JC would like to acknowledge the David Hay Memorial Fund, The University of Melbourne for financial support in writing this manuscript. Funding for this project was provided by an ARC Linkage grant 100200129 (BVB, AJV, CTON).

Declaração de ética: Experiências com animais foram conduzidos de acordo com o Código australiano para o Cuidado e Utilização de animais para fins científicos (2013). aprovação ética animal foi obtido a partir Comitê de Ética Animal da Universidade de Melbourne. Os materiais aqui são para experiências de laboratório apenas, e não destinados ao uso médico ou veterinário. 1. Os eléctrodos Preparando Nota: Um transmissor três canais é usado para implantação cirúrgica que permite 2 ERG e gravação de 1 VEP a ser realizado simultaneamente. Os três ativa e três eletrodos inativos precisam ser pré-moldado em forma de anel antes da implantação, a fim de anexar ao olho. Para fins de identificação, o fabricante tenha fechado eletrodos ativos na metade branco, metade bainhas de plástico coloridos, enquanto eletrodos inativos são cobertos em bainhas de cor completos. O eletrodo terra (claro bainha de plástico) é deixado inalterado. Para todos electr ativos e inativosconduta odes os passos 1.1, 1.2, 1.3 e 1.7. <ol><li> Destorcer o eletrodo de aço inoxidável de cadeia dupla com dois alicates de ponta fina. </li><li> Apare um dos cordões de aço inoxidável (aproximadamente a 1 cm da ponta), deixando uma cadeia simples mais linear restante para moldar o eléctrodo anular. </li><li> Dobrar o único fio de aço inoxidável para trás sobre si própria e torção, formando um anel liso na ponta do eléctrodo. </li><li> Para o ERG eletrodos ativos moda este laço ~ 0,2 - 0,5 mm de diâmetro por torção da base do loop (para a finalidade descrita aqui, forma dois eletrodos ativos desta forma para gravar ERG de ambos os olhos), e para o ERG inativos e eletrodos VEP faça o diâmetro do laço ~ 0,8 mm de diâmetro (neste exemplo, fazer isso para um eletrodo VEP ativo e todos os três eletrodos inativos). </li><li> Prenda a circular VEP eletrodo ativo em torno de um parafuso de aço inoxidável (diâmetro 0,7 mm, comprimento 3 mm) de modo que o eletrodo fica encostada à cabeça do parafuso. </li><li> Hook os 3 eletrodos inativos (2 ERG, 1 VEP) em torno de um segundo parafuso de aço inoxidável (diâmetro 0,7 mm, comprimento 3 mm). </li><li> Puxe a manga de plástico para a frente sobre as pontas afiadas da vertente dois de aço inoxidável para reduzir a irritação. </li><li> Esterilizar os transmissores de telemetria por imersão em 2% de glutaraldeído durante mais de 10 horas a cerca de 25 ° C. Em seguida, lave o transmissor com solução salina estéril 3 vezes. </li></ol> 2. Transmissor de Implantação <ol><li> Preparação animal <ol><li> Desinfectar a área cirúrgica antes da experimentação por limpeza com etanol a 70%. Autoclave todo o equipamento cirúrgico antes do uso e manutenção do equipamento em clorexidina quando não em uso durante a cirurgia. Cobrir o animal com um penso cirúrgico durante a cirurgia para manter um ambiente estéril. Garantir que todos os experimentadores usar máscaras cirúrgicas, luvas e aventais estéreis. </li><li> Induzir a anestesia com 1,5 - 2% de isoflurano, a um caudal de 3 L / min e manutennido a 1,5 - 2% em 2 L / min durante toda a cirurgia. Confirmar profundidade suficiente de anestesia pela ausência de um reflexo pedal sobre beliscar o músculo entre os dedos. </li><li> Raspar uma área de 40 mm x 30 mm no abdómen de cima da virilha ao esterno. </li><li> Raspar uma área de 30 mm x 20 milímetros sobre a testa, posterior aos olhos e anterior para os ouvidos. </li><li> Desinfectar as duas áreas raspadas. Para a área da testa desinfectar com 10% de