RESUMEN
According to different studies, between 5% and 10% of people suffer a single isolated seizure episode at some time in their life. However, little is known about the effects of a single seizure episode on cognitive function, and clinical investigations of this issue are not easy to perform. In this situation, animal models may be a reasonable choice. The aim of our study was to follow the time course of delayed effects of generalized clonic-tonic convulsions on learning and memory functions in rats. A clonic-tonic seizure episode was induced by a single i.p. injection of pentylenetetrazole (70 mg/kg). Different behavioral tests were performed between days 10 and 100 after the convulsant administration. A single seizure episode resulted in a gradual decline in short-term memory function as assessed by novel object recognition and social recognition tests. The seizure episode induced a quick increase in hippocampal cell proliferation; however, the excessive newly generated cells seemed to be eliminated by the time of obvious cognitive impairment. These observations are indicative of a slowly developing and long-lasting influence of a single seizure episode on cognitive function. A rather long time period between the seizure episode and the manifestations of cognitive decline provides a window for a possible therapeutic intervention, and an elaboration of such "post-conditioning" treatments may be a promising opportunity to prevent subsequent mental impairments in patients.
Asunto(s)
Trastornos del Conocimiento/inducido químicamente , Hipocampo/fisiopatología , Aprendizaje por Laberinto/fisiología , Reconocimiento en Psicología/fisiología , Convulsiones/inducido químicamente , Animales , Proliferación Celular , Trastornos del Conocimiento/complicaciones , Trastornos del Conocimiento/fisiopatología , Progresión de la Enfermedad , Masculino , Pentilenotetrazol , Ratas , Ratas Wistar , Convulsiones/complicaciones , Convulsiones/fisiopatologíaRESUMEN
We have previously reported that a single injection of an ultra-low dose of delta-9-tetrahydrocannabinol (THC; the psychoactive ingredient of marijuana) protected the brain from pentylenentetrazole (PTZ)-induced cognitive deficits when applied 1-7 days before or 1-3 days after the insult. In the present study we expanded the protective profile of THC by showing that it protected mice from cognitive deficits that were induced by a variety of other neuronal insults, including pentobarbital-induced deep anesthesia, repeated treatment with 3,4 methylenedioxymethamphetamine (MDMA; "ecstasy") and exposure to carbon monoxide. The protective effect of THC lasted for at least 7 weeks. The same ultra-low dose of THC (0.002 mg/kg, a dose that is 3-4 orders of magnitude lower than the doses that produce the known acute effects of the drug in mice) induced long-lasting (7 weeks) modifications of extracellular signal-regulated kinase (ERK) activity in the hippocampus, frontal cortex and cerebellum of the mice. The alterations in ERK activity paralleled changes in its activating enzyme MEK and its inactivating enzyme MKP-1. Furthermore, a single treatment with the low dose of THC elevated the level of pCREB (phosphorylated cAMP response element-binding protein) in the hippocampus and the level of BDNF (brain-derived neurotrophic factor) in the frontal cortex. These long-lasting effects indicate that a single treatment with an ultra-low dose of THC can modify brain plasticity and induce long-term behavioral and developmental effects in the brain.
Asunto(s)
Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/metabolismo , Dronabinol/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Plasticidad Neuronal/fisiología , Fármacos Neuroprotectores/farmacología , Animales , Trastornos del Conocimiento/prevención & control , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos ICR , Plasticidad Neuronal/efectos de los fármacosRESUMEN
Either protective or toxic effects of cannabinoids on cell survival have been reported extensively in the literature; however, the factors that determine the direction of the effect are still obscured. In this study we have used the neuroblastoma cell line N18TG2 that expresses CB1 cannabinoid receptors to investigate several factors that may determine the consequences of exposure to cannabinoid agonists. Cells that were grown under optimal, stressful, or differentiating conditions were exposed to cannabinoid agonists and then assayed for cell viability by measuring MTT, LDH, and caspase-3 activity. Various cannabinoid agonists (CP 55,940, ∆9-THC, HU-210, and WIN 55,212-2) failed to affect cell viability when the cells were grown under optimal conditions. On the other hand, the same agonists significantly reduced cell viability when the cells were grown under stressful conditions (glucose- and serum-free medium), while enhancing the viability of cells grown in differentiation medium (0.5% serum and 1.5% DMSO). The toxic/protective profile was not dependent on the type or the concentration of the cannabinoid agonist that was applied. The cannabinoid agonist CP 55,940 similarly affected the non-neuronal HEK-293 cells that were grown under stressful conditions only when they expressed CB1 receptors. Our results shed light on the conflicting reports regarding the protective or toxic effects of cannabinoids in vitro and indicate that cannabinoids may activate different intracellular signaling mechanisms, depending on the state of the cell, thus leading to different physiological consequences.
