C Pillot

A Heron

V Cochois

J Tardivel-Lacombe

X Ligneau

J.-C Schwartz

J.-M Arrang
email: arrang@broca.inserm.fr

A detailed mapping of the histamine H 3 receptor and its gene transcripts in rat brain

The detailed distribution of histamine H3 receptor mRNAs in rat brain was analyzed by in situ hybridization using a 33 P-labelled riboprobe and was combined for the first time with the detailed autoradiographic distribution of the receptor determined in the same animals with [ 125 I]iodoproxyfan, a selective radioligand. The signals generated on adjacent brain sections by each probe were quantified and/or rated and were compared in order to identify neuronal populations expressing the receptor. In addition, the cellular localization of the transcripts within various brain structures was analyzed in sections dipped in a photographic emulsion.

In the cerebral cortex, the strong mRNA expression in intermediate and deep layers indicates the presence of H3 receptors on several types of neurons. The binding is dense except in layer V, suggesting that H3 receptors are located on granule cells and apical dendrites of pyramidal cells. In addition to their localization on monoaminergic afferents, the dense binding in layer IV and strong mRNA expression in thalamic nuclei suggest the presence of heteroreceptors on thalamocortical projections. In the hippocampus, the strong mRNA expression but low binding in pyramidal layers of the CA1 and ventral CA3 fields suggest that H3 receptors are abundant on efferent projections of pyramidal cells. In the dentate gyrus, some binding sites in the molecular layer may correspond to H3 receptors synthesized in granule cells and coexpressed with H1 and H2 receptors in their dendrites. In the basal ganglia, H3 receptors are highly expressed in the striatal complex and olfactory tubercles but not in islands of Calleja. Some of the striatal binding sites may correspond to presynaptic receptors present on afferents. The mRNAs in cortical layer V may encode for heteroreceptors on corticostriatal neurons. The presence of mRNAs in the substantia nigra pars compacta suggests that H3 receptors are located upon nigrostriatal afferents. However, the absence of any signal in the ventral tegmental area indicates that some but not all dopaminergic neurons express H3 receptors. In addition, the homogeneous mRNA expression within the caudate putamen and nucleus accumbens suggests that many striatal H3 receptors are present on medium-sized, spiny projection neurons of both the direct and indirect movement pathways. In agreement, a dense binding, but low mRNA expression, is observed in external and internal pallidum and in substantia nigra pars reticulata. In the amygdala, the dense binding and mRNA expression indicate the presence of receptors on both afferents and projections. In the thalamus, the binding in some association nuclei may correspond to receptors present on neurons emanating from the deep cortical layers that strongly express the mRNAs, as well as receptors on the visual systems. However, the low binding and high mRNA expression in most nuclei indicate that many receptors are present upon thalamic projections. In the hypothalamus, the mRNA expression parallels the density of binding sites and is the highest in the tuberomammillary nucleus. Further investigation is needed to know if the dense binding and mRNA expression observed in other nuclei such as the paraventricular, ventromedial and medial tuberal nuclei correspond to preand/or postsynaptic receptors. mRNAs are also observed in several areas projecting to the tuberomammillary nucleus, such as the ventrolateral preoptic nucleus. In the lower brainstem, the high mRNA expression and very low binding in the locus coeruleus and raphe nuclei indicate that presynaptic rather than somatodendritic receptors regulate noradrenaline and serotonin release, respectively. A similar pattern in vestibular nuclei suggests that receptors located on projections account for the anti-vertigo properties of H3 receptor antagonists. In the cerebellum, binding is hardly detectable but a strong mRNA expression is found in most, if not all, Purkinje cells as well as in several central cerebellar nuclei, suggesting the presence of H3 receptors on efferent projections.

The present study reports the first detailed quantification and/or rating of H3 receptor mRNAs in the brain. The comparison, performed in the same animals, with the distribution of the H3 receptor protein provides evidence for the presence of H3 receptors on many neuronal perikarya, dendrites and projections. Although some localizations, mainly as auto-or heteroreceptors, are consistent with previous functional studies, the physiological role, if any, of most of these presynaptic or postsynaptic receptors remains to be established.

The histamine H3 receptor (H3R) was initially characterized as an autoreceptor controlling histamine synthesis and release [START_REF] Arrang | Autoinhibition of histamine release mediated by a novel class (H3) of histamine receptor[END_REF](Arrang et al., , 1987b)). Thereafter, it was shown to inhibit presynaptically the release of other monoamines in brain and peripheral tissues as well as of neuropeptides from unmyelinated C fibers. In neurons, the H3R mediates presynaptic inhibition of release of histamine, noradrenaline, serotonin, dopamine, glutamate, GABA and tachykinins [START_REF] Schlicker | Modulation of neurotransmitter release via histamine H3 heteroreceptors[END_REF][START_REF] Hill | International Union of Pharmacology. XIII. Classification of histamine receptors[END_REF][START_REF] Brown | The physiology of brain histamine[END_REF][START_REF] Schwartz | Histamine[END_REF], presumably by inhibiting calcium channels [START_REF] Arrang | Autoregulation of histamine release in brain by presynaptic H3-receptors[END_REF][START_REF] Takeshita | Histamine modulates high-voltage-activated calcium channels in neurons dissociated from the rat tuberomammillary nucleus[END_REF][START_REF] Brown | On the mechanism of histaminergic inhibition of glutamate release in the rat dentate gyrus[END_REF]. The inhibition mediated by H3autoreceptors expressed in the tuberomammillary nucleus (TM) constitutes a major regulatory mechanism of the activity and functions of histaminergic neurons [START_REF] Schwartz | Histamine[END_REF]. However, the physiological role of the H3Rs present on other neuronal populations remains largely unknown.

The modulation of agonist binding by guanylnucleotides [START_REF] Arrang | Subclassification of histamine receptors, H3 receptor subtypes? Localization of H3 receptors in the brain[END_REF] and the sensitivity of various H3R-mediated responses to pertussis toxin [START_REF] Clark | Sensitivity of histamine H3 receptor agonist-stimulated[END_REF][START_REF] Takeshita | Histamine modulates high-voltage-activated calcium channels in neurons dissociated from the rat tuberomammillary nucleus[END_REF] suggested that the H3R was a Gi/Go protein-coupled heptahelical receptor. This proposal was confirmed by the recent cloning of H3R cDNAs from human [START_REF] Lovenberg | Cloning and functional expression of the human histamine H3 receptor[END_REF], guinea-pig [START_REF] Tardivel-Lacombe | Cloning andcerebral expression of the guinea pig histamine H3 receptor: evidence for two isoforms[END_REF] and rat [START_REF] Morisset | High constitutive activity of native H3 receptors regulates histamine neurons in brain[END_REF][START_REF] Lovenberg | Cloning of rat histamine H3 receptor reveals distinct species pharmacological profiles[END_REF][START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF].