iodopovidona três vezes (evitar o uso de anti-sépticos à base de álcool para a área perto do olho, sendo consistente com o padrão de prática estabelecidos pela Associação dos Tecnólogos cirúrgicos). Durante o abdómen desinfecção com 10% de iodo de povidona e 70% de etanol. </li><li> Aplicar uma gota de proxymetacaine à córnea para a anestesia tópica adicional. </li><li> Aplicar uma gota de carboximetilcelulose de sódio a córnea para evitar a secagem dos olhos. </li></ol></li><li> implantação cirúrgica <ol><li> Adicione uma incisão 10 milímetrosna cabeça ao longo da linha média vertical entre as orelhas com um bisturi cirúrgico. </li><li> Adicione uma incisão cinco milímetros no abdómen através da camada de pele ao longo da linha média abaixo do esterno. </li><li> Túnel a 5 mm de diâmetro de uma cânula subcutânea da incisão abdominal para a incisão cabeça. </li><li> Alimentar os fios dos eléctrodos (3 ativos e inativos 3) do transmissor através da cânula do abdómen para a cabeça. </li><li> Deixe o eletrodo de referência com a base transmissor e cobrir a ponta do eletrodo com gaze asséptica. </li><li> Cubra as pontas dos eléctrodos (3 ativos e inativos 3) com gaze asséptica. </li><li> Fixe a cabeça do rato para uma plataforma estereotáxico. </li><li> Estender a incisão testa a 30mm de comprimento com uma tesoura cirúrgica. </li><li> Expor área cirúrgica retraindo a pele solta com 2 suturas (3 - 0) em ~ 3 e 9 horas. </li><li> Raspe o periósteo que recobre o crânio com gaze esterilizada para expor bregma, lambda e da linha média suturas. </li&gt; <li> Perfurar dois buracos através do crânio no VEP activa (7 milímetros ventral em relação ao bregma 3 milímetros lateral à linha média) e inactiva (5 mm rostral à bregma na linha média) estereotáxicas co-ordenadas. </li><li> Anexar VEP eletrodos ativos e inativos com parafusos pré-inscritos aço inoxidável (diâmetro de 0,7 mm, comprimento 3 mm) para o crânio com uma pequena chave de fenda para ~ 1 mm de profundidade para os buracos premade. Este ancora o parafuso para o osso sem danificar o tecido cortical subjacente. </li><li> Para implantar os eletrodos ativos ERG usar um 8-0 sutura para retrair temporariamente a pálpebra superior. </li><li> Inserir um 16 a 21 G por via subcutânea cânula de trás do olho através do fundo de saco conjuntival superior. </li><li> Remover o guiamento de agulhas. </li><li> Alimente o eletrodo ativo através do cateter de plástico encurtado da testa para o olho. Em seguida, remover o cateter de plástico. </li><li> Utilize uma sutura temporária (8-0), que é enfiada através do laço eléctrodo, para evitar que os electrode de retrair volta para o túnel. </li><li> Adicione uma incisão 0,5 milímetros sobre a conjuntiva superior a 0:00, 1 mm por trás do limbo. Use dissecção cega para expor o esclerótica subjacente. </li><li> Implantar um 8-0 ou 9-0 sutura imediatamente atrás do limbo na metade da espessura da esclera. </li><li> Remover sutura temporária do ERG eletrodo ativo. </li><li> Ancorar o eletrodo ativo ERG à meia escleral sutura espessura amarrando 3 nós consecutivos, garantindo a ponta do eletrodo está situado próximo ao limbo. </li><li> Feche a aba conjuntival utilizando 1 a 2 suturas interrompidas (8 - 0 a 9 - 0). Certifique-se que a conjuntiva cobre completamente o eletrodo ERG para melhorar o conforto. </li><li> Remover a pálpebra retracção sutura. </li><li> Repetir o procedimento para o olho contralateral. </li><li> Aplique o gel de cianoacrilato sobre o crânio para proteger todas as parafusos de aço inoxidável e fios de eletrodos. Garantir os eletrodos ativos ERG não são puxados muito apertado antes de fixar a enmovimentos oculares capazes. </li><li> Fechar o ferimento