Agradeço Anthony Taylor para a formação inicial em gravação no intracelular vivo e A Maxwell e David Brown para ajudar com o desenvolvimento inicial da técnica de coloração intracelular. Agradeço M. Prata ajuda com a macro collocalization. Muitos estudiosos com quem tenho colaborado desde insight para o desenvolvimento desta técnica, incluindo R. Donga, Moritani M., P. Luo, Ambalavanar R.. Esta técnica foi desenvolvida com o apoio considerável de NIH concede DE10132, DE15386 e RR017971.

1. Preparação dos animais <ol><li> Anestesiar ratos com pentobarbital sódico (50mg/kg IP) e coloque sobre uma almofada de aquecimento. Raspar a pele que recobre o crânio posterior com clippers animal. Verifique o animal para assegurar que um nível cirúrgica do nível de anestesia foi obtida por meio de testes para a ausência de um reflexo de retirada e vocalização quando os dedos são comprimidos, bem como a ausência de reflexo palpebral. Verificar o nível de anestesia a cada 15 minutos e manter um nível cirúrgico de anesthetisa por injeções de pentobarbital sódico 15mg/kg a cada 45 minutos. </li><li> Use uma técnica asséptica e fazer uma incisão na região inguinal imediatamente distal à prega formada pelo abdômen e parte interna da coxa e inserir uma cânula (1mm de diâmetro, Clay Adams) na veia femoral e da artéria femoral. Faça uma incisão mediana na região submandibular, refletem os músculos infra-hióideos. Fazer uma pequena incisão na traquéia e inserir uma cânula (2mm de diâmetro) para permitir a ventilação. Tye uma sutura em torno do trachael cannual para fixá-lo na pressão arterial sistêmica place.Monitor sistólica e diastólica através da cânula arterial. Além de monitorar a retirada e reflexo palpebral agora monitor de pressão arterial para avaliar o nível de anestesia. Administrar anestesia adicionais quando necessário através da cânula venosa. </li><li> Coloque o rato em uma moldura estereotáxica e realizar uma craniotomia para expor o cerebelo. Anexar a cânula traqueal a um ventilador de roedores. Ventilar o animal com um volume de 2 cm 3 a uma taxa de 100/min com ar umidificado. A pressão expiratória final positiva de 1 cm H 2 O deve ser mantida para evitar o colapso do pulmão. A cada 15 minutos hyperinflate dos pulmões para prevenir atelectasias. Em seguida, cobrir a superfície do cérebro com óleo mineral aquecido (30 ° C). Tenha cuidado para evitar o seio venoso grande localizado diretamente sob a junção entre o parietal e ossos interparietal. </li><li> Aplique cola de cianoacrilato a uma haste acoplada a um vibrador eletromagnético e anexar a vara no diastema da mandíbula. Mover a mandíbula sinais de comando usando o vibrador de qualquer um A saída do computador / D ou um gerador de sinal. </li><li> Coloque um siliver-prata cloreto de eletrodo de aterramento sob a pele adjacente à craniotomia. </li></ol> 2. Preparação do eletrodo <ol><li> Fabricar microeletrodos de vidro de quartzo ou de alumínio usando um extrator de microeletrodos horizontal. </li><li> Preencher o microeletrodo com biotinamide 5-10% dissolvido em 0,25 m e KCl 0.5M Tris HCl (pH 7,6) ou tetramethlyrhodamine 2% em solução salina (pH 6). Verificação da impedância eletrodo com um testador de eletrodo. Para gravar a partir de axônios de grande diâmetro tornar eletrodos com uma impedância de 60-80M Ω, por axônios de pequeno e interneurônios fazer eletrodos com impedância de 80 150MΩ. </li><li> Coloque o microeletrodo no palco cabeça do eletrômetro. Visualize o eletrodo através de um pequeno telescópio (20x com retículo em anexo) que é fixado na posição atrás da cabeça do animal. Usando o telescópio é importante porque permite que os eletrodos para ser stereotaxically posicionados com grande precisão. </li></ol> 3. Gravação eletrofisiológicos e coloração intracelular <ol><li> Faça uma pequena abertura na pia-máter e compensar o feedback capacitância de modo que quando o eletrodo toca o cérebro um sinal de realimentação é produzida. Isto permite a localização exata da superfície do cérebro. </li><li> Produzir repetido deslocamento mandibular e avançar o eletrodo no cérebro com um motor de passo. </li><li> Reconhecer impalements neuronal por quedas bruscas no potencial DC e identificar intrassomáticas impalements neuronal pela atividade sináptica em curso. Zumbindo o eletrodo com uma sobrecompensação dos capacitância ou tocando raramente produzem uma penetração celular de sucesso. Devido à alta impedância dos eletrodos, as respostas neuronal raramente são observados antes da penetração. </li><li> Depois de empalar um neurônio ea penetração é considerado estável, caracterizar a resposta neuronal usando rampa e segurar e movimentos da mandíbula sinusoidal. 