Consistent data have been reported with respect to the autoradiographic localization of H3R binding sites in rat brain using various radiolabelled agonists, i.e. [ 3 H](R)α-methylhistamine (Arrang et al., 1987a;[START_REF] Yanai | Receptor autoradiography with 11C and [ 3H]-labelledligan ds visualizedby imaging plates[END_REF][START_REF] Ryu | Ontogenetic development of histamine receptor subtypes in rat brain demonstrated by quantitative autoradiography[END_REF] and [ 3 H]N α -methylhistamine [START_REF] Cumming | High affinity histamine binding is the H3 receptor: characterization andautorad iographic localization in rat brain[END_REF](Cumming et al., , 1994;;Cumming and Gjedde, 1994), or various radiolabelled antagonists, i.e. [ 3 H]S-methylthioperamide [START_REF] Yanai | Binding characteristics of a histamine H3-receptor antagonist, [3H]Smethylthioperamide: comparison with [3H](R)K-methylhistamine binding to rat tissues[END_REF] and [ 125 I]iodophenpropit [START_REF] Jansen | Characterization of the binding of the first selective radiolabelled histamine H3-receptor antagonist, [125I]-iodophenpropit, to rat brain[END_REF][START_REF] Jansen | Characterization of histamine H3 receptors in mouse brain using the H3 antagonist [125I]iodophenpropit[END_REF]. Among these various radioligands, only [ 3 H](R)αmethylhistamine was used for a detailed mapping of rat brain [START_REF] Pollard | A detailed autoradiographic mapping of histamine H3 receptors in rat brain areas[END_REF]. However, being an agonist, its binding may be dependent on various factors including G proteins. In a preliminary study, we showed that the radiolabelled antagonist [ 125 I]iodoproxyfan was a reliable probe for autoradiography. Being iodinated, this radioligand displayed a much higher specific activity and therefore allowed a much more sensitive detection of the H3R than that obtained with [ 3 H](R)α-methylhistamine, even after a short exposure time [START_REF] Ligneau | 125I]Iodoproxyfan, a new antagonist to label andvisualize cerebral histamine H3 receptors[END_REF].

With the cloning of the H3R, a preliminary and non-rated analysis of the gene transcripts was performed by in situ hybridization [START_REF] Lovenberg | Cloning and functional expression of the human histamine H3 receptor[END_REF]. Following this cloning, evidence was obtained for the existence of H3R isoforms [START_REF] Tardivel-Lacombe | Cloning andcerebral expression of the guinea pig histamine H3 receptor: evidence for two isoforms[END_REF]Morisset et al., 2000) which revealed a differential expression in various brain regions [START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF][START_REF] Morisset | The rat H3 receptor: gene organization andmultiple isoforms[END_REF]. In the present study, in situ hybridization was used for a detailed mapping of H3R mRNAs and was combined for the first time with the autoradiographic mapping of the receptor performed in the same animals with [ 125 I]iodoproxyfan. Moreover, the densities of the signals obtained in both approaches on adjacent brain sections were rated and the complementary information provided by each method was taken into account to further identify neuronal populations expressing the receptor in brain. The rat was selected because most of the previous studies that established the localizations, signalling mechanisms and detailed pharmacology of the H3R were performed in this species.

Experimental procedures

Tissue preparation

All experiments performed in the present study conformed to the National Institutes of Health guidelines (décret no. 2001(décret no. -464, May 29, 2001, from the French Ministry of Agriculture). Male Wistar rats (Iffa-Credo, L'Arbresle, France) were killed by decapitation, their brain was removed rapidly, immediately frozen (-40°C) by immersion in monochlorodifluoromethane and stored at -70°C. Brain sections (10 µm) were prepared on a cryostat and thaw-mounted onto Superfrost slides. For in situ hybridization, sections adjacent to those prepared for incubation with [ 125 I]iodoproxyfan, were immediately fixed for 40 min at 4°C in 4% paraformaldehyde made up in 0.1 M phosphate-buffered saline, pH 7.4, and 0.1% diethylpyrocarbonate water. Sections were rinsed three times (5 min each) in 0.1 M phosphate-buffered saline, pH 7.4, dehydrated through graded ethanol and dried under a stream of cold air. All the sections were stored at -70°C until use.

Receptor binding autoradiography

Autoradiographic localization of [ 125 I]iodoproxyfan binding sites was determined essentially as described [START_REF] Ligneau | 125I]Iodoproxyfan, a new antagonist to label andvisualize cerebral histamine H3 receptors[END_REF]. In brief, sections were preincubated for 15 min in phosphate buffer in the presence of 0.1% bovine serum albumin and 1 µM S132 (a 1substituted imidazole derivative displaying a very low affinity at H3Rs and used to decrease non-specific labelling). They were then incubated for 1 h at 4°C in the same buffer with 15 pM [ 125 I]iodoproxyfan. Non-specific binding was determined in the presence of 1 µM (R)αmethylhistamine. After incubations, the sections were extensively washed, dried and apposed to a βmax Hyperfilm (Amersham, UK) for 5 days at 4°C.

In situ hybridization histochemistry

Sections were incubated at 37°C for 10 min with proteinase K (1 µg/ml), acetylated for 10 min (in 0.1 M triethanolamine, pH 8 and 0.25% acetic anhydride) at room temperature and dehydrated in graded ethanol up to 100%. Hybridization was performed overnight at 55°C in the presence of 4×10 6 d.p.m. of 33 P-labelled antisense or sense probes in hybridization buffer (50% formamide, 10% dextran sulfate, 2×standard saline citrate (SSC), 1×Denhardt's solution, 50 mM Tris-HCl buffer, 0.1% NaPPi, 0.1 mg/ml yeast tRNA, 0.1 mg/ml salmon sperm DNA, 1 mM EDTA). Subsequently, the sections were rinsed with 2×SSC for 5 min and incubated for 40 min at 37°C with RNAse A (200 µg/ml). The sections were then extensively washed in SSC, dehydrated in graded ethanol, dried and exposed for 1 week to a βmax Hyperfilm (Amersham) or dipped in liquid photographic LM1 emulsion (Amersham) for 2 weeks. Dipped sections were observed with a photomicroscope (Axiophot Zeiss, Carl Zeiss, Germany) after counterstaining of cell nuclei with Mayer's hemalun.

For the hybridization probe, a partial coding sequence of the rat H3R was amplified from striatum cDNAs using primers 1 and 2 based on the sequence of the third transmembrane domain and the third intracellular loop of the human H3R, respectively [START_REF] Lovenberg | Cloning and functional expression of the human histamine H3 receptor[END_REF] (primer 1: 5′-AGTCGGATCCAGCTACGACCGCTTCCTGTC-3′ and primer 2: 5′-AGTCAAGCTTGGAGCCCCTCTTGAGTGAGC-3′). The amplified fragment was sequenced and corresponded to nucleotides 636-1243 of the rat H3R sequence. It was previously shown to hybridize to the various H3R mRNA isoforms expressed in the brain or peripheral tissues [START_REF] Morisset | The rat H3 receptor: gene organization andmultiple isoforms[END_REF]Héron et al., 2001). After subcloning into pGEM-4Z (Promega, Charbonnières, France), 33 P-labelled antisense and sense strand RNA probes were prepared by in vitro transcription using a riboprobe kit (Promega).

Labelled structures were identified on adjacent Nissl-stained sections and are indicated in abbreviated form (see Abbreviations used in the figures) according to the atlas of [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates, 4th edn[END_REF]. The densities of [ 125 I]iodoproxyfan binding sites and the level of H3R mRNA expression in these structures were arbitrarily rated from 0 to 4+ (double-blind evaluation by four observers). In addition, sections from four separate animals were also digitized using a camera and analyzed using Starwise/Autorad 210 program (Imstar, Paris, France), and the mean optical density of pixels was determined within the main brain regions.

Radiochemicals and drugs

[ 125 I]iodoproxyfan (specific activity of 2000 Ci/mmol at activity reference date) was prepared as described [START_REF] Krause | Iododestannylation: an improved synthesis of [125I]iodoproxyfan, a specific radioligand of the histamine H3 receptor[END_REF]. S132 (3-cyclohexyl-N-[3-(1H-imidazol-1yl)propyl]propanic amide) was synthesized by Prof. W. Schunack (Freie Universität Berlin, Germany). (R)α-methylhistamine was from Bioprojet (Paris, France).

Results

The densities of [ 125 I]iodoproxyfan binding sites and H3R mRNAs in the various structures were quantified and/or arbitrarily rated and the results are given in Fig. 1 and Tables 12345.

