 5-HT1F receptor messenger RNA in rodent and primate brain. Neuropharmacology 33, 367–386. Dixon, R.A., Kobilka, B.K., Strader, D.J., Benovic, J.L., Dohlman, H. G., Frielle, T., Bolanowski, M.A., Bennett, C.D., Rands, E., Diehl, R.E., Mumford, R.A., Slater, E.E., Sigal, I.S., Caron, M.G., Lefkowitz, R.J., Strader, C.D., 1986. Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin. Nature 321, 75–79. Fargin, A., Raymond, J.R., Lohse, M.J., Kobilka, B.K., Caron, M.G., Lefkowitz, R.J., 1988. The genomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the 5-HT1A receptor. Nature 335, 358–360. Green, J.P., 1987. Nomenclature and classification of receptors and binding sites:the need for harmony. Trends Pharmacol. Sci. 8, 90–94. Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin, G.R., Mylecharane, E.J., Saxena, P.R., Humphrey, P.P., 1994. International union of pharmacology classification of receptors for 5hydroxytryptamine (serotonin). Pharmacol. Rev. 46, 157–203. Hoyer, D., Pazos, A., Probst, A., Palacios, J.M., 1986a. Serotonin receptors in the human brain. I. Characterization and autoradiographic localization of 5-HT1A recognition sites. Apparent absence of 5-HT1B recognition sites. Brain Res. 376, 85–96. Hoyer, D., Pazos, A., Probst, A., Palacios, J.M., 1986b. Serotonin receptors in the human brain. II. Characterization and autoradiographic localization of 5-HT1C and 5-HT2 recognition sites. Brain Res. 376, 97–107. Julius, D., Huang, K.N., Livelli, T.J., Axel, R., Jessell, T.M., 1990. The 5HT2 receptor defines a family of structurally distinct but functionally conserved serotonin receptors. Proc. Natl. Acad. Sci. USA 87, 928–932. Julius, D., MacDermott, A.B., Axel, R., Jessell, T.M., 1988. Molecular characterization of a functional cDNA encoding the serotonin 1c receptor. Science 241, 558–564. Kobilka, B.K., Frielle, T., Collins, S., Yang-Feng, T., Kobilka, T.S., Francke, U., Lefkowitz, R.J., Caron, M.G., 1987. An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature 329, 75–79. Kuhar, M.J., 1981. Autoradiographic localization of drug and neurotransmitter receptors in the brain. Trends Neurosci. 4, 60–64. Lefkowitz, R.J., 2004. Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends Pharmacol. Sci. 25, 413–422. Lubbert, H., Hoffman, B.J., Snutch, T.P., van Dyke, T., Levine, A.J., Hartig, P.R., Lester, H.A., Davidson, N., 1987. cDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes. Proc. Natl. Acad. Sci. USA 84, 4332–4336.
Mengod, G., Corte´s, R., Vilaro´, M.T., Hoyer, D., 2010. Distribution of of 5-HT Receptors in the central nervous system. In: Roth, B.L. (Ed.), The serotonin receptors: From molecular pharmacology to human therapeutics. Humana Press Inc., Totowa, NJ, pp. 123–138. Mengod, G., Palacios, J.M., Cortes, R., 2015. Cartography of 5-HT1A and 5-HT2A receptor subtypes in prefrontal cortex and its projections. ACS Chem. Neurosci.. Mengod, G., Vilaro´, M.T., Corte´s, R., Lo´pez-Gime´nez, J.F., Raurich, A., Palacios, J.M., 2006. Chemical neuroanatomy of 5-HT receptor subtypes in the mammalian brain. In: Bryan, L. Roth (Ed.), The Serotonin Receptors. From Molecular Pharmacology to Human Therapeutics. Humana Press, Totowa, New Jersey, pp. 319–364. Palacios, J.M., Niehoff, D.L., Kuhar, M.J., 1981. Receptor autoradiography with tritium sensitive film: potential for computerized densitometry. Neurosci. Lett. 25, 101–105. Palacios, J.M., Pazos, A., Hoyer, D., 2010. The Making of the 5-HT2C Receptor. In: Di Giovanni, G., Esposito, E., Di Matteo, V. (Eds.), 5-HT2C Receptors in the Pathophysiology of CNS Disease. Humana Press, New York, pp. 1–16. Palacios, J.M., Probst, A., Corte´s, R., 1986. Mapping receceptors in the human brain. Trends Neurosci. 9, 284–289. Paterson, L.M., Kornum, B.R., Nutt, D.J., Pike, V.W., Knudsen, G.M., 2013. 5-HT radioligands for human brain imaging with PET and SPECT. Med. Res. Rev. 33, 54–111. Pazos, A., Corte´s, R., Palacios, J.M., 1985. Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res. 346, 231–249. Pazos, A., Hoyer, D., Palacios, J.M., 1984. The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site. Eur. J. Pharmacol. 106, 539–546. Pazos, A., Palacios, J.M., 1985. Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors. Brain Res. 346, 205–230. Pazos, A., Probst, A., Palacios, J.M., 1987a. Serotonin receptors in the human brain–III. Autoradiographic mapping of serotonin1 receptors. Neuroscience 21, 97–122. Pazos, A., Probst, A., Palacios, J.M., 1987b. Serotonin receptors in the human brain–IV. Autoradiographic mapping of serotonin2 receptors. Neuroscience 21, 123–139. Pedigo, N.W., Yamamura, H.I., Nelson, D.L., 1981. Discrimination of multiple [3H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain. J. Neurochem. 36, 220–226. Peroutka, S.J., Snyder, S.H., 1979. Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid die ethylamide and [3H]spiroperidol. Mol. Pharmacol. 16 (3), 687–699. Pompeiano, M., Palacios, J.M., Mengod, G., 1992. Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding. J. Neurosci. 12, 440–453. Pompeiano, M., Palacios, J.M., Mengod, G., 1994. Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors. Mol. Brain Res. 23, 163–178. Waeber, C., Dietl, M.M., Hoyer, D., Probst, A., Palacios, J.M., 1988. Visualization of a novel serotonin recognition site (5-HT1D) in the human brain by autoradiography. Neurosci. Lett. 88, 11–16.
Please cite this article as: Palacios, J.M., Serotonin receptors in brain revisited. Brain Research (2016), http://dx.doi.org/10.1016/ j.brainres.2015.12.042
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pp:024ðcol:fig: : NILÞ brain research ] (]]]]) ]]]–]]]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Review Q2
Serotonin receptors in brain revisited
Q1
Jose´ M. Palacios Frontera Biotechnology S.L., Barcelona 08013, Spain
art i cle i nfo
ab st rac t
Article history:
In the early 1980’s, the dispute on the existence of multiplicity of receptor for neuro-
Accepted 19 December 2015
transmitter was at its height. Several subtypes of serotonin (5-HT) receptors were proposed on the basis of radioligand binding assays. In order to provide further support to the
Keywords:
existence of these receptors we performed quantitative autoradiographic mapping of the
Receptor multiplicity
binding of several ligands for the 5-HT1 receptor labeling the subtypes 5-HT1A, 5-HT1B and
Serotonin (5-HT)
5-HT1C, and characterize pharmacologically these different receptors. The results demon-
Receptor autoradiography
strated differential localization of the subtypes of 5-HT1 receptor indicating that they were
Molecular neuroanatomy
expressed by different cell populations, probably neurons, in the brain and further
Human brain
supporting their reality. Shortly afterwards, the cloning of the genes coding for these 5HT receptors, and many others, ended the dispute by demonstrating that they were different proteins. The advent of Molecular Biology provided new methodologies for the study the chemical and molecular anatomy of 5-HT receptors in brain, by visualizing cells expressing their mRNA by in situ hybridization and showed that the family of mammalian 5-HT receptors has 14 members, a figure much larger than ever suspected. Original article abstract: Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors: The distribution of serotonin-1 (5-HT1) receptors in the rat brain was studied by light microscopic quantitative autoradiography. Receptors were labeled with [3H]serotonin (5-[3H]HT), 8-hydroxy-2-[H-dipropylamino-3H]tetralin (8-OH-[3H] DPAT), [3H]LSD and [3H]mesulergine, and the densities quantified by microdensitometry with the aid of a computer-assisted image-analysis system. Competition experiments for 5-[3H]HT binding by several serotonin-1 agonizts led to the identification of brain areas enriched in each one of the three subtypes of 5-HT1 recognition sites already described (5HT1A, 5-HT1B, 5-HT1C). The existence of these'selective' areas allowed a detailed pharmacological characterization of these sites to be made in a more precise manner than has been attained in membrane-binding studies. While 5-[3H]HT labeled with nanomolar affinity all the 5-HT1 subtypes, the other 3H-labeled ligands labeled selectively 5-HT1A (8OH-[3H]DPAT), 5-HT1C ([3H]mesulergine) and both of them ([3H]LSD). Very high concentrations of 5-HT1 receptors were localized in the choroid plexus, lateroseptal nucleus, globus pallidus and ventral pallidum, dentate gyrus, dorsal subiculum, olivary pretectal nucleus, substantia nigra, reticular and external layer of the entorhinal cortex. The different fields of the hippocampus (CA1–CA4), some nuclei of the amygdaloid complex, the hypothalamic nuclei and the dorsal raphé, among others, also presented high concentrations of sites. Areas containing intermediate densities of 5-HT1 receptors included the claustrum, olfactory tubercle, accumbens, central gray and lateral cerebellar nucleus. The nucleus
