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

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 [(125)I]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 non-radioactive 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 [(125)I]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 [(125)I]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.

Localization and function of the D3 dopamine receptor.

A novel dopamine receptor has been recently cloned, which differs from D1 and D2 receptors by its sequence, localization, pharmacology and possibly signalling system, hence its designation as D3 receptor. The D3 receptor cDNA was obtained by a combination of screenings of DNA libraries and PCR polymerase chain reaction experiments. It encodes a protein with a predicted structure consisting in 7 transmembrane domains indicating that it belongs to the G-protein coupled receptor family. Its global homology is 52% with the D2 receptor but 78% if only transmembrane domains are considered. Two shorter transcript variants, in addition to the full-length cDNA were detected by PCR in various rat brain regions. The shorter transcripts are generated by alternative splicing and encode two putative proteins respectively interrupted after the second transmembrane domain and lacking the second extracytoplasmic loop. After transfection of this latter isoform into cultured cells, no dopaminergic activity could be detected. These shorter splice variants may regulate the number of active D3 receptors. The human D3 receptor was also cloned using similar approaches with rat D3 receptor cDNA probes and was found highly homologous to the rat receptor, except in the third intracytoplasmic loop. The human D3 receptor gene was assigned to the chromosome 3 at q13.3 band. Visualisation of D3 receptor mRNA in rat brain by in situ hybridization indicated a predominant expression of the message in the ventral striatum and other "limbic" areas. There is no overlap in the distributions of D2 and D3 receptor mRNAs in discrete structures, suggesting that the two transcripts are expressed by different cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA.

The messenger RNA (mRNA) of the recently characterized D3 dopamine receptor was visualized on rat brain sections using in situ hybridization with a 32P-labeled ribonucleic acid probe corresponding to a major part of the third cytoplasmic loop, a domain in which D2 and D3 dopamine receptors display little homology. For the purpose of comparison, D2 receptor mRNA was also specifically visualized on adjacent sections. The areas that expressed D2 and/or D3 receptors were also compared with those previously detected using [125I]iodosulpride, a ligand that binds to both D2 and D3 receptors with a similar affinity. The localization of D3 receptor mRNa markedly differs from that of D2 receptor mRNA. Whereas D2 receptor mRNA is expressed in all major brain areas receiving dopaminergic projections, particularly in the whole striatal complex, D3 receptor mRNA is expressed in a more restricted manner. It is mainly detected in telencephalic areas receiving dopaminergic inputs from the A10 cell group, e.g. accumbens nucleus, islands of Calleja, bed nucleus of the stria terminalis and other limbic areas such as the hippocampus and the mammillary nuclei. D2 and D3 receptor mRNAs were also detected at the level of the substantia nigra, suggesting that these receptors function as both autoreceptor and postsynaptic receptors. In several dopaminergic projection areas, e.g. ventral straitum, septal or mammillary nuclei, the distributions of D2 and D3 receptor mRNAs appeared complementary without overlap. The distribution of [125I]iodosulpride binding sites generally overlapped that of D2 or D3 receptor mRNAs, the latter being most abundant in dopaminergic areas known to be associated with cognitive and emotional functions.

Reversible and irreversible labelling of H1- and H2 -receptors using novel [125I] probes.

We have recently designed the first 125I-labelled probes specific for the histamine H1 and H2 receptors. These reversible and irreversible antagonists are among the most potent H1 and H2 ligands and have enabled investigations into the biochemical and pharmacological properties of these two receptors. In various brain animal species, the ligand binding peptide of the H1 and H2 receptors, as determined by photoaffinity labeling, resides within 56-59 kDa peptides. In contrast, in guinea pig heart, the ligand binding domain of the H1 receptor is characterized by a higher molecular weight (68 kDa), suggesting the presence of an isoform of this protein, clearly differentiable by this biochemical property but not by its pharmacology. The reversible 125I-probes allowed us to extend the pharmacology of these receptors in several biological preparations and in human brain, and to establish their interaction with G-proteins. A detailed mapping of H1 and, for the first time, of H2 receptors, has been achieved in guinea pig brain, establishing their presence in almost all brain areas. These experiments show that there is no correlation between the density of H2 receptor and the activity of adenylate cyclase sensitive to histamine suggesting a molecular heterogeneity of this receptor.

Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics.

