Name: surgical labyrinthectomy of the rat to study the vestibular system
Uploaded: 2018-05-19T13:00:00.000+00:00
Duration: 4 min 3 s
Description: Watch this Scientific Journal Video about Surgical Labyrinthectomy of the Rat to Study the Vestibular System at JoVE.com

Esta pesquisa foi apoiada por uma concessão da Coreia saúde tecnologia R & D projeto através da Coreia saúde indústria desenvolvimento Institute (KHIDI), financiado pelo Ministério da saúde & bem-estar, República da Coreia (número de concessão: HI15C2651).

Este estudo foi realizado em conformidade com o cuidado de Animal institucional e uso Comitê de Seul nacional Hospital Universitário (14-0148-C1A1), que é credenciado pela Associação para a avaliação e acreditação do cuidado de Animal de laboratório Internacional.
Nota: Os experimentos foram realizados em ratos machos Sprague-Dawley (SD) de 7-8 semanas de idade (200/250 g). Cada animal foi aclimatado para as condições de laboratório por 1 semana antes do início do experimento. Os animais foram alojados em uma sala de controle de temperatura e umidade com uma constante 12-h:12-h luz: escuro ciclo com livre acesso à comida e água.
1. vestibulotóxicos
<ol><li>Cobrir os instrumentais cirúrgicos esterilizados em autoclave com uma almofada de estéril. Desinfete a área de atuação com 70% de etanol. Use um pano estéril e manter condições estéreis durante a cirurgia.</li>
	<li>Vestir a toga, máscara (para cobrir nariz e boca), PAC (para cobrir a cabeça) e um par de luvas estéreis. Os instrumentos cirúrgicos só devem entrar em contato com superfícies estéreis.</li>
	<li>Injetar o anestésico tiletamina-eficiente (40 mg/kg de peso corporal) por via intramuscular na coxa medial ou intraperitonealmente e xilazina (10mg/kg de peso corporal) por via intramuscular ou intraperitonealmente. Nota: Outros modos de anestesia geral, incluindo a inalação de isoflurano, também podem ser usados.</li>
	<li>Coloque o rato sobre uma almofada de aquecimento (inicialmente definida a 42 ° C). Aplica a pomada lubrificante para ambos os olhos do rato para evitar ressecamento do olho enquanto sob anestesia.</li>
	<li>Coloque o rato do seu lado direito. Raspe a pele na área cirúrgica com máquina de cortar cabelo. Desinfecte o local com etanol a 70%.</li>
	<li>Injete cloridrato de lidocaína a 1% por via subcutânea em região retroauricular esquerda.</li>
	<li>Usando uma lâmina de bisturi 15 #, faça uma incisão de retroauricular ~5.0 cm. Separe o músculo e a fáscia para expor o canal auditivo externo com tesoura Iris (figura 1A). Abra ligeiramente o canal auditivo externo usando a lâmina #15 ou a tesoura de Iris (figura 1B). Expor amplamente a membrana timpânica (Figura 2A).</li>
	<li>Sob um microscópio (7,5 X ou 12,5 X), remova a membrana timpânica e ossículos com excepção do estribo, usando a pinça. Desanexe os músculos no cume lambdoidal com a lâmina #15 ou a tesoura de Iris (Figura 2B).</li>
	<li>Usando uma broca em baixa velocidade (< 3000rpm) com uma broca de diamante (2.1 ou 1,4 mm), perfurar o osso timpânica em torno do ponto de saída do nervo facial (Figura 3A). Identifica a artéria estapédica. Tome cuidado para não danificar a artéria estapédica durante o processo de perfuração.</li>
	<li>Continuar a perfuração do vestíbulo superior à artéria estapédica e expor a janela redonda e o ósseo canal semicircular lateral. Abra o canal semicircular lateral sua ampola. Continuar a perfuração no plano do canal semicircular lateral e broca da ampola do canal semicircular superior.</li>
	<li>Aspire o conteúdo do vestíbulo usando uma agulha de 18 G ou 22-G. Injetar e Aspire 1 cm3 de etanol 100% 3 x.</li>
	<li>Feche os músculos e a pele em duas camadas com suturas interrompidas simples.</li>
	<li>Não abandone um animal até que ele recuperou a consciência suficiente para manter a prostração esternal. Aquece o rato até que ele desperta usando uma almofada de aquecimento ou uma lâmpada de calor. Após o despertar, mova o rato para uma gaiola individual em uma sala com as condições ambientais normais.</li>
	<li>Administração de antibióticos (por exemplo, cefazolina 20 mg/kg, trimetoprim-sulfonamida 100 µ l por via subcutânea, ou penicilina 300.000 UI) conforme necessário no dia 1; um analgésico (não um opioide; por exemplo, Metacam, carprofeno a 2 mg/kg 5 mg/kg por via subcutânea) também pode ser dada.</li>
</ol>2. sham cirurgia
<ol><li>Para a cirurgia de Souza como um controle, executar os mesmos procedimentos cirúrgicos e pós-cirúrgicos (acordo com o ponto 1), incluindo a exposição do canal auditivo externo e a membrana timpânica (etapa 1.7), a remoção da membrana timpânica e ossículos e o desprendimento dos músculos sobre o lambdoidal ridge (passo 1.8), mas não abra o canal semicircular ou injetar o etanol.</li>
</ol>3. Verifique a perda da função Vestibular
Nota: A perda da função vestibular pode ser avaliada usando testes de qualquer função comportamental ou vestibular. 13 , 17 , 18 testes comportamentais incluem a avaliação da assimetria postural e nistagmo.
<ol><li>Identifica a assimetria postural que inclui espontânea ou evocado barril-rolamento, caindo para o lado esquerdo, ou se movendo enquanto inclinando-se para o lado lesado18. Veja o vídeo 1.</li>
	<li>Identifica o nistagmo por uma inspeção visual do movimento ocular voluntária. Quando o nistagmo espontâneo está ausente no estado de repouso, soprar ar suavemente sobre a cabeça do animal18. Veja o vídeo 2.</li>
</ol>

