AK é apoiado pela Fundação Oppenheim e RH é apoiado pela Fundação Velux. Agradecemos I. Krumbein para Buder técnica e K. de apoio histológico e J. Goldschmidt (Instituto Leibniz de Neurobiologia, Magdeburg, Alemanha) para aconselhamento técnico sobre coloração TIMM.

Todas as intervenções animais são realizados em conformidade com a Convenção Europeia para a Animal Care e Uso de Animais de Laboratório ea Demonstração ARVO para o uso de animais em Ophthalmic and Vision Research. Todos os experimentos foram aprovados pelo comitê de ética local. O procedimento de lesão ON em ratos é descrito em outra parte 9. 1. Intravítrea Manganês Injeção <ol><li> Realize o Mn 2 + injeção 24 horas antes do MR varredura com a ajuda de um assistente. Anestesiar os animais, por injecção intraperitoneal de uma solução de hidrato de cloral a 5% (420-450 mg / kg de peso corporal em PBS estéril). Para anestesia tópica adicional, aplicar uma gota de conjuncain líquido (0,4% de cloridrato de oxibuprocaína) à córnea antes da punção olho. Para injectar 15 nmol de Mn 2 + por olho preparar uma mM de MnCl2 solução 7,5, por exemplo, por diluição de 1 L de uma solução 2 M de MnCl estoque em 132 LH 2 &lt;/ Sub&gt; O. Carga 5 ul da solução final numa seringa de 5 mL de Hamilton ligada a um 34 L pequeno cubo de agulha amovível (agulha RN). </li><li> Ao iniciar com o olho direito, posicione o mouse-esquerda lado sob um microscópio binocular e gentilmente aberta e fixar o olho direito entre o polegar eo dedo indicador de sua mão esquerda. Pegar a seringa com a mão direita e segure a agulha perto da ponta. Por punção atraumática da lâmpada olho, inserir cuidadosamente a agulha para dentro do corpo vítreo a circunferência ínfero-temporal aproximadamente, 1 mm distal do limbo, poupando assim os vasos da esclera. </li><li> Em seguida, o assistente aplica-se lentamente o volume total de 2 mL, enquanto controla a escala da seringa Hamilton. Durante este procedimento, monitorizar a colocação da agulha óptima sob o microscópio e evitar a perfuração da lente ou o derramamento do líquido. Mantenha a agulha inserida estaticamente para um adicional de 30 segundos, em seguida, retirá-la lentamente para minimizar líquidoperda no local da injeção. </li><li> Durante todo o processo, deve ser tomado cuidado especial para evitar a pressão no olho. Da mesma forma, evitar tentativas hostis ou numerosas para perfurar o bulbo ocular. Desde Mn 2 + captação no CGR e transporte ao longo da ON já está saturado em 15 nmol MnCl2, isso minimiza as variações de sinal por volumes de injeção um pouco imprecisas. Para o aumento do sinal bilateral da projeção visual, repita o procedimento de injeção para o olho esquerdo. </li><li> Aplicar ofloxacina contendo (3 mg / ml), as gotas oculares e pomadas contendo pantenol uma vez após o procedimento para evitar infecções oculares e secagem do olho. Retorne os ratos para suas gaiolas em condições normais de habitação até o início da digitalização MR. </li></ol> 2. Preparação Animal para MRI <ol><li> Anestesiar o rato pela administração de uma mistura de gás isoflurano / oxigênio 2% / 98%. Monte o mouse sobre um suporte de rato em, uma posição quase horizontal sem torção. Insert-o na bobina de MR, que é ajustado, em seguida, no interior do scanner de MR. Monitore a respiração ea frequência cardíaca em sistemas apropriados. Para obter detalhes técnicos, consulte Herrmann et al 20. </li><li> Durante MRI, anestesia oferta por insuflação contínua de um 1,5% / 98,5% mistura de gás inicialmente isoflurano / oxigênio através de um evaporador ligado ao suporte da cabeça do rato por um tubo integrado. Durante a verificação, ajustar a profundidade da anestesia de acordo com os parâmetros vitais gravados (ou seja, apontar para uma taxa de respiração estável de cerca de 40 respirações por minuto). Usando um dispositivo de aquecimento de manter a temperatura da superfície do corpo estável entre 35 e 37 ° C, como medido por um sensor de temperatura posicionado no local abdominal do rato. Para obter detalhes técnicos, consulte Herrmann et al 17. </li><li> Após a verificação, libertar o rato do suporte e abastecer com oxigênio puro para acelerar a recuperação da anestesia. Para além disso, manter a temperatura do corpo estável usandouma fonte de aquecimento da luz