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1.
Eur J Pain ; 24(8): 1517-1536, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32446289

RESUMO

BACKGROUND: Unilateral injection of Complete Freund's Adjuvant (CFA) into the intra-plantar surface of the rodent hindpaw elicits chronic inflammation and hyperalgesia in the ipsilateral hindlimb. Mechanisms contributing to this hyperalgesia may act over multiple time courses and can include changes in ion channel expression and post-translational SUMOylation. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current, Ih . An HCN2-mediated increase in C-nociceptor Ih contributes to mechanical hyperalgesia in the CFA model of inflammatory pain. Changes in HCN2 post-translational SUMOylation and protein expression have not been systematically documented for a given dorsal root ganglia (DRG) throughout the time course of inflammation. METHODS: This study examined HCN2 protein expression and post-translational SUMOylation in a rat model of CFA-induced hindpaw inflammation. L5 DRG cryosections were used in immunohistochemistry experiments and proximity ligation assays to investigate HCN2 expression and SUMOylation, respectively, on days 1 and 3 post-CFA. RESULTS: Unilateral CFA injection elicited a significant bilateral increase in HCN2 staining intensity in small diameter DRG neurons on day 1 post-CFA, and a significant bilateral increase in the number of small neurons expressing HCN2 but not staining intensity on day 3 post-CFA. HCN2 channels were hyper-SUMOylated in small diameter neurons of ipsilateral relative to contralateral DRG on days 1 and 3 post-CFA. CONCLUSIONS: Unilateral CFA injection elicits unilateral mechanical hyperalgesia, a bilateral increase in HCN2 expression and a unilateral increase in post-translational SUMOylation. This suggests that enhanced HCN2 expression in L5 DRG is not sufficient for mechanical hyperalgesia in the early stages of inflammation and that hyper-SUMOylation of HCN2 channels may also be necessary. SIGNIFICANCE: Nociceptor HCN2 channels mediate an increase in Ih that is necessary for mechanical hyperalgesia in a CFA model of chronic pain, but the mechanisms producing the increase in nociceptor Ih have not been resolved. The data presented here suggest that the increase in Ih during the early stages of inflammation may be mediated by an increase in HCN2 protein expression and post-translational SUMOylation.

2.
Front Mol Neurosci ; 12: 144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31213982

RESUMO

Post-translational conjugation of Small Ubiquitin-like Modifier (SUMO) peptides to lysine (K) residues on target proteins alters their interactions. SUMOylation of a target protein can either promote its interaction with other proteins that possess SUMO binding domains, or it can prevent target protein interactions that normally occur in the absence of SUMOylation. One subclass of voltage-gated potassium channels that mediates an A-type current, IA, exists as a ternary complex comprising Kv4 pore-forming subunits, Kv channel interacting proteins (KChIP) and transmembrane dipeptidyl peptidase like proteins (DPPL). SUMOylation could potentially regulate intra- and/or intermolecular interactions within the complex. This study began to test this hypothesis and showed that Kv4.2 channels were SUMOylated in the rat brain and in human embryonic kidney (HEK) cells expressing a GFP-tagged mouse Kv4.2 channel (Kv4.2g). Prediction software identified two putative SUMOylation sites in the Kv4.2 C-terminus at K437 and K579. These sites were conserved across mouse, rat, and human Kv4.2 channels and across mouse Kv4 isoforms. Increasing Kv4.2g SUMOylation at each site by ~30% produced a significant ~22%-50% decrease in IA Gmax, and a ~70%-95% increase in channel surface expression. Site-directed mutagenesis of Kv4.2g showed that K437 SUMOylation regulated channel surface expression, while K579 SUMOylation controlled IA Gmax. The K579R mutation mimicked and occluded the SUMOylation-mediated decrease in IA Gmax, suggesting that SUMOylation at K579 blocked an intra- or inter-protein interaction involving K579. The K437R mutation did not obviously alter channel surface expression or biophysical properties, but it did block the SUMOylation-mediated increase in channel surface expression. Interestingly, enhancing K437 SUMOylation in the K579R mutant roughly doubled channel surface expression, but produced no change in IA Gmax, suggesting that the newly inserted channels were electrically silent. This is the first report that Kv4.2 channels are SUMOylated and that SUMOylation can independently regulate Kv4.2 surface expression and IA Gmax in opposing directions. The next step will be to determine if/how SUMOylation affects Kv4 interactions within the ternary complex.

