Activity Of Histaminergic Neurons Research Articles

Top-down modulation of the retinal code via histaminergic neurons of the hypothalamus.

The mammalian retina is considered an autonomous circuit, yet work dating back to Ramon y Cajal indicates that it receives inputs from the brain. How such inputs affect retinal processing has remained unknown. We confirmed brain-to-retina projections of histaminergic neurons from the mouse hypothalamus. Histamine application ex vivo altered the activity of various retinal ganglion cells (RGCs), including direction-selective RGCs that gained responses to high motion velocities. These results were reproduced in vivo with optic tract recordings where histaminergic retinopetal axons were activated chemogenetically. Such changes could improve vision of fast-moving objects (e.g., while running), which fits with the known increased activity of histaminergic neurons during arousal. An antihistamine drug reduced optomotor responses to high-speed moving stimuli in freely moving mice. In humans, the same antihistamine nonuniformly modulated visual sensitivity across the visual field, indicating an evolutionary conserved function of the histaminergic system. Our findings expose a previously unappreciated role for brain-to-retina projections in modulating retinal function.

Chemogenetic activation or inhibition of histaminergic neurons bidirectionally modulates recognition memory formation and retrieval in male and female mice.

Several lines of evidence demonstrate that the brain histaminergic system is fundamental for cognitive processes and the expression of memories. Here, we investigated the effect of acute silencing or activation of histaminergic neurons in the hypothalamic tuberomamillary nucleus (TMNHA neurons) in vivo in both sexes in an attempt to provide direct and causal evidence of the necessary role of these neurons in recognition memory formation and retrieval. To this end, we compared the performance of mice in two non-aversive and non-rewarded memory tests, the social and object recognition memory tasks, which are known to recruit different brain circuitries. To directly establish the impact of inactivation or activation of TMNHA neurons, we examined the effect of specific chemogenetic manipulations during the formation (acquisition/consolidation) or retrieval of recognition memories. We consistently found that acute chemogenetic silencing of TMNHA neurons disrupts the formation or retrieval of both social and object recognition memory in males and females. Conversely, acute chemogenetic activation of TMNHA neurons during training or retrieval extended social memory in both sexes and object memory in a sex-specific fashion. These results suggest that the formation or retrieval of recognition memory requires the tonic activity of histaminergic neurons and strengthen the concept that boosting the brain histaminergic system can promote the retrieval of apparently lost memories.

1532-P: A Novel Light-Responsive Adipose–Hypothalamus Axis in Metabolic Regulation

Light plays an essential role in life. Animals and humans possess light-sensing photoreceptors in visual and non-visual organs. We and others recently showed a non-visual role for Opsin3 (Opn3) in regulating adipose function. In this study, we discovered that direct blue light exposure to subcutaneous white adipose tissue (WAT) could reduce lipids accumulation in WAT and improve high-fat diet-induced metabolic abnormalities in an Opn3-dependent manner. Adipose tissue is an orchestrated matrix comprising high innervation that can engage in crosstalk with other organs. Particularly, it communicates with the brain via the sympathetic nervous system (SNS). Using the metabolomic approach, we found that blue light increased circulating levels of histidine, which then triggered the activation of histaminergic neurons in the hypothalamus and turned on brown adipose tissue via SNS. The administration of histidine decarboxylase antagonist blunted the effects of blue light. Taken together, these data demonstrate a novel light-responsive adipose-hypothalamus axis in metabolic regulation and provide a potential light-based therapeutic approach for obesity. Disclosure T.Tsuji: None. T.Huang: None. Y.Zhang: None. M.Lynes: None. G.Profeta: None. N.Narain: None. M.Kiebish: None. Y.Tseng: Consultant; Cellarity, LyGenesis. Funding American Heart Association (903968)

