Histamine-gated Chloride Channel Research Articles

Cys-loop ligand-gated ion channel superfamily of Pardosa pseudoannulata: Implication for natural enemy safety

Cys-loop ligand-gated channels mediate neurotransmission in insects and are receptors for many insecticides. Some insecticides acting on cysLGIC also have lethal effects on non-targeting organisms, but the mechanism of this negative effect is unclear due to information absence. The identification and analysis of cysLGIC family in Pardosa pseudoannulata, a pond wolf spider, can deepen the understanding of insecticides for natural enemy safety. Thirty-four cysLGIC genes were identified in P. pseudoannulata genome, including nicotinic acetylcholine receptors, γ-aminobutyric acid gated chloride channels, glutamate-gated chloride channels, histamine-gated chloride channels, and pH-sensitive chloride channels. The expansion of GABACls and HisCls accounts for the large number of cysLGICs in P. pseudoannulata, and the alternative splicing events in nAChR and RDL subunits enriched the diversity of the superfamily. Most cysLGIC genes show the highest expression in brain and lowest expression in the early-egg sac stage. Variable residues (R81, V83, R135, N137, F190, and W197) in P. pseudoannulata nAChR β subunits and critical differences in α6 subunit TM4 region compared with insects would apply for the insensitivity to neonicotinoids and spinosyn. In contrast, avermectin and dieldrin may be lethal to P. pseudoannulata due to the similar drugs binding sites in GluCls compared with insects. These findings will provide a valuable clue for natural enemy protection and environmentally friendly insecticide development.

Identification and pharmacological characterization of histamine-gated chloride channels in the fall armyworm, Spodoptera frugiperda

Histamine-gated chloride channels (HACls) mediate fast inhibitory neurotransmission in invertebrate nervous systems and have important roles in light reception, color processing, temperature preference and light-dark cycle. The fall armyworm, Spodoptera frugiperda is a main destructive pest of grain and row crops. However, the pharmacological characterization of HACls in S. frugiperda remain unknown. In this study, we identified two cDNAs encoding SfHACl1 and SfHACl2 in S. frugiperda. They had similar expression patterns and were most abundantly expressed in the head of larvae and at the egg stage. Electrophysiological analysis with the two-electrode voltage clamp method showed that histamine (HA) and γ-aminobutyric acid (GABA) activated inward currents when SfHACls were singly or collectively expressed with different ratios in Xenopus laevis oocytes. These channels were ≥2000-fold more sensitive to HA than to GABA. They were anion-selective channels, which were highly dependent on changes in external chloride concentrations, but insensitive to changes in external sodium concentrations. The insecticides abamectin (ABM) and emamectin benzoate (EB) also activated these channels with the EC50 to SfHACl1 lower than that to SfHACl2. And the EC50s of ABM and EB to the co-expressed channels gradually increased with increase in the injection ratio of SfHACl2 cRNA. Homology models and docking simulations revealed that HA bound to the large amino-terminal extracellular domain of SfHACl1 and SfHACl2 by forming 4 and 2 hydrogen bonds, respectively. The docking simulations of ABM and EB had similar binding sites in the transmembrane regions. Overall, these findings indicated that HACls act as targets for macrolide, and this study provides theoretical guidance for further derivatization of abamectin insecticides.

In silico identification and assessment of insecticide target sites in the genome of the small hive beetle, Aethina tumida

BackgroundThe small hive beetle, Aethina tumida, is a rapidly emerging global pest of honey bee colonies. Small hive beetle infestation can be extremely destructive, which may cause honey bees to abscond and render colony infrastructure unusable. Due to the impacts small hive beetles have on honey bees, a wide variety of physical, cultural, and chemical control measures have been implemented to manage small hive beetle infestations. The use of insecticides to control small hive beetle populations is an emerging management tactic. Currently, very little genomic information exists on insecticide target sites in the small hive beetle. Therefore, the objective of this study is to utilize focused in silico comparative genomics approaches to identify and assess the potential insecticide sensitivity of the major insecticide target sites in the small hive beetle genome.ResultsNo previously described resistance mutations were identified in any orthologs of insecticide target sites. Alternative exon use and A-to-I RNA editing were absent in AtumSC1. The ryanodine receptor in small hive beetle (Atum_Ryr) was highly conserved and no previously described resistance mutations were identified. A total of 12 nAChR subunits were identified with similar alternative exon use in other insects. Alternative exon use and critical structural features of the GABA-gated chloride channel subunits (Atum_RDL, Atum_GRD, and Atum_LCCH3) were conserved. Five splice variants were found for the glutamate-gated chloride channel subunit. Exon 3c of Atum_GluCl may be a beetle-specific alternative exon. The co-occurrence of exons 9a and 9b in the pH-sensitive chloride channel (Atum_pHCl) is a unique combination that introduces sites of post-translational modification. The repertoire and alternative exon use for histamine-gated chloride channels (Atum-HisCl), octopamine (Atum_OctR) and tyramine receptors (Atum_TAR) were conserved.ConclusionsThe recently published small hive beetle genome likely serves as a reference for insecticide-susceptible versions of insecticide target sites. These comparative in silico studies are the first step in discovering targets that can be exploited for small hive beetle-specific control as well as tracking changes in the frequency of resistance alleles as part of a resistance monitoring program. Comparative toxicity alongside honey bees is required to verify these in silico predictions.