na cabeça usando um não-absorvível 3-0 sutura. </li><li> Gire roedor para expor região abdominal. Alongar a incisão dérmica abdominal a 40 mm ao longo da linha alba com uma tesoura cirúrgica. </li><li> Adicione uma incisão 35 milímetros através da parede interior do músculo para expor a cavidade abdominal interior. </li><li> Usando duas suturas (3 - 0) anexar o corpo do transmissor para o lado direito da parede abdominal interna do animal. Evitar o contacto do fígado. </li><li> Passe o eléctrodo de terra e seguro nesta forma com uma sutura (3-0). Coloque-lo flutuando livremente na cavidade abdominal. </li><li> Feche o peritônio usando uma sutura contínua (3-0). </li><li> Feche a incisão na pele usando suturas interrompidas (3 - 0). </li></ol></li><li> Cuidados pós-operatórios <ol><li> Monitorar o animal até que ele recuperou a consciência suficiente para manter decúbito esternal. Casa do animal individualmente após a cirurgia. </li><li> administrar carprofen por via subcutânea para a analgesia (5 mg / kg) uma vez por dia durante 4 dias. </li><li> Adicionar antibióticos orais profiláticos (Enrofloxin, 5 mg / kg) para a água potável durante 7 dias pós-cirurgia. </li><li> Aplicar uma pomada anti-inflamatória a locais de incisão da pele para reduzir a irritação durante os primeiros 7 dias pós-cirurgia. </li></ol></li></ol> 3. Conduta ERG e VEP Recordings em ratos conscientes <ol><li> Escuro adaptar animal durante 12 h antes de registos de ERG e VEP </li><li> Efectuar todas as manipulações experimentais sob iluminação fraca vermelha (17,4 cd.m -2, λ max = 600 nm) </li><li> Aplicar anestesia tópica (0,5% proxymetacaine) e dilatando (0,5% de tropicamida) cai para a córnea. </li><li> Orientar o roedor consciente em um personalizado feito restrainer, claro. Nota: O comprimento deste tubo de plástico pode ser ajustado para acomodar diferentes de tamanhos ratos com o diâmetro total fixado em 60 mm. A extremidade dianteira do dispositivo é afunilada para minimizar cabeça movimento e contém perfurações para permitir a respiração normal. Esta frente afunilada permite o alinhamento e estabilização da cabeça e os olhos do rato para a abertura da esfera Ganzfeld. Note-se que o roedor tem sido aclimatizadas ao limitador (3 a 5 vezes) antes da cirurgia. </li><li> Colocar o roedor na frente da bacia Ganzfeld com os olhos alinhada com a abertura da bacia. </li><li> Ligue o transmissor habitação por meio de um ímã dentro de ~ 5 cm do transmissor. Verifique se o transmissor está ligado, verificando o LED de status na base do receptor. </li><li> Recolhe sinais a uma variedade de energias luminosas (ou seja, -5,6 a 1,52 log cd.sm -2) conforme descrito anteriormente 21. Resumidamente, médios mais sinais nos níveis de intensidade da luz de luz (~ 80 repetições) e menos nas energias mais brilhante luminosos (~ 1 repetição). Gradualmente aumentar o intervalo interstimulus de 1 a 180 segundos do mais obtuso para o nível de luz mais brilhante. </li><li> Para isolar a haste do ERGe as respostas de cone utilizar um paradigma twin-flash 8. Por exemplo, iniciar dois flashes em cd.sm 1,52 log -2 com um intervalo inter-estímulo de 500 ms no meio. </li><li> Para gravar sinais VEP, média de 20 repetições para as energias mais brilhante luminosos (ou seja, 1,52 log cd.sm -2, 5 seg intervalo inter-estímulo). </li><li> Para avaliar a estabilidade do implante, o que é avaliado por variabilidade do sinal ao longo do tempo, realizar registos de ERG e VEP 7, 10, 14, 21 e 28 dias pós-cirurgia. </li><li> Seguindo período experimental, os ratos eutanásia por meio de injecção intracardíaca de pentobarbiturate (1,5 ml / kg) depois de cetamina: xilazina anestesia (12: 1 mg / kg). </li></ol>