vermelha. </li></ol> 3. Protocolo de MRI <ol><li> O protocolo é validado por um scanner de 3 Tesla equipado com um, SNR-eficiente, pequena bobina animais dedicada (polarizada linearmente bobina Litz) com um campo de visão efetivo de 35 mm x 38 mm de diâmetro. Operar a bobina no modo de transmissão-recepção. </li><li> Com o animal na sua posição final, ajustar o tom e fósforo da bobina com o auxílio de um analisador de frequência. Ajustar manualmente a tensão de referência do transmissor e as correntes de calços para otimizar a homogeneidade ea qualidade da imagem. </li><li> Adquirir T 1 ponderados imagens 2D TSE com uma resolução de 0,5 mm x 0,5 mm x 2 mm de vista longitudinal e transversal para o planejamento. Usando os exames de RM de planejamento, adquirir as imagens MEMR em direção medição coronal, girada para ser paralela à cabeça do animal com a codificação de fase ao longo da direção esquerda-direita. Para minimizar o tempo de aquisição, use um campo retangular de vista ajustado aodimensões da cabeça reais. Empregar uma seqüência FLASH 3D mimada (VIBE 3D), utilizando os seguintes parâmetros: matriz de base 256, campo de visão 54 milímetros × 50,65 milímetros × 14,08 milímetros, utilizando 93,8% de campo de visão retangular na direção de codificação de fase, e 128 fatias de 0,11 mm espessura de corte com a resolução definida fatia para 61%. </li><li> Ative a interpolação no plano para criar imagens finais com 512 × 480 × 128, proporcionando uma resolução efetiva de 0,21 mm x 0,21 milímetros × 0,18 milímetros (0.1 mm x 0,1 mm x 0,09 milímetros interpolado), tempo de eco T E = 6,51 ms, tempo de repetição T R = 16 ms, largura de banda = 160 Hz / px, ângulo de inclinação = 22 °. Aplicar duas médias e três repetições para obter um tempo de aquisição total (T A) de cerca de 30 min. </li></ol> 4. RM Análise de Dados <ol><li> Analisar os dados usando o software fastView syngo. Para o aumento do sinal quantitativa, selecione regiões definidas of interesse em gravações de ressonância magnética planares 2D e determinar a intensidade do sinal (SI) da estrutura de reforço da (SI MEMRI), o fundo do tecido (Fundo SI), e o desvio padrão do ruído (SD N). Quando necessário, use um atlas do cérebro do rato para facilitar a orientação neuro-anatômica para LGN e estruturas SC. Calcula-se a razão de contraste-ruído (CNR) usando a fórmula: </li><li> CNR = (SI MEMRI - SI Fundo) / SD N </li><li> Quantificar três imagens consecutivas para o cálculo CNR média para cada amostra. Em animais injectados bilateralmente, analisar cada hemisfério independentemente. </li><li> Para representação de imagens horizontais, coronal e sagital, calcule reconstruções multiplanares do conjunto original de dados de MRI 3D. Estas imagens processadas não são recomendados para análise quantitativa. Para criar reconstruções 3D animadas (projeções de intensidade máxima, MIPS) da retinoProjeção tectal, use um módulo de software de pós-processamento de angiografia. </li></ol> 5. Mn 2 + Autometallography (TIMM Coloração) <ol><li> Para TIMM coloração de Mn 2 + estruturas cerebrais traçadas seguinte MEMRI, injetar uma dose de 15-150 nmol Mn 2 + ivit 24 horas antes da imagem. </li><li> Depois do MR varredura, perfuse os animais com 30 ml gelada 0,325% Na 2 S em PBS (pH 7,4). Dissecar retina e amostras congeladas na seção de congelados. </li><li> Corte seqüenciais, seções equatoriais da espessura 15 mM em um cryotome. </li><li> Realização da coloração TIMM 21, na ausência de agente fixador e crioprotecção de acordo com ANGENSTEIN et al 22. </li></ol> 6. Statistical Analysis Realizar análises estatísticas usando o teste t de Student para comparações individuais, seguido pelo post hoc ANOVA. Os dados são apresentados como média ± erro padrão. 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MEMRI do sistema visual estende técnicas neurobiológicas convencionais para avaliar a funcionalidade em condições ingênuos e patológicos. Além de oferecer uma perspectiva única sobre a integridade de um isolado CNS trato fibra, MEMRI pode ser facilmente complementada com testes comportamentais, por exemplo, optometria e tarefas de água com base visualmente, para investigar as conseqüências imediatas de um determinado paradigma para a percepção visual. Ela também liga eletrofisiológico e investiga...

