Detailed mapping of the histamine H2 receptor and its gene transcripts in guinea-pig brain

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PII:

Neuroscience Vol. 80, No. 2, pp. 321–343, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(97)00010-9

DETAILED MAPPING OF THE HISTAMINE H2 RECEPTOR AND ITS GENE TRANSCRIPTS IN GUINEA-PIG BRAIN M. L. VIZUETE,* E. TRAIFFORT,†‡ M. L. BOUTHENET,* M. RUAT,† E. SOUIL,* J. TARDIVEL-LACOMBE† and J.-C. SCHWARTZ† *Laboratoire de Physiologie, Faculte´ de Pharmacie, Universite´ Rene´ Descartes, Paris, France †Unite´ de Neurobiologie et Pharmacologie (U. 109) de l’INSERM, Centre Paul Broca, 2ter rue d’Ale´sia, 75014 Paris, France Abstract––Autoradiographic studies of the distribution of the histamine H2 receptor and its messenger RNAs were performed on serial frontal and a few sagittal sections of guinea-pig brain using [125I]iodoaminopotentidine for radioligand binding and a 33P-labelled complementary RNA probe for in situ hybridization, respectively. Both probes were validated by assessing non-specific labelling using nonradioactive competing H2 receptor ligands and a sense probe for binding sites and gene transcripts, respectively. In some areas, e.g., cerebral cortex, hippocampal complex or cerebellum, such studies were completed by identification of neurons expressing the H2 receptor messenger RNAs on emulsion-dipped sections. Nissl-stained sections from comparable levels were used to localize brain structures. In many brain areas, the distribution of the H2 receptor and its messenger RNAs appeared to parallel that known for histaminergic axons. For instance, high levels of both H2 receptor markers were detected in striatal and limbic areas known to receive abundant histaminergic projections. In contrast, in septum, hypothalamic, pontine and several thalamic nuclei, a comparatively low density of both H2 receptor markers was detected, suggesting that histamine actions in these areas are mediated by H1 and/or H3 receptors. Generally, the distribution of H2 receptor messenger RNA correlates well with that of [125I]iodoaminopotentidine binding sites, although some differences were observed. In a few regions (e.g., substantia nigra, locus coeruleus) high or moderate densities of binding sites were accompanied by a much more restricted expression of H2 receptor transcripts. Conversely, the mammillary region and the pontine nucleus exhibited higher levels of hybridization than of binding sites. In hippocampus, cerebral and cerebellar cortex there was a selective localization of the H2 receptor messenger RNA in the granule cells of dentate gyrus, pyramidal cells of the Ammon’s horn and cerebral cortex, and Purkinje cells of cerebellum, whereas [125I]iodoaminopotentidine binding sites were located in layers where the dendritic trees of these messenger RNA-expressing neurons extend. The same discrepancy between messenger RNAs and binding sites suggests that striatonigral endings are endowed with the H2 receptor. The histamine H1 and H2 receptors both appear to be present in several brain areas, in some cases in a way suggesting their potential co-expression by the same neuronal populations, e.g., in granule and pyramidal cells in the hippocampal formation. This co-expression accounts for synergic responses, e.g., on cAMP generation, previously observed upon co-stimulation of both receptor subtypes. The widespread distribution of the H2 receptor, namely in thalamic nuclei or in telencephalic areas such as most layers of the cerebral cortex, together with its excitatory role previously established in electrophysiological studies, support its alleged function in mediating the histamine-driven control of arousal mechanisms. In addition, the detection of H2 receptor expression in brainstem areas from which other monoaminergic pathways involved in the control of states of sleep and wakefulness emanate, e.g., several raphe nuclei, locus coeruleus or substantia innominata, suggests possible interrelationships between all of these systems with highly divergent projections to the thalamus and telencephalon. The present mapping of the H2 receptor and its gene transcripts should facilitate neurochemical, neurophysiological and behavioural studies aimed at clarifying the role of histaminergic systems in brain. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: histamine H2 receptor, autoradiographic mapping, [125I]iodoaminopotentidine, in situ hybridization, emulsion-dipped sections, guinea-pig brain.

Histamine is released in mammalian brain from widespread axons emanating from a group of neurons packed together in the tuberomammillary nucleus of the posterior hypothalamus and affects target cells via activation of three receptor subtypes, ‡To whom correspondence should be addressed. Abbreviations: APT, iodoaminopotentidine; HPLC, highperformance liquid chromatography; SSC, standard saline citrate.

termed H1, H2 and H3.33 Establishment of the roles of the amine in neurophysiological, neurochemical, behavioural or other studies is facilitated by the knowledge of the neuronal populations expressing these various receptor subtypes. In agreement with this, detailed mappings of H1 and H3 receptors in rodent brain were previously established.6,29 In addition, the recent cloning of an H1 receptorencoding cDNA35 allowed this information to be completed by in situ hybridization studies of the