3.
J Neurosci ; 39(4): 596-611, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30504282

RESUMO

Neurons operate within defined activity limits, and feedback control mechanisms dynamically tune ionic currents to maintain this optimal range. This study describes a novel, rapid feedback mechanism that uses SUMOylation to continuously adjust ionic current densities according to changes in activity. Small ubiquitin-like modifier (SUMO) is a peptide that can be post-translationally conjugated to ion channels to influence their surface expression and biophysical properties. Neuronal activity can regulate the extent of protein SUMOylation. This study on the single, unambiguously identifiable lateral pyloric neuron (LP), a component of the pyloric network in the stomatogastric nervous system of male and female spiny lobsters (Panulirus interruptus), focused on dynamic SUMOylation in the context of activity homeostasis. There were four major findings: First, neuronal activity adjusted the balance between SUMO conjugation and deconjugation to continuously and bidirectionally fine-tune the densities of two opposing conductances: the hyperpolarization activated current (Ih) and the transient potassium current (IA). Second, tonic 5 nm dopamine (DA) gated activity-dependent SUMOylation to permit and prevent activity-dependent regulation of Ih and IA, respectively. Third, DA-gated, activity-dependent SUMOylation contributed to a feedback mechanism that restored the timing and duration of LP activity during prolonged modulation by 5 µm DA, which initially altered these and other activity features. Fourth, DA modulatory and metamoduatory (gating) effects were tailored to simultaneously alter and stabilize neuronal output. Our findings suggest that modulatory tone may select a subset of rapid activity-dependent mechanisms from a larger menu to achieve homeostasis under varying conditions.SIGNIFICANCE STATEMENT Post-translational SUMOylation of ion channel subunits controls their interactions. When subunit SUMOylation is dysregulated, conductance densities mediated by the channels are distorted, leading to nervous system disorders, such as seizures and chronic pain. Regulation of ion channel SUMOylation is poorly understood. This study demonstrated that neuronal activity can regulate SUMOylation to reconfigure ionic current densities over minutes, and this regulation was gated by tonic nanomolar dopamine. Dynamic SUMOylation was necessary to maintain specific aspects of neuronal output while the neuron was being modulated by high (5 µm) concentrations of dopamine, suggesting that the gating function may ensure neuronal homeostasis during extrinsic modulation of a circuit.


Assuntos
Homeostase/fisiologia , Canais Iônicos/fisiologia , Palinuridae/fisiologia , Sumoilação/fisiologia , Animais , Dopamina/fisiologia , Feminino , Gânglios dos Invertebrados/fisiologia , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , Canais de Potássio Shal/fisiologia
4.
Front Mol Neurosci ; 9: 168, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28127275

RESUMO

Small Ubiquitin-like Modifier (SUMO) is a ∼10 kDa peptide that can be post-translationally added to a lysine (K) on a target protein to facilitate protein-protein interactions. Recent studies have found that SUMOylation can be regulated in an activity-dependent manner and that ion channel SUMOylation can alter the biophysical properties and surface expression of the channel. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel surface expression can be regulated in an activity-dependent manner through unknown processes. We hypothesized that SUMOylation might influence the surface expression of HCN2 channels. In this manuscript, we show that HCN2 channels are SUMOylated in the mouse brain. Baseline levels of SUMOylation were also observed for a GFP-tagged HCN2 channel stably expressed in Human embryonic kidney (Hek) cells. Elevating GFP-HCN2 channel SUMOylation above baseline in Hek cells led to an increase in surface expression that augmented the hyperpolarization-activated current (Ih) mediated by these channels. Increased SUMOylation did not alter Ih voltage-dependence or kinetics of activation. There are five predicted intracellular SUMOylation sites on HCN2. Site-directed mutagenesis indicated that more than one K on the GFP-HCN2 channel was SUMOylated. Enhancing SUMOylation at one of the five predicted sites, K669, led to the increase in surface expression and IhGmax. The role of SUMOylation at additional sites is currently unknown. The SUMOylation site at K669 is also conserved in HCN1 channels. Aberrant SUMOylation has been linked to neurological diseases that also display alterations in HCN1 and HCN2 channel expression, such as seizures and Parkinson's disease. This work is the first report that HCN channels can be SUMOylated and that this can regulate surface expression and Ih.

