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Cortical activity patterns are strongly modulated by fast synaptic inhibition mediated through ionotropic, chloride-conducting receptors. Consequently, chloride homeostasis is ideally placed to regulate activity. We therefore investigated the stability of baseline [Cl-]i in adult mouse neocortex, using in vivo two-photon imaging. We found a two-fold increase in baseline [Cl-]i in layer 2/3 pyramidal neurons, from day to night, with marked effects upon both physiological cortical processing and seizure susceptibility. Importantly, the night-time activity can be converted to the day-time pattern by local inhibition of NKCC1, while inhibition of KCC2 converts day-time [Cl-]i towards night-time levels. Changes in the surface expression and phosphorylation of the cation-chloride cotransporters, NKCC1 and KCC2, matched these pharmacological effects. When we extended the dark period by 4 h, mice remained active, but [Cl-]i was modulated as for animals in normal light cycles. Our data thus demonstrate a daily [Cl-]i modulation with complex effects on cortical excitability.
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Simportadores , Córtex Visual , Animais , Camundongos , Cloretos/metabolismo , Simportadores/metabolismo , Células Piramidais/fisiologia , Homeostase , Córtex Visual/metabolismoRESUMO
BACKGROUND: Phelan-McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by developmental delay, intellectual disability, and autistic-like behaviors and is primarily caused by haploinsufficiency of SHANK3 gene. Currently, there is no specific treatment for PMS, highlighting the need for a better understanding of SHANK3 functions and the underlying pathophysiological mechanisms in the brain. We hypothesize that SHANK3 haploinsufficiency may lead to alterations in the inhibitory system, which could be linked to the excitatory/inhibitory imbalance observed in models of autism spectrum disorder (ASD). Investigation of these neuropathological features may shed light on the pathogenesis of PMS and potential therapeutic interventions. METHODS: We recorded local field potentials and visual evoked responses in the visual cortex of Shank3∆11-/- mice. Then, to understand the impact of Shank3 in inhibitory neurons, we generated Pv-cre+/- Shank3Fl/Wt conditional mice, in which Shank3 was deleted in parvalbumin-positive neurons. We characterized the phenotype of this murine model and we compared this phenotype before and after ganaxolone administration. RESULTS: We found, in the primary visual cortex, an alteration of the gain control of Shank3 KO compared with Wt mice, indicating a deficit of inhibition on pyramidal neurons. This alteration was rescued after the potentiation of GABAA receptor activity by Midazolam. Behavioral analysis showed an impairment in grooming, memory, and motor coordination of Pv-cre+/- Shank3Fl/Wt compared with Pv-cre+/- Shank3Wt/Wt mice. These deficits were rescued with ganaxolone, a positive modulator of GABAA receptors. Furthermore, we demonstrated that treatment with ganaxolone also ameliorated evocative memory deficits and repetitive behavior of Shank3 KO mice. LIMITATIONS: Despite the significant findings of our study, some limitations remain. Firstly, the neurobiological mechanisms underlying the link between Shank3 deletion in PV neurons and behavioral alterations need further investigation. Additionally, the impact of Shank3 on other classes of inhibitory neurons requires further exploration. Finally, the pharmacological activity of ganaxolone needs further characterization to improve our understanding of its potential therapeutic effects. CONCLUSIONS: Our study provides evidence that Shank3 deletion leads to an alteration in inhibitory feedback on cortical pyramidal neurons, resulting in cortical hyperexcitability and ASD-like behavioral problems. Specifically, cell type-specific deletion of Shank3 in PV neurons was associated with these behavioral deficits. Our findings suggest that ganaxolone may be a potential pharmacological approach for treating PMS, as it was able to rescue the behavioral deficits in Shank3 KO mice. Overall, our study highlights the importance of investigating the role of inhibitory neurons and potential therapeutic interventions in neurodevelopmental disorders such as PMS.
