RESUMO
In the intermediate stages of amyotrophic lateral sclerosis (ALS), surviving motor neurons (MNs) that show intrinsic resistance to TDP-43 proteinopathy can partially compensate for the loss of their more disease-susceptible counterparts. Elucidating the mechanisms of this compensation may reveal approaches for attenuating motor impairment in ALS patients. In the rNLS8 mouse model of ALS-like pathology driven by doxycycline-regulated neuronal expression of human TDP-43 lacking a nuclear localization signal (hTDP-43ΔNLS), slow MNs are more resistant to disease than fast-fatigable (FF) MNs and can mediate recovery following transgene suppression. In the present study, we used a viral tracing strategy to show that these disease-resistant slow MNs sprout to reinnervate motor endplates of adjacent muscle fibers vacated by degenerated FF MNs. Moreover, we found that neuromuscular junctions within fast-twitch skeletal muscle (tibialis anterior, TA) reinnervated by SK3-positive slow MNs acquire resistance to axonal dieback when challenged with a second course of hTDP-43ΔNLS pathology. The selective resistance of reinnervated neuromuscular junctions was specifically induced by the unique pattern of reinnervation following TDP-43-induced neurodegeneration, as recovery from unilateral sciatic nerve crush did not produce motor units resistant to subsequent hTDP-43ΔNLS. Using cross-reinnervation and self-reinnervation surgery in which motor axons are disconnected from their target muscle and reconnected to a new muscle, we show that FF MNs remain hTDP-43ΔNLS-susceptible and slow MNs remain resistant, regardless of which muscle fibers they control. Collectively, these findings demonstrate that MN identity dictates the susceptibility of neuromuscular junctions to TDP-43 pathology and slow MNs can drive recovery of motor systems due to their remarkable resilience to TDP-43-driven degeneration. This study highlights a potential pathway for regaining motor function with ALS pathology in the advent of therapies that halt the underlying neurodegenerative process.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA , Proteinopatias TDP-43 , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Proteinopatias TDP-43/patologiaRESUMO
Catecholamines and their metabolites act as neurotransmitters in the brain and are important for nervous system function. In the current work, a highly selective and sensitive UPLC-MS/MS assay was developed for quantitation of six catecholamines and their metabolites, including dopamine, norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindolacetic acid from rat and mouse striatum as pharmacodynamic biomarkers to support neuroscience and pharmaceutical research. A fit-for-purpose strategy for method development, assay qualification and study support were adopted for this assay. A surrogate matrix (brain homogenizing solution absent of targeted analytes) was used for preparation of calibration samples and certain levels of quality control samples to avoid interference from endogenous baselines. Homogenized rodent striatum was derivatized by dansyl chloride to enhance the sensitivity, followed by liquid-liquid extraction with ethyl acetate in 96-well plate format. The lower limit of quantitation (LLOQ) was 0.2 ng/mL in tissue homogenate, equivalent to 3.2 pg/mg in brain tissue, which could be further reduced to ten times lower by changing the re-dissolving and injecting volume in the last sample purification step. Acceptable accuracy, precision, linearity, specificity, recovery, and matrix effect was obtained. Bench-top stability (2 h), freeze-thaw stability (3 cycles at -20 °C), and - 80 °C storage stability (up to 51 days) in both tissue homogenate and surrogate matrix along with autosampler stability (60 h at 4°C) all met acceptance criteria. This assay was successfully applied to measure the six analytes in striatum from mice treated with the neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an animal model of Parkinsonism for which dosing protocols can vary widely, and further confirmed the metabolic pathway of neurotoxicity by the quantification of catecholamine metabolites. Our study is the first detailed the step-by-step recovery and pointed out the key factors for the assay to simultaneously quantify these six neurotransmitters in rodent striatum with superior sensitivity.
