RESUMO
Psychostimulants regulate behavioral responses in zebrafish via epigenetic mechanisms. We have previously shown that DNA methylation and histone deacetylase (HDAC) inhibition abolish nicotine-induced conditioned place preference (CPP) but little is known about the role of histone methylation in addictive-like behaviors. To assess the influence of histone methylation on nicotine-CPP, zebrafish were treated with a histone (H3) lysine-9 (K9) dimethyltransferase G9a/GLP inhibitor, BIX-01294 (BIX), which was administered before conditioning sessions. We observed a dual effect of the inhibitor BIX: at high doses inhibited while at low doses potentiated nicotine reward. Transcriptional expression of α6 and α7 subunits of the nicotinic acetylcholine receptor and of G9a, DNA methyl transferase-3, and HDAC-1 was upregulated in zebrafish with positive scores for nicotine-CPP. Changes in relative levels of these mRNA molecules reflected the effects of BIX on nicotine reward. BIX treatment per sé did not affect transcriptional levels of epigenetic enzymes that regulate trimethylation or demethylation of H3. BIX reduced H3K9me2 protein levels in a dose-dependent manner in key structures of the reward pathway. Thus, our findings indicated that different doses of BIX differentially affect nicotine CPP via strong or weak inhibition of G9a/GLP activity. Additionally, we found that the lysine demethylase inhibitor daminozide abolished nicotine-CPP and drug seeking. Our data demonstrate that H3 methylation catalyzed by G9a/GLP is involved in nicotine-CPP induction. Dimethylation of K9 at H3 is an important epigenetic modification that should be considered as a potential therapeutic target to treat nicotine reward and perhaps other drug addictions.
Assuntos
Histona-Lisina N-Metiltransferase , Nicotina , Peixe-Zebra , Animais , Nicotina/farmacologia , Histona-Lisina N-Metiltransferase/metabolismo , Azepinas/farmacologia , Histonas/metabolismo , Recompensa , Quinazolinas/farmacologia , Receptores Nicotínicos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , MasculinoRESUMO
Nicotine induces long-term changes in the neural activity of the mesocorticolimbic reward pathway structures. The mechanisms involved in this process have not been fully characterized. The hypothesis discussed here proposed that epigenetic regulation participates in the installation of persistent adaptations and long-lasting synaptic plasticity generated by nicotine action on the mesolimbic dopamine neurons of zebrafish. The epigenetic mechanisms induced by nicotine entail histone and DNA chemical modifications, which have been described to lead to changes in gene expression. Among the enzymes that catalyze epigenetic chemical modifications, histone deacetylases (HDACs) remove acetyl groups from histones, thereby facilitating DNA relaxation and making DNA more accessible to gene transcription. DNA methylation, which is dependent on DNA methyltransferase (DNMTs) activity, inhibits gene expression by recruiting several methyl binding proteins that prevent RNA polymerase binding to DNA. In zebrafish, phenylbutyrate (PhB), an HDAC inhibitor, abolishes nicotine rewarding properties together with a series of typical reward-associated behaviors. Furthermore, PhB and nicotine alter long- and short-term object recognition memory in zebrafish, respectively. Regarding DNA methylation effects, a methyl group donor L-methionine (L-met) was found to dramatically reduce nicotine-induced conditioned place preference (CPP) in zebrafish. Simultaneous treatment with DNMT inhibitor 5-aza-2'-deoxycytidine (AZA) was found to reverse the L-met effect on nicotine-induced CPP as well as nicotine reward-specific effects on genetic expression in zebrafish. Therefore, pharmacological interventions that modulate epigenetic regulation of gene expression should be considered as a potential therapeutic method to treat nicotine addiction.