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+218a-5p,+219a-5p,+and+221-3p+regulate+vestibular+compensation.">MicroRNAs 218a-5p, 219a-5p, and 221-3p regulate vestibular compensation.</a> Scientific Reports. 7 (1), 8701 (2017).</li><li>Bergquist, F., Ludwig, M., Dutia, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+commissural+inhibitory+system+in+vestibular+compensation+in+the+rat.">Role of the commissural inhibitory system in vestibular compensation in the rat.</a> The Journal of Physiology. 586 (18), 4441-4452 (2008).</li><li>Cameron, S. A., Dutia, M. 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Os autores não têm nada para divulgar.

Esta técnica é um método útil para a criação de perda súbita, permanente e completa função vestibular. Isso poderia ser usado para estudar patologias vestibulares, como neurite vestibular, um tumor acústico e a doença de Meniere. Muitos estudos têm usado essa técnica para estudar a plasticidade neuronal dos núcleos vestibulares ou o processo central relacionado5,17,18,19.
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O órgão vestibular é essencial para o controle de equilíbrio e olho. Uma função vestibular normal depende de sinais aferentes simétricos de órgãos vestibulares nos dois ouvidos interno. Hipofunção vestibular ou perda induz tonturas, nistagmo e desequilíbrio postural. Depois de danos agudos, a função vestibular recupera espontaneamente dentro de alguns dias, um processo conhecido como compensação vestibular1,2. A compensação vestibular dos défices estáticos é um processo de recuperação relacionado com o desequilíbrio da atividade espontânea de descanso entre os núcleos vestibulares ipsilaterais e contralaterais. A compensação vestibular dos défices dinâmicos é conseguida principalmente através de substituições sensoriais e comportamentais (usando entradas visuais ou somatossensorial)3. Estes processos são atraentes para a plasticidade neuronal estudos4,5.
Vários métodos foram desenvolvidos para estudar o sistema vestibular e os mecanismos subjacentes a plasticidade neuronal durante a compensação vestibular, tais como químicos e cirúrgicos vestibulotóxicos e neurectomia vestibular5,6 ,7,8. Neurectomia vestibular é uma certa maneira de induzir a perda completa do vestibular, mas é um procedimento mais difícil e invasivo e pode induzir dano cerebral8,9. Este método requer maior habilidade cirúrgica e leva mais tempo do que vestibulotóxicos. Vestibulotóxicos químico incluindo gentamicina, arsenilato e Tetracaína, é mais fácil e podem produzir resultados fiáveis10,11,12. No entanto, a cóclea, também pode ser danificada e perda vestibular pode desenvolver ao longo do tempo11. Além disso, os efeitos das substâncias químicas no cérebro, que deve ser preservado para avaliação exata, não são claras. Vestibulotóxicos cirúrgico foi introduzido pela primeira vez em estudos com animais em 184215 e foi primeiramente relatado em ratos em 193616. Esta técnica tem sido usada, desde que, em muitos animais estuda5,17,18,19. Vestibulotóxicos cirúrgico é um método confiável e relativamente simples de específicos. 13 , 14 além disso, os sintomas de dano vestibular são vistos imediatamente após a cirurgia. Aqui, descrevemos nossa técnica cirúrgica para rato vestibulotóxicos.

