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Conflicting evidence suggest that perturbations of GABAergic neurotransmission play crucial roles in disrupting cortical neuronal network oscillations, memory, and cognitive deficits in Alzheimer's disease (AD). However, the role and impact of sex differences on GABAergic transmission in AD are not well understood. Using an APP knock-in mouse model of AD, APPNLGF mice, we studied the effects of acute diazepam administration on memory and anxiety-like behavior to unveil sex-dependent dysregulation of GABAergic neurotransmission. We also examined sex differences in GABAA receptor subunit mRNA and protein expression and the role of epigenetic regulation in hippocampus of APPNLGF mice. We found that diazepam elicited dose-dependent suppression of locomotion in wildtype and APPNLGF mice. However, a low dose, which had no significant effect in both male and female wildtype as well as female APPNLGF mice, significantly suppressed locomotion in male APPNLGF mice. Furthermore, this low dose of diazepam was more efficacious at eliciting anxiolytic-like effects in male than female APPNLGF mice. The same low dose of diazepam disrupted recognition memory exclusively in male APPNLGF mice. Biochemical analyses revealed that hippocampal α1 and α5 GABAA receptor subunits mRNA and protein expression were significantly higher in male than female APPNLGF mice and were regulated by histone H3 tri-methylation (H3K4me3) but not histone H3 acetylation. The higher sensitivity of APPNLGF males to diazepam-induced behavioral effects may potentially be due to epigenetic-dependent upregulation of hippocampal α1 and α5 GABAA receptor subunits expression compared to female APPNLGF mice. These findings suggest that dysregulation of GABAergic neurotransmission plays a significant role in memory and affective behavior, particularly in male APPNLGF mice.
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Alcohol Use Disorder (AUD) is a multifaceted relapsing disorder that is commonly comorbid with psychiatric disorders, including anxiety. Alcohol exposure produces a plethora of effects on neurobiology. Currently, therapeutic strategies are limited, and only a few treatments - disulfiram, acamprosate, and naltrexone - are available. Given the complexity of this disorder, there is a great need for the identification of novel targets to develop new pharmacotherapy. The GABAergic system, the primary inhibitory system in the brain, is one of the well-known targets for alcohol and is responsible for the anxiolytic effects of alcohol. Interestingly, GABAergic neurotransmission is fine-tuned by neuroactive steroids that exert a regulatory role on several endocrine systems involved in neuropsychiatric disorders including AUD. Mounting evidence indicates that alcohol alters the biosynthesis of neurosteroids, whereas acute alcohol increases and chronic alcohol decreases allopregnanolone levels. Our recent work highlighted that chronic alcohol-induced changes in neurosteroid levels are mediated by epigenetic modifications, e.g., DNA methylation, affecting key enzymes involved in neurosteroid biosynthesis. These changes were associated with changes in GABAA receptor subunit expression, suggesting an imbalance between excitatory and inhibitory signaling in AUD. This review will recapitulate the role of neurosteroids in the regulation of the neuroendocrine system, highlight their role in the observed allostatic load in AUD, and develop a framework from mechanisms to potential pharmacotherapy.
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Alcoholismo , Neuroesteroides , Humanos , Pregnanolona/metabolismo , Alcoholismo/tratamiento farmacológico , Receptores de GABA-A/metabolismo , Ansiedad , EtanolRESUMEN
BACKGROUND: Alcohol intoxication produces ataxia by affecting the cerebellum, which coordinates movements. Fragile X mental retardation (FMR) protein is a complex regulator of RNA and synaptic plasticity implicated in fragile X-associated tremor/ataxia syndrome, which features ataxia and increased Fmr1 mRNA expression resulting from epigenetic dysregulation of FMRP. We recently demonstrated that acute ethanol-induced ataxia is associated with increased cerebellar Fmr1 gene expression via histone modifications in rats, but it is unknown whether similar behavioral and molecular changes occur following chronic ethanol exposure. Here, we investigated the effects of chronic ethanol exposure on ataxia and epigenetically regulated changes in Fmr1 expression in the cerebellum. METHODS: Male adult Sprague-Dawley rats were trained on the accelerating rotarod and then fed with chronic ethanol or a control Lieber-DeCarli diet while undergoing periodic behavioral testing for ataxia during ethanol exposure and withdrawal. Cerebellar tissues were analyzed for expression of the Fmr1 gene and its targets using a real-time quantitative polymerase chain reaction assay. The epigenetic regulation of Fmr1 was also investigated using a chromatin immunoprecipitation assay. RESULTS: Ataxic behavior measured by the accelerating rotarod behavioral test developed during chronic ethanol treatment and persisted at both the 8-h and 24-h withdrawal time points compared to control diet-fed rats. In addition, chronic ethanol treatment resulted in up-regulated expression of Fmr1 mRNA and increased activating epigenetic marks H3K27 acetylation and H3K4 trimethylation at 2 sites within the Fmr1 promoter. Finally, measurement of the expression of relevant FMRP mRNA targets in the cerebellum showed that chronic ethanol up-regulated cAMP response element binding (CREB) Creb1, Psd95, Grm5, and Grin2b mRNA expression without altering Grin2a, Eaa1, or histone acetyltransferases CREB binding protein (Cbp) or p300 mRNA transcripts. CONCLUSIONS: These results suggest that epigenetic regulation of Fmr1 and subsequent FMRP regulation of target mRNA transcripts constitute neuroadaptations in the cerebellum that may underlie the persistence of ataxic behavior during chronic ethanol exposure and withdrawal.