5.
Artigo em Inglês | MEDLINE | ID: mdl-26539083

RESUMO

Experimental and computational studies demonstrate that different sets of intrinsic and synaptic conductances can give rise to equivalent activity patterns. This is because the balance of conductances, not their absolute values, defines a given activity feature. Activity-dependent feedback mechanisms maintain neuronal conductance correlations and their corresponding activity features. This study demonstrates that tonic nM concentrations of monoamines enable slow, activity-dependent processes that can maintain a correlation between the transient potassium current (I(A) and the hyperpolarization activated current (Ih) over the long-term (i.e., regulatory change persists for hours after removal of modulator). Tonic 5 nM DA acted through an RNA interference silencing complex (RISC)- and RNA polymerase II-dependent mechanism to maintain a long-term positive correlation between I(A) and Ih in the lateral pyloric neuron (LP) but not in the pyloric dilator neuron (PD). In contrast, tonic 5 nM 5HT maintained a RISC-dependent positive correlation between I(A) and Ih in PD but not LP over the long-term. Tonic 5 nM OCT maintained a long-term negative correlation between I(A) and Ih in PD but not LP; however, it was only revealed when RISC was inhibited. This study also demonstrated that monoaminergic tone can also preserve activity features over the long-term: the timing of LP activity, LP duty cycle and LP spike number per burst were maintained by tonic 5 nM DA. The data suggest that low-level monoaminergic tone acts through multiple slow processes to permit cell-specific, activity-dependent regulation of ionic conductances to maintain conductance correlations and their corresponding activity features over the long-term.


Assuntos
Monoaminas Biogênicas/metabolismo , Gânglios dos Invertebrados/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Palinuridae/fisiologia , Animais , Gânglios dos Invertebrados/metabolismo , Neurônios/metabolismo , Palinuridae/metabolismo
6.
PLoS One ; 10(2): e0117965, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25692473

RESUMO

Volume transmission results in phasic and tonic modulatory signals. The actions of tonic dopamine (DA) at type 1 DA receptors (D1Rs) are largely undefined. Here we show that tonic 5nM DA acts at D1Rs to stabilize neuronal output over minutes by enabling activity-dependent regulation of the hyperpolarization activated current (I h). In the presence but not absence of 5nM DA, I h maximal conductance (G max) was adjusted according to changes in slow wave activity in order to maintain spike timing. Our study on the lateral pyloric neuron (LP), which undergoes rhythmic oscillations in membrane potential with depolarized plateaus, demonstrated that incremental, bi-directional changes in plateau duration produced corresponding alterations in LP I hG max when preparations were superfused with saline containing 5nM DA. However, when preparations were superfused with saline alone there was no linear correlation between LP I hGmax and duty cycle. Thus, tonic nM DA modulated the capacity for activity to modulate LP I h G max; this exemplifies metamodulation (modulation of modulation). Pretreatment with the Ca2+-chelator, BAPTA, or the specific PKA inhibitor, PKI, prevented all changes in LP I h in 5nM DA. Calcineurin inhibitors blocked activity-dependent changes enabled by DA and revealed a PKA-mediated, activity-independent enhancement of LP I hG max. These data suggested that tonic 5nM DA produced two simultaneous, PKA-dependent effects: a direct increase in LP I h G max and a priming event that permitted calcineurin regulation of LP I h. The latter produced graded reductions in LP I hG max with increasing duty cycles. We also demonstrated that this metamodulation preserved the timing of LP's first spike when network output was perturbed with bath-applied 4AP. In sum, 5nM DA permits slow wave activity to provide feedback that maintains spike timing, suggesting that one function of low-level, tonic modulation is to stabilize specific features of a dynamic output.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Calcineurina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dopamina/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Neurônios/metabolismo , Palinuridae
7.
Front Cell Neurosci ; 8: 39, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24596543