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localization of its gene transcripts and the expressing neuronal populations to be identified. Such studies have been delayed for a long time in the case of the H2 receptors in brain by the lack of suitable probes, although the presence of this receptor subtype was characterized as early as 1975 by electrophysiological15 and biochemical studies.2 In various brain regions, the H2 receptor appears to be positively coupled with adenylyl cyclase.13,17 H2 receptor activation in hippocampal pyramidal neurons decreases a calcium-activated potassium channel16 and in thalamic relay neurons enhances the hyperpolarization-activated cation current Ih.23 Both effects seem common to a series of monoamines (catecholamines, indolamines) and may, in some cases, involve cAMP formation but not protein kinase A activation, which usually accompanies it.28 A series of observations suggests, however, that H2 receptor signalling in brain may not involve cAMP as a second messenger in all cases. Firstly, there is no strict parallelism between the regional distributions of H2 receptor binding and H2 receptor-activated adenylyl cyclase activity;31 secondly, the recombinant H2 receptor expressed in transfected fibroblasts not only activates cAMP formation but also inhibits arachidonate release36 and stimulates calcium transients.8 Labelling of the H2 receptor has remained for a long time a difficult task. [3H]Tiotidine was proposed for this purpose10 but, owing to a high non-specific binding, the radioprobe could not be reliably used in autoradiographic studies. [125I]Iodoaminopotentidine ([125I]APT) was more recently designed and shown to display high affinity (Kd z 0.3 nM), receptor specificity and specific radioactivity, allowing its use for autoradiographic detection of the H2 receptor in rodent31 or human brain21 and in peripheral tissues such as the stomach.9 The recent cloning of H2 receptor cDNAs from dog,11 rat30 and guinea-pig37 opened the way for the detection of corresponding gene transcripts by in situ hybridization. In the present work, the two technical approaches were combined, taking into account the complementary information provided by each method. The guinea-pig brain was selected for such studies in view of the high expression of the H2 receptor it displays,31 as well as to allow comparison with the distribution of H1 receptor,6 which is expressed at a higher level in the guinea-pig than in the rat brain. EXPERIMENTAL PROCEDURES

Materials Na125I (usually 2000 Ci/mmol) was from Amersham. Tiotidine was from ICI, cimetidine, burimamide and bolpyramine from SmithKline and French, ranitidine from Glaxo, and N-[2-(4-aminobenzamido)ethyl]-N* cyano-N-[3[3-(1-piperidinyl-methyl) phenoxy]propyl] guanidine (APT) from Dr Schunack (Berlin, Germany).

[125I]APT was synthesized by direct iodination of APT using the chloramine T method and then purified by high-performance liquid chromatography (HPLC).31 [125I]Iodobolpyramine was synthesized by direct iodination of bolpyramine using iodogen and Na125I, then purified by HPLC.4 Tissue preparation The results reported in this study are derived from experiments performed on tissues from 15 male guinea-pigs (300 g, Hartley, Iffa Credo, France). All efforts were made to minimize animal suffering and to reduce the number of animals used. After decapitation, guinea-pig brains were rapidly removed, frozen without any fixation by slow immersion in liquid monochlorodifluoromethane as described7 and sectioned on a cryostat (10 µm thickness). For incubation with [125I]APT, the sections were mounted on to poly--lysinecoated glass slides and stored at "20)C until use. For in situ hybridization experiments, sections from comparable levels of separate animals were mounted on to gelatin-coated slides. Brain sections were immediately fixed for 60 min at 4)C in 4% paraformaldehyde made up in 0.1 M phosphate-buffered saline, pH 7.4, and 0.1% diethyl pyrocarbonate–water. Sections were rinsed three times (5 min each) in phosphate-buffered saline, pH 7.4, dehydrated through graded ethanol, dried under a stream of cold air and stored at "80)C until needed. Receptor binding autoradiography Optimal binding conditions for [125I]APT, previously determined for brain membranes,31 were adjusted to generate optimal autoradiograms. Slide-mounted guineapig brain sections were incubated for 3 h at room temperature by dropping on to them 500 µl of 0.1 nM [125I]APT prepared in 50 mM Na2/K phosphate buffer, pH 7.5. To determine non-specific binding adjacent sections were incubated in the presence of [125I]APT and 3 µM tiotidine, a H2 receptor antagonist. After incubation, the sections were washed at 4)C in drug-free buffer (5#4 min). Then, the tissue sections were quickly dried under a stream of cold air. To assess the specificity of the H2 receptor labelling, two different competition experiments were carried out by incubating consecutive brain sections in buffer solutions containing [125I]APT alone or in the presence of increasing concentrations (corresponding to 3, 100 and 300 times the Ki values) of various H2 receptor antagonists, i.e. tiotidine, cimetidine, ranitidine and burimamide. To compare the precise localization of H2 and H1 receptors in the same brain areas, binding experiments were performed on serial sections using either [125I]APT or [125I]iodobolpyramine. The binding procedure using [125I]iodobolpyramine as ligand has been described earlier.6 In brief, tissue sections were incubated in the presence of 0.1 nM [125I]iodobolpyramine in Na2/K phosphate buffer (50 mM, pH 7.5) containing 0.1% bovine serum albumin, for 3 h at 25)C, rinsed at 25)C in drug-free buffer (5#12 min) and dried. The dried sections were apposed to 3H-ultrafilms (Amersham, France) in X-ray cassettes, generally for a two-day period. At the end of the exposure period, films were developed and photographic enlargments were obtained as described elsewhere.5 For histological identification of brain areas, the Nissl staining method was applied to tissue sections that had been used for autoradiography on to adjacent sections. In situ hybridization All prehybridization and hybridization solutions were made in 0.1% diethyl pyrocarbonate–water. The brain sections were removed from storage and treated with proteinase K (1 mg/ml in 100 mM Tris–HCl, pH 8, containing 50 mM ethylenediaminetetra-acetate) for 10 min at 37)C. After a brief dipping into water, sections were kept in 0.1 M triethanolamine, pH 8, and 0.25% acetic anhydride for