Identification of putative amine receptor complement in the eyestalk of the crayfish, Procambarus clarkii.

In decapod crustaceans, the amines dopamine, octopamine, serotonin, and histamine are known to serve as locally released and/or circulating neuromodulators. While many studies have focused on determining the modulatory actions of amines on decapod nervous systems, comparatively little is known about the identity of the receptors through which they exert their actions. Here, a crayfish, Procambarus clarkii, tissue-specific transcriptome was used to identify putative amine receptors in the eyestalk, a structure composed largely of the eyestalk ganglia, including the neuroendocrine X-organ-sinus gland system, and retina. Transcripts encoding 17 distinct putative amine receptors, three dopamine (one dopamine 1-like, one dopamine 2-like, and one dopamine/ecdysteroid-like), five octopamine (one alpha-like, three beta-like, and one octopamine/tyramine-like), three serotonin (two type-1-like and one type-7-like), and six histamine (five histamine-gated chloride channel A-like and one histamine-gated chloride channel B-like) were identified in the assembly. Comparison of the nucleotide sequence of the transcript encoding one predicted type-1-like serotonin receptor with that cloned previously from the P. clarkii nervous system shows the two sequences to be essentially identical, providing increased support for the validity of the transcripts used to deduce the proteins reported here. Reciprocal BLAST and structural/functional domain analyses support the protein family annotations ascribed to the putative P. clarkii receptors. These data represent the first large-scale description of amine receptors from P. clarkii, and as such provide a new resource for initiating gene-based studies of aminergic control of physiology/behavior at the level of receptors in this species.

Immunolocalization suggests a role of the histamine-gated chloride channel PxHCLB in spectral opponent processing in butterfly photoreceptors.

Spectrally opponent responses, that is, wavelength-dependent inversions of response polarity, have been observed at the level of photoreceptors in butterflies. As inter-photoreceptor connections have been found in the butterfly Papilio xuthus, and histamine is the only neurotransmitter so far identified in insect photoreceptors, we hypothesize that histaminergic sign-inverting synapses exist in the lamina between different spectral receptors as a mechanism for spectral opponency as in the medulla of Drosophila. Here, we localized two histamine-gated chloride channels, PxHCLA (Drosophila Ort homolog) and PxHCLB (Drosophila HisCl1 homolog), in the visual system of Papilio xuthus by using specific antisera. The antiPxHCLA labeling was associated with the membrane of nonphotoreceptor cells that are postsynaptic to photoreceptors, while the antiPxHCLB labeling overlapped with photoreceptor axons, indicating that PxHCLB is expressed at inter-photoreceptor synapses: PxHCLB is likely involved in producing spectral opponency at the first visual synapses. Color processing in Papilio may appear earlier than previously hypothesized in insect visual systems.

Physiological responses of ionotropic histamine receptors, PxHCLA and PxHCLB, to neurotransmitter candidates in a butterfly, Papilio xuthus.

