Os autores gostariam de agradecer ao Dr. Candace Hsieh para sugestões e assistência com técnicas de imagem e Doris Dr. Wu para a perícia no pinto anatomia do ouvido interno durante a embriogênese. Este trabalho foi financiado pela NSF IOS-0642346, NIH T32-DC010775, NIH T32-GM008620, NIH R01-DC010796, e GAANN DOE P200A120165.

1. Preparar os instrumentos de dissecação seguinte e Reagentes <ul><li> Fluido cerebrospinal artificial (aCSF; 130 mM de NaCl, 3 mM KCl, 1,2 mM de KH 2 PO 4, 20 mM de NaHCO3, 3 mM de HEPES, 10 mM de glicose, 2 mM de CaCl2, 1,3 mM MgSO4) infundida continuamente com 95% de O 2/5% CO 2, à temperatura ambiente. Para a perfusão, encher a 2/3 de 500 ml de boca larga frasco Nalgene com um buraco perfurado na tampa. Tanque vai ser ligado a um tubo de um borbulhador de vidro haste, que penetra o aCSF através do orifício na tampa do frasco. Regule a pressão para se obter um fluxo constante de bolhas que alcançam de cerca de 1/3 do líquido, mas não são suficientemente forte para provocar salpicos sobre o aro. Submerge 5 ml frascos de vidro (até 6) para dentro do frasco de Nalgene para separar amostras individuais uns dos outros e da turbulência do fluxo de gás. ACSF oxigenado deve ser, pelo menos, 20 min antes de qualquer incubação de tecidos. </li><li> Prato de dissecação pequeno silicone (35 mm x 10milímetro revestido Petri usando Sylgard elastômeros Kit) com dois pinos de dissecação colocados em poliestireno hexagonal pequena (47 fundo mm) pesam barco. </li><li> Dois ponta fina fórceps, um tip-fórceps curvo, numa proveta de 50 ml, e um recipiente para resíduos de tecidos. </li><li> Puxado micropipeta de vidro (1,2 OD / 0,9 ID) quebrado de aproximadamente 20-50 uM de abertura na ponta e preenchido com rodamina dextran amina (RDA, MW = 3000, Invitrogen) em uma solução de 6,25% com 0,4% de Triton X-100 em PBS . Anexar por tubos bem para picospritzer e estabilizar a micromanipulador. </li></ul> 2. Micro dissecção para revelar papila basilar no E8 4,5 <ol><li> Com pinças curvas, corte E8 embrião logo abaixo do tronco, colocando a cabeça diretamente sobre silicone revestido prato dissecção dentro pesar barco. </li><li> A utilização de pinos de dissecação, posicionar a cabeça com uma vista ventral de modo que o ouvido do lado de juros é um pouco visível, o cabeça ligeiramente inclinada dorsalmente e apontando 10-30 graus de distância da center (Figura 2A). Imediatamente proteger a cabeça com pinos. Se bilateral traçado é desejada, a cabeça do centro em 0 graus, ou uma visão completa ventral para que ambos os lados podem ser acessados. Da mesma forma, para uma injeção do lado esquerdo, fixar a cabeça de -10 a -30 graus do centro para o lado esquerdo é acessível. </li><li> Usando o copo de 50 ml, transferir 30 ml de aCSF oxigenado para o prato, totalmente submergindo o tecido. </li><li> Agarre cuidadosamente e descascar para baixo a mandíbula inferior utilizando uma pinça de ponta fina, em seguida, remover a pálpebra inferior do lado de interesse. </li><li> Remover o tecido vascular sobrejacente, incluindo qualquer paladar restante e garganta. Estes tecidos moles deverão descascar revelar a superfície lisa de desenvolver chondrocranium com as artérias vertebrais distintas na sua superfície. Até este ponto, a massa branca característica otoconial deve ser visível. </li><li> Dissecar cuidadosamente a pele ao redor do meato externo, cartilagem da articulação da mandíbula e do ouvido médio, deixando o emner ouvido intacto. Use a pinça para gentilmente cortar estas estruturas cartilaginosas. </li><li> Remover artérias vertebrais e acúmulo de sangue através de um movimento de varredura suave com as pontas finas fórceps. Sangue pode obscurecer vista e de coagulação pode ligar a abertura pipeta de