<table><tbody><tr><td>ASPIRATOR KB-012</td><td>KOH BONG &amp;amp; CO., LTD.</td><td>KB-012</td><td>Medical aspirator</td></tr><tr><td>Blade: #15</td><td>Fine Science Tools</td><td>#10015-00</td><td>Blades for #7 Scalpel Handles, #15</td></tr><tr><td>Carbon Steel Burrs</td><td>Fine Science Tools</td><td>#19007-05</td><td>shaft diameter: 2.3 mm, length: 44 mm, package of 10 burrs</td></tr><tr><td>Carl Zeiss Surgical GmbH</td><td>Carl Zeiss</td><td>#6627100863</td><td>Surgical microscope</td></tr><tr><td>Dumont #3c</td><td>Fine Science Tools</td><td>#11231-20</td><td>Standard tip 0.17 x 0.10 mm, 11 cm</td></tr><tr><td>Dumont #5SF</td><td>Fine Science Tools</td><td>#11252-00</td><td></td></tr><tr><td>Dumont #7B</td><td>Fine Science Tools</td><td>#11270-20</td><td>Serrated 0.17 x 0.10 mm, 11 cm</td></tr><tr><td>Extra Fine Bonn: straight</td><td>Fine Science Tools</td><td>#14084-08</td><td>Iris scissors, best suited for microdissection under high magnification</td></tr><tr><td>Fine Iris Scissors: straight</td><td>Fine Science Tools</td><td>#14094-11</td><td>Made from martensitic stainless steel, combined with molybdenum and vanadium</td></tr><tr><td>Finger Loop Ear Punch</td><td>Fine Science Tools</td><td>#24212-01</td><td>1 mm. Provides stability and control for researchers using the numbering system</td></tr><tr><td>Hartman</td><td>Fine Science Tools</td><td>#13002-10</td><td>Tip width: 1 cm, serrated, 10 cm</td></tr><tr><td>Short Scalpel Handle #7 Solid</td><td>Fine Science Tools</td><td>#10003-12</td><td>#7 short, 12 cm</td></tr><tr><td>Small Vessel Cauterizer</td><td>Fine Science Tools</td><td>#18000-03</td><td>Replacement tip, straight knife, keeps bleeding to a minimum and therefore provides a surgical field clear of clamps and hemostats</td></tr><tr><td>Strong 207S</td><td>SAESHIN</td><td>207S</td><td>Powerful torque at low speed, available with speed or on/off foot controller</td></tr><tr><td>Suction Tubes</td><td>JEUNGDO B&amp;amp;P CO., LTD.</td><td>H-1927-8</td><td>Frazier, 18 cm</td></tr><tr><td>VICRYL</td><td>ETHICON</td><td>W9570T</td><td>Synthetic absorbable sterile surgical suture</td></tr><tr><td>Weitlaner-Locktite</td><td>Fine Science Tools</td><td>#17012-13</td><td>Maximum spread: 4.5 cm, 2 x 3 blunt teeth, 11 cm</td></tr><tr><td>Zoletil</td><td>Virbac, France</td><td></td><td>Tiletamine-zolazepam</td></tr><tr><td>Rompun</td><td>Bayer</td><td></td><td>Xylazine</td></tr><tr><td>Rimadyl</td><td>Pfizer</td><td></td><td>Carprofen</td></tr><tr><td>Septra</td><td>Pfizer</td><td></td><td>Trimethoprim-sulfonamide&amp;nbsp;</td></tr></tbody></table>

surgical labyrinthectomy of the rat to study the vestibular system

Para estudar o sistema vestibular ou o processo de compensação vestibular, vários métodos foram desenvolvidos para causar dano vestibular, incluindo vestibulotóxicos cirúrgicos ou químicos e neurectomia vestibular. Vestibulotóxicos cirúrgico é um método relativamente simples, confiável e rápido. Aqui, descrevemos a técnica cirúrgica para rato vestibulotóxicos. Uma incisão postauricular é feita sob anestesia geral para expor o canal auditivo externo e a membrana timpânica, após o qual a membrana timpânica e ossículos são removidos sem estribo. A artéria do estribo, que está localizada entre o estribo e a janela oval, é uma estrutura vulnerável e deve ser preservada para obter um campo cirúrgico limpo. Um buraco para fenestrado o vestíbulo feito com uma broca de 2,1 mm de broca superior com o estribo. Então, 100% de etanol é injetado através deste buraco e aspirada várias vezes. Minuciosa dissecação sob um microscópio e cuidadoso controle de sangramento são essenciais para obter resultados fiáveis. Sintomas de perda vestibular, como nistagmo, inclinação de cabeça e um movimento de rolamento, são vistos imediatamente após a cirurgia. O teste de cadeira rotarod ou rotação pode ser usado para objetivamente e quantitativamente avaliar a função vestibular.

