N-methyl-D-aspartate receptor antagonists enhance histamine neuron activity in rodent brain

The modulation of histamine neuron activity by various non-competitive NMDA-receptor antagonists was evaluated by changes in tele-methylhistamine (t-MeHA) levels and histidine decarboxylase (hdc) mRNA expression induced in rodent brain. The NMDA open-channel blockers phencyclidine (PCP) and MK-801 enhanced t-MeHA levels in mouse brain by 50-60%. Ifenprodil, which interacts with polyamine sites of NR2B-containing NMDA receptors, had no effect. PCP also increased hdc mRNA expression in the rat tuberomammillary nucleus. The enhancement of t-MeHA levels elicited by MK-801 (ED50 of approximately 0.1 mg/kg) was observed in the hypothalamus, cerebral cortex, striatum and hippocampus. Control t-MeHA levels and the t-MeHA response to MK-801 were not different in male and female mice. Double immunostaining for HDC and NMDA receptor subunits showed that histamine neurons of the rat tuberomammillary nucleus express NMDA receptor subunit 1 (NR1) with NMDA receptor subunit 2A (NR2A) and NMDA receptor 2B subunit (NR2B). In addition, immunoreactivity for the neuronal glutamate transporter EAAC1 was observed near most histaminergic perikarya. Hence, these findings support the existence of histamine/glutamate functional interactions in the brain. The increase in histamine neuron activity induced by NMDA receptor antagonists further suggests a role of histamine neurons in psychotic disorders. In addition, the decrease in MK-801-induced hyperlocomotion observed in mice after administration of ciproxifan further strengthens the potential interest of H3-receptor antagonist/inverse agonists for the symptomatic treatment of schizophrenia.     

Modulation of the histaminergic system and behaviour by alpha-fluoromethylhistidine in zebrafish

The functional role of histamine (HA) in zebrafish brains was studied. Zebrafish did not display a clear circadian variation in brain HA levels. Loading of zebrafish with l-histidine increased HA concentration in the brain. A single injection of the histidine decarboxylase (HDC) inhibitor, alpha-fluoromethylhistidine (alpha-FMH), gave rise to a rapid reduction in zebrafish brain HA. Low HDC activity in the brain after injections verified the effect of alpha-FMH. A reduction in the number of histaminergic fibres but not neurones and an increased expression of HDC mRNA was evident after alpha-FMH. Automated behavioural analysis after alpha-FMH injection showed no change in swimming activity, but abnormalities were detected in exploratory behaviour examined in a circular tank. No significant behavioural changes were detected after histidine loading. The time spent for performance in the T-maze was significantly increased in the first trial 4 days after alpha-FMH injections, suggesting that lack of HA may impair long-term memory. The rostrodorsal telencephalon, considered to correspond to the mammalian amygdala and hippocampus in zebrafish, is densely innervated by histaminergic fibres. These results suggest that low HA decreases anxiety and/or affects learning and memory in zebrafish, possibly through mechanisms that involve the dorsal forebrain.     

Measurement of hypocretin/orexin content in the mouse brain using an enzyme immunoassay: the effect of circadian time,age and genetic background

The hypocretins (1 and 2) have emerged as key regulators of sleep and wakefulness. We developed a high-throughput enzyme immunoassay (EIA) to measure total brain hypocretin levels from large numbers of mice. Hypocretin levels were not altered by circadian time or age. However, significant differences in one or both hypocretin peptides were observed between different mouse strains. We studied hypocretin levels in knockout and transgenic mouse models with obesity, circadian gene mutations or monoaminergic defects. Compared to controls, only histamine receptor knockouts had lower hypocretin levels. This was most pronounced in H1 receptor knockouts suggesting the existence of a positive feedback loop between hypocretin and histaminergic neurons.     

Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant

masterblind (mbl) is a zebrafish mutation characterised by the absence or reduction in size of the telencephalon, optic vesicles and olfactory placodes. We show that inhibition of Gsk3beta in zebrafish embryos either by overexpression of dominant negative dn gsk3beta mRNA or by lithium treatment after the midblastula transition phenocopies mbl. The loss of anterior neural tissue in mbl and lithium-treated embryos is preceded by posteriorization of presumptive anterior neuroectoderm during gastrulation, which is evident from the anterior shift of marker genes Otx2 and Wnt1. Heterozygous mbl embryos showed increased sensitivity to inhibition of GSK3beta by lithium or dn Xgsk3beta that led to the loss of eyes. Overexpression of gsk3beta mRNA rescued eyes and the wild-type fgf8 expression of homozygous mbl embryos. emx1 that delineates the telencephalon is expanded and shifted ventroanteriorly in mbl embryos. In contrast to fgf8, the emx1 expression domain was not restored upon overexpression of gsk3beta mRNA. These experiments place mbl as an antagonist of the Wnt pathway in parallel or upstream of the complex consisting of Axin, APC and Gsk3beta that binds and phosphorylates beta-catenin, thereby destabilising it. mbl maps on LG 3 close to a candidate gene axin1. In mbl we detected a point mutation in the conserved minimal Gsk3beta-binding domain of axin1 leading to a leucine to glutamine substitution at position 399. Overexpression of wild-type axin1 mRNA rescued mbl completely, demonstrating that mutant axin1 is responsible for the mutant phenotype. Overexpression of mutant L399Q axin1 in wild-type embryos resulted in a dose-dependent dominant negative activity as demonstrated by the loss of telencephalon and eyes. We suggest that the function of Axin1/Mbl protein is to antagonise the Wnt signal and in doing so to establish and maintain the most anterior CNS. Our findings provide new insights into the mechanisms by which the Wnt pathway generates anteroposterior polarity of the neural plate.     

The Histaminergic Tuberomamillary Nucleus Is Involved in Appetite for Sex,Water and Amphetamine

The histaminergic system is one component of the ascending arousal system which is involved in wakefulness, neuroendocrine control, cognition, psychiatric disorders and motivation. During the appetitive phase of motivated behaviors the arousal state rises to an optimal level, thus giving proper intensity to the behavior. Previous studies have demonstrated that the histaminergic neurons show an earlier activation during the appetitive phase of feeding, compared to other ascending arousal system nuclei, paralleled with a high increase in arousal state. Lesions restricted to the histaminergic neurons in rats reduced their motivation to get food even after 24h of food deprivation, compared with intact or sham lesioned rats. Taken together, these findings indicate that the histaminergic system is important for appetitive behavior related to feeding. However, its role in other goal-directed behaviors remains unexplored. In the present work, male rats rendered motivated to obtain water, sex, or amphetamine showed an increase in Fos-ir of histaminergic neurons in appetitive behaviors directed to get those reinforcers. However, during appetitive tests to obtain sex, or drug in amphetamine-conditioned rats, Fos expression increased in most other ascending arousal system nuclei, including the orexin neurons in the lateral hypothalamus, dorsal raphe, locus coeruleus and laterodorsal tegmental neurons, but not in the ventral tegmental area, which showed no Fos-ir increase in any of the 3 conditions. Importantly, all these appetitive behaviors were drastically reduced after histaminergic cell-specific lesion, suggesting a critical contribution of histamine on the intensity component of several appetitive behaviors.     

Dcc regulates asymmetric outgrowth of forebrain neurons in zebrafish

The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates.     

Effects of thioperamide, a histamine H3 receptor antagonist, on locomotor activity and brain histamine content in mast cell-deficient W/Wv mice.

