RESUMO
Though the facilitating influence of stress on drug abuse is well documented, the mechanisms underlying this interaction have yet to be fully elucidated. The present study explores the neurobiological mechanisms underpinning the sensitized response to the psychomotor-stimulating effects of cocaine following chronic restraint stress (CRS), emphasizing the differential contribution of both subcompartments of the nucleus accumbens (NA), the core (NAcore) and shell (NAshell), to this phenomenon. Adult male Wistar rats were restrained for 2 h/day for 7 days and, 2 weeks after the last stress exposure (day 21), all animals were randomly assigned to behavioral, biochemical or neurochemical tests. Our results demonstrated that the enduring CRS-induced increase in psychostimulant response to cocaine was paralleled by an increase of extracellular dopamine levels in the NAcore, but not the NAshell, greater than that observed in the non-stress group. Furthermore, we found that CRS induced an impairment of glutamate homeostasis in the NAcore, but not the NAshell. Its hallmarks were increased basal extracellular glutamate concentrations driven by a CRS-induced downregulation of GLT-1, blunted glutamate levels in response to cocaine and postsynaptic structural remodeling in pre-stressed animals. In addition, ceftriaxone, a known GLT-1 enhancer, prevented the CRS-induced GLT-1 downregulation, increased basal extracellular glutamate concentrations and changes in structural plasticity in the NAcore as well as behavioral cross-sensitization to cocaine, emphasizing the biological importance of GLT-1 in the comorbidity between chronic stress exposure and drug abuse. A future perspective concerning the paramount relevance of the stress-induced disruption of glutamate homeostasis as a vulnerability factor to the development of stress and substance use disorders during early life or adulthood of descendants is provided.
RESUMO
Stressful experience-induced cocaine-related behaviors are associated with a significant impairment of glutamatergic mechanisms in the Nucleus Accumbens core (NAcore). The hallmarks of disrupted glutamate homeostasis following restraint stress are the enduring imbalance of glutamate efflux after a cocaine stimulus and increased basal concentrations of extracellular glutamate attributed to GLT-1 downregulation in the NAcore. Glutamate transmission is tightly linked to microglia functioning. However, the role of microglia in the biological basis of stress-induced addictive behaviors is still unknown. By using minocycline, a potent inhibitor of microglia activation with anti-inflammatory properties, we determined whether microglia could aid chronic restraint stress (CRS)-induced glutamate homeostasis disruption in the NAcore, underpinning stress-induced cocaine self-administration. In this study, adult male rats were restrained for 2 h/day for seven days (day 1-7). From day 16 until completing the experimental protocol, animals received a vehicle or minocycline treatment (30 mg/Kg/12h i.p.). On day 21, animals were assigned to microscopic, biochemical, neurochemical or behavioral studies. We confirm that the CRS-induced facilitation of cocaine self-administration is associated with enduring GLT-1 downregulation, an increase of basal extracellular glutamate and postsynaptic structural plasticity in the NAcore. These alterations were strongly related to the CRS-induced reactive microglia and increased TNF-α mRNA and protein expression, since by administering minocycline, the impaired glutamate homeostasis and the facilitation of cocaine self-administration were prevented. Our findings are the first to demonstrate that minocycline suppresses the CRS-induced facilitation of cocaine self-administration and glutamate homeostasis disruption in the NAcore. A role of microglia is proposed for the development of glutamatergic mechanisms underpinning stress-induced vulnerability to cocaine addiction.