Abbreviatons used in the figures AA AcbC AcbSh AC ACo AH AHi AM AOD AOL AOM AOP APT AStr BAOT BL BM BST CA1 CA2 CA3 Cg CG ChP CIC CL Cl CLi CM CnF CPu DA DC DG DEn DLG DM DpG DR dtg DTg DwmCb ECu Ent EPl EW FL Fr FStr GDG Gi Gl GP GrCb HL I ICj ICjM IGr IMD IMG In InG IO IP IPl LC LD LDTg LH LHb LMol LOT LP LS LSO LVe MCb

anterior amygdaloid area accumbens nucleus, core accumbens nucleus, shell anterior commissural nucleus anterior cortical amygdaloid nucleus anterior hypothalamic area amygdalohippocampal area anteromedial thalamic nucleus anterior olfactory nucleus, dorsal part anterior olfactory nucleus, lateral part anterior olfactory nucleus, medial part anterior olfactory nucleus, posterior part anterior pretectal area amygdalostriatal transition area bed nucleus of the accesory olfactory tract basolateral amygdaloid nucleus basomedial amygdaloid nucleus bed nucleus of stria terminalis field CA1 of Ammon’s horn field CA2 of Ammon’s horn field CA3 of Ammon’s horn cingulate cortex central (periaqueductal) gray choroid plexus central nucleus of inferior colliculus centrolateral thalamic nucleus claustrum caudal linear nucleus of raphe central medial thalamic nucleus cuneiform nucleus caudate–putamen (striatum) dorsal hypothalamic area dorsal cochlear nucleus dentate gyrus dorsal endopiriform nucleus dorsal lateral geniculate nucleus dorsomedial hypothalamic nucleus deep gray layer of superior colliculus dorsal raphe nucleus dorsal tegmental bundle dorsal tegmental nucleus deep white matter of cerebellar cortex external cuneate nucleus enthorinal cortex external plexiform layer of bulb Edinger–Westphal nucleus forelimb cortex frontal cortex fundus striati granular cell layer of dentate gyrus gigantocellular reticular nucleus glomerular layer of olfactory bulb globus pallidus granular cell layer of cerebellar cortex hindlimb cortex intercalated nuclei of amygdala island of Calleja island of Calleja major internal granular layer of olfactory bulb intramediodorsal thalamic nucleus amygdaloid intramedullary gray insulate cortex intermediate gray layer of superior colliculus inferior olive interpeduncular nucleus internal plexiform layer of olfactory bulb locus coeruleus laterodorsal thalamic nucleus laterodorsal tegmental nucleus lateral hypothalamic area lateral habenular nucleus lacunosum molecular layer of hippocampus nucleus of lateral olfactory tract lateral posterior thalamic nucleus lateral septal nucleus lateral superior olive lateral vestibular nucleus molecular layer of cerebellar cortex

MCPO MD Me MG Mi mlf MM MnR Mo5 Mol MPA MVe O Oc Or OT Par PaS PC PCRt PF PH Pir PLCo PMCo PMD Pn PnC PnO Po PoDG PPTg PrC PRh PrH PrS Pu PV Py Rad Re Rh RLi RPa RPn RRF RSA RSG RtTg S SGe SHy SI SN SNC SNL SNR Sol SpVe STh SuG SuVe Tem TM Tu VDB Ve VL VLG VM VMH VP VPM VTA VTg ZI

magnocellular preoptic nucleus mediodorsal thalamic nucleus medial amygdaloid nucleus medial geniculate nucleus mitral cell layer of olfactory bulb medial longitudinal fasciculus medial mammillary nucleus median raphe nucleus motor trigeminal nucleus molecular layer of hippocampus medial preoptic area medial vestibular nucleus orbital cortex occipital cortex oriens layer of hippocampus nucleus of optic tract parietal cortex parasubiculum paracentral thalamic nucleus parvocellular reticular nucleus parafascicular thalamic nucleus posterior hypothalamic area piriform cortex posterolateral cortical amygdaloid nucleus posteromedial cortical amygdaloid nucleus premammillary nucleus, dorsal part pontine nucleus pontine reticular nucleus, caudal part pontine reticular nucleus, oral part posterior thalamic nuclear group polymorph layer of hippocampus pedunculopontine tegmental nucleus precommissural nucleus perirhinal cortex prepositus hypoglossal nucleus presubiculum Purkinje cell layer of cerebellar cortex paraventricular thalamic nucleus pyramidal cell layer of hippocampus stratum radiatum of hippocampus reuniens thalamic nucleus rhomboid thalamic nucleus rostral linear nucleus of raphe raphe pallidus nucleus raphe pontis nucleus retrorubral field retrosplenial agranular cortex retrosplenial granular cortex reticulotegmental nucleus of pons subiculum suprageniculate nucleus of the pons septohypothalamic nucleus substantia innominata substantia nigra substantia nigra, compact part substantia nigra, lateral part substantia nigra, reticular part nucleus of solitary tract spinal vestibular nucleus subthalamic nucleus superficial gray layer of superior colliculus superior vestibular nucleus temporal cortex tuberomammillary nucleus olfactory tubercle nucleus of the vertical limb of the diagonal band (Broca) vestibular nucleus ventrolateral thalamic nucleus ventral lateral geniculate nucleus ventromedial thalamic nucleus ventromedial hypothalamic nucleus ventral pallidum ventral posteromedial thalamic nucleus ventral tegmental area (Tsai) ventral tegmental nucleus zona incerta