The purpose of this study was to examine the effects of thioperamide, a histamine H3 antagonist, on the locomotor activity and the brain histamine content in mast-cell-deficient W/Wv mice. Thioperamide (12.5 and 25 mg/kg) showed significant increase in the locomotor activity of W/Wv mice, measured by a photo-beam system, 1 hr after the intraperitoneal injection. However, more than 75 mg/kg of thioperamide showed not only the reduction of the locomotor activity but also the inhibition of motor coordination measured by the rotarod performance. The increase in the locomotor activity by thioperamide was blocked by i. p. pretreatment with (R)-alpha-methyl-histamine, an H3 agonist, or pyrilamine, an H1 antagonist, or zolantidine, an H2 antagonist. The brain histamine content was decreased by thioperamide (12.5-75.0 mg/kg), 1 hr after administration. Thus, the blockade of histamine H3 receptor by thioperamide showed the activation of locomotor activity of mice, which may be mediated by H1 and/or H2 receptors. The present data support the hypothesis that central histaminergic neurons may be involved in the control of state of wakefulness.     

Expression of RNA-binding protein Rbfox1l demarcates a restricted population of dorsal telencephalic neurons within the adult zebrafish brain

Rbfox RNA-binding proteins are expressed in the adult mammalian brain and are required for proper brain development and function. Studies in mice and humans have implicated Rbfox1/RBFOX1 in autism, neuronal excitation and epilepsy, and Rbfox2/RBFOX2 in cerebellar development. The zebrafish has emerged as a prominent model system for brain study, possessing neuroanatomical conservation with mammals and an extensive capacity for adult neurogenesis and plasticity. In this study, we characterize Rbfox1l and Rbfox2 expression in the adult zebrafish brain. While Rbfox2 is expressed broadly, Rbfox1l is expressed in restricted populations of neurons in the dorsal telencephalon and cerebellum. In the dorsal telencephalon, Rbfox1l is expressed in a specific population of neurons spanning Dm and Dc regions. In the cerebellum, Rbfox1l and Rbfox2 are expressed in the Purkinje cell layer, reminiscent of Rbfox1 and Rbfox2 expression in the mammalian cerebellum. Our findings motivate future studies of Rbfox function in the zebrafish brain.     

Activation of central orexin/hypocretin neurons by dietary amino acids

Hypothalamic orexin/hypocretin (orx/hcrt) neurons regulate energy balance, wakefulness, and reward; their loss produces narcolepsy and weight gain. Glucose can lower the activity of orx/hcrt cells, but whether other dietary macronutrients have similar effects is unclear. We show that orx/hcrt cells are stimulated by nutritionally relevant mixtures of amino acids (AAs), both in brain slice patch-clamp experiments, and in c-Fos expression assays following central or peripheral administration of AAs to mice in?vivo. Physiological mixtures of AAs electrically excited orx/hcrt cells through a dual mechanism involving inhibition of K(ATP) channels and activation of system-A amino acid transporters. Nonessential AAs were more potent in activating orx/hcrt cells than essential AAs. Moreover, the presence of physiological concentrations of AAs suppressed the glucose responses of orx/hcrt cells. These results suggest a new mechanism of hypothalamic integration of macronutrient signals and imply that orx/hcrt cells sense macronutrient balance, rather than net energy value, in extracellular fluid.     

Histamine Receptor and its Regulation of Energy Metabolism

In a series of studies on brain functions of histamine, probes to manipulate activities of histaminergic neuronal systems were applied to assess histaminergic function in non-obese normal, and lean and obese Zucker rats. Food intake was suppressed by both activation of H₁-receptors and inhibition of H₃-receptors in the ventromedial hypothalamic nucleus (VMH) and the paraventricular nucleus, each of which is a satiety center. Feeding circadian rhythm was decreased in its amplitude through histaminergic modulation in the hypothalamus. Histamine neurons in the mesencephalic trigeminal nucleus (Me5) were involved in regulation of masticatory functions, particularly eating speed, while histamine-containing neurons in the VMH controlled intake volume of meals. Energy deficiency in the brain enhanced satiation through histaminergic activation of VMH neurons, which in turn produced glycogenolysis in the hypothalamus to maintain homeostatic control of glucose supply. A very-low-calorie conventional Japanese diet, which is a fiber rich and low energy food source, enhanced satiation by increased mastication and because of the low energy supply of the diet. Hypothalamic histamine neurons were activated by high ambient temperature and also by interleukin-1β, an endogenous pyrogen, to maintain homeostatic thermoregulation. Behavioral and metabolic abnormalities of Zucker obese rats were mediated by a deficit in hypothalamic neuronal histamine, and the Zucker rat was evaluated as an animal model of histamine deficiency. Transplantation of the lean fetal hypothalamus into the third cerebroventricle of host obese Zuckers attenuated the abnormalities.     

