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Potencial de membrana Já em 1902, Bernstein estava lidando com as questões de um potencial de repouso no axônio de uma lula. É intrigante a considerar como essas primeiras idéias e observações de Berstein (1902) e Nernst (1888) mais tarde influenciou pesquisas em fisiologia da membrana. (Veja a revisão por Malmivuo e Plonsey, 1995; também está disponível no www <a href="http://www.bem.fi/book/">http://www.bem.fi/book/</a> ). Há ainda, até hoje, os avanços estão sendo feitas sobre a função de canal de íons e propriedades das membranas biológicas, que são muito importantes na compreensão da fisiologia celular que diz respeito à função dos tecidos, órgãos e sistemas. A comparação dos efeitos experimental e teoricamente derivadas de externa [K +] no potencial de repouso da membrana indica a influência dos íons sobre o potencial de membrana. Experimentos adicionais utilizando esta mesma preparação continuam a ser realizado para abordar questões fundamentais fisiológicas. Alguns se destacam em 1968 por Atwood e Parnas e ainda não foram totalmente resolvidos. Com as técnicas obtidos neste exercício, pode-se avançar para responder a muitas perguntas restantes em outras preparações experimentais, bem como em aplicações fisiológicas relacionadas à medicina e saúde. Nós demonstramos a utilidade de uma preparação de invertebrados modelo para tratar de questões fundamentais pertinentes a todos os animais. Com o conhecimento adquirido sobre os gradientes eletroquímicos de íons neste exercício acima, você pode agora avançar para a excitabilidade das membranas através da análise transmissão sináptica em preparações neuromusculares no lagostim. Medição Responses Synaptic Os detalhes fornecidos para a primeira parte deste laboratório, eo filme associados, deram passos-chave para registro do potencial de membrana e investigar a estrutura muscular. Na segunda parte deste laboratório, a demonstração de dissecção de gravação e transmissão sináptica no NMJs de unidades motoras fásicas e tônicas desde uma exposição de conceitos fundamentais em fisiologia. A exposição a um circuito neural, que pode em parte pode ser usada para explicar comportamentos associados, no animal intacto tem potencial não só para os alunos a investigar várias questões abertas dentro de seus exercícios de laboratório, mas também para futuras pesquisas sobre circuitos neuronais, em um poço preparação de invertebrados estabelecido (Kennedy et al, 1969;. Antonsen e Edwards, 2003) Estas preparações podem também ser usados ​​para investigar facilitação sináptica, depressão e longo prazo, a plasticidade (não foi investigada neste estudo de laboratório). Mesmo dentro de algumas espécies de lagostas, plasticidade neuronal depende das condições de estimulação experimental (Mercier e Atwood, 1989;. Cooper et al, 1998), bem como seu ambiente natural. Até que ponto a capacidade de alterar a eficácia sináptica e dinâmica muscular serve o animal continua a ser investigado. Desde lagostas alterem o seu comportamento em relação à variação sazonal e do ciclo de muda, existem diferenças relativamente a longo prazo a atividade em seus sistemas neuromuscular. Tem sido demonstrado que os terminais do motor fásica nervosas dos músculos mais garra apresentam a morfologia clássica fásica durante o inverno, mas incham e se tornam mais varicosas ao longo do comprimento do terminal durante os meses de verão (Lnenicka 1993; Lnenicka e Zhao, 1991). Alguns estudos iniciais realizados em lagostas gigantes laterais (LG) interneurônios na medula nervosa ventral demonstraram a presença de junções gap (Johnson, 1924; Watanabe e Grundfest, 1961). É bem conhecido que o CO 2 tem um efeito na comunicação elétrica por desacoplamento junções (Arellano et al, 1990). Recentemente, foi demonstrado que o cabo dos nervos e de comunicação dentro do circuito sensório-CNS-motor-músculo, como descrito neste relatório, também é sensível a exposição ao CO 2, indicando a presença de junções gap (Bierbower, 2010; Bierbower e Cooper, 2010) A atividade espontânea da 3 ª raiz motora tem sido um tema desde 1960 quando Eckert (1961) examinou se a tônica disparo órgão receptor muscular estática (MRO) dentro do mesmo segmento ou vizinhas poderiam ser responsáveis ​​para a unidade motora espontânea. Nestes estudos anteriores tornou-se evidente que a atividade foi impulsionada dentro do cordão nervoso ventral (VNC), possivelmente a partir de centros superiores (Eckert, 1961; Kennedy e Takeda, 1965a, b;. Strawn et al, 2000). Pois a presença de CO 2 parou a atividade espontânea, pode-se assumir em algum lugar da unidade para os neurônios motores pode haver junções ou unidade excitatória glutamatérgica. O NMJs são bloqueados ou apresentar diminuição da sensibilidade ao glutamato na presença de CO 2, e podem ser blocoked bem no SNC (Bierbower, 2010; Bierbower e Cooper, 2010; ver também Badre et al, 2005).. A ação de vários neuromoduladores também é facilmente estudado em vários tipos de NMJs (Cooper e Cooper, 2009; Griffis et al, 2000;. Southard et al, 2000;.. Strawn et al, 2000). Além disso, várias influências são exercidas por neuromoduladores no circuito CNS. Tem sido sugerido que a 5-HT e neurônios octopaminergic pode funcionar como &quot;ganho-setters&quot; em alterar a produção de circuitos neuronais (Ma et al, 1992;. Schneider et al, 1996;. Hörner et al, 1997;. Edwards et al., 2002). Ainda há muito trabalho a ser feito antes que possamos entender completamente os efeitos da neuromoduladores nas células-alvo individual. Dado que neuromoduladores diferentes podem trabalhar em conjunto com uma outra, a análise de sua ação mista é uma área para pesquisas futuras (Djokaj et al., 2001). Além disso, poucos estudos, especialmente nos vertebrados, enfrentar os efeitos da neuromoduladores em vias de todo o que pode regular um comportamento específico. Nessa preparação unidade sensorial-CNS-motor pode-se examinar a influência de ambos os estímulos sensoriais e neuromoduladores na atividade dos neurônios motores (Kennedy et al., 1969). Uma vez que tem sido postulado que o 5-HT desempenha um papel na regulação do estado comportamental de lagostins, lagostas, caranguejos e (Livingstone et al, 1980;.. Sneddon et al, 2000), várias tentativas foram feitas para determinar a sua concentração em o VNC, a hemolinfa e nos gânglios isolados de lagostas (Livingstone et al, 1980;. Harris-Warrick e Kravitz 1984;. Fadool et al, 1988). No entanto, houve uma variação considerável nas medidas registradas, impedindo específicas relações dose-resposta, que poderia ser responsável por ações comportamentais. A lagosta com as garras realizada em uma posição elevada e com a cauda debaixo do seu abdômen foi pensado para mostrar uma postura dominar (Livingstone et al., 1980). O estado de flexão abdominal nos lagostins não parece ser a postura que lagostas dominante, dentro de um par, exibir durante as interações sociais ou, mantendo um estatuto dominante hierárquica (Listerman et al., 2000). Lagostas submissa vai mesmo dobra seus abdômens sob a si mesmos como eles retiro de um adversário. Enfiando a cauda como também é visto como uma postura de defesa (Listerman et al., 2000). Esses comportamentos foram prontamente observadas no campo e em laboratório (Bovbjerg, 1953, 1956; Bruski e Dunham, 1987; Li et al, 2000;.. Listerman et al, 2000). Curiosamente, as posturas de comportamento observado em lagostas (Livingstone et al., 1980) são revertidos para a 5-HT e injeções octopamine na lagostas australiano, destructor Cherax (McRae, 1996). Possivelmente, respostas totalmente diferentes seria observado na preparação flexor superficial na lagostas australiano. Além disso, já que o domínio é geralmente relacionados entre tamanho lagostas, seria de esperar de um sistema de resposta muito plástico para rapidamente alteradas as condições sociais (Strawn et al., 2000). A plasticidade em resposta às neuromoduladores em invertebrados é uma área aberta de investigação. Wyttenbach, Johnson, e Hoy (1999) produziram mídia digital e um manual de laboratório para experimentações lagostas diversas envolvendo a mesma muscular apresentados neste relatório, além de preparações outras lagostas. Este é um excelente recurso para os exercícios dos alunos.