Com base na sua estrutura neuro-anatômica favorável do sistema visual de roedores oferece possibilidades únicas para avaliar compostos farmacológicos e sua capacidade de mediar neuroproteção 1 ou efeitos pró-regenerativas 2,3. Além disso, ele permite que os estudos sobre as características funcionais e neuro-anatômica de mutantes do rato, como recentemente exemplificado para camundongos sem a proteína andaimes pré-sináptico Fagote 4. Além disso, um amplo espectro de ferramentas complementares proporciona adicional que caracteriza de células da retina gânglio (RGC) e os números de axónios RGC, bem como a actividade das CGR, por exemplo, por electrorretinografia e testes comportamentais, e a determinação de rearranjos corticais por imagiologia óptica de sinais intrínsecos. Os últimos desenvolvimentos técnicos em microscopia a laser permitem a visualização em situ de RGC regeneração por tecidos profundos imagens de fluorescência em amostras inteiras de montagem de nervo óptico (ON) e cérebro. Neste histológicoabordagem iCal, tetra baseado compensação tecido em combinação com a microscopia de fluorescência de luz folha permite a resolução de fibras individuais que re-entrar no ON desaferentada e trato óptico 5. Enquanto tais técnicas pode ser superior na resolução e determinação de padrões de crescimento, eles não permitem análises repetitivas e longitudinais de acontecimentos individuais de crescimento, as quais são particularmente desejados para avaliar o processo de regeneração a longo prazo. RM contrastada tem sido empregada para a visualização minimamente invasiva da projeção retino-tectal em camundongos e ratos 6,7. Isto pode ser conseguido por entrega intra-ocular directa de iões paramagnéticos (por exemplo, Mn 2 +) para as células da retina. Como um análogo de cálcio, Mn 2 + é incorporada RGC somata através de canais de cálcio dependentes da voltagem e ativamente transportado ao longo do citoesqueleto axonal do ON intacta e trato óptico. Enquanto se acumula nos núcleos do cérebroda projecção visual, isto é, o núcleo geniculado lateral (LGN) e colículo superior (SC), a propagação transsináptica no córtex visual primário parece insignificante 8,9, embora possa ocorrer 10,11. Sob MR seqüenciamento, paramagnética Mn 2 + aumenta MR contraste principalmente pelo encurtamento T1 spin-rede tempo de relaxamento 12. Tal Mn 2 + reforçada MRI (MEMRI) tem sido aplicado com sucesso em vários estudos neuro-anatômicas e funcionais de ratos, incluindo a avaliação da regeneração axonal e degeneração após ON lesão 13,14, o mapeamento anatômico preciso da 15 projeção retino-tectal , bem como a determinação das características de transporte axonal após o tratamento farmacológico 16. Refinamentos recentes na dosagem, toxicidade e cinética de protocolos de ressonância magnética neuronais Mn 2 + absorção e transporte, bem como melhorados têm estendido a sua aplicação aos estudos sobre transgênicosratinhos 9 utilizando 3 scanners Tesla vulgarmente utilizados na prática clínica 17. Aqui, apresentamos um protocolo adequado para MEMRI longitudinal in vivo de imagens da projeção retino-tectal rato e exemplificar sua aplicabilidade avaliando Mn 2 + aumento do sinal dependente em condições neurodegeneração ingênuos e vários. Nosso protocolo coloca especial ênfase para a aquisição de dados em um MR 3 T campo magnético moderado que geralmente é mais acessível do que os scanners de animais dedicados. Em camundongos ingênuos, que ilustram como a intensidade do sinal específico do trato pode ser substancialmente e reproduzível tornar aumentou após intravítrea (ivit) Mn 2 + aplicação. Quantitativamente, Mn 2 + propagação ao longo da projeção visual ocorre independentemente do processo de envelhecimento normal (medido entre os ratos 3 e 26 meses de idade) e de aumento é refratário a estimulação visual e adaptação à escuridão. Em contraste, Mn <sup&gt; 2 + enriquecimento em centros tálamo e mesencéfalo é diminuída seguinte aguda ON lesão por esmagamento 18, bem como em NFKB1 camundongos knock-out (KO p50) que sofrem de morte espontânea RGC apoptose e na degeneração 19. Assim, em expansão para análise histológica convencional, análise MEMRI longitudinal de animais individuais permite perfis de cinética únicas de processos neurodegenerativos. Isso deve ser útil para estudos sobre a neuroproteção e regeneração axonal associados com intervenções farmacológicas ou genéticas.