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Transtorno do Espectro Autista , Comportamento Problema , Camundongos , Animais , Transtorno do Espectro Autista/genética , Proteínas do Tecido Nervoso/genética , Neurônios , Proteínas dos MicrofilamentosRESUMO
PCDH19 epilepsy (DEE9) is an X-linked syndrome associated with cognitive and behavioral disturbances. Since heterozygous females are affected, while mutant males are spared, it is likely that DEE9 pathogenesis is related to disturbed cell-to-cell communication associated with mosaicism. However, the effects of mosaic PCDH19 expression on cortical networks are unknown. We mimicked the pathology of DEE9 by introducing a patch of mosaic protein expression in one hemisphere of the cortex of conditional PCDH19 knockout mice one day after birth. In the contralateral area, PCDH19 expression was unaffected, thus providing an internal control. In this model, we characterized the physiology of the disrupted network using local field recordings and two photon Ca2+ imaging in urethane anesthetized mice. We found transient episodes of hyperexcitability in the form of brief hypersynchronous spikes or bursts of field potential oscillations in the 9-25 Hz range. Furthermore, we observed a strong disruption of slow wave activity, a crucial component of NREM sleep. This phenotype was present also when PCDH19 loss occurred in adult mice, demonstrating that PCDH19 exerts a function on cortical circuitry outside of early development. Our results indicate that a focal mosaic mutation of PCDH19 disrupts cortical networks and broaden our understanding of DEE9.
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Excitabilidade Cortical , Epilepsia , Animais , Caderinas/genética , Epilepsia/genética , Feminino , Masculino , Camundongos , Mosaicismo , ProtocaderinasRESUMO
The soil nematode community plays an important role in ecosystem services. The objective of this study was to assess the effect of Super-high density (SHD) olive orchards on the nematode community in five sites with different soils, climates, and cultivars. At each site, the SHD management system was compared to the adjacent olive orchard traditional (TRAD) system, in which the same soil management and phytosanitary measures were applied. Soil management was assessed by total organic carbon content (TOC), while the soil nematode community was evaluated using the nematode taxa abundances and soil nematode indicators. TOC was significantly decreased in the SHD olive orchard system compared to TRAD in the sites characterized by conventional tillage and mineral fertilization. The two-way ANOSIM analysis on nematode abundance showed no difference between the two olive management methods, instead showing only a significant difference per site mainly due to variabilities in plant-parasitic nematode assemblage. However, a negative impact of SHD management was evident in environments stressed by summer droughts and conventional tillage: the ratio of obligate plant-parasites to bacterivores and fungivores (Pp/(B+F)) was significantly higher in SHD than in the TRAD olive orchard system, and the prey-to-predator θ mass ratio showed the lowest values in the sites under organic fertilization or green manure. The canonical correspondence analysis showed that the free-living nematodes were only slightly affected by SHD olive orchards; instead, the presence of plant-parasitic nematodes families such as Telotylenchidae, Paratylenchidae, Meloidogynidae, and Criconematidae was favored, in comparison to Longidoridae, Heteroderidae, and Pratylenchidae.
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Mutations in the EPM2A gene encoding laforin cause Lafora disease (LD), a progressive myoclonic epilepsy characterized by drug-resistant seizures and progressive neurological impairment. To date, rodents are the only available models for studying LD; however, their use for drug screening is limited by regulatory restrictions and high breeding costs. To investigate the role of laforin loss of function in early neurodevelopment, and to screen for possible new compounds for treating the disorder, we developed a zebrafish model of LD. Our results showed the epm2a-/- zebrafish to be a faithful model of LD, exhibiting the main disease features, namely motor impairment and neuronal hyperexcitability with spontaneous seizures. The model also showed increased inflammatory response and apoptotic death, as well as an altered autophagy pathway that occurs early in development and likely contributes to the disease progression. Early administration of trehalose was found to be effective for rescuing motor impairment and neuronal hyperexcitability associated with seizures. Our study adds a new tool for investigating LD and might help to identify new treatment opportunities.
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Doença de Lafora , Animais , Doença de Lafora/tratamento farmacológico , Doença de Lafora/genética , Doença de Lafora/metabolismo , Mutação , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Convulsões , Trealose/farmacologia , Ubiquitina-Proteína Ligases/genética , Peixe-Zebra/metabolismoRESUMO
Epilepsy can be both a primary pathology and a secondary effect of many neurological conditions. Many papers show that neuroinflammation is a product of epilepsy, and that in pathological conditions characterized by neuroinflammation, there is a higher probability to develop epilepsy. However, the bidirectional mechanism of the reciprocal interaction between epilepsy and neuroinflammation remains to be fully understood. Here, we attempt to explore and discuss the relationship between epilepsy and inflammation in some paradigmatic neurological and systemic disorders associated with epilepsy. In particular, we have chosen one representative form of epilepsy for each one of its actual known etiologies. A better understanding of the mechanistic link between neuroinflammation and epilepsy would be important to improve subject-based therapies, both for prophylaxis and for the treatment of epilepsy.
