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Distribution of the 5-HT5A serotonin receptor mRNA in the human brain
Molecular Brain Research 56 Ž1998. 1–8
Research report
Distribution of the 5-HT5A serotonin receptor mRNA in the human brain Massimo Pasqualetti a , Michela Ori a , Irma Nardi a,) , Maura Castagna b, Giovanni Batista Cassano c , Donatella Marazziti c a
Laboratori di Biologia cellulare e dello sÕiluppo, Dipartimento di Fisiologia e Biochimica, UniÕersita` di Pisa, Õia Carducci 13, Ghezzano 56010, Pisa, Italy b Laboratorio di Anatomia Patologica, Dipartimento di Chirurgia, UniÕersita` di Pisa, Õia Roma 57, 56100 Pisa, Italy c Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, UniÕersita` di Pisa, Õia Roma 67, 56100 Pisa, Italy Accepted 16 December 1997
Abstract The 5-HT5A receptor is a member of a new subfamily of serotonin w5-hydroxytryptamine Ž5-HT.x receptors recently cloned from the human and rodent brain. The role of this receptor in normal brain functions as well as its possible involvement in pathological states is still to be determined. We therefore studied the regional distribution and cellular localization of 5-HT5A receptor mRNA in human brain sections from autopsy samples by in situ hybridization histochemistry, in order to obtain anatomical information which might be useful in formulating hypotheses on possible functions subserved by this receptor in the central nervous system ŽCNS.. Our results showed that the main sites of 5-HT5A mRNA expression were the cerebral cortex, hippocampus and cerebellum. In the neocortical regions, the 5-HT5A receptor mRNA was mainly distributed in the layers II–III and V–VI. In the hippocampus, the dentate gyrus and the pyramidal cell layer of the CA1 and CA3 fields expressed 5-HT5A mRNA at high levels. The broad distribution in the neocortex and hippocampus supports the view that the 5-HT5A receptor in these areas might be implicated in high cortical and limbic functions. The 5-HT5A mRNA was widely distributed in the cerebellum where it was highly expressed in the Purkinje cells, in the dentate nucleus and, at a lower level, in the granule cells. Since the cerebellum receives diffuse serotonergic afferents, this finding suggests that the 5-HT5A receptor may have an important role in mediating the effects of 5-HT on cerebellar functions. q 1998 Elsevier Science B.V. Keywords: 5-HT5A receptor mRNA expression; In situ hybridization; Human central nervous system; Cortex; Hippocampus; Cerebellum
1. Introduction The neuromodulator 5-hydroxytryptamine Žserotonin, 5-HT. has been implicated in a wide range of behavioural and physiological functions, including processes as diverse as cardiovascular control, feeding regulation, the sleep– wake cycle, learning, nociception, analgesia and some neuropsychiatric disorders w15x. The various actions of 5-HT are mediated by several receptors which have been classified as pharmacologically distinct subtypes w3,14,16x. These receptors belong to two different protein superfamilies: ligand-gated ion channel receptors Ž5-HT3 . and the G protein-coupled receptor superfamily. During recent years, however, molecular cloning studies have revealed the existence of several novel 5-HT receptors for which little or no
) Corresponding author. Fax: q39-50-878486; E-mail: [email protected]
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 0 0 3 - 5
prior pharmacological or functional data existed Ž5-HT1E , 5-HT1F , 5-HT5A , 5-HT5B , 5-HT6 and 5-HT7 .. Much effort has, therefore, been directed towards the understanding of the physiological roles of these receptors and towards identifying selective ligands to be used as putative therapeutic agents. Among these novel cloned receptors, a new subfamily, containing two members designated 5-HT5A and 5-HT5B , has been isolated in the mouse and the rat w9,17x. Recently, the human 5-HT5A receptor has also been cloned, while a 5-HT5B receptor does not seem to exist in humans w22x. According to pharmacological criteria, the 5-HT5 receptors fall into the 5-HT1-like class w21x. However, their separate classification is justified by other characteristics such as the sequence, which is less than 37% homologous to members of the 5-HT1 family and the presence of an intron between the putative transmembrane domain V and VI, in contrast with 5-HT1 receptor genes which are intronless w13x. Moreover, the expression of 5-HT5A recep-
M. Pasqualetti et al.r Molecular Brain Research 56 (1998) 1–8
2 Table 1 Characteristics of autoptic brain samples Number Age Sex
Cause of death
1 2 3 4 5 6
Pulmonary embolism Cardiac Cardiac Acute heart failure Pulmonary embolism Cardiac
46 55 60 73 78 80
Female Male Male Male Female Male
lyzed its gene expression in various regions of the brain by means of in situ hybridization. Postmortem delay Žh. 12 20 14 12 22 24
tors in mammalian cell lines has failed so far to demonstrate any G protein coupling w17,21,26x. By means of the reverse transcriptase-polymerase chain reaction ŽRT-PCR. technique, the 5-HT5A mRNA has been shown to be expressed in the adult human brain and, at a lower level, in the foetal brain, thus suggesting that the receptor is developmentally regulated w22x. As the pharmacological profile of the 5-HT5A receptor resembles that of the 5-HT1D receptor, it seems plausible that it may subserve some of the functions previously attributed to the 5-HT1D receptor family w17x. However, its precise distribution and role within the human central nervous system ŽCNS., as well as its possible involvement in pathological states, is still unknown. In order to contribute to the understanding of the function of the 5-HT5A receptor in the human CNS, we ana-
2. Materials and methods 2.1. Tissue collection and processing Human brain samples were obtained at autopsy from six subjects Žfour men and two women., aged between 46 and 80 years who were selected for the absence of any neurological or psychiatric disorders and who had not taken any psychotropic drugs Žsee Table 1.. Tissue collection, dissection and storage have been described in a previous paper w20x. Sections Ž10–15 m m thick. obtained with a microtome cryostat ŽLeitz 1720. at y208C, were thaw-mounted on organosylanized-coated slides and fixed with paraformaldehyde in a phosphatebuffered saline solution ŽPBS., rinsed twice in 1 = PBS, dehydrated in graded alcohol and processed immediately. 2.2. In situ hybridization 2.2.1. RNA probe synthesis The human 5-HT5A cDNA clone w22x was kindly provided by the Glaxo Wellcome Research and Development ŽStevenage, UK.. The 1.080 kb EcoRI–SacI insert was
Fig. 1. Dark-field photomicrographs showing the distribution of 5-HT5A receptor mRNA in human parietal cortex. ŽA. A diffuse distribution of autoradiographic grains are present through layers II–VI. ŽB. High magnification of a selected area Žindicated by arrowheads. of the parietal cortex shown in ŽA.: layer III appears more intensely labeled. Scale bars, 0.2 cm. ŽA., 0.3 mm ŽB..
