The EIGER detector for low-energy electron microscopy and photoemission electron microscopy.

EIGER is a single-photon-counting hybrid pixel detector developed at the Paul Scherrer Institut, Switzerland. It is designed for applications at synchrotron light sources with photon energies above 5 keV. Features of EIGER include a small pixel size (75 µm × 75 µm), a high frame rate (up to 23 kHz), a small dead-time between frames (down to 3 µs) and a dynamic range up to 32-bit. In this article, the use of EIGER as a detector for electrons in low-energy electron microscopy (LEEM) and photoemission electron microscopy (PEEM) is reported. It is demonstrated that, with only a minimal modification to the sensitive part of the detector, EIGER is able to detect electrons emitted or reflected by the sample and accelerated to 8-20 keV. The imaging capabilities are shown to be superior to the standard microchannel plate detector for these types of applications. This is due to the much higher signal-to-noise ratio, better homogeneity and improved dynamic range. In addition, the operation of the EIGER detector is not affected by radiation damage from electrons in the present energy range and guarantees more stable performance over time. To benchmark the detector capabilities, LEEM experiments are performed on selected surfaces and the magnetic and electronic properties of individual iron nanoparticles with sizes ranging from 8 to 22 nm are detected using the PEEM endstation at the Surface/Interface Microscopy (SIM) beamline of the Swiss Light Source.

The expression and function of Ca(2+)-sensing receptors in rat mesenteric artery; comparative studies using a model of type II diabetes.

BACKGROUND AND PURPOSE: The extracellular calcium-sensing receptor (CaR) in vascular endothelial cells activates endothelial intermediate-conductance, calcium-sensitive K(+) channels (IK(Ca)) indirectly leading to myocyte hyperpolarization. We determined whether CaR expression and function was modified in a rat model of type II diabetes. EXPERIMENTAL APPROACH: Pressure myography, western blotting, sharp microelectrode and K(+)-selective electrode recordings were used to investigate the functional expression of the CaR and IK(Ca) in rat mesenteric arteries. KEY RESULTS: Myocyte hyperpolarization to the CaR activator calindol was inhibited by Calhex 231. U46619-induced vessel contraction elevated the extracellular [K(+)] around the myocytes, and inhibition of this 'K(+) cloud' by iberiotoxin was needed to reveal calindol-induced vasodilatations. These were antagonized by Calhex 231 and significantly smaller in Zucker diabetic fatty rat (ZDF) vessels than in Zucker lean (ZL) controls. Myocyte hyperpolarizations to calindol were also smaller in ZDF than in ZL arteries. In ZDF vessels, endothelial cell CaR protein expression was reduced; IK(Ca) expression was also diminished, but IK(Ca)-generated hyperpolarizations mediated by 1-EBIO were unaffected. CONCLUSIONS AND IMPLICATIONS: The reduced CaR-mediated hyperpolarizing and vasodilator responses in ZDF arteries result from a decrease in CaR expression, rather than from a modification of IK(Ca) channels. Detection of CaR-mediated vasodilatation required the presence of iberiotoxin, suggesting a CaR contribution to vascular diameter, that is, inversely related to the degree of vasoconstriction. Compromise of the CaR pathway would favour the long-term development of a higher basal vascular tone and could contribute to the vascular complications associated with type II diabetes.

Molecular characterization of the family of choline transporter-like proteins and their splice variants.

We show here that the choline transporter-like (CTL) family is more extensive than initially described with five genes in humans and complex alternative splicing. In adult rat tissues, CTL2-4 mRNAs are mainly detected in peripheral tissues, while CTL1 is widely expressed throughout the nervous system. During rat post-natal development, CTL1 is expressed in several subpopulations of neurones and in the white matter, where its spatio-temporal distribution profile recalls that of myelin basic protein, an oligodendrocyte marker. We identified two major rat splice variants of CTL1 (CTL1a and CTL1b) differing in their carboxy-terminal tails with both able to increase choline transport after transfection in neuroblastoma cells. In the developing brain, CTL1a is expressed in both neurones and oligodendroglial cells, whereas CTL1b is restricted to oligodendroglial cells. These findings suggest specific roles for CTL1 splice variants in both neuronal and oligodendrocyte physiology.