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RG and RF are employees to the commercial funder of this research (Pfizer Neusentis and Pfizer Global Research). MI was an employee of Pfizer Global Research during this research and is currently an employee of Proteostasis Therapeutics (Cambridge, USA).

Devido à natureza minimamente invasiva da eletrofisiologia visual, registos de ERG e VEP em pacientes humanos são conduzidos sob condições conscientes e exigem o uso apenas de anestésicos tópicos para a colocação do eletrodo. Em contraste, eletrofisiologia visual em modelos animais é convencionalmente realizado sob anestesia geral para permitir a colocação do eletrodo estável, eliminando movimentos oculares e corporais voluntárias. No entanto, anestésicos gerais comumente utilizados alterar as respostas do...

O ERG e VEP são minimamente invasivos em ferramentas in vivo para avaliar a integridade das vias da retina e visuais, respectivamente, tanto no laboratório e clínica. O full-campo do ERG produz uma forma de onda característico, que pode ser dividido em componentes diferentes, com cada elemento representando diferentes classes de células da retina 1,2 percurso. O de campo total ERG forma de onda clássica consiste em um declive inicial negativa (a onda), que foi mostrado para representar fotorreceptor atividade pós exposição à luz 2-4. A uma onda é seguido por uma forma de onda positiva substancial (b-ondas), que reflecte a actividade eléctrica da retina meio predominantemente, as células bipolares de ON-5-7. Além disso, pode-se variar a energia luminosa e inter-estímulo-intervalo para isolar cone de respostas haste 8. O flash VEP representa potenciais eléctricos do tronco cerebral e córtex visual em resposta à estimulação da retina luz9,10. Esta forma de onda pode ser dividido em componentes precoce e tardio, com o componente precoce reflectindo uma actividade de neurónios da via retino-geniculo-estriado 11-13 e representando a componente de final de processamento cortical realizada de várias lâminas V1 em ratos 11,13. Portanto medição simultânea do ERG e VEP retorna avaliação abrangente das estruturas envolvidas na via visual. Atualmente, a fim de gravar eletrofisiologia em animais, a anestesia é utilizada para permitir a colocação estável de eletrodos. Tem havido tentativas de medir ERG e VEP em ratos conscientes 14-16 mas estes estudos utilizou uma configuração com fio, que pode ser complicado e pode levar ao estresse dos animais através da restrição da circulação de animais e comportamento natural 17. Com os recentes avanços na tecnologia sem fio, incluindo maior miniaturização e vida útil da bateria, agora é possível implementar uma abordagem de telemetria para ERG umd gravação VEP, diminuindo o estresse associado com as gravações com fio e melhorar a viabilidade a longo prazo. Implantes estáveis ​​totalmente internalizadas de sondas de telemetria provaram ser bem sucedido para monitoramento crônica da temperatura, pressão arterial 18, a atividade 19, bem como eletroencefalograma 20. Tais avanços na tecnologia também vai ajudar com repetibilidade e estabilidade das gravações conscientes, aumentando a utilidade da plataforma para estudos crônicos.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bioamplifier</td>
			<td>ADInstruments</td>
			<td>ML 135</td>
			<td>Amplifies ERG and VEP signals</td>
		</tr>
		<tr>
			<td>Carboxymethylcellulose sodium 1.0%</td>
			<td>Allergan</td>
			<td>CAS 0009000-11-7</td>
			<td>Maintain corneal hydration during surgery</td>
		</tr>
		<tr>
			<td>Carprofen 0.5%</td>
			<td>Pfizer Animal Health Group</td>
			<td>CAS 53716-49-7</td>
			<td>Post-surgery analgesia, given with injectable saline for fluid replenishment</td>
		</tr>
		<tr>
			<td>Chlorhexadine 0.5%</td>
			<td>Orion Laboratories</td>
			<td>27411, 80085</td>
			<td>Disinfection of surgical instrument</td>
		</tr>
		<tr>
			<td>Cyanoacrylate gel activator</td>
			<td>RS components</td>
			<td>473-439</td>
			<td>Quickly dries cyanoacrylate gel</td>
		</tr>
		<tr>
			<td>Cyanocrylate gel&#160;</td>
			<td>RS components</td>
			<td>473-423</td>
			<td>Fix stainless screws to skull</td>
		</tr>
		<tr>
			<td>Dental burr</td>
			<td>Storz Instruments, Bausch and Lomb</td>
			<td>E0824A</td>
			<td>Miniature drill head of ~0.7mm diameter for making a small hole in the skull over each hemisphere to implant VEP screws</td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Bosch</td>
			<td>Dremel 300 series</td>
			<td>Automatic drill for trepanning</td>
		</tr>
		<tr>
			<td>Enrofloxin</td>
			<td>Troy Laboratories</td>
			<td></td>
			<td>Prophylactic antibiotic post surgey</td>
		</tr>
		<tr>
			<td>Ganzfeld integrating sphere</td>
			<td>Photometric Solutions International</td>
			<td></td>
			<td>Custom designed light stimulator: 36 mm diameter, 13 cm aperture size</td>
		</tr>
		<tr>
			<td>Gauze swabs</td>
			<td>Multigate Medical Products Pty Ltd</td>
			<td>57-100B</td>
			<td>Dries surgical incision and exposed skull surface during surgery</td>
