Histamine is required for H3 receptor-mediated alcohol reward inhibition, but not for alcohol consumption or stimulation.

BACKGROUND AND PURPOSE: Conflicting data have been published on whether histamine is inhibitory to the rewarding effects of abused drugs. The purpose of this study was to clarify the role of neuronal histamine and, in particular, H3 receptors in alcohol dependence-related behaviours, which represent the addictive effects of alcohol. EXPERIMENTAL APPROACH: Alcohol-induced conditioned place preference (alcohol-CPP) was used to measure alcohol reward. Alcohol-induced locomotor stimulation, alcohol consumption and kinetics were also assessed. mRNA levels were quantified using radioactive in situ hybridization. KEY RESULTS: Low doses of H3 receptor antagonists, JNJ-10181457 and JNJ-39220675, inhibited alcohol reward in wild-type (WT) mice. However, these H3 receptor antagonists did not inhibit alcohol reward in histidine decarboxylase knock-out (HDC KO) mice and a lack of histamine did not alter alcohol consumption. Thus H3 receptor antagonists inhibited alcohol reward in a histamine-dependent manner. Furthermore, WT and HDC KO mice were similarly stimulated by alcohol. The expression levels of dopamine D1 and D2 receptors, STEP61 and DARPP-32 mRNA in striatal subregions were unaltered in HDC KO mice. No differences were seen in alcohol kinetics in HDC KO compared to WT control animals. In addition, JNJ-39220675 had no effect on alcohol kinetics in WT mice. CONCLUSIONS AND IMPLICATIONS: These data suggest that histamine is required for the H3 receptor-mediated inhibition of alcohol-CPP and support the hypothesis that the brain histaminergic system has an inhibitory role in alcohol reward. Increasing neuronal histamine release via H3 receptor blockade could therefore be a novel way of treating alcohol dependence.

Expression of histamine receptor genes Hrh3 and Hrh4 in rat brain endothelial cells.

BACKGROUND AND PURPOSE: Brain vascular endothelial cells express histamine H1 and H2 receptors, which regulate brain capillary permeability. We investigated whether H3 and H4 receptors are also expressed in these cells and may thus play a role in permeability regulation. EXPERIMENTAL APPROACH: An immortalized rat brain endothelial cell line RBE4 was used to assess the presence of H3 and H4 receptors. Reverse transcription-PCR (RT-PCR) and sequencing were used to identify the receptor mRNAs. The receptors were stimulated with histamine and immepip, and specific inverse agonists/antagonists ciproxifan and JNJ 7777120 were used to block H3 and H4 receptors, respectively. KEY RESULTS: RT-PCR of mRNA extracted from cultured immortalized RBE4 cells revealed two rat H4 receptor gene (Hrh4) transcripts, one full-length (coding sequence 1173 bp), and one with a 164 bp deletion. Also, two rat H3 receptor gene (Hrh3) isoform mRNAs were expressed in RBE4 cells, and sequencing showed they were the full-length H3 receptor and the 144 bp deletion form. Both histamine and immepip (H3 and H4 receptor agonists) activated the Erk1/2 MAPK pathway in the RBE4 cells and in vivo in brain blood vessels by activating H4 receptors, as the H4 receptor-specific inverse agonists/antagonist JNJ 7777120, but not ciproxifan, H3 receptor antagonist, dose-dependently blocked this effect in RBE4 cells. CONCLUSIONS AND IMPLICATIONS: Both Hrh3 and Hrh4 receptors are expressed in rat brain endothelial cells, and activation of the histamine H4 receptor activates the Erk1/2 cascade. H3 and H4 receptors in endothelial cells are potentially important for regulation of blood-brain barrier permeability, including trafficking of immunocompetent cells.

Multiple sites of L-histidine decarboxylase expression in mouse suggest novel developmental functions for histamine.