<ol><li> Lagostim (Procambarus clarkii). Atchafalaya Biological Supply Co., Raceland, LA., EUA. </li><li> Padrão lagostas salina: Modificado de solução de Van Harreveld (1936). (Em mM) 205 NaCl; 5,3 KCl, 13,5 CaCl 2 2H 2 O, 2,45 MgCl 2 6H 2 O, 5 e HEPES pH ajustado para 7,4. Glutamato, serotonina e dopamina são feitas em solução salina lagostim. Solução fixadora de Bouin foi usado diretamente e azul de metileno é feita em solução salina lagostim. Todos os produtos químicos são obtidos da Sigma Chemical Company (St. Louis, MO). </li><li> Ferramentas de dissecção: Fine # 5 pinças, tesoura fina, titular lâmina de faca, # 26002-20 pinos de insetos (todos obtidos a partir de Ferramentas Ciência Fine (EUA), Inc., 373-G Vintage Park Drive, Foster City, CA 94404-1139) . </li><li> Sylgard de fundo prato de vidro </li><li> Copos (para segurar soluções químicas) </li><li> Sinais elétricos são registrados na linha de uma interface PowerLab 26T para um computador (ADInstruments, Colorado Springs, CO, EUA). Nós usamos software padrão de ADInstruments chamado gráfico ou Âmbito. </li><li> Amplificador modelo 3000 AC / DC para gravações intracelulares, bem como extracelular pode ser usado. </li><li> Dissecando microscópio com função de zoom para gravações intracelular. Focal para a gravação em terminais visualizado um microscópio composto com os objetivos de pé (4 x e 20x) é usado. Um precisa de uma fonte de luz Hg. </li><li> Para gravações intracelular usamos um tubo de vidro capilar (30-31-0 catalogo de FHC, Brunswick, ME, 04011, EUA). O eletrodo intracelular deve ter uma resistência de 20 a 30 mOhm. Eletrodos intracelulares foram preenchidos com KCl 3 M. </li><li> Um eletrodo de sucção é usada para gravar sinais extracelulares. </li><li> Gaiola de Faraday. </li><li> Iluminador de Alta Intensidade (fonte de luz). </li><li> Micromanipulador </li></ol>