5 Mapa do campo receptivo do neurônio sondando a pele ao redor da cabeça e cavidade oral intra com uma sonda não-condutor como um palito de madeira. Se relevantes para o estudo, examinar a resposta do neurônio para outro funcionalmente estímulos relevantes, tais como a contração muscular e estímulos nocivos. Aferentes nociceptivos primários seis receptores neuronais responder a estímulos nociceptivos. Anesthesetics geral, tais como pentobarbital sódico não bloqueio da condução axonal em neurônios aferentes primários, mas sim diminuir a transmissão sináptica. Assim condução axonal no axônio aferente primário neuronal é preservada. </li><li> Injetar corrente contínua (DC, 1-4NA) para um tempo de injeção total de 15 70nA minutos. Monitorar a penetração do eletrodo durante a injeção de corrente e interromper o potencial de membrana se torna mais positivo do que-30mV.</li></ol> 4. Processamento de tecidos <ol><li> Eutanásia do animal por overdose de pentobarbital (140mg/kg IV) e perfundir com um enxágüe vascular (0,9% NaCl 38 ° C, contendo 500 unidades de heparina e 1 ml de xilocaína 2% seguido de paraformaldeído 4% em tampão fosfato 0,1 M (pH 7,4) . </li><li> Remover o cérebro e seção, em 50-100μm usando um vibratome em qualquer plano, frontal sagital ou horizontal. Secions são coletados livre flutuação em 25 ° C PBS. </li><li> Processo do cérebro para DAB, incubando em soro de cabra normal e 1-2% 1% Triton X-100 em PBS 0,01 M seguido de incubação em avidina biotina (1:50 Elite Vectastain). Reagir seções usando níquel DAB com H 2 O 2. Ao processo de Texas Red, seções incubar no complexo avidina-biotina (1:50) em PBS overnight a 4 ° C e incubar em 4% Texas Red avidina DCS em PBST a 4 ° C durante a noite. Contracoloração seções com uma fluorescente Nissl mancha (NeuroTrace) 20min a 23 ° C. </li><li> Se neurônio foi injetado com rodamina, visualize o neurônio injetado diretamente com um microscópio de fluorescência (Ex 545nm). </li></ol> 5. Combinando com o método de rotulagem retrógrada, imunocitoquímica, imagem confocal, análise quantitativa co-localização O sucesso de combinar esta técnica com outros métodos, é em grande parte dependente de rotulagem intracelular bom. <ol><li> O método visualizadas aqui podem ser facilmente combinados com rotulagem neuronal retrógrada. 7-10 Para fazer isso, anestesiar o animal e utilizando uma técnica asséptica, injetar um traçador neuronal, tais como peroxidase (20% peroxidase. Sigma VI, Sigma) e 1% gérmen de trigo aggultinated peroxidase (Sigma) em regiões-alvo periféricos, tais como o músculo masseter. Este marcador neuronal será retomada em axônios periféricos e transportados através de transporte axonal ao somata motoneurônio. Para o músculo masseter, 15μl de traçador é injetado no músculo, utilizando uma micro microlitro estéril 10. Os animais são então colocados em uma almofada de aquecimento e monitorado até a recuperação anestésica e, em seguida, colocados em suas gaiolas para a recuperação. Após 24h, anestesiar animais e caracterizar fisiologicamente neurônios e intracelularmente manchá-las. Então perfundir o animal, como descrito acima e cortes de tecidos processo para a presença de HRP utilizando tetrametilbenzidina (TMB) como o Chromagen e tungstato de sódio como estabilizador e intensificou-se com cobalto. Seções lugar no tungstato de sódio a 1% dissolvida em 0,1 M PBS (pH 6,5) e TMB 0,0007% dissolvida em etanol absoluto e acetona a 15 ° C por 20 min. Então reagir cortes de tecidos, adicionando 1,0 ml de 0,3% H 2 O 2 por 100 ml de solução de incubação por 60 min. Enxaguar tecido em 0,1 M PBS (pH 6,5) e coloque em diaminobenzidina 0,05% (pH 7,4), cobalto 0,02% e 0,01% H 2 O 2 0,1 M em PBS (pH 7,4) por 10 min. a 37 ° C. Processo o tecido para a visualização do neurônio injetados com biotinamide como descrito e visualizar as relações entre o neurônio sensorial rotulados e uma variedade de motoneurônios. </li><li> A técnica mostrada aqui também pode ser facilmente combinada com immnocytochemistry. 