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Suscetibilidade a Doenças , Epilepsia/etiologia , Inflamação/complicações , Animais , Biomarcadores , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/etiologia , Neoplasias Encefálicas/patologia , Terapia Combinada , Gerenciamento Clínico , Epilepsia/diagnóstico , Epilepsia/metabolismo , Epilepsia/terapia , Predisposição Genética para Doença , Humanos , Inflamação/etiologia , Doenças Neurodegenerativas/complicações , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Avaliação de Sintomas , Resultado do TratamentoRESUMO
Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.
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Corantes Fluorescentes/química , Genes Reporter , Integrases/metabolismo , Mosaicismo , Transtornos do Neurodesenvolvimento/genética , Potenciais de Ação , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Eletroencefalografia , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Transtornos do Neurodesenvolvimento/fisiopatologia , PTEN Fosfo-Hidrolase/metabolismo , Tamoxifeno/farmacologiaRESUMO
Soil plays a fundamental role in many ecological processes, throughout a complex network of above- and below-ground interactions. This has aroused increasing interest in the use of correlates for biodiversity assessment and has demonstrated their reliability with respect to proxies based on environmental data alone. Although co-variation of species richness and composition in forests has been discussed in the literature, only a few studies have explored these elements in forest plantations, which are generally thought to be poor in biodiversity, being aimed at timber production. Based on this premise our aims were 1) to test if cross-taxon congruence across different groups of organisms (bacteria, vascular plants, mushrooms, ectomycorrhizae, mycelium, carabids, microarthropods, nematodes) is consistent in artificial stands; 2) to evaluate the strength of relationships due to the existing environmental gradients as expressed by abiotic and biotic factors (soil, spatial-topographic, dendrometric variables). Correlations between groups were studied with Mantel and partial Mantel tests, while variance partition analysis was applied to assess the relative effect of environmental variables on the robustness of observed relationships. Significant cross-taxon congruence was observed across almost all taxonomic groups pairs. However, only bacteria/mycelium and mushrooms/mycelium correlations remained significant after removing the environmental effect, suggesting that a strong abiotic influence drives species composition. Considering variation partitioning, the results highlighted the importance of bacteria as a potential indicator: bacteria were the taxonomic group with the highest compositional variance explained by the predictors used; furthermore, they proved to be involved in the only cases where the variance attributed solely to the pure effect of biotic or abiotic predictors was significant. Remarkably, the co-dependent effect of all predictors always explained the highest portion of total variation in all dependent taxa, testifying the intricate and dynamic interplay of environmental factors and biotic interactions in explaining cross-taxon congruence in forest plantations.
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Biodiversidade , Meio Ambiente , Florestas , Pinus , Animais , Bactérias , Embriófitas , Agricultura Florestal , Fungos , Invertebrados , Itália , Microbiota , Microbiologia do SoloRESUMO
In the neocortex, critical periods (CPs) of plasticity are closed following the accumulation of perineuronal nets (PNNs) around parvalbumin (PV)-positive inhibitory interneurons. However, how PNNs tune cortical function and plasticity is unknown. We found that PNNs modulated the gain of visual responses and γ-oscillations in the adult mouse visual cortex in vivo, consistent with increased interneuron function. Removal of PNNs in adult V1 did not affect GABAergic neurotransmission from PV cells, nor neuronal excitability in layer 4. Importantly, PNN degradation coupled to sensory input potentiated glutamatergic thalamic synapses selectively onto PV cells. In the absence of PNNs, increased thalamic PV-cell recruitment modulated feed-forward inhibition differently on PV cells and pyramidal neurons. These effects depended on visual input, as they were strongly attenuated by monocular deprivation in PNN-depleted adult mice. Thus, PNNs control visual processing and plasticity by selectively setting the strength of thalamic recruitment of PV cells.