3+ 3-4+ Paratenial nucleus (PT) 1+ 2-3+ Intermediodorsal nucleus (IMD) 2-3+ 3+ Central nucleus (CL) 0-1+ 3+ Central medial nucleus (CM) 0-1+ 3-4+ Paracentral nucleus (PC) 0-1+ 3+ Parafascicular nucleus (PF) 0-1+ 1-2+ Reuniens nucleus (Re) 2-3+ 3+ Rhomboid nucleus (Rh) 2-3+ 3+ Thalamic reticular nucleus (Rt) 0-1+ 2-3+ Habenular nucleus (Hb) 0 1+ Zona incerta (ZI) 0-1+ 1-2+

Autoradiographic distribution of [ 125 I]iodoproxyfan binding sites

A heterogeneous labelling of sections was observed (Fig. 1) which contrasted with the extremely faint non-specific background, as evidenced in the presence of 1 µM (R)-αmethylhistamine (Fig. 2A).

The highest receptor densities were found in the cerebral cortex, tenia tecta, anterior olfactory nucleus, nucleus accumbens, caudate putamen, olfactory tubercles, external and internal segments of the globus pallidus, amygdala, substantia nigra pars reticulata and TM (Fig. 1 and Tables 1, 2, 4 and5). In the cerebral cortex, H3R binding sites were abundant in all areas. However, their laminar distribution was clearly apparent in several areas of the neocortex such as the somatosensory cortex and the auditory and visual cortices, in which a much lower density of binding sites was detected in layer V (Table 1). A laminar pattern of labelling was also observed in the hippocampal formation, a moderate binding being detected in the pyramidal layer and stratum lacunosum moleculare of the CA1 field and in the molecular layer of the dentate gyrus and subiculum (Table 1). A highly heterogeneous labelling was observed in the thalamus, with the highest binding being found in the lateral posterior, laterodorsal, paraventricular, intermediodorsal, reuniens and rhomboid nuclei (Table 3). A rather dense binding was also found in some hypothalamic nuclei, including the medial preoptic, paraventricular, supraoptic, arcuate, ventromedial, medial tuberal, terete, mammillary and tuberomammillary nuclei (Table 4). In the lower brainstem, moderate labelling was detected in the colliculi, pontine nuclei and periaqueductal gray (Table 5).

Autoradiographic distribution of H3 receptor gene transcripts

The distribution of H3R mRNAs was analyzed on adjacent sections from the same levels by in situ hybridization. Well-contrasted autoradiograms were generated with the 33 P-labelled antisense RNA probe, whereas a very faint background was seen with the corresponding sense probe (Fig. 2E). In addition, the cellular localization of H3R gene transcripts was analyzed in various brain regions by in situ hybridization in sections dipped in a photographic emulsion (Figs. 789).

The highest level of expression was found in the cerebral cortex, hippocampus, tenia tecta, nucleus accumbens, caudate putamen, olfactory tubercles, amygdala, thalamus, some hypothalamic nuclei, cerebellum and vestibular nuclei (Fig. 1, Tables 12345).

In the cerebral cortex, a laminar distribution pattern was found, the expression being stronger in the intermediate and deep layers than in the superficial layers. In all areas of the neocortex, layer V displayed an intense mRNA labelling, strong signals being observed within pyramidal cells (not shown). Strong signals were also found in pyramidal layer III in the secondary motor cortex, granular layer IV in the auditory cortex and layer VI in the somatosensory and auditory cortices (Table 1 and Fig. 7B-F). An intense mRNA labelling was also observed in the archeocortex and the distribution pattern in the cingulate and retrosplenial cortices was laminar, with a lower expression in superficial layers (Table 1).

In the hippocampic formation, a strong mRNA expression was found in CA1 and ventral CA3 pyramidal layers of Ammon's Horn as well as in the pyramidal layer of the subiculum (Table 1, Fig. 7A). A very low density of signals was detected in the stratum lacunosum moleculare of the CA1 field and no signals were detected in the other layers of Ammon's Horn. In the dentate gyrus, mRNA signals were rather dense in the granular layer, lower in the polymorph layer and not detected in the molecular layer (Table 1, Fig. 7A). An intense labelling was found in the entorhinal cortex, particularly in layers I, V and VI, and in the tenia tecta (Table 1).

In the olfactory system, a strong mRNA expression was found in the anterior olfactory nucleus. Dense signals were also observed in the amygdala and in the bed nucleus of the stria terminalis (Table 2). In the basal forebrain, a strong and homogeneous expression was detected in the caudate putamen and in the core and shell subdivisions of the nucleus accumbens (Fig. 8A andB). A high expression was found in the olfactory tubercles. A rather dense mRNA labelling was detected in the lateral and medial septal nuclei as well as in the subthalamic nucleus (Table 2). The signals were very low in the globus pallidus and substantia innominata. H3R gene transcripts were not detected in islands of Calleja (Table 2, Fig. 8C,D).

In the thalamus, strong signals were evidenced in all sensory nuclei, except the medial part of the medial geniculate nucleus, and in all motor and association nuclei (Table 3). All midline and intralaminar nuclei also expressed high mRNA densities, although a lower density was evidenced in the parafascicular nucleus. Rather dense signals were also found in the reticular nucleus (Table 3). A low to moderate mRNA expression was detected in the habenula and zona incerta (Table 3).

In the hypothalamus, a highly heterogeneous mRNA expression was observed (Table 4). In the preoptic region, high signals were observed in the medial preoptic nucleus. Rather dense signals were also evidenced in the medial preoptic area, the lateral and magnocellular preoptic nuclei. In the anterior region, a dense mRNA labelling was found in the paraventricular nucleus and in the anterior part of the anterior area. A low to moderate density of signals was observed in the supraoptic and suprachiasmatic nuclei (Table 4). In the tuberal region, a strong mRNA expression was observed in the ventromedial and terete nuclei. Rather dense signals were also found in the dorsomedial and medial tuberal nuclei (Table 4, Fig. 9B). In the mammillary region, strong signals were evidenced in the TM and rather dense signals were found in the posterior hypothalamic area and supramammillary nucleus (Table 4, Fig. 9A).

In the caudal areas, dense mRNA signals were evidenced within the locus coeruleus and the various vestibular nuclei (Table 5, Fig. 9C,F). In the cerebellum, a strong expression of H3R mRNAs was found in most, if not all, Purkinje cells of the cortex and a rather high expression was also detected in several central cerebellar nuclei including the medial cerebellar nucleus and the interposed cerebellar nuclei (Table 5, Fig. 9D,E). A moderate density of hybridization signals was detected in the periaqueductal gray, in several nuclei of the brainstem, e.g. the dorsal raphe, solitary tract, dorsal cochlear, facial and pontine nuclei, and in the colliculi and inferior olive (Table 5). Hybridization signals were low in the substantia nigra pars compacta and pars reticulata and were not detected in the ventral tegmental area (Table 5, Fig. 8E,F).

