RESUMO
Dopamine D3 R are widely expressed in basal ganglia where interact with D1 R. D3 R potentiate cAMP accumulation and GABA release stimulated by D1 R in striatonigral neurons through "atypical" signaling. During dopaminergic denervation, D3 R signaling changes to a "typical" in which antagonizes the effects of D1 R, the mechanisms of this switching are unknown. D3 nf splice variant regulates membrane anchorage and function of D3 R and decreases in denervation; thus, it is possible that D3 R signaling switching correlates with changes in D3 nf expression and increases of membranal D3 R that mask D3 R atypical effects. We performed experiments in unilaterally 6-hydroxydopamine lesioned rats and found a decrease in mRNA and protein of D3 nf, but not of D3 R in the denervated striatum. Proximity ligation assay showed that D3 R-D3 nf interaction decreased after denervation, whereas binding revealed an increased Bmax in D3 R. The new D3 R antagonized cAMP accumulation and GABA release stimulated by D1 R; however, in the presence of N-Ethylmaleimide (NEM), to block Gi protein signaling, activation of D3 R produced its atypical signaling stimulating D1 R effects. Finally, we investigated if the typical and atypical effects of D3 R modulating GABA release are capable of influencing motor behavior. Injections of D3 R agonist into denervated nigra decreased D1 R agonist-induced turning behavior but potentiated it in the presence of NEM. Our data indicate the coexistence of D3 R typical and atypical signaling in striatonigral neurons during denervation that correlated with changes in the ratio of expression of D3 nf and D3 R isoforms. The coexistence of both atypical and typical signaling during denervation influences motor behavior.
Assuntos
Receptores de Dopamina D3/metabolismo , Transdução de Sinais , Substância Negra/metabolismo , Animais , AMP Cíclico/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Masculino , Movimento , Bloqueio Nervoso , Splicing de RNA , Ratos , Ratos Wistar , Receptores de Dopamina D3/genética , Substância Negra/citologia , Substância Negra/fisiologia , Ácido gama-Aminobutírico/metabolismoRESUMO
In vivo activation of dopamine D3 receptors (D3Rs) depresses motor activity. D3Rs are widely expressed in subthalamic, striatal, and dendritic dopaminergic inputs into the substantia nigra pars reticulata (SNr). In vitro studies showed that nigral D3Rs modulate their neurotransmitter release; thus, it could be that these changes in neurotransmitter levels modify the discharge of nigro-thalamic neurons and, therefore, motor behavior. To determine how the in vitro responses correspond to the in vivo responses, we examined the effect of intra-nigral and systemic blockade of D3Rs in the interstitial content of glutamate, dopamine, and GABA within the SNr using microdialysis coupled to motor activity determinations in freely moving rats. Intranigral unilateral blockade of D3R with GR 103,691 increased glutamate, dopamine, and GABA. Increments correlated with increased ambulatory distance, non-ambulatory activity, and induced contralateral turning. Concomitant blockade of D3R with D1R by perfusion of SCH 23390 reduced the increase of glutamate; prevented the increment of GABA, but not of dopamine; and abolished behavioral effects. Glutamate stimulates dopamine release by NMDA receptors, while blockade with kynurenic acid prevented the increase in dopamine and, in turn, of GABA and glutamate. Finally, systemic administration of D3R selective antagonist U 99194A increased glutamate, dopamine, and GABA in SNr and stimulated motor activity. Blockade of intra-nigral D1R with SCH 23390 prior to systemic U 99194A diminished increases in neurotransmitter levels and locomotor activity. These data highlight the pivotal role of presynaptic nigral D3 and D1R in the control of motor activity and help to explain part of the effects of the in vivo administration of D3R agents.
Assuntos
Compostos de Bifenilo/administração & dosagem , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Piperazinas/administração & dosagem , Substância Negra/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Benzazepinas/administração & dosagem , Benzazepinas/farmacologia , Compostos de Bifenilo/farmacologia , Locomoção/efeitos dos fármacos , Masculino , Microdiálise , Piperazinas/farmacologia , Ratos , Receptores de Dopamina D3/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/efeitos dos fármacosRESUMO
Extensive damage to nigrostriatal dopaminergic neurons leads to Parkinson's disease (PD). To date, the most effective treatment has been administration of levodopa (L-DOPA) to increase dopaminergic tone. This treatment leads to responses that vary widely among patients, from predominantly beneficial effects to the induction of disabling, abnormal movements (L-DOPA induced dyskinesia (LID)). Similarly, experimental studies have shown animals with widely different degrees of LID severity. In this study, unilateral injections of 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle (MFB) produced more than 90% depletion of dopamine in both the striatum and the substantia nigra reticulata (SNr) of rats. Population analysis showed that dopamine depletion levels were clustered in a single population. In contrast, analysis of abnormal involuntary movements (AIMs) induced by L-DOPA treatment of 6-OHDA-lesioned animals yielded two populations: one with mild LID, and the other with severe LID, which are also related to different therapeutic responses. We examined whether the severity of LID correlated with changes in dopamine 3 receptor (D3R) signaling because of the following: (a) D3R expression and the induction of LID are strongly correlated; and (b) dopaminergic denervation induces a qualitative change in D3R signaling in the SNr. We found that the effects of D3R activation on cAMP accumulation and depolarization-induced [3H]-gamma-aminobutyric acid ([3H]-GABA) release were switched. L-DOPA treatment normalized the denervation-induced changes in animals with mild LID. The D3R activation caused depression of both dopamine 1 receptor (D1R)-induced increases in cAMP production and depolarization-induced [3H]-GABA release, which were reversed to their pre-denervation state. In animals with severe LID, none of the denervation-induced changes were reversed. The finding that in the absence of identifiable differences in 6-OHDA and L-DOPA treatment, two populations of animals with different D3R signaling and LIDs severity implies that mechanisms intrinsic to the treated subject determine the segregation.
