Characterization of Drosophila octopamine receptor neuronal expression using MiMIC-converted Gal4 lines.

Octopamine, the invertebrate analog of norepinephrine, is known to modulate a large variety of behaviors in Drosophila including feeding initiation, locomotion, aggression, and courtship, among many others. Significantly less is known about the identity of the neurons that receive octopamine input and how they mediate octopamine-regulated behaviors. Here, we characterize adult neuronal expression of MiMIC-converted Trojan-Gal4 lines for each of the five Drosophila octopamine receptors. Broad neuronal expression was observed for all five octopamine receptors, yet distinct differences among them were also apparent. Use of immunostaining for the octopamine neurotransmitter synthesis enzyme Tdc2, along with a novel genome-edited conditional Tdc2-LexA driver, revealed all five octopamine receptors express in Tdc2/octopamine neurons to varying degrees. This suggests autoreception may be an important circuit mechanism by which octopamine modulates behavior.

High motivation for exercise is associated with altered chromatin regulators of monoamine receptor gene expression in the striatum of selectively bred mice.

Although exercise is critical for health, many lack the motivation to exercise, and it is unclear how motivation might be increased. To uncover the molecular underpinnings of increased motivation for exercise, we analyzed the transcriptome of the striatum in four mouse lines selectively bred for high voluntary wheel running and four non-selected control lines. The striatum was dissected and RNA was extracted and sequenced from four individuals of each line. We found multiple genes and gene systems with strong relationships to both selection and running history over the previous 6 days. Among these genes were Htr1b, a serotonin receptor subunit and Slc38a2, a marker for both glutamatergic and γ-aminobutyric acid (GABA)-ergic signaling. System analysis of the raw results found enrichment of transcriptional regulation and kinase genes. Further, we identified a splice variant affecting the Wnt-related Golgi signaling gene Tmed5. Using coexpression network analysis, we found a cluster of interrelated coexpression modules with relationships to running behavior. From these modules, we built a network correlated with running that predicts a mechanistic relationship between transcriptional regulation by nucleosome structure and Htr1b expression. The Library of Integrated Network-Based Cellular Signatures identified the protein kinase C δ inhibitor, rottlerin, the tyrosine kinase inhibitor, Linifanib and the delta-opioid receptor antagonist 7-benzylidenenaltrexone as potential compounds for increasing the motivation to run. Taken together, our findings support a neurobiological framework of exercise motivation where chromatin state leads to differences in dopamine signaling through modulation of both the primary neurotransmitters glutamate and GABA, and by neuromodulators such as serotonin.

Developmental expression analysis and immunolocalization of a biogenic amine receptor in Schistosoma mansoni.

A Schistosoma mansoni G-protein coupled receptor (SmGPCR) was previously cloned and shown to be activated by the biogenic amine, histamine. Here we report a first investigation of the receptor's subunit organization, tissue distribution and expression levels in different stages of the parasite. A polyclonal antibody was produced in rabbits against the recombinant third intracellular loop (il3) of SmGPCR. Western blot studies of the native receptor and recombinant protein expressed in HEK293 cells showed that SmGPCR exists both as a monomer (65 kDa) and an apparent dimer of approximately 130 kDa These species were verified by immunoprecipitation of SmGPCR from S. mansoni extracts, using antibody that was covalently attached to agarose beads. Further investigation determined that the SmGPCR dimer was resistant to treatment with various detergents, 4 M urea and 0.1 M DTT but could be made to dissociate at acidic pH, suggesting the dimer is non-covalent in nature. Confocal immunofluorescence studies revealed significant SmGPCR immunoreactivity in sporocysts, schistosomula and adult worms but not miracidia. SmGPCR was found to be most widely expressed in the schistosomula, particularly the tegument, the subtegumental musculature and the acetabulum. In the adult stage we detected SmGPCR immunofluorescence mainly in the tubercles of male worms and, to a lesser extent, the body wall musculature. Localization in sporocysts was mainly confined to the tegument and cells within parenchymal matrices. A real-time quantitative reverse-transcription PCR analysis revealed that SmGPCR is upregulated at the mRNA level in the parasitic stages compared to the free-living miracidium and cercariae, and it is particularly elevated during early sporocyst and schistosomula development. The results identify SmGPCR as an important parasite receptor with potential functions in muscle and the tegument of S. mansoni.

Identification of critical structural determinants responsible for octopamine binding to the alpha-adrenergic-like Bombyx mori octopamine receptor.