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Fig. 1. Figs 1–10. Distribution of H2 receptor gene transcripts and H2 receptor ([125I]APT) binding sites in guinea-pig brain. Sections were hybridized with 33P-labelled antisense (Figs 1A, 2A, 3A, 4A,D, 5A,D, 6A,D, 7A, 8F) or sense (Fig. 1B) riboprobes. Sections from comparable levels of separate animals were incubated with 0.1 nM [125I]APT alone (Figs 1C, 2B, 3B, 4B,E, 5B,E, 6B,E, 7B, 8A,D, 9A,C, 10A,C) or in the presence of 3 µM of tiotidine (Figs 1D, 7D, 8C). Histological sections adjacent to these last sections were stained using the Nissl procedure (Figs 1E, 2C, 3C, 4C,F, 5C,F, 6C,F, 7C, 8B,E, 9B,D, 10B,D). 10 min, then rinsed twice for 5 min in 2#standard saline citrate (SSC), dehydrated in graded ethanol up to 100% and allowed to air dry. For hybridization, each brain section was covered with 50 µl of buffer containing 50% deionized formamide, 10% dextran sulphate, 1#Denhardt’s solution, 2#SSC, 0.1% sodium pyrophosphate, 100 µg/ml tRNA, 100 µg/ml denaturated salmon sperm DNA and 4# 106 d.p.m. of 33P-labelled antisense or sense probe. The sections were covered with Parafilm, placed in closed humid boxes and incubated for 12 h at 55)C. Following incubation, the sections were allowed to cool to room temperature in 2#SSC for 30 min, treated with RNAase A (200 µg/ml) for 1 h at 37)C, and rinsed in 2#SSC for 30 min, 0.5#SSC for 30 min at 55)C, 0.1#SSC for 30 min at 60)C and 0.1#SSC for 10 min at room temperature. Finally, the sections were dehydrated in graded ethanol containing 300 mM ammonium acetate and allowed to air dry. Autoradiograms were generated by apposing the labelled tissue to Bmax Hyperfilm (Amersham) for one month. Some sections were dipped in a photographic emulsion (LM-1, Amersham, U.K.) and stored at 4)C for 21–30 days. Emulsion-coated slides were developed in Kodak D-19 (4 min), rinsed

in water, fixed in Kodak fixer (10 min), dipped and counterstained with Mayer’s hemalum–eosin. The hybridization probes were synthesized by polymerase chain reaction and subcloned in PGEM-4Z (Promega), and 33 P-labelled antisense- or sense-strand RNA probes were prepared by in vitro transcription using a Riboprobe kit (Promega). These probes corresponded to the C-terminal end of the guinea-pig H2 receptor gene sequence (414 base pairs) from amino acid 290.37 RESULTS

Autoradiograms from frontal or sagittal sections generated with [125I]APT or with the 33P-labelled antisense-strand RNA probe, as well as stained histological sections, are shown in Figs 1–14. Figures 11–14 display enlargements of autoradiograms performed using [125I]APT or, for comparison purposes, [125I]iodobolpyramine, an H1 receptor radioligand, and in situ hybridization images of H2

Histamine H2 receptor in guinea-pig brain

325

Fig. 2.

receptor gene transcripts at a macroscopic or a cellular level in the cerebral cortex (Fig. 11), hippocampal formation (Figs 12 and 14) or cerebellar cortex (Figs 13 and 14). Labelled structures were identified on Nissl-stained sections and are indicated in abbreviated form (Abbreviations used in the figures) on photographs of

the histological sections according to the atlas of Paxinos and Watson.27 The densities of [125I]APT binding sites and the level of hybridization signals in the various structures were arbitrarily rated from 0 to 4+ (independent evaluation by two observers) and values are reported in Tables 1–4.

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Fig. 3.

[125I]Iodoaminopotentidine binding site distribution A heterogeneous labelling of sections was observed which contrasted with the extremely faint and homogeneous non-specific labelling evidenced in most areas (Fig. 1D), with an exception in some mesencephalic structures, such as substantia nigra, caudal linear and dorsal raphe nuclei, medial longi-

tudinal fasciculus and dorsal tegmental bundle; in those areas, the labelling was partially but not completely prevented in the presence of 3 µM tiotidine (Figs 7D, 8C). The highest receptor densities were observed in superficial cerebral cortex (layers I–III), CA1 (stratum radiatum, lacunosum moleculare and oriens layers) of hippocampal formation, parasubiculum

327

Fig. 4.

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Fig. 5.

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329

Fig. 6.