Key roles of the histaminergic system in sleep-wake regulation

Histamine plays an important role in mediating wakefulness in mammals. Based on the findings from gene-manipulated mice, we provide several lines of evidence showing the roles of the histaminergic system in the somnogenic effects of prostaglandin (PG) D2 and adenosine, and in the arousal effects of PGE2 and orexin. PGD2 activates DP1 receptors (R) to promote sleep by stimulating them to release adenosine. The released adenosine activates adenosine A2AR and subsequently excites the ventrolateral preoptic area (VLPO), one of the sleep centers in the anterior hypothalamus. VLPO neurons then send inhibitory signals to downregulate the histaminergic tuberomammillary nucleus (TMN), which contributes to arousal. A1R is expressed in histaminergic neurons of the rat TMN. Adenosine in the TMN inhibits the histaminergic system via A1R and promotes non–rapid eye movement sleep. Conversely, both endogenous PGE2 and orexin activate the histaminergic system through EP4R and OX-2R, respectively, to promote wakefulness via histamine H1R. Furthermore, the arousal effect of ciproxifan, H3R antagonist, depends on the activation of histaminergic systems. These findings indicate that VLPO and TMN regulate sleep and wakefulness by means of a “flip-flop” mechanism operating in an anti-coincident manner during sleep–wake state transitions.

     

Role of hypothalamic histaminergic systems in the regulation of vigilance states in cats

We have studied the effects of local injections of histaminergic and antihistaminic drugs on the sleep-waking cycle in the cat. Microinjections of alpha-fluoromethylhistidine (alpha-FMH), a specific inhibitor of histidine decarboxylase, in the ventrolateral posterior hypothalamus, where histamine-immunoreactive neurons have been recently identified, resulted in a significant decrease in wakefulness (W) and increase in deep slow wave sleep (SWS). On the other hand, microinjections of SKF-91488 (Homodimaprit), a specific inhibitor of histamine-N-methyltransferase, increased W and decreased SWS and paradoxical sleep (PS). Microinjections of histamine also produced an increase of W, while this effect was abolished by pretreatment with mepyramine, an H1-histamine receptor antagonist.     

Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth

Olfm1, a secreted highly conserved glycoprotein, is detected in peripheral and central nervous tissues and participates in neural progenitor maintenance, cell death in brain, and optic nerve arborization. In this study, we identified Olfm1 as a molecule promoting axon growth through interaction with the Nogo A receptor (NgR1) complex. Olfm1 is coexpressed with NgR1 in dorsal root ganglia and retinal ganglion cells in embryonic and postnatal mice. Olfm1 specifically binds to NgR1, as judged by alkaline phosphatase assay and coimmunoprecipitation. The addition of Olfm1 inhibited the growth cone collapse of dorsal root ganglia neurons induced by myelin-associated inhibitors, indicating that Olfm1 attenuates the NgR1 receptor functions. Olfm1 caused the inhibition of NgR1 signaling by interfering with interaction between NgR1 and its coreceptors p75NTR or LINGO-1. In zebrafish, inhibition of optic nerve extension by olfm1 morpholino oligonucleotides was partially rescued by dominant negative ngr1 or lingo-1. These data introduce Olfm1 as a novel NgR1 ligand that may modulate the functions of the NgR1 complex in axonal growth.     

