Este estudo foi financiado pelo Instituto Nacional sobre Abuso de Drogas Grants DA 006886, 032270 e DA.

<ol><li> Fio os motores de passo bipolares apertando os fios nos conectores fornecidos com a placa de motorista. As cores dos fios de motores de passo bipolares são padronizados (Figura 1). Nota: Os motores de passo descritos têm uma resolução de 1,8 ° / passo, orientada para 0,346 ° / passo. A stereotax padrão tem 3 mm/360 ° de viagem. A resolução final é 2,88 mM / passo. Os motores também são capazes de pisar fracionada. <ol><li> Conecte o fio verde para A +, conecte o fio preto para A-, conecte o fio vermelho para B + e ligar o fio azul para B-. </li></ol></li><li> Deslize os engates nos motores de passo, tomando cuidado para alinhar os furos de fixação, e prenda-os com 12x Parafusos M3 cabeça de soquete (20 mm) (Figura 2). <ol><li> Certifique-se de que os engates estão firmemente ligados aos motores. Nota: Os modelos 3D não incluem threads. As partes são rotulados com tamanho de rosca, mas que deve ser aproveitado após eles sãofabricado. </li></ol></li><li> Remova os parafusos de fixação das garras do polegar em todos os três eixos do instrumento estereotáxico usando uma pequena chave sextavada. Os apertos de polegar são segmentadas, assim transformá-las anti-horário para a remoção. Manter as anilhas de PTFE no lugar sobre o braço (Figura 3). </li><li> Parafuso a extremidade roscada de os colares sobre as barras roscadas de braços do instrumento estereotáxico (Figura 3). <ol><li> Certifique-se de que não há diferença entre os colares e anéis de vedação PTFE. Isto garante que as coordenadas são mantidas quando o robot muda de instruções. </li><li> Fixe os colares para os fios dos braços estereotáxica utilizando 3x NF10-32 (1/4 de polegada) parafusos de ponta cavada. </li><li> Deslize cada motor e acoplador sobre os colares e os braços estereotáxica. Certifique-se que os motores ficam nivelados com os braços, e os furos conjunto sobre os colares alinhar com a parte plana dos eixos do motor (Figura 4). </li><li> Fixe o couplers ao stereotax usando os furos de montagem e 6x NF10-32 (1/2 polegada) parafusos de ponta xícara (Figura 4). </li><li> Fixe os colares para os eixos do motor utilizando 3x NF10-32 (1/4 de polegada) parafusos de fixação (Figura 4). </li></ol></li><li> Prepare a placa de motorista CNC, definindo cada um dos pinos do controlador à metade do piso. Nota: Este driver de motor de passo vem como uma placa de circuito exposto. Um exemplo pode ser construído, embora não seja necessário. Além disso, podem ser utilizados um número de diferentes controladores de motor passo a passo bipolar. Se assim for, verifique se as instruções de instalação são seguidos para a placa que tiver adquirido. <ol><li> Alinhe todos os 6 pinos por motor de passo da mesma forma. Half-stepping permite o dobro da resolução passo em graus / passo (Figura 5). </li><li> Virar o pino 1 para a posição, o pino 2 para a posição desligado, o pino 3 ao em posiçãoção, o pino 4 para a posição desligado, o pino 5 para a posição, e pino 6 para a posição desligado (Figura 5). </li></ol></li><li> Ligue os motores (X - Y - Z) no driver de motor de passo, junto com a fonte de alimentação de 12 V. A colocação correta é marcado no driver. Além disso, conecte o condutor de passo a porta serial de um computador usando um DB25cable (Figura 6). </li><li> Instale o software de fresagem CNC em um computador pessoal (este terá de estar localizado em uma área cirúrgica) seguindo as instruções padrão. Uma vez instalado, abra o software para iniciar a configuração. <ol><li> Configure o software para se comunicar com os motores de passo. Nota: As seguintes instruções são para uso apenas com o driver de motor de passo TB6560. </li><li> Clique através dos menus do software como se segue. Abertas → Configuração → Ports and Pins→ Sinais de saída. Preencha o prompt para corresponder Figura 7 e clique em Aplicar. </li><li> Clique através dos menus do software como se segue. Abrir → Configuração → Ports and Pins → sinais de entrada. Preencha o prompt para corresponder Figura 8 e clique em Aplicar. </li><li> Clique através dos menus do software como se segue. Abra → Configuração → Portos e pinos → saídas do motor. Preencha o prompt para corresponder Figura 9 e clique em Aplicar. </li><li> Clique através dos menus do software como se segue. Afinação Configuração → Motor → Abrir. Preencha o prompt para corresponder Figura 10 e clique em Salvar configurações do Eixo. Repita o passo anterior para todos os 3 eixos usando os mesmos valores. </li></ol></li><li> Calibre o stereotax para a escala do software CNC. Nota: O software é projetado para máquinas de usinagem convencionais, pelo que a sua unidade de medida não será proporcional ao percurso de um instrumento estereotáxico.