Discussion

The aim of the present work was to rate and compare the detailed autoradiographic distributions of the H3R and its gene transcripts on adjacent brain sections from the same rats, using two selective probes, i.e. the radioligand [ 125 I]iodoproxyfan [START_REF] Ligneau | 125I]Iodoproxyfan, a new antagonist to label andvisualize cerebral histamine H3 receptors[END_REF]) and a 33 P-labelled riboprobe corresponding to a partial coding sequence (∼600 bp) of the rat H3R and detecting all the previously cloned functional isoforms of the receptor [START_REF] Morisset | The rat H3 receptor: gene organization andmultiple isoforms[END_REF]Héron et al., 2001). The detailed distribution that we have reported using [ 3 H](R)αmethylhistamine indicated already that the highest concentrations of the H3R were observed in the cerebral cortex and basal ganglia [START_REF] Pollard | A detailed autoradiographic mapping of histamine H3 receptors in rat brain areas[END_REF]. However, the relative H3R densities within cell layers of the cerebral cortex, cerebellum and hippocampus and in nuclei of thalamus, hypothalamus and brainstem could be determined with much more details with [ 125 I]iodoproxyfan (Figs. 3456), leading to the identification of neuronal populations expressing the H3R. Recent in situ hybridization studies have already reported a strong expression of H3R mRNAs not only within the TM, in which histaminergic perikarya are located [START_REF] Panula | The histaminergic neuronal system as revealedwith antisera against histamine[END_REF][START_REF] Tohyama | Morphology of histaminergic neurons with histidine decarboxylase as a marker[END_REF][START_REF] Wouterlood | Afferent and efferent fiber connections of histaminergic neurons in the rat brain: comparison with dopaminergic, noradrenergic and serotonergic systems[END_REF], but also in other regions of the rat brain [START_REF] Lovenberg | Cloning and functional expression of the human histamine H3 receptor[END_REF]Tardivel-Lacombe et al., 2000;[START_REF] Morisset | The rat H3 receptor: gene organization andmultiple isoforms[END_REF][START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF]. However, these studies were not as detailed as the present one, the signals obtained were not rated and some of these studies used oligoprobes for which an inherently lower sensitivity, due to a lower specific activity, is expected when compared to that of a riboprobe. The main finding of this study is that the localization of the H3R parallels that of its mRNAs in some areas, but that interesting discrepancies were observed in numerous other brain regions, revealing the expression of many H3Rs on neuronal dendrites and/or projections.

In the cerebral cortex, H3R mRNA expression is found in all layers but displays a laminar pattern which varies within the different areas (Table 1). Previous studies suggested that these mRNAs predominantly encode for isoforms with 445 and 397 amino acids [START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF][START_REF] Morisset | The rat H3 receptor: gene organization andmultiple isoforms[END_REF]. The strong mRNA labelling that we constantly observed at the cellular level in pyramidal cells of layer V (Fig. 7B-F ;[START_REF] Tardivel-Lacombe | Cloning andcerebral expression of the guinea pig histamine H3 receptor: evidence for two isoforms[END_REF], contrasts with the low binding found in this layer (except in the motor cortex). This suggests that a part of the dense binding in layers I-IV corresponds to H3Rs located upon apical dendrites of pyramidal cells present in layer V. Such an apical sorting of the H3R is supported by the intense H3R immunoreactivity recently reported within the soma and apical dendrites of layer V pyramidal cells in the mouse brain [START_REF] Chazot | Immunological identification of the mammalian H3 histamine receptor in the mouse brain[END_REF]. In addition, H3Rs may be located on granule cells, at least in layer IV, in which we easily detect mRNAs, particularly in the auditory cortex (Fig. 7E). In layer VI, the mRNA expression probably accounts for the binding in this layer (Fig. 7 and Table 1). All these findings indicate that H3Rs are present on various types of cortical neurons, in agreement with the strong decrease in cortical binding induced by quinolinic or kainic acid lesions [START_REF] Cumming | High affinity histamine binding is the H3 receptor: characterization andautorad iographic localization in rat brain[END_REF][START_REF] Pollard | A detailed autoradiographic mapping of histamine H3 receptors in rat brain areas[END_REF]. However, several of our findings are consistent with the expected presence of presynaptic H3Rs on afferents. In contrast with the moderate to strong expression previously reported in layer II with an oligoprobe [START_REF] Lovenberg | Cloning and functional expression of the human histamine H3 receptor[END_REF][START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF], a low mRNA expression was observed in this layer as well as in layer I with the riboprobe (Fig. 7B-F). The binding observed in these layers may therefore largely correspond to autoreceptors modulating histamine release from the dense network of histaminergic fibers innervating these layers [START_REF] Takeda | Origins of histamine-containing fibers in the cerebral cortex of rats studied by immunohistochemistry with histidine decarboxylase as a marker and transection[END_REF][START_REF] Steinbusch | Immunohistochemical localization of histamine in neurons and mast cells in the rat brain[END_REF][START_REF] Arrang | Autoregulation of histamine release in brain by presynaptic H3-receptors[END_REF] and to presynaptic heteroreceptors known to modulate noradrenaline and serotonin release in the cerebral cortex [START_REF] Schlicker | Modulation of neurotransmitter release via histamine H3 heteroreceptors[END_REF]. In addition, the strong mRNA expression (but very low binding) in thalamic sensorimotor nuclei and the dense binding in cortical layer IV, a major destination of glutamatergic thalamocortical projections, suggest that H3Rs are presynaptically located upon, and may regulate the activity of, thalamic relay neurons. All these cortical H3Rs may act in a synergic manner to regulate cortical activation known to be induced by histaminergic neurons, either directly by their ascending cortical projections, or indirectly via the thalamocortical system [START_REF] Lin | Brain structures andmechanisms involvedin the control of cortical activation andwakefulnes s, with emphasis on the posterior hypothalamus andhistaminergic neurons[END_REF]. Besides H3 auto-and heteroreceptors, H3Rs on cortical pyramidal neurons and thalamic relay neurons, that are both depolarized by histamine [START_REF] Steriade | Thalamocortical oscillations in the sleeping and aroused brain[END_REF][START_REF] Reiner | Mechanisms of antihistamine-induced sedation in the human brain: H1 receptor activation reduces a backgroundleakage potassium current[END_REF], may be involved in the arousal and improvement of attention induced by H3R antagonists (or inverse agonists) [START_REF] Lin | Involvement of histaminergic neurons in arousal mechanisms demonstrated with H3-receptor ligands in the cat[END_REF][START_REF] Ligneau | Neurochemical andbehavioral effects of ciproxifan, a potent histamine H3-receptor antagonist[END_REF].

In Ammon's Horn of the hippocampus, the binding is moderate and restricted to the CA1 field, in agreement with the low binding previously reported in this region [START_REF] Cumming | High affinity histamine binding is the H3 receptor: characterization andautorad iographic localization in rat brain[END_REF][START_REF] Pollard | A detailed autoradiographic mapping of histamine H3 receptors in rat brain areas[END_REF]. The small labelling of the pyramidal layer indicates the presence of some H3Rs on pyramidal cell perikarya. However, the very high mRNA expression in pyramidal layers of the CA1 and ventral CA3 fields, which may predominantly encode for the longer H3R isoform [START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF][START_REF] Morisset | The rat H3 receptor: gene organization andmultiple isoforms[END_REF], indicates that H3Rs are more abundant on the efferent projections of pyramidal cells. In agreement, a dense binding is observed within the target areas of the latter, i.e. the perirhinal cortex, tenia tecta, anterior olfactory nucleus, accumbens nucleus and anterior and dorsomedial hypothalamic nuclei. The binding in the stratum lacunosum moleculare of the CA1 field may represent receptors located upon afferents including perforant pathway fibers [START_REF] Amaral | The three-dimensional organization of the hippocampal formation: a review of anatomical data[END_REF]) and histaminergic nerve endings [START_REF] Panula | Histamine-immunoreactive nerve fibres in the rat brain[END_REF]. In the dentate gyrus, H3R mRNAs are found in the granule cell layer (Fig. 7A), whereas binding is restricted to the molecular layer (Table 1), suggesting the insertion of H3Rs in the dendrites of granule cells. It should be noted that H3Rs may be colocalized with H1 and H2 receptors in most, if not all, granule cells, since H1 and H2 receptor gene expression and binding occur with a similar pattern [START_REF] Traiffort | Guinea pig histamine H1 receptor. I. Gene cloning, characterization, andtissue distribution revealedby in situ hybridization[END_REF][START_REF] Vizuete | Detailed mapping of the histamine H2 receptor andits gene transcripts in guinea pig brain[END_REF]. However, whether H3Rs regulate the activity of granule cells and mossy fibers remains unknown. Whereas a prominent H3R immunoreactivity was detected in the granular and molecular layer of the CA3 field in the mouse hippocampus [START_REF] Chazot | Immunological identification of the mammalian H3 histamine receptor in the mouse brain[END_REF], we detect a very low binding, if any, in the CA3 field, including its stratum lucidum, which rather suggests that mossy fibers are not endowed with presynaptic H3Rs. A part of the binding in the molecular layer of the dentate gyrus is expected to represent autoreceptors on histaminergic projections [START_REF] Panula | Histamine-immunoreactive nerve fibres in the rat brain[END_REF] and heteroreceptors inhibiting glutamate release from the perforant path [START_REF] Brown | Histamine H3 receptor-mediated depression of synaptic transmission in the dendate gyrus of the rat in vitro[END_REF][START_REF] Brown | On the mechanism of histaminergic inhibition of glutamate release in the rat dentate gyrus[END_REF]. The latter is formed by axons of stellate and pyramidal cells of layers II and III of the entorhinal cortex, respectively, where we found a clear mRNA expression (Table 1).