Histamine is the only known neurotransmitter released by arthropod photoreceptors. Synaptic transmission from photoreceptors to second-order neurons is mediated by the activation of histamine-gated chloride channels (HCLs). These histaminergic synapses have been assumed to be conserved among insect visual systems. However, our understanding of the channels in question has thus far been based on studies in flies. In the butterfly Papilio xuthus, we have identified two candidate histamine-gated chloride channels, PxHCLA and PxHCLB, and studied their physiological properties using a whole-cell patch-clamp technique. We studied the responses of channels expressed in cultured cells to histamine as well as to other neurotransmitter candidates, namely GABA, tyramine, serotonin, d-/l-glutamate and glycine. We found that histamine and GABA activated both PxHCLA and PxHCLB, while the other molecules did not. The sensitivity to histamine and GABA was consistently higher in PxHCLB than in PxHCLA. Interestingly, simultaneous application of histamine and GABA activated both PxHCLA and PxHCLB more strongly than either neurotransmitter individually; histamine and GABA may have synergistic effects on PxHCLs in the regions where they co-localize. Our results suggest that the physiological properties of the histamine receptors are basically conserved among insects, but that the response to GABA differs between butterflies and flies, implying variation in early visual processing among species.

Neurotoxic Effects of Linalool and β-Pinene on Tribolium castaneum Herbst.

Effective, ethical pest control requires the use of chemicals that are highly specific, safe, and ecofriendly. Linalool and β-pinene occur naturally as major constituents of the essential oils of many plant species distributed throughout the world, and thus meet these requirements. These monoterpenes were tested as repellents against Tribolium castaneum, using the area preference method, after four hours of exposure and the effect transcriptional of genes associated with neurotransmission. Changes in gene expression of acetylcholinesterase (Ace1), GABA-gated anion channel splice variant 3a6a (Rdl), GABA-gated ion channel (Grd), glutamate-gated chloride channel (Glucl), and histamine-gated chloride channel 2 (Hiscl2) were assessed and the interaction with proteins important for the insect using in silico methods was also studied. For linalool and β-pinene, the repellent concentration 50 (RC50) values were 0.11 µL/cm2 and 0.03 µL/cm2, respectively. Both compounds induced overexpression of Hiscl2 gen in adult insects, and β-pinene also promoted the overexpression of Grd and the Ace1 gene. However, β-pinene and linalool had little potential to dock on computer-generated models for GABA-gated ion channel LCCH3, nicotinic acetylcholine receptor subunits alpha1 and alpha2, and putative octopamine/tyramine receptor proteins from T. castaneum as their respective binding affinities were marginal, and therefore the repellent action probably involved mechanisms other than direct interaction with these targets. Results indicated that β-pinene was more potent than linalool in inducing insect repellency, and also had a greater capacity to generate changes in the expression of genes involved in neuronal transmission.

Multiple Signaling Pathways Coordinately Regulate Forgetting of Olfactory Adaptation through Control of Sensory Responses in Caenorhabditis elegans.

Forgetting memories is important for animals to properly respond to continuously changing environments. To elucidate the mechanisms of forgetting, we used one of the behavioral plasticities of Caenorhabditis elegans hermaphrodite, olfactory adaptation to an attractive odorant, diacetyl, as a simple model of learning. In C. elegans, the TIR-1/JNK-1 pathway accelerates forgetting of olfactory adaptation by facilitating neural secretion from AWC sensory neurons. In this study, to identify the downstream effectors of the TIR-1/JNK-1 pathway, we conducted a genetic screen for suppressors of the gain-of-function mutant of tir-1 (ok1052), which shows excessive forgetting. Our screening showed that three proteins-a membrane protein, MACO-1; a receptor tyrosine kinase, SCD-2; and its putative ligand, HEN-1-regulated forgetting downstream of the TIR-1/JNK-1 pathway. We further demonstrated that MACO-1 and SCD-2/HEN-1 functioned in parallel genetic pathways, and only MACO-1 regulated forgetting of olfactory adaptation to isoamyl alcohol, which is an attractive odorant sensed by different types of sensory neurons. In olfactory adaptation, odor-evoked Ca2+ responses in olfactory neurons are attenuated by conditioning and recovered thereafter. A Ca2+ imaging study revealed that this attenuation is sustained longer in maco-1 and scd-2 mutant animals than in wild-type animals like the TIR-1/JNK-1 pathway mutants. Furthermore, temporal silencing by histamine-gated chloride channels revealed that the neuronal activity of AWC neurons after conditioning is important for proper forgetting. We propose that distinct signaling pathways, each of which has a specific function, may coordinately and temporally regulate forgetting by controlling sensory responses.SIGNIFICANCE STATEMENT Active forgetting is an important process to understand the whole mechanisms of memories. Recent papers have reported that the noncell autonomous regulations are required for proper forgetting in invertebrates. We found that in Caenorhabditis elegans hermaphrodite, the noncell autonomous regulations of forgetting of olfactory adaptation is regulated by three conserved proteins: a membrane protein, MACO-1; a receptor tyrosine kinase, SCD-2: and its ligand, HEN-1. MACO-1 and SCD-2/HEN-1, working in coordination, accelerate forgetting by controlling sensory responses in parallel. Furthermore, temporal regulation of neuronal activity is important for proper forgetting. We suggest that multiple pathways may coordinately and temporally regulate forgetting through control of sensory responses. This study should lead to a better understanding of forgetting in higher organisms.