<ol><li> Defina a velocidade dos motores de 1 polegada por minuto, e &quot;jog&quot; eixo Z do instrumento estereotáxico com PgUp / PgDn ao milímetro. Nota: A velocidade máxima recomendada é de corrida de 3.500 mM / seg ea velocidade máxima recomendada de corte é 500 mm / seg. </li><li> Zero do eixo Z, e Jog o estereotáxica instrumento de 1 mm. A distância percorrida no eixo Z no Mach3 é a &quot;Escala Constant&quot;. Coordenadas da máquina são determinadas multiplicando as coordenadas do crânio (mm) pela &quot;Escala Constant&quot;. </li><li> Realizar testes aleatórios de todos os 3 eixos, programando-os a viajar algumas distâncias conhecidas, e garantir os movimentos são precisos. Se a viagem stereotax está muito longe ou curto, modificar a constante de escala de acordo. Nota: Depois de escala é concluída, os scripts personalizados incluídos podem ser usados ​​para gerar g-code para cirurgias. No entanto, é altamente recomendável que os usuários se familiarizem com g-code antes de tentar gerar auto-cirurgias. Isto é imperativo para a solução de problemas e modificar cirurgias automatizadas. </li></ol></li><li> Prenda a broca micro motor para o instrumento estereotáxico usando o suporte extra grande sonda. Nota: A velocidade mínima recomendada broca é de 40.000 rpm. </li><li> Auto-gerar G-Code para uma craniotomia borda afiada com 3 furos para parafusos crânio. <ol><li> Coloque todos os scripts personalizados da tabela de software em uma única pasta em um PC. </li><li> Abra o script &quot;SharpEdgeCraniotomy.m&quot; e executar o código. </li><li> Selecione Ambos para o prompt &quot;que tipo de cirurgia você estará realizando?&quot; (Figura 11). </li><li> Selecione Personalizar para definir os cantos da janela do crânio. Preencha cada pedido para combinar Figura 12. </li><li> Definir as posições X e Y dos cantos craniotomia. Cada coordenada deve ser inserido na ordem correta, de acordo com o exemplo na Figura 13 </strong&gt;. </li><li> Insira 3 no prompt para produzir 3 buracos do crânio (Figura 14). </li><li> Selecione Definir utilizando coordenadas e insira as coordenadas de cada buraco a partir do modelo na Figura 15. Nota: Se as coordenadas precisas não são importantes, há uma opção para apontar e clicar as posições dos furos para uma imagem de um crânio de ratos. Posições será gerada automaticamente. </li><li> Definir os parâmetros de perfuração. Pela primeira cirurgia testes, aceite os valores padrão. Nota: Estes valores estão dependentes dos motores de passo, e o crânio do animal. Cada local raça de rato e alvo tem uma espessura crânio ligeiramente diferente. Para as poucas cirurgias iniciais utilizando este dispositivo, estar preparado para testar a profundidade de perfuração e remova todos os pedaços do crânio restantes manualmente. Os valores podem então ser modificados para cirurgias futuras (Figura 16). </li><li> Nomeie o g-code, que será gerado e sa automaticamenteved ao diretório de trabalho. </li></ol></li><li> Carregue o g-code no software de fresagem CNC e um crânio de teste para os instrumentos estereotáxica barras de ouvido. <ol><li> Correr manualmente a broca para Bregma usando as setas. Use uma velocidade de jog lento (~ 5 polegadas / m) para garantir a precisão. </li><li> Comece a broca rotativa de maior do que 38.000 rpm. </li><li> Imprensa CycleStart, o stereotax irá realizar muitas passagens da mesma corte, em diferentes profundidades. Entre cada passagem, o stereotax fará uma pausa, de modo que o cirurgião pode continuar ou abortar corte. Pressione o botão Continuar Ciclo (Alt-R) para continuar cortando passes. </li></ol></li></ol>