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Moléculas de Adesão Celular/metabolismo , Matriz Extracelular/metabolismo , Plasticidade Neuronal , Neurônios/fisiologia , Proteoglicanas/metabolismo , Vias Visuais/fisiologia , Animais , Camundongos , Tálamo/fisiologia , Córtex Visual/fisiologiaRESUMO
Impairments of the dialog between excitation and inhibition (E/I) is commonly associated to neuropsychiatric disorders like autism, bipolar disorders and epilepsy. Moderate levels of hyperexcitability can lead to mild alterations of the EEG and are often associated with cognitive deficits even in the absence of overt seizures. Indeed, various testing paradigms have shown degraded performances in presence of acute or chronic non-ictal epileptiform activity. Evidences from both animal models and the clinics suggest that anomalous activity can cause cognitive deficits by transiently disrupting cortical processing, independently from the underlying etiology of the disease. Here, we will review our understanding of the influence of an abnormal EEG activity on brain computation in the context of the available clinical data and in genetic or pharmacological animal models.
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Biological invasions are a global threat to biodiversity. Since the spread of invasive alien plants may have many impacts, an integrated approach, assessing effects across various ecosystem components, is needed for a correct understanding of the invasion process and its consequences. The nitrogen-fixing tree Robinia pseudoacacia (black locust) is a major invasive species worldwide and is used in forestry production. While its effects on plant communities and soils are well known, there have been few studies on soil fauna and microbes. We investigated the impacts of the tree on several ecosystem components, using a multi-trophic approach to combine evidence of soil chemical properties and soil microbial, nematode, microarthropod and plant communities. We sampled soil and vegetation in managed forests, comparing those dominated by black locust with native deciduous oak stands. We found qualitative and quantitative changes in all components analysed, such as the well-known soil nitrification and acidification in stands invaded by black locust. Bacterial richness was the only component favoured by the invasion. On the contrary, abundance and richness of microarthropods, richness of nematodes, and richness and diversity of plant communities decreased significantly in invaded stands. The invasion process caused a compositional shift in all studied biotic communities and in relationships between the different ecosystem components. We obtained clear insights into the effects of invasion of managed native forests by black locust. Our data confirms that the alien species transforms several ecosystem components, modifying the plant-soil community and affecting biodiversity at different levels. Correct management of this aggressive invader in temperate forests is urgently required.
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Ecossistema , Robinia/crescimento & desenvolvimento , Microbiologia do Solo , Solo/química , Animais , Artrópodes , Biodiversidade , Espécies Introduzidas , Nematoides , PlantasRESUMO
Intracellular chloride ([Cl-]i) and pH (pHi) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-]i and pHi, but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl- and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-]i and pHi at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-]i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-]i in response to hypercapnia and seizure activity.
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Cloretos/metabolismo , Citoplasma/metabolismo , Hipocampo/metabolismo , Imagem Óptica/métodos , Fótons , Células Piramidais/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Animais , Animais Recém-Nascidos , Hipocampo/citologia , Concentração de Íons de Hidrogênio , Camundongos , Células Piramidais/citologiaRESUMO
Serotonin-producing neurons profusely innervate brain regions via long-range projections. However, it remains unclear whether and how endogenous serotonergic transmission specifically influences regional or global functional activity. We combined designed receptors exclusively activated by designed drugs (DREADD)-based chemogenetics and functional magnetic resonance imaging (fMRI), an approach we term "chemo-fMRI," to causally probe the brain-wide substrates modulated by endogenous serotonergic activity. We describe the generation of a conditional knockin mouse line that, crossed with serotonin-specific Cre-recombinase mice, allowed us to remotely stimulate serotonergic neurons during fMRI scans. We show that endogenous stimulation of serotonin-producing neurons does not affect global brain activity but results in region-specific activation of a set of primary target regions encompassing corticohippocampal and ventrostriatal areas. By contrast, pharmacological boosting of serotonin levels produced widespread fMRI deactivation, plausibly reflecting the mixed contribution of central and perivascular constrictive effects. Our results identify the primary functional targets of endogenous serotonergic stimulation and establish causation between activation of serotonergic neurons and regional fMRI signals.