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Depresores del Sistema Nervioso Central/efectos adversos , Ataxia Cerebelosa/inducido químicamente , Cerebelo/efectos de los fármacos , Etanol/efectos adversos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Intoxicación Alcohólica/etiología , Intoxicación Alcohólica/metabolismo , Animales , Depresores del Sistema Nervioso Central/administración & dosificación , Ataxia Cerebelosa/metabolismo , Cerebelo/metabolismo , Epigénesis Genética/efectos de los fármacos , Etanol/administración & dosificación , Código de Histonas/efectos de los fármacos , Masculino , Ratas Sprague-DawleyRESUMEN
Some of the biochemical abnormalities underlying schizophrenia, involve differences in methylation and methylating enzymes, as well as other related target genes. We present results of a study of differences in mRNA expression in peripheral blood lymphocytes (PBLs) and post-mortem brains of chronic schizophrenics (CSZ) and non-psychotic controls (NPC), emphasizing the differential effects of sex and antipsychotic drug treatment on mRNA findings. We studied mRNA expression in lymphocytes of 61 CSZ and 49 NPC subjects using qPCR assays with TaqMan probes to assess levels of DNMT, TET, GABAergic, NR3C1, BDNF mRNAs, and several additional targets identified in a recent RNA sequence analysis. In parallel we studied DNMT1 and GAD67 in samples of brain tissues from 19 CSZ, 26 NPC. In PBLs DNMT1 and DNMT3A mRNA levels were significantly higher in male CSZ vs NPC. No significant differences were detected in females. The GAD1, NR3C1 and CNTNAP2 mRNA levels were significantly higher in CSZ than NPC. In CSZ patients treated with clozapine, GAD-1 related, CNTNAP2, and IMPA2 mRNAs were significantly higher than in CSZ subjects not treated with clozapine. Differences between CSZ vs NPC in these mRNAs was primarily attributable to the clozapine treatment. In the brain samples, DNMT1 was significantly higher and GAD67 was significantly lower in CSZ than in NPC, but there were no significant sex differences in diagnostic effects. These findings highlight the importance of considering sex and drug treatment effects in assessing the substantive significance of differences in mRNAs between CSZ and NPC.
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Schizophrenia is a severe neuropsychiatric disorder associated with a wide array of transcriptomic and neurobiochemical changes. Genome-wide transcriptomic profiling conducted in postmortem brain have provided novel insights into the pathophysiology of this disorder, and identified biological processes including immune/inflammatory-related responses, metabolic, endocrine, and synaptic function. However, few studies have investigated whether similar changes are present in peripheral tissue. Here, we used RNA-sequencing to characterize transcriptomic profiles of lymphocytes in 18 nonpsychotic controls and 19 individuals with schizophrenia. We identified 2819 differentially expressed transcripts (P nominal < .05) in the schizophrenia group when compared to controls. Bioinformatic analyses conducted on a subset of 293 genes (P nominal < .01 and |log2 FC| > 0.5) highlighted immune/inflammatory responses as key biological processes in our dataset. Differentially expressed genes in lymphocytes were highly enriched in gene expression profiles associated with cortex layer 5a and immune cells. Thus, we investigated whether the changes in transcripts levels observed in lymphocytes could also be detected in the prefrontal cortex (PFC, BA10) in a second replication cohort of schizophrenia subjects. Remarkably, mRNA levels detected in the PFC and lymphocytes were in strong agreement, and measurements obtained using RNA-sequencing positively correlated with data obtained by reverse transcriptase-quantitative polymerase chain reaction analysis. Collectively, our work supports a role for immune dysfunction in the pathogenesis of schizophrenia and suggests that peripheral markers can be used as accessible surrogates to investigate putative central nervous system disruptions.
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Chronic exposure to stress throughout lifespan alters brain structure and function, inducing a maladaptive response to environmental stimuli, that can contribute to the development of a pathological phenotype. Studies have shown that hypothalamic-pituitary-adrenal (HPA) axis dysfunction is associated with various neuropsychiatric disorders, including major depressive, alcohol use and post-traumatic stress disorders. Downstream actors of the HPA axis, glucocorticoids are critical mediators of the stress response and exert their function through specific receptors, i.e., the glucocorticoid receptor (GR), highly expressed in stress/reward-integrative pathways. GRs are ligand-activated transcription factors that recruit epigenetic actors to regulate gene expression via DNA methylation, altering chromatin structure and thus shaping the response to stress. The dynamic interplay between stress response and epigenetic modifiers suggest DNA methylation plays a key role in the development of stress surfeit disorders.
