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J Neurophysiol ; 104(2): 873-84, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20519576


Dopamine (DA) modifies the motor pattern generated by the pyloric network in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, by directly acting on each of the circuit neurons. The 14 pyloric neurons fall into six cell types, and DA actions are cell type specific. The transient potassium current mediated by shal channels (I(A)) is a common target of DA modulation in most cell types. DA shifts the voltage dependence of I(A) in opposing directions in pyloric dilator (PD) versus lateral pyloric (LP) neurons. The mechanism(s) underpinning cell-type specific DA modulation of I(A) is unknown. DA receptors (DARs) can be classified as type 1 (D1R) or type 2 (D2R). D1Rs and D2Rs are known to increase and decrease intracellular cAMP concentrations, respectively. We hypothesized that the opposing DA effects on PD and LP I(A) were due to differences in DAR expression patterns. In the present study, we found that LP expressed somatodendritic D1Rs that were concentrated near synapses but did not express D2Rs. Consistently, DA modulation of LP I(A) was mediated by a Gs-adenylyl cyclase-cAMP-protein kinase A pathway. Additionally, we defined antagonists for lobster D1Rs (flupenthixol) and D2Rs (metoclopramide) in a heterologous expression system and showed that DA modulation of LP I(A) was blocked by flupenthixol but not by metoclopramide. We previously showed that PD neurons express D2Rs, but not D1Rs, thus supporting the idea that cell specific effects of DA on I(A) are due to differences in receptor expression.

Dopamina/farmacologia , Rede Nervosa/fisiologia , Neurônios/efeitos dos fármacos , Potássio/metabolismo , Piloro/citologia , Receptores de Dopamina D1/fisiologia , Transdução de Sinais/efeitos dos fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Colforsina/farmacologia , AMP Cíclico/metabolismo , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica/métodos , Gânglios dos Invertebrados/citologia , Técnicas In Vitro , Isoquinolinas/farmacologia , Rede Nervosa/efeitos dos fármacos , Palinuridae , Técnicas de Patch-Clamp/métodos , Éteres Fosfolipídicos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piloro/efeitos dos fármacos , Piloro/fisiologia , Transdução de Sinais/fisiologia , Sulfonamidas/farmacologia , Tetraetilamônio/farmacologia , Tetrodotoxina/farmacologia
J Comp Neurol ; 518(3): 255-76, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19941347


Dopamine (DA) modulates motor systems in phyla as diverse as nematodes and arthropods up through chordates. A comparison of dopaminergic systems across a broad phylogenetic range should reveal shared organizing principles. The pyloric network, located in the stomatogastric ganglion (STG), is an important model for neuromodulation of motor networks. The effects of DA on this network have been well characterized at the circuit and cellular levels in the spiny lobster, Panulirus interruptus. Here we provide the first data about the physical organization of the DA signaling system in the STG and the function of D(2) receptors in pyloric neurons. Previous studies showed that DA altered intrinsic firing properties and synaptic output in the pyloric dilator (PD) neuron, in part by reducing calcium currents and increasing outward potassium currents. We performed single cell reverse transcriptase-polymerase chain reaction (RT-PCR) experiments to show that PD neurons exclusively expressed a type 2 (D(2alphaPan)) DA receptor. This was confirmed by using confocal microscopy in conjunction with immunohistochemistry (IHC) on STG whole-mount preparations containing dye-filled PD neurons. Immunogold electron microscopy showed that surface receptors were concentrated in fine neurites/terminal swellings and vesicle-laden varicosities in the synaptic neuropil. Double-label IHC experiments with tyrosine hydroxylase antiserum suggested that the D(2alphaPan) receptors received volume neurotransmissions. Receptors were further mapped onto three-dimensional models of PD neurons built from Neurolucida tracings of confocal stacks from the IHC experiments. The data showed that D(2alphaPan) receptors were selectively targeted to approximately 40% of synaptic structures in any given PD neuron, and were nonuniformly distributed among neurites.

Sistema Nervoso Entérico/metabolismo , Gânglios dos Invertebrados/metabolismo , Neurônios/metabolismo , Palinuridae/metabolismo , Receptores de Dopamina D2/genética , Sinapses/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Forma Celular/fisiologia , Dopamina/biossíntese , Sistema Nervoso Entérico/ultraestrutura , Corantes Fluorescentes , Gânglios dos Invertebrados/ultraestrutura , Trato Gastrointestinal/inervação , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Microscopia Confocal , Microscopia Imunoeletrônica , Neurônios/ultraestrutura , Palinuridae/ultraestrutura , Comunicação Parácrina/fisiologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo
J Neurochem ; 104(4): 1006-19, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17986222


Neuromodulators, such as dopamine (DA), control motor activity in many systems. To begin to understand how DA modulates motor behaviors, we study a well-defined model: the crustacean stomatogastric nervous system (STNS). The spiny lobster STNS receives both neuromodulatory and neurohormonal dopaminergic input, and extensive background information exists on the cellular and network effects of DA. However, there is a void of information concerning the mechanisms of DA signal transduction in this system. In this study, we show that Gs, Gi, and Gq are activated in response to DA in STNS membrane preparations from five crustacean species representing distant clades in the order Decapoda. Three evolutionarily conserved DA receptors mediate this response in spiny lobsters: D(1alphaPan), D(1betaPan) and D(2alphaPan). G protein coupling for these receptors can vary with the cell type. In the native membrane, the D(1alphaPan) receptor couples with Gs and Gq, the D(1betaPan) receptor couples with Gs, and the D(2alphaPan) receptor couples with Gi. All three receptors are localized exclusively to the synaptic neuropil and most likely generate global biochemical signals that alter ion channels in distant compartments, as well as local signals.