Galanin inhibition of wake-active histaminergic neurons

Histaminergic neurons are a population of wake-active neurons confined to the tuberomammillary nucleus (TMN) in the posterior hypothalamus. They form a crucial component of the ‘sleep switch’ in the brain and promote arousal when activated. The histaminergic neurons are strongly innervated by ventrolateral preoptic area (VLPO) neurons expressing galanin, a neuropeptide associated with sleep promotion. While recent studies have confirmed the importance of VLPO projections to the TMN in initiating and maintaining sleep, the ability of galanin to modulate the activity of the wake-promoting histaminergic neurons of the TMN has largely been overlooked. Therefore, we aimed to determine the mechanisms by which galanin alters the electrical excitability of genetically identified histaminergic neurons. We hypothesized that galanin inhibits histaminergic neuron activity via direct post-synaptic activation of galanin 1 receptors (GalR1). Whole-cell patch-clamp electrophysiology was performed on hypothalamic brain slices from mice expressing tdTomato fluorescence in cells expressing histidine decarboxylase (HDC), the sole enzyme required for histamine synthesis (HDCCre::tdTomato mice). RNAscope ® in situ hybridization was used to explore Gal1R mRNA expression in HDC expressing neurons. Whole-cell current-clamp recordings in male mice revealed that galanin (100nM) inhibits the majority of HDC neurons. The galanin-induced inhibition was associated with a hyperpolarization of the membrane potential and a decrease in firing frequency. Consistent with our hypothesis, a selective galanin 2 receptor (Gal2R) agonist (M1145 - 200nM) had no effect on male HDC neuron activity. RNAscope ® studies in male mice revealed co-expression of Gal1R mRNA in a large proportion of HDC-expressing neurons. Interestingly, the galanin-induced inhibition of HDC neuron electrical excitability was less pronounced in female mice. Overall, our data point to an important role of post-synaptic Gal1Rs expressed on histaminergic neurons in mediating galanin’s inhibitory effects in the TMN. Our data also suggest that sex differences exist in histaminergic neuron responsiveness to galanin. These findings are consistent with the idea that some of the sleep promoting effects of VLPO neuron activation occur through galanin’s ability to inhibit the wake-active histaminergic neurons. Sleep Research Society (SRS), Canada First Research Excellence Fund (Sentinel North Partnered Research Chair in Sleep Pharmacometabolism), Fondation Institut Universitaire de Cardiologie et de Pneumologie de Quebec (FIUCPQ), Fonds de recherche de Quebec - Sante (FRQS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antagonism of histamine H3 receptor promotes angiogenesis following focal cerebral ischemia.

Our previous study showed that H3 receptor antagonists reduced neuronal apoptosis and cerebral infarction in the acute stage after cerebral ischemia, but through an action independent of activation of histaminergic neurons. Because enhanced angiogenesis facilitates neurogenesis and neurological recovery after ischemic stroke, we herein investigated whether antagonism of H3R promoted angiogenesis after brain ischemia. Photothrombotic stroke was induced in mice. We showed that administration of H3R antagonist thioperamide (THIO, 10 mg·kg-1·d-1, i.p., from D1 after cerebral ischemia) significantly improved angiogenesis assessed on D14, and attenuated neurological defects on D28 after cerebral ischemia. Compared with wild-type mice, Hrh3-/- mice displayed more blood vessels in the ischemic boundary zone on D14, and THIO administration did not promote angiogenesis in these knockout mice. THIO-promoted angiogenesis in mice was reversed by i.c.v. injection of H3R agonist immepip, but not by H1 and H2 receptor antagonists, histidine decarboxylase inhibitor α-fluoromethylhistidine, or histidine decarboxylase gene knockout (HDC-/-), suggesting that THIO-promoted angiogenesis was independent of activation of histaminergic neurons. In vascular endothelial cells (bEnd.3), THIO (10-9-10-7 M) dose-dependently facilitated cell migration and tube formation after oxygen glucose deprivation (OGD), and H3R knockdown caused similar effects. We further revealed that H3R antagonism reduced the interaction between H3R and Annexin A2, while knockdown of Annexin A2 abrogated THIO-promoted angiogenesis in bEnd.3 cells after OGD. Annexin A2-overexpressing mice displayed more blood vessels in the ischemic boundary zone, which was reversed by i.c.v. injection of immepip. In conclusion, this study demonstrates that H3R antagonism promotes angiogenesis after cerebral ischemia, which is independent of activation of histaminergic neurons, but related to the H3R on vascular endothelial cells and its interaction with Annexin A2. Thus, H3R antagonists might be promising drug candidates to improve angiogenesis and neurological recovery after ischemic stroke.