Octopamine (OA) is a biogenic amine with a widespread distribution in the insect nervous system. OA modulates and/or regulates various behavioral patterns of insects as a neurotransmitter, neuromodulator, and neurohormone. OA receptors (OARs) belong to one of the families of G protein-coupled receptors (GPCRs). The binding of OA to OARs is coupled to the activation of the specific G proteins, which induces the release of intracellular second messengers such as cAMP and/or calcium. We previously reported the isolation of an OAR (BmOAR1) from Bombyx mori. In the study presented here, five mutated BmOAR1s were constructed with a point mutation in the putative binding crevice and expressed in HEK-293 cells. The S202A mutant receptor was found to retain the cAMP response to OA as does the wild-type receptor, but such function was impaired in the other four mutants (D103A, S198A, Y412F, and S198A/S202A). Furthermore, competition binding assays using [3H]OA and calcium mobilization assays gave results that were approximately consistent with those of the cAMP assays. Taken together, the results indicate that D103 and S198 are involved in the binding and activation of BmOAR1 with OA through electrostatic or hydrogen bond interactions, but S202 does not appear to participate in this process. Y412 seems to be involved in one of the active forms of BmOAR1. These findings should prove helpful in designing new pest control chemicals.

Molecular cloning and heterologous expression of an alpha-adrenergic-like octopamine receptor from the silkworm Bombyx mori.

A cDNA encoding an octopamine (OA) receptor (BmOAR1) was isolated from the nerve tissue of silkworm (Bombyx mori) larvae. Comparison of amino acid sequences showed that BmOAR1 is highly identical to OA receptors isolated from Periplaneta americana (Pa oa(1)), Apis mellifera (AmOA1), and Drosophila melanogaster (OAMB or DmOA1A). BmOAR1 was stably expressed in HEK-293 cells. OA above 1 microM led to an increase in intracellular cyclic AMP concentration ([cAMP](i)). The synthetic OA-receptor agonist demethylchlordimeform also elevated [cAMP](i) to the same maximal level (approximately 5-fold over the basal level) as that induced by OA. However, other biogenic amines, tyramine and dopamine, and chlordimeform were without effects. The [cAMP](i) level raised by OA was lowered by antagonists; the rank order of antagonist activity was chlorpromazine > mianserin = yohimbine. Cyproheptadine and metoclopramide had little effect. OA above 100 nM induced a transient or sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), depending on the concentration of OA. Sequence homology and functional analysis data indicate that BmOAR1 is an alpha-adrenergic-like OA receptor of B. mori.

TYRA-2 (F01E11.5): a Caenorhabditis elegans tyramine receptor expressed in the MC and NSM pharyngeal neurons.

Tyramine appears to regulate key processes in nematodes, such as pharyngeal pumping, and more complex behaviors, such as foraging. Recently, a Caenorhabditis elegans tyramine receptor, SER-2, was identified that is involved in the TA-dependent regulation of these processes. In the present study, we have identified a second C. elegans gene, tyra-2 (F01E11.5) that encodes a tyramine receptor. This is the first identification of multiple tyramine receptor genes in any invertebrate. Membranes from COS-7 cells expressing TYRA-2 bind [(3)H]tyramine with high affinity with a K(d) of 20 +/- 5 nM. Other physiologically relevant biogenic amines, such as octopamine and dopamine, inhibit [(3)H]tyramine binding with much lower affinity (K(i)s of 1.55 +/- 0.5 and 1.78 +/- 0.6 microM, respectively), supporting the identification of TYRA-2 as a tyramine receptor. Indeed, tyramine also dramatically increases GTPgammaS binding to membranes from cells expressing TYRA-2 (EC(50) of 50 +/- 13 nM) and the TA-dependent GTPgammaS binding is PTX-sensitive suggesting that TYRA-2 may couple to Galpha(i/o). Based on fluorescence from tyra::gfp fusion constructs, TYRA-2 expression appears to be exclusively neuronal in the MC and NSM pharyngeal neurons, the AS family of amphid neurons and neurons in the nerve ring, body and tail. Taken together, these results suggest that TYRA-2 encodes a second Galpha(i/o)-coupled tyramine receptor and suggests that TA-dependent neuromodulation may be mediated by multiple receptors and more complex than previously appreciated.

A family of octopamine [corrected] receptors that specifically induce cyclic AMP production or Ca2+ release in Drosophila melanogaster.