Assuntos
Nicotina , Peixe-Zebra , Animais , Epigênese Genética , Inibidores de Histona Desacetilases/farmacologia , Nicotina/farmacologia , Recompensa , Peixe-Zebra/genéticaRESUMO
In zebrafish, nicotine is known to regulate sensitivity to psychostimulants via epigenetic mechanisms. Little however is known about the regulation of addictive-like behavior by DNA methylation processes. To evaluate the influence of DNA methylation on nicotine-induced conditioned place preference (CPP), zebrafish were exposed to methyl supplementation through oral L-methionine (Met) administration. Met was found to reduce dramatically nicotine-induced CPP as well as behaviors associated with drug reward. The reduction was associated with the upregulation of DNA methyltransferases (DNMT1 and 3) as well as with the downregulation of methyl-cytosine dioxygenase-1 (TET1) and of nicotinic receptor subunits. Met also increased the expression of histone methyltransferases in nicotine-induced CPP groups. It reversed the nicotine-induced reduction in the methylation at α7 and NMDAR1 gene promoters. Treatment with the DNMT inhibitor 5-aza-2'-deoxycytidine (AZA) was found to reverse the effects of Met in structures of the reward pathway. Interestingly, Met did not modify the amount of the phospho-form of CREB (pCREB), a key factor establishing nicotine conditioning, whereas AZA increased pCREB levels. Our data suggest that nicotine-seeking behavior is partially dependent on DNA methylation occurring probably at specific gene loci, such as α7 and NMDAR1 receptor gene promoters. Overall, they suggest that Met should be considered as a potential therapeutic drug to treat nicotine addiction.
Assuntos
Comportamento Animal/fisiologia , Comportamento de Escolha , Suplementos Nutricionais , Metionina/farmacologia , Nicotina/farmacologia , Peixe-Zebra/fisiologia , 5-Metilcitosina/metabolismo , Animais , Azacitidina/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Condicionamento Clássico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Recompensa , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Injured retinas in mammals do not regenerate and heal with loss of function. The adult retina of zebrafish self-repairs after damage by activating cell-intrinsic mechanisms, which are regulated by extrinsic signal interactions. Among relevant regulatory extrinsic systems, purinergic signaling regulates progenitor proliferation during retinogenesis and regeneration and glia proliferation in proliferative retinopathies. ATP-activated P2X7 (P2RX7) and adenosine (P1R) receptors are involved in the progression of almost all retinopathies leading to blindness. Here, we examined P2RX7 and P1R participation in the retina regenerative response induced by photoreceptor damage caused by a specific dose of CoCl2 . First, we found that treatment of uninjured retinas with a potent agonist of P2RX7 (BzATP) provoked photoreceptor damage and mitotic activation of multipotent progenitors. In CoCl2 -injured retinas, blockade of endogenous extracellular ATP activity on P2RX7 caused further neurodegeneration, Müller cell gliosis, progenitor proliferation, and microglia reactivity. P2RX7 inhibition in injured retinas also increased the expression of lin28a and tnfα genes, which are related to multipotent progenitor proliferation. Levels of hif1α, vegf3r, and vegfaa mRNA were enhanced by blockade of P2RX7 immediately after injury, indicating hypoxic like damage and endothelial cell growth and proliferation. Complete depletion of extracellular nucleotides with an apyrase treatment strongly potentiated cell death and progenitor proliferation induced with CoCl2 . Blockade of adenosine P1 and A2A receptors (A2A R) had deleterious effects and deregulated normal timing for progenitor and precursor cell proliferation following photoreceptor damage. ATP via P2RX7 and adenosine via A2A R are survival extracellular signals key for retina regeneration in zebrafish.
