Name: direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway
Uploaded: 2015-01-20T15:00:00.000+00:00
Duration: 7 min 58 s
Description: Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Financiamento: Este trabalho foi apoiado por uma bolsa da Fundação Bertarelli (DJL), uma subvenção MED-EL (DJL), e um dos Institutos Nacionais de Saúde Grants DC01089 (MCB).

NOTA: Todos os procedimentos experimentais são realizados de acordo com o Cuidado e Uso Comitê Animal do Massachusetts Eye e Ear Infirmary e Harvard Medical School, que seguem as diretrizes nacionais de cuidados de animais, incluindo a Política Pública de Saúde de serviço na Humane Cuidado e Uso de Animais de Laboratório, o Guia ILAR, e da Lei do Bem-Estar Animal. Procedimentos experimentais listados abaixo exposição do DCN esquerda detalhe. Usar instrumentos esterilizados durante a realização da cirurgia de sobrevivência. 1. Primária Craniotomia e Dorsal Coclear Nucleus Exposição <ol><li> Anestesia <ol><li> Anestesiar um rato, com idade 8-12 semanas, com peso de 18-24 g, com xilazina 20 mg / kg de cetamina e 100 mg / kg via intraperitoneal. Confirme anestesia adequada pelo monitoramento da frequência cardíaca, frequência respiratória, bem como reflexo toe pitada retirada. Lugar vet pomada nos olhos para evitar a secura e sob anestesia. Uma vez adequadamente sob anestesia, cabelo shave recobre tele couro cabeludo para fornecer acesso livre para o local da cirurgia. </li></ol></li><li> Posicionamento cirúrgico <ol><li> Posicione o mouse de forma segura em um suporte estereotáxico de pequenos animais, mantido no lugar por um grampo focinho. </li><li> Certifique-se de que o grampo focinho é solto o suficiente para permitir a respiração adequada, mas firme o suficiente para imobilizar completamente a cabeça do rato. Se a cabeça do mouse é solto, o animal é provável cair fora do suporte da cabeça durante a parte craniotomia de procedimento. </li><li> Coloque auditivo de tronco cerebral implante de eletrodos em forma padrão, o que permite o monitoramento da frequência cardíaca. A freqüência respiratória deve ser monitorizada através da visualização. Note-se que a frequência cardíaca normal e taxa respiratória irá variar dependendo da idade do rato. </li><li> Um termômetro é colocado e é assegurada euthermia via colocação em um cobertor térmico homeotérmicos. </li></ol></li><li> Incisão da pele e exposição dos ossos interparietal e occipital do crânio <ol><li> Sob uma Microscope, fazer uma incisão vertical através da pele a partir da linha média, diretamente entre o pavilhão auricular, e estendendo-se até a porção caudal do occipital. Seguindo na linha média da pele incisão, deslocar a pele lateralmente. Visualize os músculos que cobrem a esquerda da face posterior do crânio. </li><li> Remover muscular que recobre a parietal esquerdo, interparietal, e os ossos occipital do crânio pela desarticulação de anexos musculares para seus respectivos ossos com um bisturi ou tesoura iris. Observar um pequeno grau de sangramento ao longo das bordas musculares corte. Minimizar isso por uma leve pressão com um aplicador com ponta de algodão para 10-15 seg. </li></ol></li><li> Sobreposta craniotomia Coclear Nucleus <ol><li> Identificar linhas de sutura relevantes, incluindo suturas sagital e lambda linhas (Figura 1, painel da esquerda). </li><li> Usando rongeurs, fazer uma craniotomia sobre o osso interparietal, à esquerda da linha média, ~ 2 milímetros caudal para a linha de sutura lambda. Esta região sobrepõe à DCN. </li><li> Seguireig craniotomia, observar uma fina camada de dura-máter que se sobrepõe ao cerebelo. Usando uma lâmina de bisturi, remover a dura-máter. (Figura 1, painel médio). NOTA: A remoção da dura-máter pode causar um pequeno grau de hemorragia. Este processo de usar a lâmina de bisturi para remover a dura destina-se a reduzir a quantidade de perda de sangue durante a aspiração cerebelo, que vai reunir na superfície do tronco cerebral e identificação obscura do DCN. Volume total de sangue de rato é de aproximadamente 1,5 ml não deve exceder&gt; de 15%. Se o sangramento for superior a esse valor, o rato deve ser fornecido com a suplementação com fluido. </li><li> Retirar sangue coagulado sobrepondo o cerebelo com o aplicador de ponta de algodão. Opcionalmente, pingar soro fisiológico delicadamente usando um ponto dental para o cerebelo para limpar o sangue para fora do sangue. </li></ol></li><li> Cerebelar Aspiração <ol><li> Usando uma sucção 5 francês, aspirar a porção mais lateral do cerebelo que se sobrepõe à esquerda DCN até o DCN é visual (Figura 1, painel da direita). Retirar cerca de 1/4 a 1/3 do cerebelo esquerdo. O principal marco adjacente à DCN é a ampola do canal semicircular superior. NOTA: A aspiração do cerebelo é o passo fundamental do protocolo. Dirigido aspiração do cerebelo é realizado com mais sucesso se ocorrer em uma única tentativa e não com várias passagens da sucção, pois isso irá causar sangramento. Para auxiliar na aspiração, definir o plano focal do microscópio na profundidade prevista para o CN, o