E-mail address: [email protected] http://dx.doi.org/10.1016/j.brainres.2015.12.042 0006-8993/& 2016 Published by Elsevier B.V.
Please cite this article as: Palacios, J.M., Serotonin receptors in brain revisited. Brain Research (2016), http://dx.doi.org/10.1016/ j.brainres.2015.12.042
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brain research ] (]]]]) ]]]–]]]
caudate-putamen and the cortex, at the different levels studied, presented receptor densities ranging from intermediate to low. Finally, in other brain areas-pons, medulla, and spinal cord-only low or very low concentrations of 5-HT1 receptors were found. From the areas strongly enriched in 5-HT1 sites, dentate gyrus and septal nucleus contained 5HT1A sites, while globus pallidus, dorsal subiculum, substantia nigra and olivary pretectal nucleus were enriched in 5-HT1B. The sites in the choroid plexus, which presented the highest density of receptors in the rat brain, were of the 5-HT1C subtype. The distribution of 5-HT1 receptors reported here is discussed in correlation with the distribution of serotoninergic neurons and fibers, the related anatomical pathways and the effects which appear to be mediated by these sites. © 1985. This article is part of a Special Issue entitled SI:50th Anniversary Issue. & 2016 Published by Elsevier B.V.
Contents Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
The work reported in our paper was conducted in Basle (Switzerland) at Sandoz, now Novartis, in a moment of tremendous change in the study of receptors for neurotransmitters. In the late 1970's and early 1980's the dispute between the supporters of a limited number of receptor for a neurotransmitter (one, at the most two), the classical isolated organ, functional pharmacologists and those, the new biochemical “radioactive” pharmacologists, postulating multiplicity of receptors for a neurotransmitter, was at its height (Palacios et al., 2010). This was driven by, in one side, the development of new relatively simple methods, such as radioligand binding techniques, Robert Lefkowitz has written: “if a single technical advance can be said to have opened the door to the molecular era of receptors, it was the development of radioligand binding methods during the 1970's” (Lefkowitz, 2004). On the other hand, the pharmaceutical industry was committed to the discover y and development of new molecules with significant potential for the treatment of diseases of the CNS, and significant economic interest. I joined Sandoz in August 1981, following a 3 years postdoctoral stay at the Department of Neuroscience, Medical School, Johns Hopkins University, working with Michael Kuhar (Kuhar, 1981), and in the exciting environment created by Sol Snyder. There I took part in the development of the technique of receptor autoradiography, a daughter of the “grind and bind” assays. This relatively simple procedure consisted, basically in labeling receptors in microtome sections of brain tissues and generating autoradiographic images of the radiolabeled sites by apposing it to photographic emulsions. The visualization of the binding sites for receptor ligands at the light microscopic level and recently developed digital computerized image analysis systems, allowing the mapping of the brain areas, nuclei and cell layers where the receptors were located and its ligands would exert their actions (Palacios et al., 1981). The methods were quantitative and made it possible to characterize pharmacologically the
sites being visualized. Together with the development in the preceding decades of histochemical and later immunohistochemically, methods for the transmitter and related proteins, our knowledge of the anatomical and cellular geography of neurotransmission in the mammalian brain made a significant leap forward. The field of serotonin receptors (5-HTRs) was a paradigmatic example of receptor complexities and promises. The existence of multiple 5-HTRs had been postulated already on the 1950's, on the basis of classical pharmacology. Peroutka and Snyder (1979) presented, in 1979, the first evidences for 5HTRs subtypes from radioligand studies.Their results showed that 5-HTRs