Assuntos
Regeneração Nervosa/fisiologia , Neurônios/patologia , Células Fotorreceptoras de Vertebrados/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animais , Morte Celular/fisiologia , Cobalto/toxicidade , Degeneração Neural/induzido quimicamente , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Peixe-ZebraRESUMO
Prior exposure to drugs of abuse may facilitate addiction. It has been described that pre-exposure to nicotine can increase or, contrarily, prevent conditioned place preference (CPP). Here, we evaluated the effect of nicotine pre-exposure on CPP performance using an original protocol mimicking smokers' behaviour in zebrafish. We simulated nicotine withdrawal at sleep time by exposing zebrafish to nicotine during daylight but not at night (D/N) for 14 days and then performed nicotine-CPP in zebrafish. D/N-nicotine-treated zebrafish obtained the highest CPP score, whereas zebrafish pre-exposed continuously to nicotine did not show nicotine-CPP. Evaluation of locomotor activity, seeking and anxiety-like behaviours supported the CPP findings. Nicotinic receptor subunit gene expression showed significant increases in the brain of zebrafish exposed to nicotine. Zebrafish exposed to D/N-nicotine showed further increases of α6- and α7-subunit expression after CPP establishment. Inhibition of histone acetylation by phenylbutyrate prevented nicotine-CPP. Transcriptional expression of epigenetic enzymes controlling histone acetylation/deacetylation and DNA methylation/demethylation was widely modified in brain portions containing reward areas of zebrafish exposed to D/N-nicotine after CPP. Zebrafish exposed to D/N-nicotine showed high levels of acetylated histone 3 and pCREB immunoreactivity differentially found in nuclei of the dopaminergic reward circuit in zebrafish homologous to the ventral tegmental area, nucleus accumbens and dorsal habenula. Our findings demonstrated that repetitive abstinent periods are risky factors for drug abuse that potentiate nicotine-environment associations and seeking. Brain modifications can persist long after nicotine use and are likely due to changes in the transcriptional expression of enzymes regulating drug reward-related gene expression via epigenetic modifications.
Assuntos
Ritmo Circadiano , Epigênese Genética/efeitos dos fármacos , Nicotina/farmacologia , Peixe-Zebra/genética , Acetilação , Envelhecimento/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Condicionamento Clássico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Lisina/metabolismo , Fenilbutiratos/farmacologia , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Nicotínicos/metabolismo , Recompensa , Análise e Desempenho de TarefasRESUMO
The adult zebrafish is considered a useful model for studying mechanisms involved in tissue growth and regeneration. We have characterized cytotoxic damage to the retina of adult zebrafish caused by the injection of cobalt chloride (CoCl2 ) into the vitreous cavity. The CoCl2 concentration we used primarily caused injury to photoreceptors. We observed the complete disappearance of cones, followed by rods, across the retina surface from 28 to 96 hr after CoCl2 injury. The loss of 30% of bipolar cells was also observed by 50 hr after lesion (hpl). CoCl2 injury provoked a strong induction of the proliferative activity of multipotent Müller glia and derived progenitors. The effect of CoCl2 on retina cells was significantly reduced by treatment with glutamate ionotropic receptor antagonists. Cone photoreceptor regeneration occurred 25 days after injury. Moreover, a single dose of CoCl2 induced vascular damage and regeneration, whereas three injections of CoCl2 administered weekly provoked neovascular-like changes 20 days after injury. CoCl2 injury also caused microglial reactivity in the optic disc, retina periphery and fibre layer. CoCl2 -induced damage enhanced pluripotency and proneural transcription factor gene expression in the mature retina 72 hpl. Tumour necrosis factor alpha, vascular endothelial growth factor (VEGF) and VEGF receptor mRNA levels were also significantly enhanced by 72 hpl. The injury paradigm we have described in this work may be useful for the discovery of signalling molecules and pathways that participate in the regenerative response and it may serve as a model to screen for compounds that could potentially treat aberrant angiogenesis.