qual será ligeiramente distai para a superfície do cerebelo. Definir o plano focal para o CN vai melhorar a visualização e garantir uma imagem nítida. </li><li> Após aspiração, de nova hemorragia, líquido cefalorraquidiano (LCR) build-up, e deslocamento cerebelo no DCN são esperados. Instilar 0,5 cc de solução salina estéril rapidamente na craniotomia para impedir a coagulação do sangue. Uma combinação de pontuar suave com um ponto dental e sucção pode então serusado para limpar um caminho para a visualização direta da DCN. Não contactar directamente a superfície do DCN. </li></ol></li></ol> 2. Microinjection Pressão para mediada-Virus Gene Transferência e recuperação cirúrgica <ol><li> Após a DCN é livre de sangue sobrejacente e CSF e é claramente visível, fazer microinjeções de pressão no DCN usando a 10 ul Hamilton seringa durante um período de 2 min. </li><li> Introduzir a agulha com um micromanipulador até que a ponta já não é visível sob a superfície do DCN. </li><li> Para um desempenho óptimo, utilizar uma agulha de calibre 33 ou 34 (com uma seringa estanque aos gases com a agulha de calibre 34) com um chanfro raso, tal como 45 °, para minimizar o trauma sem corte e localizar o volume de injecção no interior da DCN, que é uma fina e estrutura do tronco cerebral raso (&lt;300 mm de espessura). NOTA: Outros instrumentos microinjeção pode ser utilizado com base. Idealmente, a injeção instrumento deve ser flexível para permitir uma ligeira bend, se necessário, para atingir diretamente o DCN. Além disso, como o rato se move ligeiramente ao longo do processo, devido à respiração, o instrumento deve ser suficientemente robusto para resistir a pequenas quantidades de movimento. </li></ol> 3. Recuperação Cirúrgica <ol><li> Imediatamente após a injecção, re-aproximar a pele e permitir que o mouse para se recuperar por procedimento padrão de recuperação. Restante cerebelo e cicatriz irá preencher a cavidade causada pela aspiração do cerebelo. Constantemente monitorar os animais até que a consciência suficiente é recuperada para manter decúbito esternal. Monitor de frequência cardíaca, frequência respiratória, bem como a capacidade para se alimentar. NOTA: Nenhum animal que foi submetido a cirurgia é devolvido para uma gaiola com outros animais até totalmente recuperado. </li><li> Se um rato começa a andar em círculos de pós-operatório (&lt;5% dos casos), geralmente 2-4 horas após o fechamento da incisão na pele, sacrificar imediatamente o mouse, como ele provavelmente teria uma alimentação momento difícil. Tseu efeito colateral é provavelmente devido à aspiração de o cerebelo. O animal deve ser monitorado para a dor no pós-operatório, e narcóticos adequadas devem ter para garantir o conforto dos animais com base em normas institucionais. Antibióticos podem ser necessárias, se houver sinais de infecção. </li></ol> 4. Secundária Craniotomia e Coclear Nucleus Exposição <ol><li> Após 2-4 semanas de cicatrização e gene transferência de incubação mediada por vírus, re-anestesiar uma previamente injetados mouse e repita o passo 1,1-1,5 tempo de incubação Optimal varia com o tipo de gene transferido. </li><li> Remover o tecido cicatricial no local da craniotomia por uma combinação de uma pinça, bisturi, e fórceps. </li><li> Após visualizar a craniotomia, identificar uma combinação de permanecer cerebelo e tecido cicatricial que recobre a DCN. Use a 5 de sucção para aspirar francês cerebelo sobreposta / tecido cicatricial (similar à aspiração cerebelar anterior). </li><li> Após aspiração, esperar mais sangramento, CSF build-up, e os bits restantes do cerebelo. Introduzir rapidamente em solução salina a craniotomia para impedir a coagulação do sangue. Use uma combinação de suave dabbing ponto dental e sucção para limpar o caminho para a visualização direta da DCN. </li><li> Observar a superfície do DCN. Realizar experimentos de fisiologia à base de optogenética como DCN é agora acessível. Introduzir um estímulo de luz, por exemplo com uma fibra óptica (Figura 2), para conduzir uma experiência baseada Optogenetics. </li></ol> 5. Histologia <ol><li> Após a conclusão dos experimentos, sacrificar o mouse com uma overdose de cetamina. Perfundir do rato com soro fisiológico normal seguido por paraformaldeído a 4%. </li><li> Extraia o tronco cerebral a partir do crânio e pós-correção para 2 horas. Cryoprotect do tronco cerebral em 30% de sacarose por 24-48 horas. Secção do tronco cerebral utilizando um criostato padrão usando 60 mm secções. </li></ol>

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Este artigo descreve a técnica de visualização direta do DCN no modelo murino para a manipulação do sistema auditivo central. A abordagem descrita de visualização direta oferece vantagens significativas sobre a alternativa principal, que são abordagens estereotáxica. Primeiramente, a visualização direta da DCN permite confirmação imediata do local do tronco cerebral, enquanto abordagens estereotáxica não pagar a visualização direta. Em experiências que implicam o prolongados períodos de incubação, c...