Histamine mediates many types of physiologic signals in multicellular organisms. To clarify the developmental role of histamine, we have examined the developmental expression of L-histidine decarboxylase (HDC) mRNA and the production of histamine during mouse development. The predominant expression of HDC in mouse development was seen in mast cells. The HDC expression was evident from embryonal day 13 (Ed13) until birth, and the mast cells were seen in most peripheral tissues. Several novel sites with a prominent HDC mRNA expression were revealed. In the brain, the choroid plexus showed HDC expression at Ed14 and the raphe neurons at Ed15. Close to the parturition, at Ed19, the neurons in the tuberomammillary (TM) area and the ventricular neuroepithelia also displayed a clear HDC mRNA expression and histamine immunoreactivity (HA-ir). From Ed14 until birth, the olfactory and nasopharyngeal epithelia showed an intense HDC mRNA expression and HA-ir. In the olfactory epithelia, the olfactory receptor neurons (ORN) were shown to have very prominent histamine immunoreactivity. The bipolar nerve cells in the epithelium extended both to the epithelial surface and into the subepithelial layers to be collected into thick nerve bundles extending caudally toward the olfactory bulbs. Also, in the nasopharynx, an extensive subepithelial network of histamine-immunoreactive nerve fibers were seen. Furthermore, in the peripheral tissues, the degenerating mesonephros (Ed14) and the convoluted tubules in the developing kidneys (Ed15) showed HDC expression, as did the prostate gland (Ed15). In adult mouse brain, the HDC expression resembled the neuronal pattern observed in rat brain. The expression was restricted to the TM area in the ventral hypothalamus, with the main expression in the five TM subgroups called E1-E5. A distinct mouse HDC mRNA expression was also seen in the ependymal wall of the third ventricle, which has not been reported in the rat. The tissue- and cell-specific expression patterns of HDC and histamine presented in this work indicate that histamine could have cell guidance or regulatory roles in development.

Regional expression of the histamine H(2) receptor in adult and developing rat brain.

Histamine H(2) receptor expression was studied in adult and developing rat brain. Northern blot and in situ hybridizations indicated that histamine H(2) receptor messenger RNA expression is widespread and not limited to neurons in the adult rat brain. Prominent H(2) receptor expression in the adult brain was seen in the dentate gyrus, hippocampal subfields CA1-CA3, piriform cortex and in some diencephalic nuclei, e.g. in the suprachiasmatic nucleus and the red nucleus. Most of the adult brain nuclei displayed a very low H(2) receptor expression. Histamine H(2) receptor was also expressed during development in widespread areas of the central nervous system, coinciding with the transient production of histamine in the raphe neurons at embryonic day 15. From embryonic days 16 and 17 until birth, histamine H(2) receptor expression in the cortical plate coincided with the development and sprouting of histaminergic fibers into the cerebral cortex. The widespread and diffuse expression of histamine H(2) receptors in the adult rat brain suggests that the H(2) receptor modulates the excitability of neuron and astrocyte functions in many brain areas rather than mediating targeted cell-to-cell signals. During development, histamine H(2) receptor expression is seen in several target areas for the histaminergic fibers. This could indicate that histamine, through the H(2) receptor, regulates fetal development of the brain.

Identification of rat H3 receptor isoforms with different brain expression and signaling properties.