Assuntos
Catecolaminas , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Camundongos , Neurotransmissores , Ratos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
The microglial reaction is a hallmark of neurodegenerative conditions, and elements thereof may exert differential effects on disease progression, either worsening or ameliorating severity. In amyotrophic lateral sclerosis (ALS), a syndrome characterized by cytoplasmic aggregation of TDP-43 protein and atrophy of motor neurons in the cortex and spinal cord, the transcriptomic signatures of microglia during disease progression are incompletely understood. Here, we performed longitudinal RNAseq analysis of cortical and spinal cord microglia from rNLS8 mice, in which doxycycline-regulatable expression of human TDP-43 (hTDP-43) in the cytoplasm of neurons recapitulates many features of ALS. Transgene suppression in rNLS8 mice leads to functional, anatomical and electrophysiological resolution that is dependent on a microglial reaction that is concurrent with recovery rather than disease onset. We identified basal differences between the gene expression profiles of microglia dependent on localization in spinal cord or cortex. Microglia subjected to chronic hTDP-43 overexpression demonstrated transcriptomic changes in both locations. We noted strong upregulation of Apoe, Axl, Cd63, Clec7a, Csf1, Cst7, Igf1, Itgax, Lgals3, Lilrb4, Lpl and Spp1 during late disease and recovery. Importantly, we identified a distinct suite of differentially expressed genes associated with each phase of disease progression and recovery. Differentially expressed genes were associated with chemotaxis, phagocytosis, inflammation, and production of neuroprotective factors. These data provide new insights into the microglial reaction in TDP-43 proteinopathy. Genes differentially expressed during progression and recovery may provide insight into a unique instance in which the microglial reaction promotes functional recovery after neuronal insult.
Assuntos
Esclerose Lateral Amiotrófica/genética , Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA/genética , Microglia/metabolismo , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Córtex Cerebral/citologia , Quimiotaxia/genética , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Estudos Longitudinais , Camundongos , Camundongos Transgênicos , Doenças Neuroinflamatórias/genética , Neuroproteção/genética , Fagocitose , RNA-Seq , Recuperação de Função Fisiológica , Medula Espinal/citologia , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/metabolismoRESUMO
BACKGROUND AND PURPOSE: Endocannabinoids are critically involved in brain reward functions, mediated by activation of CB1 receptors, reflecting their high density in the brain. However, the recent discovery of CB2 receptors in the brain, particularly in the midbrain dopamine neurons, has challenged this view and inspired us to re-examine the roles of both CB1 and CB2 receptors in the effects of cannabis. EXPERIMENTAL APPROACH: In the present study, we used the electrical intracranial self-stimulation paradigm to evaluate the effects of various cannabinoid drugs on brain reward in laboratory rats and the roles of CB1 and CB2 receptors activation in brain reward function(s). KEY RESULTS: Two mixed CB1 / CB2 receptor agonists, Δ9 -tetrahydrocannabinol (Δ9 -THC) and WIN55,212-2, produced biphasic effects-mild enhancement of brain-stimulation reward (BSR) at low doses but inhibition at higher doses. Pretreatment with a CB1 receptor antagonist (AM251) attenuated the low dose-enhanced BSR, while a CB2 receptor antagonist (AM630) attenuated high dose-inhibited BSR. To confirm these opposing effects, rats were treated with selective CB1 and CB2 receptor agonists. These compounds produced significant BSR enhancement and inhibition, respectively. CONCLUSIONS AND IMPLICATIONS: CB1 receptor activation produced reinforcing effects, whereas CB2 receptor activation was aversive. The subjective effects of cannabis depend on the balance of these opposing effects. These findings not only explain previous conflicting results in animal models of addiction but also explain why cannabis can be either rewarding or aversive in humans, as expression of CB1 and CB2 receptors may differ in the brains of different subjects.
Assuntos
Canabinoides/farmacologia , Cannabis/química , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Recompensa , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Masculino , Ratos , Ratos Long-Evans , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/genéticaRESUMO
Therapeutic strategies are needed for the treatment of amyotrophic lateral sclerosis (ALS). One potential target is matrix metalloproteinase-9 (MMP-9), which is expressed only by fast motor neurons (MNs) that are selectively vulnerable to various ALS-relevant triggers. Previous studies have shown that reduction of MMP-9 function delayed motor dysfunction in a mouse model of familial ALS. However, given that the majority of ALS cases are sporadic, we propose preclinical testing in a mouse model which may be more clinically translatable: rNLS8 mice. In rNLS8 mice, neurodegeneration is triggered by the major pathological hallmark of ALS, TDP-43 mislocalization and aggregation. MMP-9 was targeted in 3 different ways in rNLS8 mice: by AAV9-mediated knockdown, using antisense oligonucleotide (ASO) technology, and by genetic modification. All 3 strategies preserved the motor unit during disease, as measured by MN counts, tibialis anterior (TA) muscle innervation, and physiological recordings from muscle. However, the strategies that reduced MMP-9 beyond the motor unit lead to premature deaths in a subset of rNLS8 mice. Therefore, selective targeting of MMP-9 in MNs could be beneficial in ALS, but side effects outside of the motor circuit may limit the most commonly used clinical targeting strategies.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/inervação , Músculo Esquelético/fisiopatologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1A4V). Moreover, we found that the administration of TUDCA in an hSOD1G93A mouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.