Hedgehog interacting protein in the mature brain: membrane-associated and soluble forms.

Hedgehog interacting protein (Hip) is considered as a membrane protein implicated in sequestering the hedgehog (hh) morphogens during embryonic development. Here, we demonstrate that Hip transcription also occurs in cells scattered in discrete brain areas of adult rodents and we identify the presence of membrane-associated and soluble forms of Hip in the mature brain. Moreover, we show that soluble forms of Hip, present in the conditioned medium of HEK293 cells overexpressing Hip, inhibit Sonic hedgehog (Shh)-induced differentiation of C3H10T1/2 cells, a well-characterised response associated with Shh signalling. After transfection in HEK293 cells, Hip partitions with the raft component ganglioside GM1 during density gradient centrifugation. Analysis of tagged Hip constructs reveals that the putative transmembrane domain of Hip is not cleaved suggesting that other mechanisms are implicated in the release of its soluble forms. Taken together, these data are consistent with the involvement of both membrane-associated and soluble Hip in the regulation of Shh signalling in adult neural tissues.

Intrastriatal sonic hedgehog injection increases Patched transcript levels in the adult rat subventricular zone.

The morphogen sonic hedgehog (Shh) is implicated in neural tissue patterning and the growth of brain structures during embryogenesis and postnatal development and is also present in the adult brain. Shh signals through interaction with the tumour suppressor Patched (Ptc). This receptor for Shh is associated with Smoothened (Smo), a protein with high homology to the G-protein coupled receptors. However, little is known about the transduction mechanisms implicated in Shh signalling in the adult brain. The study described here shows that injection of aminoterminal myristoylated Shh (myrShhN) into the adult rat striatum robustly increases the levels of Ptc transcripts in selective brain areas including the subventricular zone (SVZ). The adult SVZ contains cell progenitors, which can proliferate and differentiate into new neurons and glia. In the myrShhN injected animals, proliferation and differentiation of these SVZ precursor cells were not affected as demonstrated by BrdU incorporation and immunohistochemistry performed with specific antibodies for nestin (uncommitted neural progenitors), PSA-NCAM (migrating neuroblasts) or GFAP (astrocytes). Together with the presence of Smo expressing cells and amino-terminal Shh (ShhN) protein in SVZ area of untreated animals, the data presented here supports the hypothesis that the Shh pathway may be activated in the adult brain, and that a niche for Shh signalling exists within the adult SVZ.

High expression and anterograde axonal transport of aminoterminal sonic hedgehog in the adult hamster brain.

Sonic hedgehog (SHH) is considered to play an important role in tissue induction and patterning during development, particularly in determining neuronal cell fate in the ventral neural tube and in the embryonic forebrain. SHH precursor is autoproteolytically cleaved to an aminoterminal fragment (SHHN) which retains all known SHH biological activities. Here, we demonstrate the expression of a 22-kDa SHHN immunoreactive peptide in developing and adult hamster brain regions using a rabbit antiserum directed against a mouse SHHN fragment. Interestingly, SHHN was developmentally regulated with the highest expression observed in the adult brain, was resistant to Triton X-100 solubilization at 4 degrees C and partitioned with the raft component ganglioside GM1 during density gradient centrifugation. In rat brain, Shh transcripts were identified by double in situ hybridization in GABAergic neurons located in various basal forebrain nuclei including globus pallidus, ventral pallidum, medial septum-diagonal band complex, magnocellular preoptic nucleus and in cerebellar Purkinje cells as well as in motoneurons of several cranial nerve nuclei and of the spinal cord. We show that radiolabelled SHHN peptides are synthesized in the adult hamster retina and are transported axonally along the optic nerve to the superior colliculus in vivo. Our data indicate that SHHN is associated with cholesterol rich raft-like microdomains and anterogradely transported in the adult brain, and suggest that the roles of this extracellular protein are more diverse than originally thought.

Multiple subcellular localizations of PCTAIRE-1 in brain.

