This work was funded by NIH grants DA013137, DA031604, and HD043680. Partial support was provided by a NIH T32 training grant in Biomedical-Behavioral science.

Todos os protocolos de animais foram revistos e aprovados pela Comissão de animal Cuidado e Uso da Universidade da Carolina do Sul (número de garantia: A3049-01). 1. baixa densidade Embryonic Neuronal Cultura <ol><li> Preparação para a primária neurônio cultura cortical <ol><li> soluções: <ol><li> Preparar a solução da poli-L-lisina por dissolução de 5 mg de poli-L-lisina em 10 ml de tampão de borato. </li><li> Preparar uma solução de trabalho por diluição de 1 ml de poli-L-lisina em 49 ml da tampão de Borato. </li><li> Preparar tampão de borato, pH 8,4, iniciando com 395 ml de dH 2 O e em seguida a adição de todos os ingredientes (1,24 g de ácido bórico + 1,9 g de bórax). Ajustar o pH para 8,4 com NaOH 1 M. Ajustar a 400 ml e filtrar com filtro de membrana de nylon 0,2 m sob o capô. </li><li> Preparar a solução de HBSS, pH 7,2, iniciando com 445 ml de dH 2 O e a adição de todos os outros ingredientes (50 mL de Stock 10X HBSS + 1,2 g de HEPES). Ajustar o pH para 7,2 com HCl 1 N, BRIng para 500 ml e filtrar com 0,2 m de filtro de membrana de nylon sob o capô. </li><li> Prepare meio de plaqueamento: DMEM / F12 com soro de bovino fetal a 10%. Filtro com 0,2 m de filtro de membrana de nylon sob o capô. </li><li> Preparar o meio de crescimento completo: Para 50 mL de meio Neurobasal, adicionar os suplementos (500 ul de GlutaMax (100X) + 500 ul de glucose + 1 ml de B-27 (50X) + 500 ul de solução de antibiótico-antimicótico (100X) + 100 ul de 7,5% Bicarbonato de sódio. Filtro com 0,2 m de filtro de membrana de nylon sob o capô. </li></ol></li><li> Dois dias antes da cultura: <ol><li> Brasão de doze 35 mm pratos com fundo de vidro com 2 ml de solução de trabalho de poli-L-lisina, e manter sob o capô O / N. </li></ol></li><li> Um dia antes da cultura: <ol><li> Agente de revestimento vazio dos pratos e enxaguar com DDH 2 O. Permitir pratos para secar por uma hora sob o capô. </li><li> Prepare meio de plaqueamento (DMEM / F12 com soro de bovino fetal a 10% (10% da solução total). Pipeta 2 ml de meio emcada prato, e incubar O / N a 37 ° C, 5% de CO 2. Nota: É importante evitar a pratos de cultura de plástico, quando a cultura. Use de pratos com fundo de vidro (para microscópios invertidos) ou lamelas de vidro (para microscópios verticais) produz imagens nítidas e claras. Comparado a um prato de plástico de baixo, o prato com fundo de vidro evita tons indesejados ou brilho durante microscopia fluorescente. </li></ol></li></ol></li><li> Protocolo de cultura celular: <ol><li> Colocar refrigerado Tampão HBSS (100-150 ml), 100 mm pratos de petri, fórceps, tesouras, tubos de 50 ml e 15 ml tubos sob luz UV na capa. </li><li> Euthanize rato grávida de inalação letal de 5% sevoflurano sob o capô fora da sala de cultura. </li><li> Esterilizar com 70% de EtOH, tenda a pele da parte inferior do abdômen, utilizando fórceps durante o corte da cauda para cima no meio da cavity.Open abdominal do útero usando uma tesoura para expor a placenta. </li><li> Remover 8-10 E18 fetos. Place 8-10 fetos em placas de Petri contendo solução de HBSS. </li><li> Segure a parte de trás da cabeça do feto com uma pinça, em seguida, usar a tesoura para cortar a cabeça do corpo. Coloque-o em uma outra placa de petri preenchida com 5-7 ml de HBSS. Transferir prato para capuz. </li><li> Encha dois prato adicional petri 100 mm, com HBSS frio. </li><li> Peel lado crânio, e colher cérebro em uma nova placa de petri preenchida com HBSS. </li><li> Use uma pinça curvas acentuadas, cerebelo separada e tronco cerebral do cérebro, em seguida, transferir cérebro para uma nova placa de Petri com HBSS frio nele. </li><li> Use uma pinça para fixar, separar os hemisférios com uma pinça curva. Remover meninges; isolar córtex frontal e peças de transferência para marcados tubos de centrífuga de 15 ml. </li><li> Encha o tubo de 15 ml com frescos 2 ml HBSS e adicionar 20 ul de tripsina EDTA (10x mistura concentrada com 0,5% de tripsina e EDTA 5,3 mM.) A cada tubo de 15 ml. </li><li> Incubar 10-15 min à temperatura ambiente. Agite cuidadosamente tubo a cada poucos minutos, não permitem resolver. </li><li> After 10-15 min, o uso pipeta de vidro para remover velho HBSS e lavar duas vezes com HBSS novo. </li><li> Mergulhe na inibidor de tripsina (1 mg / ml, 2 