Assuntos
Neurônios Motores/citologia , Células-Tronco/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Humanos , Indóis/farmacologia , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Mutação , Células-Tronco/efeitos dos fármacos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Ácido Tauroquenodesoxicólico/farmacologiaRESUMO
Though motor neurons selectively degenerate in amyotrophic lateral sclerosis, other cell types are likely involved in this disease. We recently generated rNLS8 mice in which human TDP-43 (hTDP-43) pathology could be reversibly induced in neurons and expected that microglia would contribute to neurodegeneration. However, only subtle microglial changes were detected during disease in the spinal cord, despite progressive motor neuron loss; microglia still reacted to inflammatory triggers in these mice. Notably, after hTDP-43 expression was suppressed, microglia dramatically proliferated and changed their morphology and gene expression profiles. These abundant, reactive microglia selectively cleared neuronal hTDP-43. Finally, when microgliosis was blocked during the early recovery phase using PLX3397, a CSF1R and c-kit inhibitor, rNLS8 mice failed to regain full motor function, revealing an important neuroprotective role for microglia. Therefore, reactive microglia exert neuroprotective functions in this amyotrophic lateral sclerosis model, and definition of the underlying mechanism could point toward novel therapeutic strategies.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Neurônios Motores/patologia , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/patologia , Aminopiridinas/farmacologia , Animais , Perfilação da Expressão Gênica , Gliose/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , Camundongos Transgênicos , Músculo Esquelético/patologia , Mutação/genética , Células Mieloides/patologia , Pirróis/farmacologia , Recuperação de Função Fisiológica , Medula Espinal/patologia , Superóxido Dismutase-1/genéticaRESUMO
In order to treat progressive paralysis in ALS patients, it is critical to develop a mouse that closely models human ALS in both pathology and also in the timing of these events. We have recently generated new TDP-43 bigenic mice (called rNLS8) with doxycycline (Dox)-suppressible expression of human TDP-43 (hTDP-43) harboring a defective nuclear localization signal (hTDP-43∆NLS) under the control of the NEFH promoter. Our previous studies characterized the pathology and disease course in young rNLS8 mice following induction of neuronal hTDP-43ΔNLS. We now seek to examine if the order and timing of pathologic events are changed in aged mice. We found that the expression of hTDP-43∆NLS in 12+ month old mice did not accelerate the appearance of neuromuscular abnormalities or motor neuron (MN) death in the lumbar spinal cord (SC), though disease progression was accelerated. However, following suppression of the transgene, important differences between young and aged rNLS8 mice emerged in functional motor recovery. We found that recovery was incomplete in aged mice relative to their younger treatment matched counterparts based on gross behavioral measures and physiological recordings from the animals' gastrocnemius (GC) muscles, despite muscle reinnervation by surviving MNs. This is likely because the reinnervation most often only resulted in partial nerve and endplate connections and the muscle's junctional folds were much more disorganized in aged rNLS8 mice. We believe that these studies will be an important basis for the future design and evaluation of therapies designed to slow denervation and promote re-innervation in adult ALS patients.
RESUMO
UNLABELLED: Motor neurons (MNs) are the neuronal class that is principally affected in amyotrophic lateral sclerosis (ALS), but it is widely known that individual motor pools do not succumb to degeneration simultaneously. Because >90% of ALS patients have an accumulation of cytoplasmic TDP-43 aggregates in postmortem brain and spinal cord (SC), it has been suggested that these inclusions in a given population may trigger its death. We investigated seven MN pools in our new inducible rNLS8 transgenic (Tg) mouse model of TDP-43 proteinopathy and found striking differences in MN responses to TDP-43 pathology. Despite widespread neuronal expression of cytoplasmic human TDP-43, only MNs in the hypoglossal nucleus and the SC are lost after 8 weeks of transgene expression, whereas those in the oculomotor, trigeminal, and facial nuclei are spared. Within the SC, slow MNs survive to end stage, whereas fast fatigable MNs are lost. Correspondingly, axonal dieback occurs first from fast-twitch muscle fibers, whereas slow-twitch fibers remain innervated. Individual pools show differences in the downregulation of endogenous nuclear TDP-43, but this does not fully account for vulnerability to degenerate. After transgene suppression, resistant MNs sprout collaterals to reinnervate previously denervated neuromuscular junctions concurrently with expression of matrix metalloproteinase 9 (MMP-9), a marker of fast MNs. Therefore, although pathological TDP-43 is linked to MN degeneration, the process is not stochastic and mirrors the highly selective patterns of MN degeneration observed in ALS patients. SIGNIFICANCE STATEMENT: Because TDP-43 is the major pathological hallmark of amyotrophic lateral sclerosis (ALS), we generated mice in which mutant human TDP-43 expression causes progressive neuron loss. We show that these rNLS8 mice have a pattern of axonal dieback and cell death that mirrors that often observed in human patients. This finding demonstrates the diversity of motor neuron (MN) populations in their response to pathological TDP-43. Furthermore, we demonstrate that resistant MNs are able to compensate for the loss of their more vulnerable counterparts and change their phenotype in the process. These findings are important because using a mouse model that closely models human ALS in both the disease pathology and the pattern of degeneration is critical to studying and eventually treating progressive paralysis in ALS patients.