M. Pasqualetti et al.r Molecular Brain Research 56 (1998) 1–8
subcloned in the pGem 7ZfŽy. expression vector ŽPromega., containing T7 and SP6 RNA polymerase promoters and linearized to generate 35 S-radiolabeled antisense and sense riboprobes, as previously described w20x. Probes were degraded to optimal hybridization sizes Ž100–300 nucleotides. by alkaline hydrolysis w6x. 2.2.2. Pretreatment of slides and hybridization Pretreatment of slides and in situ hybridization were performed as already described w20x. Sections were exposed to Kodak MR X-ray film for 2–4 days and then dipped in 1:1 diluted Kodak NTB-2 emulsion with a solution of 0.6 M ammonium acetate, 2% glycerol and exposed for 4–6 weeks at 48C, developed in Kodak D19, stained with Giemsa, dehydrated and mounted. Sections were examined using bright- and dark-field light microscopy ŽZeiss..
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3. Results The anatomical distribution of the 5-HT5A mRNA was analyzed in serial histological sections of the following brain regions: cerebral cortex, hippocampus, amygdala, basal ganglia, substantia innominata Žbasal nucleus of Meynert., substantia nigra, thalamus, choroid plexus and cerebellum. Some sections of each sample were not processed for in situ hybridization, but stained with Cresyl violet to permit a detailed analysis of the cytoarchitecture. Brain areas and nuclei were identified according to DeArmond et al. w7x. As control experiments, histological sections of each analyzed brain structure were hybridized with a sense probe. In these control experiments the level of the signal was very low and was considered as unspecific background Žan example is shown in Fig. 5B.. In contrast,
Fig. 2. Dark-field photomicrographs of coronal sections of entorhinal cortex, subiculum and hippocampus showing the distribution of 5-HT5A mRNA. ŽA. A moderate level of labeling is shown by the subiculum and entorhinal cortex. ŽB. CA1, CA3 fields and the dentate gyrus of hippocampal formation are densely labeled. Bright-field high-magnification photomicrographs of the CA3 field ŽC. and the dentate gyrus ŽD.: the pyramidal neurons of the CA3 field and the granular cells of the dentate gyrus are labeled. Small cells, probably glial cells, are clearly unlabeled Žarrowhead in ŽC... DG, dentate gyrus; EC, entorhinal cortex; S, subiculum. Scale bars, 0.2 cm ŽA., 0.15 cm ŽB., 30 m m ŽC., 30 m m ŽD..
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M. Pasqualetti et al.r Molecular Brain Research 56 (1998) 1–8
hybridization with the antisense probe resulted in a localized signal distribution. We detected no significant differences in the hybridization signal in the examined autopsy samples, in spite of the different ages of the subjects andror postmortem delay. 3.1. Cortical areas The distribution of the 5-HT5A receptor mRNA was examined in the prefrontal, parietal, temporal, occipital and entorhinal cortices. All these areas showed a general labeling pattern consisting of a diffuse autoradiographic signal throughout the different layers of the neocortex. However, the labeling was preferentially concentrated in the layers II–III and V–VI ŽFig. 1A.. In particular the layer III appeared to be intensely labeled ŽFig. 1B.. A lower level of hybridization signal was found in the entorhinal cortex ŽFig. 2A.. To determine which cell types express the 5-HT5A mRNA, we studied in situ emulsion autoradiographs at the light microscopy level. All the labeled cells seemed to belong to the neuronal types, among which large pyramidal cells could be recognized. No hybridization signal was detected in small cells Ždata not shown.. 3.2. Hippocampus The 5-HT5A receptor mRNA was expressed at high levels in the hippocampal formation ŽFig. 2B–D.. The granule cell layer of the dentate gyrus and the CA1 and CA3 fields of the Hammon’s horn were strongly labeled ŽFig. 2B.. At higher magnification all the pyramidal neu-
rons of CA1 and CA3 fields and the granule cells of the dentate gyrus were labeled ŽFig. 2C and D., while small cells were not labeled ŽFig. 2C and D.. No differences in the pattern of expression were found in the rostrocaudal extent of the hippocampus Ždata not shown.. An autoradiographic signal was also present in the subiculum Žcf. Fig. 2A.. 3.3. Amygdala The amygdala was differentially labeled: a high level of 5-HT5A mRNA expression was detected in the ventrolateral part, while the dorsomedial region was not labeled ŽFig. 3A.. 3.4. Basal ganglia and substantia innominata The 5-HT5A mRNA distribution in the basal ganglia was heterogeneous. A weak hybridization signal was found in the nucleus caudatus Ždata not shown.; in the putamen, only scattered cells appeared to be labeled ŽFig. 3B.; the pars medialis of the globus pallidus was not labeled, while the pars lateralis presented a few scattered, labeled cells ŽFig. 3B.. The cell bodies of the substantia innominata showed a high density of autoradiographic grains ŽFig. 3B.: at higher magnification the majority of neuronal cells of this nucleus appeared labeled Ždata not shown.. 3.5. Cerebellum In the cerebellum, the highest level of 5-HT5A mRNA expression was detected in the Purkinje cell perikarya ŽFig.
Fig. 3. Dark-field photomicrographs from autoradiograms of coronal sections showing the 5-HT5A mRNA expression in amygdala ŽA., globus pallidus, putamen and substantia innominata ŽB.. A high hybridization signal is present in the ventrolateral part the amygdala. The pars lateralis of globus pallidus shows labeled scattered cells, while no hybridization signal can be observed in the pars medialis. Scattered cells are labeled in the putamen. The majority of cells in the substantia innominata appears densely labelled. GPL, globus pallidus pars lateralis; GPM, globus pallidus pars medialis; PU, putamen; SI, substantia innominata; VLA, ventro lateral amygdala. Scale bars, 0.2 cm ŽA.; 0.9 mm ŽB..
M. Pasqualetti et al.r Molecular Brain Research 56 (1998) 1–8
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Fig. 4. Distribution of 5-HT5A receptor mRNA in the cerebellum. ŽA. Dark-field photomicrograph of a coronal section of the cerebellar cortex: the Purkinje cells are heavily labeled. High-magnification bright-field ŽB. and dark-field ŽC. photomicrographs showing a high level of hybridization on the Purkinje cell perikaria. ŽD. A high hybridization signal is present in the dentate nucleus. ŽE. Bright-field high-magnification photomicrograph of dentate nucleus: all neuronal cell types are labeled. DN, dentate nucleus; PC, Purkinje cell. Scale bars, 0.6 mm ŽA., 500 m m ŽB., 900 m m ŽC., 0.1 mm ŽD., 30 m m ŽE..
