RESUMO
We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.
Assuntos
Nanotubos de Carbono , Encéfalo , Espaço Extracelular , Imagem Individual de Molécula , SinapsesRESUMO
Following implantation, neuroelectrode functionality is susceptible to deterioration via reactive host cell response and glial scar-induced encapsulation. Within the neuroengineering community, there is a consensus that the induction of selective adhesion and regulated cellular interaction at the tissue-electrode interface can significantly enhance device interfacing and functionality in vivo. In particular, topographical modification holds promise for the development of functionalized neural interfaces to mediate initial cell adhesion and the subsequent evolution of gliosis, minimizing the onset of a proinflammatory glial phenotype, to provide long-term stability. Herein, a low-temperature microimprint-lithography technique for the development of micro-topographically functionalized neuroelectrode interfaces in electrodeposited poly(3,4-ethylenedioxythiophene):p-toluene sulfonate (PEDOT:PTS) is described and assessed in vitro. Platinum (Pt) microelectrodes are subjected to electrodeposition of a PEDOT:PTS microcoating, which is subsequently topographically functionalized with an ordered array of micropits, inducing a significant reduction in electrode electrical impedance and an increase in charge storage capacity. Furthermore, topographically functionalized electrodes reduce the adhesion of reactive astrocytes in vitro, evident from morphological changes in cell area, focal adhesion formation, and the synthesis of proinflammatory cytokines and chemokine factors. This study contributes to the understanding of gliosis in complex primary mixed cell cultures, and describes the role of micro-topographically modified neural interfaces in the development of stable microelectrode interfaces.
Assuntos
Benzenossulfonatos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Impressão Molecular/métodos , Neuroglia/metabolismo , Polímeros/química , Animais , Astrócitos/citologia , Quimiocinas/metabolismo , Técnicas Eletroquímicas , Microeletrodos , Ratos Sprague-Dawley , Ratos WistarRESUMO
The extracellular space (ECS) and its constituents play a crucial role in brain development, plasticity, circadian rhythm, and behavior, as well as brain diseases. Yet, since this compartment has an intricate geometry and nanoscale dimensions, its detailed exploration in live tissue has remained an unmet challenge. Here, we used a combination of single-nanoparticle tracking and super-resolution microscopy approaches to map the nanoscale dimensions of the ECS across the rodent hippocampus. We report that these dimensions are heterogeneous between hippocampal areas. Notably, stratum radiatum CA1 and CA3 ECS differ in several characteristics, a difference that gets abolished after digestion of the extracellular matrix. The dynamics of extracellular immunoglobulins vary within these areas, consistent with their distinct ECS characteristics. Altogether, we demonstrate that ECS nanoscale anatomy and diffusion properties are widely heterogeneous across hippocampal areas, impacting the dynamics and distribution of extracellular molecules.
Assuntos
Encefalopatias , Espaço Extracelular , Humanos , Hipocampo , Difusão , Matriz ExtracelularRESUMO
We present the design, fabrication, and characterization of an implantable neural interface based on anisotropic magnetoresistive (AMR) magnetic-field sensors that combine reduced size and high performance at body temperature. The sensors are based on La0.67Sr0.33MnO3 (LSMO) as a ferromagnetic material, whose epitaxial growth has been suitably engineered to get uniaxial anisotropy and large AMR output together with low noise even at low frequencies. The performance of LSMO sensors of different film thickness and at different temperatures close to 37 °C has to be explored to find an optimum sensitivity of â¼400%/T (with typical detectivity values of 2 nT·Hz-1/2 at a frequency of 1 Hz and 0.3 nT·Hz-1/2 at 1 kHz), fitted for the detection of low magnetic signals coming from neural activity. Biocompatibility tests of devices consisting of submillimeter-size LSMO sensors coated by a thin poly(dimethyl siloxane) polymeric layer, both in vitro and in vivo, support their high suitability as implantable detectors of low-frequency biological magnetic signals emerging from heterogeneous electrically active tissues.
