We thank Souvik Maiti for help in designing the antagomirs. We also acknowledge Rangeetha J. Naik, Rakesh Dey, and Bijay Pattnaik for their help with experimental methods. This work was funded by the Council of Scientific and Industrial Research (BSC0123). HS, MV and RR acknowledge fellowship from the Council of Scientific and Industrial Research, India. MAS acknowledge fellowship from the University Grants Commission, India.

Nota: Todo o processo, incluindo indivíduos animais foram aprovados pelo Comitê de Ética Institucional Animais (AICE) do Instituto de Genômica e Biologia Integrativa, Nova Deli (IGIB / AEC / 10/2013). Este protocolo é adaptado especificamente para entrega direccionada de antagomir-29 no cérebro e knockdown de miR-29. 1. antagomir Design Estratégia <ol><li> Recuperar a sequência madura miRNA de miRBase 11 <a href="http://www.mirbase.org/">(http://www.mirbase.org/).</a> </li><li> Recuperar as sequências dos membros da família relacionadas miRNA usando o link &quot;Gene Família&quot; no registro miRBase <a href="http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009">(Http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009).</a> </li><li> Inverta a sequência complementar: <a href="http://www.bioinformatics.org/sms/rev_comp.html">http://www.bioinformatics.org/sms/rev_comp.html</a> proporciona uma ferramenta conveniente parainverter sequências do complemento. </li><li> Marcar as posições a serem modificados por LNA usando as seguintes diretrizes: <ol><li> Escolha resíduos timina colocados 3-4 bases com excepção para a modificação LNA, desde pirimidinas LNA são mais estáveis ​​do que 12 purinas. </li><li> Escolha resíduos de citosina separadas por 3-4 resíduos se o passo anterior produz menos do que cinco modificações. </li><li> Dar prioridade às cinco alterações à extremidade 5 &#39;do antagomir, uma vez que isto evita a sequência de semente. </li></ol></li></ol> Preparação Complexo 2. antagomir-neuropeptídeo Nota: Este é o passo mais crítico no protocolo e que necessita de padronização. Para padronizar o protocolo qualquer oligonucleótido marcado por fluorescência (FLO) pode ser usado em lugar do antagomir 9. Os péptidos devem ser de elevada pureza (qualidade para HPLC, 98% de pureza) .Sequences e carga do péptido e antagomir são dadas na Tabela 1. <ol><li> Decida o número de moles de antagomir a ser injectado com base na carga molar em antagomir, a sua toxicidade e o peso do rato. Calcular o número de moles de péptido a ser injectado com base na razão molar de carga antagomir: péptido. NOTA: Padronizar razão de carga molar de antagomir: peptídeo de toxicidade e entrega efetiva no cérebro do rato. Use diferentes razões de carga molar de oligonucleotídeo marcado por fluorescência: peptídeo para a normalização. Descobrimos que antagomir-29 e controle foram tóxicos em 4microgram / grama de peso corporal do rato. Antagomir-29 e controlo foram usadas em 2microgram / grama de peso corporal, mas esta concentração eficaz é susceptível de variar para cada microARN. </li><li> Dilui-se o péptido e antagomir em tubos de microcentrífuga separados a partir de soluções de reserva estéril com 10% de D-glicose para a concentração final desejada, como calculado no passo 1. </li><li> Manter o tubo de microcentrifugação com a solução de péptido em um vórtice no vórtex a velocidade moderada. </li><li> Adicione 1/3 do umntagomir solução para o tubo de solução de péptido, lentamente, gota a gota, ao mesmo tempo que é muito bem misturado em um misturador de vórtice. </li><li> Continue a misturar de soluções no vortex durante 1 minuto e em seguida, em seguida, deixar a mistura repousar