Asunto(s)
Cannabinoides/farmacología , Cannabinoides/toxicidad , Fármacos Neuroprotectores/farmacología , Neurotoxinas/toxicidad , Cannabinoides/agonistas , Caspasa 3/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Ciclohexanoles , Glucosa/deficiencia , Células HEK293 , Humanos , L-Lactato Deshidrogenasa/metabolismo , Receptor Cannabinoide CB1/metabolismo , Estrés Fisiológico/efectos de los fármacosRESUMEN
In the present study we investigated the signal transduction pathways leading to the activation of extracellular signal-regulated kinase (ERK) by opioid or cannabinoid drugs, when their receptors are coexpressed in the same cell-type. In N18TG2 neuroblastoma cells, the opioid agonist etorphine and the cannabinoid agonist CP-55940 induced the phosphorylation of ERK by a similar mechanism that involved activation of delta-opioid receptors or CB1 cannabinoid receptors coupled to Gi/Go proteins, matrix metalloproteases, vascular endothelial growth factor (VEGF) receptors and MAPK/ERK kinase (MEK). In HEK-293 cells, these two drugs induced the phosphorylation of ERK by separate mechanisms. While CP-55940 activated ERK by transactivation of VEGFRs, similar to its effect in N18TG2 cells, the opioid agonist etorphine activated ERK by a mechanism that did not involve transactivation of a receptor tyrosine kinase. Interestingly, the activation of ERK by etorphine was resistant to the inhibition of MEK, suggesting the possible existence of a novel, undescribed yet mechanism for the activation of ERK by opioids. This mechanism was found to be specific to etorphine, as activation of ERK by the micro-opioid receptor (MOR) agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin) was mediated by MEK in these cells, suggesting that etorphine and DAMGO activate distinct, ligand-specific, conformations of MOR. The characterization of cannabinoid- and opioid-induced ERK activation in these two cell-lines enables future studies into possible interactions between these two groups of drugs at the level of MAPK signaling.
Asunto(s)
Sistema Nervioso Central/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neuronas/metabolismo , Receptores de Cannabinoides/metabolismo , Receptores Opioides/metabolismo , Analgésicos/farmacología , Analgésicos Opioides/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Sistema Nervioso Central/citología , Ciclohexanoles/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Etorfina/farmacología , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 1/efectos de los fármacos , MAP Quinasa Quinasa 1/metabolismo , Ratones , Neuroblastoma , Neuronas/efectos de los fármacos , Ratas , Receptor Cannabinoide CB1/efectos de los fármacos , Receptor Cannabinoide CB1/metabolismo , Receptores de Cannabinoides/efectos de los fármacos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides/efectos de los fármacos , Receptores Opioides delta/efectos de los fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides mu/efectos de los fármacos , Receptores Opioides mu/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We have previously reported that an injection of a single, extremely low dose (0.001 mg/kg) of delta 9-tetrahydrocannabinal (THC, the major psychoactive ingredient of marijuana) to mice deteriorated their performance in the Morris water maze test 3 weeks later. In the present study we verify our original findings and show that the long-term cognitive deficits that are induced in mice by a low dose of THC are even more pronounced in another behavioral test-the water T-maze. This effect was abolished by the CB1 receptor antagonist SR141716A, indicating the involvement of CB1 receptors. In an attempt to find a biochemical correlate to these deleterious consequences of such a low dose of THC, we investigated its effect on the activation of extracellular signal-regulated kinase (ERK1/2) in the cerebellum and hippocampus of the mice, two brain regions that were shown to participate in spatial learning. A significant increase in ERK1/2 phosphorylation was found in the cerebellum of mice 24 h following the injection of 0.001 mg/kg THC. These findings lead to further studies into the neuronal mechanisms underlying the long-term deleterious effects of THC and should be taken into consideration when evaluating the therapeutic benefits of cannabinoid drugs.