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Neurônios Motores/fisiologia , Recuperação de Função Fisiológica/fisiologia , Proteinopatias TDP-43/patologia , Animais , Tronco Encefálico/patologia , Morte Celular/genética , Toxina da Cólera/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estimulação Elétrica , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Neurônios Motores/ultraestrutura , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Mutação/genética , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Medula Espinal/patologia , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/fisiopatologia , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
Accumulation of phosphorylated cytoplasmic TDP-43 inclusions accompanied by loss of normal nuclear TDP-43 in neurons and glia of the brain and spinal cord are the molecular hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the role of cytoplasmic TDP-43 in the pathogenesis of these neurodegenerative TDP-43 proteinopathies remains unclear, due in part to a lack of valid mouse models. We therefore generated new mice with doxycycline (Dox)-suppressible expression of human TDP-43 (hTDP-43) harboring a defective nuclear localization signal (∆NLS) under the control of the neurofilament heavy chain promoter. Expression of hTDP-43∆NLS in these 'regulatable NLS' (rNLS) mice resulted in the accumulation of insoluble, phosphorylated cytoplasmic TDP-43 in brain and spinal cord, loss of endogenous nuclear mouse TDP-43 (mTDP-43), brain atrophy, muscle denervation, dramatic motor neuron loss, and progressive motor impairments leading to death. Notably, suppression of hTDP-43∆NLS expression by return of Dox to rNLS mice after disease onset caused a dramatic decrease in phosphorylated TDP-43 pathology, an increase in nuclear mTDP-43 to control levels, and the prevention of further motor neuron loss. rNLS mice back on Dox also showed a significant increase in muscle innervation, a rescue of motor impairments, and a dramatic extension of lifespan. Thus, the rNLS mice are new TDP-43 mouse models that delineate the timeline of pathology development, muscle denervation and neuron loss in ALS/FTLD-TDP. Importantly, even after neurodegeneration and onset of motor dysfunction, removal of cytoplasmic TDP-43 and the concomitant return of nuclear TDP-43 led to neuron preservation, muscle re-innervation and functional recovery.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/fisiopatologia , Recuperação de Função Fisiológica/fisiologia , Esclerose Lateral Amiotrófica/patologia , Animais , Atrofia , Encéfalo/metabolismo , Encéfalo/patologia , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/patologia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Doxiciclina , Feminino , Degeneração Lobar Frontotemporal/patologia , Humanos , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Transtornos dos Movimentos/patologia , Transtornos dos Movimentos/fisiopatologia , Músculo Esquelético/inervação , Distribuição Aleatória , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
Angiogenesis is crucial for the success of most tissue engineering strategies. The natural inflammatory response is a major regulator of vascularization, through the activity of different types of macrophages and the cytokines they secrete. Macrophages exist on a spectrum of diverse phenotypes, from "classically activated" M1 to "alternatively activated" M2 macrophages. M2 macrophages, including the subsets M2a and M2c, are typically considered to promote angiogenesis and tissue regeneration, while M1 macrophages are considered to be anti-angiogenic, although these classifications are controversial. Here we show that in contrast to this traditional paradigm, primary human M1 macrophages secrete the highest levels of potent angiogenic stimulators including VEGF; M2a macrophages secrete the highest levels of PDGF-BB, a chemoattractant for stabilizing pericytes, and also promote anastomosis of sprouting endothelial cells in vitro; and M2c macrophages secrete the highest levels of MMP9, an important protease involved in vascular remodeling. In a murine subcutaneous implantation model, porous collagen scaffolds were surrounded by a fibrous capsule, coincident with high expression of M2 macrophage markers, while scaffolds coated with the bacterial lipopolysaccharide were degraded by inflammatory macrophages, and glutaraldehyde-crosslinked scaffolds were infiltrated by substantial numbers of blood vessels, accompanied by high levels of M1 and M2 macrophages. These results suggest that coordinated efforts by both M1 and M2 macrophages are required for angiogenesis and scaffold vascularization, which may explain some of the controversy over which phenotype is the angiogenic phenotype.