Pitolisant in an Adolescent with Prader-Willi Syndrome.

This case report evaluates the potential benefit of pitolisant in a 15-year-old female with Prader-Willi syndrome, obsessive-compulsive disorder, autism spectrum disorder, and mild intellectual disability. Due to its action on the H3 receptor, it enhances central activity of histaminergic neurons resulting in increased alertness, irrespective of the loss of orexin neurons seen in narcolepsy. Additionally, it is thought to modulate various other neurotransmitter systems including acetylcholine, norepinephrine, and dopamine. Pitolisant has the potential to improve many symptoms in patients with Prader-Willi syndrome and it appears to be well tolerated with minimal side effects observed. Therefore, the use of pitolisant should be considered in patients with Prader-Willi syndrome who fail a psychostimulant trial.

A Novel Potent and Selective Histamine H3 Receptor Antagonist Enerisant: In Vitro Profiles, In Vivo Receptor Occupancy, and Wake-Promoting and Procognitive Effects in Rodents.

Histamine H3 receptor antagonists/inverse agonists are known to enhance the activity of histaminergic neurons in the brain, thereby promoting arousal and cognition. Here, we report the in vitro and in vivo pharmacological profiles for a newly synthesized histamine H3 receptor antagonist/inverse agonist: [1-(4-{3-[(2R)-2-methylpyrrolidin-1-yl]propoxy}phenyl)-1H-pyrazol-4-yl](morpholin-4-yl)methanone monohydrochloride (enerisant hydrochloride). In vitro assays showed that enerisant was a competitive antagonist/inverse agonist with a high affinity and selectivity for human and rat histamine H3 receptors. Enerisant showed antagonist activity in vivo, as assessed using R-α-methylhistamine (a histamine H3 receptor agonist)-induced dipsogenia, and occupied the histamine H3 receptor in the frontal cortex in a dose-dependent manner. Enerisant also enhanced the extracellular levels of histamine in the posterior hypothalamus and the levels of dopamine and acetylcholine in the medial prefrontal cortex of rats. Enerisant exerted a procognitive effect or reversed scopolamine-induced cognitive impairment in a social recognition test and a novel object recognition test in rats at doses at which less than 50% of the histamine H3 receptor were occupied (0.03-0.3 mg/kg, p.o.). In contrast, higher doses (3-10 mg/kg, p.o.) at which nearly all the histamine H3 receptors were occupied were needed to exert wake-promoting effects in rats. These results indicate that enerisant is a potent and selective histamine H3 receptor antagonist/inverse agonist with the potential to promote arousal and procognition in rats. Moreover, the results also suggest that the histamine H3 receptor occupancy required to exert a pharmacological effect may vary depending on the domain that is being tested. SIGNIFICANCE STATEMENT: Enerisant is a novel histamine H3 receptor antagonist/inverse agonist that exerts wake-promoting and procognitive effects in addition to increasing the release of neurotransmitters related to these pharmacological effects in rodents. Moreover, an in vivo receptor binding study revealed that the in vivo occupancy of the histamine H3 receptor required to exert the pharmacological effects of enerisant varied, and such variations in required occupancy should be taken into account when performing dose selection in clinical studies.

0008 SUVN-G3031, A Histamine H3 Receptor Inverse Agonist Produces Robust Wake Promoting and Anticataplectic Activity in Orexin Knockout Mice