		</tr>
		<tr>
			<td>Isoflurane 99.9%</td>
			<td>Abbott Australasia Pty Ltd</td>
			<td>CAS 26675-46-7</td>
			<td>Proprietory Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for VEP electrode implant surgery</td>
		</tr>
		<tr>
			<td>Kenacomb ointment</td>
			<td>Aspen Pharma Pty Ltd</td>
			<td></td>
			<td>To reduce skin irritation and itching after surgery</td>
		</tr>
		<tr>
			<td>Luxeon LEDs</td>
			<td>Phillips Lighting Co.</td>
			<td></td>
			<td>For light stimulation, twenty 5 watt and one 1 watt LEDs, controlled by Scope software</td>
		</tr>
		<tr>
			<td>Needle (macrosurgery)</td>
			<td>World Precision Instruments</td>
			<td>501959</td>
			<td>for suturing abdominal and head surgery, used with 3-0 suture, eye needle, cutting edge 5/16 circle Size 1, 15mm</td>
		</tr>
		<tr>
			<td>Needle holder (macrosurgery)</td>
			<td>World Precision Instruments</td>
			<td>500224</td>
			<td>To hold needle during abdominal and head surgery</td>
		</tr>
		<tr>
			<td>Needle holder (microsurgery)</td>
			<td>World Precision Instruments</td>
			<td>555419NT</td>
			<td>To hold needle during ocular surgery</td>
		</tr>
		<tr>
			<td>Optiva catheter</td>
			<td>Smiths Medical International LTD</td>
			<td>16 or 21 G</td>
			<td>Guide corneal active electrodes from skull to conjunctiva</td>
		</tr>
		<tr>
			<td>Povidone iodine 10%</td>
			<td>Sanofi-Aventis</td>
			<td>CAS 25655-41-8</td>
			<td>Proprietory name: Betadine, Antiseptic to prepare the shaved skin for surgery 10%, 500 mL</td>
		</tr>
		<tr>
			<td>Powerlab data acquisition system</td>
			<td>ADInstruments</td>
			<td>ML 785</td>
			<td>Acquire signal from telemetry transmitter, paired to telemetry data converter</td>
		</tr>
		<tr>
			<td>Proxymetacaine 0.5%</td>
			<td>Alcon Laboratories&#160;</td>
			<td>CAS 5875-06-9</td>
			<td>Topical ocular analgesia</td>
		</tr>
		<tr>
			<td>Restrainer</td>
			<td>cutom made</td>
			<td></td>
			<td>Front of the restrainer is tapered to minimize head movement, length can be adjusted to accommodate different rat length, overall diameter is 60 mm.&#160;</td>
		</tr>
		<tr>
			<td>Scapel blade</td>
			<td>R.G. Medical Supplies</td>
			<td>SNSM0206</td>
			<td>For surgical incision</td>
		</tr>
		<tr>
			<td>Scissors (macrosurgery)</td>
			<td>World Precision Instruments</td>
			<td>501225</td>
			<td>for cutting tissue on the abodmen and forhead</td>
		</tr>
		<tr>
			<td>Scissors (microsurgery)</td>
			<td>World Precision Instruments</td>
			<td>501232</td>
			<td>To dissect the conjunctiva for electrode attachment</td>
		</tr>
		<tr>
			<td>Scope Software</td>
			<td>ADInstruments</td>
			<td>version 3.7.6</td>
			<td>Simultaneously triggers the stimulus via the ADI Powerlab system and collects data</td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Oster</td>
			<td>Golden A5</td>
			<td>Shave fur from surgical areas</td>
		</tr>
		<tr>
			<td>Stainless streel screws&#160;</td>
			<td>MicroFasteners</td>
			<td>L001.003CS304</td>
			<td>0.7 mm shaft diameter, 3 mm in length&#160;</td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>David Kopf</td>
			<td>Model 900</td>
			<td>A small animal stereotaxic instrument for locating the implantation landmarks on the skull</td>
		</tr>
		<tr>
			<td>Surgical drape</td>
			<td>Vital Medical Supplies</td>
			<td>GM29-612EE</td>
			<td>Ensure sterile enviornment during surgery</td>
		</tr>
		<tr>
			<td>Suture (macrosurgery)</td>
			<td>Ninbo medical needles</td>
			<td>3-0</td>
			<td>for suturing abdominal and head surgery, sterile silk braided, 60cm</td>
		</tr>
		<tr>
			<td>Suture needle (microsurgery)</td>
			<td>Ninbo medical needles</td>
			<td>8-0 or 9-0</td>
			<td>for ocular surgery including, suturing electrode to sclera and closing conjunctival wound, nylon suture, 3/8 circle 1&#215;5, 30cm</td>
		</tr>
		<tr>
			<td>Telemetry data converter&#160;</td>
			<td>DataSciences International</td>
			<td>R08</td>
			<td>allows telemetry signal to interface with data collection software</td>
		</tr>
		<tr>
			<td>Telemetry Data Exchange Matrix</td>
			<td>DataSciences International</td>
			<td></td>
			<td>Gathers data from transmitters, pair with receiver</td>
		</tr>
		<tr>
			<td>Telemetry data receiver</td>
			<td>DataSciences International</td>
			<td>RPC-1</td>
			<td>Receives telemetry data from transmitter</td>
		</tr>
		<tr>
			<td>Telemetry transmitter</td>
			<td>DataSciences International</td>
			<td>F50-EEE</td>
			<td>3 channel telemetry transmitter</td>
		</tr>
		<tr>
			<td>Tropicamide 0.5%</td>
			<td>Alcon Laboratories&#160;</td>
			<td></td>
			<td>Iris dilation</td>
		</tr>
		<tr>
			<td>Tweezers (macrosurgery)</td>
			<td>World Precision Instruments</td>
			<td>500092</td>
			<td>Manipulate tissues during abdominal and head surgery</td>
		</tr>
		<tr>
			<td>Tweezers (microsurgery)</td>
			<td>World Precision Instruments</td>
			<td>500342</td>
			<td>Manipulate tissues during ocular surgery</td>
		</tr>
	</tbody>
</table>