Assuntos
Cocaína , Animais , Cocaína/metabolismo , Ácido Glutâmico/metabolismo , Masculino , Microglia/metabolismo , Minociclina/metabolismo , Minociclina/farmacologia , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Preclinical models of stress-induced relapse to drug use have shown that the dysregulation of glutamatergic transmission within the nucleus accumbens (NA) contributes notably to the reinstatement of cocaine-seeking behavior in rodents. In this sense, there has been increasing interest in the cannabinoid type-1 receptor (CB1R), due to its crucial role in modulating glutamatergic neurotransmission within brain areas involved in drug-related behaviors. This study explored the involvement of CB1R within the NA subregions in the restraint stress-induced reinstatement of cocaine-conditioned place preference (CPP), as well as in the regulation of glutamatergic transmission, by using a pharmacological approach and the in vivo microdialysis sampling technique in freely moving rats. CB1R blockade by the antagonist/inverse agonist AM251 (5 nmol/0.5 µl/side) or CB1R activation by the agonist ACEA (0.01 fmol/0.5 µl/side), prevented or potentiated restraint stress-induced reinstatement of cocaine-CPP, respectively, after local administration into NAcore, but not NAshell. In addition, microdialysis experiments demonstrated that restraint stress elicited a significant increase in extracellular glutamate in NAcore under reinstatement conditions, with the local administration of AM251 or ACEA inhibiting or potentiating this, respectively. Interestingly, this rise specifically corresponded to the cocaine-associated CPP compartment. We also showed that this context-dependent change in glutamate paralleled the expression of cocaine-CPP, and disappeared after the extinction of this response. Taken together, these findings demonstrated the key role played by CB1R in mediating reinstatement of cocaine-CPP after restraint stress, through modulation of the context-specific glutamate release within NAcore. Additionally, CB1R regulation of basal extracellular glutamate was demonstrated and proposed as the underlying mechanism.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/etiologia , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/efeitos adversos , Ácido Glutâmico/metabolismo , Núcleo Accumbens/metabolismo , Receptor CB1 de Canabinoide/agonistas , Estresse Fisiológico , Animais , Comportamento Animal , Biomarcadores , Condicionamento Clássico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Extinção Psicológica , Espaço Extracelular/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/antagonistas & inibidores , Estresse Fisiológico/genéticaRESUMO
Actin dynamics in dendritic spines can be associated with the neurobiological mechanisms supporting the comorbidity between stress exposure and cocaine increase rewards. The actin cytoskeleton remodeling in the nucleus accumbens (NA) has been implicated in the expression of stress-induced cross-sensitization with cocaine. The present study evaluates the involvement of cofilin, a direct regulator of actin dynamics, in the impact of stress on vulnerability to cocaine addiction. We assess whether the neurobiological mechanisms that modulate repeated-cocaine administration also occur in a chronic restraint stress-induced cocaine self-administration model. We also determine if chronic stress induces alterations in dendritic spines through dysregulation of cofilin activity in the NA core. Here, we show that the inhibition of cofilin expression in the NA core using viral short-hairpin RNA is sufficient to prevent the cocaine sensitization induced by chronic stress. The reduced cofilin levels also impede a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor surface expression enhancement and promote the reduction of head diameter in animals pre-exposed to stress after a cocaine challenge in the NA core. Moreover, downregulation of cofilin expression prevents facilitation of the acquisition of cocaine self-administration (SA) in male rats pre-exposed to chronic stress without modifying performance in sucrose SA. These findings reveal a novel, crucial role for cofilin in the neurobiological mechanisms underpinning the comorbidity between stress exposure and addiction-related disorders.
RESUMO
Altered glutamate transmission within the nucleus accumbens (NAc) has been proposed as a central mechanism underlying behavioural sensitisation associated with repeated cocaine exposure. In addition to glutamate, enkephalin, an endogenous opioid peptide derived from proenkephalin, is necessary for the neuroadaptations associated with chronic cocaine. However, the influence of enkephalin on long-term changes in glutamate transmission within the NAc associated with cocaine-induced sensitisation has not been described. This study used knockout proenkephalin mice (KO) to study the influence of endogenous enkephalin on the adaptations in glutamate neurotransmission associated with repeated cocaine treatment. Wild-type (WT) and KO mice were treated with daily cocaine injections for 9 days to induce sensitisation. On days 15 and 21, the animals received a cocaine challenge and locomotor sensitisation was evaluated, and microdialysis was performed to determine accumbens glutamate content on day 21. No expression of behavioural sensitisation to cocaine was evidenced in the KO mice. Consistently, these showed no changes in glutamate transmission in the NAc associated with repeated cocaine. This study reveals the central role of enkephalin in regulating the glutamate mechanisms associated with cocaine sensitisation.
