Initial Characterization of a Transgenic Mouse with Overexpression of the Human H1-Histamine Receptor on the Heart.

There is a debate on whether H1-histamine receptors can alter contractility in the mammalian heart. We studied here a new transgenic mouse model where we increased genetically the cardiac level of the H1-histamine receptor. We wanted to know if histamine could augment or decrease contractile parameters in mice with cardiac-specific overexpression of human H1-histamine receptors (H1-TG) and compared these findings with those in littermate wild-type mice (WT). In H1-TG mice, we studied the presence of H1-histamine receptors by autoradiography of the atrium and ventricle using [3H]mepyramine. The messenger RNA for human H1-histamine receptors was present in the heart from H1-TG and absent from WT. Using in situ hybridization, we noted mRNA for the human H1-histamine receptor in cardiac cells from H1-TG. We noted that histamine (1 nM-10 µM) in paced (1 Hz) left atrial preparations from H1-TG, exerted at each concentration of histamine initially reduced force of contraction and then raised contractile force. Likewise, in spontaneously beating left atrial preparations from H1-TG, we noted that histamine led to a transient reduction in the spontaneous beating rate followed by an augmentation in the beating rate. The negative inotropic and chronotropic and the positive inotropic effects on histamine in isolated atrial muscle strips from H1-TG were attenuated by the H1-histamine receptor antagonist mepyramine. Histamine failed to exert an increased force or reduce the heartbeat in atrial preparations from WT. We concluded that stimulation of H1-histamine-receptors can decrease and then augment contractile force in the mammalian heart and stimulation of H1-histamine receptors exerts a negative chronotropic effect. SIGNIFICANCE STATEMENT: We made novel transgenic mice with cardiomyocyte-specific high expressional levels of the human H1-histamine receptor to contribute to the clarification of the controversy on whether H1-histamine receptors increase or decrease contractility and beating rate in the mammalian heart. From our data, we conclude that stimulation of H1-histamine receptors first decrease and then raise contractile force in the mammalian heart but exert solely negative chronotropic effects.

Initial characterization of a transgenic mouse with overexpression of the human D1-dopamine receptor in the heart.

Dopamine can exert effects in the mammalian heart via five different dopamine receptors. There is controversy whether dopamine receptors increase contractility in the human heart. Therefore, we have generated mice that overexpress the human D1-dopamine receptor in the heart (D1-TG) and hypothesized that dopamine increases force of contraction and beating rate compared to wild-type mice (WT). In D1-TG hearts, we ascertained the presence of D1-dopamine receptors by autoradiography using [3H]SKF 38393. The mRNA for human D1-dopamine receptors was present in D1-TG hearts and absent in WT. We detected by in-situ-hybridization mRNA for D1-dopamine receptors in atrial and ventricular D1-TG cardiomyocytes compared to WT but also in human atrial preparations. We noted that in the presence of 10 µM propranolol (to antagonize ß-adrenoceptors), dopamine alone and the D1- and D5-dopamine receptor agonist SKF 38393 (0.1-10 µM cumulatively applied) exerted concentration- and time-dependent positive inotropic effects and positive chronotropic effects in left or right atrial preparations from D1-TG. The positive inotropic effects of SKF 38393 in left atrial preparations from D1-TG led to an increased rate of relaxation and accompanied by and probably caused by an augmented phosphorylation state of the inhibitory subunit of troponin. In the presence of 0.4 µM propranolol, 1 µM dopamine could increase left ventricular force of contraction in isolated perfused hearts from D1-TG. In this model, we have demonstrated a positive inotropic and chronotropic effect of dopamine. Thus, in principle, the human D1-dopamine receptor can couple to contractility in the mammalian heart.

Pharmacological Characterization of the Zebrafish (Danio Rerio) Histamine H1 Receptor Reveals the Involvement of the Second Extracellular Loop in the Binding of Histamine.