injeção. </li><li> O ducto coclear com epitélio sensorial adjacente (papila basilar) e gânglio acústico encontra-se directamente por baixo do chondrocranium 6 e pode ser visualizada como uma projecção semelhante a dedo alongado que se prolonga a partir do ouvido interno ventralmente, com a ponta distal-mais (vértice) próximo do anterior linha média 5. Pode haver um pequeno agrupamento de sangue (vermelho) do dissecação e / ou calcificações otolíticos (branco) no vértice do ducto coclear (Figura 2B), que pode auxiliar na visualização. No ápice é a mácula lagenar, um adesivo sensitivo pensado para ser da função vestibular 7,8. </li></ol> 3. Rotulagem seletiva de CN VIII Auditivo Fibras <ol><li&gt; Coloque a pipeta de injeção perto da superfície do embrião na região da papila do ducto coclear e basilar. </li><li> Lentamente baixar a micropipeta a primeira punção do crânio. Esta região craniana é mais fino do que áreas adjacentes e requer menos força para penetrar. A micropipeta deve agora ser dentro do ducto coclear. Opcional: uma pequena injecção de rastreio corante pode ser feita aqui para ajudar a visualizar a papila basilar. </li><li> Sem reposicionar o micropipeta, continue baixando até ele apenas violações da fronteira profunda do ducto coclear (Figura 1). Realizar uma injecção e repetir em várias regiões ao longo do comprimento da papila basilar (Figuras 2C, 2D), excluindo a extremidade mais proximal onde a lagena vestibular está localizado. Com o picospritzer definido entre 10-30 psi, várias injeções são feitas. O volume do fluido injectado varia de corante, e depende do grau de rotulagem desejada. Cada injecção deverá resultar num corante fluorescente-laponto Beled de aproximadamente 200-400 um de diâmetro (Figura 2D). </li><li> Uma vez que a injecção está completa, utilize uma pinça para transecção da amostra acima do tronco cerebral e da utilização de uma pipeta de transferência para colocar a porção de caudal para um pequeno frasco dentro do recipiente de aCSF continuamente perfundidos com 95% O2 / 5% de CO 2. </li><li> Para cada embrião, 20-30 min para permitir a transferência de corantes, em função da distância do centro de rastreio desejado. </li><li> Remover o tecido e se preparar para cortes histológicos. No exemplo mostrado aqui, o tecido é imerso em paraformaldeído a 4% (em 1X PBS) durante a noite, de sacarose a 30% (em 1X PBS) durante a noite. </li></ol> 4. Contracoloração e Análise <ol><li> Contracoloração é útil para ajudar a orientar o observador a localização anatômica, bem como identificar as regiões de interesse particular. Um contracorante eficaz nesta preparação é de imunomarcação de neurofilamento (Figuras 1, 3B, 3E), o que fortemente l comabels axônios e permite a demarcação de vias axonais no SNC. </li><li> Análise deveria ser realizada por toda a região marcada, tal como as saliências podem estender várias centenas de microns ou mais ao longo do eixo rostro-caudal. Alguns exemplos de análises básicas são: taxas de erro de segmentação de tempo, e / ou alterar os padrões de divergência de projecção. RDA axónios marcados são visualizados no sistema nervoso periférico e central, com alta resolução (Figuras 3A, 3D). </li></ol>

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Estudos sobre o desenvolvimento precoce do nervo VIII têm sido limitada, em parte, devido à dificuldade em identificar os axônios embrionárias resultantes de gânglios distinto múltipla. Vários estudos exploraram os sinais moleculares orientadores auditivo e vestibular célula sensorial e destinos de células ganglionares durante o desenvolvimento precoce, 5,11,12 mas os processos que regulam a inervação central têm ainda a ser determinado. Relatórios de projeções das células ganglionares acústi...