Toxicity of Naphthalene and Benzene on Tribollium castaneum Herbst.

Naphthalene and benzene are widely-used volatile organic compounds. The aim of this research was to examine the toxicological effects of naphthalene and benzene against Tribolium castaneum as an animal model. Adult insects were exposed to these aromatic compounds to assess mortality after 4–48 h of exposure. The lethal concentration 50 (LC50) for naphthalene, naphthalin, and benzene were 63.6 µL/L, 20.0 µL/L, and 115.9 µL/L in air, respectively. Real-time polymerase chain reaction (PCR) analysis revealed expression changes in genes related to oxidative stress and metabolism [Glutathione S-Transferase (Gst), and Cytochrome P450 6BQ8 (Cyp6bq8)]; reproduction and metamorphosis [Hormone receptor in 39-like protein (Hr39), Ecdysone receptor: (Ecr), and Chitin synthase 2 (Chs2)]; and neurotransmission [Histamine-gated chloride channel 2 (Hiscl2)] in insects exposed for 4 h to 70.2 µL/L naphthalene. Adults exposed to benzene (80 µL/L; 4 h) overexpressed genes related to neurotransmission [GABA-gated anion channel (Rdl), Hiscl2, and GABA-gated ion channel (Grd)]; reproduction and metamorphosis [Ultraspiracle nuclear receptor (USP), Ecr; and Hr39]; and development (Chs2). The data presented here provides evidence that naphthalene and benzene inhalation are able to induce alterations on reproduction, development, metamorphosis, oxidative stress, metabolism, neurotransmission, and death of the insect.

Pharmacological characterization of histamine-gated chloride channels from the housefly Musca domestica

The biogenic amine histamine (HA) is not only the neurotransmitter of photoreceptors but also has important roles in mechanosensory reception, temperature preference, sleep and olfactory processing in insects. Two cDNAs (MdhclA and MdhclB) that encode HA-gated chloride channel subunits (MdHCLA and MdHCLB) were cloned from the housefly Musca domestica. The cRNAs were injected into Xenopus laevis oocytes to examine the functions and pharmacological characteristics of MdHCLA and MdHCLB channels using a two-electrode voltage clamp method. HA was used to activate MdHCLA and MdHCLB channels to evoke inward currents with EC50s of 33.1μM and 6.28μM, respectively. 2-(3-Trifluoromethylphenyl)histamine, an HA H1 receptor agonist, was a partial agonist of MdHCLB receptors with an EC50 of 49.4μM. MdHCLB channels were also activated by γ-aminobutyric acid (GABA) and monoamines, such as octopamine, serotonin (5-HT) and dopamine (DA); 5-HT and DA also acted as competitive antagonists. GABA acted as a full agonist of MdHCLB receptors with an EC50 of 1.11mM. d-Tubocurarine, cimetidine and picrotoxinin were poor inhibitors of HA- and GABA-evoked currents in MdHCLB channels. Our data show that HCLB channels are more sensitive to agonists when compared with HCLA channels. HCLB channels are also affected by antagonists but insusceptible to known insecticides that target GABA- and glutamate-gated chloride channels.

Pharyngeal pumping in Caenorhabditis elegans depends on tonic and phasic signaling from the nervous system.