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

<table><tbody><tr><td>Stereotaxic holder</td><td>Stoelting</td><td>51500</td><td></td></tr><tr><td>Homeothermic blanket</td><td>Harvard</td><td>507214</td><td></td></tr><tr><td>Scalpel blade #11</td><td>Fine Surgical Tools</td><td>10011-00</td><td></td></tr><tr><td>Iris scissor</td><td>Fine Surgical Tools</td><td>14084-08</td><td></td></tr><tr><td>5 French suction</td><td>Symmetry Surgical</td><td>2777914</td><td></td></tr><tr><td>Dental Points</td><td>Henry Schein</td><td>100-8170</td><td></td></tr><tr><td>Bone rongeur</td><td>Fine Surgical Tools</td><td>16020-14</td><td></td></tr><tr><td>10 &amp;micro;l Hamilton syringe</td><td>Hamilton&amp;nbsp;</td><td>7633-01</td><td></td></tr><tr><td>34 gauge, needle</td><td>Hamilton&amp;nbsp;</td><td>207434</td><td></td></tr><tr><td>Rongeurs</td><td>Fine Surgical Tools</td><td>16021-14</td><td></td></tr></tbody></table>

direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

A investigação sobre o uso da transferência de genes mediada por vírus para prender ou reverter a perda auditiva tem sido amplamente relegado para o sistema auditivo periférico. Poucos estudos examinaram transferência de genes para o sistema auditivo central. O núcleo dorsal coclear (DCN) do tronco cerebral, que contém neurônios de segunda ordem da via auditiva, é um local potencial de transferência de genes. Neste protocolo, uma técnica para exposição direta e máxima da DCN murino através de uma abordagem posterior fossa é demonstrada. Esta abordagem permite a qualquer cirurgia aguda ou sobrevivência. Após a visualização direta da DCN, uma série de experimentos são possíveis, incluindo a injeção de opsinas para o núcleo coclear e subsequente estimulação por uma fibra óptica acoplada a uma luz de laser azul. Outros experimentos neurofisiologia, como a estimulação elétrica e traçados injetores neurais também são viáveis. O nível de Visualização e a duração da estimulação realizável que esta abordagem seja aplicável a uma ampla gama de experiências.

Parcial Cerebellar aspiração demonstra ter acesso ao núcleo coclear Após a pele e músculo sobrejacente ao crânio são removidos, marcadores de superfície do crânio, como as linhas de sutura coronal e lamda, demonstrar a localização aproximada da craniotomia. Após craniotomia com fórceps, o cerebelo é visualizada. Aspiração cuidadosa da pequena porção de o cerebelo demonstra visualização do CN, que podem então ser injectados (Figura 1). <p cla...

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

Visualização direta do Murino Dorsal Coclear Nucleus para Optogenetic Estimulação da via auditiva

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

direct-visualization-murine-dorsal-cochlear-nucleus-for-optogenetic

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.2K visualizações.  Harvard Medical School. The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. 

Vídeo: Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy como uma abordagem cirúrgica para entrega de drogas para o ouvido interno de ratos adultos e Neonatal

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genética

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Transdução Viral seletiva de Adultos-nascido neurônios olfativos de crônica In vivo Estímulo Optogenetic

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Neurociência

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primária e Eletroporação Plasmid do Órgão de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biologia

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo Optogenetic estimulação do sistema nervoso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Um Guia para<em&gt; In vivo</em&gt; Gravação única unidade de Optogenetically Identificado Cortical Inibitória Interneurônios

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

A estimulação optogenética do nervo auditivo

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Técnica de Explante Coclear Murino Neonatal como Ferramenta de Triagem In Vitro na Pesquisa Auditiva

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Medicina

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

Preparação de fatia de cunha in vitro para imitar conectividade do circuito neuronal vivo

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Source: Fischl, M. J. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61664">In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates the process of preparing wedge-shaped brain slices in a laboratory setting. Using a vibratome, a wedge-shaped slice from a mouse&#39;s brain is crafted with varying thickness. This slice contains the auditory nerve root positioned on the thicker end, while the hearing-related neurons are located on the thinner end.

Preparando fatias cerebrais em cunha para visualizar neurônios auditivos

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Brain ...