could be classified into two classes, 5 HT1 and 5 HT2 as differentially labeled by [3H]-5-HT, [3H]-spiperone and [3H]-LSD. Further subdivisions were proposed, based in the properties of new ligand and selective compounds, subdividing 5-HT1 into 5-HT1A and 5-HT1B (Pedigo et al., 1981). We had proposed 5 HT1C based on the localization and characteristics of the sites labeled by a Sandoz compound mesulergine (Palacios et al., 2010; Pazos et al., 1984). Obviously not everybody in the neuropharmacological community was happy with the uncontrolled proliferation of 5 HTRs and the polemic took different aspects see (Palacios et al., 2010). The question Angel Pazos and I put was that if these different proposed 5-HTRs (or “sites”, as we were required to call them at the time) were really different molecular entities, they would probably be expressed by different cell populations and show different regional distributions in the brain. To do that properly we should first look at the pharmacological characteristics of the binding sites of different radioligands, using as many unlabeled displacers as necessary and construct saturation and displacement curves, all that at the microscopic level, and then analyze the differential anatomical localization of these sites throughout the brain. We generated an atlas of the rat brain showing the detailed distribution, density and molecular pharmacological
Please cite this article as: Palacios, J.M., Serotonin receptors in brain revisited. Brain Research (2016), http://dx.doi.org/10.1016/ j.brainres.2015.12.042
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characteristics of 5-HT1A, 5-HT1B and 5-HT1C (later to become 5-HT2C) in the rat brain (Pazos and Palacios, 1985). A companion paper (Pazos et al., 1985) presented similar data for 5HT2, then still considered a single population. The results showed a clear difference in the brain areas labeled or enriched in one or the other population. These differences were not random distributions but rather showed association of receptor subtypes with well-defined anatomical and functional brain areas. For example, we found 5-HT1A enriched in the components of the limbic system, while 5HT1B where predominant in the basal ganglia and the striato nigral pathway. This suggested different cellular populations expressing these receptors, its involvement in the functions of different brain functions thus adding further support to the actual existence of these receptors, and their interest as new targets for drug development. The concept of target identification did not existed at the time. Because of the limited cellular resolution of the technique, it was not possible to assign receptors to specific neuronal or glial populations. A way to overcome this limitation was to study the effects of selective lesions in the brain of the experimental animal and examine changes in receptor densities and localizations, and later studying human neuropathologies. Thanks to our collaboration with Alphonse Probst of the Pathology Institute of the University of Basel, and to the fact that receptors could stand the conditions of human postmortem period (Palacios et al., 1986), we went ahead with the characterization of human 5-HTRs (Pazos et al., 1987a, 1987b). These studies provided important information for later imaging studies of 5-HT receptors in the living human brain (Paterson et al., 2013) It is worth mentioning that all these investigations were always complemented with extensive medicinal chemistry and molecular pharmacology, including detailed membrane binding assays as well as the study signal mechanisms and function of the sites. The expertize of Daniel Hoyer, and that of many other colleagues at Sandoz, was essential for the success of the project (Hoyer et al., 1986a, 1986b). Human studies revealed differences in pharmacology that we extended to other species. In a short time the complexity of the system grew incredibly. Soon important species differences were detected affecting for example drug targets such as the 5-HT1B, not detected in man. This led eventually to the discovery of a new subtype the 5-HT1D (Waeber et al., 1988). Shortly afterwards 5-HT4 and others will come (see Mengod et al., 2006). The advent of the molecular age of receptors will change things dramatically. In 1986 Lefkowitz and colleagues (Dixon et al., 