We identified the cDNAs of three functional rat H3 receptor isoforms (H3A, H3B, and H3C) and one nonfunctional truncated H3 receptor (H3T). The H3A, H3B, and H3C receptor isoforms vary in the length of their third intracellular loop; the H3B and H3C receptor lack 32 and 48 amino acids, respectively. Transient expression of the H3A, H3B, and H3C receptors in COS-7 cells results in high affinity binding for the H3 antagonist [125I]iodophenpropit, which is displaced by selective H3 agonists and antagonists. The three isoforms differentially couple to the Gi protein-dependent inhibition of adenylate cyclase or stimulation of p44/p42 mitogen activated protein kinase (MAPK), a new signaling pathway for the H3 receptor. Whereas the H3A receptor was less effective in inhibiting forskolin-induced cAMP production compared with the H3B or H3C receptor, this isoform was more effective in the stimulation of p44/p42 MAPK. The H3 receptor isoforms also displayed differential CNS expression in key areas involved in regulation of sensory, endocrine, and cognitive functions. A differential H3 receptor isoform expression was seen in, for example, hippocampus, where a characteristic dorsoventral distribution was revealed. Differential H3 receptor expression was also characteristic for the cerebellum, indicating possible histaminergic regulation of motor functions. The identification of these new H3 receptor isoforms and their specific signaling properties adds a new level of complexity to our understanding of the role of histamine, and the H3 receptor in brain function. The heterogeneous distribution of the isoforms suggests that H3 receptor isoform-specific regulation is important in several brain functions.

Histamine in brain development and tumors.

Histamine is found in developing mammalian brain in both neurons and mast cells. Under normal conditions, histamine H1 and H2 receptors are found in neural, glial and endothelial cells, and H3 receptors at least on neurons. Experimental brain tumors display both H1 and H2 receptors, and histamine increases permeability in the tumors and in the neighboring areas. Many studies have addressed histaminergic signalling mechanisms in cell lines originating from brain tumors. However, the role of histamine in normal development of brain structures, proliferation and differentiation of neurons and glial cells, and growth of malignant tumors in situ is still poorly understood.

The histaminergic system in the brain: structural characteristics and changes in hibernation.

Histaminergic neurons in adult vertebrate brain are confined to the posterior hypothalamic area, where they are comprised of scattered groups of neurons referred to as the tuberomammillary nucleus. Histamine regulates hormonal functions, sleep, food intake, thermoregulation and locomotor activity, for example. In the zebrafish, Danio rerio, histamine was detected only in the brain, where also the histamine synthesizing enzyme L-histidine decarboxylase (HDC) was expressed. It is possible that histamine has first evolved as a neurotransmitter in the central nervous system. We established sensitive quantitative in situ hybridization methods for histamine H(1) and H(2) receptors and HDC, to study the modulation of brain histaminergic system under pathophysiological conditions. A transient increase in H(1) receptor expression was seen in the dentate gyrus and striatum after a single injection of kainic acid, a glutamate analog. H(1) antagonists are known to increase duration of convulsions, and increased brain histamine is associated with reduced convulsions in animal models of epilepsy. No HDC mRNA was detected in brain vessels by in situ hybridization, which suggests lack of histamine synthesis by brain endothelial cells. This was verified by lack of HDC mRNA in a rat brain endothelial cell line, RBE4 cells. Both H(1) and H(2) receptor mRNA was found in this cell line, and the expression of both receptors was downregulated by dexamethasone. The findings are in agreement with the concept that histamine regulates blood-brain barrier permeability through H(1) and H(2) receptor mediated mechanisms. Hibernation is characterized by a drastic reduction of central functions. The activity of most transmitter systems is maintained at a very low level. Surprisingly, histamine levels and turnover were clearly elevated in hibernating ground squirrels, and the density of histamine-containing fibers was higher than in euthermic animals. It is possible that histamine actively maintains the low activity of other transmitters during the hibernation state.

Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli.

Neuropeptides FF (NPFF), AF (NPAF), and SF (NPSF) are homologous amidated peptides that were originally identified on the basis of similarity to the molluscan neuropeptide FMRF-amide. They have been hypothesized to have wide-ranging functions in the mammalian central nervous system, including pain modulation, opiate function, cardiovascular regulation, and neuroendocrine function. We have cloned the NPFF gene from human, bovine, rat, and mouse, and show that the precursor mRNA encodes for all three of the biochemically identified peptides (NPFF, NPAF, and NPSF). We demonstrate that NPFF precursor mRNA expression by Northern analysis and map sites of expression by in situ hybridization. We confirm the validity of the in situ hybridization by showing that its distribution in the brain and spinal cord matches the distribution of NPFF and NPSF immunoreactivity. We go on to show that the mRNA levels (as measured by in situ hybridization) in the spinal cord can be up-regulated by a model for inflammatory pain (carrageenan injection), but not by a model for neuropathic pain (lumbar nerve ligation). Our results confirm the evolutionary conservation of NPFF, NPAF, and NPSF neuropeptide expression in mammalian brain. They also provide a context for the interpretation of the pain-sensitizing effects of injections of these peptides that have been previously reported. Our results support a model for the role of these peptides in pain regulation at the level of the spinal cord.