O sucesso da cirurgia foi validado por testes comportamentais. Todos os animais exibiram o comportamento típico de uma perda unilateral da função vestibular. Barril-rolamento espontâneo era evidente imediatamente após a cirurgia, sendo evocada por um sopro de ar sobre a cabeça ou um leve toque no corpo, na fase inicial de recuperação (vídeo 1). 3-d após a cirurgia, os animais movimentados mais inclinado para o lado lesado com ocasionais cai para o lado esquerdo....

Watch this Scientific Journal Video about Surgical Labyrinthectomy of the Rat to Study the Vestibular System at JoVE.com

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

Este protocolo descreve o vestibulotóxicos cirúrgico de um rato, que é um método útil para o estudo do sistema vestibular.

Vestibulotóxicos cirúrgico do rato para estudar o sistema Vestibular

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

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10.0K visualizações.  Chung-Ang University College of Medicine. Este protocolo descreve o vestibulotóxicos cirúrgico de um rato, que é um método útil para o estudo do sistema vestibular.

Vídeo: Surgical Labyrinthectomy of the Rat to Study the Vestibular System

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

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

Cirurgia estereotáxica sobrevivência em Roedores

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Biologia

A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O&#x2019;Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and S&#233;guinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."

Although there is considerable evidence that the hippocampus is involved in piloting (O&#x2019;Keefe and Nadel, 1978; O&#x2019;Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O&#x2019;Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria&#x2013;fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.

In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.

Uma demonstração em vídeo de Pilotagem Preservado por Rastreamento Scent mas Dead Reckoning alterado após a fímbria-Fornix Lesões no Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; ...

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

Avaliando os efeitos autônomos e comportamentais do movimento passivo em ratos usando movimento vertical do elevador e rotação de roda-gigante

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and ...

Comportamento

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Uma preparação semi-intactas isolada do Rato Vestibular epitélio sensorial de Eletrofisiologia e de alta resolução Microscopia de dois fótons

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Neurociência

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

A longo prazo conflito sensorial em livremente, comportando-se ratos

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

Desvendando o papel das áreas discretas do cérebro de rato na regulação da ovulação através da inativação reversível por microinjeções de tetrodotoxinas

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

Infravermelho Próximo (NIR) Luz aumenta a expressão de um marcador da função mitocondrial no mouse Vestibular Sensorial Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

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 ...

Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. Traditional temporal bone histopathology methods rely on lengthy, expensive decalcification protocols that take 9-10 months and reduce the types of tissue analysis possible due to RNA degradation. There is a critical need to develop methods to access fresh human inner ear tissue to better understand otologic diseases, such as M&#233;ni&#232;re's disease, at the cellular and molecular level. This paper describes a technique for the harvest of human vestibular end organs from a living donor under physiologic conditions. An individual with M&#233;ni&#232;re's disease and 'drops attacks' that were refractory to intratympanic gentamicin injection underwent labyrinthectomy. A traditional mastoidectomy was first performed, and the horizontal and superior semicircular canals (SCC) were identified. The mastoid cavity was filled with a balanced salt solution so that the labyrinth could be opened under more physiologic conditions to preserve cellular integrity. A zero-degree endoscope fit with a lens-cleaning sheath irrigation system was used to visualize the submerged mastoid cavity, and a 2 mm diamond burr was used to skeletonize and open the horizontal and superior SCCs, followed by the vestibule. The ampullae and portion of the canal ducts for the superior and lateral SCCs were harvested. The utricle was similarly harvested. Harvested tissue was immediately placed in an ice-cold buffer and then fixed for one hour in 4% paraformaldehyde in phosphate-buffered saline (PBS). The tissue was rinsed several times in 1x PBS and stored for 48 h at 4 &#176;C. The tissue samples underwent immunostaining with a combination of primary antibodies against tenascin-C (Calyx), oncomodulin (streolar hair cells), calretinin (Calyx and Type II hair cells), synaptic vesicle protein 2 (efferent fibers and boutons), &#946;-tubulin 1 (Calyx and afferent boutons), followed by incubation with fluorophore-conjugated secondary antibodies. The tissue samples were then rinsed and mounted for confocal microscopy examination. Images revealed the presence of ampullar and macular hair cells and neural structures. This protocol demonstrates that it is possible to harvest intact, high-quality human inner ear tissue from living donors and may provide an important tool for the study of otologic disease.

Here, we describe a technique for harvesting human vestibular end-organs under physiologic conditions during labyrinthectomy and their analysis using immunostaining.