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in vivo imaging of optic nerve fiber integrity by contrast-enhanced mri in mice

O sistema visual de roedores engloba células ganglionares da retina e seus axônios que formam o nervo óptico para entrar centros tálamo e mesencéfalo, e projeções pós-sinápticos para o córtex visual. Com base na sua estrutura anatómica distinta e acessibilidade conveniente, tornou-se a estrutura preferida para estudos sobre a sobrevivência neuronal, regeneração axonal, e plasticidade sináptica. Os recentes avanços na ressonância magnética permitiram a visualização in vivo da parte retino-tectal dessa projeção usando manganês mediada realce de contraste (MEMRI). Aqui, apresentamos um protocolo MEMRI para ilustração da projeção visual em camundongos, pelo qual resoluções de (200 mm) 3 pode ser conseguido usando scanners comuns 3 Tesla. Demonstramos como injecção intravítrea de uma única dose de 15 nmol de MnCl2 conduz a um acessório saturada de projecção intacto dentro de 24 horas. Com exceção da retina, alterações na intensidade do sinal são indedente de estimulação visual coincidiu ou envelhecimento fisiológico. Aplicamos ainda mais essa técnica para monitorar longitudinalmente degeneração axonal em resposta à lesão do nervo óptico aguda, um paradigma pelo qual Mn 2 + transporte completamente prisões no local da lesão. Por outro lado, Mn 2 + transporte ativo é quantitativamente proporcional à viabilidade, número e atividade elétrica das fibras de axônio. Para tal análise, exemplificamos Mn 2 + cinética de transporte ao longo do caminho visual em um modelo de camundongo transgênico (NF-kB p50 KO) exibindo atrofia espontânea de sensorial, incluindo visuais, projeções. Nestes ratos, MEMRI indica reduzida mas não retardada de Mn 2 + transporte, em comparação com ratinhos de tipo selvagem, revelando sinais de deficiências estruturais e / ou funcionais, por mutações de NF-kB. Em resumo, MEMRI convenientemente pontes ensaios in vivo e post mortem histologia para o characterizatina de integridade e atividade de fibras nervosas. É muito útil para estudos longitudinais sobre degeneração e regeneração axonal e investigações de ratos mutantes para fenótipos genuínos ou induzidas.

A capacidade desta técnica de imagem para avaliar com precisão a vitalidade ea funcionalidade da projeção visual depende a aplicação precisa de um atóxico Mn 2 + dosagem para o corpo vítreo e sua absorção pelas CGR. Este pressuposto importante é testado na Figura 1, onde específica Mn 2 + absorção camada é demonstrada por autometallography (coloração TIMM) 21. Secções da retina foram analisados ​​às 24 h após a aplicação de ivit ou 15 ...

Watch this Scientific Journal Video about In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice at JoVE.com

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Este vídeo ilustra um método, usando um scanner T clínica 3, por MR com contraste de imagem da projeção visual ingênuo do mouse, para estudos in vivo repetitivos e longitudinais de degeneração do nervo óptico associados com lesão por esmagamento do nervo óptico aguda e degeneração do nervo óptico crônica em camundongos knock-out (KO p50).

Imagem in vivo da fibra óptica Nerve Integrity por ressonância magnética com contraste em Ratos

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

in-vivo-imaging-optic-nerve-fiber-integrity-contrast-enhanced-mri

Research

JoVE Journal

Neuroscience

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

13.2K visualizações.  Jena University Hospital.  Este vídeo ilustra um método, usando um scanner T clínica 3, por MR com contraste de imagem da projeção visual ingênuo do mouse, para estudos in vivo repetitivos e longitudinais de degeneração do nervo óptico associados com lesão por esmagamento do nervo óptico aguda e degeneração do nervo óptico crônica em camundongos knock-out (KO p50). 

Vídeo: In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Métodos para manipulações experimentais após a transecção do nervo óptico no sistema nervoso central de mamíferos

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Neurociência

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimagem Funcional Usando rompimento da barreira hemato-encefálica ultra-sônico e Manganês-RM

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Alta resolução<em&gt; In Vivo</em&gt; Manual Protocolo Segmentação para hipocampo humano subcampos Utilizando Ressonância Magnética 3T

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultâneo<em&gt; Ex vivo</em&gt; Teste Funcional de Dois retinas por<em&gt; In vivo</em&gt; Sistema Eletrorretinograma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Visual Gravação Potencial Evocado em um modelo de rato de Experimental Nervo Óptico desmielinização

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

De alta resolução de ressonância magnética estrutural do Subcórtex Humano<I&gt; In Vivo</I&gt; E Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Em Gene Vivo Transferência de células de Schwann no nervo ciático de roedores por Eletroporação

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Na Vivo Imagem de dois fotões de neurônios Cortical em camundongos Neonatais

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.