Assuntos
Discinesias/etiologia , Discinesias/metabolismo , Levodopa/efeitos adversos , Levodopa/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Animais , AMP Cíclico/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Masculino , Feixe Prosencefálico Mediano/efeitos dos fármacos , Feixe Prosencefálico Mediano/metabolismo , Oxidopamina/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
Substantia nigra pars reticulata is the output station in basal ganglia; its GABAergic neurons control the activity of thalamo-cortical premotor nuclei, thus controlling motor behavior. D1-like and D2-like presynaptic dopamine receptors on subthalamo-nigral afferents by modulation of glutamate release change the firing rate of nigral neurons; however, their relative contribution to the control of glutamate release and their pharmacological properties have not been studied. This is important since the prevalence of the inhibition or stimulation of release determines the firing rate of nigral neurons, therefore motor activity. Here we used depolarization induced [3H]-glutamate release in slices of rat substantia nigra from reserpinized and non-reserpinized rats to explore the relative contribution of the D1-like and D2-like receptor subtypes to the control of glutamate release. We found a significant control of release by D1-like and D3R, and a modest effect of D2R. D4R exerted no effect. Dopamine showed more potency for D3R than for D1-like receptors; however, these latter enhanced release to a greater degree, as shown by the Emax. We also co-activated these to test their interaction; an antagonist interaction of D1-like with D2 and D3R, and an additive between D2 and D3R were found. Pharmacological receptor antagonist effects in release from reserpinized vs. non-reserpinized slices were similar, suggesting that endogenous dopamine stimulates receptors in the same way. These findings suggest differences in the control of glutamate release by different dopamine receptors in the substantia nigra, which could contribute to explaining the effect of dopamine and its agonists on motor behavior.
Assuntos
Ácido Glutâmico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D3/metabolismo , Substância Negra/metabolismo , Trítio/metabolismo , Animais , Dopamina/farmacologia , Relação Dose-Resposta a Droga , Masculino , Técnicas de Cultura de Órgãos , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D3/agonistas , Substância Negra/efeitos dos fármacosRESUMO
Recent studies suggested the expression of CB2 receptors in neurons of the CNS, however, most of these studies have only explored one aspect of the receptors, i.e., expression of protein, messenger RNA, or functional response, and more complete studies appear to be needed to establish adequately their role in the neuronal function. Electron microscopy studies showed the presence of CB2r in asymmetric terminals of the substantia nigra pars reticulata (SNr), and its mRNA appeared is expressed in the subthalamic nucleus. Here, we explore the expression, source, and functional effects of such receptors by different experimental approaches. Through PCR and immunochemistry, we showed mRNA and protein for CB2rs in slices and primary neuronal cultures from subthalamus. GW833972A, GW405833, and JHW 133, three CB2r agonists dose-dependent inhibited K+ -induced [3 H]-Glutamate release in slices of SNr, and the two antagonist/inverse agonists, JTE-907 and AM630, but not AM281, a CB1r antagonist, prevented GW833972A effect. Subthalamus lesions with kainic acid prevented GW833972A inhibition on release and decreased CB2r protein in nigral synaptosomes, thus nigral CB2rs originate in subthalamus. Inhibition of [3 H]-Glutamate release was PTX- and gallein-sensitive, suggesting a Gißγ -mediated effect. P/Q Ca2+ -type channel blocker, ω-Agatoxin-TK, also inhibited the [3 H]-Glutamate release, this effect was occluded with GW833972A inhibition, indicating that the ßγ subunit effect is exerted on Ca2+ channel activity. Finally, microinjections of GW833972A in SNr induced contralateral turning. Our data showed that presynaptic CB2rs inhibit [3 H]-Glutamate release in subthalamo-nigral terminals by P/Q-channels modulation through the Gißγ subunit and suggested their participation in motor behavior.
