RESUMO
Mutations in the cytoskeletal linker protein plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a plectin-organized vimentin scaffold keeps actomyosin contractility 'in-check' and maintains AJ homeostasis.
Assuntos
Actinas/metabolismo , Células Endoteliais/metabolismo , Plectina/metabolismo , Vimentina/metabolismo , Animais , Permeabilidade Capilar , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plectina/genética , Estresse MecânicoRESUMO
We describe the multigram synthesis and in vivo efficacy studies of a donepezilâhuprine hybrid that has been found to display a promising in vitro multitarget profile of interest for the treatment of Alzheimer's disease (AD). Its synthesis features as the key step a novel multigram preparative chromatographic resolution of intermediate racemic huprine Y by chiral HPLC. Administration of this compound to transgenic CL4176 and CL2006 Caenorhabditis elegans strains expressing human Aß42, here used as simplified animal models of AD, led to a significant protection from the toxicity induced by Aß42. However, this protective effect was not accompanied, in CL2006 worms, by a reduction of amyloid deposits. Oral administration for 3 months to transgenic APPSL mice, a well-established animal model of AD, improved short-term memory, but did not alter brain levels of Aß peptides nor cortical and hippocampal amyloid plaque load. Despite the clear protective and cognitive effects of AVCRI104P4, the lack of Aß lowering effect in vivo might be related to its lower in vitro potency toward Aß aggregation and formation as compared with its higher anticholinesterase activities. Further lead optimization in this series should thus focus on improving the anti-amyloid/anticholinesterase activity ratio.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Aminoquinolinas/administração & dosagem , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Indanos/administração & dosagem , Piperidinas/administração & dosagem , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Aminoquinolinas/química , Aminoquinolinas/uso terapêutico , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Modelos Animais de Doenças , Donepezila , Células Hep G2 , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Indanos/química , Indanos/uso terapêutico , Camundongos , Estrutura Molecular , Piperidinas/química , Piperidinas/uso terapêuticoRESUMO
The serum amyloid A (SAA) family of proteins is encoded by multiple genes, which display allelic variation and a high degree of homology in mammals. The SAA1/2 genes code for non-glycosylated acute-phase SAA1/2 proteins, that may increase up to 1000-fold during inflammation. The SAA4 gene, well characterized in humans (hSAA4) and mice (mSaa4) codes for a SAA4 protein that is glycosylated only in humans. We here report on a previously uncharacterized SAA4 gene (rSAA4) and its product in Rattus norvegicus, the only mammalian species known not to express acute-phase SAA. The exon/intron organization of rSAA4 is similar to that reported for hSAA4 and mSaa4. By performing 5'- and 3'RACE, we identified a 1830-bases containing rSAA4 mRNA (including a GA-dinucleotide tandem repeat). Highest rSAA4 mRNA expression was detected in rat liver. In McA-RH7777 rat hepatoma cells, rSAA4 transcription was significantly upregulated in response to LPS and IL-6 while IL-1α/ß and TNFα were without effect. Luciferase assays with promoter-truncation constructs identified three proximal C/EBP-elements that mediate expression of rSAA4 in McA-RH7777 cells. In line with sequence prediction a 14-kDa non-glycosylated SAA4 protein is abundantly expressed in rat liver. Fluorescence microscopy revealed predominant localization of rSAA4-GFP-tagged fusion protein in the ER.
Assuntos
Proteína Amiloide A Sérica/metabolismo , Animais , Linhagem Celular Tumoral , Fígado/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/genéticaRESUMO
Mouse models of Huntington's disease (HD) recapitulate many aspects of the human disease. These genetically modified mice are powerful tools that are used not only to examine the pathogenesis of the disease, but also to assess the efficacy of potential new treatments. Disappointingly, in the past few years we have seen the success of potential therapies in animal studies, subsequently followed by failure in clinical trials. We discuss here a number of factors that influence the translatability of findings from the preclinical to the clinical realm. In particular, we discuss issues related to sample size, reporting of information regarding experimental protocols and mouse models, assignment to experimental groups, incorporation of cognitive measures for early phases of the disease, environmental enrichment, surrogate measures for survival, and the use of more than one HD mouse model. Although it is reasonable to question the appropriateness of the animal models used, we argue that it is more parsimonious to assume that improvements in experimental design and publication of negative results will lead to improved translatability to the clinic and insights about HD pathophysiology.