Assuntos
Proliferação de Células/fisiologia , Cobalto/toxicidade , Neovascularização Fisiológica/fisiologia , Neuroglia/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Fatores Etários , Animais , Antimutagênicos/toxicidade , Proliferação de Células/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/patologia , Retina/efeitos dos fármacos , Retina/patologia , Peixe-ZebraRESUMO
Damage in fish activates retina repair that restores sight. The purinergic signalling system serves multiple homeostatic functions and has been implicated in cell cycle control of progenitor cells in the developing retina. We examined whether changes in the expression of purinergic molecules were instrumental in the proliferative phase after injury of adult zebrafish retinas with ouabain. P2RY1 messenger RNA (mRNA) increased early after injury and showed maximal levels at the time of peak progenitor cell proliferation. Extracellular nucleotides, mainly ADP, regulate P2RY1 transcriptional and protein expression. The injury-induced upregulation of P2RY1 is mediated by an autoregulated mechanism. After injury, the transcriptional expression of ecto-nucleotidases and ecto-ATPases also increased and ecto-ATPase activity inhibitors decreased Müller glia-derived progenitor cell amplification. Inhibition of P2RY1 endogenous activation prevented progenitor cell proliferation at two intervals after injury: one in which progenitor Müller glia mitotically activates and the second one in which Müller glia-derived progenitor cells amplify. ADPßS induced the expression of lin28a and ascl1a genes in mature regions of uninjured retinas. The expression of these genes, which regulate multipotent Müller glia reprogramming, was significantly inhibited by blocking the endogenous activation of P2RY1 early after injury. We consistently observed that the number of glial fibrillary acidic protein-BrdU-positive Müller cells after injury was larger in the absence than in the presence of the P2RY1 antagonist. Ecto-ATPase activity inhibitors or P2RY1-specific antagonists did not modify apoptotic cell death at the time of peak progenitor cell proliferation. The results suggested that ouabain injury upregulates specific purinergic signals which stimulates multipotent progenitor cell response.
Assuntos
Regulação da Expressão Gênica/fisiologia , Regeneração Nervosa/fisiologia , Células-Tronco Pluripotentes/fisiologia , Receptores Purinérgicos P2Y1/metabolismo , Retina/fisiologia , Animais , Mitose , Células-Tronco Neurais , Neurogênese/fisiologia , Retina/citologia , Transdução de Sinais/fisiologia , Regulação para Cima , Peixe-ZebraRESUMO
Screening for novel anticonvulsant drugs requires appropriate animal seizure models. Zebrafish provide small, accessible, and cost-efficient preclinical models applicable to high-throughput small molecule screening. Based on previous results in rodents, we have here examined the effects of artificial sweetener sodium cyclamate and antimicrobial agent sodium propylparaben on a model of pentylenetetrazole (PTZ)-induced seizures in zebrafish. Sodium cyclamate reduced the bursts of hyperactivity, the spasms, increased the latency to spasms, and the latency to seizure, while propylparaben increased the latency to spasms. The results show the potential of zebrafish to detect novel anticonvulsant compounds while they also demonstrate the ability of two commonly ingested chemical compounds to modify the seizure threshold when were administrated at low concentration.
Assuntos
Anticonvulsivantes/farmacologia , Ciclamatos/farmacologia , Parabenos/farmacologia , Convulsões/fisiopatologia , Animais , Modelos Animais de Doenças , Pentilenotetrazol/toxicidade , Conservantes Farmacêuticos/efeitos adversos , Conservantes Farmacêuticos/farmacologia , Tempo de Reação/efeitos dos fármacos , Convulsões/etiologia , Edulcorantes/efeitos adversos , Edulcorantes/farmacologia , Testes de Toxicidade/métodos , Peixe-ZebraRESUMO
Studies using mice and rats have demonstrated that nicotine induces a conditioned place preference (CPP), with more effective results obtained by using biased procedures. Zebrafish have also been used as a model system to identify factors influencing nicotine-associated reward by using an unbiased design. Here, we report that zebrafish exhibited putative nicotine biased CPP to an initially aversive compartment (nicotine-paired group). A counterbalanced nicotine-exposed control group did not show a significant preference shift, providing evidence that the preference shift in the nicotine-paired group was not due to a reduction of aversion for this compartment. Zebrafish preference was corroborated by behavioral analysis of several indicators of drug preference, such as time spent in the drug-paired side, number of entries to the drug-paired side, and distance traveled. These results provided strong evidence that zebrafish may actually develop a preference for nicotine, although the drug was administrated in an aversive place for the fish, which was further supported by molecular studies. Reverse transcription-quantitative real-time PCR analysis depicted a significant increase in the expression of α7 and α6 but not α4 and ß2 subunits of the nicotinic receptor in nicotine-paired zebrafish brains. In contrast, zebrafish brains from the counterbalanced nicotine group showed no significant changes. Moreover, CREB phosphorylation, an indicator of neural activity, accompanied the acquisition of nicotine-CPP. Our studies offered an incremental value to the drug addiction field, because they further describe behavioral features of CPP to nicotine in zebrafish. The results suggested that zebrafish exposed to nicotine in an unfriendly environment can develop a preference for that initially aversive place, which is likely due to the rewarding effect of nicotine. Therefore, this model can be used to screen exogenous and endogenous molecules involved in nicotine-associated reward in vertebrates.