implantation and recording of wireless electroretinogram and visual evoked potential in conscious rats

O eletrorretinograma de campo total (ERG) e potencial evocado visual (VEP) são ferramentas úteis para avaliar retina e integridade via visual, tanto em laboratórios e ambientes clínicos. Atualmente, pré-clínicos medições ERG e VEP são realizados com anestesia para assegurar a colocação de eletrodos estáveis. No entanto, a presença de anestesia foi mostrado para contaminar respostas fisiológicas normais. Para superar estes confunde anestesia, desenvolvemos uma plataforma inovadora para ensaiar ERG e VEP em ratos conscientes. Eletrodos são implantados cirurgicamente sub-conjunctivally no olho para ensaiar o ERG e epidural sobre o córtex visual para medir a VEP. A gama de amplitude e sensibilidade / parâmetros de tempo são ensaiadas, tanto para o ERG e VEP a aumentar energias luminosas. Os sinais de ERG e VEP são mostrados para ser estável e repetível para, pelo menos, 4 semanas pós-implantação cirúrgica. Esta capacidade de gravar sinais de ERG e VEP sem anestesia confunde nas s pré-clínicosreparativos deve fornecer uma tradução superior aos dados clínicos.

A resposta de fotorreceptores é analisada por montagem de uma Gaussiana retardada até ao bordo dianteiro do ramo descendente inicial da resposta do ERG no topo 2 energias luminosas (1,20, 1,52 log CSM -2) para cada animal, com base no modelo de carneiro e Pugh 22, formulada por Hood e Birch 23. Esta fórmula retorna uma amplitude e um parâmetro de sensibilidade, (Figura 1C e 1D, respectivamente). Uma função hiperból...

Watch this Scientific Journal Video about Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats at JoVE.com

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

Mostramos procedimentos de implantação e de gravação cirúrgicos para medir sinais eletrofisiológicos visuais do olho (eletrorretinograma) eo cérebro (potencial evocado visual) em ratos conscientes, que é mais análogo à condição humana, onde as gravações são realizadas sem confunde anestesia.

Implantação e Gravação do Wireless Eletrorretinograma e Visual Potencial Evocado em ratos conscientes

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

implantation-recording-wireless-electroretinogram-visual-evoked

Research

JoVE Journal

Behavior

8.6K visualizações.  University of Melbourne.  Mostramos procedimentos de implantação e de gravação cirúrgicos para medir sinais eletrofisiológicos visuais do olho (eletrorretinograma) eo cérebro (potencial evocado visual) em ratos conscientes, que é mais análogo à condição humana, onde as gravações são realizadas sem confunde anestesia. 