durante 1 min. </li><li> Repetir o passo 4 e 5 mais duas vezes para misturar o restante antagomir solução com a solução de péptido no mesmo tubo de microcentrífuga. A adição lenta é crítico para a formação de complexos mono-dispersa que são eficazes na transfecção. Adição rápida pode conduzir a agregação dos complexos e a sua precipitação. </li><li> Incubar o complexo durante 30 min à temperatura ambiente sem agitação em vórtice. Durante este tempo de incubação, aclimatar os ratos para o laboratório onde as injecções serão realizados. </li></ol> 3. Injecção da veia da cauda do Complexo antagomir-neuropeptídeo <ol><li> Para conter o animal, coloque o mouse em uma restrainer ou decapicone de tamanho adequado. </li><li> Limpar a superfície da cauda usando cotonete PRem água morna (cerca de 40 ° C) embebido-e. Isto vai promover a vasodilatação e aumentar a visibilidade da veia. </li><li> Abordagem a cauda com um pequeno volume (0,5 - 1.0 cc) seringa de insulina com um ângulo de 15-20 °. Tenha cuidado para não introduzir qualquer ar dentro da seringa. Comece na porção distal da cauda. Injectar o complexo. Remover a agulha e aplicar um toalhete anti-séptico directamente no local da injecção (aproximadamente 5-10 s) para parar qualquer sangramento. </li><li> Substituir o rato em uma gaiola ventilado individualmente no grupo de 3-5, com o carolo de milho e papel de seda como o enriquecimento. Não manter os ratos não injectados e injetadas no mesmo gaiola. </li></ol> 4. Os ensaios comportamentais Nota: Mantenha o intervalo de tempo de 3-4 horas entre injeção e comportamentais ensaios para permitir a recuperação após a injecção. Traga o mouse para uma tranqüila. Não perturbe os animais durante este período de aclimatação. <ol><li> Aclimatar o mouse para anovo ambiente durante 30 minutos e, em seguida, iniciar os seguintes ensaios comportamentais. Manter um intervalo de 10 minutos entre cada ensaio comportamental. </li><li> Clasping hindlimb: NOTA: Este teste indica disfunção motora e comprometimento neurológico. <ol><li> Levante o mouse suavemente, segurando perto da base da cauda no fundo claro. </li><li> Verifique a posição dos membros posteriores de 10-15 seg. Tipo selvagem do rato splays membros posteriores longe do abdómen, enquanto um rato atáxica tende a retrair ou ambos os membros posteriores um em direcção ao abdómen mais do que 50% do tempo de suspensão 13. </li><li> Devolver o mouse para a gaiola </li></ol></li><li> Teste Ledge: <ol><li> Levante o mouse suavemente, segurando a cauda e mantê-lo na borda de uma gaiola. </li><li> Observar o ratinho especificamente no canto da gaiola. Rato do tipo selvagem pode passar os cantos facilmente sem medo e perder o equilíbrio. Rato Ataxic perder seu equilíbrio, congelar ou treme enquanto caminhava ao longo da borda gaiola e nos cantos. </li></ol></li><li&gt; ensaio Rato foot print: NOTA: Para este ensaio manter a folha absorvente pronto, no chão de uma pista estreita (~ 70 cm de comprimento, largura ~-5-cm, com paredes de ~ 5 cm de altura). <ol><li> Segure o mouse suavemente por um lado e aplicar tinta nas patas traseiras por uma escova. </li><li> Coloque a folha absorvente sobre o chão de uma pista estreita. </li><li> Permitir mouse para caminhar ou correr sobre uma folha absorvente em uma linha reta em uma pista de decolagem de uma ponta a outra. </li><li> Repetir o processo mais duas vezes com cada rato utilizando folha absorvente fresco. </li><li> Medir a distância entre duas etapas consecutivas no movimento para a frente. Não incluem primeiro e últimos pegadas onde o animal é apenas iniciar e terminar sua corrida, respectivamente. </li></ol></li></ol>