Lack of histamine synthesis and down-regulation of H1 and H2 receptor mRNA levels by dexamethasone in cerebral endothelial cells.

The purpose of this work was to determine whether cerebral endothelial cells have the capacity to synthesize histamine or to express mRNA of receptors that specifically respond to available free histamine. The histamine concentrations and the expression of L-histidine decarboxylase (HDC) and histamine H1 and H2 receptor mRNA, both in adult rat brain and in cultured immortalized RBE4 cerebral endothelial cells, were investigated. In this study endothelial cells were devoid of any kind of detectable histamine production, both in vivo and in the immortalized RBE4 cells in culture. Both the immunostainings for histamine and the in situ hybridizations for HDC were negative, as well as histamine determinations by HPLC, indicating that endothelial cells do not possess the capacity to produce histamine. Also, glucocorticoid (dexamethasone) treatment failed to induce histamine production in the cultured cells. Although the cerebral endothelial cells lack histamine production, a nonsaturable uptake in RBE4 cells is demonstrated. The internalized histamine is detected both in the cytoplasm and in the nucleus, which could indicate a role for histamine as an intracellular messenger. Histamine H1 and H2 receptor mRNA was expressed in RBE4 cells, and glucocorticoid treatment down-regulated the mRNA levels of both H1 and H2 receptors. This mechanism may be involved in glucocorticoid-mediated effects on cerebrovascular permeability and brain edema.

Postnatal expression of H1-receptor mRNA in the rat brain: correlation to L-histidine decarboxylase expression and local upregulation in limbic seizures.

Histamine is implicated in the regulation of brain functions through three distinct receptors. Endogenous histamine in the brain is derived from mast cells and neurons, but the importance of these two pools during early postnatal development is still unknown. The expression of histamine H1-receptor in the rat brain was examined using in situ hybridization during postnatal development and in adults. For comparison, the expression of L-histidine decarboxylase (HDC) in the two pools was revealed. H1-receptor was evenly expressed throughout the brain on the first postnatal days, but resembled the adult, uneven pattern already on postnatal day 5 (P5). HDC was expressed in both mast cells and tuberomammillary neurons from birth until P5, after which the mast cell expression was no more detectable. In adult rat brain, high or moderate levels of H1-receptor expression were found in the hippocampus, zona incerta, medial amygdaloid nucleus and reticular thalamic nucleus. In most areas of the adult brain the expression of H1-receptor mRNA correlates well with binding data and histaminergic innervation. A notable exception is the hypothalamus, with high fibre density but moderate or low H1-receptor expression. Systemic kainic acid administration induced increased expression of H1-receptor mRNA in the caudate-putamen and dentate gyrus, whereas no change was seen in the hippocampal subfields CA1-CA3 or in the entorhinal cortex 6 h after kainic acid injections. This significant increase supports the concept that histaminergic transmission, through H1-receptor, is involved in the regulation of seizure activity in the brain.

In situ detection of H1-receptor mRNA and absence of apoptosis in the transient histamine system of the embryonic rat brain.