In the basal ganglia, high densities of both H3R binding sites and mRNAs are found in the caudate putamen, nucleus accumbens and olfactory tubercles. High densities of H3R mRNAs were already reported in the striatum from various species including rat [START_REF] Lovenberg | Cloning and functional expression of the human histamine H3 receptor[END_REF], guinea-pig [START_REF] Tardivel-Lacombe | Cloning andcerebral expression of the guinea pig histamine H3 receptor: evidence for two isoforms[END_REF] and human [START_REF] Anichtchik | Distribution andmod ulation of histamine H3 receptors in basal ganglia and frontal cortex of healthy controls andpatients with Parkinson's disease[END_REF] and may predominantly encode for the shorter H3R isoform [START_REF] Morisset | The rat H3 receptor: gene organization andmultiple isoforms[END_REF][START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF]. Consistent with lesion experiments indicating that the vast majority of striatal H3Rs were present on intrinsic neurons [START_REF] Cumming | High affinity histamine binding is the H3 receptor: characterization andautorad iographic localization in rat brain[END_REF][START_REF] Pollard | A detailed autoradiographic mapping of histamine H3 receptors in rat brain areas[END_REF]Ryu et al., 1994aRyu et al., ,b, 1995[START_REF] Ryu | The effect of dopamine D1 receptor stimulation on the up-regulation of histamine H3-receptors following destruction of the ascending dopaminergic neurones[END_REF][START_REF] Anichtchik | Modulation of histamine H3 receptors in the brain of 6-hydroxydopamine-lesioned rats[END_REF], H3R mRNAs are densely and homogeneously distributed within the caudate putamen and nucleus accumbens when analyzed at the cellular level (Fig. 8A,B). This observation suggests that most H3Rs expressed in the striatal complex are present on GABAergic medium-sized spiny neurons which represent over 90% of striatal neuronal cells and project to the pallidum and substantia nigra [START_REF] Gerfen | The neostriatal mosaic: multiple levels of compartmental organization[END_REF][START_REF] Parent | Functional anatomy of the basal ganglia. I. The cortical-basal ganglia-thalamo-cortical loop[END_REF][START_REF] Kawaguchi | Neostriatal cell subtypes andtheir functional roles[END_REF]. Consistent with this hypothesis, the high densities of H3Rs found in the human striatum and globus pallidus [START_REF] Martinez-Mir | Three histamine receptors (H1, H2 andH 3) visualizedin the brain of human and non-human primates[END_REF][START_REF] Anichtchik | Distribution andmod ulation of histamine H3 receptors in basal ganglia and frontal cortex of healthy controls andpatients with Parkinson's disease[END_REF] are significantly lower in Huntington's disease [START_REF] Goodchild | Distribution of histamine H3-receptor binding in the normal human basal ganglia: comparison with Huntington's and Parkinson's disease cases[END_REF], which is characterized by degeneration of medium-sized, spiny projection neurons in the striatum [START_REF] Sharp | Neurobiology of Huntington's disease[END_REF]. However, the presence of H3Rs on other striatal elements cannot be ruled out. Recently, an intense bipolar H3R immunoreactivity was reported at the level of medium-sized neuronal cell somata, possibly indicating the presence of presynaptic H3Rs located on the inputs from the cholinergic interneurons [START_REF] Chazot | Immunological identification of the mammalian H3 histamine receptor in the mouse brain[END_REF]. Moreover, some binding sites are expected from functional studies to represent presynaptic H3Rs upon striatal afferents. H3autoreceptors are known to modulate histamine release from rat striatal slices [START_REF] Arrang | Autoregulation of histamine release in brain by presynaptic H3-receptors[END_REF]. The very high mRNA expression that we observe in cortical layer V may encode for presynaptic H3Rs regulating glutamate release from corticostriatal neurons [START_REF] Doreulee | Histamine H3 receptors depress synaptic transmission in the corticostriatal pathway[END_REF]. Although previous studies using an oligoprobe failed to detect any significant H3R mRNA expression within the dopaminergic neurons [START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF][START_REF] Anichtchik | Distribution andmod ulation of histamine H3 receptors in basal ganglia and frontal cortex of healthy controls andpatients with Parkinson's disease[END_REF], mRNA hybridization signals were clearly observed at the cellular level with our riboprobe in the substantia nigra pars compacta (Fig. 8E), suggesting that H3Rs previously reported to modulate dopamine synthesis [START_REF] Molina-Hernandez | Histamine H3-receptor activation inhibits dopamine synthesis in rat striatum[END_REF] and release [START_REF] Schlicker | Histamine inhibits dopamine release in the mouse striatum via presynaptic H3 receptors[END_REF] are presynaptically located on nigrostriatal afferents. However, the apparent absence of any signal in the ventral tegmental area (VTA) (Fig. 8F) shows that not all dopaminergic neurons express H3Rs. This observation is consistent with the previous finding that H3Rs may have no important role in the regulation of dopamine neuron activity in vivo [START_REF] Oishi | Is monoamine turnover in the brain regulated by histamine H3 receptors?[END_REF][START_REF] Imaizumi | The behavioral and biochemical effects of thioperamide, a histamine H3-receptor antagonist, in a light dark test measuring anxiety in mice[END_REF]. In addition, in contrast with the high expression observed in the caudate putamen and nucleus accumbens, the absence of mRNA signals in islands of Calleja suggests that neurons targeted by mesolimbic dopaminergic pathways in this projection area do not either express H3Rs.

Consistent with the hypothesis that H3Rs are present on spiny, striatal projection neurons, a dense binding was detected not only in the substantia nigra pars reticulata, but also in both external and internal segments of the globus pallidus. In addition, this binding was always accompanied by a low mRNA expression which contrasted with the prominent mRNA expression recently reported in the human globus pallidus [START_REF] Anichtchik | Distribution andmod ulation of histamine H3 receptors in basal ganglia and frontal cortex of healthy controls andpatients with Parkinson's disease[END_REF]. This finding is consistent with the H3R-mediated inhibition of [ 3 H]GABA release in rat substantia nigra pars reticulata [START_REF] Garcia | Histamine H3 receptor activation selectively inhibits dopamine D1 receptor-dependent [3H]-Q-aminobutyric acidrelease from depolarisation-stimulatedslices of rat substantia nigra pars reticulata[END_REF] and suggests the presence of H3Rs presynaptically located not only on striatonigral, but also on striatopallidal projections of both the 'direct' and 'indirect' movement pathways. In addition, the very low mRNA expression in the pallidum and the very low binding in the subthalamic nucleus and motor thalamic nuclei, the target structures of external and internal pallidum, respectively (Tables 1 and3), reveal a very low density of H3Rs on pallidal efferents. However, the activity of the 'indirect' movement pathway may also be modulated by presynaptic H3Rs present on excitatory projections from the subthalamic nucleus to the substantia nigra, two regions that express mRNAs and very dense binding, respectively.