Zebrafish genes for neuropeptide Y and peptide YY reveal origin by chromosome duplication from an ancestral gene linked to the homeobox cluster

Neuropeptide Y (NPY) and peptide YY (PYY) are related 36-amino acid peptides. NPY is widely distributed in the nervous system and has several physiological roles. PYY serves as an intestinal hormone as well as a neuropeptide. We report here cloning of the npy and pyy genes in zebrafish (Danio rerio). NPY differs at only one to four amino acid positions from NPY in other jawed vertebrates. Zebrafish PYY differs at three positions from PYY from other fishes and at 10 positions from mammals. In situ hybridization showed that neurons containing NPY mRNA have a widespread distribution in the brain, particularly in the telencephalon, optic tectum, and rhombencephalon. PYY mRNA was found mainly in brainstem neurons, as reported previously for vertebrates as divergent as the rat and the lamprey, suggesting an essential role for PYY in these neurons. PYY mRNA was observed also in the telencephalon. These results were confirmed by immunocytochemistry. As in the human, the npy gene is located adjacent to homeobox (hox) gene cluster A (copy a in zebrafish), whereas the pyy gene is located close to hoxBa. This suggests that npy and pyy arose from a common ancestral gene in a chromosomal duplication event that also involved the hox gene clusters. As zebrafish has seven hox clusters, it is possible that additional NPY family genes exist or have existed. Also, the NPY receptor system seems to be more complex in zebrafish than in mammals, with at least two receptor genes without known mammalian orthologues.     

Narcolepsy with hypocretin/orexin deficiency, infections and autoimmunity of the brain

The loss of hypothalamic hypocretin/orexin (hcrt) producing neurons causes narcolepsy with cataplexy. An autoimmune basis for the disease has long been suspected and recent results have greatly strengthened this hypothesis. Narcolepsy with hcrt deficiency is now known to be associated with a Human Leukocyte Antigen (HLA) and T-cell receptor (TCR) polymorphisms, suggesting that an autoimmune process targets a single peptide unique to hcrt-cells via specific HLA-peptide-TCR interactions. Recent data have shown a robust seasonality of disease onset in children and associations with Streptococcus Pyogenes, and influenza A H1N1-infection and H1N1-vaccination, pointing towards processes such as molecular mimicry or bystander activation as crucial for disease development. We speculate that upper airway infections may be common precipitants of a whole host of CNS autoimmune complications including narcolepsy.     

Effects of α-fluoromethylhistidine (FMH), an irreversible inhibitor of histidine decarboxylase,on development of brain histamine and catecholamine systems in the neonatal rat

Daily administration of FMH to neonatal rats produced long-lasting inhibition of histidine decarboxylase in hypothalamus and cerebral cortex and led to depletion of histamine in both brain regions. The onset of depletion was more rapid in cerebral cortex, a region in which non-neurotransmitter pools of histamine predominate in early postnatal life, appearing as early as postnatal day 3; depletion in the hypothalamus, a region rich in histaminergic neuronal projections, appeared later. No effects were seen on body or brain growth, nor was development of other biogenic amine systems affected. FMH thus provides a selective probe for examining the role of histamine in brain development.     