6 Como exemplo, immunocytochemically processo do cérebro para sinaptofisina para localizar com precisão as sinapses nos neurônios marcados (Fig. 3). Para fazer isso, incubar seções do cérebro do rato em anticorpos anti-sinaptofisina (1:10.000) para 2 dias a 4 ° C e incubar em anti-camundongo FITC (1:400) por 1h a 23 ° C. </li><li> Neurônios marcados com marcadores fluorescentes ou processados ​​para imagens fluorescentes são ideais para imagens confocal. Figura 3 é um exemplo de uma secção óptica obtidos por microscopia confocal através de um bouton axônio. (Fig. 3). Gerar animações de neurônios rotuladas através da aquisição de várias secções ópticas através do neurônio rotulada (Fig. 4). </li><li> Neurônios marcados com este método pode ser usado para a análise de co-localização quantitativa. Para fazer isso, use uma macro software disponíveis publicamente em conjunto com o NIH Imagem (encontrado em http://phy.ucsf.edu/ ° IDL / colocalization.htm). Localize sinaptofisina dentro de terminais de axônio único, combinando rotulagem intracelular com imunocitoquímica sinaptofisina 6. </li></ol> 6. Resultados representativos: Uma visão geral dos resultados representativos que podem ser obtidos através deste método são ilustrados na Figura 1. Este único neurônio do tronco cerebral foi eletrofisiologicamente registrados durante o movimento da mandíbula e, como pode ser visto claramente, a resposta deste neurônio (Figura 1 inferior esquerdo, azul claro) foi modulada durante o movimento. Este neurônio foi injetado com biotinamide após caracterização eletrofisiológica e posteriormente processadas para visualização. O neurônio reconstruído (Figura 1 médio, verde) pode ser realted de um marco anatômico, neste caso, o núcleo motor do trigêmeo designado (contorno vermelho). Com base na resposta neuronal durante o movimento ea reconstrução deste neurônio pode ser identificado como um neurônio secundário do fuso muscular aferentes. A figura 2 ilustra um exemplo representativo da resposta fisiológica de um neurônio durante o deslocamento da mandíbula. A resposta do neurônio é representado como frequência de disparo instantâneo. Note-se que a resposta neuronal imita o deslocamento mandibular, indicando que esse neurônio particular oferece feedback sensorial relacionado à posição mandibular. Figura 3 é uma imagem de alta ampliação de um axônio intracelularmente manchada combinado com coloração para sinaptofisina e uma mancha de Nissl. Observe a co-localização de sinaptofisina (amarelo) dentro da bouton axônio. Figura 4 é uma animação de um neurônio único, caracterizado fisiologicamente e intracelularmente rotulados. <img src="/files/ftp_upload/2650/2650fig1.jpg" alt="Figura 1"/> A Figura 1. Visão geral do método. Superior esquerdo: deslocamento mandibular. Gravação Intracelular meio (verde) de único neurônio (amarelo). A morfologia deste neurônio foi reconstruída após a gravação intracelular e injeção. Contorno vermelho indica a localização do núcleo motor do trigêmeo. Canto inferior esquerdo: resposta fisiológica deste neurônio durante o movimento da mandíbula. <img src="/files/ftp_upload/2650/2650fig2.jpg" alt="Figura 2"/> Figura 2. Resposta fisiológica Representante de um neurônio único músculo sensoriais gravadas in vivo durante o movimento da mandíbula. Observe a semelhança da resposta neuronal com deslocamento da mandíbula. <img src="/files/ftp_upload/2650/2650fig3.jpg" alt="Figura 3"/> Figura 3. Terminal arborização axonal com boutons sináptica (inchaços vermelhos) de um neurônio sensorial intracelularmente manchada que responderam durante muscular sondagem. Processamento imunocitoquímicos subseqüentes para sinaptofisina mostra localização de sinaptofisina no bouton axonal (amarelo). Verde é uma mancha fluorescente Nissl. Figura 4. Animation do músculo axônio neurônio fusiforme aferentes primários cuja resposta fisiológica foi gravado in vivo durante o movimento mandibular, o axônio foi então intracelularmente manchadas e processados ​​para visualização. <a href="/files/ftp_upload/2650/collateral4.avi">Download de um vídeo de alta resolução da Figura 4 aqui</a> <a href="/files/ftp_upload/2650/medres.mov">Download de um vídeo de média resolução Figura 4 aqui</a>