<ol>
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O uso de equipamentos de cirurgia automatizada ajuda a eliminar alguns dos problemas mais comuns na pesquisa em neurociência. Primeiro, os caminhos da ferramenta são 100% reproduzível. Cada corte tem a garantia de ser no mesmo local em relação ao bregma. Em segundo lugar, ele deve reduzir o erro experimentador. Embora muitos pesquisadores são cirurgiões altamente qualificados, é preciso uma quantidade excepcional de prática para se tornar até mesmo um cirurgião competente. Este dispositivo permitirá que os n...

Cirurgia estereotáxica roedor é usado em uma ampla variedade de aplicações, incluindo a lesão neuroscience 1, iontoforese 2, o implante de microfio, 3, 4 estimulação, e imagiologia crânio fina 5. No entanto, existem grandes obstáculos enfrentados por aqueles que desejam aplicar essas técnicas, incluindo a curva de aprendizagem para a realização de cirurgia estereotáxica precisos e alta probabilidade de erro humano. Erros humanos incluem medição e falhas de cálculo, bem como a baixa precisão e reprodutibilidade dos movimentos humanos. Em um esforço para reduzir os erros de confusão, os cirurgiões estereotáxicos se beneficiariam de um sistema que garante que todos os procedimentos cirúrgicos são realizados de forma idêntica entre os indivíduos. A redução de erros também é um método pelo qual os investigadores podem minimizar o uso de matérias de origem animal, o objetivo principal dos Institutos Nacionais de Saúde para experimentos com animais 6. Em um mundo ideal, todo scirurgias tereotactic seria perfeitamente replicável dentro experimentos e entre laboratórios. Para resolver esse problema, as empresas desenvolveram novas stereotaxics ultra-precisos e displays digitais para leitura de medições. Para remover os erros de deslocação humanos, micro manipuladores e stereotaxics motorizados foram produzidas comercialmente, mas seu alto custo pode ser proibitivo para um laboratório com um orçamento limitado. Além disso, o software é totalmente exclusiva, e não pode ser modificada pelo investigador para acomodar um novo tipo de cirurgia. Uma solução acessível para o problema de erro humano é construir uma stereotax robótico do modelo existente de um laboratório, utilizando-se equipamento padrão da indústria CNC. Por causa de uma comunidade florescente hobby CNC, os materiais são significativamente menos caro do que o equipamento científico. Isto permite construir um instrumento estereotáxico precisas CNC, o que também é altamente flexível e barato. Com um conhecimento básico de usinagem CNC e G-Code, indivíduosals pode programar qualquer cirurgia estereotáxica que eles imaginam, sem as limitações de software proprietário. E, a fim de acelerar a produção de g-code para cirurgias simples, este protocolo inclui um software que permite ao usuário projetar cirurgias (craniotomia borda afiada, de janelas crânio fino, furo de perfuração e implantes rebaixamento) dentro de apontar e clicar menus. Estes programas de saída de um g-code concluído que pode ser executado diretamente de software CNC. Ao todo, um upgrade estereotáxica motorizada é ideal para quem tem interesse em aumentar a precisão ea replicabilidade das cirurgias, mantendo a flexibilidade e baixo custo de uma plataforma de código aberto.