The zebrafish (Danio rerio) histamine H1 receptor gene (zfH1R) was cloned in 2007 and reported to be involved in fish locomotion. Yet, no detailed characterization of its pharmacology and signaling properties have so far been reported. In this study, we pharmacologically characterized the zfH1R expressed in HEK-293T cells by means of [3H]-mepyramine binding and G protein-signaling assays. The zfH1R [dissociation constant (KD), 0.7 nM] displayed similar affinity for the antagonist [3H]-mepyramine as the human histamine H1 receptor (hH1R) (KD, 1.5 nM), whereas the affinity for histamine is 100-fold higher than for the human H1R. The zfH1R couples to Gαq/11 proteins and activates several reporter genes, i.e., NFAT, NFÏ°B, CRE, VEGF, COX-2, SRE, and AP-1, and zfH1R-mediated signaling is prevented by the Gαq/11 inhibitor YM-254890 and the antagonist mepyramine. Molecular modeling of the zfH1R and human H1R shows that the binding pockets are identical, implying that variations along the ligand binding pathway could underly the differences in histamine affinity instead. Targeting differentially charged residues in extracellular loop 2 (ECL2) using site-directed mutagenesis revealed that Arg21045x55 is most likely involved in the binding process of histamine in zfH1R. This study aids the understanding of the pharmacological differences between H1R orthologs and the role of ECL2 in histamine binding and provides fundamental information for the understanding of the histaminergic system in the zebrafish. SIGNIFICANCE STATEMENT: The use of the zebrafish as in vivo models in neuroscience is growing exponentially, which asks for detailed characterization of the aminergic neurotransmitter systems in this model. This study is the first to pharmacologically characterize the zebrafish histamine H1 receptor after expression in HEK-293T cells. The results show a high pharmacological and functional resemblance with the human ortholog but also reveal interesting structural differences and unveils an important role of the second extracellular loop in histamine binding.

Zebrafish embryonically exposed to valproic acid present impaired retinal development and sleep behavior.

Prenatal exposure to valproic acid (VPA), a drug widely used to treat epilepsy and bipolar disorder, is an environmental risk factor for autism spectrum disorder (ASD). VPA has been used to reproduce the core symptoms of ASD in animal model organisms, including zebrafish. Visual system functioning is essential in the interpretation of social conditions and plays an important role of several behavioral responses. We hypothesized that behavioral deficits displayed by ASD patients may involve impaired visual processing. We used zebrafish as model organism to investigate the visual system after embryonic exposure to VPA using histological, behavioral and gene expression analysis. We analyzed the pineal gland of zebrafish and sleep-like behavior to study how VPA exposure alters photo-sensibility of zebrafish. VPA-exposed zebrafish showed a delay in the development of the retina and optic nerve, which normalized at five days post fertilization. At larval stage, VPA-exposed zebrafish showed sleep disturbances associated with a reduced number of serotonin-producing cells of the pineal gland. In addition, the number of hypocretin/orexin (hcrt) expressing neurons in the rostral hypothalamus at 6 and 14 days post fertilization was reduced. In conclusion, we demonstrated that although VPA exposure leads to a delay in visual system development, it does not affect larval visual function. The novel finding that VPA alters significantly cells involved in sleep regulation and the sleep-like state itself may be relevant for understanding sleep disturbances in ASD patients.

The Roles of Histamine Receptor 1 (hrh1) in Neurotransmitter System Regulation, Behavior, and Neurogenesis in Zebrafish.