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Poliestireno Pesar </td><td> Fisher Scientific </td><td> 02-202-101 </td><td></td></tr><tr><td> Prato de Petri, 35 X 10 mm </td><td> Fisher Scientific </td><td> 50820644 </td><td> Use para fazer prato dissecção de silicone </td></tr><tr><td> Sylgard Kit elastômero de silicone </td><td> Instrumentos de Precisão mundo </td><td> SYLG184 </td><td> Brasão Petri para fazer prato dissecção </td></tr><tr><td> Pinos de dissecação </td><td> Vário </td><td></td><td> Embrião mantém no local durante a dissecção </td></tr><tr><td> NaCL </td><td> Vário </td><td></td><td> parte da receita aCSF </td></tr><tr><td> KCl </td><td> Vário </td><td></td><td> parte da receita aCSF </td&gt; </tr><tr><td> KH 2 PO 4 </td><td> Vário </td><td></td><td> parte da receita aCSF </td></tr><tr><td> NaHCO3 </td><td> Vário </td><td></td><td> parte da receita aCSF </td></tr><tr><td> Glicose </td><td> Vário </td><td></td><td> parte da receita aCSF </td></tr><tr><td> CaCl 2 </td><td> Vário </td><td></td><td> parte da receita aCSF </td></tr><tr><td> MgSO4 </td><td> Vário </td><td></td><td> parte da receita aCSF </td></tr><tr><td> Recipiente para aCSF. Sugerem translúcido boca larga frasco de Nalgene, 500 ml (16 onças) com tampa. </td><td> CPLabSafety </td><td> QP-PLC-03717 </td><td> Abertura do orifício na parte superior da tampa para haste de vidro bubling penetrar líquido </td></tr><tr><td> Vazio 5 ml frasco de vidro ou frasco transparente e comparável </td><td> Farmacêutica americana Partners, Inc </td><td> 6332300105 </td><td&gt; Utilização durante a incubação aCSF para manter as amostras separadas umas das outras e do fluxo de borbulhamento </td></tr><tr><td> Tanque de carbogénio (95% O2 / 5% CO 2) ligada a um tubo de borbulhador </td><td> Vário </td><td></td><td> Anexar por tubulação para borbulhador haste de vidro para perfusão aCSF </td></tr><tr><td> Vidro tronco borbulhador </td><td> Vário </td><td></td><td> Para infundir carbogênio em aCSF </td></tr><tr><td> Curvo-ponta fórceps </td><td> Instrumentos de Precisão mundo </td><td> 501008 </td><td> Para remover a cabeça do embrião do ovo </td></tr><tr><td> Dois bem-ponta fórceps </td><td> Instrumentos de Precisão mundo </td><td> 501985 </td><td> Para micro-dissecção </td></tr><tr><td> 50 ml Proveta </td><td> vário </td><td></td><td></td></tr><tr><td> Rodamina Dextran Amine (RDA) </td><td> Invitrogen </td><td> vário </td><td> Fluorescente axônio traçador </td></tr><tr><td> Triton X-100 </td><td> ICN Biomedicals </td><td></td><td></td></tr><tr><td> Solução salina tamponada com fosfato, (1X PBS) </td><td> Vário </td><td></td><td> Reagente de laboratório padrão </td></tr><tr><td> Fina parede capilares de vidro, 1,2 OD, 0,9 ID 4 &quot;(100 mm) de comprimento </td><td> Instrumentos de Precisão mundo </td><td> TW120F-4 </td><td> Carregar com a RDA. Cada capilar faz duas micropipetas de vidro </td></tr><tr><td> Agulha / Pipetar extrator </td><td> David Kopf Instruments </td><td> Modelo 720 </td><td> Configurações usadas: Calor 16,4, 2,2 Solenóide </td></tr><tr><td> Picospritzer </td><td> Parker Instrumentation </td><td> vário </td><td> Anexar por tubos de vidro fino para micropipeta </td></tr><tr><td> Micromanipulador </td><td> Narishige </td><td> vário </td><td></td></tr><tr><td> Microscópio de dissecção com fluorescência </td><td> Vário </td><td></td><td></td&gt; </tr><tr><td> Paraformaldeído a 4% </td><td> Vário </td><td></td><td> Reagente de laboratório padrão </td></tr><tr><td> anticorpo anti-neurofilamento, opcional </td><td> Millipore </td><td> AB1991 </td><td> Siga o protocolo recomendado pelo fabricante histológica </td></tr><tr><td> Criostato e materiais associados para seccionamento </td><td> Leica </td><td> vário </td><td></td></tr><tr><td> Microscópio de epifluorescência para imagiologia </td><td> Zeiss, vários </td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