Rhythmic movements are ubiquitous in animal locomotion, feeding, and circulatory systems. In some systems, the muscle itself generates rhythmic contractions. In others, rhythms are generated by the nervous system or by interactions between the nervous system and muscles. In the nematode Caenorhabditis elegans, feeding occurs via rhythmic contractions (pumping) of the pharynx, a neuromuscular feeding organ. Here, we use pharmacology, optogenetics, genetics, and electrophysiology to investigate the roles of the nervous system and muscle in generating pharyngeal pumping. Hyperpolarization of the nervous system using a histamine-gated chloride channel abolishes pumping, and optogenetic stimulation of pharyngeal muscle in these animals causes abnormal contractions, demonstrating that normal pumping requires nervous system function. In mutants that pump slowly due to defective nervous system function, tonic muscle stimulation causes rapid pumping, suggesting tonic neurotransmitter release may regulate pumping. However, tonic cholinergic motor neuron stimulation, but not tonic muscle stimulation, triggers pumps that electrophysiologically resemble typical rapid pumps. This suggests that pharyngeal cholinergic motor neurons are normally rhythmically, and not tonically active. These results demonstrate that the pharynx generates a myogenic rhythm in the presence of tonically released acetylcholine, and suggest that the pharyngeal nervous system entrains contraction rate and timing through phasic neurotransmitter release.

Cys-loop ligand-gated ion channel gene discovery in the Locusta migratoria manilensis through the neuron transcriptome

As an ideal model, Locusta migratoria manilensis (Meyen) has been widely used in the study of endocrinological and neurobiological processes. Here we created a large transcriptome of the locust neurons, which enriched ion channels whose potential for functional genetic experiments is currently limited. With high-throughput Illumina sequencing technology, we obtained more than 50 million raw reads, which were assembled into 61,056 unique sequences with average size of 737bp. Among the unigenes, a total 24,884 sequences had significant similarities with proteins in the five public databases (NR, SwissProt, GO, COG and KEGG) with a cut-off E-value of 10−5 using BLASTx. Moreover, the number of potential genes of the cys-loop ligand-gated ion channels (LGICs) was manually curated, including 39 putative nicotinic acetylcholine receptors (nAChRs), 6 putative γ-aminobutyric acid (GABA) gated anion channels, 21 putative glutamate-gated chloride channels (GluCls) and 1 histamine-gated chloride channels (HisCls). In addition, the full-length of 11 nAChRs subunits (9 alpha and 2 beta) were obtained by RACE technique that would be helpful to further studies on nAChR neurochemistry and pharmacological aspects. To our knowledge, this is the first study to characterize the locust neuron transcriptome, which will provide a useful resource especially for future studies on the neuro-function and behavior of the locust.

Inducible and titratable silencing ofCaenorhabditis elegansneurons in vivo with histamine-gated chloride channels

Recent progress in neuroscience has been facilitated by tools for neuronal activation and inactivation that are orthogonal to endogenous signaling systems. We describe here a chemical-genetic approach for inducible silencing of Caenorhabditis elegans neurons in intact animals, using the histamine-gated chloride channel HisCl1 from Drosophila and exogenous histamine. Administering histamine to freely moving C. elegans that express HisCl1 transgenes in neurons leads to rapid and potent inhibition of neural activity within minutes, as assessed by behavior, functional calcium imaging, and electrophysiology of neurons expressing HisCl1. C. elegans does not use histamine as an endogenous neurotransmitter, and exogenous histamine has little apparent effect on wild-type C. elegans behavior. HisCl1-histamine silencing of sensory neurons, interneurons, and motor neurons leads to behavioral effects matching their known functions. In addition, the HisCl1-histamine system can be used to titrate the level of neural activity, revealing quantitative relationships between neural activity and behavioral output. We use these methods to dissect escape circuits, define interneurons that regulate locomotion speed (AVA, AIB) and escape-related omega turns (AIB), and demonstrate graded control of reversal length by AVA interneurons and DA/VA motor neurons. The histamine-HisCl1 system is effective, robust, compatible with standard behavioral assays, and easily combined with optogenetic tools, properties that should make it a useful addition to C. elegans neurotechnology.

Histamine-HisCl1 Receptor Axis Regulates Wake-Promoting Signals in Drosophila melanogaster

Histamine and its two receptors, histamine-gated chloride channel subunit 1 (HisCl1) and ora transientless (Ort), are known to control photoreception and temperature sensing in Drosophila. However, histamine signaling in the context of neural circuitry for sleep-wake behaviors has not yet been examined in detail. Here, we obtained mutant flies with compromised or enhanced histamine signaling and tested their baseline sleep. Hypomorphic mutations in histidine decarboxylase (HDC), an enzyme catalyzing the conversion from histidine to histamine, caused an increase in sleep duration. Interestingly, hisCl1 mutants but not ort mutants showed long-sleep phenotypes similar to those in hdc mutants. Increased sleep duration in hisCl1 mutants was rescued by overexpressing hisCl1 in circadian pacemaker neurons expressing a neuropeptide pigment dispersing factor (PDF). Consistently, RNA interference (RNAi)-mediated depletion of hisCl1 in PDF neurons was sufficient to mimic hisCl1 mutant phenotypes, suggesting that PDF neurons are crucial for sleep regulation by the histamine-HisCl1 signaling. Finally, either hisCl1 mutation or genetic ablation of PDF neurons dampened wake-promoting effects of elevated histamine signaling via direct histamine administration. Taken together, these data clearly demonstrate that the histamine-HisCl1 receptor axis can activate and maintain the wake state in Drosophila and that wake-activating signals may travel via the PDF neurons.