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Mapeamento Encefálico/métodos , Encéfalo/fisiologia , Imageamento por Ressonância Magnética/métodos , Neurônios Serotoninérgicos/fisiologia , Transmissão Sináptica , Animais , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacocinética , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
Environmental enrichment (EE) has a remarkable impact on brain development. Continuous exposure to EE from birth determines a significant acceleration of visual system maturation both at retinal and cortical levels. A pre-weaning enriched experience is sufficient to trigger the accelerated maturation of the visual system, suggesting that factors affected by EE during the first days of life might prime visual circuits towards a faster development. The search for such factors is crucial not only to gain a better understanding of the molecular hierarchy of brain development but also to identify molecular pathways amenable to be targeted to correct atypical brain developmental trajectories. Here, we showed that IGF-1 levels are increased in the visual cortex of EE rats as early as P6 and this is a crucial event for setting in motion the developmental program induced by EE. Early intracerebroventricular (i.c.v.) infusion of IGF-1 in standard rats was sufficient to mimic the action of EE on visual acuity development, whereas blocking IGF-1 signaling by i.c.v. injections of the IGF-1 receptor antagonist JB1 prevented the deployment of EE effects. Early IGF-1 decreased the ratio between the expression of NKCC1 and KCC2 cation/chloride transporters, and the reversal potential for GABAAR-driven Cl- currents (ECl) was shifted toward more negative potentials, indicating that IGF-1 is a crucial factor in accelerating the maturation of GABAergic neurotransmission and promoting the developmental switch of GABA polarity from excitation to inhibition. In addition, early IGF-1 promoted a later occurring increase in its own expression, suggesting a priming effect of early IGF-1 in driving post-weaning cortical maturation.
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Fator de Crescimento Insulin-Like I/fisiologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Córtex Visual/crescimento & desenvolvimento , Córtex Visual/fisiologia , Animais , Meio Ambiente , Neurônios GABAérgicos/fisiologia , Fator de Crescimento Insulin-Like I/administração & dosagem , Fator de Crescimento Insulin-Like I/metabolismo , Parvalbuminas/metabolismo , Ratos , Ratos Long-Evans , Receptores de GABA/fisiologia , Acuidade Visual/fisiologia , Córtex Visual/metabolismo , Cotransportadores de K e Cl-RESUMO
Two-photon excitation spectroscopy is a powerful technique for the characterization of the optical properties of genetically encoded and synthetic fluorescent molecules. Excitation spectroscopy requires tuning the wavelength of the Ti:sapphire laser while carefully monitoring the delivered power. To assist laser tuning and the control of delivered power, we developed an Arduino Due based tool for the automatic acquisition of high quality spectra. This tool is portable, fast, affordable and precise. It allowed studying the impact of scattering and of blood absorption on two-photon excitation light. In this way, we determined the wavelength-dependent deformation of excitation spectra occurring in deep tissues in vivo.
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Montecristo Island is an integral natural reserve of the Tuscan Archipelago National Park (Central Italy), characterized by a peculiar assemblage of flora and fauna, with several endemic taxa, and also with a high number of alien species. During a soil survey, we found an alien Oscheius tipulae Lam & Webster, 1971 isolate, phylogenetically close to others from South America. In this article, we examined the possible pathways of introduction of this nematode. Because of the high number of alien plants in this protected area and the low desiccation survival ability of O. tipulae, we hypothesized that the presence of this alien nematode isolate may be related to the soil of introduced plants, although historical association with plant-associated invertebrates is also possible. Further studies with more populations and marker molecules are necessary to investigate the distribution of O. tipulae and the possible impact on this natural reserve.
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Krabbe disease (KD) is a neurodegenerative disorder caused by the lack of ß- galactosylceramidase enzymatic activity and by widespread accumulation of the cytotoxic galactosyl-sphingosine in neuronal, myelinating and endothelial cells. Despite the wide use of Twitcher mice as experimental model for KD, the ultrastructure of this model is partial and mainly addressing peripheral nerves. More details are requested to elucidate the basis of the motor defects, which are the first to appear during KD onset. Here we use transmission electron microscopy (TEM) to focus on the alterations produced by KD in the lower motor system at postnatal day 15 (P15), a nearly asymptomatic stage, and in the juvenile P30 mouse. We find mild effects on motorneuron soma, severe ones on sciatic nerves and very severe effects on nerve terminals and neuromuscular junctions at P30, with peripheral damage being already detectable at P15. Finally, we find that the gastrocnemius muscle undergoes atrophy and structural changes that are independent of denervation at P15. Our data further characterize the ultrastructural analysis of the KD mouse model, and support recent theories of a dying-back mechanism for neuronal degeneration, which is independent of demyelination.