O pintainho embrionário é um modelo largamente utilizado para o estudo das projecções periféricas e centrais de células ganglionares. No sistema auditivo, rotulagem seletiva de axônios auditivos dentro do nervo craniano VIII aumentaria o estudo do centro de desenvolvimento circuito auditivo. Esta abordagem é difícil porque múltiplos órgãos sensoriais do ouvido interno, contribuir para o nervo VIII 1. Além disso, os marcadores que distinguem de forma confiável auditivo versus grupos vestibulares de axónios no interior do nervo VIIIth aviária ainda têm de ser identificados. Vias auditivas e vestibulares não podem ser distinguidas funcionalmente em embriões precoces, como sensoriais evocadas respostas não estão presentes antes dos circuitos são formados. Centralmente projetando axônios VIII foram traçadas em alguns estudos, mas auditivo rotulagem axônio foi acompanhada de rotulagem de outros componentes do nervo VIII 2,3. Aqui, nós descrevemos um método para o rastreio anterógrada do gânglio acústico para selectivamente labeaxónios auditivas l dentro do nervo VIIIth desenvolvimento. Em primeiro lugar, após o esvaziamento parcial da região cefálica anterior de um embrião de pinto com 8 dias de imersão em fluido cerebrospinal artificial oxigenado, o ducto coclear é identificado por anatómicas. Em seguida, uma micropipeta de vidro fino puxado está posicionado para injectar uma pequena quantidade de rodamina dextran amina na região adesiva e profunda adjacente onde as células ganglionares acústicos estão localizados. Dentro de 30 minutos após a injeção, os axônios auditivas são rastreados centralmente na parte posterior do cérebro e posteriormente pode ser visualizado a seguir à preparação histológica. Este método proporciona uma ferramenta útil para estudos do desenvolvimento da formação periférica de circuito auditivo central.

Os componentes do nervo VIII e a anatomia do nervo em si são complexas e tortuosas (Figuras 1, 3). Seletivamente o rastreio fibras resultantes a partir de células do gânglio acústicos, os segmentos do nervo VIII, bem como aferentes auditivos primários dentro do tronco cerebral podem ser rastreados de forma limpa e distinguidos dos seus homólogos vestibulares (Figuras 2, 3). Do mesmo modo, esta técnica pode ser utilizada para estudar as projecções periféricas das células gangl...

Watch this Scientific Journal Video about Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve at JoVE.com

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Aqui descrevemos uma técnica de microdissecção seguido de injeção de corante fluorescente no gânglio acústico de embriões de galinha cedo para rastreamento seletivo das fibras auditivas axônios no nervo e rombencéfalo.

Rastreamento seletiva de fibras no nervo auditivo embrionário aviária Vestibulococlear

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

selective-tracing-auditory-fibers-avian-embryonic-vestibulocochlear

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.1K visualizações.  University of California, Irvine. Aqui descrevemos uma técnica de microdissecção seguido de injeção de corante fluorescente no gânglio acústico de embriões de galinha cedo para rastreamento seletivo das fibras auditivas axônios no nervo e rombencéfalo.

