Este estudo foi financiado pelo Nacional Natural Science Foundation da China (projeto código n º 81373557; n. º 81403327).

Este estudo foi aprovado pelo Comitê de ética em China Academy de chinês ciências médicas (número de referência 20160014). Todos os procedimentos foram conduzidos de acordo com os institutos nacionais de saúde guia para o cuidado e o uso de animais de laboratório (National Academy Press, Washington, D.C., 1996). Quatro ratos macho adulto (250-280 g de peso) foram utilizados neste estudo. Todos os animais foram alojados em um ciclo claro/escuro de 12 h com umidade e temperatura controlada e permitiu o livre acesso à comida e água. Os instrumentos e os materiais utilizados no presente estudo foram mostrou na Figura 1.  Antes da cirurgia, todos os instrumentos, tais como armação estereotáxica e pipeta de vidro, foram limpas com etanol a 70%.
1. cirurgias
<ol><li>Determine a coordenar área de interesse em VPM usando um atlas estereotáxica9 (Figura 2A).</li>
	<li>Prepare uma 1 µ l microseringa equipada com uma micropipeta de vidro (com um diâmetro de ponta de aproximadamente 10-20 µm) (Figura 1) e testá-lo com parafina líquida.</li>
	<li>Anestesia os ratos com 7% hidrato de cloral (0,7 mL/100 g) por injeção intraperitoneal.</li>
	<li>Uma vez confirmada anestesia profunda com cauda beliscar e pedal reflexo de retirada, raspar a parte superior da cabeça do animal com um barbeador elétrico e esfregue o local cirúrgico 3 vezes com 10% de iodopovidona seguida de etanol a 70%, respectivamente.</li>
	<li>Coloque o rato em dispositivo estereotáxica colocando barras de orelha contundente para as orelhas e coloque os incisivos superiores o rato está no suporte da boca (Figura 1E) e em seguida, aplique pomada oftálmica nos olhos.</li>
	<li>Limpe a pele cabeça do local cirúrgico novamente usando etanol a 70%. Use luvas cirúrgicas estéreis e toalhas para manter a cirurgia sob condições estéreis.</li>
	<li>Faça uma incisão sagital na pele com um bisturi ao longo da sutura sagital (Figura 1F).</li>
	<li>Raspe o músculo e o periósteo para longe do crânio utilizando aplicadores com ponta de algodão estéril durante a cirurgia para controle do sangramento (Figura 1F).</li>
	<li>Usando coordenadas predefinidas de um atlas (Figura 2A), determine a localização (ponto de Bregma mm-3,30, 2,6 mm direito mediana) de craniotomia (Figura 1).</li>
	<li>Executar uma craniotomia usando uma broca de rebarba com um pouco de ponta redonda (#106) (Figura 1 H) e continuar a perfuração para sobre uma profundidade de 1 mm dentro de alguns minutos até atingir as meninges (Figura 1I).</li>
	<li>Impostos especiais de consumo a dura-máter usando um microfórceps para expor o córtex cerebral o local da injeção (Figura 1I).</li>
	<li>Alterar a parafina líquida na microseringa com solução a 10% BDA (peso molecular 10.000, em água destilada) (Figura 1J).</li>
	<li>Monte a seringa no aparelho de microinjeção e conectar-se com uma microbomba (Figura 1 K). Nota: O volume injetado é dependente da velocidade da microbomba. Aqui ele foi ajustado para 30 nL/min (Figura 1 L).</li>
	<li>Sob um estereomicroscópio, insira uma micropipeta de vidro manualmente com o aparelho de microinjeção a VPM através da superfície cortical do cérebro na profundidade de 5,8 mm (Figura 1 M).</li>