mg Inibidor de tripsina em 2 ml de HBSS), misture bem e deixe descansar 5 min. Lavar duas vezes com HBSS fresco. </li><li> Usando uma pipeta de vidro com bulbo de borracha, pedaços de cérebro Triturar 10-15 vezes muito lentamente, de modo a evitar bubbles.Triturate novamente utilizando uma pipeta calibrada e uma ponta estéril com um diâmetro de ponta reduzida muito lentamente até à homogeneidade. </li><li> Transferência dissociada células a densidade desejada para pré-preparados pratos de cultura revestidos (50 células / mm2). Incubar O / N a 37 ° C, 5% de CO2. </li><li> Após 24 h, substitua meio DMEM / F12 com meio Neurobasal sem soro crescimento completo feitos na hora. </li><li> Manter as células em todos os momentos em CO 2/95% ar ambiente incubadora humidificada de 5% de, pelo menos, 12-14 dias antes da experimentação. Suplemento as células a cada 5-6 dias com meio Neurobasal fresco substituindo aproximadamente 50% do meio velho. </li></ol></li></ol> 2. Labeling fluorescentes e Imunocitoquï¿½ica Nota: A marcação imunofluorescente de culturas de células corticais primárias foi realizado em pratos de vidro de fundo de cultura de células de 35 mm com um volume de trabalho de 1 ml. <ol><li> Lava-se a placa com fundo de vidro duas vezes com tampão fosfato salino pH 7,4 (PBS). </li><li> Fixar as células com paraformaldeído a 4% durante 15 min à TA. </li><li> Lavar as células duas vezes com PBS e permeabilizar com 0,1% de Triton X-100 em PBS durante 5 min. </li><li> Tratar as células durante 20 minutos à TA com uma coloração específica F-actina, AlexaFluor 488 faloidina (01:40, 25 ul de faloidina em 1 mL de PBS). </li><li> Lavar as células duas vezes com PBS e bloquear com soro de cabra normal a 10% à temperatura ambiente durante 1-2 h. </li><li> Dilui-se o anticorpo policlonal anti-galinha MAP2 (1: 2500) em PBS com soro de cabra normal a 2% e incubar O / N a 4 o C. </li><li> Após a incubação, lavar duas vezes em PBS. </li><li> Dilui-se o anticorpo secundário (R AlexaEd cabra-594 conjugado anti-IgG de galinha (1: 500) com soro de cabra normal a 2% e incubar durante 2 horas à temperatura ambiente. </li><li> Lavar com PBS e adicionar 10 mL / prato de Hoescht corante. </li><li> Incubar 3 min à temperatura ambiente e lava-se duas vezes com PBS. Nota: As células marcadas agora pronto para o passo 3 (contagem de pontos lacrimais F-actina). </li><li> Preservar amostra de células com 100 ml de reagente Antifade como um agente de lamínula e manter a 4 ° C no escuro para armazenamento a longo prazo. Nota: faloidina é relativamente estável em PBS a 4 ° C no escuro durante 1 semana e fluorescência podem ser preservados com antifade reagentes lamínulas. No entanto, imagens óptimas são obtidas a partir de 1-3 dias após coloração com faloidina desde pode começar a difundir para fora com o armazenamento prolongado. </li></ol> Contando puncta 3. F-actina <ol><li> Ligue microscópio de fluorescência e mudar para 20 × objetiva. microscópio software Open, e configurar programa em 1280 × 960 pixels tamanho da imagem, e 0,1resolução de imagem / pixel 7 mm a 1 × zoom. </li><li> Adquirir imagens de co-marcado F-actina / neurônios MAP2 sob verde (495 nm) / vermelho (613 nm) canais fluorescentes. </li><li> Escolha 5 Green (F-actina) / vermelho (MAP2) immunolabeled / Blue (Hoescht) imagens fluorescentes de neurônio individual com mandris dendríticas claramente definidos. </li><li> Identificar os F-actina estruturas ricas em segunda ordem segmento dendríticas (gama de comprimento 25-75 mm) com imunofluorescência MAP2 contínua. </li><li> Gire a região selecionada de imagens para um nível horizontal. Copie e cole como uma nova imagem. </li><li> Subtrair o fundo das imagens, usando uma ajustes consistentes para cada prato. </li><li> Contar o puncta verde brilhante F-actina e traçar o comprimento do segmento dendrítica selecionados manualmente por observadores independentes treinados. Exportar os dados para um arquivo de planilha. </li><li> Calcula-se a densidade dividindo F-actina total de pontos lacrimais marcada (N) por o comprimento (L) da MAP2 marcado dendritos. Dados expressos como o número de F-acpontos lacrimais estanho por 10 uM de dendrite Nota: puncta (tamanho ≤1.5 uM) de fluorescência F-actina com uma intensidade de pico de pelo menos 50% superior à média de intensidade de coloração no eixo dendríticas foram incluídos em cada segmento seleccionado dendrítica. </li></ol>