4A–C.. A lower hybridization signal was found in the granule layer, as clearly shown in X-ray film autoradiographs ŽFig. 5A.. Due to the high density of Giemsa-stained cells, the labeling on the granule layer was, in fact, undetectable in emulsion autoradiographs Žcf. Fig. 4A–C.. No
labeling was present in the molecular layer. This pattern of mRNA expression was found in the cortex of both the hemispheres and vermis Žcf. Figs. 4 and 5. with no rostrocaudal regional differences. The dentate nucleus was highly labeled: as observed at higher magnification, the hybridiza-
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M. Pasqualetti et al.r Molecular Brain Research 56 (1998) 1–8
Fig. 5. X-ray film autoradiograms of coronal sections of the vermis cerebellar cortex. ŽA. The hybridization signal is present in the Purkinje cell perikaria Žarrow. and in the granule cell layer. ŽB. An adjacent section of cerebellar cortex hybridized with a sense probe: the level of the signal is very low and considered as unspecific background. Scale bars, 0.18 cm.
tion signal was concentrated only on neuronal cell types ŽFig. 4D and E.. 3.6. Other brain areas 5-HT5A transcripts were not detectable in some thalamic nuclei such as the dorsomedial, lateral dorsal and ventral posterolateral, as well as in substantia nigra and choroid plexus Ždata not shown.. 4. Discussion In the present study, we determined the expression of mRNA encoding the 5-HT5A receptor subtype in postmortem human brain tissues, by using in situ hybridization. Our results confirm that the 5-HT5A receptor mRNA is expressed in different regions of the brain and provide the first detailed data as to its regional and cellular distribution. The main sites of 5-HT5A mRNA expression were represented by the cerebral cortex, amygdala, hippocampus and cerebellum, while a lower level of expression was found in the other examined regions. The distribution of 5-HT5A mRNA in the human brain was found to be generally similar to that in the mouse brain w21x, although some differences were observed.
In the cerebral cortex, the 5-HT5A receptor mRNA was mainly distributed in the layers II–III and V–VI. When these areas were analyzed at higher magnification, it was evident that cells with pyramidal morphology expressed the receptor mRNA. In particular, a high level of expression was found in pyramidal cells of the layer III of the prefrontal, occipital and parietal cortices. However, the possibility of other neuronal types expressing the receptor mRNA could not be excluded. The presence of 5-HT5A mRNA expression in pyramidal cells of different neocortical areas would suggest that these receptors might play a role in several brain functions, such as those involving highly integrative activities. In the hippocampal formation, the dentate gyrus and the pyramidal cell layer of the CA1 and CA3 fields appeared to express the 5-HT5A mRNA at a high level. The 5-HT5A mRNA was present in the granule cells of the dentate gyrus and in the pyramidal cells of the Hammon’s horn. The pattern of 5-HT5A mRNA expression parallels that of the 5-HT1A mRNA w4,20x, suggesting that these two receptors may be co-expressed in the pyramidal cells of the CA1 and CA3 fields and in the granule cells of the dentate gyrus. Our previous observation that the 5-HT1D a and the 5-HT2A receptor mRNA are expressed only in the pyramidal cells of CA3 and CA1 fields respectively w20x, seems to indicate that different combinations of receptor transcripts might exist in these hippocampal areas. Although any
M. Pasqualetti et al.r Molecular Brain Research 56 (1998) 1–8
definitive conclusion with regard to the presence of multiple 5-HT receptors in the same cell will require doublelabeling techniques, these results would suggest that the balance between different receptors might be relevant to the functioning of one area, rather than the level of activity of one receptor only. In addition to the hippocampus, 5-HT5A mRNA expression was found in other areas belonging to the limbic system, such as the amygdala and entorhinal cortex. As this system is assumed to be involved in learning, memory and impulsivity w25x, 5-HT5A may be one of the candidate receptors regulating these functions. The 5-HT5A mRNA was widely distributed in the cerebellum where it appeared to be highly expressed in the Purkinje cell perikarya, in the dentate nucleus and, at a lower level, in the granule cells. To our knowledge, this is the first report of a 5-HT receptor localized in all the main cell types which control both cerebellar inputs and outputs. In mice, the 5-HT5A mRNA seems to be expressed only in the granule cell layer w21x, while the Purkinje cells represent one of the main sites of expression of the 5-HT1B receptor, which is the rodent homologue of the human 5-HT1D b w12x. A difference in the expression of the 5-HT5A receptor would seem therefore to exist between human and mouse cerebellum, although this possibility requires further investigation. Although the cerebellum has traditionally been viewed as a structure that contributes primarily to motor coordination and control, there is now new and growing evidence that it is also involved in some aspects of cognitive functions w1,10x. Animal studies and psychometric tests in psychiatric patients have even suggested that the cerebellum may contribute to emotional processing w2,24x. This new concept with regard to cerebellar functions has found an anatomical substrate in the presence of cerebellar outputs gaining access to the prefrontal cortex w18x. Since the cerebellum receives diffuse serotonergic innervation by afferents originating from raphe nuclei, it is tempting to speculate that 5-HT may modulate such nonmotor or cognitive cerebellar functions through specific receptors. The wide distribution of 5-HT5A mRNA in the cerebellum makes this receptor a putative candidate for involvement in the cerebellar contribution to cognition or other emotional processes. This hypothesis is in agreement with the observation that the 5-HT5A mRNA is expressed in the cortex and in limbic structures such as the hippocampal formation, amygdala and entorhinal cortex, while it is poorly represented in the basal ganglia and absent from the substantia nigra, which are both implicated in the control of motor activity. Recently, in order to study the function of the 5-HT5A receptor, knockout mice lacking this receptor have been generated by homologous recombination w11x. Interestingly, these mutant mice, which develop normally, were found to have enhanced exploratory activity in a novel environment as compared with controls, suggesting that
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the 5-HT5A receptors might be involved in exploratory behaviour w11,8x. It is also worth noting that 5-HT5A receptor transcripts were detected in the substantia innominata, one of the cholinergic nuclei of the basal forebrain ŽBF. which projects to the cortex w27x. The cholinergic system has been implicated in various behavioural and mental functions w23x and 5-HT afferents have been reported to form synapses with BF neurons, suggesting that 5-HT may influence the functional state of the cholinergic projections to the cerebral cortex and other brain areas. w5,19x. Although the localization of the 5-HT5A receptor in cholinergic cells deserves further investigation using double labeling techniques, the presence of 5-HT5A transcripts in the same area as cholinergic neurons indicates that this receptor might mediate the action of 5-HT on cholinergic system. The identification of a specific 5-HT receptor on cholinergic neurons may have some relevance in view of the possibility of manipulating their activity by means of serotonergic drugs. Although our data support the notion that the 5-HT5A receptors could play an important role in various human brain functions, further studies on its pharmacological properties and on its second messenger pathway are necessary in order to provide a definition of its physiological role, as well as of its possible implication in the pathophysiology of the CNS.