Transient and Specific Inactivation of Drosophila Neurons In Vivo Using a Native Ligand-Gated Ion Channel

A key tool in neuroscience is the ability to transiently inactivate specific neurons on timescales of milliseconds to minutes. In Drosophila, there are two available techniques for accomplishing this (shibire(ts) and halorhodopsin [1-3]), but both have shortcomings [4-9]. Here we describe a complementary technique using a native histamine-gated chloride channel (Ort). Ort is the receptor at the first synapse in the visual system. It forms large-conductance homomeric channels that desensitize only modestly in response to ligand [10]. Many regions of the CNS are devoid of histaminergic neurons [11, 12], raising the possibility that Ort could be used to artificially inactivate specific neurons in these regions. To test this idea, we performed in vivo whole-cell recordings from antennal lobe neurons misexpressing Ort. In these neurons, histamine produced a rapid and reversible drop in input resistance, clamping the membrane potential below spike threshold and virtually abolishing spontaneous and odor-evoked activity. Every neuron type in this brain region could be inactivated in this manner. Neurons that did not misexpress Ort showed negligible responses to histamine. Ort also performed favorably in comparison to the available alternative effector transgenes. Thus, Ort misexpression is a useful tool for probing functional connectivity among Drosophila neurons.

The cys-loop ligand-gated ion channel gene family of Tetranychus urticae: Implications for acaricide toxicology and a novel mutation associated with abamectin resistance

The cys-loop ligand-gated ion channel (cysLGIC) super family of Tetranychus urticae, the two-spotted spider mite, represents the largest arthropod cysLGIC super family described to date and the first characterised one within the group of chelicerates. Genome annotation, phylogenetic analysis and comparison of the cysLGIC subunits with their counterparts in insects reveals that the T. urticae genome encodes for a high number of glutamate- and histamine-gated chloride channel genes (GluCl and HisCl) compared to insects. Three orthologues of the insect γ-aminobutyric acid (GABA)-gated chloride channel gene Rdl were detected. Other cysLGIC groups, such as the nAChR subunits, are more conserved and have clear insect orthologues. Members of cysLGIC family mediate endogenous chemical neurotransmission and they are prime targets of insecticides. Implications for toxicology associated with the identity and specific features of T. urticae family members are discussed. We further reveal the accumulation of known and novel mutations in different GluCl channel subunits (Tu_GluCl1 and Tu_GluCl3) associated with abamectin resistance in T. urticae, and provide genetic evidence for their causality. Our study provides useful toxicological insights for the exploration of the T. urticae cysLGIC subunits as putative molecular targets for current and future chemical control strategies.

Histamine-gated ion channels in mammals?

There is ample pharmacological and physiological evidence for yet unidentified histamine receptors in mammalian brain that are linked to a Cl− conductance. In invertebrates, two histamine-gated chloride channels (HisCl α1 and α2) are already well known. HisCl channels are members of the Cys-loop receptor superfamily of ligand-gated ion channels and are closely related to the mammalian GABAA and glycine receptors (GlyR). Indeed, they share particularly strong homology within the ligand binding and ion channel domains. Here we discuss the possibility that mammalian HisCl channels might exist among the known GABAA or GlyR subunits. Studies published to date support this hypothesis, including evidence for direct histamine gating of GABAA β homomers, histamine potentiation of GABAA αβ and αβγ heteromeric receptors, and GABAA receptor blockade by some antihistamines. We explore what is known about the binding-site structure, function and pharmacology of invertebrate HisCl channels and other histamine binding sites to support and inform a broader search for HisCl channels among the mammalian GABAA and GlyR subunits. The discovery and identification of HisCl-like channels in mammals would not only enhance understanding of inhibitory signaling and histamine function in the mammalian brain, but also provide new avenues for development of therapeutic compounds targeting this novel histamine site. This commentary is therefore intended to foster consideration of a novel and potentially important target of histamine and histaminergic drugs in the CNS.

