Agradecemos ao Dr. Dan Verde para a prestação de apoio técnico durante o desenvolvimento destas técnicas experimentais. Este trabalho foi financiado pelo NIH Grant R01-EY016849A e da Família Smith New Investigator Award.

Parte I: Fabricação de tungstênio-in Glass-Eletrodos <ol><li> Um anel de grafite é colocado em um copo cheio de uma solução concentrada de nitrato de sódio (0.71g/mL) e hidróxido de potássio (0.34g/mL) dissolvido em água 1, eo copo é colocado sob o braço de costura de uma máquina de costura modificado . </li><li> Fio de tungstênio (0,5 mm de diâmetro, comprimento 6cm) é gravado para o braço de costura para que ele seja parcialmente imerso (1-2cm) em solução. </li><li> 2V são aplicados em todo o fio de tungstênio, anexando uma liderança positiva para o braço de costura e uma liderança negativa para o anel de grafite. </li><li> A máquina de costura é ligado e se o fio de tungstênio é mergulhado repetidamente na solução a uma taxa de aproximadamente 4Hz para 2 min, o que eletroliticamente grava o metal a uma ponta afiada. </li><li> O fio gravado é colocado em um micromanipulador manual eo ponto alinhado sob a orientação microscópica (40x) com o eixo de uma pipeta de vidro (0,25 mm OD, 0,175 milímetros ID) puxado para uma ponta de 1 milímetro com um puxador de vidro. Para impedir o movimento durante a inserção do fio, o vidro está temporariamente preso ao estágio do microscópio com putty. </li><li> O arame é cuidadosamente avançado na extremidade unpulled do vidro, para baixo o eixo, e através da abertura 1 Hm Um pequeno pedaço de vidro é visto como quebrar a ponta no vídeo, indicando um ajuste apertado do tungstênio no vidro. O fio deve se estender em torno de 5-10mm além da ponta para as gravações de ratos de fibra óptica. </li><li> Cianoacrilato é aplicada ao final unpulled do vidro e após o endurecimento da impedância elétrica é medido, onde eletrodos 1MΩ geralmente funcionam melhor. </li></ol> Parte II: Setup estereotáxica e Gravações Train de Spike <ol><li> O sistema de exibição de estímulo é ilustrado no vídeo, junto com o aparelho estereotáxico e microdrive para o posicionamento e diminuindo o eletrodo no cérebro. </li><li> Para realizar gravações de trem da retina spike o rato é anestesiado e, mais tarde paralisado. Procedimentos-padrão (não ilustrado) são usados ​​para a inserção cirúrgica de uma cânula veia femoral para a entrega da droga e uma cânula (Y-tubo) traqueal para ventilação mecânica durante a paralisia. Os experimentos são terminal para o animal. </li><li> Após os procedimentos de preparação, o animal é colocado sobre o cobertor de aquecimento ea cabeça é fixada na posição estereotáxica, como definido pelo cérebro de rato atlas 2, com orelha e barras de dente. </li><li> A sonda retal é inserido, que controla a manta de aquecimento através de uma unidade de controle homeotérmicos que mantém a temperatura corporal a 37 ° C. </li><li> Agulha de metal são usados ​​como leva a medir a freqüência cardíaca e eletrocardiograma monitor. </li><li> A pele é aberto na parte superior do crânio e da interseção de placas de crânio (bregma) é exposto. Um quadro de trave de design personalizado é fixado no local, um círculo é marcado no crânio em torno begma através de um buraco na trave e no travessão está temporariamente removidos. </li><li> O sistema é calibrado estereotáxica, posicionando a agulha guia sobre bregma. Um buraco de 5 mm é no crânio perfurado no local marcado e osso é removida, expondo o cérebro. Um anel fina de massa é colocada em torno da abertura no crânio para formar uma vedação em torno do buraco na trave, que é reposto no lugar. </li><li> Se o animal tem estado em um avião estável de anestesia por algum tempo, é injetado com paralítico a fim de impedir os movimentos dos olhos durante a coleta de dados. Após a respiração pára o animal está sob ventilação mecânica. Um programa personalizado faixas parâmetros ventilatórios e fisiológicos durante todo o experimento e informa o pesquisador se a ação compensatória é necessário nenhum parâmetro deve ir para fora dos níveis normais. </li><li> A agulha guia é atrasaram com o eletrodo de tungstênio e baixou para o cérebro. Após a penetração do cérebro do buraco barra está cheia de agar (não