Assuntos
Modelos Animais de Doenças , Doença de Huntington/terapia , Pesquisa Translacional Biomédica/métodos , Animais , Transtornos Cognitivos/etiologia , Humanos , Doença de Huntington/complicações , CamundongosRESUMO
Drug-induced Behavioral Signature Analysis (DBSA), is a machine learning (ML) method for in silico screening of compounds, inspired by analytical methods quantifying gene enrichment in genomic analyses. When applied to behavioral data it can identify drugs that can potentially reverse in vivo behavioral symptoms in animal models of human disease and suggest new hypotheses for drug discovery and repurposing. We present a proof-of-concept study aiming to assess Drug-induced Behavioral Signature Analysis (DBSA) as a systematic approach for drug discovery for rare disorders. We applied Drug-induced Behavioral Signature Analysis to high-content behavioral data obtained with SmartCube®, an automated in vivo phenotyping platform. The therapeutic potential of several dozen approved drugs was assessed for phenotypic reversal of the behavioral profile of a Huntington's Disease (HD) murine model, the Q175 heterozygous knock-in mice. The in silico Drug-induced Behavioral Signature Analysis predictions were enriched for drugs known to be effective in the symptomatic treatment of Huntington's Disease, including bupropion, modafinil, methylphenidate, and several SSRIs, as well as the atypical antidepressant tianeptine. To validate the method, we tested acute and chronic effects of tianeptine (20 mg/kg, i. p.) in vivo, using Q175 mice and wild type controls. In both experiments, tianeptine significantly rescued the behavioral phenotype assessed with the SmartCube® platform. Our target-agnostic method thus showed promise for identification of symptomatic relief treatments for rare disorders, providing an alternative method for hypothesis generation and drug discovery for disorders with huge disease burden and unmet medical needs.
RESUMO
Tuberous sclerosis complex (TSC) is a monogenic disorder characterized by hyperactivation of the mTOR signaling pathway and developmental brain malformations leading to intractable epilepsy. Although treatment with the recently approved mTOR inhibitor, everolimus, results in clinically relevant seizure suppression in up to 40% of TSC patients, seizures remain uncontrolled in a large number of cases, underscoring the need to identify novel treatment targets. The MEK-ERK signaling pathway has been found to be aberrantly activated in TSC and inhibition of MEK-ERK activity independently of mTOR rescued neuronal dendrite overgrowth in mice modeling TSC neuropathology. Here, we evaluated the efficacy of MEK-ERK inhibition on seizures in two mouse models of TSC. We found that treatment with the MEK inhibitor PD0325901 (mirdametinib) significantly reduced seizure activity in both TSC mouse models. These findings support inhibiting MEK-ERK activity as a potential alternative strategy to treat seizures in TSC.
Assuntos
Esclerose Tuberosa , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Convulsões/tratamento farmacológico , Transdução de Sinais , Esclerose Tuberosa/complicações , Esclerose Tuberosa/tratamento farmacológicoRESUMO
We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.