<ol>
	<li>Heller, E. 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=The+biochemical+anatomy+of+cortical+inhibitory+synapses.">The biochemical anatomy of cortical inhibitory synapses.</a> PLoS One. 7, e39572 (2012).</li><li>Craig, A. M., Blackstone, C. D., Huganir, R. L., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+clustering+of+glutamate+and+gamma-aminobutyric+acid+receptors+opposite+terminals+releasing+the+corresponding+neurotransmitters.">Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters.</a> Proc Natl Acad Sci USA. 91, 12373-12377 (1994).</li><li>Johnson, O. L., Ouimet, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+role+for+actin+in+dendritic+spine+proliferation.">A regulatory role for actin in dendritic spine proliferation.</a> Brain Res. 1113, 1-9 (2006).</li><li>Rao, A., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+between+the+actin+cytoskeleton+and+the+postsynaptic+density+of+dendritic+spines.">Signaling between the actin cytoskeleton and the postsynaptic density of dendritic spines.</a> Hippocampus. 10 (5), 527-541 (2000).</li><li>Matus, A., Ackermann, M., Pehling, G., Byers, H. R., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+actin+concentrations+in+brain+dendritic+spines+and+postsynaptic+densities.">High actin concentrations in brain dendritic spines and postsynaptic densities.</a> Proc Natl Acad Sci USA. 79, 7590-7594 (1982).</li><li>Allison, D. W., Gelfand, V. I., Spector, I., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+actin+in+anchoring+postsynaptic+receptors+in+cultured+hippocampal+neurons:+differential+attachment+of+NMDA+versus+AMPA+receptors.">Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors.</a> J Neurosci. 18, 2423-2436 (1998).</li><li>Sekino, Y., Kojima, N., Shirao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+actin+cytoskeleton+in+dendritic+spine+morphogenesis.">Role of actin cytoskeleton in dendritic spine morphogenesis.</a> Neurochem Int. 51, 92-104 (2007).</li><li>Zhang, W., Benson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+synapse+development+defined+by+dependence+on+F-actin.">Stages of synapse development defined by dependence on F-actin.</a> J Neurosci. 21 (14), 5169-5181 (2001).</li><li>Bertrand, S. J., Aksenova, M. V., Mactutus, C. F., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+Tat+protein+variants:+Critical+role+for+the+cysteine+region+in+synaptodendritic+injury.">HIV-1 Tat protein variants: Critical role for the cysteine region in synaptodendritic injury.</a> Exp Neurol. 248, 228-235 (2013).</li><li>Bertrand, S. J., Mactutus, C. F., Aksenova, M. V., Espensen-Sturges, T. D., Booze, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptodendritic+recovery+following+HIV+Tat+exposure:+neurorestoration+by+phytoestrogens.">Synaptodendritic recovery following HIV Tat exposure: neurorestoration by phytoestrogens.</a> J Neurochem. 128 (1), 140-151 (2014).</li><li>Lau, P. M., Zucker, R. S., Bentley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+filopodia+by+direct+local+elevation+of+intracellular+calcium+ion+concentration.">Induction of filopodia by direct local elevation of intracellular calcium ion concentration.</a> J Cell Biol. 145, 1265-1275 (1999).</li></ol>

None of the authors have conflicts of interest to declare.

Neste protocolo, descrevemos a cultura de neurônios corticais de rato em baixa densidade em pratos com fundo de vidro de 35 mm, o que nos permite identificar dendritos de neurônios individuais. Em seguida, usamos faloidina e coloração MAP2 para detectar alterações dendríticas. Em seguida, foi utilizado um software especializado para quantificar mudanças na puncta F-actina. Para determinar mudanças na puncta F-actina toda a rede neuronal de um neurônio individual deve ser claramente...