mostrado), eo conjunto microdrive é preso à trave para a estabilidade mecânica. O eletrodo de tungstênio é então lentamente avançado (2μm/sec) a agulha guia até a ponta é de cerca de 8-9mm abaixo do crânio, onde o quiasma óptico está localizado. </li><li> Trens de Spike pertencentes a células ganglionares da retina são identificados por sua resposta a estímulos visuais. Após o isolamento de uma única fibra óptica do campo receptivo da célula registrado é mapeado no espaço visual e suas propriedades de resposta são caracterizados com grades à deriva ou outros padrões visuais de interesse. A estabilidade do sistema permite a única unidade de gravações de várias horas 3. </li></ol> Materiais Adulto ratos Brown-Norway (250-400g) foram comprados de um fornecedor comercial e alojados em um mercado regulamentado (12/12) ciclo claro / escuro. A anestesia foi induzida com uma injeção intraperitoneal de cloridrato de ketamina e xilazina (70 e 2 mg / kg, respectivamente, Henry Schein Inc) e mantidos para a duração da experiment com uma infusão intravenosa de ketamina e xilazina (30 e 1 mg / kg / hr) misturado com dextrose, soro fisiológico, e trietiodeto galamina (40 mg / kg / hr, Fischer Inc) através de um cateter (0,13 milímetros OD, Small Parts Inc ) na veia femoral direita. Galamina está incluída na mistura para paralisar os movimentos dos olhos. A taxa de infusão da bomba (WPI Inc) foi ajustada conforme necessário para manter um avião estável de anestesia, avaliada pela freqüência cardíaca e variabilidade da pressão arterial. Depois de paralisia, o animal era ventilado mecanicamente (Harvard Apparatus, Modelo 683) através de uma cânula traqueal em 60-80 respirações / min (2cc volume), com a taxa ajustada conforme necessário para manter end-tidal CO 2 medido com uma linha de in- capnometer (Novametrix Inc, modelo 710Sp) a 30%. Temperatura corporal foi regulamentada através de um sistema homeotérmicos cobertor controle (Harvard Apparatus Inc). Temperatura corporal, end-tidal freqüência cardíaca CO 2, e pressão arterial foram continuamente monitorizadas durante todo o experimento por um programa LABVIEW. Estímulos visuais foram apresentados em um Sony Multiscan 17e monitor CRT (média de luminância de 30 cd / m 2) rodando a 100 Hz com uma resolução de 800x600 pixels. Aquisição de dados e saída do monitor foram controlados com software personalizado escrito em Matlab e LabView em conjunto com um processador de imagem de vídeo (Cambridge Research Systems Inc, Bits + +) e caixa de ferramentas Psicofísica 4. O animal viu a tela de estímulo (40,4 x 30,2 cm) a uma distância de 16,5 centímetros por meio de lentes de contato (Ocular Instruments Inc), e solução oftálmica foi aplicado periodicamente durante o experimento para manter os olhos úmidos. O campo visual acessível a estimulação foi maximizada por inverter a montagem do animal em um aparelho estereotáxico (Stoelting Co) 14 centímetros elevada acima da superfície de uma mesa flutuante (TMC Inc) e configuração do monitor em um trenó personalizados que se movia sobre a mesa, juntamente um arco ± 100 graus sobre o nariz. Isto proporcionou um campo de exibição repositionable que se estendeu de 60 graus acima e 35 graus abaixo do nível dos olhos e ± 42 graus lateralmente do centro de monitor. Electodes tungstênio foram fabricadas com fios obtidos a partir de pequenas peças Inc e vidro de borosilicato personalizado obtido a partir de Friedrich e Inc. Dimmock Um padrão micropippete extrator (Sutter Instruments, modelo P-97) foi usado para dar forma a ponta de vidro. O testador da impedância do eletrodo foi comprado de Bak-Electronics. Eletrodos foram avançadas com um microdrive sistema motorizado (Newport Inc, StepperMike). Electrocardiograma e sinais de nervo óptico foram amplificados e filtrados com um amplificador de alta impedância de entrada multieletrodo (FHC Inc, X-Cell 3x4). Batimentos cardíacos foram detectados com um discriminador janela (WPI Inc, modelo 121). Potenciais de ação do nervo foram detectados e hora com resolução de 0,1 ms por um pico digitais discriminador (FHC Inc, APM). O discriminador pico foi fechado por um sinal de disparo do processador de imagem de vídeo para que os tempos estavam trancadas pico ao estímulo.