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The authors have nothing to disclose.

Here we demonstrate a widely accessible methodology to study the effects of miRNA modulation. Currently, most attempts at in vivo characterization of miRNA functions involve the creation of knockout mice or a transgenic that expresses a miRNA sponge. Most miRNAs, even the cell type specific ones are expressed in more than one organ. For instance, miRNAs initially thought to be specific to the hematopoietic system are also expressed in the brain, due to the presence of microglia. Thus even a cell type specifi...

MicroRNAs surgiram como novos alvos terapêuticos devido ao seu papel universal na regulação da expressão gênica e evidência direta para o envolvimento na doença. MiRNAs estão sendo ativamente explorados por seu potencial como fármaco atinge 1,2. Além disso, alterações na expressão de miARN estão associados com várias doenças e 3 simulação da variação por perturbação expressão artificial de miARN pode ser usado para estudar as vias celulares envolvidas na manifestação da doença. Entrega específica do tecido de miRNA drogas que alvejam é atualmente um grande desafio para o desenvolvimento de drogas com base miRNA. Antagomirs e imita miARN são agentes promissores para perturbar os níveis de miARN 4-6. No entanto, as características especiais que melhoram a sua especificidade e eficácia tem que ser incorporado no desenho de antagomirs antes que eles possam ser utilizados in vivo para a perturbação da expressão miARN. Os microRNAs são especialmente relevantes<html> como alvos em neurodegenerativa atualmente incurável e doenças neuro-desenvolvimento. A barreira hemato-encefálica coloca uma restrição para a entrega de antagomirs no cérebro. Injecções estereotáxicas são amplamente utilizados em modelos de roedores para entregar moléculas para locais específicos no cérebro 7. Ele requer habilidade, grandes investimentos na instrumentação e no tempo. Injecções estereotáxicas são invasivos, envolver cirurgia, causar pelo menos lesões menores e são restritas a entrega local. A utilização de péptidos de células de penetração com uma preferência para os neurónios de segmentação pode contrariar estas limitações, uma vez que podem ser entregues por via trans-vascular, mas romper a barreira hemato-encefálica. Tal péptido derivado da glicoproteína do vírus da raiva (RVG), foi previamente utilizado para entregar siRNA contra vírus de encefalite japonesa em ratinhos 8. Descobrimos que utilizando o péptido para antagomir entrega, miRNAs pode ser efetivamente derrubado no cérebro do rato 9. ontent &quot;&gt; O segundo grande desafio de miRNA knock-down é oriundo da pequena dimensão dos miRNAs ea presença de isoformas de sequências estreitamente relacionadas. Tomamos o exemplo de mmu-miR-29 da família que consiste em três isoformas estreitamente relacionadas, miR-29a , b e c. antagomirs são também geralmente modificado ao longo da espinha dorsal para aumentar a sua estabilidade e torná-las resistentes ao ataque por nucleases. Locked Nucleic Acid (LNAs) oferecem uma vantagem adicional que eles melhoram a estabilidade térmica e mesmo conduzir a alvejar degradação ao longo e para além impedimento estérico 10. Introduzindo modificações ao longo de toda a coluna vertebral pode ser eficaz, mas dispendioso. Temos anteriormente visto que as modificações para além de um número óptimo pode não aumentar ainda mais a eficácia. O desenho do antagomir envolve, por conseguinte, a modificação óptima do antagomir. Para o complexo antagomir de forma não covalente com o péptido neurotrópico, um hepta- carregada para o ramal de nona-arginina é utilizada. D-Argininaresíduos são utilizados, uma vez que conferem uma maior estabilidade, que não são susceptíveis a clivagem por proteases. Hepta para trechos nona-arginina agir agentes celulares penetrante como eficientes, embora eles não conferem especificidade tipo de célula. Por ligação covalente do péptido ao ligante RVG nona-arginina, um neurotrópico, célula penetrando péptido foi gerado. Os resíduos carregados positivamente do péptido interagir com a estrutura de ácido nucleico carregado negativamente, para formar complexos. Estes complexos podem ser utilizados para transfectar eficazmente ADN ou ARN em células em cultura e in vivo para os tecidos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer or Decapicone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow runway</td>
			<td></td>
			<td></td>
			<td>~70-cm-long, ~5-cm-wide with ~5-cm-high walls.</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently labelled oligonucleotides (siGLO)</td>
			<td>GE Healthcare Dharmacon INC</td>
			<td>D0016300120</td>
			<td></td>
		</tr>
		<tr>
			<td>10% sterile D-glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-29</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-control</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuropeptide RVG</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td>Neuropeptide RVM</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple alternative to stereotactic injection for brain specific knockdown of mirna

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

Usando o processo aqui apresentado, os complexos de oligonucleótido marcado por fluorescência 50microgram (FLO) e ~ 850microgram RVG péptido de 01:15 proporção molar de carga (FLO: péptido) foram preparados e injectados apenas uma vez através da veia da cauda. Complexo de não-neurotrópico Matrix VÍRUS DA RAIVA (RVM) e FLO péptido foi utilizado como um controlo de entrega. No dia seguinte, os ratos cérebro e fígado foram isoladas e suspensões de células isoladas foram preparadas. As células foram observad...

Watch this Scientific Journal Video about A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA at JoVE.com

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

Uma alternativa simples para injeção estereotáxica para Brain Knockdown específico de miRNA

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

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Neuroscience

12.6K visualizações.  CSIR-Institute of Genomics and Integrative Biology, New Delhi, India. MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

Vídeo: A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

O Vermicelli e Testes Handling Capellini: Simple medidas quantitativas da função forepaw dexterous em ratos e camundongos

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Neurociência

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Entrega de genes ao cérebro pós-natal do rato por Non-ventricular Injection sHRNA e Eletroporação

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Cirurgia estereotáxica para lesão excitotóxica de áreas específicas do cérebro do rato adulto

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Combinado optogenética e Freeze-fratura Replica imunomarcação para examinar Arranjo específicos de entrada de glutamato receptores no mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Um método de injeção Alternativa e validado para acessar o espaço sub-retiniano<i&gt; via</i&gt; Uma abordagem transcleral Posterior

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Uma maneira simples para medir alterações na Recompensa comportamento de busca Usando<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Estereotáxica guiada por Atlas Laser Microdissection de captura de regiões do cérebro afetadas por lesão traumática

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Simples geração de uma cultura de alto rendimento de neurônios induzidas de fibroblastos de pele humana adulta

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Combinação de Optogenetics com microRNAs Artificial para caracterizar os efeitos do Gene Knockdown na função pré-sináptica dentro circuitos neuronais intacta

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Uma abordagem alternativa para estudar eventos primários na neurodegeneração usando fatias de cérebro de rato Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...