Assuntos
Corpo Estriado/metabolismo , Ácido Glutâmico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Substância Negra/metabolismo , Animais , Canais de Cálcio/metabolismo , Células Cultivadas , Corpo Estriado/efeitos dos fármacos , Relação Dose-Resposta a Droga , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Neurotransmissores/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Cultura Primária de Células , RNA Mensageiro/metabolismo , Ratos Wistar , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Substância Negra/efeitos dos fármacos , Núcleo Subtalâmico/efeitos dos fármacos , Núcleo Subtalâmico/metabolismo , Técnicas de Cultura de Tecidos , TrítioRESUMO
Striatal dopamine D2 receptors activate the PLCâ¯ââ¯IP3â¯ââ¯Calcineurin-signaling pathway to modulate the neural excitability of En+ Medium-sized Spiny GABAergic neurons (MSN) through the regulation of L-type Ca2+ channels. Presynaptic dopaminergic D2 receptors modulate GABA release at striatopallidal terminals through L-type Ca2+ channels as well, but their signaling pathway is still undetermined. Since D2 receptors are Gi/o-coupled and negatively modulate adenylyl cyclase (AC), we investigated whether presynaptic D2 receptors modulate GABA release through the same signaling cascade that controls excitability in the striatum or by the inhibition of AC and decreased PKA activity. Activation of D2 receptors stimulated formation of [3H]IP1 and decreased Forskolin-stimulated [3H]cAMP accumulation in synaptosomes from rat Globus Pallidus. D2 receptor activation with Quinpirole in the presence of L 745,870 decreased, in a dose-dependent manner, K+-induced [3H]GABA release in pallidal slices. The effect was prevented by the pharmacological blockade of Gi/o ßγ subunit effects with Gallein, PLC with U 73122, IP3 receptor activation with 4-APB, Calcineurin with FK506. In addition, when release was stimulated with Forskolin to activate AC, D2 receptors also decreased K+-induced [3H]GABA release, an effect occluded with the effect of the blockade of PKA with H89 or stimulation of release with the cAMP analog 8-Br-cAMP. These data indicate that D2 receptors modulate [3H]GABA release at striatopallidal terminals by activating the PLCâ¯ââ¯IP3â¯ââ¯Calcineurin-signaling cascade, the same one that modulates excitability in soma. Additionally, D2 receptors inhibit release when AC is active. Both mechanisms appear to converge to regulate the activity of presynaptic L-type Ca2+ channels.
Assuntos
Corpo Estriado/metabolismo , Globo Pálido/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de Dopamina D2/metabolismo , Ácido gama-Aminobutírico/metabolismo , Adenilil Ciclases/metabolismo , Animais , Calcineurina/metabolismo , Corpo Estriado/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Globo Pálido/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Fosfoinositídeo Fosfolipase C/metabolismo , Potássio/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos , TrítioRESUMO
Because activation of D2 receptors reverses the neurochemical effects of cannabinoids, we examined whether increasing dopaminergic tone in the globus pallidus (GPe) switches cannabinoid induced depression of synaptic transmission. GABAergic synaptic currents evoked in pallidal neurons by stimulation of striatal projections (IPSCs) were depressed by perfusion with the CB1R agonist ACEA. Coactivation of D2Rs with quinpirole converted the depression into stimulation. Pretreatment with pertussis toxin (PTX) to limit Gi/o protein coupling also switched the CB1R-induced depression of IPSCs. The stimulation of IPSCs was blocked by the selective PKA blocker H89. Changes in the paired pulse ratio during both inhibitory and stimulatory responses indicate that the effects are due to changes in transmitter release. Postsynaptic depolarization induces endocannabinoid release that inhibits transmitter release (DSI). When D2Rs were activated with quinpirole, depolarization increased transmission instead of depressing it. This increase was blocked by AM251. We also examined the effects of CB1R/D2R coactivation on cAMP accumulation in the GPe to further verify that the AC/PKA cascade is involved. CB1R/D2R coactivation converted the inhibition of cAMP seen when each receptor is stimulated alone into a stimulation. We also determined the effects on turning behavior of unilateral injection of ACEA into the GPe of awake animals and its modification by dopamine antagonists. Blockade of D2 family receptors with sulpiride antagonized the motor effects of ACEA. We show, for the first time, that cannabinoid-inhibition of synaptic transmission in the GPe becomes a stimulation after D2Rs or PTX treatment and that the switch is probably relevant for the control of motor behavior.
