Este trabalho foi financiado pela empresa de seguros de vida &quot;AG2R-La Mondiale-&quot;, Ecole des Neurociências de Paris (PEV), a Agence Nationale de la Recherche &quot;ANR-09-NEUR-004&quot; no quadro de &quot;NEURON ERA-NET &quot;do 7 º PQ programa pela Comissão Europeia, e da Fundação Pasteur. Sebastien Wagner foi apoiado pela Fundação Letten.

1. Calibração óptico: Power LED de medição <ol><li> Anexar uma matriz de LEDs para um dissipador de calor ativa refrigerada por uma ventoinha e apor este aparato LED / dissipador de calor para uma lente colimadora. </li><li> Troque a lâmpada usada em iluminação com LED brightfield / heatsink / fan / aparelho lente. Este aparelho deve ser cuidadosamente posicionado de modo que o feixe colimado LED viaja ao longo de um caminho reto em direção à lente óptica condensador. Verifique se o dissipador de calor / ventilador está devidamente aterrado para um terreno comum do sistema. </li><li> Unidade de matriz LED com uma fonte de alimentação que pode dar pulsos rápidos e quadrado da corrente. Esta fonte de alimentação pode ser controlado por um pulso TTL 5V proveniente de um gerador de pulsos. </li><li> Centro do feixe colimado ao longo do caminho da luz definida entre o diafragma de campo ea lente condensador. Idealmente, o feixe de LED será ligeiramente sobrecarregue o diafragma de campo totalmente aberto. Tipicamente, um feixe mais firmemente colimado LED vai encher esta abertura menos, e vai produzir morpoder e à custa de uniformidade. Em nossa configuração, aumentamos a uniformidade de luz, escolhendo uma lente colimadora que projetam uma imagem ligeiramente ampliada da matriz de LED no seu plano conjugado no diafragma consenser. </li><li> Atingir a iluminação Kohler, concentrando-se o condensador, para que a imagem do diafragma de campo (o diafragma mais próximo da fonte de luz) é focada na câmara de slice (fig. 1). Um tecido de papel fino lente pode agir como uma tela de projeção para visualizar a imagem focalizada do diafragma de campo em outras profundidades. </li><li> Perfurar uma série de furos de diâmetro conhecido em um material opaco. Coloque um desses pequenos furos sobre o sensor óptico de um poder metros. Coloque o medidor de energia no palco exemplar e centro do medidor de energia sobre a imagem focada do diafragma de campo simplesmente movendo o medidor de energia até que dá uma leitura máxima. Apor metros o poder nesta posição. </li><li> Totalmente aberta todas as aberturas (diafragma e Aberturacampo diafragma). Sistematicamente mover a câmara anexada fatia metros / potência em relação ao caminho óptico óptico e calcular a uniformidade de potência óptica dentro da área iluminada. Construir o enredo uniformidade para o seu sistema. Se o microscópio está configurada corretamente com iluminação Kohler centrado em foco o objetivo, a potência máxima deve ser diretamente abaixo do objetivo, e as regiões fora deste foco agora deve receber uma quantidade conhecida de energia de acordo com o enredo uniformidade. </li><li> Para cada tamanho de pinhole, construir uma curva padrão de potência óptica versus superfície pinhole. Ao ajustar a corrente de entrada para a matriz de LED, produzir esta curva em múltiplos níveis de potência e de cada curva de calcular a potência por mm2. Se a matriz de LEDs é para ser usado para correção óptica, certifique-se de introduzir elementos de óptica necessários para remendar (condensadores, pinholes e filtros) para saber a quantidade de luz é transmitido sob iluminação patching. </li><li> Lançando o medidor de potência paraenfrentar o objetivo, calcular a intensidade de iluminação em 470nm quando a lâmpada de mercúrio está ligado. </li><li> Adicionar uma fatia ao vivo para a câmara, e reconstruir as curvas padrão para determinar a potência de luz transmitida através do tecido cerebral de espalhamento. </li></ol> 2. Procedimento corte e Eletrofisiologia Uma parte: Preparação Slice <ol><li> Anestesiar (60 mg / kg cetamina e Xilazina 2mg/kg) e decapitar o mouse. Dissecar o cérebro no líquido cerebral espinhal artificial (ACSF, em mM: 124 NaCl, KCl 3, 1,3 MgSO 4, 26 NaHCO 3, 1,25 NaHPO 4, 20 glicose, 2 CaCl 2; ~ 310mOsm, pH 7,4, quando borbulhou com uma mistura de O2 de 95% e 5% CO 2 5,1), tomando cuidado para não danificar os bulbos olfatórios. Separar os dois hemisférios e colocar em agar com a superfície ventral, mesmo com uma borda (para seções horizontal). </li><li> Cola o ágar e superfície dorsal de cada