O principal objetivo deste estudo é desenvolver um método fiável de medição (estimativa) de integridade sináptica da rede dendrítica neuronal. Aqui nós descrevemos quantificação de puncta F-actina em ratos primário neurônios cultivados usando uma combinação de coloração faloidina e detecção imunocitoquímica (ICC) de dendrites com posterior análise usando software especializado (NIS-Elements). Falotoxina rotulados têm afinidade semelhante tanto para grandes e pequenos filamentos (F-actina), mas não se ligam a monomérica actina globular (G-actina), ao contrário de alguns anticorpos de actina 1. A ligação não específica de faloidina é insignificante, proporcionando assim fundo mínima durante imagiologia celular. Faloidina é muito menor do que os anticorpos que seria normalmente usado para marcar as proteínas celulares para a microscopia de fluorescência, o que permite muito mais intensa marcação da actina F por faloidina. Assim, imagens detalhadas de localização F-actina em neurônios pode serobtida através do uso de faloidina etiquetada. Phalloidin (F-actina) coloração das dendrites neuronais gera discretos &quot;pontos quentes&quot; ou brilhante &quot;puncta&quot;, que representam uma variedade de estruturas dendríticas, incluindo espinhas maduras, sinapses não-espinhosos 2 e espinhas imaturos. Espinhos imaturos incluem filopódios finas e algumas formas de morfologia remendo, e pode representar o início de spinogenesis 3. Espinhos imaturos e patches não-espinhosos falta PSD95 4. Mudanças na produção de chumbo F-actina para alterações subsequentes não só espinhos, mas também estruturas dendríticas adicionais, tornando assim faloidina uma ferramenta importante para a investigação de integridade synaptodendritic 5-7. Em geral, o número de (F-actina) puncta faloidina-positivos refletem um equilíbrio entre as sinapses ativas (excitatórios e inibitórios), dinâmica de actina e estabilidade sinapse 8. Embora seja importante para o estudo específico tYpes de sinapses (isto é, espinhas excitatórios), quando o alvo de um tratamento é desconhecido, é necessário primeiro estimar a integridade geral de uma variedade de estruturas dendríticas. Uma vez que a actina F é um componente importante das espinhas dendriticas e outras estruturas, incluindo sinapses inibitórias, um número alterado de pontos lacrimais F-actina pode indicar um synaptopathy. Este synaptopathy pode então ser mais investigada para alterações mais específicos. O nosso método de quantificação para a detecção de vários tipos / sinápticas estruturas produz uma estimativa global das alterações sinápticas dendríticas (aumentos e diminuições) após vários tratamentos experimentais.

<table border="0" cellpadding="0" cellspacing="0" width="626">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:211px;">35 mm Glass Bottom Dishes No. 1.5 coverglass</td>
			<td style="width:155px;">MatTek Corporation</td>
			<td style="width:121px;">P35G-1.5-20-C</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:211px;">DMEM/F12 medium</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">10565-018</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Trypsin-EDTA</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">15400-054</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:211px;">Poly-L-Lysine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:121px;">P9155</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:211px;">Boric acid</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:121px;">B0252</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:211px;">Borax</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:121px;">B9876</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:211px;">GlutaMax</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">35050-061</td>
			<td style="width:139px;">100X</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:211px;">Glucose</td>
			<td style="width:155px;">VWR</td>
			<td style="width:121px;">101174Y</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:211px;">HBSS</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:121px;">H4641</td>
			<td style="width:139px;">10X</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:211px;">Neurobasal medium</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">21103-049</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:211px;">B-27 supplement</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">17504-044</td>
			<td style="width:139px;">50X</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:211px;">Antibiotic-Antimycotic solution</td>
			<td style="width:155px;">Cellgro</td>
			<td style="width:121px;">30004CI</td>
			<td style="width:139px;">100X</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:211px;">Sodium Bicarbonate</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">25080</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Vannas Scissors</td>
			<td style="width:155px;">World Precision Instruments</td>
			<td style="width:121px;">500086</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Iris Scissors</td>
			<td style="width:155px;">World Precision Instruments</td>
			<td style="width:121px;">500216</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:211px;">Iris Forceps</td>
			<td style="width:155px;">World Precision Instruments</td>
			<td style="width:121px;">15914</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:211px;">Dumont #7 Forceps</td>
			<td style="width:155px;">World Precision Instruments</td>
			<td style="width:121px;">14097</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:211px;">Dumont #5 Forceps</td>
			<td style="width:155px;">World Precision Instruments</td>
			<td style="width:121px;">14095</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:211px;">ProLong Gold</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">P36930</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:211px;">Paraformaldehyde&#160;</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:121px;">P6148</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:211px;">Cover glass</td>
			<td style="width:155px;">VWR</td>
			<td style="width:121px;">631-0137</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:211px;">AlexaFluor 488 Phalloidin</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">A12379</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Normal horse serum</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">26050-070</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:211px;">Chicken polyclonal anti-MAP2</td>
			<td style="width:155px;">abcam</td>
			<td style="width:121px;">Ab92434</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:211px;">Alexa Red 594-conjugated goat anti-chicken IgG</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">A11042</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:211px;">NucBlue Live cell stain ReadyProbes Reagent Hoescht 33342</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:121px;">R37605</td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">NIS-Elements software package</td>
			<td style="width:155px;">Nikon Instruments</td>
			<td style="width:121px;"></td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:211px;">Nikon Eclipse TE2000-E inverted fluorescent computer-controlled microscope&#160;</td>
			<td style="width:155px;">Nikon Instruments</td>
			<td style="width:121px;"></td>
			<td style="width:139px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:211px;">0.2 &#956;m Nalgene nylon membrane filter</td>
			<td style="width:155px;">Fishersci</td>
			<td style="width:121px;">151-4020</td>
			<td style="width:139px;"></td>
		</tr>
	</tbody>
</table>