Acknowledgements We thank Glaxo Wellcome Research and Development for kindly supplying the 5-HT5A cDNA clone. We also thank Fabio Cini for his technical assistance and Dr. Augusto Innocenti for his help. This work was supported by a grant from Murst and Ravizza Farmaceutici.
References w1x G. Allen, R.B. Buxton, E.C. Wong, E. Courchesne, Attentional activation of the cerebellum independent of motor involvement, Science 275 Ž1997. 1940–1943. w2x G.G. Berntson, M.W. Torello, The paleocerebellum and the integration of behavioral function, Physiol. Psychol. 10 Ž1982. 2–12. w3x F.G. Boess, I.L. Martin, Molecular biology of 5-HT receptors, Neuropharmacology 33 Ž1994. 275–317. w4x P.W.J. Burnet, S.L. Eastwood, K. Lacey, P.J. Harrison, The distribution of 5-HT1A and 5-HT2A receptor mRNA in human brain, Brain Res. 676 Ž1995. 157–168. w5x J.-C. Cassel, H. Jeltsch, Serotonergic modulation of cholinergic function in the central nervous system: cognitive implications, Neuroscience 69 Ž1995. 1–41. w6x K.H. Cox, D.V. DeLeon, L.M. Angerer, R.C. Angerer, Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes, Dev. Biol. 101 Ž1984. 485–502. w7x S.J. DeArmond, M.M. Fusco, M.M. Dewey, Structure of the Human Brain, Oxford Univ. Press, Oxford, 1989, 202 pp.
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w8x S.C. Dulawa, R. Graihle, R. Hen, M.A. Geyer, Characterization of exploratory behavior and sensorimotor gating in wild type and serotonin 5A knockout mice, Soc. Neurosci. Abstr. Ž1997. 482.20. w9x M.G. Erlander, T.W. Lovenberg, B.M. Baron, L. Lecea, P.E. Danielson, M. Racke, A.L. Slone, B.W. Siegel, P.E. Foye, K. Cannon, J.E. Burns, J.G. Sutcliffe, Two members of a distinct subfamily of 5-hydroxytryptamine receptors differentially expressed in rat brain, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 3452–3456. w10x J.A. Fiez, Cerebellar contribution to cognition, Neuron 16 Ž1996. 13–15. w11x R. Grailhe, C. Waeber, D. Brunner, X. Zhuang, Y. Huang, M.A. Moskowitz, R. Hen, The 5-HT5A receptors: localization in the CNS and increased exploratory activity in 5-HT5A knockout mice, Soc. Neurosci. Abstr. Ž1997. 482.19. w12x P.R. Hartig, T.A. Branchek, R.L. Weinshank, A subfamily of 5-HT1D receptor genes, Trends Pharmacol. Sci. 13 Ž1992. 152–159. w13x D. Hoyer, D.E. Clarke, J.R. Fozard, P.R. Hartig, G.R. Martin, E.J. Mylecharane, P.R. Saxena, P.A. Humphrey, VII. International union of pharmacology classification of receptor for 5-hydroxytryptamine Žserotonin., Pharmacol. Rev. 46 Ž1994. 157–203. w14x D. Hoyer, G. Martin, 5-HT receptor classification and nomenclature: towards a harmonization with the human genome, Neuropharmacology 36 Ž1997. 419–428. w15x B.L. Jacobs, C.A. Formal, Serotonin and behaviour: a general hypothesis, in: F.E. Bloom, H.Y. Meltzer ŽEds.., Psychopharmacology the Fourth Generation of Progress, Raven Press, New York, 1995, pp. 461–469. w16x G.R. Martin, P.P.A. Humphrey, Receptors for 5-hydroxytryptamine: current perspectives on classification and nomenclature, Neuropharmacology 33 Ž1994. 261–273. w17x H. Matthes, U. Boschert, N. Amlaiky, R. Grailhe, J.-L. Plassat, F. Muscatelli, M.-G. Mattei, R. Hen, Mouse 5-hydroxytryptamine5A and 5-hydroxytryptamine5B receptors define a new family of sero-
w18x
w19x
w20x
w21x
w22x
w23x w24x
w25x
w26x
w27x
tonin receptors: cloning, functional expression, and chromosomal localization, Mol. Pharmacol. 43 Ž1993. 313–319. F.A. Middleton, P.L. Strick, Anatomical evidence for cerebellar and basal ganglia involvement in higher cognitive function, Science 266 Ž1994. 458–461. T.A. Milner, E. Veznedaroglu, Serotonin-containing terminals synapse on septohippocampal neurons in the rat, J. Neurosci. Res. 36 Ž1993. 260–271. M. Pasqualetti, I. Nardi, H. Ladinsky, D. Marazziti, G.B. Cassano, Comparative anatomical distribution of serotonin 1A, 1Da and 2A receptor mRNA in human brain postmortem, Mol. Brain Res. 39 Ž1996. 223–233. J.L. Plassat, U. Boschert, N. Amlaik, R. Hen, The mouse 5-HT5 receptor reveals a remarkable heterogeneity within the 5-HT1D receptor family, EMBO J. 11 Ž1992. 4779–4786. S. Rees, I. den Daas, S. Foord, S. Goodson, D. Bull, G. Kilpatrick, M. Lee, Cloning and characterization of the human 5-HT5A serotonin receptor, FEBS Lett. 355 Ž1994. 242–246. M. Sarter, J.P. Bruno, Cognitive functions of cortical acetylcholine: toward a unifying hypothesis, Brain Res. Rev. 23 Ž1997. 28–46. Schmahmann, J.D., in: M.L. Bauman, T.L. Kemper ŽEds.., The Neurobiology of Autism, Johns Hopkins Univ. Press, Baltimore, 1994, pp. 195–226. Squire, L., The hippocampus and the neuropsychology of memory, in: W. Seifert ŽEd.., Neurobiology of the Hippocampus, Academic Press, London, 1983, pp. 491–511. W. Wisden, E.M. Parker, C.D. Mahle, D.A. Grisel, H.P. Nowak, F.D. Yocca, C.C. Felder, P.H. Seeburg, M.M. Voigt, Cloning and characterization of the rat 5-HT5B receptor. Evidence that the 5-HT5B receptor couples to a G protein in mammalian cell membranes, FEBS lett. 333 Ž1993. 25–31. N.C. Woolf, Cholinergic system in mammalian brain and spinal cord, Prog. Neurobiol. 37 Ž1991. 475–524.