A dopamine receptor has been characterized which differs in its pharmacology and signalling system from the D1 or D2 receptor and represents both an autoreceptor and a postsynaptic receptor. The D3 receptor is localized to limbic areas of the brain, which are associated with cognitive, emotional and endocrine functions. It seems to mediate some of the effects of antipsychotic drugs and drugs used against Parkinson's disease, that were previously thought to interact only with D2 receptors.

Histamine H1-receptor in heart: unique electrophoretic mobility and autoradiographic localization.

Histamine H1-receptors, visualized in the guinea pig heart by autoradiography using [125I]iodobolpyramine as a specific probe, are abundant in the nodal tissue and cardiac vessels but also occur heterogeneously in the myocardium. Following photoaffinity labeling with [125I]iodoazidophenpyramine and electrophoresis, the ligand binding domain of the heart H1-receptor was shown to be present on a major 68-kDa and a less abundant 54- to 58-kDa protein. The 68-kDa protein displayed a molecular size higher in heart than in all other tissues (56 kDa). This indicates the existence of at least two isoforms of the H1-receptor; the cardiac isoform, however, was pharmacologically indistinguishable from the common isoform studied in cerebellar membranes using available ligands. Its distinct electrophoretic properties suggest that the cardiac isoform may have a unique function.

Reversible and irreversible labeling and autoradiographic localization of the cerebral histamine H2 receptor using [125I]iodinated probes.

Iodoaminopotentidine (I-APT)--i.e., N-[2-(4-amino-3-iodobenzamido)ethyl]-N'-cyano-N''-(3-[3- (1-piperidinylmethyl)phenoxy]propyl)guanidine--represents one of the most potent H2-receptor antagonists known so far. In membranes of guinea pig brain 125I-APT bound reversibly, selectively, and with high affinity (Kd = 0.3 nM) to a homogeneous population of sites unambiguously identified as H2 receptors by inhibition studies conducted with a large panel of antagonists. 125I-APT binding was also inhibited by histamine, and the effect was modulated by a guanyl nucleotide, which is consistent with the association of the H2 receptor with a guanine nucleotide binding regulatory protein. The low nonspecific binding of 125I-APT generated high contrast autoradiographic pictures in brain sections and established the precise distribution of H2 receptors. Their highly heterogeneous distribution and laminated pattern in some areas--e.g., cerebral and hippocampal cortices--suggest their major association with neuronal elements. These localizations were more consistent than those of H1 receptors with the distribution of histaminergic projections, indicating that H2 receptors mediate a larger number of postsynaptic actions of histamine--e.g., in striatum. Colocalizations of H1 and H2 receptors in some areas account for their known synergistic interactions in cAMP formation induced by histamine. The distribution of 125I-APT binding sites did not strictly parallel that of the H2-receptor-linked adenylate cyclase activity, which may reflect heterogeneity among H2 receptors. After UV irradiation and SDS/PAGE analysis, [125I]iodoazidopotentidine (125I-AZPT), a photoaffinity probe derived from 125I-APT, was covalently incorporated in several peptides, among which the labeling of two peptides of 59 and 32 kDa was prevented by H2 antagonists, suggesting that they correspond to H2-receptor binding peptides or proteolysis products of the latter. These probes should be useful for sensitive radioassays, localization, purification, and molecular studies of the H2 receptor, which were previously impracticable.

Ontogeny of dopaminergic D-2 receptors in the rat nervous system: characterization and detailed autoradiographic mapping with [125I]iodosulpride.