Assuntos
Campos Magnéticos , Próteses e Implantes , Anisotropia , PolímerosRESUMO
The increasing engineering of carbon-based nanomaterials as components of neuroregenerative interfaces is motivated by their dimensional compatibility with subcellular compartments of excitable cells, such as axons and synapses. In neuroscience applications, carbon nanotubes (CNTs) have been used to improve electronic device performance by exploiting their physical properties. Besides, when manufactured to interface neuronal networks formation in vitro, CNT carpets have shown their unique ability to potentiate synaptic networks formation and function. Due to the low optical transparency of CNTs films, further developments of these materials in neural prosthesis fabrication or in implementing interfacing devices to be paired with in vivo imaging or in vitro optogenetic approaches are currently limited. In the present work, we exploit a new method to fabricate CNTs by growing them on a fused silica surface, which results in a transparent CNT-based substrate (tCNTs). We show that tCNTs favor dissociated primary neurons network formation and function, an effect comparable to the one observed for their dark counterparts. We further adopt tCNTs to support the growth of intact or lesioned entorhinal-hippocampal complex organotypic cultures (EHCs). Through immunocytochemistry and electrophysiological field potential recordings, we show here that tCNTs platforms are suitable substrates for the growth of EHCs and we unmask their ability to significantly increase the signal synchronization and fiber sprouting between the cortex and the hippocampus with respect to Controls. tCNTs transparency and ability to enhance recovery of lesioned brain cultures, make them optimal candidates to implement implantable devices in regenerative medicine and tissue engineering.
Assuntos
Giro Denteado/citologia , Giro Denteado/fisiologia , Córtex Entorrinal/citologia , Córtex Entorrinal/fisiologia , Nanotubos de Carbono , Animais , Animais Recém-Nascidos , Microscopia Eletrônica de Varredura/métodos , Técnicas de Cultura de Órgãos/métodos , Ratos , Ratos WistarRESUMO
By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry, and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system.
Assuntos
Técnicas de Cultura de Órgãos , Baço/anatomia & histologia , Animais , Anexina A5/análise , Quimiocinas/farmacologia , Quimiotaxia/efeitos dos fármacos , Corantes , Citocinas/biossíntese , Citocinas/genética , Citometria de Fluxo , Corantes Fluorescentes , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microtomia/instrumentação , Microtomia/métodos , Mitógenos/farmacologia , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos Específicos , Manejo de Espécimes/métodos , Baço/química , Baço/citologia , Baço/fisiologia , Coloração e Rotulagem/métodos , Azul TripanoRESUMO
Understanding neural physiopathology requires advances in nanotechnology-based interfaces, engineered to monitor the functional state of mammalian nervous cells. Such interfaces typically contain nanometer-size features for stimulation and recording as in cell-non-invasive extracellular microelectrode arrays. In such devices, it turns crucial to understand specific interactions of neural cells with physicochemical features of electrodes, which could be designed to optimize performance. Herein, versatile flexible nanostructured electrodes covered by arrays of metallic nanowires are fabricated and used to investigate the role of chemical composition and nanotopography on rat brain cells in vitro. By using Au and Ni as exemplary materials, nanostructure and chemical composition are demonstrated to play major roles in the interaction of neural cells with electrodes. Nanostructured devices are interfaced to rat embryonic cortical cells and postnatal hippocampal neurons forming synaptic circuits. It is shown that Au-based electrodes behave similarly to controls. Contrarily, Ni-based nanostructured electrodes increase cell survival, boost neuronal differentiation, and reduce glial cells with respect to flat counterparts. Nonetheless, Au-based electrodes perform superiorly compared to Ni-based ones. Under electrical stimulation, Au-based nanostructured substrates evoke intracellular calcium dynamics compatible with neural networks activation. These studies highlight the opportunity for these electrodes to excite a silent neural network by direct neuronal membranes depolarization.
Assuntos
Estimulação Elétrica/instrumentação , Nanotecnologia/instrumentação , Nanofios/química , Neurônios , Sinapses/fisiologia , Animais , Sinalização do Cálcio , Células Cultivadas , Córtex Cerebral/citologia , Desenho de Equipamento , Feminino , Hipocampo/citologia , Microeletrodos , Neurônios/citologia , Neurônios/fisiologia , Ratos , Ratos WistarRESUMO
Carbon nanotube (CNT)-modified surfaces unequivocally demonstrate their biocompatibility and ability to boost the electrical activity of neuronal cells cultured on them. Reasons for this effect are still under debate. However, the intimate contact at the membrane level between these thready nanostructures and cells, in combination with their unique electrical properties, seems to play an important role. The entire existing literature exploiting the effect of CNTs on modulating cellular behavior deals with cell cultures grown on purified multiwalled carbon nanotubes (MWNTs) deposited on a supporting surface via drop-casting or mechanical entrapment. Here, for the first time, it is demonstrated that CNTs directly grown on a supporting silicon surface by a chemical vapor deposition (CVD)-assisted technique have the same effect. It is shown that primary neuronal cells developed above a carpet of CVD CNTs form a healthy and functional network. The resulting neuronal network shows increased electrical activity when compared to a similar network developed on a control glass surface. The low cost and high versatility of the here presented CVD-based synthesis process, together with the possibility to create on supporting substrate patterns of any arbitrary shape of CNTs, open up new opportunities for brain-machine interfaces or neuroprosthetic devices.