Research report
Distribution of the 5-HT5A serotonin receptor mRNA in the human brain Massimo Pasqualetti a , Michela Ori a , Irma Nardi a,) , Maura Castagna b, Giovanni Batista Cassano c , Donatella Marazziti c a
Laboratori di Biologia cellulare e dello sÕiluppo, Dipartimento di Fisiologia e Biochimica, UniÕersita` di Pisa, Õia Carducci 13, Ghezzano 56010, Pisa, Italy b Laboratorio di Anatomia Patologica, Dipartimento di Chirurgia, UniÕersita` di Pisa, Õia Roma 57, 56100 Pisa, Italy c Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, UniÕersita` di Pisa, Õia Roma 67, 56100 Pisa, Italy Accepted 16 December 1997
Abstract The 5-HT5A receptor is a member of a new subfamily of serotonin w5-hydroxytryptamine Ž5-HT.x receptors recently cloned from the human and rodent brain. The role of this receptor in normal brain functions as well as its possible involvement in pathological states is still to be determined. We therefore studied the regional distribution and cellular localization of 5-HT5A receptor mRNA in human brain sections from autopsy samples by in situ hybridization histochemistry, in order to obtain anatomical information which might be useful in formulating hypotheses on possible functions subserved by this receptor in the central nervous system ŽCNS.. Our results showed that the main sites of 5-HT5A mRNA expression were the cerebral cortex, hippocampus and cerebellum. In the neocortical regions, the 5-HT5A receptor mRNA was mainly distributed in the layers II–III and V–VI. In the hippocampus, the dentate gyrus and the pyramidal cell layer of the CA1 and CA3 fields expressed 5-HT5A mRNA at high levels. The broad distribution in the neocortex and hippocampus supports the view that the 5-HT5A receptor in these areas might be implicated in high cortical and limbic functions. The 5-HT5A mRNA was widely distributed in the cerebellum where it was highly expressed in the Purkinje cells, in the dentate nucleus and, at a lower level, in the granule cells. Since the cerebellum receives diffuse serotonergic afferents, this finding suggests that the 5-HT5A receptor may have an important role in mediating the effects of 5-HT on cerebellar functions. q 1998 Elsevier Science B.V. Keywords: 5-HT5A receptor mRNA expression; In situ hybridization; Human central nervous system; Cortex; Hippocampus; Cerebellum
1. Introduction The neuromodulator 5-hydroxytryptamine Žserotonin, 5-HT. has been implicated in a wide range of behavioural and physiological functions, including processes as diverse as cardiovascular control, feeding regulation, the sleep– wake cycle, learning, nociception, analgesia and some neuropsychiatric disorders w15x. The various actions of 5-HT are mediated by several receptors which have been classified as pharmacologically distinct subtypes w3,14,16x. These receptors belong to two different protein superfamilies: ligand-gated ion channel receptors Ž5-HT3 . and the G protein-coupled receptor superfamily. During recent years, however, molecular cloning studies have revealed the existence of several novel 5-HT receptors for which little or no
) Corresponding author. Fax: q39-50-878486; E-mail: [email protected]
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 0 0 3 - 5
prior pharmacological or functional data existed Ž5-HT1E , 5-HT1F , 5-HT5A , 5-HT5B , 5-HT6 and 5-HT7 .. Much effort has, therefore, been directed towards the understanding of the physiological roles of these receptors and towards identifying selective ligands to be used as putative therapeutic agents. Among these novel cloned receptors, a new subfamily, containing two members designated 5-HT5A and 5-HT5B , has been isolated in the mouse and the rat w9,17x. Recently, the human 5-HT5A receptor has also been cloned, while a 5-HT5B receptor does not seem to exist in humans w22x. According to pharmacological criteria, the 5-HT5 receptors fall into the 5-HT1-like class w21x. However, their separate classification is justified by other characteristics such as the sequence, which is less than 37% homologous to members of the 5-HT1 family and the presence of an intron between the putative transmembrane domain V and VI, in contrast with 5-HT1 receptor genes which are intronless w13x. Moreover, the expression of 5-HT5A recep-
M. Pasqualetti et al.r Molecular Brain Research 56 (1998) 1–8
2 Table 1 Characteristics of autoptic brain samples Number Age Sex
Cause of death
1 2 3 4 5 6
Pulmonary embolism Cardiac Cardiac Acute heart failure Pulmonary embolism Cardiac
46 55 60 73 78 80
Female Male Male Male Female Male
lyzed its gene expression in various regions of the brain by means of in situ hybridization. Postmortem delay Žh. 12 20 14 12 22 24
tors in mammalian cell lines has failed so far to demonstrate any G protein coupling w17,21,26x. By means of the reverse transcriptase-polymerase chain reaction ŽRT-PCR. technique, the 5-HT5A mRNA has been shown to be expressed in the adult human brain and, at a lower level, in the foetal brain, thus suggesting that the receptor is developmentally regulated w22x. As the pharmacological profile of the 5-HT5A receptor resembles that of the 5-HT1D receptor, it seems plausible that it may subserve some of the functions previously attributed to the 5-HT1D receptor family w17x. However, its precise distribution and role within the human central nervous system ŽCNS., as well as its possible involvement in pathological states, is still unknown. In order to contribute to the understanding of the function of the 5-HT5A receptor in the human CNS, we ana-
2. Materials and methods 2.1. Tissue collection and processing Human brain samples were obtained at autopsy from six subjects Žfour men and two women., aged between 46 and 80 years who were selected for the absence of any neurological or psychiatric disorders and who had not taken any psychotropic drugs Žsee Table 1.. Tissue collection, dissection and storage have been described in a previous paper w20x. Sections Ž10–15 m m thick. obtained with a microtome cryostat ŽLeitz 1720. at y208C, were thaw-mounted on organosylanized-coated slides and fixed with paraformaldehyde in a phosphatebuffered saline solution ŽPBS., rinsed twice in 1 = PBS, dehydrated in graded alcohol and processed immediately. 2.2. In situ hybridization 2.2.1. RNA probe synthesis The human 5-HT5A cDNA clone w22x was kindly provided by the Glaxo Wellcome Research and Development ŽStevenage, UK.. The 1.080 kb EcoRI–SacI insert was
Fig. 1. Dark-field photomicrographs showing the distribution of 5-HT5A receptor mRNA in human parietal cortex. ŽA. A diffuse distribution of autoradiographic grains are present through layers II–VI. ŽB. High magnification of a selected area Žindicated by arrowheads. of the parietal cortex shown in ŽA.: layer III appears more intensely labeled. Scale bars, 0.2 cm. ŽA., 0.3 mm ŽB..