In the developing brain, histamine is one of the first neurotransmitters to appear. The concentration of histamine in the prenatal brain is fivefold that of adult levels. During the prenatal development a large transiently histamine-immunoreactive cell population distinct from the adult histaminergic system can be found within a subpopulation of the developing serotonergic raphe nuclei neurons. Also histamine-immunoreactive nerve fibers are widely distributed already during the prenatal development extending to the diencephalon, the thalamus, the cortex, and the spinal cord. Large numbers of histamine-containing mast cells also migrate into the brain during the late prenatal life. The wide distribution and high prenatal concentrations imply important functions for the histaminergic system during intrauterine development. However, little is known about the actual functions of histamine during development, and which of the histamine receptors are present in the prenatal rat brain is currently unknown. In the present study, we used in situ hybridization to study the distribution of H1-receptor (H1R) mRNA in the embryonic rat brain and spinal cord. H1R mRNA could be detected in rat brain and in spinal cord on embryonic day (E) 14, and the expression pattern seemed to partially localize in areas containing histamine-immunoreactive nerve fibers through E14-E20. H1R mRNA was also detected by reverse transcriptase polymerase chain reaction from embryonic brain samples and by Northern hybridization. The possible involvement of apoptosis in the disappearance of the developing transiently histaminergic system was studied by using apoptosis detection based on the terminal dUTP nick end labeling (TUNEL) technique and with c-Fos immunostaining. Although histamine immunoreactivity disappears dramatically from the developing raphe nuclei after E18, only occasional apoptotic nuclei could be seen in the histamine-immunoreactive cell bodies. The presence of H1R mRNA during the embryonic development renders it possible that histamine could exert an H1R-specific function at the time of the embryonic histamine peak.

Development of the histaminergic neurons and expression of histidine decarboxylase mRNA in the zebrafish brain in the absence of all peripheral histaminergic systems.

The histamine-storing neural system in adult and developing zebrafish (Danio rerio) was studied with immunocytochemical and chromatographical methods. Furthermore, the gene for histidine decarboxylase was partially cloned and its expression mapped with in situ hybridization. The histamine-storing neurons were only seen in the caudal hypothalamus, around the posterior recess of the diencephalic ventricle. Almost all parts of the brain, except the cerebellum, contained at least some histamine-immunoreactive fibres. The ascending projections had the rostral part of the dorsal telencephalon as a major target. Descending projections terminated in the torus semicircularis, central grey and inferior olive. A prominent innervation of the optic tectum, which has not been reported in other fish, was seen. The in situ hybridization gave a strong signal in cells with the same anatomical position as the histamine-immunoreactive neurons. The first histamine-immunoreactive neurons appeared in the ventral hypothalamus at about 85 h post-fertilization, and at 90 h, immunoreactive fibres terminated in the dorsal telencephalon. The embryonic histamine production described in mammals was lacking in this species. Both immunocytochemical and chromatographical studies indicated that histamine is absent in all other parts of the zebrafish body, and no specific hybridization was seen in any other part of the fish than the hypothalamus. The zebrafish could therefore be a very useful model for pharmacological in vivo studies of the histaminergic system of the brain, since the powerful peripheral actions of histamine should be lacking in this species.

Expression of histidine decarboxylase and cellular histamine-like immunoreactivity in rat embryogenesis.