Abstract Introduction Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness, sleep paralysis, hallucinations, and in some cases episodes of cataplexy. Results from animal studies indicate the involvement of deficient orexin transmission in narcolepsy which can be circumvented by the activation of histaminergic neurons. SUVN-G3031 is a potent and selective histamine H3 receptor inverse agonist with hKi of 8.7 nM and shows less than 50% inhibition at 1 µM against 70 other targets. SUVN-G3031 exhibited excellent pharmacokinetic properties and brain penetration in preclinical species. Oral administration of SUVN-G3031 produces significant increase in histamine, dopamine and norepinephrine levels in the rat cortex. Long-term safety studies in animals have been successfully completed without any concern for further development of SUVN-G3031. In the present study, the effects of SUVN-G3031 were evaluated in orexin knockout mice, a reliable animal model of narcolepsy as a proof-of-concept study for the treatment of narcolepsy with and without cataplexy. Methods Male orexin knockout mice (10 - 15 weeks old, 25 - 35 g at the time of surgery) were implanted with telemetric device for simultaneous monitoring of electroencephalography (EEG) and electromyography. Animals were allowed surgical recovery of 3 weeks prior to EEG recording. Effects of SUVN-G3031 (3 and 10 mg/kg, p.o.) were evaluated during active period of animals. Results SUVN-G3031 produced significant increase in wakefulness with concomitant decrease in non-rapid eye movement sleep in orexin knockout mice. SUVN-G3031 also significantly decreased the number of cataplectic episodes in orexin knockout mice. Conclusion Results from the current preclinical study provide a strong basis for the utility of SUVN-G3031 for the treatment of narcolepsy with and without cataplexy. SUVN-G3031 is currently being evaluated in a Phase 2 study as monotherapy for the treatment of narcolepsy with and without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). Support None

Melanocortin regulation of histaminergic neurons via perifornical lateral hypothalamic melanocortin 4 receptors

ObjectiveHistaminergic neurons of the tuberomammillary nucleus (TMN) are wake-promoting and contribute to the regulation of energy homeostasis. Evidence indicates that melanocortin 4 receptors (MC4R) are expressed within the TMN. However, whether the melanocortin system influences the activity and function of TMN neurons expressing histidine decarboxylase (HDC), the enzyme required for histamine synthesis, remains undefined. MethodsWe utilized Hdc-Cre mice in combination with whole-cell patch-clamp electrophysiology and in vivo chemogenetic techniques to determine whether HDC neurons receive metabolically relevant information via the melanocortin system. ResultsWe found that subsets of HDC-expressing neurons were excited by melanotan II (MTII), a non-selective melanocortin receptor agonist. Use of melanocortin receptor selective agonists (THIQ, [D-Trp8]-γ-MSH) and inhibitors of synaptic transmission (TTX, CNQX, AP5) indicated that the effect was mediated specifically by MC4Rs and involved a glutamatergic dependent presynaptic mechanism. MTII enhanced evoked excitatory post-synaptic currents (EPSCs) originating from electrical stimulation of the perifornical lateral hypothalamic area (PeFLH), supportive of melanocortin effects on the glutamatergic PeFLH projection to the TMN. Finally, in vivo chemogenetic inhibition of HDC neurons strikingly enhanced the anorexigenic effects of intracerebroventricular administration of MTII, suggesting that MC4R activation of histaminergic neurons may restrain the anorexigenic effects of melanocortin system activation. ConclusionsThese experiments identify a functional interaction between the melanocortin and histaminergic systems and suggest that HDC neurons act naturally to restrain the anorexigenic effect of melanocortin system activation. These findings may have implications for the control of arousal and metabolic homeostasis, especially in the context of obesity, in which both processes are subjected to alterations.

The effect of mastication on food intake, satiety and body weight

As mastication is the major component of the oral processing of solid foods a better understanding of its influence on ingestion, digestion and metabolism may lead to new approaches to improve health. A growing number of studies provide evidence that mastication may influence energy balance through several routes: activation of histaminergic neurons, reducing eating rate, altered digestion kinetics, and changes in macronutrient availability. Indeed, accumulating evidence indicates that increasing the number of masticatory cycles before swallowing reduces food intake and increases satiety. However, while slowing eating rate has been shown to limit weight gain in children and adolescents it is not clear that slowing eating rate by increasing the number of masticatory cycles or slowing mastication rate is a viable method to aid weight management ([10], [15]). Further research is required to determine the influence of mastication on energy balance and how it could be manipulated to aid weight management. This narrative review will provide a brief overview of the effect of mastication on food intake, satiety and body weight.

Hypothalamic Tuberomammillary Nucleus Neurons: Electrophysiological Diversity and Essential Role in Arousal Stability.