Assuntos
Doença de Huntington , Camundongos , Humanos , Animais , Lactente , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Agregados Proteicos , Proteína Huntingtina/genética , Proteína Huntingtina/líquido cefalorraquidiano , Modelos Animais de Doenças , Corpo Estriado/metabolismo , Progressão da DoençaRESUMO
The ability to calculate whether small molecules will cross the blood-brain barrier (BBB) is an important task for companies working in neuroscience drug discovery. For a decade, scientists have relied on relatively simplistic rules such as Pfizer's central nervous system multiparameter optimization models (CNS-MPO) for guidance during the drug selection process. In parallel, there has been a continued development of more sophisticated machine learning models that utilize different molecular descriptors and algorithms; however, these models represent a "black box" and are generally less interpretable. In both cases, these methods predict the ability of small molecules to cross the BBB using the molecular structure information on its own without in vitro or in vivo data. We describe here the implementation of two versions of Pfizer's algorithm (Pf-MPO.v1 and Pf-MPO.v2) and compare it with a Bayesian machine learning model of BBB penetration trained on a data set of 2296 active and inactive compounds using extended connectivity fingerprint descriptors. The predictive ability of these approaches was compared with 40 known CNS active drugs initially used by Pfizer as their positive set for validation of the Pf-MPO.v1 score. 37/40 (92.5%) compounds were predicted as active by the Bayesian model, while only 30/40 (75%) received a desirable Pf-MPO.v1 score ≥4 and 33/40 (82.5%) received a desirable Pf-MPO.v2 score ≥4, suggesting the Bayesian model is more accurate than MPO algorithms. This also indicates machine learning models are more flexible and have better predictive power for BBB penetration than simple rule sets that require multiple, accurate descriptor calculations. Our machine learning model statistics are comparable to recent published studies. We describe the implications of these findings and how machine learning may have a role alongside more interpretable methods.
Assuntos
Barreira Hematoencefálica , Sistema Nervoso Central , Teorema de Bayes , Fármacos do Sistema Nervoso Central , Aprendizado de MáquinaRESUMO
BACKGROUND: Human health is closely interconnected with its microbiome. Resilient microbiomes in, on, and around the human body will be key for safe and successful long-term space travel. However, longitudinal dynamics of microbiomes inside confined built environments are still poorly understood. Herein, we used the Hawaii Space Exploration Analog and Simulation IV (HI-SEAS IV) mission, a 1 year-long isolation study, to investigate microbial transfer between crew and habitat, in order to understand adverse developments which may occur in a future outpost on the Moon or Mars. RESULTS: Longitudinal 16S rRNA gene profiles, as well as quantitative observations, revealed significant differences in microbial diversity, abundance, and composition between samples of the built environment and its crew. The microbiome composition and diversity associated with abiotic surfaces was found to be rather stable, whereas the microbial skin profiles of individual crew members were highly dynamic, resulting in an increased microbiome diversity at the end of the isolation period. The skin microbiome dynamics were especially pronounced by a regular transfer of the indicator species Methanobrevibacter between crew members within the first 200 days. Quantitative information was used to track the propagation of antimicrobial resistance in the habitat. Together with functional and phenotypic predictions, quantitative and qualitative data supported the observation of a delayed longitudinal microbial homogenization between crew and habitat surfaces which was mainly caused by a malfunctioning sanitary facility. CONCLUSIONS: This study highlights main routes of microbial transfer, interaction of the crew, and origins of microbial dynamics in an isolated environment. We identify key targets of microbial monitoring, and emphasize the need for defined baselines of microbiome diversity and abundance on surfaces and crew skin. Targeted manipulation to counteract adverse developments of the microbiome could be a highly important strategy to ensure safety during future space endeavors. Video abstract.