RESUMO

Long-term intrinsic and synaptic plasticity must be coordinated to ensure stability and flexibility in neuronal circuits. Coordination might be achieved through shared transduction components. Dopamine (DA) is a well-established participant in many forms of long-term synaptic plasticity. Recent work indicates that DA is also involved in both activity-dependent and -independent forms of long-term intrinsic plasticity. We previously examined DA-enabled long-term intrinsic plasticity in a single identified neuron. The lateral pyloric (LP) neuron is a component of the pyloric network in the crustacean stomatogastric nervous system (STNS). LP expresses type 1 DA receptors (D1Rs). A 1 h bath application of 5 nM DA followed by washout produced a significant increase in the maximal conductance (G max) of the LP transient potassium current (I A) that peaked ~4 h after the start of DA application; furthermore, if a change in neuronal activity accompanied the DA application, then a persistent increase in the LP hyperpolarization activated current (I h) was also observed. Here, we repeated these experiments with pharmacological and peptide inhibitors to determine the cellular processes and signaling proteins involved. We discovered that the persistent, DA-induced activity-independent (I A) and activity-dependent (I h) changes in ionic conductances depended upon many of the same elements that enable long-term synaptic plasticity, including: the D1R-protein kinase A (PKA) axis, RNA polymerase II transcription, RNA interference (RNAi), and mechanistic target of rapamycin (mTOR)-dependent translation. We interpret the data to mean that increasing the tonic DA concentration enhances expression of a microRNA(s) (miRs), resulting in increased cap-dependent translation of an unidentified protein(s).

8.
BMC Neurosci ; 14: 143, 2013 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-24225021

RESUMO

BACKGROUND: Dopamine (DA) can produce divergent effects at different time scales. DA has opposing immediate and long-term effects on the transient potassium current (IA) within neurons of the pyloric network, in the Panulirus interruptus stomatogastric ganglion. The lateral pyloric neuron (LP) expresses type 1 DA receptors (D1Rs). A 10 min application of 5-100 µM DA decreases LP IA by producing a decrease in IA maximal conductance (Gmax) and a depolarizing shift in IA voltage dependence through a cAMP-Protein kinase A (PKA) dependent mechanism. Alternatively, a 1 hr application of DA (≥5 nM) generates a persistent (measured 4 hr after DA washout) increase in IA Gmax in the same neuron, through a mechanistic target of rapamycin (mTOR) dependent translational mechanism. We examined the dose, time and protein dependencies of the persistent DA effect. RESULTS: We found that disrupting normal modulatory tone decreased LP IA. Addition of 500 pM-5 nM DA to the saline for 1 hr prevented this decrease, and in the case of a 5 nM DA application, the effect was sustained for >4 hrs after DA removal. To determine if increased cAMP mediated the persistent effect of 5nM DA, we applied the cAMP analog, 8-bromo-cAMP alone or with rapamycin for 1 hr, followed by wash and TEVC. 8-bromo-cAMP induced an increase in IA Gmax, which was blocked by rapamycin. Next we tested the roles of PKA and guanine exchange factor protein activated by cAMP (ePACs) in the DA-induced persistent change in IA using the PKA specific antagonist Rp-cAMP and the ePAC specific agonist 8-pCPT-2'-O-Me-cAMP. The PKA antagonist blocked the DA induced increases in LP IA Gmax, whereas the ePAC agonist did not induce an increase in LP IA Gmax. Finally we tested whether extracellular signal regulated kinase (Erk) activity was necessary for the persistent effect by co-application of Erk antagonists PD98059 or U0126 with DA. Erk antagonism blocked the DA induced persistent increase in LP IA. CONCLUSIONS: These data suggest that dopaminergic tone regulates ion channel density in a concentration and time dependent manner. The D1R- PKA axis, along with Erk and mTOR are necessary for the persistent increase in LP IA induced by high affinity D1Rs.