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	<li>Cuellar, C. A., Tapia, J. A., Juarez, V., Quevedo, J., Linares, P., Marinez, L., Manjarrez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+sinusoidal+electrical+waves+along+the+spinal+cord+during+a+fictive+motor+task.">Propagation of sinusoidal electrical waves along the spinal cord during a fictive motor task.</a> J. Neurosci. 29, 798-810 (2010).</li><li>Frigon, A., Gossard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+specialized+rhythm-generating+mechanisms+in+the+adult+mammalian+spinal+cord.">Evidence for specialized rhythm-generating mechanisms in the adult mammalian spinal cord.</a> J. Neurosci. 30, 7061-7071 (2010).</li><li>Wang, W., Chan, S. S., Heldman, D. A., Moran, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+cortical+representation+of+hand+translation+and+rotation+during+reaching.">Motor cortical representation of hand translation and rotation during reaching.</a> J. Neurosci. 30, 958-962 (2010).</li><li>Ma, C., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+investigating+cortical+control+of+stand+and+squat+in+conscious+behavioral+monkeys.">A method for investigating cortical control of stand and squat in conscious behavioral monkeys.</a> J. Neurosci. Meth. 192, 1-6 (2010).</li><li>Dessem, D., Donga, R., Luo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary-+and+secondary-like+jaw-muscle+spindle+afferents+have+characteristic+topographic+distributions.">Primary- and secondary-like jaw-muscle spindle afferents have characteristic topographic distributions.</a> J. Neurophysiol. 77, 2925-2944 (1997).</li><li>Dessem, D., Moritaini, A., Ambalavanar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+craniofacial+muscle+primary+afferent+neurons+synapse+in+both+the+rostral+and+caudal+brain+stem.">Nociceptive craniofacial muscle primary afferent neurons synapse in both the rostral and caudal brain stem.</a> J. 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Neurol. 128, 451-459 (1999).</li><li>Luo, P., Wong, R., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+of+jaw-muscle+spindle+afferents+to+the+caudal+brainstem+in+rats+demonstrated+using+intracellular+biotinamide.">Projection of jaw-muscle spindle afferents to the caudal brainstem in rats demonstrated using intracellular biotinamide.</a> J. Comp. Neurol. 358, 63-78 (1995).</li><li>Luo, P., Dessem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+anatomy+of+physiologically+identified+jaw-muscle+spindle+afferent+terminations+onto+retrogradely+labeled+jaw-elevator+motoneurons+in+the+rat.">Ultrastructural anatomy of physiologically identified jaw-muscle spindle afferent terminations onto retrogradely labeled jaw-elevator motoneurons in the rat.</a> J. Comp. Neurol. 406, 384-401 (1999).</li><li>Hassani, O. K., Henny, P., Lee, M. G., Jones, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+intermingled+with+orexin+and+MCH+neurons+in+the+lateral+hypothalamus+discharge+maximally+during+sleep.">GABAergic neurons intermingled with orexin and MCH neurons in the lateral hypothalamus discharge maximally during sleep.</a> Eur. J. Neurosci. 32, 448-457 (2010).</li><li>Inokawa, H., Yamada, H., Matsumoto, N., Muranishi, M., Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juxtacellular+labeling+of+tonically+active+neurons+and+phasically+active+neurons+in+the+rat+striatum.">Juxtacellular labeling of tonically active neurons and phasically active neurons in the rat striatum.</a> Neuroscience. 168, 395-404 (2010).</li></ol>