Vídeo: Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura da embrionárias mouse cocleares explantes e Gene Transferência de Eletroporação

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Biologia do Desenvolvimento

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

Gravações pós-sinápticos em Dendrites aferente entrar em contato com células ciliadas da cóclea: Release multivesiculares Monitoramento em uma fita Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

Neurociência

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

Preparação e Cultura do Fatias de frango auditivo de tronco cerebral

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

A transferência de genes para o frango auditivo órgão por<em&gt; In Ovo</em&gt; Micro-eletroporação

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

Em Ovo Electroporation no Brainstem Auditivo da Galinha

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

In Ovo e Ex Ovo Métodos para estudar o desenvolvimento do ouvido interno aviário

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology differentially oriented along a tonotopic (i.e., frequency) axis. The tonotopic axis permits the segregated encoding of high-frequency sounds in the rostral-medial plane and low-frequency encoding in caudo-lateral regions. Traditionally, coronal brainstem slices of embryonic tissue permit the study of relative individual iso-frequency lamina. Although sufficient to investigate anatomical and physiological questions pertaining to individual iso-frequency regions, the study of tonotopic variation and its development across larger auditory brainstem areas is somewhat limited. This protocol reports brainstem slicing techniques from chicken embryos that encompass larger gradients of frequency regions in the lower auditory brainstem. The utilization of different slicing methods for chicken auditory brainstem tissue permits electrophysiological and anatomical experiments within one brainstem slice, where larger gradients of tonotopic properties and developmental trajectories are better preserved than coronal sections. Multiple slicing techniques allow for improved investigation of the diverse anatomical, biophysical, and tonotopic properties of auditory brainstem microcircuits.

Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections.

Fatiando o tronco auditivo de frango embrionário para avaliar gradientes tonotópicos e microcircuitos

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology ...

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ABRs in measuring auditory neural synchrony and estimating hearing sensitivity in other vertebrate model systems, methods for recording ABRs in the chicken have not been reported in nearly four decades. Chickens provide a robust animal research model because their auditory system is near functional maturation during late embryonic and early hatchling stages. We have demonstrated methods used to elicit one or two-channel ABR recordings using subdermal needle electrode arrays in chicken hatchlings. Regardless of electrode recording configuration (i.e., montage), ABR recordings included 3-4 positive-going peak waveforms within the first 6 ms of a suprathreshold click stimulus. Peak-to-trough waveform amplitudes ranged from 2-11 &#181;V at high-intensity levels, with positive peaks exhibiting expected latency-intensity functions (i.e., increase in latency as a function of decreased intensity). Standardized earphone position was critical for optimal recordings as loose skin can occlude the ear canal, and animal movement can dislodge the stimulus transducer. Peak amplitudes were smaller, and latencies were longer as animal body temperature lowered, supporting the need for maintaining physiological body temperature. For young hatchlings (&#60;3 h post-hatch day 1), thresholds were elevated by ~5 dB, peak latencies increased ~1-2 ms, and peak to trough amplitudes were decreased ~1 &#181;V compared to older hatchlings. This suggests a potential conductive-related issue (i.e., fluid in the middle ear cavity) and should be considered for young hatchlings. Overall, the ABR methods outlined here permit accurate and reproducible recording of in-vivo auditory function in chicken hatchlings that could be applied to different stages of development. Such findings are easily compared to human and mammalian models of hearing loss, aging, or other auditory-related manipulations.

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

Avaliação da Resposta Auditiva do Brainstem em Filhotes de Frango

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

Dissecando a Função Autônoma Celular da Proteína de Retardo Mental do X Frágil em um Circuito Auditivo por Eletroporação In Ovo

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

Isolamento e Cultivo de Somatas Vestibulares e Gangliões Espirais de Roedores Neonatais para Gravações de Patch-Clamp

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...