Cloning and embryonic expression of zebrafish neuropilin genes

Neuropilin (Nrp), a cell surface receptor for class 3 semaphorins and for certain heparin forms of vascular endothelial growth factors, functions in many biological processes including axon guidance, neural cell migration and angiogenesis in the development of the nervous system and the cardiovascular system. To understand the role of neuropilins in zebrafish embryogenesis, we have cloned three zebrafish neuropilin homologues, nrp1b, nrp2a and nrp2b. Based on synteny, zebrafish nrp1b and the previously cloned nrp1a are orthologous to human nrp1, and zebrafish nrp2a and 2b orthologous to human nrp2. We have characterized the expression patterns of these four zebrafish neuropilin genes in wild type embryos from the beginning of somitogenesis to 48 h post-fertilization. Zebrafish nrp1a is expressed in the neural tube including telencephalon, epithalamus, cells along the axonal trajectory of the posterior commissure and the medial longitudinal fascicle, hindbrain neurons, vagus motor neurons and spinal motoneurons. Zebrafish nrp1b is expressed in the nose, the cranial neural crest cell (NCC) derived tissue underlying the hypothalamus, endothelial precursors and the trunk and tail vasculature. Zebrafish nrp2a is expressed in telencephalon, anterior pituitary, oculomotor and trochlear motor neurons, cells along the supra-optic and posterior commissures, hindbrain rhombomere 1, hindbrain neurons, cranial NCCs and sclerotome. Zebrafish nrp2b is expressed in telencephalon, thalamus, hypothalamus, epiphysis, cells along the anterior and posterior commissures, post-optic and supra-optic commissures and the olfactory axonal trajectory, hindbrain neurons, cranial NCCs, somites and spinal cord neurons.     

Ethanol Induces Sedation and Hypnosis via Inhibiting Histamine Release in Mice

Ethanol is one of the most highly abused psychoactive compounds worldwide and induces sedation and hypnosis. The histaminergic system is involved in the regulation of sleep/wake function and is a crucial player in promoting wakefulness. To explore the role and mechanism of the histaminergic system in ethanol-induced sedation and hypnosis, we recorded locomotor activity (LMA) and electroencephalography (EEG)/electromyography (EMG) in mice using an infrared ray passive sensor recording system and an EEG/EMG recording system, respectively, after administration of ethanol. In vivo microdialysis coupled with high performance liquid chromatography and fluorometry technology were used to detect histamine release in the mouse frontal cortex (FrCx). The results revealed that ethanol significantly suppressed LMA of histamine receptor 1 (H₁R)-knockout (KO) and wild-type (WT) mice in the range of 1.5–2.5 g/kg, but suppression was remarkably stronger in WT mice than in H₁R-KO mice. At 2.0 and 2.5 g/kg, ethanol remarkably increased non-rapid eye movement sleep and decreased wakefulness, respectively. Neurochemistry experimental data indicated that ethanol inhibited histamine release in the FrCx in a dose-dependent manner. These findings suggest that ethanol induces sedation and hypnosis via inhibiting histamine release in mice.

     

Ontogeny of Histaminergic Neurotransmission in the Rat Brain: Concomitant Development of Neuronal Histamine, H-1 Receptors, and H-1 Receptor-Mediated Stimulation of Phospholipid Turnover

The ontogeny of histaminergic neurotransmission in the rat brain was studied by assessing development of histamine levels in brain regions, along with H-1 receptor binding of [3H]mepyramine and H-1 receptor-mediated cellular events. In the hypothalamus, which is rich in histaminergic innervation, levels of the amine were low at birth, increased sharply at 8 days of age, and reached adult concentrations shortly thereafter; this pattern is typical of most neurotransmitters. In contrast, regions poor in neuronal histamine showed an initially high histamine level and a subsequent decline with development, as is known to occur during general growth of tissues. The developmental profile of H-1 receptor binding sites resembled that of the neuronal histamine pool, and the increases with age resulted from changes in the number of binding sites without alterations in Kd. Cellular responses to H-1 receptor activation were assessed by determining the stimulation of phospholipid turnover evoked by intracisternally administered histamine, a process that has been shown to involve only the neuronal compartment. Again, the developmental profile was superimposable upon that of H-1 receptor binding and that of hypothalamic histamine levels. These studies indicate that ontogeny of histaminergic neurotransmission is a coordinated process, with simultaneous development of neuronal histamine, its key biosynthetic enzyme, and H-1 receptors coupled directly to cellular events.