Histamine H2 receptor in guinea-pig brain

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Fig. 7.

330

331

Fig. 8.

Histamine H2 receptor in guinea-pig brain

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Fig. 9.

and presubiculum, basal ganglia (striatum, nucleus accumbens, islands of Calleja, olfactory tubercle, ventral pallidum), olfactory, central and basolateral amygdala, bed nucleus of stria terminalis, superficial

gray layer of superior colliculus, substantia nigra, ventral tegmental area and inferior olive. Moderate labelling was found in deep layers of cerebral cortex, claustrum and endopiriform nuclei,

Histamine H2 receptor in guinea-pig brain

333

Fig. 10.

molecular and polymorph layers of dentate gyrus, internal granular, plexiform and mitral cell layers of olfactory bulb, anterior olfactory nucleus, globus pallidus, substantia innominata, medial amygdala, intralaminar, midline and median nuclear groups of

thalamus, geniculate nuclei, zona incerta, lateral and posterior area of hypothalamus, deep gray layer of superior colliculus, optic tract nuclei, dorsal tegmental area, raphe nuclei, central gray, subthalamic nuclei, locus coeruleus, molecular layer of

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Fig. 11. Distribution of: (A) H1 receptor binding sites, (C) H2 receptor binding sites and (D,E) H2 receptor mRNA in occipital cortex. Adjacent histological sections were stained using the Nissl procedure (B). H1 and H2 receptors are distributed in a laminar way. H1 receptors are distributed in higher density in superficial (I–III) than in deep layers (V–VI) and layer IV is very faint to undetectable, whereas the highest H1 receptor density is in layer IV. (D,E) Absence of H2 gene receptor transcripts in layers I and II, high transcript density in layers III and V, and a very low signal in layer IV. (D,F) Sections were hybridized with 33P-labelled antisense probe and dipped in a photographic emulsion. (F) Bright-field photomicrograph in which the autoradiographic grains appear dark. Labelling is observed in large pyramidal cells, whereas only scarce granular cells are labelled.

Fig. 12. Distribution of: (A) H1 receptor binding sites, (D) H2 receptor binding sites and (C) H2 receptor mRNA in hippocampus. Adjacent histological sections were stained using the Nissl procedure (B). H1 and H2 receptors display a laminated pattern of distribution. The most abundant expression of H1 receptor binding sites is in the dentate gyrus (molecular and polymorph layers), whereas that of the H2 receptor is in field CA1 of Ammon’s horn (lacunosum molecular layer, oriens layer and stratum radiatum). H2 receptor mRNAs are found in the pyramidal cell layer of Ammon’s horn and in the granular cell layer of the dentate gyrus.

Histamine H2 receptor in guinea-pig brain 335

Fig. 13. Distribution of: (A) H1 receptor or (D) H2 receptor binding sites and (C) H2 receptor mRNAs in cerebellar cortex. Adjacent histological sections were stained using the Nissl procedure (B). The molecular layer is densely labelled by H1 and H2 receptor ligands. H2 receptor mRNAs are found in the Purkinje and granular cell layers of the cerebellar cortex.

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Fig. 14. Cellular level visualization of H2 receptor mRNA in: (A–C) hippocampus and (D,E) cerebellar cortex. Sections were hybridized with 33P-labelled antisense probe and dipped in a photographic emulsion. (B,C,E) Bright-field photomicrographs in which the autoradiographic grains appear dark. Labelling is observed in all granular cells of the dentate gyrus (B), all pyramidal cells of Ammons’s horn (C), and Purkinje and granular cells of the cerebellar cortex (E).

cerebellar cortex, dorsal cochlear and vestibular nuclei. A low H2 receptor density was detected in CA2, CA3 (stratum radiatum, lacunosum moleculare and oriens layers) of hippocampal formation, subiculum, glomerular layer of olfactory bulb, septal area, anterior and lateral, ventral and posterior nuclear groups of thalamus, habenular nuclei, anterior and intermediate nuclei of hypothalamus, mammillary and tuberomammillary nuclei, inferior colliculus, pontine nuclei and solitary tract nucleus. A quite low labelling was observed in layer IV of visual and primary somatosensory cortices, whereas pyramidal cell layer of Ammon’s horn, granular cell

layer of dentate gyrus, granular and Purkinje cell layers of cerebellar cortex appeared to be devoid of H2 receptor binding sites. H2 receptor messenger RNA distribution The distribution of H2 receptor mRNA was determined on sections from comparable levels in separate animals using in situ hybridization. One of the images obtained with the 33P-labelled sense-strand RNA probe used as a control is shown, in which only the hippocampal formation (Fig. 1B) and cerebellar cortex (not shown) displayed a moderate labelling (Fig. 1B). The highest levels of expression were found

Dentate gyrus Granular layer Molecular layer Polymorph layer Subicular complex Presubiculum and parasubiculum Subiculum

