Agradecemos ao Dr. Sanika Chirwa para revisão crítica do manuscrito. Este trabalho foi financiado pelo National Institutes of Health (DA026947, DA021471, e NS071122).

1. Equipamentos e Suprimentos <ol><li> Montar uma gaiola de Faraday em cima da mesa de vibração anti (TMI) para diminuir o ruído de fundo. </li><li> O simultânea patch clamp sistema de gravação amperometria requer um microscópio invertido com excelentes óptica DIC e uma lente de longa distância de trabalho. Ligue o microscópio farol para uma bateria de carro. Esta fonte de luz DC para o sistema irá reduzir ainda mais o ruído eléctrico. </li><li> Micromanipuladores hidráulicos (Siskiyou) nova redução de ruído. Em nossa configuração, usamos um manipulador destro para a gravação de células inteiras, eo canhoto para amperometria. </li></ol> 2. Prepare Eletrodos para gravação <ol><li> Puxe eletrodos patch usando pipetas de quartzo em um extrator P-2000 (Sutter). Nossa puxar dura cerca de 5 segundos, com dois ciclos de calor. Desta vez, o calor tem resultado em resistência consistente (3-4 MQ) nas nossas pipetas remendo de célula completa. </li><li> Preencha o eletrodo com opipeta solução contendo 2 mM de dopamina e montá-lo no manipulador direita. Envolva o recipiente que contém a solução de pipeta contendo DA com folha de alumínio. Manter em gelo. A dopamina é oxidável. Manter a solução em gelo, protegida da luz diminuirá a taxa de oxidação da dopamina. </li><li> Remover suavemente a ProCFE (Dagan) de baixo ruído do eléctrodo de fibra de carbono amperométrico a partir da caixa de armazenamento, preencher com mercúrio, para o adaptador de montagem amperométrico (conforme representado na Figura 1) e, em seguida, montar no maniupulator direita. Proteger a ponta da fibra de carbono de danos, segurando a extremidade da fibra de carbono. Inspecione o eletrodo com um microscópio de laboratório para assegurar a dica é limpo e intacto. </li><li> Examinar a integridade do eléctrodo amperométrico, colocando o eléctrodo de vidro em um prato de Petri contendo solução de fundo externa. Gravar uma corrente de linha de base na ausência de dopamina. Adicionar 10 ul de uma solução 1 mM de DA para o prato. A re eletrodo bom amperométricocabos de um aumento da corrente de oxidante. Repetir esta etapa, no início e no fim de cada experiência para fazer o eléctrodo amperométrico certo funciona adequadamente. </li></ol> 3. Prepare a cultura primária neuronal dos neurônios de dopamina ou células de engenharia para expressar transportador de dopamina em pratos de Petri com fundo de vidro <ol><li> Lavar cuidadosamente as células ou neurônios de dopamina três vezes com solução externa quente. </li><li> Monte o fundo de vidro placa de Petri para o palco microscópio. </li></ol> 4. Visualize celular e executar Experimento <ol><li> Encontrar o ponto correto focal para visualizar claramente as células. Colocar uma pressão positiva sobre o eléctrodo de patch. Então, traga os dois eléctrodos para baixo para dentro da solução, e perto da célula. </li><li> Posicionar o eléctrodo amperométrico para a célula seguinte (no lado esquerdo), e o eléctrodo de adesivo no lado direito. </li><li> Alcançar uma vedação gigaohm na célula com o eléctrodo de patch. Ruptura do selo com a sucçãoobter uma configuração de célula inteira. </li><li> 5-8 min para permitir que a diálise da solução interna contendo dopamina para dentro da célula. </li><li> Use desejado passo de tensão ou protocolo de rampa. Simultaneamente adquirir dados a partir de ambos os @ patch pipette @ e ao eléctrodo amperométrico para medir correntes de células inteiras e o transporte reverso de dopamina através do transportador de dopamina, enquanto o potencial de membrana é controlada através da pipeta patch. </li></ol>