Assuntos
Condicionamento Psicológico/fisiologia , Nicotina/farmacologia , Peixe-Zebra/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Reação em Cadeia da Polimerase em Tempo Real , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
RATIONALE: Prior exposure to drugs of abuse may increase or decrease the reinforcing effects of the drug in later consumptions. Based on the initial locomotor activity (LA) response to an acute drug administration or to novelty in an open-field arena, animals can be classified as low or high LA responders (LR or HR). Few studies have used this classification with nicotine, and the results are controversial. Some authors suggested that nicotine can induce conditioned-place preference (CPP) following prior nicotine exposure, whereas others suggested that previous nicotine exposure extinguishes nicotine-CPP. OBJECTIVE: To explore if the administration of nicotine in a novel environment without explicit behavioral consequences to classify animals in low and high nicotine responders (LNR and HNR) could affect the establishment of nicotine CPP in male Sprague-Dawley rats. RESULTS: Prior exposure to a single dose of nicotine (0.4 mg/kg, subcutaneously) induced CPP in LNR rats after 14 days of conditioning (seven-trial) but not after two or eight conditioning days. In contrast, HNR rats did not show CPP under any condition. In addition, our results indicated that previous exposure to nicotine decreased its rewarding effects in eight conditioning days CPP (four-trial), which can be regularly established without prior exposure to nicotine. CONCLUSION: The results suggested that response to a single exposure to nicotine predicts the acquisition of nicotine preference in a 14-day conditioning protocol only for LNR rats. Thus, our findings demonstrated the relevance of using LNR and HNR classification when the individual susceptibility to nicotine preference is studied.
Assuntos
Condicionamento Psicológico/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Nicotina/farmacologia , Recompensa , Animais , Injeções Subcutâneas , Masculino , Nicotina/administração & dosagem , Ratos , Ratos Sprague-Dawley , Reforço Psicológico , Fatores de TempoRESUMO
Epigenetic mechanisms have recently been shown to be involved in the long-term effects of drugs of abuse. A well described epigenetic mechanism modulating transcriptional activity consists in the binding to DNA of methyl-CpG binding proteins, such as MeCP2, recruiting histone deacetylases (HDACs). Nicotine causes long-term changes in the brain, but little is known concerning the mechanisms involved in nicotine-preference. Using a nicotine-conditioned place preference protocol, we demonstrate here that the histone deacetylase inhibitor phenylbutyrate was able to dramatically reduce the preference for nicotine, without altering the aversive properties of the drug. We measured immunohistochemically the acetylation of lysine-9 of histone H3, and the expression of phosphorylated cAMP-response element-binding protein, HDAC2 and methyl-CpG-binding protein 2 in the striatum and prefrontal cortex of rats displaying nicotine-preference or aversion and treated with phenylbutyrate. We show that, at the dose administered, the inhibitor was effective in inhibiting HDAC activity. The data suggest that phosphorylated cAMP-response element-binding protein participates in the establishment of conditioned place preference, but not in the reduction of nicotine-preference in response to phenylbutyrate. Moreover, striatal expression of HDAC2 in response to phenylbutyrate mirrored the behavioral effects of the inhibitor, suggesting that HDAC2 is involved in promoting synaptic plasticity underlying the preference for nicotine.