Assuntos
Cocaína , Animais , Encefalinas/genética , Ácido Glutâmico , Camundongos , Microdiálise , Núcleo AccumbensRESUMO
Enkephalin expression is high in mesocorticolimbic areas associated with psychostimulant-induced behavioral and neurobiological effects, and may also modulate local neurotransmission in this circuit network. Psychostimulant drugs, like amphetamine and cocaine, significantly increase the content of enkephalin in these brain structures, but we do not yet understand the specific significance of this drug-induced adaptation. In this review, we summarize the neurochemical and molecular mechanism of psychostimulant-induced enkephalin activation in mesocorticolimbic brain areas, and the contribution of this opioid peptide in the pivotal neuroadaptations and long-term behavioral changes underlying psychostimulant addiction. There is evidence suggesting that adaptive changes in enkephalin content in the mesocorticolimbic circuit, induced by acute and chronic psychostimulant administration, may represent a key initial step in the long-term behavioral and neuronal plasticity induced by these drugs.
RESUMO
The axonal microtubule-associated protein tau is a well-known regulator of microtubule stability in neurons. However, the putative interplay between tau and End-binding proteins 1 and 3 (EB1/3), the core microtubule plus-end tracking proteins, has not been elucidated yet. Here, we show that a cross-talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E-115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down-regulation leads to a reduction of EB1/3 comet length, as observed in shRNA-stably depleted neuroblastoma cells and TAU-/- neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over-expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E-115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non-neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull-down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross-talk between a classical microtubule-associated protein and a core microtubule plus-end tracking protein may contribute to the fine-tuned regulation of microtubule dynamics and stability during neuronal differentiation. We describe here a novel function for tau as a direct regulator of End binding (EB) proteins in differentiating neuronal cells. EB1/3 cellular mobility and localization in extending neurites and axons is modulated by tau levels and localization. We provide new evidence of the interplay between classical microtubule-associated proteins (MAPs) and "core" microtubule plus-end tracking proteins (+TIPs) during neuronal development.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Camundongos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Neuritos/metabolismo , Plasmídeos , RNA Interferente Pequeno/genéticaRESUMO
This study investigated the consequence of repeated stress on actin cytoskeleton remodeling in the nucleus accumbens (NAc) and prefrontal cortex (Pfc), and the involvement of this remodeling in the expression of stress-induced motor cross-sensitization with cocaine. Wistar rats were restrained daily (2 h) for 7 days and, 3 weeks later, their NAc and Pfc were dissected 45 min after acute saline or cocaine (30 mg/kg i.p.). F-actin, actin-binding proteins (ABP) and GluR1 were quantified by Western blotting, and dendritic spines and postsynaptic density (PSD) size measured by electron microscopy. In the NAc from the stress plus cocaine group we observed a decrease in the phosphorylation of two ABPs, cofilin and cortactin, and an increase in the PSD size and the surface expression of GluR1, consistent with a more highly branched actin cytoskeleton. The Pfc also showed evidence of increased actin polymerization after stress as an increase was observed in Arp2, and in the number of spines. Inhibiting actin cycling and polymerization with latrunculin A into the NAc, but not the Pfc, inhibited the expression of cross-sensitization to cocaine (15 mg/kg i.p.) and restored the expression of GluR1 to control levels. This study shows that a history of repeated stress alters the ability of a subsequent cocaine injection to modulate dendritic spine morphology, actin dynamics and GluR1 expression in the NAc. Furthermore, by regulating GluR1 expression in the NAc, elevated actin cycling contributes to the expression of cross-sensitization between stress and cocaine, while stress-induced changes in the Pfc were not associated with cross-sensitization.
Assuntos
Citoesqueleto de Actina/metabolismo , Sensibilização do Sistema Nervoso Central , Cocaína/farmacologia , Núcleo Accumbens/fisiologia , Córtex Pré-Frontal/fisiologia , Estresse Psicológico/fisiopatologia , Citoesqueleto de Actina/efeitos dos fármacos , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cortactina/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Expressão Gênica , Masculino , Atividade Motora , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Fosforilação , Polimerização , Densidade Pós-Sináptica/metabolismo , Densidade Pós-Sináptica/ultraestrutura , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Restrição Física , Estresse Psicológico/metabolismo , Tiazolidinas/farmacologiaRESUMO
Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization.