Assuntos
Astronautas , Meio Ambiente Extraterreno , Microbiota , Pele/microbiologia , Voo Espacial , Astronave , Adulto , Ambiente Construído , Feminino , Havaí , Humanos , Masculino , Microbiota/genética , RNA Ribossômico 16S/genéticaRESUMO
Consumer wearables and sensors are a rich source of data about patients' daily disease and symptom burden, particularly in the case of movement disorders like Parkinson's disease (PD). However, interpreting these complex data into so-called digital biomarkers requires complicated analytical approaches, and validating these biomarkers requires sufficient data and unbiased evaluation methods. Here we describe the use of crowdsourcing to specifically evaluate and benchmark features derived from accelerometer and gyroscope data in two different datasets to predict the presence of PD and severity of three PD symptoms: tremor, dyskinesia, and bradykinesia. Forty teams from around the world submitted features, and achieved drastically improved predictive performance for PD status (best AUROC = 0.87), as well as tremor- (best AUPR = 0.75), dyskinesia- (best AUPR = 0.48) and bradykinesia-severity (best AUPR = 0.95).
RESUMO
Triple reuptake inhibitors (TRIs) that block the dopamine transporter (DAT), norepinephrine transporter, and serotonin transporter are being developed as a new class of antidepressant that may have better efficacy and fewer side effects compared with traditional antidepressants. We describe a novel TRI, 2-[4-(4-chlorophenyl)-1-methylpiperidin-3-ylmethylsulfanyl]-1-(3-methylpiperidin-1-yl)-ethanone (JZAD-IV-22), that inhibits all three monoamine transporters with approximately equal potency in vitro. (+/-)-1-(3,4-dichlorophenyl)-3-azabicyclo-[3.1.0]hexane hydrochloride (DOV 216,303), a TRI shown to be an effective antidepressant in a clinical trial, shows reuptake inhibition similar to that of JZAD-IV-22 in vitro. Furthermore, both JZAD-IV-22 and DOV 216,303 increase levels of dopamine, norepinephrine, and serotonin in the mouse prefrontal cortex when administered by peripheral injection. JZAD-IV-22 and DOV 216,303 exhibited antidepressant-like efficacy in the mouse forced-swim and tail-suspension tests at doses that increased neurotransmitter levels. Because development of DAT inhibitors could be hindered by abuse liability, both JZAD-IV-22 and DOV 216,303 were compared in two assays that are markers of abuse potential. Both JZAD-IV-22 and DOV 216,303 partially substituted for cocaine in a drug discrimination assay in rats, and high doses of DOV 216,303 produced locomotor sensitization in mice. JZAD-IV-22 showed no evidence of sensitization at any dose tested. These results demonstrate that JZAD-IV-22 is a TRI with antidepressant-like activity similar to that of DOV 216,303. The striking feature that distinguishes the two TRIs is that locomotor sensitization, a common underlying feature of drugs of abuse, is seen with DOV 216,303 but is completely lacking in JZAD-IV-22. These findings may have implications for the potential for abuse liability in humans.
Assuntos
Antidepressivos/farmacologia , Locomoção/efeitos dos fármacos , Inibidores da Captação de Neurotransmissores/farmacologia , Piperidinas/farmacologia , Inibidores da Captação Adrenérgica/farmacologia , Inibidores da Captação Adrenérgica/uso terapêutico , Animais , Antidepressivos/uso terapêutico , Compostos Aza/efeitos adversos , Compostos Aza/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/citologia , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cocaína/farmacologia , Corpo Estriado/citologia , Depressão/prevenção & controle , Discriminação Psicológica , Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Inibidores da Captação de Dopamina/uso terapêutico , Elevação dos Membros Posteriores , Hipotálamo/citologia , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Inibidores da Captação de Neurotransmissores/uso terapêutico , Norepinefrina/metabolismo , Piperidinas/uso terapêutico , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Natação , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
Phenotyping mouse model systems of human disease has proven to be a difficult task, with frequent poor inter- and intra-laboratory replicability, particularly in behavioral domains such as social and cognitive function. However, establishing robust animal model systems with strong construct validity is of fundamental importance as they are central tools for understanding disease pathophysiology and developing therapeutics. To complete our studies of mouse model systems relevant to autism spectrum disorder (ASD), we present a replication of the main findings from our two published studies of five genetic mouse model systems of ASD. To assess the intra-laboratory robustness of previous results, we chose the two model systems that showed the greatest phenotypic differences, the Shank3/F and Cntnap2, and repeated assessments of general health, activity and social behavior. We additionally explored all five model systems in the same framework, comparing all results obtained in this three-yearlong effort using informatics techniques to assess commonalities and differences. Our results showed high intra-laboratory replicability of results, even for those with effect sizes that were not particularly large, suggesting that discrepancies in the literature may be dependent on subtle but pivotal differences in testing conditions, housing enrichment, or background strains and less so on the variability of the behavioral phenotypes. The overall informatics analysis suggests that in our behavioral assays we can separate the set of tested mouse model system into two main classes that in some aspects lie on opposite ends of the behavioral spectrum, supporting the view that autism is not a unitary concept.