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			<td height="44" style="height:44px;width:141px;">1x Standard U Frame Stereotax</td>
			<td style="width:137px;">Kopf</td>
			<td style="width:117px;"><a href="http://www.kopfinstruments.com/Stereotaxic/900.html">Kopf</a></td>
			<td style="width:355px;">This protocol should work with most existing stereotaxic devices.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:141px;">3x 12 V, 1.6 A, 233 oz-inch Geared Bipolar Stepper Motor</td>
			<td style="width:137px;">Phidgets</td>
			<td style="width:117px;"><a href="http://www.robotshop.com/geared-bipolar-stepper-motor-3v-233-oz-in.html">Robot Shop</a></td>
			<td style="width:355px;">Any high torque geared stepper motor should do.&#160;</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:141px;">1x 3 Axis CNC Stepper Motor Driver Board Controller</td>
			<td style="width:137px;">Toshiba</td>
			<td style="width:117px;"><a href="http://www.ebay.com/itm/CNC-Router-3-Axis-TB6560-3-5A-Stepper-Motor-Driver-Board-For-Engraving-Machine-/300981888819">Ebay</a></td>
			<td style="width:355px;">Any 3 Axis CNC driver should do. Linked Item includes Mach3 CNC software.&#160;</td>
		</tr>
		<tr height="94">
			<td height="94" style="height:94px;width:141px;">2x Arm Couplers: medial-lateral (ML) &#38; dorsal-ventral (DV)</td>
			<td style="width:137px;">custom machined</td>
			<td style="width:117px;"><a href="http://eden.rutgers.edu/~mrcoffey/images/3D%20Models%20-%20CNC%20Stereotax%20Parts.zip">Part Drawings</a></td>
			<td style="width:355px;">These must be machined by your local machine shop. (costs will vary)</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:141px;">1x anterior-posterior (AP) Coupler</td>
			<td style="width:137px;">custom machined</td>
			<td style="width:117px;"><a href="http://eden.rutgers.edu/~mrcoffey/images/3D%20Models%20-%20CNC%20Stereotax%20Parts.zip">Part Drawings</a></td>
			<td style="width:355px;">These must be machined by your local machine shop. (costs will vary)</td>
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		<tr height="127">
			<td height="172" rowspan="3" style="height:172px;width:141px;">3x Motor to Stereotax Collar</td>
			<td rowspan="3" style="width:137px;">custom machined</td>
			<td style="width:117px;"><a href="http://eden.rutgers.edu/~mrcoffey/images/3D%20Models%20-%20CNC%20Stereotax%20Parts.zip">Part Drawings</a></td>
			<td rowspan="3" style="width:355px;">These must be machined by your local machine shop. (costs will vary)</td>
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		<tr height="23">
			<td height="23" style="height:23px;width:117px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:117px;"><a href="http://eden.rutgers.edu/~mrcoffey/images/Collar.html">View in Browser</a></td>
		</tr>
		<tr height="43">
			<td height="65" rowspan="2" style="height:65px;width:141px;">12x NF10-32 Cup Point Set Screws</td>
			<td rowspan="2" style="width:137px;">McMaster Carr</td>
			<td style="width:117px;"><a href="http://www.mcmaster.com/#92313A829">&#189;&#8221; Length</a></td>
			<td rowspan="2" style="width:355px;">You will need 6 of each.</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:117px;"><a href="http://www.mcmaster.com/#92313A825">&#188;&#8221; Length</a></td>
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			<td height="46" style="height:46px;width:141px;">12x M3 Socket Head Screws (20 mm)</td>
			<td style="width:137px;">McMaster Carr</td>
			<td style="width:117px;"><a href="http://www.mcmaster.com/#92855A319">20mm Length</a></td>
			<td style="width:355px;">You will need 4 for each motor</td>
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			<td height="48" style="height:48px;width:141px;">1x Micro-Motor Drill&#160;</td>
			<td style="width:137px;">Buffalo Dental</td>
			<td style="width:117px;"><a href="http://www.buffalodental.com/pdfnew/x50.pdf">X50</a></td>
			<td style="width:355px;">Any Micromotor drill will work.&#160; At least 38,000 rpm recommended</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:141px;">1x 12 V DC Power Supply</td>
			<td style="width:137px;">12 Volt Adapters</td>
			<td style="width:117px;"><a href="http://www.12vadapters.com/adapter/power-supply/12v/7-amp-7a.html">12v DC &#8211; 7 Amp</a></td>
			<td style="width:355px;">Any 12 V DC PSU should work (ensure amperage rating is higher than the sum of the motors&#8217; amperage).</td>
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		<tr height="44">
			<td height="44" style="height:44px;width:141px;">1x Extra Large Probe Holder</td>
			<td style="width:137px;">Stoelting</td>
			<td><a href="http://www.stoeltingco.com/neuroscience/accessories/stereotaxic-probe-holders/extra-large-probe-holder-551.html">Stoelting</a></td>
			<td style="width:355px;"></td>
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			<td height="44" style="height:44px;width:141px;">1x Grade B Rat Skull</td>
			<td style="width:137px;">Skulls Unlimited</td>
			<td><a href="http://www.skullsunlimited.com/record_variant.php?id=4009">Skulls Unlimited</a></td>
			<td style="width:355px;"></td>
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			<td height="21" style="height:21px;width:141px;">Mach 3 Mill</td>
			<td style="width:137px;">ArtSoft USA</td>
			<td style="width:117px;"><a href="http://www.machsupport.com/ccount/click.php?id=1">Trial Download</a></td>
			<td style="width:355px;">Any Standard CNC controlling software should work.</td>
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			<td height="44" style="height:44px;width:141px;">Surgery Designer</td>
			<td style="width:137px;">Kevin Coffey &#38; David Barker</td>
			<td style="width:117px;"><a href="http://www.mathworks.com/matlabcentral/fileexchange/40282-robot-assisted-stereotactic-surgery">MATLAB File Exchange</a></td>
			<td style="width:355px;">These codes are available to modify. We accept no responsibility for your use or modification of code.</td>
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building an open-source robotic stereotaxic instrument

Este protocolo inclui os desenhos e software necessários para a atualização de um instrumento estereotáxico existente para um (CNC) instrumento estereotáxico robótico para cerca de US $ 1.000 (excluindo uma broca), utilizando motores de passo padrão da indústria e CNC software de controle. Cada eixo possui controle de velocidade variável e podem ser operadas simultaneamente ou independentemente. A flexibilidade do robô e sistema de codificação aberta (g-code) torná-lo capaz de realizar tarefas personalizadas que não são suportadas por sistemas comerciais. Suas aplicações incluem, mas não estão limitados a, abertura de furos, craniotomias borda afiada, desbaste crânio e eletrodos ou cânula diminuindo. A fim de agilizar a escrita de g-codificação para cirurgias simples, desenvolvemos scripts personalizados que permitem aos indivíduos para projetar uma cirurgia sem nenhum conhecimento de programação. No entanto, para os usuários a tirar o máximo proveito do stereotax motorizada, seria benéfico para ser entendido em programação matemática e G-Coding (prog simplesbatendo para usinagem CNC). A velocidade de perfuração recomendada é maior do que 40.000 rpm. A resolução do motor de passo é de 1,8 ° / Step, voltado para 0,346 ° / Step. A stereotax padrão tem uma resolução de 2,88 mM / passo. A velocidade máxima recomendada de corte é 500 mm / seg. A velocidade máxima recomendada é de corrida de 3.500 mM / seg. O tamanho recomendado máximo broca é HP 2.

O resultado final da cirurgia projetado nos métodos será um crânio de ratos com uma craniotomia borda afiada, e 3 buracos do crânio (Figura 17). Note-se que o crânio utilizado para demonstrar a cirurgia foi muito maior do que o crânio de ratos prototípico. A craniotomia aresta viva pode ser utilizado para inserir uma matriz microfio, dentro do cérebro, para a gravação de alta densidade neurais. O stereotax CNC também pode ser utilizado para baixar a matriz com uma grande precisão. Software e...

Watch this Scientific Journal Video about Building An Open-source Robotic Stereotaxic Instrument at JoVE.com

Building An Open-source Robotic Stereotaxic Instrument

Este protocolo inclui os desenhos e software necessário para atualizar um instrumento estereotáxico existente para um robô (numérico controlado por computador; CNC) instrumento estereotáxico para cerca de US $ 1.000 (excluindo uma broca).

Construir um Robotic estereotáxica Instrumento Open-source

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for ...

building-an-open-source-robotic-stereotaxic-instrument

Research

JoVE Journal

Neuroscience

14.7K visualizações.  Rutgers, The State University of New Jersey.  Este protocolo inclui os desenhos e software necessário para atualizar um instrumento estereotáxico existente para um robô (numérico controlado por computador; CNC) instrumento estereotáxico para cerca de US $ 1.000 (excluindo uma broca). 

Vídeo: Building An Open-source Robotic Stereotaxic Instrument

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

Cirurgia estereotáxica de roedores e animais Melhorias Resultado do Bem-Estar de Neurociência Comportamental

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

Neurociência

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 ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Infusão estereotáxica de oligomica beta-amilóide no ratinho Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Não restrição EEG Radiotelemetria: Epidural e Deep intracerebral estereotáxica EEG Colocação de eletrodos

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.

Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

De alta densidade Electroencefalografia Aquisição em um modelo de roedor Usando de baixo custo e de código aberto Recursos

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Uma abordagem alternativa para estudar eventos primários na neurodegeneração usando fatias de cérebro de rato Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).

Cirurgia estereotástrica para implante de matrizes de microeletrodos no Marmoset comum (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the ...

An Open-Source Virtual Reality System for the Measurement of Spatial Learning in Head-Restrained Mice

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and optical imaging tools while delivering precise sensory stimuli to a behaving animal. Recently, human and rodent studies using virtual reality (VR) environments have shown VR to be an important tool for uncovering the neural mechanisms underlying spatial learning in the hippocampus and cortex, due to the extremely precise control over parameters such as spatial and contextual cues. Setting up virtual environments for rodent spatial behaviors can, however, be costly and require an extensive background in engineering and computer programming. Here, we present a simple yet powerful system based upon inexpensive, modular, open-source hardware and software that enables researchers to study spatial learning in head-restrained mice using a VR environment. This system uses coupled microcontrollers to measure locomotion and deliver behavioral stimuli while head-restrained mice run on a wheel in concert with a virtual linear track environment rendered by a graphical software package running on a single-board computer. The emphasis on distributed processing allows researchers to design flexible, modular systems to elicit and measure complex spatial behaviors in mice in order to determine the connection between neural circuit activity and spatial learning in the mammalian brain.

Here, we present a simplified open-source hardware and software setup for investigating mouse spatial learning using virtual reality (VR). This system displays a virtual linear track to a head-restrained mouse running on a wheel by utilizing a network of microcontrollers and a single-board computer running an easy-to-use Python graphical software package.

Um Sistema de Realidade Virtual de Código Aberto para a Medição da Aprendizagem Espacial em Camundongos Contidos na Cabeça

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and ...

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/Microneurography/APTrack&amp;source=gmail&amp;ust=1680789829274000&amp;usg=AOvVaw0nMk9J_EI2jUn8SlmC5JJm" href="https://github.com/Microneurography/APTrack" target="_blank">https://github.com/Microneurography/APTrack</a>). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials during&#160;in vivo&#160;human microneurography, for the first time, and during&#160;ex vivo&#160;mouse electrophysiological recordings of C-fibers and A&#948;-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and A&#948;-fiber nociceptors.

Rastreamento de limiar elétrico de circuito fechado em tempo fechado de código aberto para pesquisa translacional da dor

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in ...