hemisfério cortical à vibrAtome chuck, e, lentamente, encher a banheira com gelo frio ACSF. Fatia da superfície ventral em 300 seções M, transferindo cada seção para aquecido (34-36 ° C) e ACSF oxigenado, permitindo-lhes recuperar para 30-45 minutos. </li></ol> Parte B: Medição patch soltas do Threshold para Spike <ol start="3"><li> Depois de trazer as fatias à temperatura ambiente por 30 minutos, coloque delicadamente uma fatia na câmara de gravação da câmara de microscópio sob perfusão constante de ACSF oxigenado. Em risco de excessivamente estimulante ChR2 neurônios infectados, a presença de EYFP-ChR2 pode ser confirmado sob epifluorescência (fig. 2) </li><li> Puxe eletrodos de vidro em um extrator da pipeta (Sutter P-97). Preencha este eletrodo com ACSF. Quando colocada no banho ACSF a resistência da ponta deve ficar entre 70-10 Mohms. </li><li> Sob iluminação fluorescente, localizar a fatia de um neurônio ChR2-EYFP saudável com morfologia maduro. Também localizar essa soma neurônio sob a ótica patching. </li><li> Com pressão positiva de luz atravessou a ponta do eletrodo, menor o eletrodo de patch para o neurônio identificados fluorescente. Quando o contato da membrana é feita de forma rápida liberação de pressão positiva e aplique uma pequena quantidade e breve de sucção através da ponta. Um selo giga-ohm deve ser feita entre a membrana plasmática e as paredes do eletrodo patch. </li><li> Mesmo que um giga-selo não é formado, se o eletrodo está suficientemente perto de uma atividade dos neurônios fluorescentes spiking deve produzir um potencial campo mensuráveis ​​local. Ativar ChR2 neste neurônio, piscando doses diferentes de luz. Porque a luz da dose é uma função tanto do poder LED e duração, calcular a quantidade de luz que é necessário evocar um potencial de ação em múltiplos poderes e durações (fig. 2b). Também observar o quanto spiking ocorre sob a iluminação de mercúrio da lâmpada. </li></ol> 3. Resultados representativos: Em nosso microscópio (Olympus BX51WI), o nosso LED é in linha com duas aberturas e uma lente condensador, mantendo assim o caminho óptico original da fábrica instalada lâmpada de arco. Fechando tanto o diafragma de campo e Abertura do diafragma, podemos alcançar contraste brightfield suficientes para gravações de patch-clamp (fig. 1b). Com todos os diafragmas totalmente aberta expomos a fatia ao poder máximo de luz para channelrodopsina ativação (fig. 1a). Em nosso microscópio, essa configuração patching produz luz de densidade que é de aproximadamente três ordens de magnitude menor do que a densidade de campo máxima cheia (4,1 μW / mm 2 versus 6,88 mW / mm 2). Vemos etiquetagem robusta do adulto-nascido grânulo bulbo olfatório e semanas após a infecção neurônios periglomerular lentiviral de migrar neuroblastos no fluxo migratório rostral (fig. 2) A gravação solta-patch a partir de um único adulto-nascido ChR2-EYFP expressar célula granular indica que a 5 ms na estimulação máximahum alimentação (6,88 mW / mm 2) é suficiente para evocar spiking (fig. 2b). Desde nível de expressão varia entre as células, a quantidade de luz que passa o limiar a espiga vai variar e deve ser descrito estatisticamente para cada tipo de célula de interesse. <img src="/files/ftp_upload/3379/3379fig1.jpg" alt="Figura 1" /> Figura 1. LED de configuração matriz para full-campo fotoestimulação e patch-clamp eletrofisiologia fatia. Para ativar channelrodopsina (ChR2) projetamos um feixe colimado através de aberturas dos fundos aberta e óptica condensador (a). Esta configuração pode ser alterada em óptica de alto contraste patching por fechar completamente o diafragma de campo ea largura da modulação do diafragma de campo (b). Abreviaturas: a. ventilador, dissipador de calor b., c. matriz de LEDs, d. colimação lente, espelho e., f. diafragma de campo, g. diafragma de abertura, h. lente condensadora, i. sample palco, j. objetivo. <img src="/files/ftp_upload/3379/3379fig2.jpg" alt="Figura 2" /> Figura 2. Imagem de uma fatia horizontal de 300μm bulbo olfatório de patch clamp e de todo o campo fotoestimulação (a). Lentivirally células infectadas pelo adulto-nascido grânulo expressar ChR2-EYFP pode ser visto irradiando a partir do núcleo do bulbo olfatório. A luz da dose necessária para evocar spiking pode ser encontrada, aumentando a duração do flash LED (b). O limite para essa célula granular foi 5ms em intensidade LED completa (2.43mW/mm 2). Escala em (a) = 500μm, escala em (b) = 50ms.