We developed a selective antibody to a synthetic peptide corresponding to an N-terminal sequence of the PCTAIRE-1 protein. In rodent brain extracts it recognized only the protein doublet characteristic of PCTAIRE-1, and this signal is completely abolished by preincubation of the antibody with the immunopeptide. Immunolabeling experiments done with this PCTAIRE-1-specific antibody reveal that the protein is widely distributed in the rodent brain as are the mRNAs visualized using an antisense riboprobe corresponding to the entire PCTAIRE-1 open reading frame. Two types of PCTAIRE-1 protein localizations were observed: first a diffuse labeling of almost all brain regions, particularly intense in the molecular layer of the cerebellum and the mossy fiber region of the hippocampus, and second a spot-like localization in the nuclei of large neurons such as cerebellar Purkinje cells and pyramidal cells of the hippocampus. Colocalization with the B23 protein allows one to identify these compartments as nucleoli. Our results suggest a nucleolar function of PCTAIRE-1 in large neurons and a role in regions containing important granule cell projections.

Distribution of bone morphogenetic protein and bone morphogenetic protein receptor transcripts in the rodent nervous system and up-regulation of bone morphogenetic protein receptor type II in hippocampal dentate gyrus in a rat model of global cerebral ischemia.

Bone morphogenetic proteins belong to the transforming growth factor-beta superfamily and act through serine/threonine kinase type I and type II receptors such as bone morphogenetic protein receptor type I and type II. In order to further understand the roles that these factors exert in the nervous system, we have examined the expression pattern of seven bone morphogenetic proteins and bone morphogenetic protein receptor type I and II transcripts in the brain and spinal cord of rodent. Whereas bone morphogenetic protein receptor type I expression was low in rat brain, in situ hybridization studies performed with specific digoxigenin-labelled riboprobes revealed the presence of bone morphogenetic protein receptor type II-positive cells throughout the brain, with a notable localization in dopaminergic cells of the substantia nigra. Bone morphogenetic protein receptor type II transcripts were also expressed by large motoneuron-like cells located in the ventral horn of the spinal cord and by sensory neurons of dorsal root ganglia. In addition, we observed a significant up-regulation of bone morphogenetic protein receptor type II in the granule cells of the dentate gyrus 48 h after transient global cerebral ischemia in rat suggesting that modulation of this receptor intervenes during neuronal plasticity or repair that occur upon brain injury. Among the potential ligands for this receptor, bone morphogenetic protein-6 and bone morphogenetic protein-7 were expressed in meninges and the choroid plexus, while bone morphogenetic protein-4-expressing cells were spatially and temporally regulated in myelinated structures during development and in the adult suggesting its expression in oligodendrocytes. These data clearly indicate that besides their roles in bone and embryonic tissues, bone morphogenetic proteins and their receptors may have also important functions in adult neural tissues.

N1-Arylsulfonyl-N2-(1-aryl)ethyl-3-phenylpropane-1,2-diamines as novel calcimimetics acting on the calcium sensing receptor.

The synthesis and calcimimetic properties of N1-arylsulfonyl-N2-(1-aryl)ethyl-3-phenylpropane-1,2-diamines are described. The most active compound of the series (3n, used at 10 microM) produced 97+/-11% of the maximal stimulation of [3H]IP production obtained by 10 mM Ca2+ in CHO cells expressing the calcium sensing receptor (CaSR). This calcimimetic activity was due to a specific interaction of this compound with the CaSR.

Developmental and adult expression of rat calcium-sensing receptor transcripts in neurons and oligodendrocytes.

The calcium-sensing receptor (CaSR) is a member of a growing family of heptahelical receptors with an unusually large extracellular domain. To further delineate its functions in neurons and glia, we have investigated the expression pattern of CaSR transcripts in the postnatal and adult rat brain, spinal cord and dorsal root ganglia by in situ hybridization. CaSR-expressing cells were spatially and temporally regulated in myelinated structures with a caudo-rostral pattern that paralleled that of myelin basic protein, a marker of myelination, with a downregulation observed in the adult. Double-labelling studies demonstrated that CaSR mRNA colocalizes with myelin basic protein-expressing cells within fibre tracts, suggesting that CaSR is expressed by mature oligodendrocytes. In cultured rat oligodendrocytes, Ca2+ induced stimulation of phosphatidylinositol hydrolysis with an EC50 of 1.4 mM and increased intracellular calcium. NPS R-568 (1 microM), a calcimimetic, significantly stimulates the inositol phosphate response, whereas a less potent stereoisomer, NPS S-568 (1 microM), was without effect. These data suggest that a functional CaSR is expressed in mature oligodendrocytes with a potential role in myelination. CaSR expression was also developmentally regulated in neurons of the orbital cortex and in the CA2 region of the hippocampus, and present in olfactory nuclei, hypothalamic areas and in the area postrema through postnatal days to adulthood. This expression is consistent with a role of CaSR in olfactory or gustatory signal integration, and with the regulation of fluid and mineral homeostasis. CaSR expression in a subpopulation of small cells in dorsal root ganglia suggests additional roles for extracellular Ca2+ in sensory nerves.