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subcloned in the pGem 7ZfŽy. expression vector ŽPromega., containing T7 and SP6 RNA polymerase promoters and linearized to generate 35 S-radiolabeled antisense and sense riboprobes, as previously described w20x. Probes were degraded to optimal hybridization sizes Ž100–300 nucleotides. by alkaline hydrolysis w6x. 2.2.2. Pretreatment of slides and hybridization Pretreatment of slides and in situ hybridization were performed as already described w20x. Sections were exposed to Kodak MR X-ray film for 2–4 days and then dipped in 1:1 diluted Kodak NTB-2 emulsion with a solution of 0.6 M ammonium acetate, 2% glycerol and exposed for 4–6 weeks at 48C, developed in Kodak D19, stained with Giemsa, dehydrated and mounted. Sections were examined using bright- and dark-field light microscopy ŽZeiss..
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3. Results The anatomical distribution of the 5-HT5A mRNA was analyzed in serial histological sections of the following brain regions: cerebral cortex, hippocampus, amygdala, basal ganglia, substantia innominata Žbasal nucleus of Meynert., substantia nigra, thalamus, choroid plexus and cerebellum. Some sections of each sample were not processed for in situ hybridization, but stained with Cresyl violet to permit a detailed analysis of the cytoarchitecture. Brain areas and nuclei were identified according to DeArmond et al. w7x. As control experiments, histological sections of each analyzed brain structure were hybridized with a sense probe. In these control experiments the level of the signal was very low and was considered as unspecific background Žan example is shown in Fig. 5B.. In contrast,
Fig. 2. Dark-field photomicrographs of coronal sections of entorhinal cortex, subiculum and hippocampus showing the distribution of 5-HT5A mRNA. ŽA. A moderate level of labeling is shown by the subiculum and entorhinal cortex. ŽB. CA1, CA3 fields and the dentate gyrus of hippocampal formation are densely labeled. Bright-field high-magnification photomicrographs of the CA3 field ŽC. and the dentate gyrus ŽD.: the pyramidal neurons of the CA3 field and the granular cells of the dentate gyrus are labeled. Small cells, probably glial cells, are clearly unlabeled Žarrowhead in ŽC... DG, dentate gyrus; EC, entorhinal cortex; S, subiculum. Scale bars, 0.2 cm ŽA., 0.15 cm ŽB., 30 m m ŽC., 30 m m ŽD..
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hybridization with the antisense probe resulted in a localized signal distribution. We detected no significant differences in the hybridization signal in the examined autopsy samples, in spite of the different ages of the subjects andror postmortem delay. 3.1. Cortical areas The distribution of the 5-HT5A receptor mRNA was examined in the prefrontal, parietal, temporal, occipital and entorhinal cortices. All these areas showed a general labeling pattern consisting of a diffuse autoradiographic signal throughout the different layers of the neocortex. However, the labeling was preferentially concentrated in the layers II–III and V–VI ŽFig. 1A.. In particular the layer III appeared to be intensely labeled ŽFig. 1B.. A lower level of hybridization signal was found in the entorhinal cortex ŽFig. 2A.. To determine which cell types express the 5-HT5A mRNA, we studied in situ emulsion autoradiographs at the light microscopy level. All the labeled cells seemed to belong to the neuronal types, among which large pyramidal cells could be recognized. No hybridization signal was detected in small cells Ždata not shown.. 3.2. Hippocampus The 5-HT5A receptor mRNA was expressed at high levels in the hippocampal formation ŽFig. 2B–D.. The granule cell layer of the dentate gyrus and the CA1 and CA3 fields of the Hammon’s horn were strongly labeled ŽFig. 2B.. At higher magnification all the pyramidal neu-
rons of CA1 and CA3 fields and the granule cells of the dentate gyrus were labeled ŽFig. 2C and D., while small cells were not labeled ŽFig. 2C and D.. No differences in the pattern of expression were found in the rostrocaudal extent of the hippocampus Ždata not shown.. An autoradiographic signal was also present in the subiculum Žcf. Fig. 2A.. 3.3. Amygdala The amygdala was differentially labeled: a high level of 5-HT5A mRNA expression was detected in the ventrolateral part, while the dorsomedial region was not labeled ŽFig. 3A.. 3.4. Basal ganglia and substantia innominata The 5-HT5A mRNA distribution in the basal ganglia was heterogeneous. A weak hybridization signal was found in the nucleus caudatus Ždata not shown.; in the putamen, only scattered cells appeared to be labeled ŽFig. 3B.; the pars medialis of the globus pallidus was not labeled, while the pars lateralis presented a few scattered, labeled cells ŽFig. 3B.. The cell bodies of the substantia innominata showed a high density of autoradiographic grains ŽFig. 3B.: at higher magnification the majority of neuronal cells of this nucleus appeared labeled Ždata not shown.. 3.5. Cerebellum In the cerebellum, the highest level of 5-HT5A mRNA expression was detected in the Purkinje cell perikarya ŽFig.
Fig. 3. Dark-field photomicrographs from autoradiograms of coronal sections showing the 5-HT5A mRNA expression in amygdala ŽA., globus pallidus, putamen and substantia innominata ŽB.. A high hybridization signal is present in the ventrolateral part the amygdala. The pars lateralis of globus pallidus shows labeled scattered cells, while no hybridization signal can be observed in the pars medialis. Scattered cells are labeled in the putamen. The majority of cells in the substantia innominata appears densely labelled. GPL, globus pallidus pars lateralis; GPM, globus pallidus pars medialis; PU, putamen; SI, substantia innominata; VLA, ventro lateral amygdala. Scale bars, 0.2 cm ŽA.; 0.9 mm ŽB..
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Fig. 4. Distribution of 5-HT5A receptor mRNA in the cerebellum. ŽA. Dark-field photomicrograph of a coronal section of the cerebellar cortex: the Purkinje cells are heavily labeled. High-magnification bright-field ŽB. and dark-field ŽC. photomicrographs showing a high level of hybridization on the Purkinje cell perikaria. ŽD. A high hybridization signal is present in the dentate nucleus. ŽE. Bright-field high-magnification photomicrograph of dentate nucleus: all neuronal cell types are labeled. DN, dentate nucleus; PC, Purkinje cell. Scale bars, 0.6 mm ŽA., 500 m m ŽB., 900 m m ŽC., 0.1 mm ŽD., 30 m m ŽE..