Assuntos
Dopamina/metabolismo , Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Canais de Potássio/metabolismo , Transdução de Sinais/fisiologia , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Gânglios dos Invertebrados/fisiologia , Palinuridae , Técnicas de Patch-Clamp , Piloro/inervação , Piloro/metabolismo , Receptores Dopaminérgicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo
9.
Front Neural Circuits ; 7: 169, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24155696

RESUMO

Neuromodulators alter network output and have the potential to destabilize a circuit. The mechanisms maintaining stability in the face of neuromodulation are not well described. Using the pyloric network in the crustacean stomatogastric nervous system, we show that dopamine (DA) does not simply alter circuit output, but activates a closed loop in which DA-induced alterations in circuit output consequently drive a change in an ionic conductance to preserve a conductance ratio and its activity correlate. DA acted at low affinity type 1 receptors (D1Rs) to induce an immediate modulatory decrease in the transient potassium current (IA) of a pyloric neuron. This, in turn, advanced the activity phase of that component neuron, which disrupted its network function and thereby destabilized the circuit. DA simultaneously acted at high affinity D1Rs on the same neuron to confer activity-dependence upon the hyperpolarization activated current (Ih) such that the DA-induced changes in activity subsequently reduced Ih. This DA-enabled, activity-dependent, intrinsic plasticity exactly compensated for the modulatory decrease in IA to restore the IA:Ih ratio and neuronal activity phase, thereby closing an open loop created by the modulator. Activation of closed loops to preserve conductance ratios may represent a fundamental operating principle neuromodulatory systems use to ensure stability in their target networks.


Assuntos
Potenciais de Ação/fisiologia , Dopamina/farmacologia , Condução Nervosa/fisiologia , Neurônios/fisiologia , Receptores Dopaminérgicos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Gânglios dos Invertebrados/efeitos dos fármacos , Gânglios dos Invertebrados/fisiologia , Condução Nervosa/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Neurônios/efeitos dos fármacos , Palinuridae
10.
Dev Neurobiol ; 73(4): 274-96, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23008269

RESUMO

Retinotopic maps can undergo compression and expansion in response to changes in target size, but the mechanism underlying this compensatory process has remained a mystery. The discovery of ephrins as molecular mediators of Sperry's chemoaffinity process allows a mechanistic approach to this important issue. In Syrian hamsters, neonatal, partial (PT) ablation of posterior superior colliculus (SC) leads to compression of the retinotopic map, independent of neural activity. Graded, repulsive EphA receptor/ephrin-A ligand interactions direct the formation of the retinocollicular map, but whether ephrins might also be involved in map compression is unknown. To examine whether map compression might be directed by changes in the ephrin expression pattern, we compared ephrin-A2 and ephrin-A5 mRNA expression between normal SC and PT SC using in situ hybridization and quantitative real-time PCR. We found that ephrin-A ligand expression in the compressed maps was low anteriorly and high posteriorly, as in normal animals. Consistent with our hypothesis, the steepness of the ephrin gradient increased in the lesioned colliculi. Interestingly, overall levels of ephrin-A2 and -A5 expression declined immediately after neonatal target damage, perhaps promoting axon outgrowth. These data establish a correlation between changes in ephrin-A gradients and map compression, and suggest that ephrin-A expression gradients may be regulated by target size. This in turn could lead to compression of the retinocollicular map onto the reduced target. These findings have important implications for mechanisms of recovery from traumatic brain injury.


Assuntos
Efrinas/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Colículos Superiores/crescimento & desenvolvimento , Colículos Superiores/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Axônios/fisiologia , Mapeamento Encefálico , Clonagem Molecular , Cricetinae , Efrina-A2/biossíntese , Efrina-A2/genética , Efrina-A5/biossíntese , Efrina-A5/genética , Efrinas/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Mesocricetus , Dados de Sequência Molecular , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Plasticidade Neuronal/fisiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
11.
J Neurosci ; 31(45): 16387-97, 2011 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-22072689