membrane potentials, synaptic responses, neuronal circuitry, neuromodulation and muscle histology using the crayfish: student laboratory exercises

O objetivo deste relatório é ajudar a desenvolver uma compreensão dos efeitos causados ​​por gradientes de íons através de uma membrana biológica. Dois aspectos que influenciam o potencial de uma célula de membrana e que nos dirigimos nesses experimentos são: (1) a concentração de iões de K + na parte externa da membrana, e (2) a permeabilidade da membrana aos íons específicos. O lagostim músculos extensores abdominal estão em grupos com alguns sendo tônica (lento) e outros fásica (rápido) em seus fenótipos bioquímicos e fisiológicos, bem como em sua estrutura, os neurônios motores que inervam estes músculos são proporcionalmente diferentes nas características funcionais. Nós usamos estes músculos, bem como o músculo flexor superficial, tônico abdominal para demonstrar as propriedades na transmissão sináptica. Além disso, introduzimos um sensório-motoras CNS-circuito neurônio músculo para demonstrar o efeito de estimulação sensorial cuticular, bem como a influência de neuromoduladores sobre certos aspectos do circuito. Com as técnicas obtidos neste exercício, pode-se começar a responder a muitas perguntas restantes em outras preparações experimentais, bem como em aplicações fisiológicas relacionadas à medicina e saúde. Nós demonstramos a utilidade dos preparativos invertebrados modelo para tratar de questões fundamentais pertinentes a todos os animais.

Watch this Scientific Journal Video about Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises at JoVE.com

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

Os experimentos demonstram um método fácil para que os estudantes ganham experiência em examinar a estrutura muscular, respostas sináptica, os efeitos de gradientes de íons e permeabilidade em potenciais de membrana. Além disso, um sensório-motoras CNS-músculo-circuito é apresentado para mostrar um meio para testar os efeitos de compostos em um circuito neuronal.

Potenciais de membrana, Responses Synaptic, circuitos neuronais, neuromodulação e Histologia Muscle Usando a lagosta: Exercícios Laboratório Student

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

membrane-potentials-synaptic-responses-neuronal-circuitry

Research

JoVE Journal

Neuroscience

58.4K visualizações.  University of Kentucky. Os experimentos demonstram um método fácil para que os estudantes ganham experiência em examinar a estrutura muscular, respostas sináptica, os efeitos de gradientes de íons e permeabilidade em potenciais de membrana. Além disso, um sensório-motoras CNS-músculo-circuito é apresentado para mostrar um meio para testar os efeitos de compostos em um circuito neuronal.

Vídeo: Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Gravação de respostas comportamentais à Reflexão em Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Neurociência

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

Gravações fisiológicos de NMJs Output High and Low no músculo extensor Crayfish Leg

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

Muscular órgãos receptores no abdômen Crayfish: Um Exercício em Laboratório Student Propriocepção

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

Experimentação fisiológicas com o intestino posterior Crayfish: Um Exercício Laboratório de Student

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análise quantitativa de Reposição da vesícula sináptica Piscina em Cultured neurônios granulares do cerebelo usando Dyes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Difusão lateral e Exocitose de proteínas de membrana em neurônios cultivados avaliada através da recuperação de fluorescência e fluorescência perda de fotobranqueamento

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Rápido Micro-iontoforese de glutamato e GABA: uma ferramenta útil para investigar Integração Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

Propriocepção e tensão Receptores em Caranguejo Limbs: Exercícios Laboratório Student

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

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 ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Cultivo In Vivo-gosto murino astrócitos usando o protocolo AWESAM rápida, simples e barato

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...