Histamine receptors mediate important physiological processes and take part in the pathophysiology of different brain disorders. Histamine receptor 1 (HRH1) is involved in the development of neurotransmitter systems, and its role in neurogenesis has been proposed. Altered HRH1 binding and expression have been detected in the brains of patients with schizophrenia, depression, and autism. Our goal was to assess the role of hrh1 in zebrafish development and neurotransmitter system regulation through the characterization of hrh1-/- fish generated by the CRISPR/Cas9 system. Quantitative PCR, in situ hybridization, and immunocytochemistry were used to study neurotransmitter systems and genes essential for brain development. Additionally, we wanted to reveal the role of this histamine receptor in larval and adult fish behavior using several quantitative behavioral methods including locomotion, thigmotaxis, dark flash and startle response, novel tank diving, and shoaling behavior. Hrh1-/- larvae displayed normal behavior in comparison with hrh1+/+ siblings. Interestingly, a transient abnormal expression of important neurodevelopmental markers was evident in these larvae, as well as a reduction in the number of tyrosine hydroxylase 1 (Th1)-positive cells, th1 mRNA, and hypocretin (hcrt)-positive cells. These abnormalities were not detected in adulthood. In summary, we verified that zebrafish lacking hrh1 present deficits in the dopaminergic and hypocretin systems during early development, but those are compensated by the time fish reach adulthood. However, impaired sociability and anxious-like behavior, along with downregulation of choline O-acetyltransferase a and LIM homeodomain transcription factor Islet1, were displayed by adult fish.

Angiopoietin 1 and integrin beta 1b are vital for zebrafish brain development.

Introduction: Angiopoietin 1 (angpt1) is essential for angiogenesis. However, its role in neurogenesis is largely undiscovered. This study aimed to identify the role of angpt1 in brain development, the mode of action of angpt1, and its prime targets in the zebrafish brain. Methods: We investigated the effects of embryonic brain angiogenesis and neural development using qPCR, in situ hybridization, microangiography, retrograde labeling, and immunostaining in the angpt1sa14264, itgb1bmi371, tekhu1667 mutant fish and transgenic overexpression of angpt1 in the zebrafish larval brains. Results: We showed the co-localization of angpt1 with notch, delta, and nestin in the proliferation zone in the larval brain. Additionally, lack of angpt1 was associated with downregulation of TEK tyrosine kinase, endothelial (tek), and several neurogenic factors despite upregulation of integrin beta 1b (itgb1b), angpt2a, vascular endothelial growth factor aa (vegfaa), and glial markers. We further demonstrated that the targeted angpt1sa14264 and itgb1bmi371 mutant fish showed severely irregular cerebrovascular development, aberrant hindbrain patterning, expansion of the radial glial progenitors, downregulation of cell proliferation, deficiencies of dopaminergic, histaminergic, and GABAergic populations in the caudal hypothalamus. In contrast to angpt1sa14264 and itgb1bmi371 mutants, the tekhu1667 mutant fish regularly grew with no apparent phenotypes. Notably, the neural-specific angpt1 overexpression driven by the elavl3 (HuC) promoter significantly increased cell proliferation and neuronal progenitor cells but decreased GABAergic neurons, and this neurogenic activity was independent of its typical receptor tek. Discussion: Our results prove that angpt1 and itgb1b, besides regulating vascular development, act as a neurogenic factor via notch and wnt signaling pathways in the neural proliferation zone in the developing brain, indicating a novel role of dual regulation of angpt1 in embryonic neurogenesis that supports the concept of angiopoietin-based therapeutics in neurological disorders.

Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish.

AIM: We aimed at identifying potential roles of vesicular monoamine transporter 2, also known as Solute Carrier protein 18 A2 (SLC18A2) (hereafter, Vmat2), in brain monoamine regulation, their turnover, behaviour and brain development using a novel zebrafish model. METHODS: A zebrafish strain lacking functional Vmat2 was generated with the CRISPR/Cas9 system. Larval behaviour and heart rate were monitored. Monoamines and their metabolites were analysed with high-pressure liquid chromatography. Amine synthesising and degrading enzymes, and genes essential for brain development, were analysed with quantitative PCR, in situ hybridisation and immunocytochemistry. RESULTS: The 5-bp deletion in exon 3 caused an early frameshift and was lethal within 2 weeks post-fertilisation. Homozygous mutants (hereafter, mutants) displayed normal low locomotor activity during night-time but aberrant response to illumination changes. In mutants dopamine, noradrenaline, 5-hydroxytryptamine and histamine levels were reduced, whereas levels of dopamine and 5-hydroxytryptamine metabolites were increased, implying elevated monoamine turnover. Consistently, there were fewer histamine, 5-hydroxytryptamine and dopamine immunoreactive cells. Cellular dopamine immunostaining, in wild-type larvae more prominent in tyrosine hydroxylase 1 (Th1)-expressing than in Th2-expressing neurons, was absent in mutants. Despite reduced dopamine levels, mutants presented upregulated dopamine-synthesising enzymes. Further, in mutants the number of histidine decarboxylase-expressing neurons was increased, notch1a and pax2a were downregulated in brain proliferative zones. CONCLUSION: Lack of Vmat2 increases monoamine turnover and upregulates genes encoding amine-synthesising enzymes, including histidine decarboxylase. Notch1a and pax2a, genes implicated in stem cell development, are downregulated in mutants. The zebrafish vmat2 mutant strain may be a useful model to study how monoamine transport affects brain development and function, and for use in drug screening.