Assuntos
Macrófagos/citologia , Neovascularização Fisiológica , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Becaplermina , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-sis/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Selective neuronal loss is the hallmark of neurodegenerative diseases. In patients with amyotrophic lateral sclerosis (ALS), most motor neurons die but those innervating extraocular, pelvic sphincter, and slow limb muscles exhibit selective resistance. We identified 18 genes that show >10-fold differential expression between resistant and vulnerable motor neurons. One of these, matrix metalloproteinase-9 (MMP-9), is expressed only by fast motor neurons, which are selectively vulnerable. In ALS model mice expressing mutant superoxide dismutase (SOD1), reduction of MMP-9 function using gene ablation, viral gene therapy, or pharmacological inhibition significantly delayed muscle denervation. In the presence of mutant SOD1, MMP-9 expressed by fast motor neurons themselves enhances activation of ER stress and is sufficient to trigger axonal die-back. These findings define MMP-9 as a candidate therapeutic target for ALS. The molecular basis of neuronal diversity thus provides significant insights into mechanisms of selective vulnerability to neurodegeneration.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Neurônios Motores/metabolismo , Doenças Neurodegenerativas/genética , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Fatores Etários , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Toxina da Cólera/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Metaloproteinase 9 da Matriz/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Denervação Muscular , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Neurodegenerativas/patologia , Fosfopiruvato Hidratase/metabolismo , Superóxido Dismutase/genética , Fatores de Transcrição/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
Our previous studies have shown that the selective dopamine D(3) receptor antagonists SB-277011A or NGB 2904 significantly attenuate cocaine self-administration under a progressive-ratio reinforcement schedule and cocaine-, methamphetamine- or nicotine-enhanced brain stimulation reward. However, the poor bioavailability of SB-277011A has limited its potential use in humans. In the present study, we investigated the effects of the novel D(3) receptor antagonist PG01037 on methamphetamine self-administration, methamphetamine-associated cue-induced reinstatement of drug seeking and methamphetamine-enhanced brain stimulation reward. Rats were allowed to intravenously self-administer methamphetamine under fixed-ratio 2 and progressive-ratio reinforcement conditions, and then the effects of PG01037 on methamphetamine self-administration and cue-induced reinstatement were assessed. Additional groups of rats were trained for intracranial electrical brain stimulation reward and the effects of PG01037 and methamphetamine on brain stimulation reward were assessed. Acute intraperitoneal administration of PG01037 (3, 10, 30 mg/kg) failed to alter methamphetamine or sucrose self-administration under fixed-ratio 2 reinforcement, but significantly lowered the break-point levels for methamphetamine or sucrose self-administration under progressive-ratio reinforcement. In addition, PG01037 significantly inhibited methamphetamine-associated cue-triggered reinstatement of drug-seeking behavior and methamphetamine-enhanced brain stimulation reward. These data suggest that the novel D(3) antagonist PG01037 significantly attenuates the rewarding effects as assessed by progressive-ratio self-administration and brain stimulation reward, and inhibits methamphetamine-associated cue-induced reinstatement of drug-seeking behavior These findings support the potential use of PG01037 or other selective D(3) antagonists in the treatment of methamphetamine addiction.
Assuntos
Benzamidas/farmacologia , Condicionamento Operante/efeitos dos fármacos , Antagonistas de Dopamina/farmacologia , Inibidores da Captação de Dopamina/antagonistas & inibidores , Metanfetamina/antagonistas & inibidores , Piridinas/farmacologia , Receptores de Dopamina D3/antagonistas & inibidores , Animais , Sinais (Psicologia) , Inibidores da Captação de Dopamina/administração & dosagem , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Comportamento de Procura de Droga/efeitos dos fármacos , Estimulação Elétrica/métodos , Extinção Psicológica/efeitos dos fármacos , Masculino , Metanfetamina/administração & dosagem , Metanfetamina/farmacologia , Ratos , Ratos Long-Evans , Esquema de Reforço , Recompensa , Autoadministração , Sacarose/administração & dosagem , Sacarose/farmacologiaRESUMO
The success of methadone in treating opiate addiction has suggested that long-acting agonist therapies may be similarly useful for treating cocaine addiction. Here, we examined this hypothesis, using the slow-onset long-acting monoamine reuptake inhibitor 31,345, a trans-aminotetralin analog, in a variety of addiction-related animal models, and compared it with methadone's effects on heroin's actions in the same animal models. Systemic administration of 31,345 produced long-lasting enhancement of electrical brain-stimulation reward (BSR) and extracellular nucleus accumbens (NAc) dopamine (DA). Pretreatment with 31,345 augmented cocaine-enhanced BSR, prolonged cocaine-enhanced NAc DA, and produced a long-term (24-48 h) reduction in cocaine self-administration rate without obvious extinction pattern, suggesting an additive effect of 31,345 with cocaine. In contrast, methadone pretreatment not only dose-dependently inhibited heroin self-administration with an extinction pattern but also dose-dependently inhibited heroin-enhanced BSR and NAc DA, suggesting functional antagonism by methadone of heroin's actions. In addition, 31,345 appears to possess significant abuse liability, as it produces dose-dependent enhancement of BSR and NAc DA, maintains a low rate of self-administration behavior, and dose-dependently reinstates drug-seeking behavior. In contrast, methadone only partially maintains self-administration with an extinction pattern, and fails to induce reinstatement of drug-seeking behavior. These findings suggest that 31,345 is a cocaine-like slow-onset long-acting monoamine transporter inhibitor that may act as an agonist therapy for cocaine addiction. However, its pattern of action appears to be significantly different from that of methadone. Ideal agonist substitutes for cocaine should fully emulate methadone's actions, that is, functionally antagonizing cocaine's action while blocking monoamine transporters to augment synaptic DA.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Cocaína/farmacologia , Dopamina/metabolismo , Heroína/antagonistas & inibidores , Metadona/farmacologia , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/antagonistas & inibidores , Tetra-Hidronaftalenos/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Preparações de Ação Retardada , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estimulação Elétrica/métodos , Extinção Psicológica/efeitos dos fármacos , Heroína/farmacologia , Masculino , Núcleo Accumbens/metabolismo , Ratos , Ratos Long-Evans , Autoadministração , Tetra-Hidronaftalenos/administração & dosagemRESUMO
Pharmacological activation of group II metabotropic glutamate (mGlu2 and mGlu3) receptors inhibits reward-seeking behavior and/or rewarding efficacy induced by drugs (cocaine, nicotine) or natural rewards (food, sucrose). In the present study, we investigated whether elevation of brain N-acetylaspartylglutamate (NAAG), an endogenous group II mGlu receptor agonist, by the NAAG peptidase inhibitor 2-PMPA attenuates cocaine's rewarding effects, as assessed by intravenous cocaine self-administration and intracranial electrical brain-stimulation reward (BSR) in rats. Systemic administration of 2-PMPA (10, 30, 100 mg/kg, i.p.) or intranasal administration of NAAG (100, 300 microg/10 microl/nostril) significantly inhibited intravenous cocaine self-administration under progressive-ratio (PR), but not under fixed-ratio 2 (FR2), reinforcement conditions. In addition, 2-PMPA (1, 10, 30 mg/kg, i.p) or NAAG (50, 100 microg/10 microl/nostril) significantly inhibited cocaine-enhanced BSR, but not basal BSR. Pretreatment with LY341495 (1 mg/kg, i.p.), a selective mGlu2/3 receptor antagonist, prevented the inhibitory effects produced by 2-PMPA or NAAG in both the self-administration and BSR paradigms. In vivo microdialysis demonstrated that 2-PMPA (10, 30, 100 mg/kg) dose-dependently attenuated cocaine-enhanced extracellular dopamine (DA) in the nucleus accumbens (NAc). 2-PMPA alone inhibited basal NAc DA release, an effect that was prevented by LY341495. These findings suggest that systemic administration of 2-PMPA or intranasal administration of NAAG inhibits cocaine's rewarding efficacy and cocaine-enhanced NAc DA - likely by activation of presynaptic mGlu2/3 receptors in the NAc. These data suggest a potential utility for 2-PMPA or NAAG in the treatment of cocaine addiction.
Assuntos
Comportamento Aditivo/terapia , Encéfalo/fisiologia , Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Estimulação Elétrica/métodos , Fármacos Neuroprotetores/metabolismo , Recompensa , Animais , Comportamento Aditivo/fisiopatologia , Comportamento Aditivo/psicologia , Encéfalo/efeitos dos fármacos , Condicionamento Operante/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Injeções Intravenosas/métodos , Masculino , Microdiálise , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/antagonistas & inibidores , Compostos Organofosforados/farmacologia , Ratos , Ratos Long-Evans , Esquema de Reforço , AutoadministraçãoRESUMO
The opiate-receptor antagonist naloxone was administered to rats after passive-avoidance training either alone or in combination with forced-swim stress. A retention test revealed that while naloxone enhanced retention when administered alone, it impaired retention when administered in combination with forced-swim stress. The findings provide evidence for a "protective" endogenous opioid-based system that, when not blocked pharmacologically, limits enhancement or impairment of retention under conditions of mild and intense stress, respectively.