	<li>Pressão-injetar um 100 nL 10% BDA para a VPM durante um período de 3 min (35 nL/min) com uma microbomba (Figura 1 L, M).</li>
	<li>Após a injeção, manter a pipeta no lugar por um adicional 5 min e depois retirar lentamente.</li>
	<li>Suture a ferida com rosca estéril (Figura 1N). Siga as guias de Comitê de cuidados animais locais para analgesia pré e pós-operatória.</li>
	<li>Coloque o rato em uma área de recuperação de calor até que recupere a consciência e está totalmente recuperado.</li>
	<li>Retorna o rato recuperado para sua gaiola.</li>
</ol>2. perfusions e seções
<ol><li>Depois de um tempo de sobrevivência de geralmente de 10 dias, injete o animal com uma overdose de uretano de 10% (2 mL/100 g) por injeção intraperitoneal de induzir a eutanásia.</li>
	<li>Perfundir os ratos experimentais de capuz (Figura 1O).<ol>
<li>Uma vez que a respiração para, usando uma tesoura e pinça, abra a cavidade torácica do rato para acessar o coração. Inserir um cateter intravenoso para o ventrículo esquerdo para a aorta e em seguida, abra a orelha direita.</li>
		<li>Primeiro perfundir com 0,9% fosfato salino (PBS) a temperatura fisiológica (37 ° C) cerca de 1-2 min até que o sangue sair do coração é claro e depois continuar com paraformaldeído 4% de 250-300 mL de tampão de fosfato 0,1 M (PB, pH 7,4).</li>
	</ol>
</li>
	<li>Após a perfusão, faça uma incisão na pele da cabeça e abrir o crânio e então dissecar o cérebro de rato. Fixe o cérebro dissecado em paraformaldeído 4% para 2 h à temperatura ambiente (26 ° C), então crioproteção em 30% de sacarose em PBS 0,1 M (pH 7,4) por 3 dias a 4 ° C até que o cérebro está imerso na solução (Figura 1, P).</li>
	<li>Uma vez que o cérebro está imersa na solução, dividem o cérebro em três blocos em direção coronal em matrizes de cérebro (Figura 1T). O bloco central contém a VPM e S1.</li>
	<li>Corte o bloco central do cérebro em 40 µm em um palco de congelação micrótomo sistema na direção coronal deslizante. Colete essas seções ordenadas em um prato de 6-poços com PBS 0,1 M (pH 7,4) (figuras 1R, S).</li>
</ol>3. padrão ABC coloração
Nota: Enciclopédia seções flutuantes de cada terceira seção coronal do cérebro foram utilizadas para visualizar a rotulagem de BDA com padrão ABC procedimento10.
<ol><li>Enxague as seções em PBS 0,1 M por cerca de 1 min.</li>
	<li>Incubar as secções em solução a 1% ABC em PB 0,1 M (pH 7,4) contendo 0,3% Triton X-100 por 1h à temperatura ambiente.</li>
	<li>Lave as seções três vezes em tampão Tris de 50mm (pH 7,4).</li>
	<li>Manche as seções em uma solução contendo 0,02% 3, tetrahydrochloride 3'-diaminobenzidine (DAB) e 0,01% H2O2 em tampão Tris de 50 mM para cerca de 2-5 min à temperatura ambiente.</li>
	<li>Lave as seções três vezes em tampão Tris de 50mm (pH 7,4).</li>
	<li>Monte as seções em corrediças do microscópio usando técnicas histoquímicas padrão (Figura 1T; ver III de arquivo de vídeo suplementar, perfusão e seções).</li>
	<li>Seque as seções no ar durante a noite em temperatura ambiente.</li>
	<li>Desidrate as seções brevemente em uma série de soluções de álcool (50%, 70%, 95%, 100%). Mergulhe os slides em cada solução para cerca de 15 s. Não permita que os slides secar entre cada etapa.</li>
	<li>Limpe as seções em xilol três vezes, cerca de 20 min.</li>
	<li>Coloque 2 ou 3 gotas do bálsamo sobre as fatias, em seguida, coloque as lamelas nas seções.</li>