III. Hippocampus Ammon’s horn Pyramidal cell layer Lacunosum moleculare and oriens layers, stratum radiatum

Perirhinal areas Claustrum, endopiriform nuclei

II. Allo and periallo cortices Cingulate, retrosplenial, insulate and orbital cortices

I. Isocortex Forelimb and hindlimb areas, occipital and parietal cortices Frontal and temporal cortices

Brain structures

0

III 4+ 2–3+

4+ 1+

II 4+

3–4+ 2+

I 4+

0 2+ 2+

VI 2–3+

0–1+ 2–3+

IV

4+ 0 2–3+

V 3–4+

4+

I–II–III

4+ 4+

I–II–III

3–4+ 1+

IV 1+ 2–3+

2–3+

VI

2+ 2+

VI

0 0

III 4+

3–4+

V

3–4+ 3–4+

V

CA1 CA2, CA3

II 1+

I 0–1+

4+

1+ 1+

IV

4+

1+

0–1+

III

4+ 4+

III

IV 2–3+

[125I]Iodoaminopotentidine binding sites

CA1, CA2, CA3

II

1+ 1+

0–1+ 0–1+ I

II

I

H2 receptor mRNA

Table 1. Distribution of H2 receptor messenger RNA and [125I]iodoaminopotentidine binding sites in cerebral cortex and hippocampus

V–VI 3+

2–3+

V–VI

2–3+ 2–3+

V–VI

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Table 2. Distribution of H2 receptor messenger RNA and [125I]iodoaminopotentidine binding sites in olfactory system, basal forebrain and amygdaloid complex Brain structures

H2 receptor mRNA

[125I]Iodoaminopotentidine binding sites

0 1+ 4+ 4+ 2–3+

2–3+ 1+ 2+ 2+ 2–3+

I. Olfactory system Olfactory bulb Plexiform layer Glomerular layer Internal granular layer Mitral cell layer Anterior olfactory nucleus II. Basal forebrain Septal area Lateral septal nucleus, septohypothalamic nucleus, nucleus of the vertical limb of the diagonal band Basal ganglia Accumbens nucleus Caudate–putamen (striatum) Globus pallidus Islands of Calleja Substantia innominata Olfactory tubercle Ventral pallidum

1–2+

1–2+

4+ 3–4+ 1+ 4+ 1+ 4+ 3–4+

3–4+ 4+ 2+ 4+ 2+ 4+ 3+

III. Amygdaloid complex Olfactory amygdala Medial amygdala Central amygdala Basolateral amygdala Bed nucleus, stria terminalis

3+ 2–3+ 3–4+ 3+ 3–4+

3+ 2–3+ 3–4+ 3+ 3–4+

Table 3. Distribution of H2 receptor messenger RNA and [125I]iodoaminopotentidine binding sites in thalamus and hypothalamus H2 receptor mRNA

[125I]Iodoaminopotentidine binding sites

I. Thalamus Anterior and lateral nuclear groups Intralaminar nuclear group Midline and median nuclear groups Ventral and posterior nuclear groups Geniculate nuclei Habenular nuclei Zona incerta

1–2+ 2–3+ 2–3+ 1+ 2–3+ 2–3+ 2–3+

1–2+ 2–3+ 2–3+ 1+ 2–3+ 1+ 2+

II. Hypothalamus Anterior nuclei Lateral nuclei Intermediate nuclei Posterior nuclei Mammillary nuclei Tuberomammillary nuclei

2+ 2+ 1–2+ 2+ 2–3+ 2–3+

1–2+ 2+ 1–2+ 2+ 1–2+ 1+

Brain structures

in pyramidal cell layers III and V of cerebral cortex, pyramidal cell layer of Ammon’s horn, granular cell layer of dentate gyrus, parasubiculum and presubiculum, internal granular and mitral cell layers of olfactory bulb, basal ganglia (nucleus accumbens, striatum, islands of Calleja, olfactory tubercle, ventral pallidum), olfactory, central and basolateral amygdala, bed nucleus of stria terminalis, superficial gray layer of superior colliculus, optic tract nuclei, granular and Purkinje cell layers of cerebellar cortex, and inferior olive. Moderate expression was detected in layer VI of cerebral cortex, claustrum and endopiriform

nuclei, polymorph layer of dentate gyrus, subiculum, anterior olfactory nucleus, medial amygdala, intralaminar, midline and median nuclear groups of thalamus, geniculate and habenular nuclei, zona incerta, anterior, lateral and posterior area of hypothalamus, mammillary and tuberomammillary nuclei, deep gray layer of superior colliculus, dorsal tegmental area, central gray, subthalamic and pontine nuclei, and dorsal cochlear nuclei. Low to very low hybridization signals were observed in layers II and IV of cerebral cortex, glomerular layer of olfactory bulb, septal area, globus pallidus, substantia innominata, anterior and lateral,

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M. L. Vizuete et al. Table 4. Distribution of H2 receptor messenger RNA and [125I]iodoaminopotentidine binding sites in caudal areas

Brain structures Inferior colliculus Superficial gray layer, superior colliculus Deep gray layer, superior colliculus Optic tract nuclei Dorsal tegmental area Raphe nuclei Central gray Substantia nigra pars compacta Substantia nigra pars reticulata Ventral tegmental area Subthalamic nuclei Locus coeruleus Pontine nuclei Cerebellar cortex, granular layer Cerebellar cortex, molecular layer Cerebellar cortex, Purkinje cell layer Dorsal cochlear nuclei Inferior olive Solitary tract nucleus Vestibular nuclei