Assuntos
Transtorno do Espectro Autista/genética , Comportamento Animal , Modelos Animais de Doenças , Informática/métodos , Animais , Transtorno do Espectro Autista/fisiopatologia , Peso Corporal , Feminino , Informática/normas , Aprendizagem , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Reprodutibilidade dos Testes , Comportamento SocialRESUMO
Mechanistic target of rapamycin (mTOR) regulates cell proliferation, growth and survival, and is activated in cancer and neurological disorders, including epilepsy. The rapamycin derivative ("rapalog") everolimus, which allosterically inhibits the mTOR pathway, is approved for the treatment of partial epilepsy with spontaneous recurrent seizures (SRS) in individuals with tuberous sclerosis complex (TSC). In contrast to the efficacy in TSC, the efficacy of rapalogs on SRS in other types of epilepsy is equivocal. Furthermore, rapalogs only poorly penetrate into the brain and are associated with peripheral adverse effects, which may compromise their therapeutic efficacy. Here we compare the antiseizure efficacy of two novel, brain-permeable ATP-competitive and selective mTORC1/2 inhibitors, PQR620 and PQR626, and the selective dual pan-PI3K/mTORC1/2 inhibitor PQR530 in two mouse models of chronic epilepsy with SRS, the intrahippocampal kainate (IHK) mouse model of acquired temporal lobe epilepsy and Tsc1GFAP CKO mice, a well-characterized mouse model of epilepsy in TSC. During prolonged treatment of IHK mice with rapamycin, everolimus, PQR620, PQR626, or PQR530; only PQR620 exerted a transient antiseizure effect on SRS, at well tolerated doses whereas the other compounds were ineffective. In contrast, all of the examined compounds markedly suppressed SRS in Tsc1GFAP CKO mice during chronic treatment at well tolerated doses. Thus, against our expectation, no clear differences in antiseizure efficacy were found across the three classes of mTOR inhibitors examined in mouse models of genetic and acquired epilepsies. The main advantage of the novel 1,3,5-triazine derivatives is their excellent tolerability compared to rapalogs, which would favor their development as new therapies for TORopathies such as TSC.
Assuntos
Epilepsias Parciais/tratamento farmacológico , Everolimo/uso terapêutico , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Sirolimo/uso terapêutico , Esclerose Tuberosa/tratamento farmacológico , Animais , Modelos Animais de Doenças , Epilepsias Parciais/fisiopatologia , Everolimo/farmacologia , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Masculino , Camundongos , Camundongos Knockout , Resultado do Tratamento , Esclerose Tuberosa/fisiopatologiaRESUMO
Huntington's disease (HD) is one of the few neurodegenerative diseases with a known genetic cause, knowledge that has enabled the creation of animal models using genetic manipulations that aim to recapitulate HD pathology. The study of behavioral and neuropathological phenotypes of these HD models, however, has been plagued by inconsistent results across laboratories stemming from the lack of standardized husbandry and testing conditions, in addition to the intrinsic differences between the models. We have compared different HD models using standardized conditions to identify the most robust phenotypic differences, best suited for preclinical therapeutic efficacy studies. With a battery of tests of sensory-motor function, such as the open field and prepulse inhibition tests, we replicate previous results showing a strong and progressive behavioral deficit in the R6/2 line with an average of 129 CAG repeats in a mixed CBA/J and C57BL/6J background. We present the first behavioral characterization of a new model, an R6/2 line with an average of 248 CAG repeats in a pure C57BL/6J background, which also showed a progressive and robust phenotype. The BACHD in a FVB/N background showed robust and progressive behavioral phenotype, while the YAC128 full-length model on either an FVB/N or a C57BL/6J background generally showed milder deficits. Finally, the Hdh(Q111) knock-in mouse on a CD1 background showed very mild deficits. This first extensive standardized cross-characterization of several HD animal models under standardized conditions highlights several behavioral outcomes, such as hypoactivity, amenable to standardized preclinical therapeutic drug screening.