In the amygdaloid complex, where no data were yet available, the high mRNA expression that we observe suggests that not all binding sites correspond to autoreceptors, in spite of a dense histaminergic innervation [START_REF] Panula | The histaminergic neuronal system as revealedwith antisera against histamine[END_REF][START_REF] Wouterlood | Afferent and efferent fiber connections of histaminergic neurons in the rat brain: comparison with dopaminergic, noradrenergic and serotonergic systems[END_REF][START_REF] Tohyama | Morphology of histaminergic neurons with histidine decarboxylase as a marker[END_REF], and that a major part is located upon amygdala projections. In addition to its role in emotional learning, amygdala plays a key role in the coordinated regulation of attentional processes via projections to cholinergic systems [START_REF] Gallagher | The amygdala complex: multiple roles in associative learning and attention[END_REF][START_REF] Davis | Neurotransmission in the rat amygdala related to fear and anxiety[END_REF]. The latter, located in the forebrain and mesopontine tegmentum, are activated by histamine [START_REF] Khateb | Cholinergic nucleus basalis neurons are excitedby histamine in vitro[END_REF][START_REF] Lin | Histaminergic descending inputs to the mesopontine tegmentum and their role in the control of cortical activation andwakefulnes s in the cat[END_REF] and are involved in the maintenance of cortical activation and wakefulness by histaminergic neurons [START_REF] Lin | Brain structures andmechanisms involvedin the control of cortical activation andwakefulnes s, with emphasis on the posterior hypothalamus andhistaminergic neurons[END_REF]. H3Rs on both amygdala and hypothalamic projections to cholinergic neurons may therefore be involved in the arousal and attention induced by H3R inverse agonists [START_REF] Ligneau | Neurochemical andbehavioral effects of ciproxifan, a potent histamine H3-receptor antagonist[END_REF]Morisset et al., 2000a) and may account for the binding observed in the substantia innominata, pontine nuclei (Tables 2 and5) and lateral dorsal tegmentum (Cumming et al., 1994).

In the thalamus, the heterogeneous binding that we described using [ 3 H](R)α-methylhistamine [START_REF] Pollard | A detailed autoradiographic mapping of histamine H3 receptors in rat brain areas[END_REF] is highlighted with [ 125 I]iodoproxyfan which allows a much more detailed analysis. Although the distribution of binding sites roughly parallels histaminergic innervation [START_REF] Steinbusch | Immunohistochemical localization of histamine in neurons and mast cells in the rat brain[END_REF][START_REF] Inagaki | Organization of histaminergic fibres in the rat brain[END_REF][START_REF] Panula | Histamine-immunoreactive nerve fibres in the rat brain[END_REF], strong discrepancies in most nuclei between a low binding and very high mRNA expression reveal that many H3Rs are located upon projections such as the thalamocortical projections (Table 3). However, the binding in dorsal association nuclei may correspond to presynaptic H3Rs located upon corticothalamic neurons emanating from cortical layers V or VI, in which we detect a high mRNA expression (Fig. 6). In addition, the dense binding in the laterodorsal and lateroposterior nuclei suggests the involvement of H3Rs in visual systems. This hypothesis is further strengthened by the presence of binding sites in the main retinal primary projection areas, e.g. the superior colliculus and dorsal lateral geniculate nucleus (Tables 3 and5), that are dramatically up-regulated following visual deprivation [START_REF] Nakagawa | Markedincrease in[END_REF].

In the hypothalamus, the highest mRNA expression was found in the TM where it was suggested to encode for all isoforms of the receptor [START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF]. We also confirm that this expression is accompanied by a high density of binding sites [START_REF] Pollard | A detailed autoradiographic mapping of histamine H3 receptors in rat brain areas[END_REF]. Some of these sites seem to be located upon the histaminergic perikarya, since histamine synthesis is regulated in the posterior hypothalamus (Arrang et al., 1987b) and somatodendritic H3 autoreceptors inhibit the firing of TM neurons [START_REF] Haas | Electrophysiology of histamine receptors[END_REF][START_REF] Diewald | Histaminergic system in co-cultures of hippocampus andposterior hypothalamus: a morphological andelectrophysiolog ical study in the rat[END_REF] by modulating high voltage-activated calcium channels [START_REF] Takeshita | Histamine modulates high-voltage-activated calcium channels in neurons dissociated from the rat tuberomammillary nucleus[END_REF]. However, it is very likely that a significant fraction of the binding that we observe in the TM is located on afferent connections. In agreement, mRNA expression is rather high in several brain areas projecting to the TM, e.g. the subiculum, septum-diagonal band complex, preoptic region, lateral hypothalamus, and raphe nuclei [START_REF] Ericson | Brainstem afferents to the tuberomammillary nucleus in the rat brain with special reference to monoaminergic innervation[END_REF][START_REF] Ericson | Origin of neuronal inputs to the region of the tuberomammillary nucleus of the rat brain[END_REF][START_REF] Wouterlood | Afferent and efferent fiber connections of histaminergic neurons in the rat brain: comparison with dopaminergic, noradrenergic and serotonergic systems[END_REF] (Tables 1, 2, 4 and5). However, further studies are needed to know if mRNAs that we detected in the ventrolateral preoptic nucleus and lateral hypothalamic area encode for H3Rs regulating the activity of sleep-active GABAergic [START_REF] Sherin | Activation of ventrolateral preoptic neurons during sleep[END_REF][START_REF] Sherin | Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat[END_REF]Yang and Hatton, 1997) and orexin-containing [START_REF] Chemelli | Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation[END_REF][START_REF] Lin | The sleep disorder canine narcolepsy is causedby a mutation in the hypocretin (orexin) receptor 2 gene[END_REF][START_REF] Lu | Differential distribution andregulation of OX1 andOX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting[END_REF] projections, respectively, two major inputs to the TM involved in the control of arousal [START_REF] Schwartz | Histamine[END_REF]. The mRNA expression level in the other hypothalamic nuclei parallels the density of binding sites, suggesting the presence of H3Rs on perikarya, dendrites and/or short axons. However, many of these nuclei exhibit a very dense histaminergic innervation and it is therefore hard to predict the presynaptic and/or postsynaptic localization of hypothalamic H3Rs. For example, the rather high mRNA expression suggests that the binding sites that we detect in the supraoptic and paraventricular nuclei correspond to H3Rs coexpressed in magnocellular neurons with H1Rs involved in the regulation by histamine of vasopressin and oxytocin secretion [START_REF] Kjaer | Dehydration stimulates hypothalamic gene expression of histamine synthesis enzyme: importance for neuroendocrine regulation of vasopressin and oxytocin secretion[END_REF][START_REF] Luckman | Evidence for the involvement of histaminergic neurones in the regulation of the rat oxytocinergic system during pregnancy and parturition[END_REF]. However, these binding sites may also correspond to presynaptic autoreceptors known to regulate the activation of these magnocellular neurons by endogenous histamine [START_REF] Vizuete | Endogenous histamine induces c-fos expression within paraventricular andsupraoptic nuclei[END_REF]. The function and cellular distribution of the receptors corresponding to the dense binding and strong mRNA expression that we detect in several nuclei of the tuberal region, such as the ventromedial nucleus, requires also further investigation.