Histaminergic (HA) neurons, found in the posterior hypothalamic tuberomammillary nucleus (TMN), extend fibers throughout the brain and exert modulatory influence over numerous physiological systems. Multiple lines of evidence suggest that the activity of HA neurons is important in the regulation of vigilance despite the lack of direct, causal evidence demonstrating its requirement for the maintenance of arousal during wakefulness. Given the strong correlation between HA neuron excitability and behavioral arousal, we investigated both the electrophysiological diversity of HA neurons in brain slices and the effect of their acute silencing in vivo in male mice. For this purpose, we first validated a transgenic mouse line expressing cre recombinase in histidine decarboxylase-expressing neurons (Hdc-Cre) followed by a systematic census of the membrane properties of both HA and non-HA neurons in the ventral TMN (TMNv) region. Through unsupervised hierarchical cluster analysis, we found electrophysiological diversity both between TMNv HA and non-HA neurons, and among HA neurons. To directly determine the impact of acute cessation of HA neuron activity on sleep-wake states in awake and behaving mice, we examined the effects of optogenetic silencing of TMNv HA neurons in vivo We found that acute silencing of HA neurons during wakefulness promotes slow-wave sleep, but not rapid eye movement sleep, during a period of low sleep pressure. Together, these data suggest that the tonic firing of HA neurons is necessary for the maintenance of wakefulness, and their silencing not only impairs arousal but is sufficient to rapidly and selectively induce slow-wave sleep.SIGNIFICANCE STATEMENT The function of monoaminergic systems and circuits that regulate sleep and wakefulness is often disrupted as part of the pathophysiology of many neuropsychiatric disorders. One such circuit is the posterior hypothalamic histamine (HA) system, implicated in supporting wakefulness and higher brain function, but has been difficult to selectively manipulate owing to cellular heterogeneity in this region. Here we use a transgenic mouse to interrogate both the characteristic firing properties of HA neurons and their specific role in maintaining wakefulness. Our results demonstrate that the acute, cell type-specific silencing of HA neurons during wakefulness is sufficient to not only impair arousal but to rapidly and selectively induce slow-wave sleep. This work furthers our understanding of HA-mediated mechanisms that regulate behavioral arousal.

Hypocretin/orexin loss changes the hypothalamic immune response

Hypocretin, also known as orexin, maintains the vigilance state and regulates various physiological processes, such as arousal, sleep, food intake, energy expenditure, and reward. Previously, we found that when wild-type mice and hypocretin/ataxin-3 littermates (which are depleted of hypothalamic hypocretin-expressing neurons postnatally) were administered lipopolysaccharide (LPS), the two genotypes exhibited significant differences in their sleep/wake cycle, including differences in the degree of increase in sleep periods and in recovery from sickness behaviour. In the present study, we examined changes in the hypothalamic vigilance system and in the hypothalamic expression of inflammatory factors in response to LPS in hypocretin/ataxin-3 mice. Peripheral immune challenge with LPS affected the hypothalamic immune response and vigilance states. This response was altered by the loss of hypocretin. Hypocretin expression was inhibited after LPS injection in both hypocretin/ataxin-3 mice and their wild-type littermates, but expression was completely abolished only in hypocretin/ataxin-3 mice. Increases in the number of histidine decarboxylase (HDC)-positive cells and in Hdc mRNA expression were found in hypocretin/ataxin-3 mice, and this increase was suppressed by LPS. Hypocretin loss did not impact the change in expression of hypothalamic inflammatory factors in response to LPS, except for interferon gamma and colony stimulating factor 3. The number of c-Fos-positive/HDC-positive cells in hypocretin/ataxin-3 mice administered LPS injections was elevated, even during the rest period, in all areas, suggesting that there is an increase in the activity of histaminergic neurons in hypocretin/ataxin-3 mice following LPS injection. Taken together, our results suggest a novel role for hypocretin in the hypothalamic response to peripheral immune challenge. Our findings contribute to the understanding of the pathophysiology of narcolepsy.