RESUMO

The phases at which network neurons fire in rhythmic motor outputs are critically important for the proper generation of motor behaviors. The pyloric network in the crustacean stomatogastric ganglion generates a rhythmic motor output wherein neuronal phase relationships are remarkably invariant across individuals and throughout lifetimes. The mechanisms for maintaining these robust phase relationships over the long-term are not well described. Here we show that tonic nanomolar dopamine (DA) acts at type 1 DA receptors (D1Rs) to enable an activity-dependent mechanism that can contribute to phase maintenance in the lateral pyloric (LP) neuron. The LP displays continuous rhythmic bursting. The activity-dependent mechanism was triggered by a prolonged decrease in LP burst duration, and it generated a persistent increase in the maximal conductance (G(max)) of the LP hyperpolarization-activated current (I(h)), but only in the presence of steady-state DA. Interestingly, micromolar DA produces an LP phase advance accompanied by a decrease in LP burst duration that abolishes normal LP network function. During a 1 h application of micromolar DA, LP phase recovered over tens of minutes because, the activity-dependent mechanism enabled by steady-state DA was triggered by the micromolar DA-induced decrease in LP burst duration. Presumably, this mechanism restored normal LP network function. These data suggest steady-state DA may enable homeostatic mechanisms that maintain motor network output during protracted neuromodulation. This DA-enabled, activity-dependent mechanism to preserve phase may be broadly relevant, as diminished dopaminergic tone has recently been shown to reduce I(h) in rhythmically active neurons in the mammalian brain.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Fenômenos Biofísicos/efeitos dos fármacos , Dopamina/farmacologia , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Periodicidade , Potenciais de Ação/fisiologia , Análise de Variância , Anestésicos Locais/farmacologia , Animais , Fenômenos Biofísicos/fisiologia , Biofísica , Cloreto de Cádmio/farmacologia , Estimulação Elétrica/métodos , Gânglios dos Invertebrados/citologia , Técnicas In Vitro , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neurônios/fisiologia , Palinuridae , Técnicas de Patch-Clamp , Piloro/efeitos dos fármacos , Piloro/fisiologia , Tetrodotoxina/farmacologia , Fatores de Tempo
12.
J Neurosci ; 31(37): 13046-56, 2011 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-21917788

RESUMO

Neuromodulatory effects can vary with their mode of transmission. Phasic release produces local and transient increases in dopamine (DA) up to micromolar concentrations. Additionally, since DA is released from open synapses and reuptake mechanisms are not nearby, tonic nanomolar DA exists in the extracellular space. Do phasic and tonic transmissions similarly regulate voltage-dependent ionic conductances in a given neuron? It was previously shown that DA could immediately alter the transient potassium current (I(A)) of identified neurons in the stomatogastric ganglion of the spiny lobster Panulirus interruptus. Here we show that DA can also persistently alter I(A), and that the immediate and persistent effects of DA oppose one another. The lateral pyloric (LP) neuron exclusively expresses type 1 DA receptors (D1Rs). Micromolar DA produces immediate depolarizing shifts in the voltage dependence of LP I(A), whereas tonic nanomolar DA produces a persistent increase in LP I(A) maximal conductance (G(max)) through a translation-dependent mechanism involving target of rapamycin (TOR). The pyloric dilator (PD) neuron exclusively expresses D2Rs. Micromolar DA produces an immediate hyperpolarizing shift in PD I(A) voltage dependence of activation, whereas tonic DA persistently decreases PD I(A) G(max) through a translation-dependent mechanism not involving TOR. The persistent effects on I(A) G(max) do not depend on LP or PD activity. These data suggest a role for tonic modulators in the regulation of voltage-gated ion channel number; and furthermore, that dopaminergic systems may be organized to limit the amount of change they can impose on a circuit.


Assuntos
Dopamina/fisiologia , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Biossíntese de Proteínas/fisiologia , Receptores de Dopamina D1/fisiologia , Receptores de Dopamina D2/fisiologia , Animais , Dopamina/farmacologia , Relação Dose-Resposta a Droga , Gânglios dos Invertebrados/efeitos dos fármacos , Gânglios dos Invertebrados/fisiologia , Palinuridae , Biossíntese de Proteínas/efeitos dos fármacos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/agonistas , Serina-Treonina Quinases TOR/fisiologia
13.
Brain Res ; 1348: 42-54, 2010 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-20558147