Assuntos
Dopamina/metabolismo , Endocanabinoides/metabolismo , Globo Pálido/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Moduladores de Receptores de Canabinoides/farmacologia , AMP Cíclico/metabolismo , Globo Pálido/efeitos dos fármacos , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos Wistar , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptores de Dopamina D2/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
In striatonigral projections activation of dopamine D3 receptors (D3Rs) potentiates the stimulation of GABA release and cAMP production caused by activation of dopamine D1 receptors (D1Rs). Cytoplasmic [Ca(2+)] in the terminals controls this response by modulating CaMKII, an enzyme that depresses D3R action. To examine the effects of dopamine deprivation on D3R signaling we investigated their function in striatonigral terminals of hemiparkinsonian rats. Denervation switched the signaling cascade initiated by D3R activation. In the non-lesioned side activation of D3R potentiated the stimulatory effects of D1R activation on cAMP production and K(+)-depolarization induced [(3)H] GABA release. In contrast, in the denervated side the stimulatory effects of both D1R activation and forskolin administration were blocked by D3R activation. In non-lesioned slices, D3R responses were inhibited by the activation of CaMKII produced by K(+)-depolarization (via increased Ca(2+) entry). The CaMKII-induced inhibition was blocked by the selective inhibitor KN-62. In denervated tissues the response to D3R stimulation was not modified either by K(+) depolarization or by blocking CaMKII with KN-62. Immunoblotting studies showed that depolarization-induced CaMKII binding to the D3 receptor and CaMKII phosphorylation were suppressed in denervated tissues. We also determined calmodulin expression with PCR and immunoblot techniques. Both techniques showed that calmodulin expression was depressed in the lesioned side. In sum, our studies show that dopaminergic denervation switches the D3R signaling cascade and depresses CaMKII signaling through a process that appears to involve reduced calmodulin levels. Since calmodulin is a major cytoplasmic Ca(2+) buffer our findings suggest that abnormal Ca(2+) buffering may be an important component of the abnormalities observed during dopaminergic denervation.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Corpo Estriado/metabolismo , Neurônios Dopaminérgicos/metabolismo , Receptores de Dopamina D3/metabolismo , Substância Negra/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Corpo Estriado/efeitos dos fármacos , AMP Cíclico/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Masculino , Feixe Prosencefálico Mediano/fisiopatologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Oxidopamina , Fosforilação/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais , Substância Negra/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Ácido gama-Aminobutírico/metabolismoRESUMO
CaMKIIα is expressed at high density in the nucleus accumbens where it binds to postsynaptic D3 receptors inhibiting their effects. In striatonigral projections, activation of presynaptic D3 receptors potentiates D1 receptor-induced stimulation of cAMP production and GABA release. In this study we examined whether the presynaptic effects of D3 receptor stimulation in the substantia nigra reticulata (SNr) are modulated by Ca²âº activation of CaMKIIα. In SNr synaptosomes two procedures that increase cytoplasmic Ca²âº, ionomycin and Kâº-depolarization, blocked the additional stimulation of cAMP accumulation produced by coactivating D3 and D1 dopamine receptors. The selective CaMKIIα inhibitor KN-62 reversed the blockade produced by ionomycin and Kâº-depolarization. Incubation in either Ca²-free solutions or with the selective Ca²âº blocker nifedipine, also reversed the blocking effects of Kâº-depolarization. Immunoblot studies showed that Kâº-depolarization increased CaMKIIα phosphorylation in a KN-62 sensitive manner and promoted CaMKIIα binding to D3 receptors. In Kâº-depolarized tissues, D3 receptors potentiated D1 receptor-induced stimulation of [³H]GABA release only when CaMKIIα was blocked with KN-62. In the presence of this inhibitor, the selective D3 agonist PD 128,907 reduced the ED50 for the D1 agonist SKF 38393 from 56 to 4 nM. KN-62 also enhanced the effects of dopamine on depolarization induced [³H]GABA release. KN-62 changed ED50 for dopamine from 584 to 56 nM. KN-62 did not affect D1 and D4 receptor responses. These experiments show that in striatonigral projections, CaMKIIα inhibits the action of D3 receptors in a Ca²âº dependent manner blocking their modulatory effects on GABA release. These findings suggest a mechanism through which the frequency of action potential discharge in presynaptic terminals regulates dopamine effects.
Assuntos
Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de Dopamina D3/metabolismo , Substância Negra/metabolismo , Animais , Bloqueadores dos Canais de Cálcio , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Concentração Osmolar , Fosforilação/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D3/agonistas , Substância Negra/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
The firing rate of substantia nigra reticulata (SNr) neurons is modulated by GABA release from striatonigral and pallidonigral projections. This release is, in turn, modulated by dopamine acting on dopamine D1 receptors at striatonigral terminals and D4 receptors at pallidonigral terminals. In addition, striatal neurons that express D1 receptors also express D3 receptors. In this study we analyzed the possible significance of D3 and D1 receptor colocalization in striatonigral projections. We found that these receptors coprecipitate in SNr synaptosomes suggesting their close association in this structure. D1 agonist SKF 38393 administered alone increased mIPSC frequency in SNr slices and cAMP production in SNr synaptosomes, however, the selective D3 agonist PD 128,907 increased mIPSC frequency and cAMP production only when D1 receptors were concurrently stimulated. The D1 antagonist SCH 23390 blocked completely the effects of the concurrent administration of these agonists while the selective D3 antagonist GR 103691 blocked only the potentiating effects of PD 128,907. These findings further indicate that D1 and D3 receptors are localized in the same structure. The D4 agonist PD 168,077 decreased mIPSCs frequency without changing amplitude, an effect that was blocked by the selective D4 antagonist L 745,870. The effects of D4 receptor stimulation disappeared after lesioning the globus pallidus. D3 agonist PD 128,907 did not reduce mIPSC frequency even in neurons that responded to D4 agonist. In sum, activation of D3 receptors in SNr potentiates the stimulation of transmitter release and cAMP production caused by D1 receptor activation of striatonigral projections while it is without effects in terminals, probably of pallidal origin, that are inhibited by activation of D4 receptors.