Experimentos em animais foram realizados de acordo com o guidlines e regulação estabelecidas no Guia para o Cuidado e Uso de Animais de Laboratório (NIH Publication No. 86-23, revisado em 1985) e da Universidade de Maryland Animal Care e Use Comitê.

O método ilustrado aqui é uma poderosa técnica que fornece informação importante sobre a função dos neurônios individuais e como a resposta de neurônios individuais contribui para circuitos neuronais. 9 Este conhecimento é fundamental para a compreensão da função sensório-motor. A maior força desta técnica é que permite a determinação é de um grande número de parâmetros sobre um neurônio, incluindo a fisiologia, a morfologia ea morfologia e distribuição sináptica. Quando combinado co...

<table border="1"><tbody><tr><th> Nome do reagente ou equipamento </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> vibrador eletromagnético </td><td> Ling Dynamic Systems </td><td> V101 </td><td></td></tr><tr><td> gerador de sinal </td><td> Sistemas de Medição </td><td> PFG605 </td><td> capaz de produzir o sinal de saída trapezoidal </td></tr><tr><td> eletrodo de vidro </td><td> Sutter Instruments </td><td> AF100-68-10 </td><td> com filamento </td></tr><tr><td> extrator eletrodo </td><td> Sutter Instruments </td><td> Modelo P-2000 ou P-80 </td><td></td></tr><tr><td> biotinamide </td><td> Vector Laboratories </td><td> SP-1120 </td><td> armazenados a 4 ° C </td></tr><tr><td> Texas Red avidina DCS </td><td> Vector Laboratories </td><td> A-2016 </td><td></td></tr><tr><td> tetramethlyrhodamine </td><td> Sondas moleculares </td><td> D-3308 </td><td> 3000 o peso molecular, lisina fixable </td></tr><tr><td> rato de anticorpos anti-sinaptofisina </td><td> Chemicon </td><td> MAB5258 </td><td></td></tr><tr><td> fluorescentes mancha Nissl </td><td> Neurotrace, Molecular Probes </td><td> N-21480 </td><td></td></tr><tr><td> testador de eletrodo </td><td> Winston Eletrônica </td><td> BL-1000-B </td><td> para medir a impedância do eletrodo </td></tr><tr><td> eletrômetro </td><td> Axon Instruments </td><td> Axoprobe 1A, 2B Axoclamp </td><td></td></tr></tbody></table>

physiological, morphological and neurochemical characterization of neurons modulated by movement