<ol>
	<li>Nissant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+promotes+synaptic+plasticity+in+the+olfactory+bulb.">Adult neurogenesis promotes synaptic plasticity in the olfactory bulb.</a> Nature Neuroscience. 12, 728-730 (2009).</li><li>Apicella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidal+Cells+in+Piriform+Cortex+Receive+Convergent+Input+from+Distinct+Olfactory+Bulb+Glomeruli.">Pyramidal Cells in Piriform Cortex Receive Convergent Input from Distinct Olfactory Bulb Glomeruli.</a> Journal of Neuroscience. 30, 14255-14260 (2010).</li><li>Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=genetically+targeted+optical+control+of+neural+activity.">genetically targeted optical control of neural activity.</a> Nature. 8, 1263-1263 (2005).</li><li>Bardy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=where+new+inhibitory+neurons+release+neurotransmitters+in+the+adult+olfactory+bulb.">where new inhibitory neurons release neurotransmitters in the adult olfactory bulb.</a> The Journal of Neuroscience. 30, 17023-17034 (2010).</li><li>Grubb, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+of+the+first+synapse+in+olfaction:+development+and+adult+neurogenesis.">Functional maturation of the first synapse in olfaction: development and adult neurogenesis.</a> The Journal of neuroscience. 28, 2919-2932 (2008).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit-breakers:+optical+technologies+for+probing+neural+signals+and+systems.">Circuit-breakers: optical technologies for probing neural signals and systems.</a> Nature reviews. Neuroscience. 8, 577-581 (2007).</li><li>Grossman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-site+optical+excitation+using+ChR2+and+micro-LED+array.">Multi-site optical excitation using ChR2 and micro-LED array.</a> Journal of neural engineering. 7, 16004-16004 (2010).</li><li>Dhawale, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-redundant+odor+coding+by+sister+mitral+cells+revealed+by+light+addressable+glomeruli+in+the+mouse.">Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse.</a> Nature neuroscience. 13, 1404-1412 (2010).</li><li>Weick, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+control+of+transplantable+human+ESC-derived+neurons+via+optogenetic+targeting.">Functional control of transplantable human ESC-derived neurons via optogenetic targeting.</a> Stem cells. 28, 2008-2016 (2010).</li><li>Toni, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+born+in+the+adult+dentate+gyrus+form+functional+synapses+with+target+cells.">Neurons born in the adult dentate gyrus form functional synapses with target cells.</a> Nature Neuroscience. 11, 901-907 (2008).</li><li>Grubb, M. S., Burrone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2+Localised+to+the+Axon+Initial+Segment.">Channelrhodopsin-2 Localised to the Axon Initial Segment.</a> PLoS ONE. 5, e13761-e13761 .</li><li>Tye, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amygdala+circuitry+mediating+reversible+and+bidirectional+control+of+anxiety.">Amygdala circuitry mediating reversible and bidirectional control of anxiety.</a> Nature. 471, 358-362 (2011).</li></ol>

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Nos últimos anos temos visto uma explosão na popularidade de ferramentas para pesquisa em neurociência optogenetic 6. Como resultado, é cada vez mais importante para diminuir a barreira de entrada para laboratórios que desejam começar a usar estas novas ferramentas. Aqui nós descrevemos como conduzir uma adaptação simples e de baixo custo e calibração de um equipamento de patch-clamp convencionais, de modo que ele pode fazer de campo total estimulação óptica de channelrodopsina-expressando neur?...