Vídeo: Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The Successive Alleys Test of Anxiety in Mice and Rats

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones. Handley, Mithani and File et al. performed the first studies on the plus-maze design we use today, and in 1987 Lister published a design for use with mice. 
Time spent on, and entries into, the open arms are an index of anxiety; the lower these indices, the more anxious the mouse is. Alternatively, a mouse that spends most of its time in the closed arms is classed as anxious. 
One of the problems of the plus-maze is that, while time spent on, and entries into, the open arms is a fairly unambiguous measure of anxiety, time in the central area is more difficult to interpret, although time spent here has been classified as &#x201C;decision making&#x201D;. In many tests central area time is a considerable part of the total test time.
Shepherd et al. produced an ingenious design to eliminate the central area, which they called the &#x201C;zero maze&#x201D;. However, although used by several groups, it has never been as widely adopted as the plus-maze. 
In the present article I describe a modification of the plus-maze design that not only eliminates the central area but also incorporates elements from other anxiety tests, such as the light-dark box and emergence tests. It is a linear series of four alleys, each having increasing anxiogenic properties. It has given similar results to the plus-maze in general. Although it may not be more sensitive than the plus-maze (more data is needed before a firm conclusion can be reached on this point), it provides a useful confirmation of plus-maze results which would be useful when, for example, only a single example of a mutant mouse was available, as, for example, in ENU-based mutagenesis programs.

The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

O Becos Teste sucessiva de ansiedade em camundongos e ratos

The plus-maze was derived from the early work of Montgomery. He observed that rats tended to avoid the open arms of a maze, preferring the enclosed ones.  ...

Comportamento

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have developed visual cognitive tasks for rats using the Floor Projection Maze1,2&#160;that are optimized for visual abilities of rats permitting stronger comparisons of experimental findings with other species.
In order to investigate neural correlates of learning and memory, we have integrated electrophysiological recordings into fully automated cognitive tasks on the Floor Projection Maze1,2. Behavioral software interfaced with an animal tracking system allows monitoring of the animal's behavior with precise control of image presentation and reward contingencies for better trained animals. Integration with an in vivo electrophysiological recording system enables examination of behavioral correlates of neural activity at selected epochs of a given cognitive task.
We describe protocols for a model system that combines automated visual presentation of information to rodents and intracranial reward with electrophysiological approaches. Our model system offers a sophisticated set of tools as a framework for other cognitive tasks to better isolate and identify specific mechanisms contributing to particular cognitive processes.

We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

Tarefas cognitivas Visuais automatizadas para gravação Atividade Neural Usando uma projeção Piso Labirinto

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have ...

Methods to Explore the Influence of Top-down Visual Processes on Motor Behavior

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are deliberately planned, while others spontaneously occur below conscious awareness. The deliberate component of this dichotomy has been studied extensively in several contexts, while the spontaneous component remains largely under-explored. Moreover, how perceptual processes modulate these movement classes is still unclear. In particular, a currently debated issue is whether the visuomotor system is governed by the spatial percept produced by a visual illusion or whether it is not affected by the illusion and is governed instead by the veridical percept. Bistable percepts such as 3D depth inversion illusions (DIIs) provide an excellent context to study such interactions and balance, particularly when used in combination with reach-to-grasp movements. In this study, a methodology is developed that uses a DII to clarify the role of top-down processes on motor action, particularly exploring how reaches toward a target on a DII are affected in both deliberate and spontaneous movement domains.

It is unclear how top-down signals from the ventral visual stream affect movement. We developed a paradigm to test motor behavior towards a target on a 3D depth inversion illusion. Significant differences are reported in both deliberate, goal-directed movements and automatic actions under illusory and veridical viewing conditions.

Métodos para pesquisar a influência de cima para baixo processos visuais em Comportamento Motor

Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

The Hole Board Modificado - Medir Comportamento, Cognição e Interação Social em camundongos e ratos

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Conscious and Non-conscious Representations of Emotional Faces in Asperger's Syndrome

Several neuroimaging studies have suggested that the low spatial frequency content in an emotional face mainly activates the amygdala, pulvinar, and superior colliculus especially with fearful faces1-3. These regions constitute the limbic structure in non-conscious perception of emotions and modulate cortical activity either directly or indirectly2. In contrast, the conscious representation of emotions is more pronounced in the anterior cingulate, prefrontal cortex, and somatosensory cortex for directing voluntary attention to details in faces3,4. Asperger&#39;s syndrome (AS)5,6 represents an atypical mental disturbance that affects sensory, affective and communicative abilities, without interfering with normal linguistic skills and intellectual ability. Several studies have found that functional deficits in the neural circuitry important for facial emotion recognition can partly explain social communication failure in patients with AS7-9. In order to clarify the interplay between conscious and non-conscious representations of emotional faces in AS, an EEG experimental protocol is designed with two tasks involving emotionality evaluation of either photograph or line-drawing faces. A pilot study is introduced for selecting face stimuli that minimize the differences in reaction times and scores assigned to facial emotions between the pretested patients with AS and IQ/gender-matched healthy controls. Information from the pretested patients was used to develop the scoring system used for the emotionality evaluation. Research into facial emotions and visual stimuli with different spatial frequency contents has reached discrepant findings depending on the demographic characteristics of participants and task demands2. The experimental protocol is intended to clarify deficits in patients with AS in processing emotional faces when compared with healthy controls by controlling for factors unrelated to recognition of facial emotions, such as task difficulty, IQ and gender.