Assuntos
Comportamento Animal , Modelos Animais de Doenças , Doença de Huntington , Atividade Motora , Envelhecimento , Animais , Feminino , Técnicas de Introdução de Genes , Genótipo , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora/genética , Proteínas do Tecido Nervoso/genética , Testes Neuropsicológicos , Proteínas Nucleares/genética , Fenótipo , Caracteres SexuaisRESUMO
The dopamine D4 receptor (D4R) is predominantly expressed in the prefrontal cortex, a brain area that integrates motor, rewarding, and cognitive information. Because participation of D4Rs in executive learning is largely unknown, we challenged D4R knockout mice (Drd4(-/-)) and their wild-type (WT) littermates, neonatally treated with 6-hydroxydopamine (6-OHDA; icv) or vehicle in two operant learning paradigms. A continuous reinforcement task, in which one food-pellet was delivered after every lever press, showed that 6-OHDA-treated mice (hypodopaminergic) WT mice pressed the reinforcing lever at much lower rates than normodopaminergic WT mice. In contrast, Drd4(-/-) mice displayed increased lever pressing rates, regardless of their dopamine content. In another study, mice were trained to solve an operant two-choice task in which a first showing lever was coupled to the delivery of one food pellet only after a second lever emerged. Interval between presentation of both levers was initially 12 s and progressively shortened to 6, 2, and finally 0.5 s. Normodopaminergic WT mice obtained a pellet reward in more than 75% of the trials at 12, 6, and 2 s, whereas hypodopaminergic WT mice were severely impaired to select the reward-paired lever. Absence of D4Rs was not detrimental in this task. Moreover, hypodopaminergic Drd4(-/-) mice were as efficient as their normodopaminergic Drd4(-/-) siblings in selecting the reward-paired lever. In summary, hypodopaminergic mice exhibit severe impairments to retrieve rewards in two operant positive reinforcement tasks, but these deleterious effects are totally prevented in the absence of functional D4Rs.
Assuntos
Dopamina/deficiência , Córtex Pré-Frontal/metabolismo , Receptores de Dopamina D4/deficiência , Recompensa , Adrenérgicos/toxicidade , Animais , Condicionamento Operante , Masculino , Camundongos , Camundongos Knockout , Oxidopamina/toxicidadeRESUMO
RATIONALE: Timing deficits are characteristic of developmental and neurodegenerative disorders that are accompanied by cognitive impairment. A prominent theory of this interval timing posits an internal clock whose pace is modulated by the neurotransmitter dopamine. OBJECTIVES: We tested two hypotheses about the pharmacology of interval timing in mice: (1) that general cognitive enhancers should increase, and cognitive disruptors should decrease temporal precision and (2) that acutely elevated dopamine should speed this internal clock and produce overestimation of elapsing time. MATERIALS AND METHODS: C3H mice were tested in the peak procedure, a timing task, following acute administration of two putative cognitive enhancers (atomoxetine and physostigmine), two cognitive disruptors (scopolamine and chlordiazepoxide [CDP]), or two dopamine agonists (D: -amphetamine and methamphetamine). RESULTS: The first hypothesis received strong support: temporal precision worsened with both cognitive disruptors, but improved with both cognitive enhancers. The two dopamine agonists also produced underestimation of elapsing time-congruent with the slowing of an internal clock and inconsistent with a dopamine-driven clock. CONCLUSION: Our results suggest that interval timing has potential as an assay for generalized cognitive performance and that the dopamine-clock hypothesis needs further refinement.