Assuntos
Neurônios GABAérgicos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D3/metabolismo , Receptores de Dopamina D4/metabolismo , Substância Negra/metabolismo , Animais , Agonistas de Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Masculino , Terminações Pré-Sinápticas/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Wistar , Substância Negra/efeitos dos fármacosRESUMO
Dopamine D4 receptors are localized in the GABAergic projections that globus pallidus (GP) neurons send to the reticular nucleus of the thalamus (RTN), the substantia nigra reticulata (SNr) and the subthalamic nucleus (STN). Deficient D4 function in this network could lead to hyperactivity and thus be important in generating some of the symptoms of ADHD (attention deficit hyperactivity disorder), a condition associated with polymorphisms of dopamine D4 receptors. It is then, unexpected that systemic injections of D4 ligands have no significant effects on the motor activity of normal rats. We further examined this issue by microinjecting D4 ligands and psychostimulant drugs in relevant structures. Interstitial dopamine overflow in the RTN was increased by reverse microdialysis of both methylphenidate and methamphetamine. Intranuclear injections in the RTN of methylphenidate, methamphetamine and the selective D4 agonist PD 168,077 reduced motor activity. Intraperitoneal injection of the D4 antagonist L 745,870 blocked the effects of these intranuclear injections. Similarly, intranuclear injections of PD 168,077 in the SNr inhibited motor activity, an effect that was also blocked by intraperitoneal L 745,870. In rats with 6-OHDA induced hemiparkinsonism, intraperitoneal PD 168,077 produced ipsilateral turning behavior that was blocked by L 745,870. Our results suggest that diminished D4 signaling in GP projections could lead to increased traffic through the relay nuclei of the thalamus and hyperactivity. Hence this basal-ganglia-thalamus network may be one of the targets of the beneficial effects that psychostimulant drugs have in disorders associated with D4 receptor abnormalities. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.
Assuntos
Neurônios GABAérgicos/efeitos dos fármacos , Globo Pálido/metabolismo , Atividade Motora/efeitos dos fármacos , Receptores de Dopamina D4/metabolismo , Substância Negra/metabolismo , Núcleos Talâmicos/metabolismo , Animais , Benzamidas/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Dopamina/metabolismo , Neurônios GABAérgicos/metabolismo , Globo Pálido/efeitos dos fármacos , Masculino , Metanfetamina/farmacologia , Metilfenidato/farmacologia , Microdiálise , Atividade Motora/fisiologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Norepinefrina/metabolismo , Piperazinas/farmacologia , Ratos , Ratos Wistar , Substância Negra/efeitos dos fármacos , Núcleos Talâmicos/efeitos dos fármacosRESUMO
L-DOPA treatment induces abnormal involuntary movements (AIMs) in Parkinson's patients and experimental animals. We examined the relationship between the development of AIMs (dyskinesia) and changes in [(3)H]-GABA release and cAMP signaling in striatonigral terminals of rats with unilateral 6-OHDA lesions. Analysis of AIMs scores in hemiparkinsonian rats treated with L-DOPA for 20 days was fitted by the sum of two Gaussian distributions showing the presence of two populations: one with mild and the other with severe dyskinesia. cAMP signaling was evaluated in the two populations by determining changes in cAMP formation, Gα(olf) and adenylyl cyclase type V/VI levels. In animals that were not treated with L-DOPA, all the parameters were significantly increased in the denervated side. In the animals that had mild dyskinesia, L-DOPA treatment normalized these parameters. In contrast, in the animals in which l-DOPA treatment induced severe dyskinesia all the parameters, except for Gα(olf) levels, were significantly higher in the denervated side. Similarly, D1-stimulated [(3)H]-GABA release was not elevated in L-DOPA-treated animals with mild dyskinesia but was increased in animals with severe dyskinesia. Changes in Gα(olf) and adenylyl cyclase type V/VI levels in the striatum paralleled the response in the SNr. The linkage between the changes in [(3)H]-GABA release and cAMP activity was further evaluated with the selective adenylyl cyclase V/VI antagonist NKY80. This inhibitor blocked the increases of both [(3)H]-GABA release and cAMP production. These results indicate that increased expression of adenylyl cyclase V/VI is a major determinant of increased GABAergic transmission in the substantia nigra pars reticulata of animals in which L-DOPA induces severe dyskinesia.