An EEG experimental protocol is designed to clarify the interplay between conscious and non-conscious representations of emotional faces in patients with Asperger&#39;s syndrome. The technique suggests that patients with Asperger&#39;s syndrome have deficits in non-conscious representation of emotional faces, but have comparable performance in conscious representation with healthy controls.

Representações conscientes e não-conscientes de Faces emocionais na síndrome de Asperger

Several neuroimaging studies have suggested that the low spatial frequency content in an emotional face mainly activates the amygdala, pulvinar, and ...

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Manipulação de epileptiforme Electrocorticograms (ECoGs) e do sono em ratos e camundongos por Acupuntura

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

Uma rede sem fio, Interface bidirecional para gravação na Vivo e estimulação da atividade Neural em livremente, comportando-se ratos

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

Using Electroencephalography Measurements and High-quality Video Recording for Analyzing Visual Perception of Media Content

This article explores a method to detect differences in visual perception in humans. The method used is based on the psychological (or "cognitive") function of eyeblinks. Participants' eyeblinks are detected and acquired while watching videos specifically created for the investigation. The detection and acquisition of eyeblinks are carried out with the help of a 20-channel electroencephalographic (EEG) wireless device. The international 10-20 system for electrode placement is followed. A high-definition (HD) video camera is used to record participants' facial expressions, for contrast purposes. Instead of using pre-existing media content, purpose-made video content has been created following specific criteria of interest for this investigation, with stimuli enabling researchers to manage the precise parameters of interest. Otherwise, results could be contaminated with uncontrolled variables. The synchronization of the presentation of video stimuli with EEG recordings needs to be done in milliseconds. Analysis of collected data is performed with robust software for working with big matrices. Statistically significant differences in eyeblink rate related to media professionalization and editing style are found with the reported experimental procedures.

We present detection, acquisition, and analysis of eyeblink rates while watching media content.

Usando medições de Eletroencefalografia e gravação de vídeo de alta qualidade para analisar a percepção Visual de conteúdo de mídia

This article explores a method to detect differences in visual perception in humans. The method used is based on the psychological (or "cognitive") ...

Eye Tracking During Visually Situated Language Comprehension: Flexibility and Limitations in Uncovering Visual Context Effects

The present work is a description and an assessment of a methodology designed to quantify different aspects of the interaction between language processing and the perception of the visual world. The recording of eye-gaze patterns has provided good evidence for the contribution of both the visual context and linguistic/world knowledge to language comprehension. Initial research assessed object-context effects to test&#160;theories of modularity in language processing. In the introduction, we describe how subsequent investigations have taken the role of the wider visual context in language processing as a research topic in its own right, asking questions such as how our visual perception of events and of speakers contributes to&#160;comprehension informed by&#160;comprehenders&#39; experience. Among the examined aspects of the visual context are actions, events, a speaker&#39;s gaze, and emotional facial expressions, as well as spatial object configurations. Following an overview of the eye-tracking method and its different applications, we list the key steps of the methodology in the protocol, illustrating how to successfully use it to study visually-situated language comprehension. A final section presents three sets of representative results and illustrates the benefits and limitations of eye tracking for investigating the interplay between the perception of the visual world and language comprehension.

The present article reviews an eye-tracking methodology for studies on language comprehension. To obtain reliable data, key steps of the protocol must be followed. Among these are the correct set-up of the eye tracker (e.g., ensuring good quality of the eye and head images) and accurate calibration.

Olho de monitoramento durante a compreensão de linguagem visualmente situado: Flexibilidade e limitações no descobrindo contexto Visual Effects

The present work is a description and an assessment of a methodology designed to quantify different aspects of the interaction between language processing ...

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Visual condicionamento clássico em madeira formigas

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...