RESUMO

There is ample evidence linking octopamine (OA) and tyramine (TA) to several neurophysiological functions in arthropods. In our laboratory we use the freshwater prawn Macrobrachium rosenbergii to study the neural basis of aggressive behavior. As a first step towards understanding the possible role of these amines and their receptors in the modulation of interactive behaviors, we have cloned a putative octopamine/tyramine receptor. The predicted sequence of the cloned OA/TA(Mac) receptor consists of 1,579 base pairs (bp), with an open reading frame of 1,350bp that encodes a 450 amino acid protein. This putative protein displays sequence identities of 70% to an Aedes aegypti mosquito TA receptor, followed by 60% to a Stegomyia aegypti mosquito OA receptor, 59% and 58% to the migratory locust TA-1 and -2 receptors respectively, and 57% with the silkworm OA receptor. We also mapped the OA/TA(Mac) receptor distribution by in-situ hybridization to the receptor's mRNA, and by immunohistochemistry to its protein. We observed stained cell bodies for the receptor's mRNA, mainly in the midline region of the thoracic and in the abdominal ganglia, as well as diffuse staining in the brain ganglia. For the receptor's protein, we observed extensive punctate staining within the neuropil and on the membrane of specific groups of neurons in all ganglia throughout the CNS, including the brain, the midline region and neuropiles of the thoracic ganglia, and ventral part and neuropiles of the abdominal ganglia. The same pattern of stained cells was observed on the thoracic and abdominal ganglia in both in-situ hybridization and immunohistochemistry experiments. Diffuse staining observed with in-situ hybridization also coincides with punctate staining observed in brain, SEG, thoracic, and abdominal ganglia in immunohistochemical preparations. This work provides the first step towards characterizing the neural networks that mediate octopaminergic signaling in prawn.


Assuntos
Sistema Nervoso Central/metabolismo , Octopamina/metabolismo , Palaemonidae/anatomia & histologia , Receptores de Amina Biogênica/metabolismo , Animais , Evolução Biológica , Sistema Nervoso Central/anatomia & histologia , Clonagem Molecular/métodos , Gânglios/metabolismo , Neurópilo/metabolismo , Octopamina/genética , Filogenia , RNA Mensageiro/metabolismo , Receptores de Amina Biogênica/genética
14.
J Neurophysiol ; 104(2): 873-84, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20519576

RESUMO

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.


Assuntos
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
15.
J Comp Neurol ; 518(3): 255-76, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19941347

RESUMO

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.


Assuntos
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
16.
J Neurophysiol ; 99(6): 2844-63, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18400960

RESUMO

A fundamental question in systems biology addresses the issue of how flexibility is built into modulatory networks such that they can produce context-dependent responses. Here we examine flexibility in the serotonin (5-HT) response system that modulates the cycle frequency (cf) of a rhythmic motor output. We found that depending on the preparation, the same 5-min bath application of 5-HT to the pyloric network of the California spiny lobster, Panulirus interruptus, could produce a significant increase, decrease, or no change in steady-state cf relative to baseline. Interestingly, the mean circuit output was not significantly different among preparations prior to 5-HT application. We developed pharmacological tools to examine the preparation-to-preparation variability in the components of the 5-HT response system. We found that the 5-HT response system consisted of at least three separable components: a 5-HT(2betaPan)-like component mediated a rapid decrease followed by a sustained increase in cf; a 5-HT(1alphaPan)-like component produced a small and usually gradual increase in cf; at least one other component associated with an unknown receptor mediated a sustained decrease in cf. The magnitude of the change in cf produced by each component was highly variable, so that when summed they could produce either a net increase, decrease, or no change in cf depending on the preparation. Overall, our research demonstrates that the balance of opposing components of the 5-HT response system determines the direction and magnitude of 5-HT-induced change in steady-state cf relative to baseline.


Assuntos
Gânglios dos Invertebrados/citologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Serotonina/fisiologia , Transdução de Sinais/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Animais , Butaclamol/farmacologia , Linhagem Celular Transformada , Antagonistas de Dopamina/farmacologia , Interações Medicamentosas , Gânglios dos Invertebrados/fisiologia , Humanos , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Rede Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Palinuridae , Piperazinas/farmacologia , Receptor 5-HT1A de Serotonina/genética , Receptores 5-HT2 de Serotonina/genética , Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção/métodos
17.
J Exp Biol ; 211(Pt 1): 92-105, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18083737