Assuntos
Adenilil Ciclases/biossíntese , Discinesia Induzida por Medicamentos/metabolismo , Levodopa/toxicidade , Transtornos Parkinsonianos/enzimologia , Substância Negra/efeitos dos fármacos , Regulação para Cima/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Discinesia Induzida por Medicamentos/enzimologia , Discinesia Induzida por Medicamentos/patologia , Inibidores Enzimáticos/farmacologia , Masculino , Oxidopamina/toxicidade , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/patologia , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Substância Negra/enzimologia , Substância Negra/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
GABA(B) receptors decrease the release of GABA from the striatal terminals within the pars reticulata of the substantia nigra by opposing the increase in the release caused by dopamine D1 receptors. The dopamine D1 receptors also increase the release of glutamate from subthalamic terminals in the pars reticulata. Because GABA(B) receptors decrease the glutamate release from these terminals, we have explored if the effect of GABA(B) receptors also opposed the effect of the dopamine D1 receptors. The effect of baclofen, a selective GABA(B)-receptor agonist, was tested on the release of [³H]glutamate caused by highly (40 mM) concentrated K(+) solutions in slices of the pars reticulata. Baclofen decreased (the concentration causing 50% inhibition, IC50, was 8.15 µM) the increase in the release of the [³H]glutamate caused by the dopamine D1 receptors and it also decreased (IC50 was 0.51 µM) this release in the absence of the activation of the dopamine D1 receptors. The GABA(B) receptors appear then to inhibit glutamate release in two ways; one dependent on the activation of the dopamine D1 receptors and the other independent of such activation. The protein kinase A-inhibitor H89 blocked the increase in the release of the [³H]glutamate caused by the dopamine D1 receptors, though it did not block the dopamine D1 receptor-independent baclofen inhibition of the release. This finding indicates that this inhibition was not via the protein kinase A signal-transduction pathway. N-ethylmaleimide, an alkylating agent that inactivates pertussis toxin-sensitive Gi proteins, eliminated both the dopamine D1 receptor-dependent and -independent baclofen inhibition, showing that both were mediated by these proteins. The injection of baclofen into the pars reticulata of unanesthetized rats caused contralateral rotation, suggesting a reduced glutamate release from the subthalamic terminals, thereby stopping the inhibition of the premotor thalamic nuclei, causing locomotion. Our data suggest that GABA(B) receptors restrain the excitatory input from the subthalamic nucleus and stimulate motor behavior.
Assuntos
Ácido Glutâmico/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Inibição Neural/efeitos dos fármacos , Neurônios/metabolismo , Receptores de GABA-B/fisiologia , Substância Negra/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Agonistas de Dopamina/farmacologia , Agonistas dos Receptores de GABA-B/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Técnicas In Vitro , Masculino , Atividade Motora/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Especificidade de Órgãos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Substância Negra/efeitos dos fármacos , Núcleo Subtalâmico/efeitos dos fármacos , Núcleo Subtalâmico/metabolismo , TrítioRESUMO
Neurons of the globus pallidus express dopamine D4 receptors that can modulate transmitter release by their axon terminals. Indeed, GABA release from pallidal terminals in the subthalamic nucleus and in the reticular nucleus of the thalamus is inhibited by activation of D4 receptors. Here we investigated whether GABA release by pallidal projections to the substantia nigra reticulate (SNr) is also modulated by D4 receptors. Dopamine-stimulated depolarization-induced GABA release in slices of the SNr; however, after selective blockade of D1 receptors, dopamine inhibited release. The selective D4 agonist PD 168,077 (IC(50) = 5.30 nM) mimicked the inhibition of release while the selective D4 antagonist L-745,870 blocked the inhibition. To identify the source of D1 and D4 modulated terminals, we unilaterally injected kainic acid in either the GP or the striatum. After lesions of the pallidum, the D4 induced inhibition of release was blocked while the D1 induced stimulation was still significant. Lesions of the striatum had the converse effects. We conclude that release of dopamine in the SNr enhances GABA release mainly through activation of D1 receptors in striatonigral projections and inhibits release mainly through activation of D4 receptors in pallidonigral projections. Because deficient D4 receptor signaling in globus pallidus terminals will lead to disinhibition of impulse traffic through the thalamus we speculate that the D4 abnormalities observed in ADHD patients may be important in the generation of the syndrome.