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Metilación de ADN , Epigénesis Genética , Estrés Psicológico , Humanos , Estrés Psicológico/genética , Estrés Psicológico/metabolismoRESUMEN
Environmental factors, including substance abuse and stress, cause long-lasting changes in the regulation of gene expression in the brain via epigenetic mechanisms, such as DNA methylation. We examined genome-wide DNA methylation patterns in the prefrontal cortex (PFC, BA10) of 25 pairs of control and individuals with alcohol use disorder (AUD), using the Infinium® MethylationEPIC BeadChip. We identified 5254 differentially methylated CpGs (pnominal < 0.005). Bioinformatic analyses highlighted biological processes containing genes related to stress adaptation, including the glucocorticoid receptor (encoded by NR3C1). Considering that alcohol is a stressor, we focused our attention on differentially methylated regions of the NR3C1 gene and validated the differential methylation of several genes in the NR3C1 network. Chronic alcohol drinking results in a significant increased methylation of the NR3C1 exon variant 1H, with a particular increase in the levels of 5-hydroxymethylcytosine over 5-methylcytosine. These changes in DNA methylation were associated with reduced NR3C1 mRNA and protein expression levels in PFC, as well as other cortico-limbic regions of AUD subjects when compared with controls. Furthermore, we show that the expression of several stress-responsive genes (e.g., CRF, POMC, and FKBP5) is altered in the PFC of AUD subjects. These stress-response genes were also changed in the hippocampus, a region that is highly susceptible to stress. These data suggest that alcohol-dependent aberrant DNA methylation of NR3C1 and consequent changes in other stress-related genes might be fundamental in the pathophysiology of AUD and lay the groundwork for treatments targeting the epigenetic mechanisms regulating NR3C1 in AUD.
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Alcoholismo , Receptores de Glucocorticoides , Alcoholismo/genética , Metilación de ADN/genética , Epigénesis Genética/genética , Hipocampo/metabolismo , Humanos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismoRESUMEN
The cerebellum is widely known as a motor structure because it regulates and controls motor learning, coordination, and balance. However, it is also critical for non-motor functions such as cognitive processing, sensory discrimination, addictive behaviors and mental disorders. The cerebellum has the highest relative abundance of neuronal nitric oxide synthase (nNos) and is sensitive to ethanol. Although it has been demonstrated that the interaction of γ-aminobutyric acid (GABA) and nitric oxide (NO) might play an important role in the regulation of ethanol-induced cerebellar ataxia, the molecular mechanisms through which ethanol regulates nNos function to elicit this behavioral effect have not been studied extensively. Here, we investigated the dose-dependent effects of acute ethanol treatment on motor impairment using the rotarod behavioral paradigm and the alterations of nNos mRNA expression in cerebellum, frontal cortex (FC), hippocampus and striatum. We also examined the link between acute ethanol-induced motor impairment and nNos by pharmacological manipulation of nNos function. We found that acute ethanol induced a dose-dependent elevation of ethanol blood levels which was associated with the impairment of motor coordination performance and decreased expression of cerebellar nNos. In contrast, acute ethanol increased nNos expression in FC but did not to change the expression for this enzyme in striatum and hippocampus. The effects of acute ethanol were attenuated by l-arginine, a precursor for NO and potentiated by 7-nitroindazole (7-NI), a selective inhibitor of nNos. Our data suggests that differential regulation of nNos mRNA expression in cerebellum and frontal cortex might be involved in acute ethanol-induced motor impairment.
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Trastornos del Sistema Nervioso Inducidos por Alcohol/metabolismo , Ataxia Cerebelosa/metabolismo , Etanol/efectos adversos , Óxido Nítrico Sintasa de Tipo I/metabolismo , Trastornos Psicomotores/metabolismo , Trastornos del Sistema Nervioso Inducidos por Alcohol/inducido químicamente , Animales , Arginina/farmacología , Ataxia Cerebelosa/inducido químicamente , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Inhibidores Enzimáticos/farmacología , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/metabolismo , Indazoles/farmacología , Masculino , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Trastornos Psicomotores/inducido químicamente , Ratas Sprague-DawleyRESUMEN
BACKGROUND: The cerebellum is fundamental for motor coordination and therefore crucial in ethanol (EtOH)-induced ataxia. EtOH contributes to cerebellar pathophysiology. Fragile-X mental retardation protein (FMRP) is a complex regulator of RNA and synaptic plasticity implicated in fragile-X tremor and ataxia syndrome, a phenotype featuring increased Fmr1 mRNA expression. Recent studies have implicated glutamatergic targets of FMRP in hereditary cerebellar ataxias including the main cerebellar excitatory amino acid (Eaa1) transporter and a subtype of metabotropic glutamate receptor (Grm5). However, EtOH-induced changes in cerebellar Fmr1 expression and its epigenetic regulation have not been investigated. Here, we examined the effects of acute EtOH exposure on ataxic behavior, gene expression, and epigenetic regulation of the Fmr1 gene and its glutamatergic targets in the rat cerebellum. METHODS: Male adult Sprague Dawley rats received acute EtOH (2 g/kg) intraperitoneally 1 hour prior to ataxic behavioral testing on the accelerating rotarod and were sacrificed immediately thereafter. Cerebellar tissues were analyzed for gene expression and epigenetic regulation of the Fmr1 gene and its glutamatergic targets in the rat cerebellum using real-time quantitative polymerase chain reaction (PCR) and chromatin immunoprecipitation. RESULTS: Acute EtOH exposure caused marked ataxia on the accelerating rotarod test compared with saline-treated controls. This ataxic response was associated with increases in mRNA levels of Fmr1, postsynaptic density 95 (Psd95), Eaa1, and Grm5 in the cerebellum. In addition, we found increased H3K27 acetylation both at the promoter region of Fmr1 and at a proposed cyclic adenosine monophosphate (cAMP) response-element binding (CREB) site downstream of the Fmr1 transcription start site. Furthermore, acute EtOH exposure significantly increased Creb1 and the histone acetyltransferases (HAT) CREB binding protein (Cbp), and p300 mRNA transcripts. CONCLUSIONS: Overall, EtOH regulates cerebellar Fmr1 expression most likely via HAT-mediated increase in histone acetylation. We propose that FMRP regulation of glutamatergic transcripts plays an important role in disrupting the excitatory-inhibitory balance in the cerebellum underlying EtOH-induced ataxia.