In the lower brainstem, strong discrepancies are observed between the distributions of the H3R and its mRNAs (Table 5). The densest histaminergic innervation is found in the nucleus of the solitary tract [START_REF] Inagaki | Organization of histaminergic fibres in the rat brain[END_REF] where we detect a fairly high mRNA expression but very low binding which contrasts with the rather dense binding previously reported with a radiolabelled agonist (Cumming et al., 1994). Histaminergic fibers also innervate vestibular nuclei, particularly the medial vestibular nucleus [START_REF] Takeda | Histaminergic projection from the posterior hypothalamus to the medial vestibular nucleus of rats and its relation to motion sickness[END_REF][START_REF] Steinbusch | Distribution of histaminergic neurons and fibers in rat brain. Comparison with noradrenergic and serotonergic innervation of the vestibular system[END_REF]. In agreement with the very low binding (Table 5), previous studies suggested the presence of H3Rs only on these projections and not on vestibular perikarya themselves [START_REF] Serafin | Medial vestibular nucleus in the guinea-pig: histaminergic receptors. I. An in vitro study[END_REF][START_REF] Yabe | Medial vestibular nucleus in the guinea-pig: histaminergic receptors. II. An in vivo study[END_REF][START_REF] Wang | Effects of histamine andbetahistine on rat medial vestibular nucleus neurones: possible mechanism of action of anti-histaminergic drugs in vertigo and motion sickness[END_REF]. However, the high level of mRNAs that we observe in the vestibular nuclei, including the medial vestibular nucleus, suggests the presence of H3Rs on their projections. Besides autoreceptors, these H3Rs may explain that systemic administrations of H3R antagonists or inverse agonists strongly decrease the horizontal vestibular ocular reflex in the guinea-pig [START_REF] Yabe | Medial vestibular nucleus in the guinea-pig: histaminergic receptors. II. An in vivo study[END_REF] and facilitate vestibular compensation in the cat [START_REF] Tighilet | Betahistine dihydrochloride treatment facilitates vestibular compensation in the cat[END_REF], thereby suggesting the potential interest of these compounds as anti-vertigo or anti-motion sickness drugs. The high mRNA expression in the locus coeruleus and raphe nuclei is consistent with previous findings and was recently suggested to encode for the shorter isoforms of the receptor [START_REF] Lovenberg | Cloning and functional expression of the human histamine H3 receptor[END_REF][START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF]. However, the very low binding observed in both areas (Table 5) suggests that presynaptic rather than somatodendritic heteroreceptors are involved in the H3Rmediated inhibition of noradrenaline and serotonin release, respectively [START_REF] Schlicker | Modulation of neurotransmitter release via histamine H3 heteroreceptors[END_REF]. In addition, the rather high mRNA expression in the facial nuclei (Table 5) may encode for H3Rs present on the chorda tympani nerve, a branch of the facial nerve which is involved in the activation of histaminergic neurons by leptin [START_REF] Morimoto-Ishizuka | The role of the chorda tympani nerve in the activation of the rat hypothalamic histaminergic system by leptin[END_REF].

In the cerebellum, hybridization performed at the cellular level shows a strong mRNA expression not only in most, if not all, Purkinje cells, but also in several central cerebellar nuclei, suggesting an important functional role of H3Rs in this brain region. The signals observed in Purkinje cells may correspond to the shorter isoform [START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF] and accounts for the pronounced immunoreactivity detected in Purkinje cell bodies [START_REF] Chazot | Immunological identification of the mammalian H3 histamine receptor in the mouse brain[END_REF]. The hybridization signals in the granule cells were hardly detectable, making doubtful the high level of expression reported in these cells with an oligoprobe [START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF]. However, the high mRNA expression that we observed in the guinea-pig [START_REF] Tardivel-Lacombe | Cloning andcerebral expression of the guinea pig histamine H3 receptor: evidence for two isoforms[END_REF] and low H3R immunoreactivity reported in the mouse [START_REF] Chazot | Immunological identification of the mammalian H3 histamine receptor in the mouse brain[END_REF] suggest that the H3R expression in cerebellar granule cells varies among animal species. The very low binding, if any, that we observe in the cerebellar cortex suggests that H3Rs present on Purkinje cells are expressed at the level of their efferent projections rather than on their soma or dendrites. In addition, although they express the three gene transcripts, H3Rs may not colocalize with H1 and H2 receptors on Purkinje cells, since a high density of H1 and H2 receptors [START_REF] Traiffort | Guinea pig histamine H1 receptor. I. Gene cloning, characterization, andtissue distribution revealedby in situ hybridization[END_REF][START_REF] Vizuete | Detailed mapping of the histamine H2 receptor andits gene transcripts in guinea pig brain[END_REF], but a low H3R binding and immunoreactivity [START_REF] Chazot | Immunological identification of the mammalian H3 histamine receptor in the mouse brain[END_REF], are observed in the molecular layer which contains Purkinje cell dendrites.

Conclusion

The present study provides evidence for the expression of H3Rs upon many neuronal cell types. If some localizations are consistent with previous functional studies, the physiological role of many of the receptors present on neuronal perikarya, dendrites and/or projections remains to be understood. Moreover, many of these localizations do not parallel the histaminergic innervation, and the question then arises as to how these receptors are activated. We showed recently that native H3Rs in rodent brain display high constitutive activity [START_REF] Morisset | High constitutive activity of native H3 receptors regulates histamine neurons in brain[END_REF]. It can be therefore hypothesized that the receptors present upon the various neuronal populations described in this study, may be spontaneously active in the absence of histamine. In addition, as already mentioned, the various H3R isoforms are recognized by [ 125 I]iodoproxyfan and the riboprobe used in this study [START_REF] Ligneau | Distinct pharmacology of rat andhuman histamine H3 receptors: role of two amino acids in the third transmembrane domain[END_REF][START_REF] Morisset | The rat H3 receptor: gene organization andmultiple isoforms[END_REF] but the expression pattern and relative ratios of their transcripts vary among brain regions [START_REF] Morisset | The rat H3 receptor: gene organization andmultiple isoforms[END_REF][START_REF] Drutel | Identification of rat H3 receptor isoforms with different brain expression and signaling properties[END_REF]. When available, selective probes for the corresponding proteins will allow to investigate the distributions of these H3R isoforms and thus to identify further the neuronal populations in which they are expressed.

Fig. 1 .

 1 Fig. 1. Distribution of H3R ([ 125 I]iodoproxyfan) binding sites and H3R mRNAs in rat brain. The autoradiographic signals were measured as the mean optical density (OD) of pixels obtained with an image analyzer in the whole brain region, including all layers and/or nuclei. Results are means±S.E.M. of the values from four animals (4-10 sections per animal).

  Fig. 2. Distribution of H3R ([ 125 I]iodoproxyfan) binding sites and H3R gene transcripts in rat brain sagittal sections. Sections were incubated with 15 pM [ 125 I]iodoproxyfan alone (total binding, B) or in the presence of 1 µM (R)α-methylhistamine (non-specific binding, A). The laterality of the sections is 2.4 mm (Paxinos and Watson, 1998) (C). Sections from the same level of separate animals were hybridized with 33 P-labelled antisense (D) or sense (E) riboprobes.

Fig. 3 .

 3 Fig. 3. Autoradiographic distribution of [ 125 I]iodoproxyfan binding sites and H3R gene transcripts on serial frontal sections of the rat brain. H3R binding sites (left) and mRNAs (right) were visualized on sections using [ 125 I]iodoproxyfan alone or a 33 P-labelled antisense riboprobe, respectively. The brain structures were identified at the corresponding anteriorities indicated by the bregma distance in mm (reproduced with permission from Paxinos and Watson, Academic Press, 1998).