[125I]Iodosulpride, a highly selective and sensitive probe for dopamine D-2 receptors, was used to study the expression of these receptors in binding studies performed on membranes and serial autoradiographic sections, throughout pre- and postnatal developmental periods. D-2 receptors were first detected autoradiographically in sensory and sympathetic ganglia at the embryonic age of 12 days, i.e. much earlier than in previous studies. In membrane binding studies, D-2 receptors were found to be modulated by guanylnucleotides as early as at embryonic day 15, suggesting that they were already functionally coupled to a regulatory G protein. The overall development of D-2 receptors in the central nervous system occurred according to a caudorostral gradient and was accompanied by a slightly but significantly increased affinity for dopamine, possibly related to the late expression of a D-2 receptor subclass. The ontogeny of D-2 receptors was compared to that of tyrosine hydroxylase immunoreactivity as reported by others and taken as an index of dopaminergic innervation. Despite some variations due to experimental conditions, this comparison resulted in the definition of various situations. In some major projection areas (e.g. caudate putamen at embryonic day 14) there was a simultaneous appearance of both dopaminergic markers whereas in most others (e.g. n. accumbens or olfactory tubercles at embryonic day 20) the appearance of D-2 receptors was preceded by 1-4 days by that of tyrosine hydroxylase immunoreactivity. However, in a few projection areas (e.g. the bed nucleus of the stria terminalis at embryonic day 21), D-2 receptors appeared 3-4 days earlier than tyrosine hydroxylase immunoreactivity. In areas of dopaminergic perikarya, e.g. substantia nigra and ventral tegmental area, where they largely correspond to somatodendritic autoreceptors, D-2 receptors appeared at embryonic days 17 and 21 respectively, i.e. 3-8 days after tyrosine hydroxylase immunoreactivity, suggesting that dopamine synthesis and release is not feedback regulated by autoreceptors at initial developmental stages. In areas where D-2 receptors are present in the absence of any established dopaminergic innervation (e.g. discrete layers of the hippocampus, cerebellum, parietal cortex or in cranial nerve nuclei), they generally appeared at a late stage, i.e. during the second or even the third postnatal week. Finally, there was transient and roughly concomitant expression of both D-2 receptors and tyrosine hydroxylase immunoreactivity in some areas such as spinal ganglia or the lateral ventricle floor, consistent with a possible development function of dopamine mediated by D-2 receptors.

Detailed immunoautoradiographic mapping of enkephalinase (EC 3.4.24.11) in rat central nervous system: comparison with enkephalins and substance P.

The metallopeptidase enkephalinase known to participate in the inactivation of endogenous enkephalins and, possibly, other neuropeptides such as tachykinins, was visualized by autoradiography using a [125I]iodinated monoclonal antibody. A detailed mapping of the enzyme in rat brain and spinal cord was established on 10-micron serial sections prepared in a frontal plane as well as a few sections in a sagittal plane. On adjacent sections, and for the purpose of comparison, substance P-like and enkephalin-like immunoreactivities were also visualized by autoradiography using a 125I-monoclonal antibody and a polyclonal antibody detected by a secondary 125I-anti-rabbit antibody respectively. Histological structures were identified on adjacent Nissl-stained sections. Using the highly sensitive 125I-probe, enkephalinase immunoreactivity was found to be distributed in a markedly heterogeneous manner in all areas of the central nervous system. Immunoreactivity was undetectable in white matter areas, for example the corpus callosum or fornix, and had a laminar pattern in, for example, the cerebral cortex or hippocampal formation. Hence, although immunodetection was not performed at the cellular level, a major neuronal localization of the peptidase is suggested. The latter is consistent with the detection of a strong immunoreactivity in a pathway linking the striatum to the globus pallidum, the entopeduncular nucleus and the substantia nigra, as well as with a series of biochemical and lesion data. The strong immunoreactivity also present in choroid plexuses and ependymal cells as well as in the intermediate lobe and in scattered cells of the anterior lobe of the pituitary suggests that populations of glial and endocrine cells also express the peptidase. The highest density of enkephalinase immunoreactivity was observed in basal ganglia and limbic areas (caudate putamen, globus pallidus, nucleus accumbens, olfactory tubercles) as well as in areas involved in pain control mechanisms (superficial layers of the spinal nucleus of the trigeminal nerve or of the dorsal horn of the spinal cord) which also display the highest immunoreactivities for both enkephalins and substance P (except in globus pallidus for the latter). These localizations account for the opioid-like analgesic and motor effects of enkephalinase inhibitors inasmuch as a selective or predominant participation of the peptidase in enkephalin inactivation is assumed. A number of other areas appear richly endowed in both enkephalinase and enkephalins whereas substance P is hardly detectable. This is particularly the case for the olfactory bulb, bed nucleus of the accessory olfactory tract, the cerebellum (where enkephalinase mainly occurs in the molecular layer) and the hippocampal formation (namely in the molecular layer of the dentate gyrus).(ABSTRACT TRUNCATED AT 400 WORDS)

A detailed mapping of histamine H1-receptors in guinea-pig central nervous system established by autoradiography with [125I]iodobolpyramine.