<ol>
	<li>Michael, A. C., Ikeda, M., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+contributing+to+the+recovery+of+striatal+releasable+dopamine+following+MFB+stimulation.">Mechanisms contributing to the recovery of striatal releasable dopamine following MFB stimulation.</a> Brain Res. 421, 325-335 (1987).</li><li>Gonon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+and+extrasynaptic+excitatory+action+of+dopamine+mediated+by+D1+receptors+in+the+rat+striatum+in+vivo.">Prolonged and extrasynaptic excitatory action of dopamine mediated by D1 receptors in the rat striatum in vivo.</a> J. Neurosci. 17, 5972-5978 (1997).</li><li>Sulzer, D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+quantal+size+by+presynaptic+mechanisms.">Regulation of quantal size by presynaptic mechanisms.</a> Rev. Neurosci. 11, 159-212 (2000).</li><li>Napolitano, A., Cesura, A. M., Da Prada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+monoamine+oxidase+and+catechol+O-methyltransferase+in+dopaminergic+neurotransmission.">The role of monoamine oxidase and catechol O-methyltransferase in dopaminergic neurotransmission.</a> J. Neural. Transm. Suppl. 45, 35-45 (1995).</li><li>Sulzer, D., Maidment, N. T., Rayport, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+other+weak+bases+act+to+promote+reverse+transport+of+dopamine+in+ventral+midbrain+neurons.">Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons.</a> J. Neurochem. 60, 527-535 (1993).</li><li>Salahpour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+amphetamine-induced+hyperactivity+and+reward+in+mice+overexpressing+the+dopamine+transporter.">Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4405-4410 (2008).</li><li>Khoshbouei, H., Wang, H., Lechleiter, J. D., Javitch, J. A., Galli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+dopamine+efflux.+A+voltage-sensitive+and+intracellular+Na+-dependent+mechanism.">Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism.</a> J. Biol. Chem. 278, 12070-12077 (2003).</li><li>Goodwin, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+methamphetamine+differentially+affect+dopamine+transporters+in+vitro+and+in.">Amphetamine and methamphetamine differentially affect dopamine transporters in vitro and in.</a> , 284-2978 (2009).</li><li>Kahlig, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+induces+dopamine+efflux+through+a+dopamine+transporter+channel.">Amphetamine induces dopamine efflux through a dopamine transporter channel.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 3495-3500 (2005).</li><li>Swant, J., Chirwa, S., Stanwood, G., Khoshbouei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine+reduces+LTP+and+increases+baseline+synaptic+transmission+in+the+CA1+region+of+mouse+hippocampus.">Methamphetamine reduces LTP and increases baseline synaptic transmission in the CA1 region of mouse hippocampus.</a> PLoS One. 5, e11382 (2010).</li><li>Gnegy, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++regulates+amphetamine-induced+dopamine+efflux+and+currents+mediated+by+the+human+dopamine+transporter.">Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter.</a> Mol. Pharmacol. 66, 137-143 (2004).</li></ol>

Não há conflitos de interesse declarados.

Simultânea de tensão-clamp e amperometria tem os seguintes benefícios. Todos os tipos de células são acessíveis e podem ser utilizados para a gravação. A identificação das células neuronais ou onde as gravações são feitas é simples e directa. Em particular, se a célula é marcado por fluorescência por adição de um marcador fluorescente para a proteína de interesse, o experimentador pode facilmente seleccionar a célula alvo ou neuronal. A configuração experimental permite a distribuição uniforme ...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Equipamento </td></tr><tr><td> Anti-vibração tabela w / gaiola de Faraday </td><td> Técnico Manufacturing Corporation </td><td> 63-500 série </td><td> usamos modelo 63-543 </td></tr><tr><td> Invertido microscópio Nikon TE-2000 </td><td> Nikon </td><td> descontinuada </td><td> agora Eclipse Ti </td></tr><tr><td> Dois amplificadores de baixo ruído axopatch 200b </td><td> Molecular Devices </td><td></td><td> 800-635-5577 </td></tr><tr><td> Um andar de entrada 203-CV BU </td><td> Molecular Devices </td><td></td><td> 800-635-5578 </td></tr><tr><td> HL-1-U titular pipeta </td><td> Molecular Devices </td><td></td><td> 800-635-5579 </td></tr><tr><td> Digidata 1440A conversor A / D </td><td> Molecular Devices </td><td></td><td> 800-635-5580 </td></tr><tr><td> Dois manipuladores Siskyou, deixarame destro </td><td> Siskiyou </td><td> MX6600R MX6600L </td><td> 877-313-6418 </td></tr><tr><td> Laser pipeta extrator </td><td> Sutter Instruments </td><td> P-2000 </td><td> 888-883-0128 </td></tr><tr><td> Baixo ruído do eletrodo de fibra de carbono amperométrico </td><td> ProCFE </td><td></td><td> <a href="http://www.dagan.com" target="_blank">www.dagan.com</a> </td></tr><tr><td> Baixo ruído de quartzo pipeta </td><td> Sutter Instruments </td><td> QF100-70-7,5 </td><td> 888-883-0128 </td></tr><tr><td> 12-volts bateria de carro </td><td></td><td></td><td> amplamente disponíveis </td></tr><tr><td> Carregador de bateria automotora </td><td></td><td></td><td> amplamente disponíveis </td></tr><tr><td></td><td></td><td></td><td> Reagente </td></tr><tr><td> Cloreto de sódio (NaCl) </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3375 </td><td></td></tr><tr><td> Dextrose </td><td> Sigma </td><td> G7528 </td><td></td></tr><tr><td> Sulfato de magnésio (MgSO 4) </td><td> Sigma </td><td> M2643 </td><td></td></tr><tr><td> Fosfato de potássio monobásico (KH 2 PO 4) </td><td> Sigma </td><td> P5655 </td><td></td></tr><tr><td> Cloreto de potássio (KCl) </td><td> Sigma </td><td> P9333 </td><td></td></tr><tr><td> Di-hidrato de cloreto de cálcio (CaCl 2 • 2H 2 0) </td><td> Sigma </td><td> 223506 </td><td></td></tr><tr><td> Hexa-hidrato de cloreto de magnésio (MgCl2 • 6H 2 0) </td><td> Sigma </td><td> M2670 </td><td></td></tr><tr><td> EGTA </td><td> Sigma </td><td> E0396 </td><td></td></tr></tbody></table>

single cell measurement of dopamine release with simultaneous voltage-clamp and amperometry