Histaminergic signaling in the central nervous system of Daphnia and a role for it in the control of phototactic behavior

Daphnia magna and Daphnia pulex are well-established model organisms in the fields of ecotoxicology and toxicogenomics. Among the many assays used for determining the effects of environmental and anthropogenic stressors on these animals is monitoring for changes in their phototactic behavior. In most arthropods, histamine has been shown to play a key role in the visual system. Currently, nothing is known about histaminergic signaling in either D. magna or D. pulex. Here, a combination of immunohistochemistry and genome mining was used to identify and characterize the histaminergic systems in these daphnids. In addition, a behavioral assay was used to assess the role of histamine in their phototactic response to ultraviolet (UV) light exposure. An extensive network of histaminergic somata, axons and neuropil was identified via immunohistochemistry within the central nervous system of both daphnids, including labeling of putative photoreceptors in the compound eye and projections from these cells to the brain. Mining of the D. pulex genome using known Drosophila melanogaster proteins identified a putative ortholog of histidine decarboxylase (the rate-limiting biosynthetic enzyme for histamine), as well as two putative histamine-gated chloride channels (hclA and hclB orthologs). Exposure of D. magna to cimetidine, an H2 receptor antagonist known to block both hclA and hclB in D. melanogaster, inhibited their negative phototactic response to UV exposure in a reversible, time-dependent manner. Taken collectively, our results show that an extensive histaminergic system is present in Daphnia species, including the visual system, and that this amine is involved in the control of phototaxis in these animals.

Cys-Loop Ligand-Gated Chloride Channels in Dorsal Unpaired Median Neurons ofLocusta migratoria

In insects, inhibitory neurotransmission is generally associated with members of the cys-loop ligand-gated anion channels, such as the glutamate-gated chloride channel (GluCl), the GABA-gated chloride channels (GABACl), and the histamine-gated chloride channels (HisCl). These ionotropic receptors are considered established target sites for the development of insecticides, and therefore it is necessary to obtain a better insight in their distribution, structure, and functional properties. Here, by combining electrophysiology and molecular biology techniques, we identified and characterized GluCl, GABACl, and HisCl in dorsal unpaired median (DUM) neurons of Locust migratoria. In whole cell patch-clamp recordings, application of glutamate, GABA, or histamine induced rapidly activating ionic currents. GluCls were sensitive to ibotenic acid and blocked by picrotoxin and fipronil. The pharmacological profile of the L. migratoria GABACl fitted neither the vertebrate GABA(A) nor GABA(C) receptor and was similar to the properties of the cloned Drosophila melanogaster GABA receptor subunit (Rdl). The expression of Rdl-like subunit-containing GABA receptors was shown at the molecular level using RT-PCR. Sequencing analysis indicated that the orthologous GABACl of D. melanogaster CG10357-A is expressed in DUM neurons of L. migratoria. Histamine-induced currents exhibited a fast onset and desensitized completely on continuous application of histamine. In conclusion, within the DUM neurons of L. migratoria, we identified three different cys-loop ligand-gated anion channels that use GABA, glutamate, or histamine as their neurotransmitter.

Functional consequences of mutations in the Drosophila histamine receptor HCLB

The gene hclB encodes a histamine-gated chloride channel subunit in Drosophila melanogaster. Mutations in hclB lead to defects in the visual system and altered sensitivity to the action of ivermectin. To investigate whether this member of the Cys-loop receptors is common across the Insecta, we analysed the genomes of seven other insect species (Diptera, Hymenoptera, Coleoptera) and revealed orthologues of hclB in all of them. Sequence comparisons showed high identity levels between the orthologues, indicating similar constraints and conserved function between the species. Two D. melanogaster mutants, hclBT1 (P293S) and hclBT2 (W111*, a null mutation) were tested for the lapse into, and recovery from, paralysis induced by high temperature or the anaesthetic action of halothane. At 41°C, the hclBT2 flies lapsed into coma faster than wild-type or the hclBT1 flies, while both mutants recovered more slowly. A substantially impaired recovery rate was also observed in hclBT1 after anaesthesia with halothane. Enhanced synaptic signalling at low-intensity light stimuli was registered on electroretinograms recorded from the two mutant strains. Our results suggest that HCLB may play an essential and conserved role in insect neurophysiology.