Assuntos
Camundongos Endogâmicos C3H/fisiologia , Camundongos Endogâmicos C3H/psicologia , Tempo de Reação/efeitos dos fármacos , Animais , Cloridrato de Atomoxetina , Comportamento Animal , Clordiazepóxido/farmacologia , Cognição/efeitos dos fármacos , Cognição/fisiologia , Transtornos Cognitivos/fisiopatologia , Transtornos Cognitivos/psicologia , Condicionamento Operante/efeitos dos fármacos , Dextroanfetamina/farmacologia , Agonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Masculino , Metanfetamina/farmacologia , Camundongos , Nootrópicos/farmacologia , Fisostigmina/farmacologia , Propilaminas/farmacologia , Psicofisiologia/métodos , Tempo de Reação/fisiologia , Escopolamina/farmacologia , Cloreto de Sódio/administração & dosagem , Estatística como Assunto/métodos , Fatores de Tempo , Percepção do Tempo/fisiologiaRESUMO
Dopamine has been critically implicated in learning and motivation, although its precise role remains to be determined. In order to investigate the involvement of dopamine in learning and motivation for a food reward, we used dopamine transporter knockdown mice (DAT KD) that have chronically elevated levels of extracellular dopamine. The present study demonstrates that chronically elevated dopamine enhances tendency to work for a food reward without apparent effects on Pavlovian and operant learning for this reward. The increase in dopamine is associated with elevated levels of dynorphin and Fos B expression in the dorsal caudate-putamen and the core but not the shell subregion of the nucleus accumbens. These data suggest that motivation to work, but not learning, for a food reward appears to be under the critical influence of tonic dopaminergic activity in discrete brain areas relevant for a reward-directed behavior.
Assuntos
Dopamina/metabolismo , Alimentos , Aprendizagem/fisiologia , Motivação , Recompensa , Animais , Aprendizagem por Associação/fisiologia , Comportamento Animal , Contagem de Células/métodos , Comportamento de Escolha/fisiologia , Condicionamento Clássico/fisiologia , Condicionamento Operante/fisiologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/deficiência , Dinorfinas/genética , Dinorfinas/metabolismo , Ingestão de Alimentos/genética , Privação de Alimentos/fisiologia , Regulação da Expressão Gênica/genética , Hibridização In Situ/métodos , Camundongos , Camundongos Knockout , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de TempoRESUMO
The causal contribution of glial pathology to Huntington disease (HD) has not been heavily explored. To define the contribution of glia to HD, we established human HD glial chimeras by neonatally engrafting immunodeficient mice with mutant huntingtin (mHTT)-expressing human glial progenitor cells (hGPCs), derived from either human embryonic stem cells or mHTT-transduced fetal hGPCs. Here we show that mHTT glia can impart disease phenotype to normal mice, since mice engrafted intrastriatally with mHTT hGPCs exhibit worse motor performance than controls, and striatal neurons in mHTT glial chimeras are hyperexcitable. Conversely, normal glia can ameliorate disease phenotype in transgenic HD mice, as striatal transplantation of normal glia rescues aspects of electrophysiological and behavioural phenotype, restores interstitial potassium homeostasis, slows disease progression and extends survival in R6/2 HD mice. These observations suggest a causal role for glia in HD, and further suggest a cell-based strategy for disease amelioration in this disorder.