Assuntos
Receptores de Dopamina D1/fisiologia , Receptores de Dopamina D4/fisiologia , Substância Negra/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Dopamina/metabolismo , Dopamina/farmacologia , Agonistas de Dopamina/farmacologia , Globo Pálido/fisiologia , Técnicas In Vitro , Masculino , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D4/agonistas , Receptores de Dopamina D4/antagonistas & inibidores , Substância Negra/efeitos dos fármacos , Substância Negra/fisiopatologiaRESUMO
The motor effects of cannabinoids in the globus pallidus appear to be caused by increases in interstitial GABA. To elucidate the mechanism of this response, we investigated the effect of the selective cannabinoid type 1 receptor (CB1) cannabinoid agonist arachidonyl-2-chloroethylamide (ACEA) on [(3)H]GABA release in slices of the rat globus pallidus. ACEA had two effects: concentrations between 10(-8) and 10(-6) M stimulated release, whereas higher concentrations (IC(50) approximately 10(-6) M) inhibited it. Another cannabinoid agonist, WIN-55,212-2, also had bimodal effects on release. Studies of cAMP production indicate that under conditions of low G(i/o), availability the coupling of CB1 receptors with G(i/o) proteins can be changed into CB1:G(s/olf) coupling; therefore, we determined the effects of conditions that limit G(i/o) availability on [(3)H]GABA release. Blockers of G(i/o) protein interactions, pertussis toxin and N-ethylmaleimide, transformed the inhibitory effects of ACEA on GABA release into stimulation. It also has been suggested that stimulation of D2 receptors can reduce G(i/o) availability. Blocking D2 receptors with sulpiride [(S)-5-aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamidersqb] or depleting dopamine with reserpine inhibited the ACEA-induced stimulation of release. Thus, the D2 dependence of stimulation is consistent with the proposal that D2 receptors reduce G(i/o) proteins available for binding to the CB1 receptor. In summary, CB1 receptor activation has dual effects on GABA release in the globus pallidus. Low concentrations stimulate release through a process that depends on activation of dopamine D2 receptors that may limit G(i/o) protein availability. Higher concentrations of cannabinoid inhibit GABA release through mechanisms that are independent of D2 receptor activation.
Assuntos
Benzoxazinas/farmacologia , Canabinoides/farmacologia , Globo Pálido/metabolismo , Globo Pálido/fisiologia , Morfolinas/farmacologia , Naftalenos/farmacologia , Ácido gama-Aminobutírico/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Canabinoides/agonistas , Globo Pálido/efeitos dos fármacos , Masculino , Toxina Pertussis/farmacologia , Ratos , Ratos Wistar , Reserpina/farmacologia , TrítioRESUMO
It has been proposed that striatonigral GABAergic transmission in the substantia nigra reticulata (SNr) is enhanced during Parkinson's disease and subsequent L-DOPA treatment. To evaluate this proposal we determined the effects of activating dopamine D1 receptors on depolarization induced [(3)H]-GABA release and on [(3)H]-cAMP accumulation in slices of SNr of rats with unilateral 6-OHDA lesions with and without l-DOPA treatment. Denervation increased depolarization induced D1-stimulated [(3)H]-GABA release, while repeated L-DOPA treatment further enhanced this response. Both also enhanced the effects of forskolin on [(3)H]-cAMP production and [(3)H]-GABA release, while neither modified the stimulating effects of 8-Br-cAMP on the release. These results shown that, after 6-OHDA lesions and l-DOPA treatment, cAMP signaling is enhanced. Furthermore, the results suggest that activation of sites in the signaling cascade downstream of cAMP synthesis is not required to increase release.
Assuntos
AMP Cíclico/metabolismo , Lateralidade Funcional/fisiologia , Levodopa/uso terapêutico , Transtornos Parkinsonianos , Receptores de Dopamina D1/fisiologia , Substância Negra/metabolismo , Ácido gama-Aminobutírico/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Análise de Variância , Animais , Antiparkinsonianos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Dopaminérgicos/farmacologia , Masculino , Oxidopamina , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/patologia , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Wistar , Substância Negra/efeitos dos fármacos , Fatores de Tempo , Trítio/metabolismoRESUMO
The effects of D1/5 dopamine agonists on spontaneous excitatory postsynaptic currents (sEPSCs) were studied in neurons of the rat globus pallidus using whole-cell recordings in the presence of TTX and bicuculline. In this condition, CNQX abolished the sEPSCs, indicating that they were solely mediated by AMPA receptors. SKF 38393, a D1-like agonist, increased the frequency but not the amplitude of the sEPSCs, suggesting a presynaptic site of action. The increase in frequency was blocked by SCH 23390, a D1/5 antagonist. Quinpirole, a D2-like agonist, decreased the frequency but did not affect the amplitude of the synaptic currents. SKF 38393 increased the frequency of sEPSCs currents, even in presence of quinpirole, indicating that D1/5- and D2-like receptors independently modulate glutamate release upon a single neuron. The results suggest that the dopaminergic control of the glutamate transmission in the globus pallidus may play a role in processing cortical information in the indirect pathway of the basal ganglia.