</ol>4. dupla coloração fluorescente para BDA e elementos básicos de Neural no córtex Cerebral
Nota: em contraste, coloração fluorescente duplo foi realizado para observar a correlação da BDA rotulagem e elementos neurais básicos nas secções adjacentes ao acima usado com streptavidin-AF594 e counterstained com fluorescente Nissl mancha AF500/525.
<ol><li>Enxague as seções em PBS 0,1 M por cerca de 1 min.</li>
	<li>Incubar as seções em uma solução mista de streptavidin-AF594 (1: 500) e AF500/525 verde fluorescente Nissl mancha (1:1, 000) em PBS 0,1 M (pH 7,4) contendo 0,3% Triton X-100 para 2 h à temperatura ambiente.</li>
	<li>Lave as seções três vezes em PB 0,1 M (pH 7,4).</li>
	<li>Monte as seções em corrediças do microscópio usando técnicas histoquímicas padrão. Seque as seções no ar por cerca de 1h.</li>
	<li>Aplica as lamelas para as seções fluorescentes com glicerina 50% em água destilada antes de observação.</li>
</ol>5. dupla coloração fluorescente para BDA e interneurônios no córtex Cerebral
Nota: Coloração fluorescente duplo foi realizado para observar a correlação de BDA rotular e interneurônios nas secções representativas no destino cortical com streptavidin-AF594 e PV-imunoquímica.
<ol><li>Enxague as seções representativas em PBS 0,1 M (pH 7,4) por cerca de 1 min.</li>
	<li>Incubar as seções em uma solução de bloqueio contendo 3% de soro de cabra normal e 0,3% Triton X-100 em PBS 0,1 M durante 30 min.</li>
	<li>Transferir as seções para uma solução de rato anticorpo monoclonal anti-PV IgG (1:1, 000) em 0,1 M PBS (pH 7,4) contendo soro de cabra normal de 1% e 0,3% Triton X-100 para uma noite a 4 ° C.</li>
	<li>No dia seguinte, lave as seções três vezes em PBS 0,1 M (pH 7,4).</li>
	<li>Expor as seções de uma solução mista de anticorpo secundário de cabra anti-mouse-AF488 (1: 500), streptavidin-AF594 (1: 500) e 4' 6-diamidino-2-phenylindole dicloridrato (DAPI, 01:40, 000) em 0,1 M PBS (pH 7,4) com 1% cabra normal soro e 0.3% Triton X-100 por 1h.</li>
	<li>Repita as etapas de 4.3 a 4.5.</li>
</ol>6. observação
<ol><li>Com imagens do VPM, thalamocortical axônios e corticothalamic os neurônios.<ol>
<li>Observar as amostras fluorescentes com um sistema da imagem latente confocal equipado com lentes de objectivos (4x, at: 0,13; 10 x, at: x 0.40; e 40, at: 0,95). Use a excitação e emissão de comprimentos de onda de 405 (azul), 488 (verde) e 559 nm (vermelho). Nota: Aqui, o pinhole confocal é 152 µm (4x, 10x) e 105 µm (40 x). A resolução espacial de captura de imagem é 1024 × 1024 pixels (4x, 10x) e 640 × 640 pixels (40 x).</li>
		<li>Pegue vinte imagens em quadros sucessivos de 2 µm de cada seção para a espessura de 40 µm (série Z).</li>
		<li>Integrar as imagens em uma única imagem em foco com a sistema de software para análise tridimensional da seguinte forma de processamento de imagem confocal: conjunto Iniciar plano focal → final conjunto plano focal → etapa definir tamanho → escolha profundidade padrão → imagem captura → Z série.</li>
	</ol>
</li>
	<li>Tomar brightfield imagens por um microscópio óptico equipado com uma câmera digital (4x, at: 0,13; 10 x, at: x 0.40; e 40, at: 0,95 lentes). Use um tempo de exposição de fotografia de uso de 500 ms. software de edição para ajustar o brilho e o contraste das imagens e adicionar rótulos.</li>
</ol>