Asunto(s)
Conducta Animal/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/psicología , Dronabinol/farmacología , Alucinógenos/farmacología , Animales , Western Blotting , Dronabinol/administración & dosificación , Inducción Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Alucinógenos/administración & dosificación , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Piperidinas/farmacología , Desempeño Psicomotor/efectos de los fármacos , Pirazoles/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , RimonabantRESUMEN
Delta(9)-Tetrahydrocannabinol (THC) was shown to exert either neuroprotective or neurotoxic effects. Based on our in vitro studies and on pharmacokinetic considerations, we have recently presented a hypothesis that explains this dual activity of THC. This explanation is based on the assumption that extremely low doses of cannabinoids are neurotoxic. The present study verifies this assumption and shows that a single injection of 0.001 mg/kg THC (3-4 orders of magnitude lower than conventional doses) significantly affected the performance of mice in the Morris water maze test 3 weeks later. The THC-injected mice showed both longer escape latencies and lower scores in the probe tests compared to their matched controls, indicating the induction of cognitive deficits.
Asunto(s)
Trastornos del Conocimiento/inducido químicamente , Dronabinol/toxicidad , Análisis de Varianza , Animales , Conducta Animal , Relación Dosis-Respuesta a Droga , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Tiempo de Reacción/efectos de los fármacos , Factores de TiempoRESUMEN
Agonist-induced regulation of cannabinoid CB1 receptors was examined in HEK-293 cells transfected with CB1 receptors and in neuroblastoma N18TG2 cells that naturally express CB1 receptors. In HEK-293 cells, CB1 receptors internalization proceeded, in parallel, via clathrin-coated pits and caveolae. Simultaneous disruption of both pathways induced compensatory endocytic mechanism(s). In N18TG2 cells, endocytosis was not mediated by caveolae-like membrane domains. Heterologous, opioid-induced, downregulation of CB1 receptors was evident in HEK-293 but not N18TG2 cells. The data demonstrate the existence of multiple pathways of CB1 receptors regulation.
Asunto(s)
Cannabinoides/metabolismo , Regulación hacia Abajo/fisiología , Receptores de Droga/metabolismo , Animales , Cannabinoides/farmacología , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Humanos , Ratones , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Receptores de Cannabinoides , Receptores de Droga/agonistas , Células Tumorales CultivadasRESUMEN
Extensive in vitro and in vivo studies have shown that cannabinoid drugs have neuroprotective properties and suggested that the endocannabinoid system may be involved in endogenous neuroprotective mechanisms. On the other hand, neurotoxic effects of cannabinoids in vitro and in vivo were also described. Several possible explanations for these dual, opposite effects of cannabinoids on cellular fate were suggested, and it is conceivable that various factors may determine the final outcome of the cannabinoid effect in vivo. In the current review, we focus on one of the possible reasons for the dual neuroprotective/neurotoxic effects of cannabinoids in vivo, namely, the opposite effects of low versus high doses of cannabinoids. While many studies reported neuroprotective effects of the conventional doses of cannabinoids in various experimental models for acute brain injuries, we have shown that a single administration of an extremely low dose of Δ(9) -tetrahydrocannabinol (THC) (3-4 orders of magnitude lower than the conventional doses) to mice induced long-lasting mild cognitive deficits that affected various aspects of memory and learning. These findings led to the idea that this low dose of THC, which induces minor damage to the brain, may activate preconditioning and/or postconditioning mechanisms and thus will protect the brain from more severe insults. Indeed, our recent findings support this assumption and show that a pre- or a postconditioning treatment with extremely low doses of THC, several days before or after brain injury, provides effective long-term cognitive neuroprotection. The future therapeutical potential of these findings is discussed.