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Ataxia/inducido químicamente , Depresores del Sistema Nervioso Central/efectos adversos , Cerebelo/efectos de los fármacos , Etanol/efectos adversos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Animales , Ataxia/metabolismo , Cerebelo/metabolismo , Epigénesis Genética , Regulación de la Expresión Génica , Código de Histonas , Masculino , Ratas Sprague-Dawley , Prueba de Desempeño de Rotación con Aceleración ConstanteRESUMEN
Corroborating evidence indicate that the downregulation of GABAA receptor subunit expression may underlie tolerance to the anticonvulsant and anxiolytic actions of benzodiazepine (BZ) ligands that act as full allosteric modulators (FAMs) of GABA actions at a variety of GABAA receptor subtypes. We and others have shown that 10-14 days treatment with increasing doses of diazepam (a FAM) resulted in anticonvulsant tolerance and decreased the expression of the α1 GABAA receptor subunit mRNA and protein in frontal cortex. In addition, we have also shown that long-term treatment with imidazenil, a partial allosteric modulator of GABA action at selective GABAA receptor subtypes, fail to change the expression of the α1 subunit mRNA or induce tolerance to its anticonvulsant or anxiolytic action. However, little is known regarding the potential role of epigenetic mechanisms on long-term BZ-induced downregulation of GABAA receptor subunit. Therefore, we examined the role of histone acetylation and DNA methylation mechanisms on long-term diazepam-induced downregulation of the α1 subunit mRNA expression in rat frontal cortex. We found that 10 days treatment with increasing doses of diazepam but not imidazenil decreased the expression of the α1 GABAA receptor subunit mRNA and promoter acetylation in frontal cortex. In addition, we also found that 10 days treatment with diazepam but not imidazenil increased the expression of histone deacetylase (HDAC) 1 and 2 in frontal cortex. Thus, the increased expression of HDAC1 and HDAC2 (class 1 HDACs) and consequently increased histone deacetylation mechanism of this class 1 HDACs, may underlie long-term diazepam-induced decreased expression of the α1 GABAA receptor subunit mRNA in frontal cortex.
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Ansiolíticos/farmacología , Anticonvulsivantes/farmacología , Diazepam/farmacología , Histonas/metabolismo , Corteza Prefrontal/efectos de los fármacos , Receptores de GABA-A/genética , Acetilación , Animales , Metilación de ADN , Regulación hacia Abajo , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Masculino , Corteza Prefrontal/metabolismo , Ratas Sprague-DawleyRESUMEN
Background: Cerebellum is an area of the brain particularly sensitive to the effects of acute and chronic alcohol consumption. Alcohol exposure decreases cerebellar Purkinje cell output by increasing GABA release from Golgi cells onto extrasynaptic α6/δ-containing GABAA receptors located on glutamatergic granule cells. Here, we studied whether chronic alcohol consumption induces changes in GABAA receptor subunit expression and whether these changes are associated with alterations in epigenetic mechanisms via DNA methylation. Methods: We used a cohort of postmortem cerebellum from control and chronic alcoholics, here defined as alcohol use disorders subjects (n=25/group). S-adenosyl-methionine/S-adenosyl-homocysteine were measured by high-performance liquid chromatography. mRNA levels of various genes were assessed by reverse transcriptase-quantitative polymerase chain reaction. Promoter methylation enrichment was assessed using methylated DNA immunoprecipitation and hydroxy-methylated DNA immunoprecipitation assays. Results: mRNAs encoding key enzymes of 1-carbon metabolism that determine the S-adenosyl-methionine/S-adenosyl-homocysteine ratio were increased, indicating higher "methylation index" in alcohol use disorder subjects. We found that increased methylation of the promoter of the δ subunit GABAA receptor was associated with reduced mRNA and protein levels in the cerebellum of alcohol use disorder subjects. No changes were observed in α1- or α6-containing GABAA receptor subunits. The expression of DNA-methyltransferases (1, 3A, and 3B) was unaltered, whereas the mRNA level of TET1, which participates in the DNA demethylation pathway, was decreased. Hence, increased methylation of the δ subunit GABAA receptor promoter may result from alcohol-induced reduction of DNA demethylation. Conclusion: Together, these results support the hypothesis that aberrant DNA methylation pathways may be involved in cerebellar pathophysiology of alcoholism. Furthermore, this work provides novel evidence for a central role of DNA methylation mechanisms in the alcohol-induced neuroadaptive changes of human cerebellar GABAA receptor function.