The Histamine System in Zebrafish Brain: Organization, Receptors, and Behavioral Roles.

Three of the four histamine receptors have been identified in zebrafish. Whereas only one histamine receptor 1 gene (hrh1) is known, two copies of histamine receptor 2 (hrh2a and hrh2b) have been identified. Although initially only one gene encoding for histamine receptor 3 (hrh3) was recognized in zebrafish, the genome database contains information for two more hrh3-like genes, whereas no genes corresponding for histamine receptor 4 with expression mainly in the immune system have been identified. Hrh1 and hrh3 show prominent uneven expression in the zebrafish brain, with the strongest expression in the dorsal telencephalon. Quantitatively significant expression of hrh1, hrh2, and hrh3 can also be found in several peripheral organs. Whereas antagonists of hrh1, hrh2, and hrh3 all affect the locomotor activity of zebrafish larvae, interpretation of the data is hampered by a lack of information on receptor binding and signaling characteristics. Zebrafish mutants lacking any of the three histamine receptors have shown modest behavioral phenotypes, possibly due to genetic compensation. None of the receptor mutant fish have shown significant sleep phenotypes. Adult zebrafish lacking hrh3 display decreased locomotor activity. The zebrafish histamine system shows significant life-long plasticity: presenilin 1 mutant zebrafish develop an abnormally large number of histamine neurons and increased thigmotaxis and anxiety-related phenotype. Overexpression of histidine decarboxylase (hdc) in larval zebrafish is associated with an increased number of hypocretin neurons, whereas translation inhibition of hdc or exposure to α-fluoromethylhistidine leads to decreased numbers of hypocretin neurons. Current pharmacological evidence suggests that this may be mediated by hrh1. Further studies using acute, e.g., pharmacogenetic or optogenetic manipulation of selected components of brain circuits, are required to understand the full range of physiological functions of zebrafish histamine receptors.

GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis.

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant (gch1-/-), using CRISPR/Cas technology. gch1-/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1-/- larvae improved survival without ameliorating the motor phenotype. RNAseq of gch1-/- larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1-/- The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.

Abnormal brain development of monoamine oxidase mutant zebrafish and impaired social interaction of heterozygous fish.

Monoamine oxidase (MAO) deficiency and imbalanced levels of brain monoamines have been associated with developmental delay, neuropsychiatric disorders and aggressive behavior. Animal models are valuable tools to gain mechanistic insight into outcomes associated with MAO deficiency. Here, we report a novel genetic model to study the effects of mao loss of function in zebrafish. Quantitative PCR, in situ hybridization and immunocytochemistry were used to study neurotransmitter systems and expression of relevant genes for brain development in zebrafish mao mutants. Larval and adult fish behavior was evaluated through different tests. Stronger serotonin immunoreactivity was detected in mao+/- and mao-/- larvae compared with their mao+/+ siblings. mao-/- larvae were hypoactive, and presented decreased reactions to visual and acoustic stimuli. They also had impaired histaminergic and dopaminergic systems, abnormal expression of developmental markers and died within 20 days post-fertilization. mao+/- fish were viable, grew until adulthood, and demonstrated anxiety-like behavior and impaired social interactions compared with adult mao+/+ siblings. Our results indicate that mao-/- and mao+/- mutants could be promising tools to study the roles of MAO in brain development and behavior. This article has an associated First Person interview with the first author of the paper.

Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.

LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

Histamine receptors, agonists, and antagonists in health and disease.

Histamine in the brain is produced by a group of tuberomamillary neurons in the posterior hypothalamus and a limited number of mast cells in different parts of the brain. Four G-protein-coupled receptors mediate the effects of histamine. Two of these receptors, H3 and H4 receptors, are high-affinity receptors in the brain and immune system, respectively. The two classic histamine receptors, H1 receptor and H2 receptor, are well known as drug targets for allergy and gastric ulcer, respectively. These receptors have lower affinity for histamine than the more recently discovered H3 and H4 receptors. The H1 and H2 receptors are important postsynaptic receptors in the brain, and they mediate many of the central effects of histamine on, e.g., alertness and wakefulness. H3 receptor is a pre- and postsynaptic receptor, which regulates release of histamine and several other neurotransmitters, including serotonin, GABA, and glutamate. H4 receptor is found in cerebral blood vessels and microglia, but its expression in neurons is not yet well established. Pitolisant, a H3 receptor antagonist, is used to treat narcolepsy and hypersomnia. H1 receptor antagonists have been used to treat insomnia, but its use requires precautions due to potential side effects. H2 receptor antagonists have shown efficacy in treatment of schizophrenia, but they are not in widespread clinical use. H4 receptor ligands may in the future be tested for neuroimmunological disorders and potentially neurodegenerative disorders in which inflammation plays a role, but clinical tests have not yet been initiated.

The bullies are the leaders of the next generation: Inherited aminergic neurotransmitter system changes in socially dominant zebrafish, Danio rerio.

We studied the social hierarchy in zebrafish and assessed differences in neurotransmitters and behavior in the F1 generation offspring of dominant and subordinate zebrafish (Danio rerio). We used behavioral assays to study locomotion, ability to complete cognitive tasks, social interaction and aggression. To study the neurochemical changes, we applied quantitative polymerase chain reaction, high pressure liquid chromatography and immunohistochemistry. Social hierarchies were formed both by males and females when animals were kept in same sex pairs in the dyadic dominant-subordinate hierarchy test. The offspring of dominant animals were the leaders in social interactions, however aggression in the mirror-test was not altered in any group. Serotonin and noradrenaline levels were lower in the F1 generation subordinate animals when compared with dominant animals, but not compared with animals that were naïve to social hierarchy. The mRNA level of the rate-limiting enzyme in histamine synthesis, histidine decarboxylase, was significantly lower in dominant and subordinate larval zebrafish when compared with control animals. In the dominant adult zebrafish tyrosine hydroxylase 1 mRNA level was lower compared with control animals, whereas tyrosine hydroxylase 2 mRNA was not different. The result was verified with immunohistochemistry. There were gender specific differences between the dominant and subordinate animals, where the dominant females performed better in cognitive tasks such as the T-maze than subordinate females. This was not observed in males, as the behavior of the dominant and subordinate males did not differ. These results add to the understanding of the plastic nature of the central nervous system and show that neurochemical features in aminergic neurotransmitter systems are associated with social leadership and dominance.

Cerebral Dopamine Neurotrophic Factor Regulates Multiple Neuronal Subtypes and Behavior.