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Selecionar um traçador adequado é um passo fundamental para uma experiência bem sucedida de rastreamento neural. Na família de BDA, alto peso molecular BDA (10.000 peso molecular) foi recomendado preferencialmente ser transportado pela via anterógrada neural em contraste com baixo peso molecular BDA (3.000 peso molecular)2,3 , 11 , 12 , 13. no entanto, mu...

Alto peso molecular BDA (10.000 peso molecular), um marcador altamente sensível, tem sido utilizado para rastreamento de vias neurais no sistema nervoso central por mais de 20 anos1. Embora o uso do BDA é um trato neural comum técnica de rastreamento, a qualidade da rotulagem BDA pode ser afetada em animais por vários fatores1,2,3. Nosso estudo recente indicou que a estrutura ideal do BDA rotulagem é não só associada com um tempo de sobrevivência de pós-injeção adequada, mas também correlacionada com a coloração do método4. Até agora, convencional complexo avidina-biotina-peroxidase (ABC), isotiocianato de fluoresceína-streptavidin e streptavidin-AF594 coloração métodos foram usados para revelar a rotulagem BDA em estudos anteriores2,3, 4,5. Em comparação, a coloração fluorescente para BDA pode ser facilmente executada.
A fim de estender a aplicação de alto peso molecular, BDA, um protocolo refinado foi introduzido no presente estudo. Após a injeção do BDA para a VPM do tálamo em cérebro de ratos, BDA rotulagem foi revelada pelo método regular de coloração padrão do ABC, bem como manchando duplo fluorescente, que foi realizado para observar a correlação da BDA rotulagem e básico elementos neurais ou interneurônios no destino cortical com streptavidin-AF594 e histoquímica de Nissl fluorescente ou PV-imunoquímica, respectivamente. Através de vias neurais recíprocas entre VPM e o córtex somatossensorial primário (S1)6,7,8, focamos nossa observação na BDA rotulagem nos axônios thalamocortical projetada e corticothalamic somas de célula projetada em S1. Por este processo, que esperávamos fornecer não somente um protocolo detalhado para a obtenção da alta qualidade da rotulagem neural com alto peso molecular BDA, mas também um protocolo refinado sobre a combinação de rotulagem de BDA fluorescente e outros marcadores fluorescentes neurais com histoquímica ou imunoquímica. Esta abordagem é preferível estudar os circuitos neurais locais e suas características químicas sob um laser confocal, microscopia eletrônica de varredura.

<table>
	<tbody>
		<tr>
			<td>Biotinylated dextran amine (BDA)</td>
			<td>Molecular Probes</td>
			<td>D1956</td>
			<td>10,000 molecular weight</td>
		</tr>
		<tr>
			<td>Streptavidin-Alexa Fluor 594</td>
			<td>Molecular Probes</td>
			<td>S32356</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>500/525 green fluorescent Nissl stain</td>
			<td>Molecular Probes</td>
			<td>N21480</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Brain stereotaxis instrument</td>
			<td>Narishige</td>
			<td>SR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Thermo</td>
			<td>Microm International GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal imaging</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Drill</td>
			<td>Saeyang Microtech</td>
			<td>Marathon-N7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley</td>
			<td>Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences</td>
			<td>SCKX (JUN) 2012-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>superfrost plus microscope slides</td>
			<td>Thermo</td>
			<td>#4951PLUS-001</td>
			<td>25x75x1mm</td>
		</tr>
		<tr>
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improving the application of high molecular weight biotinylated dextran amine for thalamocortical projection tracing in the rat