H2 receptor mRNA

[125I]Iodoaminopotentidine binding sites

1–2+ 3+ 2+ 3+ 2–3+ 1–2+ 2+ 1–2+ 0–1+ 1–2+ 2+ 1+ 2–3+ 3+ 0 4+ 2–3+ 3+ 1+ 1–2+

1+ 4+ 2+ 2+ 2–3+ 3+ 2+ 3+ 3+ 3+ 2+ 2+ 1+ 0 2–3+ 0 2–3+ 4+ 1+ 2+

ventral and posterior nuclear groups of thalamus, intermediate nuclei of hypothalamus, inferior colliculus, raphe nuclei, substantia nigra, ventral tegmental area, locus coeruleus, solitary tract and vestibular nuclei. The signal hybridization was quite undetectable in lamina I of cerebral cortex and was absent in lacunosum moleculare, oriens layers and stratum radiatum of Ammon’s horn, molecular layer of dentate gyrus, plexiform layers of olfactory bulb and molecular layer of cerebellar cortex. DISCUSSION

The aim of the present work was to map and compare the distributions of the H2 receptor and its gene transcripts in guinea-pig brain. For this purpose two highly selective probes were used, i.e. the radioligand [125I]APT31 and 33P-labelled RNA corresponding to the 3* end of the guinea-pig H2 receptor nucleotide sequence.37 The specificity of signals generated by in situ hybridization was confirmed by using a sense probe (Fig. 1B). The selectivity of [125I]APT for the H2 receptor was shown by the almost negligible labelling remaining in the presence of tiotidine, a potent and chemically unrelated H2 receptor antagonist (Figs 1D, 7D, 8C). Moreover, the labelling was also prevented by three other H2 receptor antagonists used at different concentrations in relation to their respective Ki values (data not shown). Distribution of the H2 receptor and histaminergic fibres The present study provides evidence for a widespread but highly contrasted distribution of the H2 receptor among guinea-pig brain areas, which reflects to a certain extent the distribution of histaminergic

axons.1 For instance, the amygdaloid area, particularly the basal nucleus of stria terminalis, which receives the most abundant histaminergic innervation in the brain,1,3 also contains abundant [125I]APT binding sites. There were, however, two opposite types of discrepancies between the distributions of [125I]APT binding sites and histaminergic axons. First, in some areas, a relatively low density of binding sites contrasts with a high level of histaminergic innervation such as in the septal area and various hypothalamic, thalamic or pontine nuclei. In almost all of these areas, histamine may induce its actions predominantly via H1 or H3 receptors, which are abundant as shown by autoradiography.6,26,29 Nevertheless, in the preoptic area of hypothalamus, where the H2 receptor appears to be rather less abundant than the H1 or H3 subtypes, histaminergic innervation seems to involve both H1 and H2 receptors in sleep–wake control.20 Moreover, in the nucleus of the vertical limb of the diagonal band, in which a very low signal is found, an H2 receptor-mediated stimulation of cholinergic neurons was reported.24 A second type of discrepancy concerns areas such as Ammon’s horn, where the very high H2 receptor density contrasts with a rather moderate histaminergic innervation. Distribution of the H2 receptor and its gene transcripts In most brain areas, the distributions of the H2 receptor and its gene transcripts appear to be consistent with, however, some interesting discrepancies. Thus, in the cerebral cortex, where a rather high H2 receptor-mediated activation of adenylyl cyclase activity occurs,2,12,17 the H2 receptor is distributed in a laminar way and in higher density in superficial (I–III) than in deep layers (IV–VI).

Histamine H2 receptor in guinea-pig brain

Moreover, the highly precise labelling allows one to note a very faint to quite undetectable labelling in the thin lamina IV of primary somatosensory and visual cortices. This distribution is complementary to that revealed in the case of the H1 receptor, for which the highest density of binding sites is localized in this last layer6 (Fig. 11). In contrast, in situ hybridization reveals an absence of H2 receptor gene transcripts in lamina I, the labelling being observed mainly in large pyramidal cells of layers III, V and VI, and, to a limited extent, in granular cells of layers II and IV (Fig. 11). Thus, it appears very likely that the presence of the binding sites in lamina I, where the network of histaminergic varicose fibres is the densest,1 corresponds to the expression of the receptor on dendrites of pyramidal cells. Cortical neurons, particularly pyramidal cells, studied with microiontophoretic techniques, generally show H2 receptor-mediated inhibitions, but an excitatory response was also recorded in the entorhinal cortex.14 In the same manner, the patterns of H2 receptor binding and mRNA appear to be different in the various layers of the olfactory bulb. Indeed, the H2 receptor seems to be synthesized almost exclusively in the mitral and internal granular cell layers, whereas the receptor protein seems to be expressed in all olfactory bulb layers. This expression appears to be higher than that of H1 or H3 receptors6,29 and suggests that, there, the high density of histaminergic terminals1 might affect target neurons principally via the H2 receptor. In the hippocampal formation as well as cerebellar cortex, the relative localizations of the H2 receptor and its gene transcripts are similar to that previously observed for the H1 receptor.35 Thus, the gene transcripts are expressed in all pyramidal cells of Ammon’s horn (Fig. 14A,C) and Purkinje or granule cells of cerebellar cortex (Fig. 14D,E), whereas the receptor is expressed in the molecular layers of the same areas, which contain the dendritic trees of the mRNA-containing neurons (Figs 12D, 13D). In the same way, the very high expression of H2 receptor mRNA in granule cells of the dentate gyrus, without any significant [125I]APT binding in these cells, may correspond to the perikaryal synthesis of the H2 receptor transported and expressed in dendrites or mossy fibre endings of these neurons in the molecular layer of the gyrus and CA3 region, respectively. In the substantia nigra pars reticula, the high density of [125I]APT binding sites, not accompanied by corresponding H2 receptor mRNA, indicates that the receptor is present on extrinsic afferents. In agreement with this, quinoleic acid lesions in striatum result in decreases in nigral H2 receptor binding, evidencing the presence of the H2 receptor on striatonigral fibre endings.22 However, the detection of the two H2 receptor markers in the substantia nigra pars compacta suggests that dopaminergic neurons

