RESUMO
Commonly used methods to visualize the biological structure of brain tissues at subcellular resolution are confocal microscopy and two-photon microscopy. Both require slicing the sample into sections of a few tens of micrometers. The recent developments in X-ray microtomography enable three-dimensional imaging at sub-micrometer and isotropic resolution with larger biological samples. In this work, we developed and compared original microtomography methods and staining protocols to improve the contrast for in vitro mouse neuron imaging. Using Golgi's method to stain neurons randomly, we imaged the whole set of mouse brain structures. For specific and nonrandom neuron labeling, we conjugated 20 nm gold nanoparticles to antibodies used in the immunohistochemistry (IHC) method, using anti-NeuN to label specifically neuronal nuclei. We applied an original subtraction dual-energy method for microtomography in the vicinity of the Au L-III absorption edge and compared image reconstructions to confocal microscopy images acquired on the same samples. The results show the possibility to characterize the 3D entire brain structure of mice. They demonstrated a high contrast and neuron detection improvement by applying the dual-energy method coupled to IHC staining.
Assuntos
Encéfalo/ultraestrutura , Imageamento Tridimensional/métodos , Nanopartículas Metálicas , Neuroimagem/métodos , Neurônios/ultraestrutura , Microtomografia por Raio-X/métodos , Animais , Ouro , CamundongosRESUMO
Background: Diverse forms of long-term potentiation (LTP) have been described, but one of the most investigated is encountered in the glutamatergic synapses of the hippocampal cornu Ammonis (CA1) subfield. However, little is known about synaptic plasticity in wildlife populations. Laboratory animals are extremely inbred populations that have been disconnected from their natural environment and so their essential ecological aspects are entirely absent. Proechimys are small rodents from Brazil's Amazon rainforest and their nervous systems have evolved to carry out specific tasks of their unique ecological environment. It has also been shown that long-term memory duration did not persist for 24-h in Proechimys, in contrast to Wistar rats, when both animal species were assessed by the plus-maze discrimination avoidance task and object recognition test. Methods: In this work, different protocols, such as theta burst, single tetanic burst or multiple trains of high frequency stimulation (HFS), were used to induce LTP in hippocampal brain slices of Proechimys and Wistar rats. Results: A protocol-independent fast decay of early-phase LTP at glutamatergic synapses of the CA1 subfield was encountered in Proechimys. Long-term depression (LTD) and baseline paired-pulse facilitation (PPF) were investigated but no differences were found between animal species. Input/output (I/O) relationships suggested lower excitability in Proechimys in comparison to Wistar rats. Bath application of d-(-)-2-amino-5-phosphonopentanoicacid (D-AP5) and CNQX prevented the induction of LTP in both Proechimys and Wistar. However, in marked contrast to Wistar rats, LTP induction was not facilitated by the GABAA antagonist in the Amazon rodents, even higher concentrations failed to facilitate LTP in Proechimys. Next, the effects of GABAA inhibition on spontaneous activity as well as evoked field potentials (FPs) were evaluated in CA1 pyramidal cells. Likewise, much lower activity was detected in Proechimys brain slices in comparison to those of the Wistar rats. Conclusions: These findings suggest a possible high inhibitory tone in the CA1 network mediated by GABAA receptors in the Amazon rodents. Currently, neuroscience research still seeks to reveal molecular pathways that control learning and memory processes, Proechimys may prove useful in identifying such mechanisms in complement to traditional animal models.
Assuntos
Região CA1 Hipocampal/fisiologia , Potenciação de Longa Duração/fisiologia , Rede Nervosa/fisiologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Diazepam/farmacologia , Antagonistas GABAérgicos/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Rede Nervosa/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Picrotoxina/farmacologia , Ratos , Ratos Wistar , Especificidade da EspécieRESUMO
The role of GABAergic neurotransmission on epileptogenesis has been the subject of speculation according to different approaches. However, it is a very complex task to specifically consider the action of the GABAa neurotransmitter, which, in its dependence on the intracellular level of Cl-, can change its effect from inhibitory to excitatory. We have developed a computational model that represents the dentate gyrus and is composed of three different populations of neurons (granule cells, interneurons and mossy cells) that are mutually interconnected. The interconnections of the neurons were based on compensation theory with Hebbian and anti-Hebbian rules. The model also incorporates non-synaptic mechanisms to control the ionic homeostasis and was able to reproduce ictal discharges. The goal of the work was to investigate the hypothesis that the observed aberrant sprouting is promoted by GABAa excitatory action. Conjointly with the abnormal sprouting of the mossy fibres, the simulations show a reduction of the mossy cells connections in the network and an increased inhibition of the interneurons as a response of the neuronal network to control the activity. This finding contributes to increasing the changes in the connectivity of the neuronal circuitry and to increasing the epileptiform activity occurrences.