4A–C.. A lower hybridization signal was found in the granule layer, as clearly shown in X-ray film autoradiographs ŽFig. 5A.. Due to the high density of Giemsa-stained cells, the labeling on the granule layer was, in fact, undetectable in emulsion autoradiographs Žcf. Fig. 4A–C.. No
labeling was present in the molecular layer. This pattern of mRNA expression was found in the cortex of both the hemispheres and vermis Žcf. Figs. 4 and 5. with no rostrocaudal regional differences. The dentate nucleus was highly labeled: as observed at higher magnification, the hybridiza-
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Fig. 5. X-ray film autoradiograms of coronal sections of the vermis cerebellar cortex. ŽA. The hybridization signal is present in the Purkinje cell perikaria Žarrow. and in the granule cell layer. ŽB. An adjacent section of cerebellar cortex hybridized with a sense probe: the level of the signal is very low and considered as unspecific background. Scale bars, 0.18 cm.
tion signal was concentrated only on neuronal cell types ŽFig. 4D and E.. 3.6. Other brain areas 5-HT5A transcripts were not detectable in some thalamic nuclei such as the dorsomedial, lateral dorsal and ventral posterolateral, as well as in substantia nigra and choroid plexus Ždata not shown.. 4. Discussion In the present study, we determined the expression of mRNA encoding the 5-HT5A receptor subtype in postmortem human brain tissues, by using in situ hybridization. Our results confirm that the 5-HT5A receptor mRNA is expressed in different regions of the brain and provide the first detailed data as to its regional and cellular distribution. The main sites of 5-HT5A mRNA expression were represented by the cerebral cortex, amygdala, hippocampus and cerebellum, while a lower level of expression was found in the other examined regions. The distribution of 5-HT5A mRNA in the human brain was found to be generally similar to that in the mouse brain w21x, although some differences were observed.
In the cerebral cortex, the 5-HT5A receptor mRNA was mainly distributed in the layers II–III and V–VI. When these areas were analyzed at higher magnification, it was evident that cells with pyramidal morphology expressed the receptor mRNA. In particular, a high level of expression was found in pyramidal cells of the layer III of the prefrontal, occipital and parietal cortices. However, the possibility of other neuronal types expressing the receptor mRNA could not be excluded. The presence of 5-HT5A mRNA expression in pyramidal cells of different neocortical areas would suggest that these receptors might play a role in several brain functions, such as those involving highly integrative activities. In the hippocampal formation, the dentate gyrus and the pyramidal cell layer of the CA1 and CA3 fields appeared to express the 5-HT5A mRNA at a high level. The 5-HT5A mRNA was present in the granule cells of the dentate gyrus and in the pyramidal cells of the Hammon’s horn. The pattern of 5-HT5A mRNA expression parallels that of the 5-HT1A mRNA w4,20x, suggesting that these two receptors may be co-expressed in the pyramidal cells of the CA1 and CA3 fields and in the granule cells of the dentate gyrus. Our previous observation that the 5-HT1D a and the 5-HT2A receptor mRNA are expressed only in the pyramidal cells of CA3 and CA1 fields respectively w20x, seems to indicate that different combinations of receptor transcripts might exist in these hippocampal areas. Although any
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definitive conclusion with regard to the presence of multiple 5-HT receptors in the same cell will require doublelabeling techniques, these results would suggest that the balance between different receptors might be relevant to the functioning of one area, rather than the level of activity of one receptor only. In addition to the hippocampus, 5-HT5A mRNA expression was found in other areas belonging to the limbic system, such as the amygdala and entorhinal cortex. As this system is assumed to be involved in learning, memory and impulsivity w25x, 5-HT5A may be one of the candidate receptors regulating these functions. The 5-HT5A mRNA was widely distributed in the cerebellum where it appeared to be highly expressed in the Purkinje cell perikarya, in the dentate nucleus and, at a lower level, in the granule cells. To our knowledge, this is the first report of a 5-HT receptor localized in all the main cell types which control both cerebellar inputs and outputs. In mice, the 5-HT5A mRNA seems to be expressed only in the granule cell layer w21x, while the Purkinje cells represent one of the main sites of expression of the 5-HT1B receptor, which is the rodent homologue of the human 5-HT1D b w12x. A difference in the expression of the 5-HT5A receptor would seem therefore to exist between human and mouse cerebellum, although this possibility requires further investigation. Although the cerebellum has traditionally been viewed as a structure that contributes primarily to motor coordination and control, there is now new and growing evidence that it is also involved in some aspects of cognitive functions w1,10x. Animal studies and psychometric tests in psychiatric patients have even suggested that the cerebellum may contribute to emotional processing w2,24x. This new concept with regard to cerebellar functions has found an anatomical substrate in the presence of cerebellar outputs gaining access to the prefrontal cortex w18x. Since the cerebellum receives diffuse serotonergic innervation by afferents originating from raphe nuclei, it is tempting to speculate that 5-HT may modulate such nonmotor or cognitive cerebellar functions through specific receptors. The wide distribution of 5-HT5A mRNA in the cerebellum makes this receptor a putative candidate for involvement in the cerebellar contribution to cognition or other emotional processes. This hypothesis is in agreement with the observation that the 5-HT5A mRNA is expressed in the cortex and in limbic structures such as the hippocampal formation, amygdala and entorhinal cortex, while it is poorly represented in the basal ganglia and absent from the substantia nigra, which are both implicated in the control of motor activity. Recently, in order to study the function of the 5-HT5A receptor, knockout mice lacking this receptor have been generated by homologous recombination w11x. Interestingly, these mutant mice, which develop normally, were found to have enhanced exploratory activity in a novel environment as compared with controls, suggesting that
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the 5-HT5A receptors might be involved in exploratory behaviour w11,8x. It is also worth noting that 5-HT5A receptor transcripts were detected in the substantia innominata, one of the cholinergic nuclei of the basal forebrain ŽBF. which projects to the cortex w27x. The cholinergic system has been implicated in various behavioural and mental functions w23x and 5-HT afferents have been reported to form synapses with BF neurons, suggesting that 5-HT may influence the functional state of the cholinergic projections to the cerebral cortex and other brain areas. w5,19x. Although the localization of the 5-HT5A receptor in cholinergic cells deserves further investigation using double labeling techniques, the presence of 5-HT5A transcripts in the same area as cholinergic neurons indicates that this receptor might mediate the action of 5-HT on cholinergic system. The identification of a specific 5-HT receptor on cholinergic neurons may have some relevance in view of the possibility of manipulating their activity by means of serotonergic drugs. Although our data support the notion that the 5-HT5A receptors could play an important role in various human brain functions, further studies on its pharmacological properties and on its second messenger pathway are necessary in order to provide a definition of its physiological role, as well as of its possible implication in the pathophysiology of the CNS.