Cerebral dopamine neurotrophic factor (CDNF) protects dopaminergic neurons against toxic damage in the rodent brain and is in clinical trials to treat Parkinson's disease patients. Yet the underlying mechanism is poorly understood. To examine its significance for neural circuits and behavior, we examined the development of neurotransmitter systems from larval to male adult mutant zebrafish lacking cdnf Although a lack of cdnf did not affect overall brain dopamine levels, dopaminergic neuronal clusters showed significant abnormalities. The number of histamine neurons that surround the dopaminergic neurons was significantly reduced. Expression of tyrosine hydroxylase 2 in the brain was elevated in cdnf mutants throughout their lifespan. There were abnormally few GABA neurons in the hypothalamus in the mutant larvae, and expression of glutamate decarboxylase was reduced throughout the brain. cdnf mutant adults showed a range of behavioral phenotypes, including increased sensitivity to pentylenetetrazole-induced seizures. Shoaling behavior of mutant adults was abnormal, and they did not display social attraction to conspecifics. CDNF plays a profound role in shaping the neurotransmitter circuit structure, seizure susceptibility, and complex behaviors in zebrafish. These findings are informative for dissecting the diverse functions of this poorly understood factor in human conditions related to Parkinson's disease and complex behaviors.SIGNIFICANCE STATEMENT A zebrafish lacking cdnf grows normally and shows no overt morphologic phenotype throughout the life span. Remarkably, impaired social cohesion and increased seizure susceptibility were found in adult cdnf KO fish conceivably associated with significant changes of dopaminergic, GABAergic, and histaminergic systems in selective brain areas. These findings suggest that cdnf has broad effects on regulating neurogenesis and maturation of transmitter-specific neuronal types during development and throughout adulthood, rather than ones restricted to the dopaminergic systems.

Increased Sensitivity of Mice Lacking Extrasynaptic δ-Containing GABAA Receptors to Histamine Receptor 3 Antagonists.

Histamine/gamma-aminobutyric acid (GABA) neurons of posterior hypothalamus send wide projections to many brain areas and participate in stabilizing the wake state. Recent research has suggested that GABA released from the histamine/GABA neurons acts on extrasynaptic GABAA receptors and balances the excitatory effect of histamine. In the current study, we show the presence of vesicular GABA transporter mRNA in a majority of quantified hypothalamic histaminergic neurons, which suggest vesicular release of GABA. As histamine/GABA neurons form conventional synapses infrequently, it is possible that GABA released from these neurons diffuses to target areas by volume transmission and acts on extrasynaptic GABA receptors. To investigate this hypothesis, mice lacking extrasynaptic GABAA receptor δ subunit (Gabrd KO) were used. A pharmacological approach was employed to activate histamine/GABA neurons and induce histamine and presumably, GABA, release. Control and Gabrd KO mice were treated with histamine receptor 3 (Hrh3) inverse agonists ciproxifan and pitolisant, which block Hrh3 autoreceptors on histamine/GABA neurons and histamine-dependently promote wakefulness. Low doses of ciproxifan (1 mg/kg) and pitolisant (5 mg/kg) reduced locomotion in Gabrd KO, but not in WT mice. EEG recording showed that Gabrd KO mice were also more sensitive to the wake-promoting effect of ciproxifan (3 mg/kg) than control mice. Low frequency delta waves, associated with NREM sleep, were significantly suppressed in Gabrd KO mice compared with the WT group. Ciproxifan-induced wakefulness was blocked by histamine synthesis inhibitor α-fluoromethylhistidine (αFMH). The findings indicate that both histamine and GABA, released from histamine/GABA neurons, are involved in regulation of brain arousal states and δ-containing subunit GABAA receptors are involved in mediating GABA response.

Genetic lack of histamine upregulates dopamine neurotransmission and alters rotational behavior but not levodopa-induced dyskinesia in a mouse model of Parkinson's disease.

The brain histaminergic and dopaminergic systems closely interact, and some evidence also suggests significant involvement of histamine in Parkinson's disease (PD), where dopaminergic neurons degenerate. To further investigate histamine-dopamine interactions, particularly in the context of PD, a genetic lack of histamine and a mouse model of PD and levodopa-induced dyskinesia were here combined. Dopaminergic lesions were induced in histidine decarboxylase knockout and wildtype mice by 6-hydroxydopamine injections into the medial forebrain bundle. Post-lesion motor dysfunction was studied by measuring drug-induced rotational behavior and dyskinesia. Striatal tissue from both lesioned and naïve animals was used to investigate dopaminergic, serotonergic and histaminergic biomarkers. Histamine deficiency increased amphetamine-induced rotation but did not affect levodopa-induced dyskinesia. qPCR measurements revealed increased striatal expression of D1 and D2 receptor, DARPP-32, and H3 receptor mRNA, and synaptosomal release experiments in naïve mice indicated increased dopamine release. A lack of histamine thus causes pre- and postsynaptic upregulation of striatal dopaminergic neurotransmission which may be reflected in post-lesion motor behavior. Disturbances or manipulations of the histaminergic system may thus have significant consequences for dopaminergic neurotransmission and motor behavior in both healthy and disease conditions. The findings also represent new evidence for the complex interplay between dopamine and histamine within the nigrostriatal pathway.