O papel dos neurônios individuais e sua função nos circuitos neuronais é fundamental para a compreensão dos mecanismos neuronais das funções sensoriais e motoras. A maioria das investigações de mecanismos sensório-motor dependem, quer o exame de neurônios ao mesmo tempo um animal é 1,2 estática ou atividade neuronal registro extracelular durante um movimento. 3,4 Embora esses estudos forneceram a base fundamental para a função sensório-motor, que não quer avaliar as informações funcionais que ocorre durante um movimento ou estão limitados na sua capacidade de caracterizar plenamente o fenótipo anatomia, fisiologia e neuroquímica do neurônio. A técnica é mostrada aqui, que permite a caracterização extensa de neurônios individuais durante um em movimento vivo. Esta técnica pode ser usada não só para estudar neurônios aferentes primários, mas também para caracterizar os motoneurônios e interneurônios sensório-motor. Inicialmente, a resposta de um único neurônio é gravado usando métodos eletrofisiológicos durante vários movimentos da mandíbula seguido por determinação do campo receptivo para o neurônio. Um traçador neuronal é, então, intracelularmente injetado no neurônio e do cérebro é processada de modo que o neurônio pode ser visualizado com microscopia de luz, elétron ou confocal (Fig. 1). A morfologia detalhada do neurônio é caracterizado depois reconstruída de modo que a morfologia neuronal pode ser correlacionada com a resposta fisiológica do neurônio (Figs. 2,3). Nesta comunicação detalhes importantes e dicas importantes para a implementação bem sucedida dessa técnica são fornecidos. Informações adicionais valiosas pode ser determinada para o neurônio em estudo através da combinação deste método com outras técnicas. Rotulagem neuronal retrógrada pode ser utilizada para determinar os neurônios com que as sinapses dos neurônios rotuladas, permitindo assim a determinação detalhada dos circuitos neuronais. Imunocitoquímica pode ser combinado com este método para examinar neurotransmissores no neurônio rotulados e para determinar os fenótipos químicos de neurônios com que as sinapses dos neurônios marcados. O neurônio marcado também podem ser processados ​​para microscopia eletrônica para determinar as características ultra-estruturais e micro circuitos do neurônio rotulados. Geral esta técnica é um método poderoso para caracterizar completamente os neurônios durante em movimento vivo, permitindo assim uma visão substancial sobre o papel do neurônio em função sensório-motor.

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Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

A técnica é descrita para quantificar a resposta fisiológica em vivo dos neurônios de mamíferos durante o movimento e correlacionar a fisiologia do neurônio com a morfologia neuronal, fenótipo neuroquímico e microcircuitos sináptica.

Caracterização fisiológica, morfológica e neuroquímicos de Neurônios Modulada por Movimento

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

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13.7K visualizações.  University of Maryland. A técnica é descrita para quantificar a resposta fisiológica em vivo dos neurônios de mamíferos durante o movimento e correlacionar a fisiologia do neurônio com a morfologia neuronal, fenótipo neuroquímico e microcircuitos sináptica.

Vídeo: Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Monitoramento Eye Movement of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

Neurociência

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Coerência entre a função cerebral cortical e Desempenho neurocognitiva durante condições de gravidade mudou

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Determinação rápida do limiar térmico nociceptivo em ratos diabéticos

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Odorante induzidas respostas registradas a partir de neurônios receptores olfativos utilizando a técnica de sucção da pipeta

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Detecção de palmitoylation proteína em neurônios cultivados por imunoprecipitação e Acil-Biotina Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

Medição de cálcio total em neurônios por Electron Probe X-ray Microanálise

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Lineage-reprogramação de células pericyte derivados do Adulto Cérebro Humano em neurônios induzidos

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Eletrofisiológica e Caracterização Morfológica de Neuronal Microcircuits em aguda fatias do cérebro do Usando Emparelhados patch-clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Funcional e morfológica de Avaliação de Diafragma Innervation por frênico Neurônios Motores

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Imunocoramento Secções biocitina-preenchidos e processados ​​para neuroquímicos Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...