Assuntos
Doença de Huntington/patologia , Neuroglia/patologia , Animais , Comportamento Animal , Quimera/metabolismo , Cognição , Cruzamentos Genéticos , Progressão da Doença , Feminino , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Proteína Huntingtina/metabolismo , Receptores de Hialuronatos/metabolismo , Masculino , Camundongos , Atividade Motora , Neostriado/patologia , Neuroglia/metabolismo , Neurônios/metabolismo , Fenótipo , Transplante de Células-Tronco , Análise de SobrevidaRESUMO
To study the functions of 5-HT4 receptors, a null mutation was engineered in the corresponding gene. 5-HT4 receptor knock-out mice displayed normal feeding and motor behaviors in baseline conditions but abnormal feeding and locomotor behavior in response to stress and novelty. Specifically, stress-induced hypophagia and novelty-induced exploratory activity were attenuated in the knock-out mice. In addition, pentylenetetrazol-induced convulsive responses were enhanced in the knock-out mice, suggesting an increase in neuronal network excitability. These results provide the first example of a genetic deficit that disrupts the ability of stress to reduce feeding and body weight and suggest that 5-HT4 receptors may be involved in stress-induced anorexia and seizure susceptibility.
Assuntos
Receptores 5-HT4 de Serotonina/fisiologia , Convulsões/etiologia , Estresse Fisiológico/complicações , Animais , Comportamento Animal , Peso Corporal , Convulsivantes , Ingestão de Alimentos , Meio Ambiente , Marcação de Genes , Locomoção , Camundongos , Camundongos Knockout , Rede Nervosa/fisiopatologia , Pentilenotetrazol , Receptores 5-HT4 de Serotonina/genética , Convulsões/induzido quimicamente , Convulsões/fisiopatologiaRESUMO
Huntington's Disease (HD) is a progressive neurodegenerative disorder that causes motor, cognitive, and psychiatric symptoms. In these experiments, we tested if operant training at an early age affected adult cognitive deficits in the zQ175 KI Het (zQ175) mouse model of HD. In Experiment 1 we trained zQ175 mice in a fixed-ratio/progressive ratio (FR/PR) task to assay learning and motivational deficits. We found pronounced deficits in response rates and task engagement in naïve adult zQ175 mice (32-33 weeks age), while deficits in zQ175 mice trained from 6-7 weeks age were either absent or less severe. When those mice were re-tested as adults, FR/PR performance deficits were absent or otherwise less severe than deficits observed in naïve adult zQ175 relative to wild type (WT) mice. In Experiment 2, we used a Go/No-go operant task to assess the effects of early cognitive testing on response inhibition deficits in zQ175 mice. We found that zQ175 mice that began testing at 7-8 weeks did not exhibit deficits in Go/No-go testing, but when re-tested at 28-29 weeks age exhibited an initial impairment that diminished with training. These transient deficits were nonetheless mild relative to deficits observed among adult zQ175 mice without prior testing experience. In Experiment 3 we trained mice in a two-choice visual discrimination test to evaluate cognitive flexibility. As in prior experiments, we found performance deficits were mild or absent in mice that started training at 6-9 weeks of age, while deficits in naive mice exposed to training at 28-29 weeks were severe. Re-testing mice at 28-29 weeks age, were previously trained starting at 6-9 weeks, revealed that deficits in learning and cognitive flexibility were absent or reduced relative to effects observed in naive adults. In Experiment 4, we tested working memory deficits with a delayed non-match to position (DNMTP) test. Mice with prior experience exhibited mild working memory deficits, with males zQ175 exhibiting no deficits, and females performing significantly worse than WT mice at a single delay interval, whereas naive zQ175 exhibited severe delay-dependent deficits at all intervals exceeding 1 s. In sum, these experiments indicate that CAG-dependent impairments in motivation, motor control, cognitive flexibility, and working memory are sensitive to the environmental enrichment and experience. These findings are of clinical relevance, as HD carrier status can potentially be detected at an early age.