Acknowledgements We thank Glaxo Wellcome Research and Development for kindly supplying the 5-HT5A cDNA clone. We also thank Fabio Cini for his technical assistance and Dr. Augusto Innocenti for his help. This work was supported by a grant from Murst and Ravizza Farmaceutici.
References w1x G. Allen, R.B. Buxton, E.C. Wong, E. Courchesne, Attentional activation of the cerebellum independent of motor involvement, Science 275 Ž1997. 1940–1943. w2x G.G. Berntson, M.W. Torello, The paleocerebellum and the integration of behavioral function, Physiol. Psychol. 10 Ž1982. 2–12. w3x F.G. Boess, I.L. Martin, Molecular biology of 5-HT receptors, Neuropharmacology 33 Ž1994. 275–317. w4x P.W.J. Burnet, S.L. Eastwood, K. Lacey, P.J. Harrison, The distribution of 5-HT1A and 5-HT2A receptor mRNA in human brain, Brain Res. 676 Ž1995. 157–168. w5x J.-C. Cassel, H. Jeltsch, Serotonergic modulation of cholinergic function in the central nervous system: cognitive implications, Neuroscience 69 Ž1995. 1–41. w6x K.H. Cox, D.V. DeLeon, L.M. Angerer, R.C. Angerer, Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes, Dev. Biol. 101 Ž1984. 485–502. w7x S.J. DeArmond, M.M. Fusco, M.M. Dewey, Structure of the Human Brain, Oxford Univ. Press, Oxford, 1989, 202 pp.
8
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w8x S.C. Dulawa, R. Graihle, R. Hen, M.A. Geyer, Characterization of exploratory behavior and sensorimotor gating in wild type and serotonin 5A knockout mice, Soc. Neurosci. Abstr. Ž1997. 482.20. w9x M.G. Erlander, T.W. Lovenberg, B.M. Baron, L. Lecea, P.E. Danielson, M. Racke, A.L. Slone, B.W. Siegel, P.E. Foye, K. Cannon, J.E. Burns, J.G. Sutcliffe, Two members of a distinct subfamily of 5-hydroxytryptamine receptors differentially expressed in rat brain, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 3452–3456. w10x J.A. Fiez, Cerebellar contribution to cognition, Neuron 16 Ž1996. 13–15. w11x R. Grailhe, C. Waeber, D. Brunner, X. Zhuang, Y. Huang, M.A. Moskowitz, R. Hen, The 5-HT5A receptors: localization in the CNS and increased exploratory activity in 5-HT5A knockout mice, Soc. Neurosci. Abstr. Ž1997. 482.19. w12x P.R. Hartig, T.A. Branchek, R.L. Weinshank, A subfamily of 5-HT1D receptor genes, Trends Pharmacol. Sci. 13 Ž1992. 152–159. w13x D. Hoyer, D.E. Clarke, J.R. Fozard, P.R. Hartig, G.R. Martin, E.J. Mylecharane, P.R. Saxena, P.A. Humphrey, VII. International union of pharmacology classification of receptor for 5-hydroxytryptamine Žserotonin., Pharmacol. Rev. 46 Ž1994. 157–203. w14x D. Hoyer, G. Martin, 5-HT receptor classification and nomenclature: towards a harmonization with the human genome, Neuropharmacology 36 Ž1997. 419–428. w15x B.L. Jacobs, C.A. Formal, Serotonin and behaviour: a general hypothesis, in: F.E. Bloom, H.Y. Meltzer ŽEds.., Psychopharmacology the Fourth Generation of Progress, Raven Press, New York, 1995, pp. 461–469. w16x G.R. Martin, P.P.A. Humphrey, Receptors for 5-hydroxytryptamine: current perspectives on classification and nomenclature, Neuropharmacology 33 Ž1994. 261–273. w17x H. Matthes, U. Boschert, N. Amlaiky, R. Grailhe, J.-L. Plassat, F. Muscatelli, M.-G. Mattei, R. Hen, Mouse 5-hydroxytryptamine5A and 5-hydroxytryptamine5B receptors define a new family of sero-
w18x
w19x
w20x
w21x
w22x
w23x w24x
w25x
w26x
w27x
tonin receptors: cloning, functional expression, and chromosomal localization, Mol. Pharmacol. 43 Ž1993. 313–319. F.A. Middleton, P.L. Strick, Anatomical evidence for cerebellar and basal ganglia involvement in higher cognitive function, Science 266 Ž1994. 458–461. T.A. Milner, E. Veznedaroglu, Serotonin-containing terminals synapse on septohippocampal neurons in the rat, J. Neurosci. Res. 36 Ž1993. 260–271. M. Pasqualetti, I. Nardi, H. Ladinsky, D. Marazziti, G.B. Cassano, Comparative anatomical distribution of serotonin 1A, 1Da and 2A receptor mRNA in human brain postmortem, Mol. Brain Res. 39 Ž1996. 223–233. J.L. Plassat, U. Boschert, N. Amlaik, R. Hen, The mouse 5-HT5 receptor reveals a remarkable heterogeneity within the 5-HT1D receptor family, EMBO J. 11 Ž1992. 4779–4786. S. Rees, I. den Daas, S. Foord, S. Goodson, D. Bull, G. Kilpatrick, M. Lee, Cloning and characterization of the human 5-HT5A serotonin receptor, FEBS Lett. 355 Ž1994. 242–246. M. Sarter, J.P. Bruno, Cognitive functions of cortical acetylcholine: toward a unifying hypothesis, Brain Res. Rev. 23 Ž1997. 28–46. Schmahmann, J.D., in: M.L. Bauman, T.L. Kemper ŽEds.., The Neurobiology of Autism, Johns Hopkins Univ. Press, Baltimore, 1994, pp. 195–226. Squire, L., The hippocampus and the neuropsychology of memory, in: W. Seifert ŽEd.., Neurobiology of the Hippocampus, Academic Press, London, 1983, pp. 491–511. W. Wisden, E.M. Parker, C.D. Mahle, D.A. Grisel, H.P. Nowak, F.D. Yocca, C.C. Felder, P.H. Seeburg, M.M. Voigt, Cloning and characterization of the rat 5-HT5B receptor. Evidence that the 5-HT5B receptor couples to a G protein in mammalian cell membranes, FEBS lett. 333 Ž1993. 25–31. N.C. Woolf, Cholinergic system in mammalian brain and spinal cord, Prog. Neurobiol. 37 Ž1991. 475–524.