Gânglios dos Invertebrados/química , Gânglios dos Invertebrados/metabolismo , Palinuridae , Receptores Dopaminérgicos/análise , Receptores Dopaminérgicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Estômago/inervação , Animais , Linhagem Celular , Mucosa Gástrica/metabolismo , Humanos , Receptores Acoplados a Proteínas-G/análise , Estômago/química
Artigo em Inglês | MEDLINE | ID: mdl-17134931


The pyloric network is an important model system for understanding neuromodulation of rhythmic motor behaviors like breathing or walking. Dopamine (DA) differentially modulates neurons within the pyloric network. However, while the electrophysiological actions of DA have been well characterized, nothing is known about the signaling events that mediate its effects. We have begun a molecular characterization of DA receptors (DARs) in this invertebrate system. Here, we describe the cloning and characterization of the lobster D(2) receptor, D(2 alpha Pan). We found that when expressed in HEK cells, the D(2 alpha Pan) receptor is activated by DA, but not other monoamines endogenous to the lobster nervous system. This receptor positively couples with cAMP through multiple Gi/o proteins via two discrete pathways: 1) a G alpha mediated inhibition of adenylyl cyclase (AC), leading to a decrease in cAMP and 2) a G beta gamma-mediated activation of phospholipase C beta (PLC beta), leading to an increase in cAMP. Alternate splicing alters the potency and efficacy of the receptor, but does not affect monoamine specificity. Finally, we show that arthropod D(2) receptor coupling with cAMP varies with the cellular milieu.

AMP Cíclico/metabolismo , Dopaminérgicos/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Palinuridae/metabolismo , Receptores de Dopamina D2/metabolismo , Transdução de Sinais/fisiologia , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Dopamina/farmacologia , Humanos , Dados de Sequência Molecular , Palinuridae/genética , Receptores de Dopamina D2/genética , Transdução de Sinais/efeitos dos fármacos
Comp Biochem Physiol B Biochem Mol Biol ; 143(3): 294-301, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16426885


Dopamine (DA) differentially modulates identified neurons in the crustacean stomatogastric nervous system (STNS). While the electrophysiological actions of DA have been well characterized, little is known about the dopaminergic transduction cascades operating in this system. As a first step toward illuminating the molecular underpinnings of dopaminergic signal transduction in the crustacean STNS, we have cloned and characterized two type-one DA receptors (DARs) from the spiny lobster (Panulirus interruptus): D(1alphaPan) and D(1betaPan). We found that the structure and function of these arthropod DARs are well conserved across species. Using a heterologous expression system, we determined that DA, but not serotonin, octopamine, tyramine or histamine activates these receptors. When stably expressed in HEK cells, the D(1alphaPan) receptor couples with Gs, and DA elicits an increase in [cAMP]. The D(1betaPan) receptor responds to DA with a net increase in [cAMP] that is mediated by Gs and Gz.

Palinuridae/fisiologia , Receptores de Dopamina D1/metabolismo , Animais , Células Cultivadas , Clonagem Molecular , Sequência Conservada , AMP Cíclico/metabolismo , Dopamina/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Dados de Sequência Molecular , Palinuridae/genética , Palinuridae/metabolismo , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/genética
J Neurosci ; 24(13): 3421-35, 2004 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-15056722


The stomatogastric nervous system (STNS) is a premiere model for studying modulation of motor pattern generation. Whereas the cellular and network responses to monoamines have been particularly well characterized electrophysiologically, the transduction mechanisms that link the different monoaminergic signals to specific intracellular responses are presently unknown in this system. To begin to elucidate monoaminergic signal transduction in pyloric neurons, we used a bioinformatics approach to predict the existence of 18 monoamine receptors in arthropods, 9 of which have been previously cloned in Drosophila and other insects. We then went on to use the two existing insect databases to clone and characterize the 10th putative arthropod receptor from the spiny lobster, Panulirus interruptus. This receptor is most homologous to the 5-HT2 subtype and shows a dose-dependent response to 5-HT but not to any of the other monoamines present in the STNS. Through a series of pharmacological experiments, we demonstrate that this newly described receptor, 5-HT2betaPan, couples with the traditional G(q) pathway when expressed in HEK293 cells, but not to G(s) or G(i/o). Moreover, it is constitutively active, because the highly conserved DRY motif in transmembrane region 3 has evolved into DRF. Site-directed mutagenesis that reverts the motif back to DRY abolishes this agonist-independent activity. We further demonstrate that this receptor most likely participates in the modulation of stomatogastric motor output, because it is found in neurites in the synaptic neuropil of the stomatogastric ganglion as well as in the axon terminals at identified pyloric neuromuscular junctions.

Neurotransmissores/metabolismo , Palinuridae/fisiologia , Receptores de Neurotransmissores/metabolismo , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Aminas Biogênicas/farmacologia , Aminas Biogênicas/fisiologia , Linhagem Celular , Clonagem Molecular , Biologia Computacional/métodos , Sequência Conservada/fisiologia , Sistema Digestório/inervação , Drosophila/genética , Evolução Molecular , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Rede Nervosa/metabolismo , Rede Nervosa/fisiologia , Palinuridae/genética , Proteína Quinase C/metabolismo , Receptores de Neurotransmissores/agonistas , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Fosfolipases Tipo C/metabolismo