GABAergic Inhibition of Histaminergic Neurons Regulates Active Waking But Not the Sleep–Wake Switch or Propofol-Induced Loss of Consciousness

The activity of histaminergic neurons in the tuberomammillary nucleus (TMN) of the hypothalamus correlates with an animal's behavioral state and maintains arousal. We examined how GABAergic inputs onto histaminergic neurons regulate this behavior. A prominent hypothesis, the "flip-flop" model, predicts that increased and sustained GABAergic drive onto these cells promotes sleep. Similarly, because of the histaminergic neurons' key hub-like place in the arousal circuitry, it has also been suggested that anesthetics such as propofol induce loss of consciousness by acting primarily at histaminergic neurons. We tested both these hypotheses in mice by genetically removing ionotropic GABA(A) or metabotropic GABA(B) receptors from histidine decarboxylase-expressing neurons. At the cellular level, histaminergic neurons deficient in synaptic GABA(A) receptors were significantly more excitable and were insensitive to the anesthetic propofol. At the behavioral level, EEG profiles were recorded in nontethered mice over 24 h. Surprisingly, GABAergic transmission onto histaminergic neurons had no effect in regulating the natural sleep-wake cycle and, in the case of GABA(A) receptors, for propofol-induced loss of righting reflex. The latter finding makes it unlikely that the histaminergic TMN has a central role in anesthesia. GABA(B) receptors on histaminergic neurons were dispensable for all behaviors examined. Synaptic inhibition of histaminergic cells by GABA(A) receptors, however, was essential for habituation to a novel environment.

Sleep and Anesthesia

Sleep and Anesthesia

The histamine H3 receptor: from discovery to clinical trials with pitolisant

The third histamine receptor was discovered in 1983 by a traditional pharmacological approach, consisting of assessing the inhibitory effect of histamine on its own release from depolarized rat brain slices. The same in vitro test was used to design, in 1987, the first highly selective and potent H3-autoreceptor ligands, the antagonist thioperamide and the agonist (R)alphamethylhistamine which enhances and inhibits, respectively, the activity of histaminergic neurons in brain. The use of these research tools was instrumental in establishing the main functions of cerebral histaminergic neurons, namely their role in maintenance of wakefulness, attention, learning and other cognitive processes. In 1990, the cloning of the gene of the H3-receptor, a member of the superfamily of heptahelical receptors coupled to G proteins, paved the way to the demonstration of the high constitutive activity of the receptor, including its native form, and its participation in the tonic control of histamine release; it also facilitated the development of H3-receptor inverse agonist programs in many drug companies. Pitolisant (BF2.649, 1-{3-[3-(4-chlorophenyl)propoxy]propyl}piperidine, hydrochloride) is the first inverse agonist to be introduced in the clinics. Its wake-promotion activity was evidenced in excessive diurnal sleepiness of patients with narcolepsy, Parkinson's disease or Obstructive Sleep Apnea/Hypopnea, in which this activity is characterized by a mean decrease of the Epworth Sleepiness Scale by about five units. The procognitive activity of this novel class of drugs may also find therapeutic applications in dementias, schizophrenia or attention deficit hyperactivity disorder.

Innervation of histamine neurons in the caudal part of the arcuate nucleus of hypothalamus and their activation in response to food deprivation under scheduled feeding

It has been well established that histaminergic neurons innervate densely the anterior hypothalamus and regulate several functions through the histamine H₁ receptor (H1R). However, the physiological function of the histaminergic neurons in other regions including the posterior hypothalamus has not been fully investigated. Recently, we have found a selective c-Fos expression in the caudal part of the arcuate nucleus of the hypothalamus (cARC) by food deprivation under scheduled feeding in rats. In this study, we histochemically examined the correlation of this c-Fos expression with the activation of histaminergic neurons in this region using an anti-H1R antibody. Strong H1R immunoreactivity was observed in the perikarya of the c-Fos positive cells. Abundant histamine-containing fibers were also found in the cARC and in the area between the cARC and the tuberomammillary nucleus (TM), where the histaminergic neuronal cell bodies are exclusively distributed. Our morphological observations suggest that c-Fos expression in the cARC by food deprivation under scheduled feeding is caused by the activation of histaminergic neurons projected from the TM.