[125I]Iodobolpyramine, a potent and selective histamine H1-receptor antagonist derived from mepyramine, was used to generate light microscopic autoradiograms on sections of guinea-pig brain and spinal cord. Histamine H1-receptors were labelled with high sensitivity over a low background as determined using mianserin or other H1-receptor antagonists as competing agents. An atlas of H1-receptors was established using five sagittal sections and 39 frontal sections, the latter serially prepared at 50 micron intervals. Labelled areas were identified by comparison with corresponding, classically stained sections and their density was rated according to an arbitrary scale. Autoradiographic grains were detected in a large variety of gray matter areas whereas they were generally absent from white matter areas. In the cerebral cortex, H1-receptors are present in all areas and layers with a higher density in lamina IV. In the hippocampal formation, H1-receptors display a laminated pattern of distribution and are the most abundant in the dentate gyrus (hilus and molecular layer) and in several areas of the subiculum and commissural complex. In the amygdaloid complex, the highest densities are found in the medial group of nuclei. In the basal forebrain, the striatum is moderately labelled whereas the nucleus accumbens, islands of Calleja and most septal nuclei are highly labelled. In the thalamus, H1-receptors are present in high density, particularly in the anterior, median and lateral groups of nuclei. In the hypothalamus the labelling is highly heterogeneous with high densities in, for example, medial preoptic area, dorsomedial, ventromedial and most posterior nuclei, including the tuberomammillary complex in which histaminergic perikarya and short axons are present.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation of two probes for the localisation of enkephalinase in rat brain: [3H]thiorphan and a 125I-labeled monoclonal antibody.

We studied the binding of two radioactive probes, i.e. [3H]thiorphan and a 125I-labeled monoclonal antibody raised against the rabbit kidney enzyme, to enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase) from rat cerebral membranes. [3H]Thiorphan binding at equilibrium to striatal membranes was monophasic with a KD (0.7 nM) and a pharmacology consistent with a selective labeling of the enzyme. The ratio of Vmax/Bmax was in the same range as the Kcat of the enzyme purified from peripheral tissues. The monoclonal antibody immunoprecipitated to a similar extent the solubilised enkephalinase activity and [3H]thiorphan binding sites from striatum. The regional distributions of binding sites for the two probes established either on isolated membranes or autoradiographic sections were highly heterogeneous and similar to that of enkephalinase activity. Hence the two probes appear to label membrane-bound enkephalinase in rat brain but, from a technical point of a view, the 125I-monoclonal antibody is a more sensitive and flexible tool.

A detailed mapping of dopamine D-2 receptors in rat central nervous system by autoradiography with [125I]iodosulpride.

The benzamide derivative [125I]iodosulpride was used to generate light microscopic autoradiograms on sections of rat brain and spinal cord. Sites specifically labelled by [125I]iodosulpride over a low background correspond to dopamine D-2 receptors as shown by their pharmacology established by densitometric analysis of 11 typical areas from autoradiograms generated in the presence of five dopamine-competing agents. An atlas of D-2 receptors was established using 1 horizontal, 6 sagittal and 30 frontal sections, the latter serially prepared at 0.5-1 mm intervals. Labelled areas were identified by comparison with corresponding, classically stained sections. When their density, rated according to an arbitrary scale, was then compared to that previously reported for dopamine innervation, evaluated from distributional maps of dopamine histofluorescence or tyrosine hydroxylase immunoreactivity, three situations were found. In areas corresponding to cells of origin and established projection fields of the mesostriatal, mesolimbocortical, diencephalospinal and periglomerular systems the density of D-2 receptors generally paralleled that of dopamine innervation. D-2 receptors in substantia nigra (pars compacta or reticulata) and ventral tegmental area were strongly reduced after injections of the neurotoxin 6-hydroxydopamine into the medial forebrain bundle, suggesting their major localization on dendrites and perikarya of dopamine neurons. Most other described dopamine cell group areas also contained D-2 receptors. In contrast many areas without established dopamine innervation contained D-2 receptors, sometimes in high density. This was the case for large areas of the cerebral cortex (layers I-III and V-VI) outside the established projection fields of the mesocortical system, the cerebellum (moleculare layer and dense patches within lobule 9), the hippocampal formation (lacunosum moleculare layer), several septal, thalamic and hypothalamic nuclei, large tectal areas, numerous brainstem areas (including cranial nerve nuclei), etc. This situation might correspond to areas with minor and still undetected dopamine innervation or to a localization of D-2 receptors on cells (or cell parts) not receiving dopamine inputs. Finally several well-established dopaminergic areas did not reveal any D-2 receptor labelling. This was particularly the case in the hypothalamus (areas of origin or termination of the tuberohypophyseal and incertohypothalamic dopamine systems) but also in the hippocampal formation (alveus, fimbria, hilus dentate gyrus), amygdaloid complex (anterior, basolateral, medial nuclei).(ABSTRACT TRUNCATED AT 400 WORDS)

[125I]Iodobolpyramine, a highly sensitive probe for histamine H1-receptors in guinea-pig brain.