Assuntos
Memória/efeitos dos fármacos , Memória/fisiologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Estresse Psicológico/fisiopatologia , Análise de Variância , Animais , Aprendizagem da Esquiva , Eletrochoque , Masculino , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/fisiopatologia , Testes Neuropsicológicos , Distribuição Aleatória , Ratos , Ratos Long-Evans , Natação , Fatores de TempoRESUMO
Tobacco use is a global problem with serious health consequences. Though some treatment options exist, there remains a great need for new effective pharmacotherapies to aid smokers in maintaining long-term abstinence. In the present article, we first discuss the neural mechanisms underlying nicotine reward, and then review various mechanism-based pharmacological agents for the treatment of nicotine dependence. An oversimplified hypothesis of addiction to tobacco is that nicotine is the major addictive component of tobacco. Nicotine binds to alpha4beta2 and alpha7 nicotinic acetylcholine receptors (nAChRs) located on dopaminergic, glutamatergic and GABAergic neurons in the mesolimbic dopamine (DA) system, which causes an increase in extracellular DA in the nucleus accumbens (NAc). That increase in DA reinforces tobacco use, particularly during the acquisition phase. Enhanced glutamate transmission to DA neurons in the ventral tegmental area appears to play an important role in this process. In addition, chronic nicotine treatment increases endocannabinoid levels in the mesolimbic DA system, which indirectly modulates NAc DA release and nicotine reward. Accordingly, pharmacological agents that target brain acetylcholine, DA, glutamate, GABA, or endocannabonoid signaling systems have been proposed to interrupt nicotine action. Furthermore, pharmacokinetic strategies that alter plasma nicotine availability, metabolism and clearance also significantly alter nicotine's action in the brain. Progress using these pharmacodynamic and pharmacokinetic agents is reviewed. For drugs in each category, we discuss the mechanistic rationale for their potential anti-nicotine efficacy, major findings in preclinical and clinical studies, and future research directions.
Assuntos
Sistemas de Liberação de Medicamentos , Nicotina/efeitos adversos , Tabagismo/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ensaios Clínicos como Assunto , Dopamina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Nicotina/farmacocinética , Nicotina/farmacologia , Agonistas Nicotínicos/efeitos adversos , Agonistas Nicotínicos/farmacocinética , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Tabagismo/fisiopatologiaRESUMO
Varenicline, a partial alpha4beta2 and full alpha7 nicotinic receptor agonist, has been shown to inhibit nicotine self-administration and nicotine-induced increases in extracellular dopamine in the nucleus accumbens. In the present study, we investigated whether varenicline inhibits nicotine-enhanced electrical brain-stimulation reward (BSR), and if so, which receptor subtypes are involved. Systemic administration of nicotine (0.25-1.0 mg/kg, i.p.) or varenicline (0.03-3 mg/kg, i.p.) produced biphasic effects, with low doses producing enhancement (e.g., decreased BSR threshold), and high doses inhibiting BSR. Pretreatment with low dose (0.03-1.0 mg/kg) varenicline dose-dependently attenuated nicotine (0.25 or 0.5 mg/kg)-enhanced BSR. The BSR-enhancing effect produced by varenicline was blocked by mecamylamine (a high affinity nicotinic receptor antagonist) or dihydro-beta-erythroidine (a relatively selective nicotinic alpha4-containing receptor antagonist), but not methyllycaconitine (a selective alpha7 receptor antagonist), suggesting an effect mediated by activation of alpha4beta2 receptors. This suggestion is supported by findings that the alpha4beta2 receptor agonist SIB-1765F produced a dose-dependent enhancement of BSR, while pretreatment with SIB-1765F attenuated nicotine (0.5 mg/kg)-enhanced BSR. In contrast, the selective alpha7 receptor agonist ARR-17779, altered neither BSR itself nor nicotine-enhanced BSR, at any dose tested. These findings suggest that: 1) varenicline inhibits nicotine-enhanced BSR, supporting its use as a smoking cessation aid; and 2) varenicline-enhanced BSR by itself and varenicline's anti-nicotine effects are mediated by activation of alpha4beta2, but not alpha7, receptors.
Assuntos
Benzazepinas/farmacologia , Condicionamento Operante/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Quinoxalinas/farmacologia , Receptores Nicotínicos/metabolismo , Recompensa , Animais , Condicionamento Operante/fisiologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica/métodos , Masculino , Feixe Prosencefálico Mediano/efeitos dos fármacos , Feixe Prosencefálico Mediano/fisiologia , Antagonistas Nicotínicos/farmacologia , Ratos , Ratos Long-Evans , VareniclinaRESUMO
The metabotropic glutamate receptor 7 (mGluR7) has received much attention as a potential target for the treatment of epilepsy, major depression, and anxiety. In this study, we investigated the possible involvement of mGluR7 in cocaine reward in animal models of drug addiction. Pretreatment with the selective mGluR7 allosteric agonist N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082; 1-20 mg/kg, i.p.) dose-dependently inhibited cocaine-induced enhancement of electrical brain-stimulation reward and intravenous cocaine self-administration under both fixed-ratio and progressive-ratio reinforcement conditions, but failed to alter either basal or cocaine-enhanced locomotion or oral sucrose self-administration, suggesting a specific inhibition of cocaine reward. Microinjections of AMN082 (1-5 microg/microl per side) into the nucleus accumbens (NAc) or ventral pallidum (VP), but not dorsal striatum, also inhibited cocaine self-administration in a dose-dependent manner. Intra-NAc or intra-VP co-administration of 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP, 5 microg/microl per side), a selective mGluR7 allosteric antagonist, significantly blocked AMN082's action, suggesting an effect mediated by mGluR7 in these brain regions. In vivo microdialysis demonstrated that cocaine (10 mg/kg, i.p.) priming significantly elevated extracellular DA in the NAc or VP, while decreasing extracellular GABA in VP (but not in NAc). AMN082 pretreatment selectively blocked cocaine-induced changes in extracellular GABA, but not in DA, in both naive rats and cocaine self-administration rats. These data suggest: (1) mGluR7 is critically involved in cocaine's acute reinforcement; (2) GABA-, but not DA-, dependent mechanisms in the ventral striatopallidal pathway appear to underlie AMN082's actions; and (3) AMN082 or other mGluR7-selective agonists may be useful in the treatment of cocaine addiction.
Assuntos
Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Globo Pálido/metabolismo , Receptores de Ácido Caínico/metabolismo , Recompensa , Ácido gama-Aminobutírico/metabolismo , Administração Oral , Animais , Comportamento Animal , Compostos Benzidrílicos/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Estimulação Elétrica/métodos , Masculino , Microdiálise/métodos , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Long-Evans , Receptores de Ácido Caínico/agonistas , Receptores de Ácido Caínico/antagonistas & inibidores , Esquema de Reforço , Autoadministração/métodos , Sacarose/administração & dosagem , Edulcorantes/administração & dosagem , Receptor de GluK3 CainatoRESUMO
We have previously reported that selective dopamine (DA) D3 receptor antagonists are effective in a number of animal models of drug addiction, but not in intravenous drug self-administration, suggesting a limited ability to modify drug reward. In the present study, we evaluated the actions ofS33138, a novel partially selective D3 receptor antagonist, in animal models relevant to drug addiction. S33138, at doses of 0.156 or 0.625 mg/kg (i.p.), attenuated cocaine-enhanced brain-stimulation reward (BSR), and the highest dose tested (2.5 mg/kg) produced a significant aversive-like rightward shift in BSR rate-frequency reward functions. Further, S33138 produced biphasic effects on cocaine self-administration, i.e., a moderate dose (2.5 mg/kg, p.o.) increased, while a higher dose (5 mg/kg, p.o.) inhibited, cocaine self-administration. The increase in cocaine self-administration likely reflects a compensatory response to a partial reduction in drug reward after S33138. In addition, S33138 (0.156-2.5 mg/kg, p.o.) also dose-dependently inhibited cocaine-induced reinstatement of drug-seeking behavior. The reduction in cocaine-enhanced BSR and cocaine-triggered reinstatement produced by lower effective doses (e.g., 0.156 or 0.625 mg/kg) of 533138 is unlikely due to impaired locomotion, as lower effective doses of S33138 decreased neither Ymax levels in the BSR paradigm, rotarod performance, nor locomotion. However, the higher doses (2.5 or 5 mg/kg) of S33138 also significantly inhibited sucrose self-administration and rotarod performance, suggesting non-D3 receptor-mediated effects on non-drug reward and locomotion. These data suggest that lower doses of S33138 interacting essentially with D3 receptors have pharmacotherapeutic potential in treatment of cocaine addiction, while higher doses occupying D2 receptors may influence locomotion and non-drug reward.