An electric lobe suppressor for a yeast choline transport mutation belongs to a new family of transporter-like proteins.

Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3. 5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2-4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

Discrete localizations of hedgehog signalling components in the developing and adult rat nervous system.

Sonic hedgehog (Shh), a morphogen molecule implicated in embryonic tissue patterning, displays inductive, proliferative, neurotrophic and neuroprotective activities on various neural cells. Shh might exert its biological functions through binding to patched (Ptc) associated with smoothened (Smo), leading to downstream activation of target genes such as the transcription factor Gli1. We have performed a detailed localization of cells expressing transcripts of Shh, Ptc, Smo and Gli1 in brain and spinal cord of the adult rat as well as in the developing cerebellum. In the adult, Shh-positive cells were mainly observed in forebrain structures, in the Purkinje cells of the cerebellum and in motor neurons. Ptc-positive cells were frequently observed in brain areas devoid of any Shh transcripts, except in the median eminence or the facial nucleus, suggesting local Shh signalling. Interestingly, Smo transcripts were predominantly present within circumventricular organs, in granular cells of the dentate gyrus and in neurons of the reticular thalamic nucleus. The presence of Shh, Ptc and Smo transcripts in hypothalamic areas may indicate a role of Shh signalling in the modulation of neuroendocrine functions. The expression pattern of these three genes as well as of Gli1, and their developmental regulation in the cerebellum, suggest a possible role for Hedgehog signalling in the control of various cell populations within the cerebellum, particularly in granule cell proliferation and/or differentiation that might be impaired in proliferative states such as medulloblastomas.

Two novel odorant receptor families expressed in spermatids undergo 5'-splicing.

We report the identification of two novel families of odorant receptor (OdR)-like proteins, termed spermatid chemoreceptors (SCRs), in rat spermatids of the testis. The full-length genomic clones encode seven transmembrane domain receptors that share 35-40% identity with certain OdRs and are among the most divergent members of the OdR superfamily based on phylogenetic analysis. RNase protection assays and in situ hybridization studies confirmed the expression of SCRs in spermatids, the post-meiotic, differentiating cell population in the testis. SCR transcripts were undetectable in the prepubertal testis but were readily identified in spermatids of sexually maturing and mature testis. Rapid amplification of cDNA end-polymerase chain reaction and genomic clone sequencing led to the discovery that SCRs are spliced upstream of their presumptive starting methionines. 5'-Splicing of OdRs may regulate the expression of functional chemoreceptors.

Characterization of 5-ht6 receptor and expression of 5-ht6 mRNA in the rat brain during ontogenetic development.

We have determined the pharmacological characteristics of the rat 5-ht6 receptor stably expressed in CHO cells. Moreover, using RT-PCR experiments the in vivo expression of the gene encoding this receptor was studied in rat at various embryonic days (ED) starting from ED10 to birth (PN0) and at post-natal days (PN) up to PN36. The pharmacological analysis of the [3H]5-HT binding in stably transfected CHO cells expressing rat 5-ht6 receptors revealed the presence of a single class of high affinity saturable binding sites for 5-HT corresponding to an affinity constant: Kd=27.2+/-3.4 nM. This receptor also exhibited a high affinity for a number of typical and atypical antipsychotics, tricyclic antidepressant drugs and ergot alkaloïds. In stably transfected CHO cells, serotonin elicited a potent stimulation of adenylyl cyclase activity which was blocked by antipsychotic and antidepressant drugs. These results confirm the hypothesis that 5-ht6 receptors may correspond to an important target for atypical antipsychotics and reveal an original pharmacological profile for this receptor. The study of the ontogeny of the 5-ht6 mRNA in rat developing brain showed that 5-ht6 mRNA were first detectable with a high level on ED12, slighly decreased up to ED17 and then remained stable at high level until the adult age. The ontogenetic pattern of 5-ht6 mRNA expression appeared to correlate with the occurence of the first cell bodies of serotonergic neurons; the early expression of 5-ht6 mRNA and the fact that this receptor is positively coupled to the production of cAMP may suggest a role for 5-ht6 receptor in the early growth process involving the serotonergic system.