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themselves may be endowed with the receptor. In addition, areas known to contain noradrenergic, serotoninergic and cholinergic cell bodies, e.g., locus coeruleus, raphe nuclei and substantia innominata, appear to synthesize (at a low level) the H2 receptor. This observation, together with the previous demonstration of H2 receptor-mediated inhibition of dorsal raphe serotoninergic neurons19 and excitation of nucleus basalis cholinergic neurons projecting to the cerebral cortex,18 would be consistent with the idea that histamine controls all major ascending aminergic systems in brain by interacting with the H2 receptor. This hypothesis remains to be verified, however, for instance by co-hybridization studies using probes for the RNAs of the H2 receptor and selective markers of the monoaminergic neurons. Such interactions are of obvious interest to understand how histaminergic neurons exert their best substantiated function, i.e. the control of sleep–wake cycles in conjunction with other monoaminergic neurons.

H2 receptor distribution and biological responses mediated by this receptor In many other brain areas, the presence of the H2 receptor is in agreement with many previously reported responses mediated by the same receptor. For instance, H2 receptor-mediated activation of cAMP formation occurs in slices or membranes of several brain areas and is highest in guinea-pig hippocampus, where [125I]APT binding sites are abundant.31 Nevertheless, the lack of strict parallelism between H2 receptor binding and H2 receptoractivated adenylyl cyclase suggests that the receptor may mediate cAMP-independent responses in neurons.31 In agreement, the heterologously expressed H2 receptors in Chinese hamster ovary cells are coupled not only positively with adenylyl cyclase but also negatively with phospholipase A2,36 and whereas many H2 receptor-mediated electrophysiological responses, e.g., the block of a calciumactivated potassium conductance in hippocampal neurons, namely pyramidal cells, involve cAMP, other cAMP-independent responses were described (reviewed in Ref. 14). In the dorsal lateral geniculate relay neurons, where both [125I]APT binding and H2 receptor hybridization signals are detected, H2 receptor activation has an excitatory effect, resulting in a switch in the activity of these thalamic neurons, rendering them more responsive to sensory inputs and constituting a potential cellular mechanism of histaminergic control of arousal.23 The same type of mechanism seems responsible for the arousal controlled by other highly divergent ascending aminergic systems, e.g., cholinergic and catecholaminergic pathways. The role of histamine in arousal is well established by a variety of approaches, e.g., monitoring the activity of

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histaminergic neurons during the nycthemeral cycle, lesioning these neurons, using synthesis inhibitors, and receptor subtype selective agonists or antagonists (reviewed in Refs 32, 33). In the vestibular nuclei, where both markers are also detected, histamine has an excitatory effect, mediated by the H2 receptor.34 In hippocampal formation and cerebellar cortex, where apparently most, if not all, granule, pyramidal or Purkinje cells synthesize the H2 receptor, a colocalization with the H1 receptor appears to be likely, since H1 receptor gene expression and binding occur according to a similar pattern in these cerebral areas.6,26,35 In hippocampus, this co-localization would be consistent with the large H1 receptor-mediated increase in the cAMP response in slices as compared to that induced by stimulation of the H2 receptor alone: only the latter receptor is positively coupled to the cyclase and the amplification of the cAMP signal through the H1 receptor seems to be due to the augmented intracellular level of Ca2+ it mediates within the same neurons.25 In addition, involvement of both H1 and H2 receptor stimulation in depolarization of thalamic23 or cholinergic basalis18 neurons is in agreement with the co-existence of both receptors in these latter cerebral areas, even if their

co-localization in the same neurons remains to be demonstrated. In some limbic brain areas, e.g., bed nucleus of stria terminalis, nucleus accumbens or olfactory tubercles, high densities of H2 receptor markers are accompanied by high densities of H3 receptor binding sites.29 The localizations of the H3 receptor on striatonigral neurons, evidenced by a combination of lesion and autoradiographic studies, suggest that H2 and H3 receptors might also be co-localized in some neuronal populations, but the functional significance of this situation is not known.22,29

CONCLUSIONS

Some of the localizations of the H2 receptor demonstrated here are consistent with major alleged functions of histaminergic neurons in brain. Among them, the role of histaminergic neurons in the maintenance of wakefulness is probably the best substantiated function, by a variety of experimental evidence and supported by the widespread occurrence of the H2 receptor in diencephalic and telencephalic areas to which these neurons project in a highly divergent manner.32,33

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