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Leucodistrofia de Células Globoides/patologia , Animais , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Neurônios Motores/ultraestrutura , Músculo Esquelético/inervação , Músculo Esquelético/ultraestrutura , Atrofia Muscular/patologia , Degeneração Neural/patologia , Junção Neuromuscular/ultraestrutura , Nervo Isquiático/ultraestruturaRESUMO
Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is an invasive pest recently reported in Europe whose spread into new areas has caused severe economic damage to many agricultural crops. There are serious concerns about the currently available chemical insecticides because of their low efficacy in controlling the species and their environmental impact; so, several studies have focused on environmentally safe strategies. The sterile insect technique (SIT), which requires colony maintenance in laboratory and production of large numbers of live animals, can be utilized in pest management programs and could be integrated with other control strategies if the potential risks associated with the rearing and maintenance of the insect line under laboratory conditions are given sufficient attention. In this regard, the ability to cryobiologically preserve such stocks would be of substantial value. Important prerequisites for long-term cryopreservation are determination of the embryonic stages, identification of specific embryonic stages, and knowledge of development time. This paper describes the main visible markers for the different stages of embryonic development and determines the timing of development at 25°C. D. suzukii embryogenesis lasts 23-25 h at 25°C and can be divided into 17 stages defined by specific morphological markers. The point at which 50% of embryos are at Stage 14 and 50% are at Stage 15, the most tolerant stages for cryopreservation treatment, as ascertained for Drosophila melanogaster Meigen in prior studies, is reached in 14-15 h. The efficiency of this procedure might be impaired by the retention of eggs in the oviducts, making it impossible to determine the stage of embryonic development for â¼25% of laid eggs.
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Criopreservação , Drosophila/citologia , Embrião não Mamífero , Controle de Insetos/métodos , Animais , Drosophila/embriologia , Microscopia de Interferência , Temperatura , Fatores de TempoRESUMO
The maturation of excitatory transmission comes about through a developmental period in which dendritic spines are highly motile and their number, form and size are rapidly changing. Surprisingly, although these processes are crucial for the formation of cortical circuitry, little is known about possible alterations of these processes in brain disease. By means of acute in vivo 2-photon imaging we show that the dynamic properties of dendritic spines of layer V cortical neurons are deeply affected in a mouse model of Rett syndrome (RTT) at a time around P25 when the neuronal phenotype of the disease is still mild. Then, we show that 24h after a subcutaneous injection of IGF-1 spine dynamics is restored. Our study demonstrates that spine dynamics in RTT mice is severely impaired early during development and suggest that treatments for RTT should be started very early in order to reestablish a normal period of spine plasticity.
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Espinhas Dendríticas/patologia , Espinhas Dendríticas/fisiologia , Plasticidade Neuronal , Células Piramidais/patologia , Células Piramidais/fisiologia , Síndrome de Rett/patologia , Síndrome de Rett/fisiopatologia , Animais , Camundongos , Camundongos KnockoutRESUMO
It has been demonstrated that the complex sensorimotor and social stimulation achieved by rearing animals in an enriched environment (EE) can reinstate juvenile-like plasticity in the adult cortex. However, it is not known whether EE can affect thalamocortical transmission. Here, we recorded in vivo field potentials from the visual cortex evoked by electrical stimulation of the dorsal lateral geniculate nucleus (dLGN) in anesthetized rats. We found that a period of EE during adulthood shifted the input-output curves and increased paired-pulse depression, suggesting an enhanced synaptic strength at thalamocortical terminals. Accordingly, EE animals showed an increased expression of the vesicular glutamate transporter 2 (vGluT-2) in geniculocortical afferents to layer IV. Rats reared in EE also showed an enhancement of thalamocortical long-term potentiation (LTP) triggered by theta-burst stimulation (TBS) of the dLGN. To monitor the functional consequences of increased LTP in EE rats, we recorded visual evoked potentials (VEPs) before and after application of TBS to the geniculocortical pathway. We found that responses to visual stimulation were enhanced across a range of contrasts in EE animals. This was accompanied by an up-regulation of the intracortical excitatory synaptic marker vGluT-1 and a decrease in the expression of the vesicular GABA transporter (vGAT), indicating a shift in the excitation/inhibition ratio. Thus, in the adult rat, EE enhances synaptic strength and plasticity of the thalamocortical pathway associated with specific changes in glutamatergic and GABAergic neurotransmission. These data provide novel insights into the mechanisms by which EE shapes the adult brain.