Assuntos
Axônios/enzimologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Cinética , Quinases Lim/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
In non-neuronal cells, inactivation of protein kinase D (PKD) blocks fission of trans-Golgi network (TGN) transport carriers, inducing the appearance of long tubules filled with cargo. We now report on the function of PKD1 in neuronal protein trafficking. In cultured hippocampal pyramidal cells, the transferrin receptor (TfR) and the low-density receptor-related protein (LRP) are predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive PKD1 or its depletion by RNA interference treatment dramatically and selectively alter the intracellular trafficking and membrane delivery of TfR- and LRP-containing vesicles, without inhibiting exit from the TGN or inducing Golgi tubulation. After PKD1 suppression, dendritic membrane proteins are mispackaged into carriers that transport VAMP2; these vesicles are distributed to both axons and dendrites, but are rapidly endocytosed from dendrites and preferentially delivered to the axonal membrane. A kinase-defective mutant of PKD1 lacking the ability to bind diacylglycerol and hence its Golgi localization does not cause missorting of TfR or LRP. These results suggest that in neurons PKD1 regulates TGN-derived sorting of dendritic proteins and hence has a role in neuronal polarity.
Assuntos
Dendritos/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Neurônios/fisiologia , Proteínas Quinases/fisiologia , Receptores da Transferrina/metabolismo , Animais , Células Cultivadas , Dendritos/efeitos dos fármacos , Embrião de Mamíferos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Proteína Quinase C , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Fatores de Tempo , Transfecção/métodos , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Gravação de Videoteipe/métodos , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismoRESUMO
How a neuron becomes polarized remains largely unknown. Results obtained with a function-blocking antibody and an siRNA targeting the insulin-like growth factor-1 (IGF-1) receptor suggest that an essential step in the establishment of hippocampal neuronal polarity and the initiation of axonal outgrowth is the activation of the phosphatidylinositol 3-kinase (PI3k)-Cdc42 pathway by the IGF-1 receptor, but not by the TrkA or TrkB receptors.
Assuntos
Polaridade Celular , Hipocampo/citologia , Neurônios/citologia , Receptor IGF Tipo 1/metabolismo , Animais , Células Cultivadas , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptor IGF Tipo 1/genética , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Activity-prompted dendritic remodeling leads to calcium-influx-dependent activation of signaling pathways within minutes and gene transcription within hours. However, dendrite growth continues for days and requires extension and stabilization of the cytoskeleton in nascent processes. In addition to binding microtubules, microtubule-associated proteins (MAPs) associate with the actin cytoskeleton, anchor ion channels and signaling complexes, and modulate synaptic growth. MAP2 is predominantly dendritic. MAP1B is at postsynaptic densities (PSD) and modulates ion channel activity, in addition to affecting axon growth. Less is known about MAP1A, but it is also enriched in dendrites at input locations, including PSDs where MAP1A associates with channel complexes and the calcium sensor caldendrin. MAP1A rescued hearing loss in tubby mice. Here we show that MAP1A becomes enriched in dendrites concurrently with dendritic branching and synapse formation in the developing brain; that synaptic activity is required for establishing mature MAP1A expression levels; and that MAP1A expression is required for activity-dependent growth, branching, and stabilization of the dendritic arbor.
Assuntos
Dendritos/fisiologia , Expressão Gênica , Hipocampo/citologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia , Animais , Hipocampo/crescimento & desenvolvimento , Immunoblotting , Imuno-Histoquímica , Microscopia de Fluorescência , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-DawleyRESUMO
Coordinated microtubule and microfilament changes are essential for the morphological development of neurons; however, little is know about the underlying molecular machinery linking these two cytoskeletal systems. Similarly, the indispensable role of RhoGTPase family proteins has been demonstrated, but it is unknown how their activities are specifically regulated in different neurites. In this paper, we show that the cytoplasmic dynein light chain Tctex-1 plays a key role in multiple steps of hippocampal neuron development, including initial neurite sprouting, axon specification, and later dendritic elaboration. The neuritogenic effects elicited by Tctex-1 are independent from its cargo adaptor role for dynein motor transport. Finally, our data suggest that the selective high level of Tctex-1 at the growth cone of growing axons drives fast neurite extension by modulating actin dynamics and also Rac1 activity.
Assuntos
Actinas/metabolismo , Axônios/metabolismo , Dineínas/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas rac1 de Ligação ao GTP/fisiologia , Animais , Axônios/ultraestrutura , Polaridade Celular , Células Cultivadas , Clonagem Molecular , Cones de Crescimento/metabolismo , Hipocampo/citologia , Neuritos/fisiologia , Ratos , Região do Complexo-t do GenomaRESUMO
In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.