Depois de sua libertação para o sináptica de dopamina fissura, exerce suas propriedades biológicas através de suas metas pré-e pós-sináptica 1. O sinal de dopamina é terminada por difusão 2-3, enzimas extracelulares 4, e transportadores de membrana 5. O transportador de dopamina, localizado na fenda peri-sináptica dos neurônios dopaminérgicos limpa as aminas liberados através de um fluxo de dopamina para dentro (captação). O transportador de dopamina pode também funcionar no sentido inverso para libertar aminas a partir de dentro para fora, num processo denominado de transporte para o exterior ou o efluxo de dopamina 5. . Mais de 20 anos atrás Sulzer et al relataram o transportador de dopamina pode funcionar em dois modos de atividade: para frente (captação) e reverso (efluxo) 5. O neurotransmissor libertado através de efluxo através do transportador pode deslocar uma grande quantidade de dopamina para o espaço extracelular, e tem sido demonstrado que desempenham um papel importante na regulação da dopamina extracelular homeostasis 6. Aqui descreve-se como patch clamp e gravação simultânea amperometria pode ser usado para medir a dopamina libertada através do mecanismo de efluxo com resolução de milissegundos do tempo em que o potencial de membrana é controlado. Por isso, células inteiras actuais e oxidativa (amperométrico) sinais são medidos simultaneamente utilizando um amplificador Axopatch 200B (Molecular Devices, com um filtro passa-conjunto de filtros de Bessel de 1.000 Hz para gravação de células inteiras de corrente). Para a gravação de um amperometria eléctrodo de fibra de carbono está ligado a um segundo amplificador (Axopatch 200B), e é colocado adjacente à membrana de plasma e mantido a 700 mV. As células inteiras e oxidativa (amperométrico) as correntes podem ser registados e a relação de corrente-tensão pode ser gerado utilizando um protocolo de passo de tensão. Ao contrário da calibração habitual amperométrico, que requer a conversão de concentração, a corrente é relatado directamente sem ter em conta o volume de 7 eficaz. Assim, os dados resultantesrepresentar um limite inferior para o efluxo de dopamina porque alguns transmissor é perdido para a solução a granel.

Patch clamp combinado com amperometria pode medir a voltagem-dependente mediada DAT DA efluxo 2A. Figura mostra uma configuração experimental representativo e gravação de DAT mediada DA efluxo quando o meio intracelular e o potencial de membrana são fixadas por uma pipeta de remendo de células inteiras. Utilizando esta técnica, as células que expressam proteínas de YFP-DAT são voltagem fixa com uma pipeta de células inteiras remendo enquanto um eléctrodo amperométrico é colocada sobre a me...

Watch this Scientific Journal Video about Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry at JoVE.com

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

A técnica amperométrica mede a libertação de dopamina a partir de uma única célula, detectando a corrente de oxidante produzido pela oxidação da dopamina espontânea. Braçadeira de tensão simultânea e metodologia amperometria revelar a relação mecanicista entre a &quot;atividade&quot; geral do transportador de dopamina eo papel regulador da atividade no transporte reverso da dopamina.

Medição única célula de liberação de dopamina com simultânea de tensão-clamp e amperometria

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

single-cell-measurement-dopamine-release-with-simultaneous-voltage

Research

JoVE Journal

Neuroscience

13.7K visualizações.  University of Florida. A técnica amperométrica mede a libertação de dopamina a partir de uma única célula, detectando a corrente de oxidante produzido pela oxidação da dopamina espontânea. Braçadeira de tensão simultânea e metodologia amperometria revelar a relação mecanicista entre a "atividade" geral do transportador de dopamina eo papel regulador da atividade no transporte reverso da dopamina.

Vídeo: Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

Mapeamento funcional com MEG e EEG em simultâneo

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

Neurociência

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Pré-sináptica de dopamina Dynamics em fatias de cérebro estriado com Fast-scan voltametria cíclica

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

De uma única célula perfil de imaturos e maduros neurônios da retina

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

A gravação ótica da atividade neural supraliminares com resolução de uma única célula e um único pico

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Eletroencefalografia simultânea, em tempo real de medição da concentração de lactato e Manipulação optogenética da atividade neuronal do córtex cerebral de roedores

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

Estimulação Cerebral Profunda com fMRI simultâneo em Roedores

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Preparação de Neuronal co-culturas com precisão única célula

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

Detecção simultânea de c-Fos Ativação de mesolímbico e mesocortical dopamina Sites Recompensa Após Naive açúcar e gordura ingestão em ratos

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Método eletrofisiológico para gravações de tensão de célula inteira de<em&gt; Drosophila</em&gt; Fotorreceptores

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Músculo levantador Auris Longus Preparação para exame da transmissão Neuromuscular mamíferos sob condições de grampo de tensão

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...