<table border="1">
	<tbody>
		<tr>
			<td>Material</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue No.</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>100 mg/ml</td>
			<td>Imalg&egrave;ne 1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>2%</td>
			<td>Rompun</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbps;</td>
			<td>Sigma </td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>P5405</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>M1880</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5761</td>
		</tr>
		<tr>
			<td>NaHPO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5011</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>G7021</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>C7902</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>A9539</td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>P-97</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>1.5 mm O.D./1.17 mm I.D.</td>
			<td>Harvard Apparatus</td>
			<td>GC150T-10</td>
		</tr>
		<tr>
			<td>LED array</td>
			<td>&nbsp;</td>
			<td>Bridgelux</td>
			<td>BXRA-C2000</td>
		</tr>
		<tr>
			<td>Collimating lens</td>
			<td>40 mm beam diameter</td>
			<td>Thor Labs</td>
			<td>LEDC1</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>2.8 amp</td>
			<td>A1W Electronik</td>
			<td>HKO2800</td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>&nbsp;</td>
			<td>Thor Labs</td>
			<td>PM 100</td>
		</tr>
		<tr>
			<td>Heatsink </td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Fan</td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>&nbsp;</td>
			<td>Leica</td>
			<td>VT1200S</td>
		</tr>
	</tbody>
</table>

using affordable led arrays for photo-stimulation of neurons

Eletrofisiologia fatia padrão tem permitido aos pesquisadores sondar os componentes individuais dos circuitos neurais gravando respostas elétricas de células individuais em resposta a elétrica ou farmacológica 1,2 manipulações. Com a invenção de métodos para controle de neurônios geneticamente opticamente alvo (optogenética), os pesquisadores agora têm um nível sem precedentes de controle sobre grupos específicos de neurônios na preparação fatia padrão. Em particular, fotossensível channelrodopsina-2 (ChR2) permite que os pesquisadores para ativar os neurônios com 3,4 luz. Ao combinar calibração cuidadosa de LED com base em fotoestimulação de ChR2 com eletrofisiologia fatia padrão, somos capazes de sonda com maior detalhe o papel de adulto-nascido interneurônios no bulbo olfativo, o primeiro relé central do sistema olfativo. Usando a expressão viral de ChR2-YFP especificamente no adulto-nascido neurônios, podemos controlar seletivamente jovens adultos-nascido neurônios em um ambiente de mais umd neurônios maduros. Nosso controle óptico usa um sistema simples e barato LED, e vamos mostrar como esse sistema pode ser calibrado para entender como muita luz é necessária para evocar spiking atividade de neurônios individuais. Por isso, lampejos de luz azul pode controlar remotamente o padrão de disparo de ChR2-transduzidas células recém-nascido.

Watch this Scientific Journal Video about Using Affordable LED Arrays for Photo-Stimulation of Neurons at JoVE.com

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Adulto-nascido neurônios expressando ChR2 pode ser manipulado em preparações fatia eletrofisiológicos, a fim de examinar sua contribuição para a função olfativa de circuitos neurais.

Acessível usando Arrays LED para Foto Estimulação de Neurônios

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

using-affordable-led-arrays-for-photo-stimulation-of-neurons

Research

JoVE Journal

Neuroscience

18.5K visualizações.  Institut Pasteur and Centre National de la Recherche Scientifique (CNRS). Adulto-nascido neurônios expressando ChR2 pode ser manipulado em preparações fatia eletrofisiológicos, a fim de examinar sua contribuição para a função olfativa de circuitos neurais.

Vídeo: Using Affordable LED Arrays for Photo-Stimulation of Neurons

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Escalável Fluidic Arrays Injector para Viral Segmentação de Intact Circuitos 3-D do cérebro

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Neurociência

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 ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Optogenética ativação de neurônios Zebrafish somatossensoriais usando chef-tdTomato

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

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 ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imagiologia espinhas dendríticas dos neurônios do hipocampo de rato primária usando Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Produção e Isolamento de axônios de neurônios sensoriais para análise bioquímica Usando filtros porosos

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Controlo óptico de uma proteína neuronal utilizando um ácido Unnatural Amino geneticamente codificados em neurónios

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Imagens ao vivo da proteína GLUT4 tráfico de Mouse de neurônios hipotálamo primário usando microscopia de deconvolução

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Um modelo in vitro para estudar a agregação Tau usando transdução mediada por Lentivirus de neurônios humanos

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Implante crônico de matrizes flexíveis múltiplas do elétrodo do polímero

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...