<ol>
	<li>Levick, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+tungsten+microelectrode.">Another tungsten microelectrode.</a> Med Biol Eng. 10 (4), 510-515 (1972).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rat brain in stereotaxic coordinates. , (1998).</li><li>Freeman, D. K., Heine, W. F., Passaglia, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maintained+discharge+of+rat+retinal+ganglion+cells.">The maintained discharge of rat retinal ganglion cells.</a> Vis Neurosci. 25 (4), 535-548 (2008).</li><li>Brainard, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+psychophysics+toolbox.">The psychophysics toolbox.</a> Spat Vis. 10, 433-436 (1997).</li></ol>

Todos os procedimentos experimentais foram aprovados pelo Institutional Animal Care e do Comitê Use Universidade de Boston.

Gravações de fibra óptica são uma abordagem atrativa para abordar questões experimental sobre a codificação e transmissão de informação da retina que requerem um olho intacto. Além disso, as propriedades de sinalização de ambos os olhos pode ser estudada em praticamente no mesmo estado fisiológico se o eletrodo está posicionado no quiasma óptico e das vias onde a atividade de cruzadas e descruzadas fibras do nervo óptico pode ser gravado com um único eletrodo de penetração. Gravações de fibra ópti...

single-unit in vivo recordings from the optic chiasm of rat

Informações sobre o mundo visual é transmitida para o cérebro em seqüências de potenciais de ação em axônios de células ganglionares da retina que compõem o nervo óptico.<em&gt; In vivo</em&gt; Gravações de trens de células ganglionares da espiga em diversos modelos animais revelaram muito do que se sabe sobre como o início dos processos do sistema visual e codifica a informação visual. No entanto, essas gravações, têm sido raros em um dos modelos animais mais comuns, o rato, possivelmente devido à dificuldade em detectar picos disparados por axônios de pequeno diâmetro. Os muitos modelos de doenças da retina envolvendo ratos motivar a necessidade de caracterizar as propriedades funcionais de células ganglionares, sem perturbar o olho, como com intra-ocular ou<em&gt; In vitro</em&gt; Gravações. Aqui, nós demonstramos um método para a gravação de células ganglionares trens pico do quiasma óptico do rato anestesiado. Primeiro mostramos como fabricar tungstênio em vidro eletrodos que pode pegar a partir de atividade elétrica dos axônios gânglio única célula de rato. Os eletrodos superar todos os comerciais que temos tentado. Em seguida, ilustrar o nosso sistema de design personalizado para estereotáxica<em&gt; In vivo</em&gt; Experimentos visuais neurofisiologia e nossos procedimentos para a preparação de animais e colocação de eletrodos fiável e estável no quiasma óptico.

Watch this Scientific Journal Video about Single-unit In vivo Recordings from the Optic Chiasm of Rat at JoVE.com

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Células ganglionares da retina transmitir informações visuais do olho para o cérebro com seqüências de potenciais de ação. Aqui, demonstramos como gravar os potenciais de ação das células ganglionares única<em&gt; In vivo</em&gt; De ratos anestesiados.

Única unidade de In vivo Gravações do quiasma óptico de rato

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

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Research

JoVE Journal

Neuroscience

Single-unit In vivo Recordings from the Optic Chiasm of Rat

11.6K visualizações.  Boston University. Células ganglionares da retina transmitir informações visuais do olho para o cérebro com seqüências de potenciais de ação. Aqui, demonstramos como gravar os potenciais de ação das células ganglionares única In vivo De ratos anestesiados.

Vídeo: Single-unit In vivo Recordings from the Optic Chiasm of Rat

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

As gravações eletrofisiológicas da via fibra Gigante da D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Neurociência

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

Um Procedimento para Implantação de Arrays organizado de microfios para gravações a única unidade em Awake, comportando-se Animais

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

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

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

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

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

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

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

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Técnica minimamente invasiva para a injeção em Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Visual Gravação Potencial Evocado em um modelo de rato de Experimental Nervo Óptico desmielinização

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

Determinação dos fotorreceptores celular Sensibilidade espectral em um inseto modelo de<em&gt; In Vivo</em&gt; Gravações intracelulares

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Agudo<em&gt; In Vivo</em&gt; Gravações eletrofisiológicas de Potenciais de campo locais e atividade de múltiplas unidades a partir do caminho hiperdiretivo em ratos anestesiados

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Gravações simultâneas de potenciais de campo corticais Local, eletrocardiograma, eletromiograma e respirar o ritmo de um rato andando livremente

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined curved surfaces, and images of real-world objects (with and without disparity) in a similar manner as described in the ventral visual stream. In addition, parieto-frontal areas are believed to convert visual object information into appropriate motor outputs, such as the pre-shaping of the hand during grasping. To better characterize object selectivity in the cortical network involved in visuomotor transformations, we provide a battery of tests intended to analyze the visual object selectivity of neurons in parieto-frontal regions.

A detailed protocol to analyze object selectivity of parieto-frontal neurons involved in visuomotor transformations is presented.

Investigar as representações do objeto no fluxo Dorsal de Visual de Macaque usando gravações unitárias

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined ...