We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

Todos os procedimentos experimentais descritos neste protocolo foram aprovados pelo Animal Care da Universidade Hebraica e do Comitê Use. 1. Preparar as soluções e ferramentas para corte <ol><li> Prepare 1 litro de solução fisiológica padrão (SPS) que contém os iões descritas na Tabela 1. <ol><li> Prepara-se uma solução de estoque contendo os sais em 10 vezes a concentração final previamente em uma garrafa de vidro de armazenamento preenchido com 1000 ml de água desionizada (condutividade de 0,055 mS / cm). Adicionar sais (NaCl, KCl, KH 2 PO 4, e MgSO4), e agita-se até estar completamente dissolvido. Guardar a solução estoque em 4 ° C. </li><li> Adicione a solução final SPS no dia do experimento. Acrescentam-se 100 ml da solução de estoque de 700 ml de água purificada, dissolver a glicose (3,6 g) e NaHCO3 (2,18 g) usando uma barra magnética e, em seguida, adicionar água purificada até 1 L. </li><li> Equilibrar as solutisobre ele por gaseamento com 95% O2 / 5% de CO 2 durante ~ 20 min. Após isto, adicionar 2 ml de uma solução M de CaCl2. </li></ol></li><li> Reúna as seguintes ferramentas (Figura 1). <ol><li> Prepare grandes tesoura ou outro instrumento de decapitação, pequenas tesouras cirúrgicas para a abertura do crânio, uma pinça de ponta fina para elevação do crânio para expor o cérebro, bisturi com lâmina para dissecar a parte desejada do cérebro para ser cortada e uma pequena espátula para manipular o partes cerebrais dissecados. </li><li> Além disso, prepare-se pequenos pedaços de papel de filtro para remover o excesso de líquido do cérebro, dois pequenos pratos de Petri para o cérebro para ser dissecado em dois copos de vidro de 500 ml para conter água aquecida purificada e SPS, e cola de cianoacrilato para colar do cérebro para o fase de corte. </li><li> Prepare uma escova pequena para lidar com as fatias durante o corte e uma pipeta de Pasteur de vidro de boca larga para mover as fatias do banho de corte para o cha de recuperaçãomber. Desinfetar as ferramentas para impedir o crescimento de bactérias nos banhos quentes fatia. Além disso, usar luvas de laboratório limpas. </li></ol></li><li> Prepare uma fatia de recuperação câmara submersa tal como descrito por Gibb e Edwards 15. Continuamente gás do SPS nele com 95% O2 / 5% de CO2 e manter a 36 ° C. Certifique-se de que o gaseamento não resulta em pequenas bolhas de ar no banho que pode danificar fatias. </li><li> Quente ~ 300 ml da SPS a 36 ° C sobre uma placa de aquecimento com agitador magnético ou em um banho de água. Se uma placa de aquecimento é usado, tome cuidado para evitar que a solução do superaquecimento, o que pode resultar na precipitação de íons. No caso de isso acontecer, SPS frescos quentes em vez de arrefecimento do antigo. </li><li> Traga ~ 500 ml de água purificada para ferver com uma chaleira eléctrica e, num copo, misture 300 ml de com água fria para obter água um pouco mais quente do que a temperatura de corte. Utilize este aqueceu água purificada durante a decapitação e usar o remaining da água fervida mais tarde para a manutenção da temperatura fisiológica do banho de corte. </li><li> Anestesiar o rato por injecção de 0,1 ml de pentobarbital (60 mg / ml) por via intraperitoneal. Depois de alguns minutos, verificar se o animal não responder à forte do dedo do pé ou da cauda pitadas de verificar a falta de sensação. </li></ol> 2. Dissecando o cérebro <ol><li> Decapitar o mouse rapidamente cortando o pescoço com as tesouras grandes, perto da parte de trás do crânio, e deixar a cabeça cair no copo com água purificada aquecida para lavar o excesso de sangue. </li><li> Exponha o forame magno no crânio sob os músculos do pescoço e da pele, possivelmente removendo mais do pescoço com as pequenas tesouras. </li><li> Puxar a pele na parte superior do crânio de ser capaz de ver claramente as suturas cranianas, a fim de orientar a abertura do crânio. </li><li> Corte o crânio aberto, inserindo a ponta inferior das pequenas tesouras através do forame magno e imediatamente transformá-los em direção ao lado lateral. Com cuidado, cortar ao longo do bordo lateral do osso parietal até uma localização atrás dos olhos e da sutura fronto-parietal; em seguida, virar-se para o centro do crânio e cortar através da linha média para o outro lado do crânio (ver linha tracejada verde na Figura 2A). </li><li> Utilizando uma pinça de ponta fina, levante o crânio do canto fronto-parietal e puxe-o na diagonal para cima e para os lados para expor o cérebro (ver amarelo seta tracejada na Figura 2A). Certifique-se de que o crânio não está ligado a qualquer osso ou pele da cabeça, de modo que o cérebro vai ficar no lugar quando o crânio é levantado. No caso em que o cérebro está em movimento, com o crânio, utilizar as pequenas tesouras para remover qualquer tecido conjuntivo entre o crânio e o cérebro. </li><li> Quando o cérebro é exposta, não permitem que o cérebro a ser seco. Assim, execute os passos seguintes, mantendo o crânio eo cérebro em uma placa de Petri cheia com aquecido, gaseados SPS. </li><li> Retire a parte do cérebro usada para o eXperiment a partir do resto do cérebro utilizando um bisturi e uma espátula. No caso de utilizar o cerebelo, separar da parte frontal do cérebro por um único corte através do mesencéfalo e ponte (ao longo das linhas vermelhas nas Figuras 2B-D) e movê-lo para uma pequena placa de Petri que contém frescos, gaseados SPS. </li><li> Remover o tronco cerebral para formar uma base recta e ampla para a colagem do cérebro a fase de corte ao cortar o cerebelo no plano horizontal. Resumidamente colocar o cerebelo em um pedaço húmido de papel de filtro de modo que o lado que foi cortado a partir do prosencéfalo está voltada para baixo. <ol><li> Cortar o tronco cerebral com um bisturi para formar a base (tal como indicado com a linha azul na Figura 2B), e devolver o cerebelo para o SPS. Quando são necessários outros planos de corte, cortar o bloco cerebelo em conformidade (linhas azuis nas Figuras 2C e D). </li></ol></li></ol> 3. Cortando o Cérebro <ol><li> Prepare o estágio de corte, verificando que é dry, antes da aplicação de uma pequena gota de supercola no meio do palco. </li><li> Levante o bloco cérebro do SPS com uma espátula com a correta voltada para cima, e retire todo o excesso de líquido do cérebro usando pequenos pedaços de papel de filtro. Então, com um único movimento, deslize o cérebro da espátula sobre a gota de cola no palco. </li><li> Para evitar que o cérebro de secagem e a cola de correr para cima sobre os lados do tecido, aplicar algumas gotas de SPS com uma pipeta de Pasteur e colocar a fase de corte na câmara de corte. Alinhar o bloco de cérebro de modo a que as regiões de interesse (por exemplo, do córtex cerebelar) estão virados para a lâmina e, portanto, vai ser submetido a menor quantidade de pressão mecânica antes de ser cortado. </li><li> Corte cada fatia de acordo com as instruções do fabricante, enquanto constantemente gaseificação do RPU na câmara de corte, bem como mantendo a temperatura a 34-37 ° C. Manter a temperatura da câmara de corte, neste intervalo, preenchendo o external câmara do cortador com água purificada aquecida e substituindo a água sempre que a temperatura cai muito baixa. Nota: Os parâmetros de corte devem ser experimentalmente encontrado para cada tipo de tecido e dos neurônios que estão sendo examinados. No caso de o Campden SMZ 700 fatiador com lâminas de cerâmica e células cerebelares de Golgi, usar 0,75 milímetros amplitude de vibração a 65 Hz e velocidade de 0,05 mm / seg avançando; para os neurónios cerebelares nucleares maior amplitude e frequência (1 mm e 90 Hz, respectivamente) e menor velocidade de avanço (0,01-0,02 mm / seg) são recomendados. Para tanto Golgi e os neurônios nucleares do cerebelo, espessura do corte deve ser de 300 mm. </li><li> Durante o corte, não permita que as fatias de dobrar sobre si mesmos. Para evitar que isto delicadamente apoiando-os com uma escova macia, de preferência tocando a fatia apenas em regiões que não são de interesse para o experimento. Tome cuidado para evitar que a escova de tocar a lâmina vibratome; especialmente no caso das lâminas de cerâmica, até mesmo um ligeiro contactopode danificar a lâmina. </li><li> Mova cada fatia da câmara de corte para a recuperação / exploração câmara usando um de boca larga, fogo-polido Pasteur de vidro pipeta. </li><li> Deixe as fatias descansar na câmara durante pelo menos 1 hora antes do início da experiência. Isto permite a degradação de tecidos e células danificadas ou a morrer e, portanto, resulta numa superfície de corte mais limpo. Durante a primeira hora, manter a temperatura da RPU na câmara no intervalo de 34-36 ° C; garantir que as fatias não são tocados por bolhas de ar que podem danificar as fatias ou torná-los flutuar. </li></ol> 4. Experiência <ol><li> Após 1 hora, deixar a temperatura da câmara de recuperação de arrefecer até à temperatura ambiente (17-25 ° C). NOTA: Este pode prolongar o período de tempo durante o qual as fatias pode ser utilizado por abrandar o crescimento de bactérias, bem como o metabolismo celular. Este período depende da região do cérebro e do tipo de célula. Por exemplo, certos tipos de células de Golgi pode ser encontrado para ser saudável em fatias8 h após o corte ao passo que outros tipos durar apenas 6 horas. </li><li> Depois de 1 hora se passou desde o corte, use as fatias para vários experimentos eletrofisiológicos. </li></ol>

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The authors declare that they have no competing financial interests.

Nós demonstramos um método para a preparação de fatias cerebrais agudas a partir de ratos em vez de temperatura fisiológica gelada. Demonstrou-se 11 que a qualidade das fatias obtidas em condições de calor é superior quando em comparação com as preparadas com condições de frio, desde que a lâmina tem fatiador de vibração vertical mínima. Cortando na temperatura fisiológica pode impedir artefatos fisiológicos causados ​​pela baixa temperatura, como as relacion...

O objectivo do método apresentado é obter de alta qualidade fatias cerebrais agudas para experiências in vitro electrofisiológicas, especialmente quando se utiliza adulto ou mesmo animais mais velhos. O método de fatiamento cerebral aguda, como descrito por Skrede e Westgaard 1 em duas frases elegantes, tornou-se um dos fundamentos da pesquisa em neurociência moderna e é empregado em inúmeras variações em todo o mundo. A qualidade das fatias é reflectido no número de neurónios por fatia vivo, o período de tempo durante o qual as células de manter as suas propriedades electrofisiológicas e morfológicas, bem como na integridade do tecido. Além disso, a duração máxima para gravações estáveis ​​depende da qualidade das fatias. Assim, ao longo das décadas, o método de corte original foi desenvolvido por grupos de pesquisa individuais para melhorar a recuperação após o corte fatia 2-10, muitas vezes por meio de modificações complexas de composição de cortar osoluções de recuperação r (como a adição de ácido ascórbico, tioureia ou mesmo de H 2 O 2), bem como intra-cardíaca de pré-perfusão do animal com soluções fisiológicas arrefecidos. Tal como foi recentemente mostrado 11, temperatura fisiológica durante o corte parece ser mais benéfico do que o arrefecimento para a saúde neuronal; a melhora é mais marcante quando se trabalha com adultos (2-8 meses) roedores. Evitar mudanças dramáticas de temperatura impede artefatos devido a processos dependentes da temperatura nas células, como plasticidade e 13 canais iónicos cinética 13,14. Tais mudanças podem influenciar a tensão de membrana e sinalização intracelular de cálcio, limiar pico e pico de forma. O método &quot;quente&quot; fatia aguda preparação aqui apresentado é um processo geral para a obtenção de alta qualidade fatias cerebrais agudas a partir de qualquer região do cérebro, incluindo no cerebelo, córtex e no hipocampo, núcleos do tronco cerebral 16 &lt;/ Sup&gt;, bem como o bolbo olfactivo, tanto em ratos e ratinhos. Notavelmente, o procedimento temperatura fatiamento fisiológico requer que a lâmina de corte vibra quase perfeitamente na horizontal e é, sem quaisquer defeitos estruturais. Tal precisão pode não ser possível com modelos de cortador de idosos; em tais casos, é recomendável a realização da preparação fatia em condições de frio como a temperatura baixa parece tornar o tecido mais resistente a danos mecânicos, mesmo que à custa de aberrações metabólicas.

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			<td height="26" style="height:26px;width:140px;">Name</td>
			<td style="width:73px;">Company</td>
			<td style="width:69px;">Catalog #</td>
			<td style="width:292px;">Comments</td>
		</tr>
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			<td height="26" style="height:26px;width:140px;">Pentobarbital</td>
			<td style="width:73px;">CTS</td>
			<td style="width:69px;">170066</td>
			<td style="width:292px;">Concentration: 60 mg / ml&#160; in physiological saline.</td>
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			<td height="40" style="height:41px;width:140px;">Big scissors&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">14001-16</td>
			<td style="width:292px;">Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&#160;</td>
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		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Iris scissors&#160;</td>
			<td style="width:73px;">Prestige medical</td>
			<td style="width:69px;">48,148</td>
			<td style="width:292px;">Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5 - 2 cm</td>
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			<td height="26" style="height:27px;width:140px;">Fine tip forceps&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">11254-20</td>
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			<td height="26" style="height:27px;width:140px;">Scalpel&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">91003-12</td>
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			<td height="26" style="height:27px;width:140px;">Scalpel blade #11</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">10011-00</td>
			<td style="width:292px;"></td>
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			<td height="26" style="height:27px;width:140px;">Small spatula&#160;</td>
			<td style="width:73px;">Fisher&#160;</td>
			<td style="width:69px;">2350</td>
			<td style="width:292px;"></td>
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			<td height="26" style="height:27px;width:140px;">Filter paper</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">Any laboratory brand can be used.</td>
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			<td height="40" style="height:41px;width:140px;">Petri dishes</td>
			<td style="width:73px;">Duroplan</td>
			<td style="width:69px;">Z231509-1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Glass beakers&#160;</td>
			<td style="width:73px;">SCHOT</td>
			<td style="width:69px;">10022846</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Pasteur pipette&#160;</td>
			<td style="width:73px;">Maple Leaf Brand</td>
			<td style="width:69px;">14672-029</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Super glue&#160;</td>
			<td style="width:73px;">LOCTITE</td>
			<td style="width:69px;">4091361/1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Slicer</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7000-smz</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Ceramic slicing blade</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7550-1-C</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Magnetic heater/stirrer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up the SPS for the procedure</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Electric kettle</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up water for temperature control</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:56px;width:140px;">Slice recovery chamber + heating unit</td>
			<td style="width:73px;">Warner instruments</td>
			<td style="width:69px;">&#160;BSC-HT +&#160; BSC-BUW</td>
			<td style="width:292px;">Home-built models may also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Thermometer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For monitoring SPS temperature during dissection and slicing</td>
		</tr>
	</tbody>
</table>

slice it hot: acute adult brain slicing in physiological temperature

Aqui apresentamos um protocolo para a preparação de fatias cerebrais agudas. Este procedimento é um elemento crítico para experiências de patch-clamp electrofisiológicas que determina em grande parte a qualidade dos resultados. Demonstrou-se que omitindo o passo de arrefecimento durante o processo de corte é benéfico na obtenção de cortes e células saudáveis, especialmente quando se lida com as estruturas cerebrais altamente mielinizadas de animais maduros. Embora o mecanismo preciso pelo qual temperatura elevada apoia a saúde neural só se pode especular em cima, é lógico que, sempre que possível, a temperatura em que o corte é realizado deve ser perto de condições fisiológicas para evitar artefatos de temperatura relacionados. Outra vantagem importante deste método é a simplicidade do processo e, por conseguinte, o tempo de preparação curto. No método demonstrado ratinhos adultos são usadas, mas o mesmo procedimento pode ser aplicado com ratos mais jovens, bem como os ratos. Além disso, os seguintes cl remendoamp experiência é realizada em fatias horizontais do cerebelo, mas o mesmo procedimento pode também ser utilizado em outros planos, bem como outras áreas do cérebro posterior.

Fatias preparados do modo descrito pode ser usado para várias experiências electrofisiológicas e Optogenetic. Na Figura 3A e 3C, que mostram um exemplo representativo de uma fatia horizontal cerebelar e uma fatia cerebral cortical coronal, respectivamente, vistas sob interferência diferencial (DIC) ópticas. Na fatia cerebelar, vários tipos de neurônios do cerebelo pode ser facilmente reconhecido por sua forma e localização das células do corpo, permitindo alvo registros eletro...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Agudo Adulto Cérebro Cortando na temperatura fisiológica

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

slice-it-hot-acute-adult-brain-slicing-in-physiological-temperature

Research

JoVE Journal

Neuroscience

25.6K visualizações.  Hebrew University of Jerusalem. In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Vídeo: Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Estado de dependência-Efeitos sobre a TMS: Um olhar sobre Comportamento fosfeno Motive

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

Neurociência

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imagem Pheromone Sensing em um Preparação Slice rato Vomeronasal aguda do tecido

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Cirurgia estereotáxica para lesão excitotóxica de áreas específicas do cérebro do rato adulto

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Detecção de palmitoylation proteína em neurônios cultivados por imunoprecipitação e Acil-Biotina Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Cérebro Fatia Biotinilação: Uma<em&gt; Ex Vivo</em&gt; Abordagem medir Região específicas do tráfego de proteínas de membrana plasmática em adultos Neurônios

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Lineage-reprogramação de células pericyte derivados do Adulto Cérebro Humano em neurônios induzidos

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

Análise em profundidade Physiological de populações de células definido no aguda Tissue Fatias do mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Gravação de plasticidade sináptica em fatias Hippocampal agudas, mantidas em um pequeno volume reciclagem - perfusão- e sistema de câmara de submersão-tipo

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Um gradiente de temperatura de ensaio para determinar preferências térmicas de larvas de Drosophila

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Culturas de fatias flutuantes de curto prazo do cérebro humano adulto

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...