Assuntos
Aprendizagem da Esquiva/fisiologia , Condicionamento Psicológico/fisiologia , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/biossíntese , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Nicotina/farmacologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Comportamento de Escolha/efeitos dos fármacos , Comportamento de Escolha/fisiologia , Condicionamento Psicológico/efeitos dos fármacos , Corpo Estriado/enzimologia , Histona Desacetilases/biossíntese , Masculino , Fenilbutiratos/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE: Experimental evidence indicates that nicotine causes long-lasting changes in the brain associated with behavior. Although much has been learned about factors participating in this process, less is known concerning the mechanisms and brain areas involved in nicotine preference. OBJECTIVES: The objective of this study is to examine the participation of brain structures during the development of nicotine-conditioned place preference (CPP). METHODS: To identify brain regions activated in CPP, we have measured the levels of phosphorylated cyclic AMP response element binding protein (pCREB) and Fos protein using a behavioral CPP and conditioned place aversion (CPA) paradigms. RESULTS: Rats developed reliable and robust CPP and also CPA. During nicotine preference and reinstatement behaviors, a significant increase of both pCREB and Fos protein expression occurs in the nucleus accumbens (NAc) and ventral tegmental area (VTA) and also in the prefrontal cortex (PFC), dorsal striatum (DStr), amygdala, and hippocampus. These increases were abolished by the administration of mecamylamine or by a CPA protocol, showing a specific activation of pCREB in drug preference animals, mediated by nicotinic receptors. Specifically in the VTA, nicotine-induced preference and reinstatement of the preference caused the activation of dopaminergic and GABAergic cells in different proportions. CONCLUSION: The results indicate that the phosphorylation of CREB and expression of Fos protein, as indicators of neural activity, accompany the acquisition and maintenance of nicotine-induced CPP but not CPA in mesolimbic areas (NAc, VTA, PFC, and DStr) as well as in memory consolidation structures (hippocampus and amygdala) and nicotinic receptor are involved in this process. Taken together, these studies identify the brain regions where pCREB activity is essential for nicotine preference.
Assuntos
Encéfalo/efeitos dos fármacos , Proteína de Ligação a CREB/metabolismo , Condicionamento Operante/efeitos dos fármacos , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Proteínas Oncogênicas v-fos/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Contagem de Células/métodos , Condicionamento Operante/fisiologia , Extinção Psicológica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Masculino , Mecamilamina/farmacologia , Antagonistas Nicotínicos/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Since protein kinase C (PKC) is known to be activated in the olfactory bulb and in several limbic areas related to odor processing, we determined whether an olfactory stimulus was able to modulate the activity of PKC in animals with bilateral entorhinal cortex lesion. The translocation of PKC from the cytosol to the membrane was studied using the phorbol ester 12,13-dibutyrate ([3H]PDBu) binding in control and bilateral entorhinal cortex (EC) lesioned rats. The lesion of EC per se did not significantly affect [3H]PDBu binding in any of the brain structures analyzed, while odor stimulation induced it in both control and EC-lesioned groups in the external plexiform layer of the olfactory bulb. In contrast, an odor-induced increase of [3H]PDBu binding in internal glomerular layer of the olfactory bulb was only observed in EC lesioned animals. Similar results were obtained in the piriform cortex. In both CA1 and CA3 hippocampal subfields, odor stimulation induced an increase of [3H]PDBu binding in both control and EC-lesioned animals, the increase being potentiated only in CA1 of lesioned rats. The dentate gyrus and the amygdala exhibited a similar pattern of [3H]PDBu binding, showing a significant increase exclusively in EC-lesioned animals after odor stimulation. The results strongly suggest that the EC plays a key role in odor processing. PKC appears to play an important role in responding to the activation of lipid second messengers, which have been described to be involved in the processing of odor stimuli in several structures of the olfactory pathway.