Assuntos
Giro Denteado , Modelos Neurológicos , Neurogênese , Estado Epiléptico , Transmissão Sináptica , Ácido gama-Aminobutírico/metabolismo , Giro Denteado/metabolismo , Giro Denteado/patologia , Giro Denteado/fisiopatologia , Humanos , Interneurônios/metabolismo , Interneurônios/patologia , Rede Nervosa/metabolismo , Rede Nervosa/patologia , Rede Nervosa/fisiopatologia , Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Estado Epiléptico/fisiopatologia , Sinapses/metabolismo , Sinapses/patologiaRESUMO
Brain injuries are often associated with the later development of epilepsy. Evidence suggests that morphological and functional changes occur in the remaining neural tissue during a silent (or latent) period in which no seizures are expressed. It is believed that this silent (reorganization) period may provide a therapeutic window for modifying the natural history of disease progression. Here we provide evidence that biperiden, a muscarinic anticholinergic agent, is able to alter disease progression in an animal model of epilepsy. We observed that biperiden was capable of slowing the manifestation of the first spontaneous epileptic seizure and effectively reduced the severity and number of recurrent, spontaneous epileptic seizures during the animals' lifespan. Biomolecular (microdialysis) and electrophysiological (extracellular field recordings) studies determined that biperiden was capable of elevating the threshold of hippocampal excitability, thereby making the hippocampal glutamatergic pathways less responsive to stimuli when high concentrations of potassium were used in vivo or in vitro. Notably, there was no hindrance of long-term memory or learning (a potential problem given the amnestic nature of biperiden). We conclude that biperiden has antiepileptogenic potential and may represent an opportunity for the prevention of post-traumatic epilepsy.
Assuntos
Biperideno/uso terapêutico , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Agonistas Muscarínicos/toxicidade , Antagonistas Muscarínicos/uso terapêutico , Pilocarpina/toxicidade , Potenciais de Ação/efeitos dos fármacos , Animais , Sistema Nervoso Autônomo/efeitos dos fármacos , Sistema Nervoso Autônomo/fisiopatologia , Doença Crônica , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Epilepsia/patologia , Comportamento Exploratório/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Hipocampo/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/metabolismoRESUMO
Sudden unexpected death in epilepsy (SUDEP) is a major cause of premature death related to epilepsy. The causes of SUDEP remain unknown, but cardiac arrhythmias and asphyxia have been suggested as a major mechanism of this event. Inflammation has been implicated in the pathogenesis of both epilepsy and ventricular arrhythmia, with interleukin-6 (IL-6) being recognized as a crucial orchestrator of inflammatory states. Our group previously reported that levels of IL-6 were increased in the hearts of epileptic rats. In this scenario, anti-inflammatory actions are among the beneficial effects of fish oil dietary supplementation. This investigation revealed that elevated levels of IL-6 in the heart were markedly reduced in epileptic rats that were treated in the long-term with fish oil, suggesting protective anti-inflammatory actions against dangerously high levels of IL-6. Based on these findings, our results suggest beneficial effects of long-term intake of fish oil in reducing the inflammation associated with chronic epilepsy.
RESUMO
The excitability of neuronal networks is strongly modulated by changes in pH. The origin of these changes, however, is still under debate. The high complexity of neural systems justifies the use of computational simulation to investigate mechanisms that are possibly involved. Simulated neuronal activity includes non-synaptic epileptiform events (NEA) induced in hippocampal slices perfused with high-K(+) and zero-Ca(2+), therefore in the absence of the synaptic circuitry. A network of functional units composes the NEA model. Each functional unit represents one interface of neuronal/extracellular space/glial segments. Each interface contains transmembrane ionic transports, such as ionic channels, cotransporters, exchangers and pumps. Neuronal interconnections are mediated by gap-junctions, electric field effects and extracellular ionic fluctuations modulated by extracellular electrodiffusion. Mechanisms investigated are those that change intracellular and extracellular ionic concentrations and are able to affect [H(+)]. Our simulations suggest that the intense fluctuations in intra and extracellular concentrations of Na(+), K(+) and Cl(-) that accompany NEA are able to affect the combined action of the Na(+)/H(+) exchanger (NHE), [HCO(-)(3)]/Cl(-) exchanger (HCE), H(+) pump and the catalytic activity of intra and extracellular carbonic anhydrase. Cellular volume changes and extracellular electrodiffusion are responsible for modulating pH.