Histamine, histamine H3 receptor, and alcohol use disorder.

Alcohol use disorder is associated with several mental, physical, and social problems. Its treatment is difficult and often requires a combination of pharmacological and behavioural therapy. The brain histaminergic system, one of the wake-active systems that controls whole-brain activity, operates through three neuronal GPCRs. The histamine H3 receptor (Hrh3), which is expressed in many brain areas involved in alcohol drinking and alcohol reward, can be targeted with a number of drugs developed initially for cognitive disorders and/or disorders related to sleep, wakefulness, and alertness. In all rodent alcohol drinking models tested so far, H3 receptor antagonists have reduced alcohol drinking and alcohol-induced place preference and cue-induced alcohol reinstatement. Several H3 receptor antagonists tested and found to be safe for humans could be subjected to clinical tests to treat alcohol use disorder. Preference should be given to short-acting drugs to avoid the sleep problems associated with the wake-maintaining effects of the drugs. LINKED ARTICLES: This article is part of a themed section on New Uses for 21st Century. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.3/issuetoc.

LIN28B affects gene expression at the hypothalamic-pituitary axis and serum testosterone levels.

Genome-wide association studies (GWAS) have recurrently associated sequence variation nearby LIN28B with pubertal timing, growth and disease. However, the biology linking LIN28B with these traits is still poorly understood. With our study, we sought to elucidate the mechanisms behind the LIN28B associations, with a special focus on studying LIN28B function at the hypothalamic-pituitary (HP) axis that is ultimately responsible for pubertal onset. Using CRISPR-Cas9 technology, we first generated lin28b knockout (KO) zebrafish. Compared to controls, the lin28b KO fish showed both accelerated growth tempo, reduced adult size and increased expression of mitochondrial genes during larval development. Importantly, data from the knockout zebrafish models and adult humans imply that LIN28B expression has potential to affect gene expression in the HP axis. Specifically, our results suggest that LIN28B expression correlates positively with the expression of ESR1 in the hypothalamus and POMC in the pituitary. Moreover, we show how the pubertal timing advancing allele (T) for rs7759938 at the LIN28B locus associates with higher testosterone levels in the UK Biobank data. Overall, we provide novel evidence that LIN28B contributes to the regulation of sex hormone pathways, which might help explain why the gene associates with several distinct traits.

Abnormal behavior, striatal dopamine turnover and opioid peptide gene expression in histamine-deficient mice.

Hypothalamic histaminergic neurons regulate a variety of homeostatic, metabolic and cognitive functions. Recent data have suggested a modulatory role of histamine and histamine receptors in shaping striatal activity and connected the histaminergic system to neuropsychiatric disorders. We characterized exploratory behavior and striatal neurotransmission in mice lacking the histamine producing enzyme histidine decarboxylase (Hdc). The mutant mice showed a distinct behavioral pattern during exploration of novel environment, specifically, increased frequency of rearing seated against the wall, jumping and head/body shakes. This behavioral phenotype was associated with decreased levels of striatal dopamine and serotonin and increased level of dopamine metabolite DOPAC. Gene expression levels of dynorphin and enkephalin, opioids released by medium spiny neurons of striatal direct and indirect pathways respectively, were lower in Hdc mutant mice than in control animals. A low dose of amphetamine led to similar behavioral and biochemical outcomes in both genotypes. Increased striatal dopamine turnover was observed in Hdc KO mice after treatment with dopamine precursor l-Dopa. Overall, our study suggests a role for striatal dopamine and opioid peptides in formation of distinct behavioral phenotype of Hdc KO mice.