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Alcoholismo/patología , Carbono/metabolismo , Cerebelo/metabolismo , Metilación de ADN/genética , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Alcoholismo/genética , Cromatografía Líquida de Alta Presión , Estudios de Cohortes , Femenino , Expresión Génica/fisiología , Humanos , Inmunoprecipitación , Masculino , Persona de Mediana Edad , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Cambios Post Mortem , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Epigenetic mechanisms such as DNA methylation play an important role in regulating the pathophysiology of alcoholism. Chronic alcohol exposure leads to behavioral changes as well as decreased expression of genes associated with synaptic plasticity. In the liver, it has been documented that chronic alcohol exposure impairs methionine synthase (Ms) activity leading to a decrease in S-adenosyl methionine/S-adenosyl homocysteine (SAM/SAH) ratio which results in DNA hypomethylation; however, it is not known whether similar alterations of SAM and SAH levels are also produced in brain. METHODS: Male adult Sprague Dawley rats were fed chronically with Lieber-DeCarli ethanol (EtOH) (9% v/v) or control diet. The EtOH-diet-fed rats were withdrawn for 0 and 24 hours. The cerebellum and liver tissues were dissected and used to investigate changes in one-carbon metabolism, SAM, and SAH levels. RESULTS: We found that chronic EtOH exposure decreased SAM levels, SAM/SAH ratio, Ms, methylene tetrahydrofolate reductase, and betaine homocysteine methyltransferase (Bhmt) expression and increased methionine adenosyltransferase-2b (Mat2b) but not Mat2a expression in the liver. In contrast, chronic EtOH exposure decreased SAH levels, increased SAM/SAH ratio and the expression of Mat2a and S-adenosyl homocysteine hydrolase, while the levels of SAM or Bhmt expression in cerebellum remained unaltered. However, in both liver and cerebellum, chronic EtOH exposure decreased the expression of Ms and increased Mat2b expression. All chronic EtOH-induced changes of one-carbon metabolism in cerebellum, but not liver, returned to near-normal levels during EtOH withdrawal. CONCLUSIONS: These results indicate a decreased "methylation index" in liver and an increased "methylation index" in cerebellum. The opposing changes of the "methylation index" suggest altered DNA methylation in liver and cerebellum, thus implicating one-carbon metabolism in the pathophysiology of alcoholism.
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Consumo de Bebidas Alcohólicas/metabolismo , Encéfalo/metabolismo , Carbono/metabolismo , Metilación de ADN/fisiología , Etanol/administración & dosificación , Hígado/metabolismo , Animales , Encéfalo/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Transferasas del Grupo 1-Carbono/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
The chemical warfare nerve agent, soman irreversibly inhibits acetylcholinesterase (AChE) leading to hypercholinergy and seizures which trigger glutamate toxicity and status epilepticus ultimately resulting in neuropathology and neurobehavioral deficits. The standard emergency treatment comprising of anticholinergic, AChE reactivator and anticonvulsant does not completely protect against soman toxicity. We have evaluated imidazenil, a new anticonvulsant imidazo benzodiazepine with high affinity and intrinsic efficacy at α5-, α2-, and α3- but low intrinsic efficacy at α1-containing GABA(A) receptors and is devoid of cardiorespiratory depression, sedative/hypnoitc and amnestic actions and does not elicit tolerance and dependence liabilities unlike diazepam, for protection against soman toxicity. Guinea pigs implanted with bipotential radiotelemetry probes for recording EEG and ECG were administered with 26 µg/kg pyridostigmine bromide 30 min prior to 2× LD(50) soman exposure and 1 min later treated with a combination of 2mg/kg atropine sulfate and 25mg/kg 2-pralidoxime and various doses of imidazenil. Intramuscular administration of imidazenil, dose-dependently protected against 2× LD(50) of soman toxicity up to 1mg/kg. Further increase in the dose of imidazenil to 2.5mg/kg was less effective than 1mg/kg probably due to non-specific actions at sites other than GABA(A) receptors. Compared to vehicle group, 1mg/kg imidazenil treatment showed optimal increase in survival rate, reduction in behavioral manifestations and high power of EEG spectrum as well as neuronal necrosis. These data suggest that imidazenil is an effective anticonvulsant for medical countermeasure against soman-induced toxicity.