The brain H 3-receptor as a novel therapeutic target for vigilance and sleep–wake disorders

Brain histaminergic neurons play a prominent role in arousal and maintenance of wakefulness (W). H 3-receptors control the activity of histaminergic neurons through presynaptic autoinhibition. The role of H 3-receptor antagonists/inverse agonists (H 3R-antagonists) in the potential therapy of vigilance deficiency and sleep–wake disorders were studied by assessing their effects on the mouse cortical EEG and sleep–wake cycle in comparison to modafinil and classical psychostimulants. The H 3R-antagonists, thioperamide and ciproxifan increased W and cortical EEG fast rhythms and, like modafinil, but unlike amphetamine and caffeine, their waking effects were not accompanied by sleep rebound. Conversely, imetit (H 3R-agonist) enhanced slow wave sleep and dose-dependently attenuated ciproxifan-induced W, indicating that the effects of both ligands involve H 3-receptor mechanisms. Additional studies using knockout (KO) mice confirmed the essential role of H 3-receptors and histamine-mediated transmission in the wake properties of H 3R-antagonists. Thus ciproxifan produced no increase in W in either histidine-decarboxylase (HDC, histamine-synthesizing enzyme) or H 1- or H 3-receptor KO-mice whereas its waking effects persisted in H 2-receptor KO-mice. These data validate the hypothesis that H 3R-antagonists, through disinhibition of H 3-autoreceptors, enhancing synaptic histamine that in turn activates postsynaptic H 1-receptors promoting W. Interestingly amphetamine and modafinil, despite their potent arousal effects, appear unlikely to depend on histaminergic mechanism as their effects still occurred in HDC KO-mice. The present study thus distinguishes two classes of wake-improving agents: the first acting through non-histaminergic mechanisms and the second acting via histamine and supports brain H 3-receptors as potentially novel therapeutic targets for vigilance and sleep–wake disorders.

BF2.649 [1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, Hydrochloride], a Nonimidazole Inverse Agonist/Antagonist at the Human Histamine H3 Receptor: Preclinical Pharmacology

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity approximately 50% higher than that of ciproxifan. Its in vitro potency was approximately 6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

Specific activation of histaminergic neurons during daily feeding anticipatory behavior in rats

When food is available during a restricted and predictable time of the day, animals show increased locomotor and food searching behaviors before the anticipated daily meal. We had shown that histamine-containing neurons are the only aminergic neurons related to arousal that become active in anticipation of an upcoming meal. To further map, the brain regions involved in the expression of the feeding-anticipatory behavior, we quantified the expression of Fos in hypothalamic areas involved in arousal. We found that nearly 35% of the histamine neurons from the tuberomammillary nucleus were Fos-immunoreactive immediately before mealtime. One hour before this transient increase in Fos-immunoreactivity, we found a similarly brief increase of fos mRNA in the tuberomammillary nucleus. In contrast, the activation of two types of perifornical hypothalamic neurons followed meal onset by 1–2 h. One neuron type was orexin/hypocretin-immunoreactive, while the other type was neither orexin nor melanin concentrating hormone-immunoreactive. The present work indicates that the increased locomotor activity that anticipates mealtime coincides with the activation of the tuberomammillary nucleus, and that the behavioral activation during the consummatory phase of feeding coincides more closely with the delayed activation of the perifornical hypothalamic area.

Wake-related activity of tuberomammillary neurons in rats

Histaminergic neurons of the tuberomammillary nucleus (TMN) are hypothesized to promote wakefulness, but little is known about the activity of these cells during spontaneous behavior. We measured histaminergic neuron activity in the dorsomedial, ventrolateral, and caudal TMN at four different times using Fos and adenosine deaminase immunohistochemistry and recordings of sleep/wake behavior. Because circadian factors could influence neuronal activity, we then assessed TMN neuron activity in predominantly sleeping or awake animals, all killed at the same time of day. In both experiments, Fos expression in histaminergic neurons of all three TMN subnuclei was higher during periods of wakefulness. These results demonstrate that histaminergic neurons throughout the TMN are wake-active, and this activity is largely independent of the time of day.