RESUMO

Serotonin (5-HT) plays important roles in the maintenance and modulation of neural systems throughout the animal kingdom. The actions of 5-HT have been well characterized for several crustacean model circuits; however, a dissection of the serotonergic transduction cascades operating in these models has been hampered by the lack of pharmacological tools for invertebrate receptors. Here we provide pharmacological profiles for two 5-HT receptors from the swamp crayfish, Procambarus clarkii: 5-HT(2beta) and 5-HT(1alpha). In so doing, we also report the first functional expression of a crustacean 5-HT(1) receptor, and show that it inhibits accumulation of cAMP. The drugs mCPP and quipazine are 5-HT(1alpha) agonists and are ineffective at 5-HT(2beta). Conversely, methiothepin and cinanserin are antagonists of 5-HT(2beta) but do not block 5-HT(1alpha). A comparison of these two receptors with their orthologs from the California spiny lobster, Panulirus interruptus, indicates conservation of protein structure, signaling and pharmacology. This conservation extends beyond crustacean infraorders. The signature residues that form the ligand-binding pocket in mammalian 5-HT receptors are found in the crustacean receptors. Similarly, the protein domains involved in G protein coupling are conserved between the two crustacean receptors and other characterized arthropod and mammalian 5-HT receptors. Considering the apparent conservation of pharmacological properties between crustacean 5-HT receptors, these tools could be applicable to related crustacean physiological preparations.


Assuntos
Astacoidea/metabolismo , Sequência Conservada , Palinuridae/metabolismo , Receptores 5-HT1 de Serotonina/química , Receptores 5-HT2 de Serotonina/química , Transdução de Sinais , Aminas , Sequência de Aminoácidos , Animais , Astacoidea/efeitos dos fármacos , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Humanos , Camundongos , Dados de Sequência Molecular , Palinuridae/efeitos dos fármacos , Receptores 5-HT1 de Serotonina/metabolismo , Receptores 5-HT2 de Serotonina/metabolismo , Análise de Sequência de Proteína , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos
18.
J Neurochem ; 104(4): 1006-19, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17986222

RESUMO

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.


Assuntos
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
19.
Endocrinology ; 148(11): 5339-47, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17702843

RESUMO

Melanocortins are implicated in the control of energy intake/expenditure. Centrally administered melanotan II (MTII), a synthetic melanocortin 3/4-receptor agonist, decreases adiposity beyond that accountable by food intake decreases. Melanocortin-4 receptor (MC4-R) mRNA is expressed on sympathetic nervous system (SNS) outflow neurons to white adipose tissue (WAT) in Siberian hamsters, suggesting a role in lipid mobilization. Therefore, we tested whether third ventricular injections of MTII increased sympathetic drive to WAT and interscapular brown adipose tissue (IBAT) using norepinephrine turnover (NETO) as a measure of sympathetic drive. We also tested for MTII-induced changes in lipolysis-related WAT gene expression (beta3-adrenoceptors, hormone sensitive lipase) and IBAT thermogenesis (beta3-adrenoceptor, uncoupling protein-1). Finally, we tested whether third ventricularly injected MTII, a highly selective MC4-R agonist (cyclo[beta-Ala-His-D-Phe-Arg-Trp-Glu]NH2) increased or agouti-related protein decreased IBAT temperature in hamsters implanted with sc IBAT temperature transponders. Centrally administered MTII provoked differential sympathetic drives to WAT and IBAT (increased inguinal WAT, dorsosubcutaneous WAT and IBAT NETO, but not epididymal WAT and retroperitoneal WAT NETO). MTII also increased circulating concentrations of the lipolytic products free fatty acids and glycerol but not plasma catecholamines, suggesting lipid mobilization via WAT SNS innervation and not via adrenal medullary catecholamines. WAT or IBAT gene expression was largely unaffected by acute MTII treatment, but IBAT temperature was increased by MTII and the MC4-R agonist and decreased by agouti-related protein. Collectively, this is the first demonstration of central melanocortin agonist stimulation of WAT lipolysis through the SNS and confirms melanocortin-induced changes in BAT thermogenesis.


Assuntos
Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/inervação , Peptídeos Cíclicos/farmacologia , Receptores de Melanocortina/agonistas , Sistema Nervoso Simpático/metabolismo , alfa-MSH/análogos & derivados , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo Branco/metabolismo , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Temperatura Corporal/efeitos dos fármacos , Cricetinae , Epinefrina/sangue , Ácidos Graxos não Esterificados/sangue , Glicerol/sangue , Leptina/sangue , Masculino , Norepinefrina/sangue , Norepinefrina/metabolismo , Phodopus , alfa-MSH/farmacologia
20.
Artigo em Inglês | MEDLINE | ID: mdl-17134931

RESUMO

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.


Assuntos
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
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