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Benzodiazepinas/uso terapéutico , Sustancias para la Guerra Química/toxicidad , Imidazoles/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Síndromes de Neurotoxicidad/tratamiento farmacológico , Síndromes de Neurotoxicidad/etiología , Soman/toxicidad , Acetilcolinesterasa/sangre , Acetilcolinesterasa/metabolismo , Análisis de Varianza , Animales , Atropina/uso terapéutico , Peso Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Reactivadores de la Colinesterasa/uso terapéutico , Modelos Animales de Enfermedad , Esquema de Medicación , Electrocardiografía/métodos , Electroencefalografía/métodos , Cobayas , Dosificación Letal Mediana , Masculino , Antagonistas Muscarínicos/uso terapéutico , Compuestos de Pralidoxima/uso terapéutico , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Telemetría , Factores de TiempoRESUMEN
The clinical use of diazepam or midazolam to control organophosphate (OP) nerve agent-induced seizure activity is limited by their unwanted effects including sedation, amnesia, withdrawal, and anticonvulsant tolerance. Imidazenil is an imidazo-benzodiazepine derivative with high intrinsic efficacy and selectivity for α2-, α3-, and α5- but low intrinsic efficacy for α1-containing GABA(A) receptors. We have previously shown that imidazenil is more efficacious than diazepam at protecting rats and mice from diisopropyl fluorophosphate (DFP)-induced seizures and neuronal damage without producing sedation. In the present study, we compared the tolerance liability of imidazenil and diazepam to attenuate the seizure activity and neurotoxic effects of DFP. Rats received protracted (14 days) oral treatment with increasing doses of imidazenil (1-4 mg/kg), diazepam (5-20 mg/kg), or vehicle. Eighteen hours after the last dose of the protracted treatment schedule, rats were tested for anticonvulsant tolerance after a 30 min pretreatment with a single test dose of imidazenil (0.5 mg/kg) or diazepam (5 mg/kg) prior to a DFP challenge (1.5 mg/kg). The anticonvulsant (modified Racine score scale) and neuroprotective (fluoro-jade B staining) effects of diazepam were significantly reduced in protracted diazepam-treated animals whereas the effects of imidazenil were not altered in protracted imidazenil-treated animals. The present findings indicate that protracted imidazenil treatment does not produce tolerance to its protective action against the neurotoxic effects of OP exposure.
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Anticonvulsivantes/uso terapéutico , Benzodiazepinas/uso terapéutico , Imidazoles/uso terapéutico , Isoflurofato/toxicidad , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Convulsiones , Animales , Inhibidores de la Colinesterasa/toxicidad , Diazepam/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fluoresceínas , Masculino , Compuestos Orgánicos , Ratas , Ratas Endogámicas F344 , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Convulsiones/patología , Estadísticas no ParamétricasRESUMEN
Diazepam (DZ), the preferred anticonvulsant benzodiazepine (BZ) for the treatment of organophosphate (OP) nerve agent-induced seizures and neuronal damage, has been associated with unwanted effects such as sedation, amnesia, cardiorespiratory depression, anticonvulsant tolerance, and dependence liability. In a search for safer and more effective anticonvulsant BZs against OP-induced seizure and neuronal damage, we have previously shown that imidazenil (IMD), a low-intrinsic efficacy positive allosteric modulator of gamma-aminobutyric acid (GABA) action at α1-containing GABA(A) receptors, which has high intrinsic efficacy at α2-, α3-, and α5-containing GABA(A) receptors, is more potent and longer lasting than DZ pretreatment at protecting rats from diisopropyl fluorophosphate (DFP)-induced electrocorticographic (ECoG) seizures and neuronal damage. The effects of IMD were observed at doses that are devoid of sedative, amnestic, and anticonvulsant tolerance actions. In the present study, we compared the anticonvulsant and neuroprotective effects of a combination of atropine (2 mg/kg, ip) and pyridine-2-aldoxime methochloride (2-PAM, 20 mg/kg, ip) with IMD (0.5 mg/kg, ip) or midazolam (MDZ, 0.5-2 mg/kg, ip) administered after the onset of DFP (1.5 mg/kg, sc)-induced seizure activity. The severity of DFP-induced ECoG seizures was assessed by continuous radio telemetry recordings in unrestrained and freely moving rats. Furthermore, the extent of neuronal damage was evaluated using a neuron-specific nuclear protein immunolabeling and fluoro-jade B staining procedure. We report here that IMD is more efficacious and longer lasting than sedating doses of MDZ in protecting rats from DFP-induced ECoG seizures and neuronal damage.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Benzodiazepinas/uso terapéutico , Encéfalo/efectos de los fármacos , Imidazoles/uso terapéutico , Isoflurofato/toxicidad , Midazolam/uso terapéutico , Neuronas/efectos de los fármacos , Convulsiones/prevención & control , Animales , Anticonvulsivantes/administración & dosificación , Antígenos Nucleares/metabolismo , Benzodiazepinas/administración & dosificación , Encéfalo/metabolismo , Encéfalo/patología , Relación Dosis-Respuesta a Droga , Imidazoles/administración & dosificación , Masculino , Midazolam/administración & dosificación , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Endogámicas F344 , Receptores de GABA/metabolismo , Convulsiones/inducido químicamente , Convulsiones/patología , Telemetría , Factores de TiempoRESUMEN
Recent evidence suggests that alpha1-containing GABA(A) receptors mediate the sedative, amnestic, and to some extent the anticonvulsant actions of non-selective benzodiazepine (BZ) receptor ligands, such as diazepam (DZ). Anxiolytic and in part, anticonvulsant actions of BZ ligands are mediated by alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. This has resulted in increasing interest in developing BZ ligands with selective actions at GABA(A) receptors, including alpha2-, alpha3-, and alpha5-subunits, but devoid of efficacy at alpha1-containing receptors. To refine their spectrum of pharmacological actions, efforts are being made to minimize unwanted effects such as sedation, amnesia, and tolerance liabilities. A prototype for such BZ ligands is imidazenil (IMD), an imidazo-benzodiazepine carboxylic acid derivative that elicits potent anticonvulsant and anxiolytic actions at doses virtually devoid of sedative, cardio-respiratory depressant and amnestic effects, and anticonvulsant tolerance liability. To define the pharmacological profile of IMD and its derivatives, we compared the anticonflict (anxiolytic), anti-proconflict (antipanic), anti-bicuculline (BIC), and maximal electroshock seizure (MES) effects, and the suppression of locomotor activity by imidazo-benzodiazepine carboxylic acid derivatives to those of DZ and bretazenil (BTZ). We report here that IMD and one of its derivatives (RO 25-2775) possess dose-dependent anticonflict, anti-proconflict, and anti-BIC actions but failed to suppress locomotor activity. Like DZ, the other IMD derivatives (enazenil, RO 25-2776, and RO 25-2847) not only elicit dose-dependent anticonflict, anti-proconflict, anti-BIC, anti-MES effects but also suppress locomotor activity. In contrast, none of the IMD derivatives studied shows any similarity to BTZ, which elicits anticonflict, anti-proconflict actions and suppresses locomotor activity but is virtually inactive against BIC-induced tonic-clonic convulsions.