Asunto(s)
Encéfalo/efectos de los fármacos , Cannabinoides/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Cannabinoides/efectos adversos , Relación Dosis-Respuesta a Droga , Humanos , Fármacos Neuroprotectores/efectos adversos , Síndromes de Neurotoxicidad/etiologíaRESUMEN
Preconditioning, a phenomenon where a minor noxious stimulus protects from a subsequent more severe insult, and post-conditioning, where the protective intervention is applied following the insult, offer new insight into the neuronal mechanism(s) of neuroprotection and may provide new strategies for the prevention and treatment of brain damage. We have previously reported that a single administration of an extremely low dose of Δ(9)-tetrahydrocannabinol (THC; the psychoactive ingredient of marijuana) to mice induced minor long-lasting cognitive deficits. In the present study we examined the possibility that such a low dose of THC will protect the mice from more severe cognitive deficits induced by the epileptogenic drug pentylenetetrazole (PTZ). THC (0.002 mg/kg, a dose that is 3-4 orders of magnitude lower than the doses that induce the conventional effects of THC) was administered 1-7 days before, or 1-3 days after the injection of PTZ (60 mg/kg). The consequences of this treatment were studied 3-7 weeks later by various behavioral tests that evaluated different aspects of memory and learning. We found that a single administration of THC either before or after PTZ abolished the PTZ-induced long-lasting cognitive deficits. Biochemical studies indicated a concomitant reduction in phosphorylated-ERK (extracellular signal-regulated kinase) in the cerebella of mice 7 weeks following the injection of THC. Our results suggest that a pre- or post-conditioning treatment with extremely low doses of THC, several days before or after brain injury, may provide safe and effective long-term neuroprotection.
Asunto(s)
Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/prevención & control , Dronabinol/administración & dosificación , Antagonistas del GABA/toxicidad , Pentilenotetrazol/toxicidad , Psicotrópicos/administración & dosificación , Análisis de Varianza , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Esquema de Medicación , Interacciones Farmacológicas , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Pruebas Neuropsicológicas , Tiempo de Reacción/efectos de los fármacos , Reconocimiento en Psicología/efectos de los fármacos , Factores de TiempoRESUMEN
Prolonged exposure of opioid receptors to agonists leads to their regulation by the classical process of clathrin-dependent internalization, followed by their intracellular degradation (down regulation). We have previously shown that the opioid agonist etorphine induced an additional process of down regulation of mu-opioid receptors (MOR) that occurred in intact MOR-transfected HEK-293 cells, as well as in isolated membranes. In the present study we show that etorphine similarly down regulated rat kappa-opioid receptors (KORs), which do not undergo the classical process of internalization and down regulation. This process was resistant to inhibitors of clathrin-coated pit formation (hypertonic sucrose, mono-dansyl-cadaverine) and was mainly mediated by membranous serine- and amino-peptidases. We further show that various opioid ligands, besides etorphine, induced down regulation of either KOR or MOR in isolated membranes. The ability of the various opioid ligands to induce membrane-delimited KOR or MOR down regulation did not correlate to their classical pharmacological profile, suggesting functional selectivity of the effect. Levorphanol, but not its stereoisomer dextrophan, induced membrane-delimited down regulation of both KOR and MOR, indicating that stereoselective binding to the receptor was necessary to initiate the process. Our findings that this proteolytic regulation of opioid receptors occurs not only in isolated membranes but also in intact cells and that it occurs even when the receptors are resistant to the conventional process of down regulation indicate its possible physiological role in the regulation of opioid activity.
Asunto(s)
Membrana Celular/enzimología , Células Epiteliales/enzimología , Péptido Hidrolasas/fisiología , Proteolisis/efectos de los fármacos , Receptores Opioides/metabolismo , Analgésicos Opioides/farmacología , Animales , Unión Competitiva/fisiología , Membrana Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Etorfina/farmacología , Células HEK293 , Humanos , Antagonistas de Narcóticos , Ratas , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/metabolismo , Fracciones SubcelularesRESUMEN
A single administration of an extremely low dose (0.002 mg/kg) of Delta9-tetrahydrocannabinol (THC; the psychoactive ingredient of marijuana) to ICR mice induced long-term cognitive deficits that lasted for at least 5 months. The behavioral deficits were detected by several tests that evaluated different aspects of memory and learning, including spatial navigation and spatial and non-spatial recognition. Our findings point to possible deficits in attention or motivation that represent a common upstream cognitive process that may affect the performance of the mice in the different behavioral assays. Similar ultra-low doses of THC (3-4 orders of magnitude lower than doses that are known to evoke the acute effects of THC) also induced sustained activation of extracellular-regulated kinase (ERK1/2) in the cerebellum, indicating that a single injection of such low doses of the cannabinoid drug can stimulate neuronal regulatory mechanisms. The relevance of these findings to the behavioral consequences of chronic exposure to marijuana is discussed.