In invertebrates, the biogenic-amine octopamine is an important physiological regulator. It controls and modulates neuronal development, circadian rhythm, locomotion, 'fight or flight' responses, as well as learning and memory. Octopamine mediates its effects by activation of different GTP-binding protein (G protein)-coupled receptor types, which induce either cAMP production or Ca(2+) release. Here we describe the functional characterization of two genes from Drosophila melanogaster that encode three octopamine receptors. The first gene (Dmoa1) codes for two polypeptides that are generated by alternative splicing. When heterologously expressed, both receptors cause oscillatory increases of the intracellular Ca(2+) concentration in response to applying nanomolar concentrations of octopamine. The second gene (Dmoa2) codes for a receptor that specifically activates adenylate cyclase and causes a rise of intracellular cAMP with an EC(50) of approximately 3 x 10(-8) m octopamine. Tyramine, the precursor of octopamine biosynthesis, activates all three receptors at > or = 100-fold higher concentrations, whereas dopamine and serotonin are non-effective. Developmental expression of Dmoa genes was assessed by RT-PCR. Overlapping but not identical expression patterns were observed for the individual transcripts. The genes characterized in this report encode unique receptors that display signature properties of native octopamine receptors.

Aminergic receptors in astrogliotic plaques from patients with multiple sclerosis.

Cultured astrocytes express a spectrum of neurotransmitter receptors. However, little is known about these receptors in situ. We previously reported the absence of beta(2) adrenergic receptors on astrocytes in multiple sclerosis (MS). Here we used [(3)H]-radioligands and receptor autoradiography to screen for a variety of other aminergic receptors in six silent chronic astrogliotic plaques in brain tissue obtained from five patients with MS. Dopamine D(1) and histamine H(1) receptors were absent. We detected specific binding for cholinergic muscarinic receptors > dopamine D(2), alpha(1-) and alpha(2)-adrenergic receptors > 5-HT(1A), 5-HT(1B/D), 5-HT(2A), 5-HT(2c), 5-HT(4), and dopamine D(3) receptors. Radiotracers for these aminergic receptors might be useful for studying astrogliosis in patients with MS, and compounds acting at some of these receptors may have potential to modulate astroglial function in MS.

Cultured insect mushroom body neurons express functional receptors for acetylcholine, GABA, glutamate, octopamine, and dopamine.

Fluorescence calcium imaging with fura-2 and whole cell, patch-clamp electrophysiology was applied to cultured Kenyon cells (interneurons) isolated from the mushroom bodies of adult crickets (Acheta domesticus) to demonstrate the presence of functional neurotransmitter receptors. In all cells investigated, 5 microM acetylcholine (ACh, n = 52) evoked an increase in intracellular free calcium ([Ca2+]i). Similar effects were observed in response to 10 microM nicotine. The ACh response was insensitive to atropine (50 microM) but was reduced by mecamylamine (50 microM) and alpha-bungarotoxin (alpha-bgt, 10 microM). ACh-induced inward ion currents (n = 28, EACh approximately 0 mV) were also blocked by 1 microM mecamylamine and by 1 microM alpha-bgt. Nicotine-induced inward currents desensitized more rapidly than ACh responses. Thus functional alpha-bgt-sensitive nicotinic ACh receptors are abundant on all Kenyon cells tested, and their activation leads to an increase in [Ca2+]i. gamma-Aminobutyric acid (GABA, 100 microM) triggered a sustained decrease in [Ca2+]i. Similar responses were seen with a GABAA agonist, muscimol (100 microM), and a GABAB agonist, 3-APPA (1 mM), suggesting that more than one type of GABA receptor can affect [Ca2+]i. This action of GABA was not observed when the extracellular KCl concentration was lowered. All cells tested (n = 26) with patch-clamp electrophysiology showed picrotoxinin (PTX)-sensitive, GABA-induced (30-100 microM) currents with a chloride-sensitive reversal potential. Thus, an ionotropic PTX-sensitive GABA receptor was found on all Kenyon cells tested. Most (61%) of the 54 cells studied responded to -glutamate (100 microM) application either with a biphasic increase in [Ca2+]i or with a single, delayed, sustained [Ca2+]i increase. Nearly all cells tested (95%, n = 19) responded to (100 microM) -glutamate with rapidly desensitizing, inward currents that reversed at approximately -30 mV. Dopamine (100 microM) elicited either a rapid or a delayed increase in [Ca2+]i in 63% of the 26 cells tested. The time course of these responses varied greatly among cells. Dopamine failed to elicit currents in patch-clamped cells (n = 4). A brief decrease in [Ca2+]i was induced by octopamine (100 microM) in approximately 54% of the cells tested (n = 35). However, when extracellular CaCl2 was lowered, octopamine triggered a substantial increase in [Ca2+]i in 35% of the cells tested (n = 26). No octopamine-elicited currents were detected in patched-clamped cells (n = 10).