Asunto(s)
Ansiolíticos/farmacología , Anticonvulsivantes/farmacología , Benzodiazepinas/farmacología , Imidazoles/farmacología , Animales , Ansiolíticos/metabolismo , Anticonvulsivantes/metabolismo , Conducta Animal/efectos de los fármacos , Benzodiazepinas/metabolismo , Bicuculina/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Hipnóticos y Sedantes/metabolismo , Hipnóticos y Sedantes/farmacología , Imidazoles/metabolismo , Ligandos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Pánico/efectos de los fármacos , Subunidades de Proteína/metabolismo , Ratas , Ratas Endogámicas F344 , Receptores de GABA-A/metabolismo , Sistema Respiratorio/efectos de los fármacosRESUMEN
Organophosphate (OP)-nerve agent poisoning may lead to prolonged epileptiform seizure activity, which can result in irreversible neuronal brain damage. A timely and effective control of seizures with pharmacological agents can minimize the secondary and long-term neuropathology that may result from this damage. Diazepam, the current anticonvulsant of choice in the management of OP poisoning, is associated with unwanted effects such as sedation, amnesia, cardio-respiratory depression, anticonvulsant tolerance, and dependence liabilities. In search for an efficacious and safer anticonvulsant benzodiazepine, we studied imidazenil, a potent anticonvulsant that is devoid of sedative action and has a low intrinsic efficacy at alpha1- but is a high efficacy positive allosteric modulator at alpha5-containing GABA(A) receptors. We compared the potency of a combination of 2 mg/kg, i.p. atropine with: (a) imidazenil 0.05-0.5 mg/kg i.p. or (b) equipotent anti-bicuculline doses of diazepam (0.5-5 mg/kg, i.p.), against diisopropyl fluorophosphate (DFP; 1.5 mg/kg, s.c.)-induced status epilepticus and its associated neuronal damage. The severity and frequency of seizure activities were determined by continuous radio telemetry recordings while the extent of neuronal damage and neuronal degeneration were assessed using the TUNEL-based cleaved DNA end-labeling technique or neuron-specific nuclear protein (NeuN)-immunolabeling and Fluoro-Jade B (FJB) staining, respectively. We report here that the combination of atropine and imidazenil is at least 10-fold more potent and longer lasting than the combination with diazepam at protecting rats from DFP-induced seizures and the associated neuronal damage or ongoing degeneration in the anterior cingulate cortex, CA1 hippocampus, and dentate gyrus. While 0.5 mg/kg imidazenil effectively attenuated DFP-induced neuronal damage and the ongoing neuronal degeneration in the anterior cingulate cortex, dentate gyrus, and CA1 hippocampus, 5 mg/kg or a higher dose of diazepam is required to produce similar protective effects. These finding suggests that imidazenil, a non-sedating anticonvulsant BZ ligand, is a more potent, effective, and safer drug than diazepam in protecting rats from DFP-induced seizures and the associated neuronal damage and/or ongoing neuronal degeneration.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Benzodiazepinas/uso terapéutico , Inhibidores de la Colinesterasa/toxicidad , Diazepam/uso terapéutico , Imidazoles/uso terapéutico , Isoflurofato/toxicidad , Neuronas/efectos de los fármacos , Convulsiones/prevención & control , Animales , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/farmacología , Conducta Animal/efectos de los fármacos , Benzodiazepinas/administración & dosificación , Benzodiazepinas/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Fragmentación del ADN/efectos de los fármacos , Diazepam/administración & dosificación , Diazepam/farmacología , Electroencefalografía , Imidazoles/administración & dosificación , Imidazoles/farmacología , Etiquetado Corte-Fin in Situ , Masculino , Neuronas/patología , Ratas , Ratas Endogámicas F344 , Convulsiones/inducido químicamente , Convulsiones/patología , Estado Epiléptico/patología , Estado Epiléptico/prevención & control , TelemetríaRESUMEN
Diisopropyl fluorophosphate (DFP) causes neurotoxicity related to an irreversible inhibition of acetylcholinesterase (AChE). Management of this intoxication includes: (i) pretreatment with reversible blockers of AChE, (ii) blockade of muscarinic receptors with atropine, and (iii) facilitation of GABA(A) receptor signal transduction by benzodiazepines. The major disadvantage associated with this treatment combination is that it must to be repeated frequently and, in some cases, protractedly. Also, the use of diazepam (DZP) and congeners includes unwanted side effects, including sedation, amnesia, cardiorespiratory depression, and anticonvulsive tolerance. To avoid these treatment complications but safely protect against DFP-induced seizures and other CNS toxicity, we adopted the strategy of administering mice with (i) small doses of huperzine A (HUP), a reversible and long-lasting (half-life approximately 5 h) inhibitor of AChE, and (ii) imidazenil (IMI), a potent positive allosteric modulator of GABA action selective for alpha(5)-containing GABA(A) receptors. Coadministration of HUP (50 microg/kg s.c., 15 min before DFP) with IMI (2 mg/kg s.c., 30 min before DFP) prevents DFP-induced convulsions and the associated neuronal damage and mortality, allowing complete recovery within 18-24 h. In HUP-pretreated mice, the ED(50) of IMI to block DFP-induced mortality is approximately 10 times lower than that of DZP and is devoid of sedation. Our data show that a combination of HUP with IMI is a prophylactic, potent, and safe therapeutic strategy to overcome DFP toxicity.