Assuntos
Dopamina/metabolismo , Globo Pálido/metabolismo , Ácido Glutâmico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores de Dopamina D1/metabolismo , Transmissão Sináptica/fisiologia , Animais , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Antagonistas GABAérgicos/farmacologia , Globo Pálido/efeitos dos fármacos , Masculino , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de AMPA/efeitos dos fármacos , Receptores de AMPA/metabolismo , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Transmissão Sináptica/efeitos dos fármacosRESUMO
GABA(B) receptors inhibit and dopamine D1 receptors stimulate the release of GABA from striatal terminals in the pars reticulata of the substantia nigra. Here we have studied the interaction between both classes of receptors by exploring the effect of GABA(B) receptors upon the stimulation of depolarization-induced [(3)H]GABA release induced by the activation of D1 receptors in slices of the pars reticulata of the rat substantia nigra. The activation of GABA(B) receptors with baclofen (100 microM) inhibited by 48+/-8% the evoked [(3)H]GABA release in normal slices but did not modify the release in slices from reserpine-treated rats, indicating that the inhibition was dependent on endogenous dopamine. The inhibitory effect of baclofen was also abolished by the D1 receptor antagonist SCH 23390 (1 microM), indicating a D1 receptor-dependence of the baclofen inhibition. Baclofen dose-dependently inhibited (IC(50)=3.6 microM) the stimulation of release induced by the D1 agonist SKF 38393 (1 microM). Baclofen also blocked the stimulation of release induced by forskolin but not that induced by 8-Br-cAMP, indicating that the inhibitory effect was exerted before cAMP synthesis. N-ethylmaleimide (NEM), a selective inactivator of PTX-sensitive G-proteins, abolished the baclofen inhibition of the SKF 38393-induced stimulation of the release without affecting the stimulation induced by the D1 agonist, suggesting that the baclofen effect was mediated by Galpha(i/o) proteins. These results might have relevance in the control motor disorders associated with D1 receptor supersensitivity.
Assuntos
Agonistas GABAérgicos/farmacologia , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de GABA-B/efeitos dos fármacos , Substância Negra/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Baclofeno/análogos & derivados , Baclofeno/farmacologia , Benzazepinas/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Interpretação Estatística de Dados , Antagonistas de Dopamina/farmacologia , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Masculino , Ratos , Ratos Wistar , Receptores de Dopamina D1/metabolismo , Reserpina/farmacologia , Substância Negra/efeitos dos fármacosRESUMO
We examined modulation of [(3)H]GABA uptake in slices of the rat globus pallidus because stimulation of adenosine A(2A) receptors increases extracellular GABA in this structure. Pharmacological analysis showed that GAT-1 is the main transporter present in these slices. Both adenosine and the A(2A) agonist CGS 21680 reduced GABA uptake. Antagonist ZM 241385 prevented these effects. Agents that increase protein kinase A activity like forskolin and 8-bromo-cAMP also inhibited GABA uptake. The inhibition of uptake produced by these substances and by CGS 21680 was prevented by the protein kinase A blocker H-89. The protein phosphatase blocker okadaic acid reduced uptake; this effect and the response to CGS 21680 were not additive. The effective concentrations of adenosine (EC(50)=15.2microM) are within the range measured in the interstitial fluid under some physiological conditions. Thus, inhibition of uptake may be important in increasing interstitial GABA during endogenous adenosine release.
Assuntos
Agonistas do Receptor A2 de Adenosina , Proteínas da Membrana Plasmática de Transporte de GABA/fisiologia , Globo Pálido/metabolismo , Ácido gama-Aminobutírico/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Animais , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Interpretação Estatística de Dados , Inibidores Enzimáticos/farmacologia , Globo Pálido/efeitos dos fármacos , Técnicas In Vitro , Isoquinolinas/farmacologia , Masculino , Ácido Okadáico/farmacologia , Fenetilaminas/farmacologia , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Triazinas/farmacologia , Triazóis/farmacologiaRESUMO
The effects of activating dopaminergic D1 and D2 class receptors of the subthalamic projections that innervate the pars reticulata of the subtantia nigra (SNr) were explored in slices of the rat brain using the whole cell patch-clamp technique. Excitatory postsynaptic currents (EPSCs) that could be blocked by 6-cyano-7-nitroquinoxalene-2,3-dione and D-(-)-2-amino-5-phosphonopentanoic acid were evoked onto reticulata GABAergic projection neurons by local field stimulation inside the subthalamic nucleus in the presence of bicuculline. Bath application of (RS)-2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine hydrochloride (SKF-38393), a dopaminergic D1-class receptor agonist, increased evoked EPSCs by approximately 30% whereas the D2-class receptor agonist, trans-(-)-4aR-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo(3,4-g)quinoline (quinpirole), reduced EPSCs by approximately 25%. These apparently opposing actions were blocked by the specific D1- and D2-class receptor antagonists: R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetra-hydro-1H-3-benzazepinehydrochloride (SCH 23390) and S-(-)-5-amino-sulfonyl-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-methoxybenzamide (sulpiride), respectively. Both effects were accompanied by changes in the paired-pulse ratio, indicative of a presynaptic site of action. The presynaptic location of dopamine receptors at the subthalamonigral projections was confirmed by mean-variance analysis. The effects of both SKF-38393 and quinpirole could be observed on terminals contacting the same postsynaptic neuron. Sulpiride and SCH 23390 enhanced and reduced the evoked EPSC, respectively, suggesting a constitutive receptor activation probably arising from endogenous dopamine. These data suggest that dopamine presynaptically modulates the subthalamic projection that targets GABAergic neurons of the SNr. Implications of this modulation for basal ganglia function are discussed.