quantification of filamentous actin (f-actin) puncta in rat cortical neurons

proteína actina filamentosa (F-actina) desempenha um papel importante na spinogenesis, plasticidade sináptica, e estabilidade sináptica. Mudanças nas estruturas ricas F-actina dendríticas sugerem alterações na integridade sináptica e conectividade. Aqui nós fornecemos um protocolo detalhado para a cultura de rato primária neurônios corticais, coloração faloidina para puncta F-actina, e técnicas de quantificação subsequentes. Em primeiro lugar, o córtex frontal de embriões de rato E18 são dissociados em cultura celular de baixa densidade, em seguida, os neurónios cultivados in vitro durante pelo menos 12-14 dias. Após o tratamento experimental, os neurônios corticais estão manchadas com AlexaFluor 488 faloidina (para rotular o puncta F-actina dendrítica) e proteína associada a microtúbulos 2 (MAP2; para validar as células neuronais e integridade dendrítica). Finalmente, software especializado é usado para analisar e quantificar dendrites neuronais selecionados aleatoriamente. F-actina estruturas ricas são identificados na segunda ordem ramos dendríticas (variação de comprimento 25-75 &amp;# 181; m) com imunofluorescência MAP2 contínua. O protocolo apresentado aqui vai ser um método útil para investigar mudanças nas estruturas sinapse dendríticas subsequentes aos tratamentos experimentais.

Nos presentes métodos, primeiro cultura rato neurônios corticais em baixa densidade em pratos com fundo de vidro de 35 mm, o que nos permite identificar os dendritos de neurônios individuais. Na Figura 1, o contraste interferência diferencial (DIC) imagens mostram as alterações morfológicas no desenvolvimento de rato fetal neurônios corticais nos dias 4, 6, 10, 14, 21 e 27 in vitro. Note-se que o comprimento e o número de dendritos aumentar com a matura...

Watch this Scientific Journal Video about Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons at JoVE.com

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Quantificação de filamentosas actina (F-actina) puncta no Rat neurônios corticais

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

quantification-filamentous-actin-f-actin-puncta-rat-cortical

Research

JoVE Journal

Neuroscience

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

12.0K visualizações.  University of South Carolina. Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Vídeo: Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

No útero Eletroporação seguido por Cultura Neuronal primário para estudar a função do gene no subconjunto dos neurônios corticais

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Neurociência

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection e Cultura Primária de Neurônios do hipocampo do rato embrionárias e Cortical

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

Rotulagem F-actina Termina farpado com Rodamina-actina em permeabilizadas Cones de Crescimento Neuronal

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Determinação de Membrana Mitocondrial Espécies de Oxigênio Reativas Potencial e em neurônios de rato vivo Cortical

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar Co-cultura de neurônios de rato Primária Cortical e Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Vivo em dois fótons de imagem de experiência dependentes de alterações moleculares em neurônios corticais

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

Juxtasomal biocitina Labeling para estudar a função Estrutura de Relacionamento Individual neurônios corticais

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantificação de Endosome e Moosidades de Lisosoma em Neurônios Cultivados Usando Sondas Fluorescentes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Na Vivo Imagem de dois fotões de neurônios Cortical em camundongos Neonatais

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Geração de neuroesferas do hipocampal preliminar misturado e dos neurônios corticais isolados de E14-E16 embrião do rato de Sprague Dawley

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...