Asunto(s)
Benzodiazepinas/farmacología , Imidazoles/farmacología , Isoflurofato/antagonistas & inhibidores , Isoflurofato/toxicidad , Sesquiterpenos/farmacología , Alcaloides , Animales , Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/prevención & control , Quimioterapia Combinada , Dosificación Letal Mediana , Locomoción/efectos de los fármacos , Masculino , Ratones , Convulsiones/inducido químicamente , Convulsiones/prevención & controlRESUMEN
Whereas advances in the molecular biology of GABA(A) receptor complex using knock-out and knock-in mice have been valuable in unveiling the structure, composition, receptor assembly, and several functions of different GABA(A) receptor subtypes, the mechanism(s) underlying benzodiazepine (BZ) tolerance and withdrawal remain poorly understood. Studies using specific GABA(A) receptor subunit knock-in mice suggest that tolerance to sedative action of diazepam requires long-term activation of alpha1 and alpha5 GABA(A) receptor subunits. We investigated the role of long-term activation of these GABA(A) receptor subunits during anticonvulsant tolerance using high affinity and high intrinsic efficacy ligands for GABA(A) receptors expressing the alpha5 subunit (imidazenil) or alpha1 subunit (zolpidem), and a non-selective BZ recognition site ligand (diazepam). We report here that long-term activation of GABA(A) receptors by zolpidem and diazepam but not by imidazenil elicits anticonvulsant tolerance. Although anticonvulsant cross-tolerance occurs between diazepam and zolpidem, there is no cross-tolerance between imidazenil and diazepam or zolpidem. Furthermore, diazepam or zolpidem long-term treatment decreased the expression of mRNA encoding the alpha1 GABA(A) receptor subunit in prefrontal cortex by 43% and 20% respectively. In addition, diazepam but not zolpidem long-term treatment produced a 30% increase in the expression of the alpha5 GABA(A) receptor subunit mRNA in prefrontal cortex. In contrast, imidazenil which is devoid of anticonvulsant tolerance does not elicit significant changes in the expression of alpha1 or alpha5 GABA(A) receptor subunit. These findings suggest that long-term activation of GABA(A) receptors containing the alpha1 or other subunits but not the alpha5 receptor subunit is essential for the induction of anticonvulsant tolerance.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Benzodiazepinas/uso terapéutico , Diazepam/uso terapéutico , Moduladores del GABA/uso terapéutico , Imidazoles/uso terapéutico , Piridinas/uso terapéutico , Convulsiones/tratamiento farmacológico , Animales , Conducta Animal/efectos de los fármacos , Bicuculina , Umbral Diferencial/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Imidazoles/farmacología , Masculino , Ratas , Ratas Endogámicas F344 , Convulsiones/inducido químicamente , ZolpidemRESUMEN
Flumazenil (FLU), a specific benzodiazepine (BZ) receptor antagonist has been used in the treatment of acute BZ intoxication or the alleviation of BZ-induced withdrawal syndrome on the basis of its weak partial agonist action at GABA(A) receptors. However, given to patients, FLU can worsen diazepam-induced withdrawal syndrome by lowering seizure threshold. We therefore investigated whether imidazenil, a selective positive allosteric modulator of GABA action at GABA(A) receptors containing alpha5 subunit, can antagonize diazepam-induced sedative action and suppression of locomotor activity without affecting diazepam anti-bicuculline action. We report here that while FLU (16.5 micromol/kg) showed no effect on locomotor activity and bicuculline-induced convulsion, it completely antagonized diazepam (10.5 micromol/kg) anti-bicuculline action and the suppression of locomotor activity. However, imidazenil (0.76 micromol/kg) elicited anti-bicuculline action and was dose-dependently antagonized by FLU (16.5 and 33 micromol/kg). Furthermore, imidazenil showed no effect on path length traveled but slightly decreased (40%) horizontal activity when compared to diazepam (85%), and maintained the anti-bicuculline action of diazepam to a threshold level similar to that observed with diazepam. Whereas cross-tolerance between BZs has been reported in animals and humans, we previously reported the absence of cross-tolerance between imidazenil and diazepam. Thus, we suggest that imidazenil might be more effective than FLU at alleviating the withdrawal syndrome associated with long-term BZ administration.