Diamine oxidase induces neurite outgrowth in chick dorsal root ganglia by a nonenzymatic mechanism.

The enzyme diamine oxidase (DAO) catalyzes the oxidative deamination of histamine, diamines, and polyamines. DAO has been localized to several tissues, including thymus, kidney, intestine, seminal vesicles, placenta, and pregnancy plasma. DAO is not constitutively expressed in the mammalian brain, but it becomes detectable following focal injury. Although the physiologic role of DAO remains unknown, the observation that it is present at the interface between rapidly dividing and quiescent cells in several tissues suggests that it might be involved in regulating cell division or differentiation at tissue boundaries. In addition, the observation that DAO is expressed in the brain following injury suggests that the protein might play a role in the CNS response to focal neuronal damage. To test that hypothesis, we assessed the ability of purified DAO to alter the pattern of neuronal differentiation and nerve growth in vitro. In chick dorsal root ganglion explant cultures, purified porcine DAO induced neurite outgrowth in the low nanomolar range. Addition of aminoguanidine, which inhibits DAO enzyme activity, did not inhibit the protein's neurotrophic activity. These findings suggest that DAO can function as a neurotrophic ligand independent of its enzymatic activity.

Regional distribution of Sonic Hedgehog, patched, and smoothened mRNA in the adult rat brain.

In vertebrates, Sonic Hedgehog (Shh), Desert Hedgehog (Dhh), and Indian Hedgehog (Ihh) genes encode a family of morphogen proteins that are implicated in a wide range of signaling activities, particularly during embryonic development. These secreted proteins are proposed to mediate their effects on target cells by interacting with their putative receptor, Patched (Ptc), and with a seven-pass transmembrane protein, Smoothened (Smo). However, the roles that these signaling molecules may play in adult tissues, particularly in brain, are not yet clearly defined. Therefore, we investigated the expression of these genes in adult rat tissues. Northern blot analysis revealed expression of Shh, Dhh, and Ihh genes in peripheral tissues, whereas Shh transcript was also identified in brain. It is interesting that northern blot analysis with probes derived from the mouse Ptc and Smo genes revealed the expression of a 7.9-kb and a 3.7-kb transcript, respectively, in all brain tissues examined. In situ hybridization experiments using specific digoxigenin-labeled riboprobes showed expression of Ptc and Smo transcripts in discrete brain areas. Shh-positive cells were observed in restricted regions of the brain. Within the cerebellum, Shh, Ptc, and Smo transcripts were colocalized in the Purkinje cell layer. These data suggest that, besides its roles in determining cell fate and patterning during embryogenesis, the hedgehog signaling pathway may have also important roles in the adult brain.

Effects of divalent cations and of a calcimimetic on adrenocorticotropic hormone release in pituitary tumor cells.

The calcium sensing receptor (CaSR), a member of the G-protein coupled receptor family, is expressed on a variety of cell types and responds to extracellular calcium. We have characterized pharmacological properties of (+/-)NPS 568, a calcimimetic, toward cloned rat brain extracellular Ca2+-sensing receptor (CaSR) expressed in Chinese hamster ovary (CHO) cells and constitutive mouse CaSR in AtT-20 cells. In the presence of 1.3 mM Ca2+, the calcimimetic displayed a potency in the micromolar range in augmenting the inositol phosphates (IP) response in both cell lines and behaved as a full agonist. (+/-)NPS 568 stimulated formation of arachidonic acid release in CHO(CaSR) with a similar potency. The IP dose response curves of (+/-)NPS 568 were shifted to the left in the presence of increasing Ca2+, indicating that the potency of the drug is dependent on extracellular Ca2+ in both cells. In AtT-20 cells, Ca2+ and Ba2+, two CaSR agonists, induced a potent stimulation of adrenocorticotropic hormone (ACTH) secretion. In the presence of 1.8 mM Ca2+, (+/-)NPS 568 led to a dose dependent secretion of ACTH with an EC50 of 0.3 microM and a maximal effect comparable to Ca2+. The similar potency of the calcimimetic on IP and ACTH responses and the sensitivity of these responses to extracellular Ca2+ indicate that the Ca2+-sensing receptor expressed in AtT-20 cells is implicated in ACTH release. These data further characterize the pharmacology of the Ca2+-sensing receptor and argue for a role for extracellular Ca2+ and CaSRs in controlling ACTH secretion, a hormone implicated in several types of stress.