In this study we investigated the developmental expression of histidine decarboxylase (HDC) mRNA and the distribution of histamine-immunoreactive (histamine-ir) cells in the rat embryonic tissues. We applied Northern blot analysis, in situ hybridization with synthetic oligonucleotide probes complementary to the rat HDC cDNA, and indirect histamine immunocytochemistry. Northern blot analysis revealed the appearance of a major (2.6 KB) HDC mRNA species in liver on embryonic Day 14. Its hybridization level peaked on Day E18, when two minor (1.6 and 3.5 KB) mRNA species were also present. During the periparturition period, a rapid decrease in HDC RNA was apparent, as the 2.6 KB mRNA species was expressed at a low level on postnatal Day P1. The embryonic liver expressed HDC on days E14-E20. On days E18 and E20, the periosteum and the epiphyseal growth plates of the endochondrally ossificating bones, and some striated muscle cells, showed hybridization signal for HDC. Histamine immunoreactivity was detected in many epithelial and neuronal cell types during embryogenesis. An intense histamine immunoreaction appeared first in essentially all cells of the liver parenchyma on day E12. This parenchymal histamine immunoreactivity disappeared by birth, after which this immunofluorescence in liver was restricted to a few scattered mast cells until adulthood. Some neurons in the peripheral sensory, sympathetic and cranial nerve ganglia were histamine-immunoreactive from day E16 to birth. In addition, many immunoreactive nerve fibers were detected in the gastrointestinal muscularis externa, mesentery, salivary glands, kidney, lung, and muscle tissue. We conclude that during rat embryogenesis histamine is produced and stored transiently by cells in liver, developing bone, and a few striated muscle cells, in addition to previously reported neurons in rat brain. Many peripheral neurons, epithelial cells, and mast cells display histamine immunoreactivity during rat embryogenesis but are devoid of detectable HDC mRNA with the current method. It remains possible that histamine is formed by another enzyme or is taken up from the extracellular space. The results support the concept that a significant proportion of histamine is formed and stored by embryonic cells other than mast cells.

Time-course study of 1,25-(OH)2-vitamin D3 induction of homologous receptor and c-myc in nontransformed and transformed C3H/10T1/2 cell clones.

1. We have used 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to investigate autoregulation of homologous receptor and the control of c-myc mRNA and protein expression in C3H/10T1/2 cells. 2. 10 nM 1,25-(OH)2D3 stimulated 1,25-(OH)2D3 receptor (VDR) synthesis in both non-transformed C3H/10T1/2 Cl 8 and in chemically transformed C3H/10T1/2 Cl 16 cells within 4 hr of treatment. Maximal induction was observed between 8 and 24 hr. 3. Two VDR mRNA transcripts, 2.7 and 4.8 kb, were present in both cell types. There were parallel changes in VDR specific mRNA levels and cellular VDR concentration in the C3H/10T1/2 Cl 8 cells indicating that the increase in receptor concentrations was dependent on de novo mRNA synthesis. 4. The increase in VDR mRNA concentration in the chemically transformed C3H/10T1/2 Cl 16 cells was maximal already at 4 hr, preceding the maximal increase in receptor concentration by 4-6 hr. 5. Analysis of c-myc mRNA levels also showed cell line specificity. 6. The c-myc mRNA level increased 2.1-fold with 10 nM 1,25-(OH)2D3 treatment in C3H/10T1/2 Cl 8 cells after 12 hr while the C3H/10T1/2 Cl 16 cells had maximal c-myc mRNA level after 1 hr. 7. The relative amount of c-myc mRNA remained higher than that of unstimulated controls the next 10-12 hr in C3H/10T1/2 Cl 16 cells. 8. The c-myc protein levels were not affected by 1,25-(OH)2D3 treatment in either cell line as detected by Western blot analysis. 9. Our data suggest that 1,25-(OH)2D3 mediated induction of VDR does not require prior c-myc protein synthesis in the C3H/10T1/2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A hidden break in the 28.0S rRNA from Diphyllobothrium dendriticum.

Nondenatured and denatured total RNA from the tapeworm Diphyllobothrium dendriticum (Cestoda) was analysed by agarose gel electrophoresis. It was found that the large subunit ribosomal RNA (lrRNA) is 28.0S and the small subunit ribosomal RNA (srRNA) is 19.5S. Following denaturation the 28.0S rRNA was disrupted into a 19.5S subfragment and a 20.7S subfragment due to the presence of a centrally located hidden break. By hybridization of Northern blot membranes with oligonucleotide probes specific for the 5'- and 3'-ends of the lrRNA respectively, we have shown that the 19.5S subfragment is from the 5'-end (the alpha-subfragment) and the 20.7S subfragment from the 3'-end (the beta-subfragment) of the 28.0S rRNA of D. dendriticum.