1986) reported the cloning of the gene coding for the beta 2 adrenergic receptor, the first G-protein coupled receptor (GPCR) to be cloned. The following year the same group reported the cloning of a gen coding for a receptor very similar to the beta 2, G21 (Kobilka et al., 1987), which was identified as the 5-HT1A in 1988 (Fargin et al., 1988). In the meantime the 5-HT1C was also cloned (Julius et al., 1988; Lubbert et al., 1987) and from there the 5-HT2 (Julius et al., 1990) demonstrating that the 5-HT1C receptor was in fact a 5HT2C, showing the family of 5 HT2 receptors to be also a multiple one. The entry of the molecular biological techniques into the receptor field signaled the end of the dispute
3
“one transmitter one receptor, or several?” The binding sites were now gen products, proteins with well-defined structural characteristics and properties, looking for a function, selective ligands and a therapeutical utility. Through the use of molecular gene techniques more and more genes coding for new 5-HTRs were identified, while new ligand studies kept postulating new receptors, although not always simultaneous. Only in the 5-HT1 receptor family up to 6 different receptors were defined by 1993. The naming of receptors became terribly complex (Green, 1987), until Hoyer et al. (1994) decided to put some order. The 1994 classification paper became a classic. The “fishing” for new receptors continued. At the end, up to 14 different 5-HTRs were characterized, all of them GCPRs with the exception of the 5-HT3 receptors a ligand-gated ion channel receptor. Some of these receptors presented different molecular forms, reaching a number that we would have not dream of, even in our more lysergic dreams. For us, those interested in the molecular cartography of the brain, the advent of molecular biology into neuropharmacology represented a golden opportunity to expand our geographical activities to the mapping of the mRNAs coding for the different receptors using in situ hybridization, which Guadalupe Mengod brought to the team. In situ hybridization was a technique well fitted for receptor mapping, using similar tissue preparation, generating results in a format similar to receptor autoradiography and amenable to the same type of image analysis. Additionally it offered the opportunity for double or even triple labeling in the same section what aided to identify the type of cell expressing the mRNA for a receptor (Mengod et al., 2015). Now we could correlate the distribution of cells, mainly neurons, expressing the different mRNAs, and which we could phenotype, with that of the binding sites of ligands reputed as specific for a given receptor. The correlative studies of binding sites and mRNA expression beautifully confirmed some of the observations made in lesion studies and human brain lesions, and pointed to specific neuronal populations, including long projecting neurons as well as interneurons throughout the brain (Bruinvels et al., 1994; Pompeiano et al., 1992, 1994). The picture was that of a complex system, where several receptors populations are co-expressed by a single neuronal population and distributed in different cell layers or brain nuclei (for a review see Mengod et al. (2010). Thirty years later many of the main questions we put ourselves in the beginning and those raised by the new findings still remain to be answered. For example: Why so many different receptors for a single transmitter? How does it work serotonergic transmission, at the end of the day? Are then, selective compounds better than non-selective, clinically? How do we explain the different actions of 5-HTRs? Answering these questions will probably need at least 50 more years of Brain Research!
Acknowledgments In addition to those mentioned in the text, I would like to express my gratitude to all technicians, M. Rigo, R. Lenher, and K-H Wiederhold, graduate students, particularly Roser
Please cite this article as: Palacios, J.M., Serotonin receptors in brain revisited. Brain Research (2016), http://dx.doi.org/10.1016/ j.brainres.2015.12.042
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Cortés, Christian Waeber and Maria Pompeiano, postdocs, coworkers and collaborators that contributed so much to this field with their invaluable work in my laboratory in Basel.
references
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