[125I]Iodobolpyramine is a novel 125I-ligand for histamine H1-receptors, synthesised using the 125I-Bolton Hunter reagent (2000 Ci/mmol) for acylation of an aminopentyl analogue of mepyramine. Its specific binding varied linearly with the concentration of guinea-pig cerebellar membranes and represented about 80% of the total. Selective interaction with H1-receptors was demonstrated by estimation of Ki values of known agonists and antagonists and confirmed by the low affinity of histamine H2- and H3-receptor antagonists and of non-histaminergic agents. At 25 degrees C, [125I]iodobolpyramine exhibited a slow association rate (180-240 min to reach equilibrium) and a slow dissociation rate (t1/2 = 201 min). Kinetic and saturation data yielded KD values of 0.05 and 0.15 nM, respectively, indicating that it is among the most potent H1-receptor antagonists known. The sensitivity for detecting H1-receptors in guinea-pig cerebellum using [125I]iodobolpyramine was increased 50-fold relative to use of [3H]mepyramine. Well-contrasted autoradiograms of guinea-pig brain, obtained after a short exposure time, confirmed previous H1-receptor localisation established with [3H]mepyramine and revealed new localisations, e.g. in cerebral cortex and nucleus accumbens.

Localisation and pharmacological characterisation of D-2 dopamine receptors in rat cerebral neocortex and cerebellum using [125I]iodosulpride.

Dopamine D-2 receptors were characterised in membranes from all areas of rat cerebral cortex and from cerebellum tested, by using [125I]iodosulpride, a highly selective ligand. The IC50 values of a large variety of dopaminergic and non-dopaminergic agents against [125I]iodosulpride binding in parietal cortex or cerebellum were highly correlated with their IC50 values against [125I]iodosulpride (or [3H]domperidone) binding in striatum. Moreover in the presence of a guanylnucleotide, 5'-guanylylimidodiphosphate (GppNHp), the dopamine inhibition curves of [125I]iodosulpride binding were shifted similarly to the right in the three regions. The density of D-2 receptors in cerebellum and in most areas of the cerebral cortex represented about 1% and 2%, respectively, of their density in striatum whereas the corresponding value in frontal cortex was 4%. Labelling on autoradiographic sections was localised to superficial (I-III) and deep layers (V) of the parietal cortex and to the molecular layer of the cerebellum.

Autoradiographic localisation of 3H-apomorphine binding sites in rat brain.

The best experimental conditions for a selective binding of 3H-apomorphine to dopamine receptors on cryostat sections were first selected by liquid scintillation quantification of the bound radioactivity. In the corpus striatum, a specific binding occurred with a half-maximal saturation concentration of about 1 nM and a maximal capacity of 180 fmol/mg of slice protein, both values in agreement with previous binding data on either membranes or slices incubated in a physiological medium. Inhibition with domperidone was clearly biphasic, indicating two classes of sites corresponding to the D-2 and D-3 sites as previously defined on membranes. When 3H-apomorphine was used at low concentrations (0.8-1.5 nM), a condition ensuring a preferential labelling of D-2 sites, rather well contrasted autoradiographic pictures were generated. The major dopaminergic projection fields in telencephalon (caudate-putamen, nucleus accumbens, olfactory tubercles) were visualised as well as other catecholaminergic regions such as the superficial gray layer of superior colliculi. Within the striatum, differences in density of these sites were observed in three perpendicular planes and confirmed by a computer densitometric image analysis. Labelling of areas of origin of the cerebral dopaminergic neurons in substantia nigra or ventral tegmental area were also observed. When a higher concentration of 3H-apomorphine (3.5 nM) was used in the presence of domperidone, another, but autoradiographically less distinct subclass of sites (D-3 sites) was demonstrated.