Amina de dextrano de biotinilado de alto peso molecular (BDA) tem sido usada como um marcador altamente sensível neuroanatômica por muitas décadas. Desde que a qualidade da sua rotulagem foi afetada por vários fatores, aqui, nós fornecemos um protocolo refinado para a aplicação de alto peso molecular BDA para estudar a rotulagem neural ideal no sistema nervoso central. Após a injeção estereotáxica de BDA para o núcleo ventral póstero-medial (VPM) do tálamo em ratos através de uma pipeta de vidro delicado, BDA foi manchado com fluorescente streptavidin-Alexa (AF) 594 e counterstained com fluorescente Nissl mancha AF500/525. Sobre o fundo de coloração de Nissl do verde, o vermelho BDA etiquetando, incluindo organismos de célula neuronais e axonal terminais, mais distintamente foi demonstrada no córtex somatossensorial. Além disso, a dupla coloração fluorescente para BDA e o parvalbumin de proteína ligadora de cálcio (PV) foi realizado para observar a correlação de BDA rotular e interneurônios PV-positivas no destino cortical, proporcionando a oportunidade de estudar o local neural circuitos e suas características químicas. Assim, este método refinado não só é adequado para a visualização de alta qualidade neural de rotulagem com o alto peso molecular BDA através de vias neurais recíprocas entre o tálamo e o córtex cerebral, mas também permitirá a simultânea demonstração de outros marcadores neurais com fluorescente histoquímica ou imunoquímica.

Sobrevivência da injeção de pós 10 dias da BDA para o VPM foi suficiente para produzir intenso neural rotulagem nas áreas corticais correspondentes ipsilateral ao lado de injeção (Figura 2). ABC convencional e fluorescentes mancha procedimentos para BDA revelaram o padrão semelhante de rotulagem neural sobre a S1, incluindo anterogradely rotulado thalamocortical axônios e rotulado retrogradely corticothalamic neurônios (Figura 2...

Watch this Scientific Journal Video about Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat at JoVE.com

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Aqui, apresentamos um protocolo refinado para efetivamente revelar biotinilado amina de dextrano (BDA) rotulagem com uma fluorescente método através de um caminho neural recíproco de coloração. É apropriado para analisar a estrutura fina do BDA, rotulagem e distinguindo-a das outros elementos neurais sob microscópio confocal laser.

Melhorar a aplicação de alto Peso Molecular Biotinylated dextrano amina para projeção Thalamocortical rastreamento em ratos

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

improving-application-high-molecular-weight-biotinylated-dextran

Research

JoVE Journal

Neuroscience

8.9K visualizações.  China Academy of Chinese Medical Sciences. Aqui, apresentamos um protocolo refinado para efetivamente revelar biotinilado amina de dextrano (BDA) rotulagem com uma fluorescente método através de um caminho neural recíproco de coloração. É apropriado para analisar a estrutura fina do BDA, rotulagem e distinguindo-a das outros elementos neurais sob microscópio confocal laser.

Vídeo: Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Relação progressiva Respondendo para Alimentos gordurosos e High-açúcar Palatable em Ratos

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

Neurociência

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualização de Propriocepção em<em&gt; Drosophila</em&gt; As larvas e pupas

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsináptica Rastreamento de Alvos periféricos com vírus da pseudo-Seguido por cólera toxina e Biotinilado Dextran aminas Duplo Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

Projeto e fabricação de Ultraleve Peso, ajustável Sondas Multi-eletrodo para gravações eletrofisiológicas em Ratos

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Condicional Genetic transsináptica Tracing no Embryonic do cérebro do rato

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Um de uma etapa simples protocolo de dissecação para Whole mount-Preparação do Adulto<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sublimação da DAN Matrix para a Detecção e Visualização de gangliosídeos no cérebro de um rato do tecido por MALDI imagem Espectrometria de Massa

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

Avaliação simultânea de Cerebral Hemodinâmica e de espalhamento de luz Propriedades do<em&gt; In Vivo</em&gt; Cérebro de Rato Utilizando Imagem de Reflexão Difusa Multispectral

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

Visualização do Padrão de Projeção Axonal de Neurônios de Motor Embrionários em<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.