Fig. 4 .

 4 Fig. 4. Autoradiographic distribution of [ 125 I]iodoproxyfan binding sites and H3R gene transcripts on serial frontal sections of the rat brain. H3R binding sites (left) and mRNAs (right) were visualized on sections using [ 125 I]iodoproxyfan alone or a 33 P-labelled antisense riboprobe, respectively. The brain structures were identified at the corresponding anteriorities indicated by the bregma distance in mm (reproduced with permission from Paxinos and Watson, Academic Press, 1998).

Fig. 5 .

 5 Fig. 5. Autoradiographic distribution of [ 125 I]iodoproxyfan binding sites and H3R gene transcripts on serial frontal sections of the rat brain. H3R binding sites (left) and mRNAs (right) were visualized on sections using [ 125 I]iodoproxyfan alone or a 33 P-labelled antisense riboprobe, respectively. The brain structures were identified at the corresponding anteriorities indicated by the bregma distance in mm (reproduced with permission from Paxinos and Watson, Academic Press, 1998).

Fig. 6 .

 6 Fig. 6. Autoradiographic distribution of [ 125 I]iodoproxyfan binding sites and H3R gene transcripts on serial frontal sections of the rat brain. H3R binding sites (left) and mRNAs (right) were visualized on sections using [ 125 I]iodoproxyfan alone or a 33 P-labelled antisense riboprobe, respectively. The brain structures were identified at the corresponding anteriorities indicated by the bregma distance in mm (reproduced with permission from Paxinos and Watson, Academic Press, 1998).

Fig. 7 .

 7 Fig. 7. Cellular localization of H3R gene transcripts within the hippocampus and cerebral cortex as revealed by in situ hybridization using a photographic emulsion (dark field photomicrographs). (A) localization of H3R mRNAs in the hippocampus. The arrows indicate the pyramidal cell layer of the CA1 field and the granule cell layer of the dentate gyrus (GrDG). (B-F) Localization of H3R mRNAs in the cerebral cortex (B, primary motor cortex; C, secondary motor cortex; D, somatosensory cortex; E, auditory cortex; F, visual cortex). The cortical layers, identified on a Nissl-stained adjacent section, are indicated on the lefthand side.

Fig. 8 .

 8 Fig. 8. Cellular localization of H3R gene transcripts in the basal ganglia. Localization of H3R mRNAs within the caudate putamen (A), nucleus accumbens (B), islands of Calleja (C, D), substantia nigra pars compacta (E) and ventral tegmental area (F), as revealed by in situ hybridization using a photographic emulsion (dark field photomicrographs).

Fig. 9 .

 9 Fig. 9. Cellular localization of H3R gene transcripts in the hypothalamus and caudal areas. Localization of H3R mRNAs in the TM (A), ventromedial and dorsomedial hypothalamic nuclei (B), locus coeruleus (C), cerebellar cortex (D), central cerebellar nuclei (E) and medial vestibular nucleus (F).

Table 1 .

 1 Distribution of [ 125 I]iodoproxyfan binding sites and H3R mRNAs in the rat cerebral cortex and

	hippocampus
	Brain structures	[ 125 I]iodoproxyfan binding sites	H3R mRNAs
	I. Neocortex	Cortical layers	Cortical layers
	Primary motor cortex (M1)	I,II,III,V: 4+, VI: 3+	I,II,III: 1-2+, V: 4+, VI: 2+
	Secondary motor cortex (M2)	I,II: 2-3+, III: 4+, V: 3+, VI: 2+	I,II: 1-2+, III: 4+, V: 3+, VI: 2+
	Somatosensory cortex (S1, S2)	I,II,III,IV: 3-4+, V: 1-2+, VI: 2-3+ I,II,III,IV: 1-2+, V: 4+, VI: 3+
	Auditory cortex (Au1)	I,II,III,IV: 4+, V: 2+, VI: 3+	I,II,III: 1-2+, IV,V: 4+, VI: 3+
	Visual cortex (V1, V2)	I,II,III,IV: 3-4+, V: 2+, VI: 3-4+	I,II,III,IV: 1-2+, V: 4+, VI: 2+
	II. Allo-and periallocortices
	Prefrontal:
	Cingulate cortex (Cg1, Cg2)	3-4+	4+ (I,II: 2+)
	Agranular insular cortex (AI)	3-4+	4+
	Orbitofrontal cortex (LO, MO, VLO, VO) 3-4+	4+
	Retrosplenial cortex (RSA, RSGa,b)	1-2+	3+ (I,II: 1-2+)
	Perirhinal cortex (PRh)	3-4+	3-4+
	Piriform cortex (Pir)	2+	4+
	III. Hippocampic formation
	Ammon's Horn
	Stratum oriens (so)	0	0
	Pyramidal cell layer	CA1: 1+, CA2/CA3: 0	CA1: 4+, CA2: 1+, CA3ventral: 3+
	Stratum lucidum (sl) of CA3	0-1+	0
	Stratum radiatum (sr)	0	0
	Stratum lacunosum moleculare (s l-m)	2+ (CA1)	0-1+ (CA1)
	Dentate gyrus
	Granular layer (GrDG)	0	2+
	Molecular layer (ml)	2+	0
	Polymorph layer (PoDG)	0	1+
	Subiculum (S)	2+ (molecular layer)	3+ (pyramidal cell layer)
	Entorhinal cortex (Ent)	3-4+	I: 4+, II,III,IV: 2+, V,VI: 4+
	Tenia tecta (TT)	3-4+	4+

Thalamic midline and intralaminar nuclei

Paraventricular nucleus (PV, PVA, PVP)

Table 4 .

 4 Distribution of [ 125 I]iodoproxyfan binding sites and H3R mRNAs in the rat hypothalamus

	Brain structures	[ 125 I]iodoproxyfan binding sites	H3R mRNAs
	Preoptic region
	Periventricular zone
	Periventricular nucleus (Pe)	1+	1+
	Vascular organ of the lamina terminalis (VOLT)	1+	1+
	Median preoptic nucleus (MnPO)	1+	1+
	Anteroventral periventricular nucleus (AVPe)	1+	1+
	Medial zone
	Medial preoptic area (MPA)	2+	2+
	Medial preoptic nucleus (MPO)	2+	3-4+
	Anterodorsal preoptic nucleus (ADP)	1+	1+
	Parastrial nucleus (PS)	1+	1+
	Lateral zone
	Lateral preoptic nucleus (LPO)	1+	2+
	Magnocellular preoptic nucleus (MCPO)	0-1+	2+
	Ventrolateral preoptic nucleus (VLPO)	1+	1+
	Anterior region
	Periventricular zone
	Suprachiasmatic nucleus (SCh)	1+	1+
	Paraventricular nucleus (PaAP)	2+	3+
	Medial zone
	Anterior hypothalamic area (AH)	1+	1+
	Anterior area, anterior part (AHA)	2+	3+
	Anterior area, central part (AHC)	1+	1-2+
	Anterior area, posterior part (AHP)	1+	1-2+
	Lateral zone
	Lateral hypothalamic area (LH)	1+	1-2+
	Supraoptic nucleus (SO)	1-2+	1-2+
	Tuberal region
	Periventricular zone
	Arcuate nucleus (Arc)	1-2+	1+
	Medial zone
	Tuber cinereum area (TC)	1+	1-2+
	Ventromedial nucleus (VMH)	2-3+	3-4+
	Dorsomedial nucleus (DM)	1-2+	2+

Table 5 .

 5 Distribution of [ 125 I]iodoproxyfan binding sites and H3R mRNAs in the rat caudal areas