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.

Stimulation of cell proliferation by calcium and a calcimimetic compound.

Some mesenchymal cells respond to stimulation by specific cations with increased cell proliferation. In the present study we have investigated whether the parathyroid/kidney/brain calcium-sensing receptor (PCaR) can mediate such mitogenic responses. We have expressed the recombinant rat PCaR in CCL39 hamster fibroblasts, which do not express a detectable endogenous cation sensor. The transfected cells responded to increased extracellular calcium concentrations ([Ca2+]e) with strong inositol phosphate (IP) formation, which was insensitive to pertussis toxin treatment of cells. We could not detect negative coupling of the receptor to adenylyl cyclase. The calcimimetic NPS R-568 left-shifted the concentration-response curve for [Ca2+]e-induced IP formation and increased the maximal response. In [3H]thymidine incorporation experiments, increasing [Ca2+]e from 1 to 4 mM was found to stimulate DNA synthesis weakly, but significantly. A strong potentiation of this response was observed in the presence of NPS R-568. [Ca2+]e and NPS R-568 also synergized to increase cell numbers in cultures maintained in defined medium. In contrast to our expectations, no significant stimulation of IP formation or cell proliferation could be observed after stimulation of cells with the reported PCaR agonist gadolinium (Gd3+) or with aluminum (Al3+), which stimulates osteoblast proliferation. Gd3+ actually inhibited IP formation stimulated by increased [Ca2+]e as well as by thrombin and AlF4-, indicating toxicity. However, submaximal receptor stimulation by Gd3+ was evident when intracellular calcium transients were measured in fluo-3-loaded cells. Our data show that PCaR can stimulate cell proliferation when expressed in an appropriate cellular context. However, it is unlikely that PCaR mediates the strong mitogenic effects elicited by the cations Gd3+ and Al3+ observed in osteoblasts.

Quantitative RT-PCR distribution of serotonin 5-HT6 receptor mRNA in the central nervous system of control or 5,7-dihydroxytryptamine-treated rats.

Possible adaptive changes of the recently cloned serotonin 5-HT6 receptor after the selective lesion of serotoninergic neurons by an intracerebral administration of 5,7-dihydroxytryptamine were investigated using competitive RT-PCR (reverse transcription followed by polymerase chain reaction) for the measurement of 5-HT6-mRNA in various areas of the rat central nervous system. In control rats, 5-HT6-mRNA was the most abundant in the nucleus accumbens, followed by the olfactory tubercle and the striatum. High levels of 5-HT6-mRNA were also found in the hypothalamus and the hippocampus, whereas the cerebral cortex, the substantia nigra, and the spinal cord contained moderate levels of the transcript. Low but easily quantifiable levels of 5-HT6-mRNA were measured in the ventral tegmental area, the anterior raphe area, and the cerebellum. In addition, moderate to low levels of this mRNA were also found in dorsal root ganglia and the pituitary gland. Three weeks after the microinfusion of 5,7-dihydroxytryptamine into the anteroventral vicinity of the dorsal raphe nucleus in nomifensine-pretreated rats, the levels of serotonin transporter-mRNA were reduced by 90% in the anterior raphe area, as expected of the extensive lesion of serotoninergic neurons. In contrast, quantitative determinations of the 5-HT6-mRNA in this area as well as in the nucleus accumbens, the striatum, and the hippocampus indicated that its levels were not significantly different in 5,7-dihydroxytryptamine-treated rats and in controls. These data showed that the 5-HT6 receptor: 1) is not an autoreceptor, and 2) exhibits probably no up regulation in postsynaptic target cells after the selective degeneration of serotoninergic projections.