Esta pesquisa foi possível em parte pelo generoso financiamento do programa Knight Campus Undergraduate Scholars e da Phil and Penny Knight Foundation.

Todos os métodos aqui detalhados foram realizados em conformidade com o protocolo IRB e aprovação (University of Oregon IRB protocol number 10182017.017). O consentimento informado foi obtido de todos os sujeitos.
1. Aparelhos de alcance
<ol><li>Coloque um tablet gráfico grande em uma área de trabalho.</li>
	<li>Use uma estrutura de alumínio ajustável de 80 a 20 para posicionar o monitor de tarefas de 6 a 8 acima do tablet em paralelo, com a tela voltada para cima (para obter uma planta, confira aqui: https://github.com/greenhouselab/Reach_TMS e Figura 1 Suplementar). NOTA: Esta configuração permite que os participantes alcancem o tablet e adquiram alvos apresentados no monitor de tarefas enquanto ocluem a visão do braço que está a alcançar.</li>
	<li>Use a configuração descrita em Kim et al.3 como referência.</li>
</ol>2. Interfaces da máquina
<ol><li>Ligue o tablet ao computador através de uma porta USB. Conecte o monitor de tarefas ao computador através da porta HDMI. Conecte a porta TMS traseira ao computador através de um cabo DB-9.</li>
	<li>Conecte o sistema EMG ao computador por meio de um DAQ da placa PCI-6220. Conecte o fotodiodo ao sistema EMG através de um cabo BNC.</li>
</ol>3. Sensor de fotodiodo
<ol><li>Conecte um sensor de fotodiodo ao cabo BNC. Prenda o sensor de fotodiodo com fita adesiva no canto superior direito do monitor de tarefas, com o sensor voltado para a tela, a ≤ 1 cm de distância. NOTA: Isso registrará o tempo dos estímulos apresentados no monitor de tarefas como dados analógicos em um canal de entrada independente.</li>
</ol>4. Software
<ol><li>Baixe o VETA Toolbox26 (https://github.com/greenhouselab/Veta) para o MATLAB 2018 para interagir com o hardware para coleta de dados.</li>
	<li>Baixe o código de tarefa de alcance (https://github.com/greenhouselab/Reach_TMS) desenvolvido para o controle de parâmetros experimentais e interface com o tablet.</li>
</ol>5. Triagem dos participantes e consentimento informado
<ol><li>Rastreie o assunto em busca de contraindicações para TMS. Os critérios de exclusão incluem uma história pessoal ou familiar de convulsão, cefaleia, trauma cerebral, desmaios, estresse crônico ou ansiedade, problemas com o sono e qualquer medicação neuroativa. Critérios de exclusão adicionais incluem quaisquer implantes de metal no cérebro ou crânio e qualquer uso recreativo de drogas ou álcool nas 24 horas anteriores ao teste. Os critérios de inclusão incluíram destro e idade entre 18 e 35 anos.</li>
	<li>Fornecer uma explicação por escrito do procedimento e dos riscos associados, esclarecendo quaisquer outras questões que o participante possa ter.</li>
	<li>Obter o consentimento informado dos participantes.</li>
</ol>6. Configuração do assunto
<ol><li>Posicione o sujeito em uma cadeira confortável de frente para o tablet. Certifique-se de que os joelhos estão flexionados a 90° com as pernas sob a mesa.</li>
	<li>Prepare a pele e coloque eletrodos EMG.<ol><li>Use lixa de grão fino para abrasar suavemente a pele no local do primeiro interósseo dorsal direito (IED), extensor radial do carpo e músculos deltoideos anteriores, bem como a proeminência C4 na base do pescoço, para detectar artefatos elétricos produzidos pelo pulso TMS. NOTA: Os locais de gravação muscular podem ser adaptados com base nas necessidades do usuário.</li>
		<li>Esfregue cada área abrasada com uma almofada de preparação de álcool uma vez por local do eletrodo para limpar.</li>
		<li>Coloque um eletrodo EMG em cada local. Certifique-se de que os eletrodos funcionem perpendicularmente às fibras musculares. Coloque o eletrodo terra na proeminência óssea do cotovelo direito.</li>
		<li>Prenda cada eletrodo com fita adesiva.</li>
	</ol></li>
	<li>Verifique a qualidade da gravação EMG. Use a caixa de ferramentas VETA para visualizar todos os traços EMG e garantir que eles estejam livres de artefatos. Se os vestígios de EMG forem barulhentos, certifique-se de que o solo esteja devidamente colocado e que todos os eletrodos façam contato adequado com a pele.</li>
</ol>7. Estimulação magnética transcraniana
<ol><li>Ligue a máquina TMS.</li>
	<li>Encontre o ponto quente TMS do músculo IDE direito através da estimulação do M1 esquerdo.<ol><li>Coloque a bobina ~5 cm lateral e 2 cm anterior ao vértice da cabeça, orientada ~45° para fora da linha média.</li>
		<li>Administrar pulsos de EMT uma vez a cada 4 s enquanto reposiciona a bobina em incrementos de aproximadamente 5 mm no plano anteroposterior e médio-lateral.</li>
		<li>Começando com 30% de saída máxima do estimulador, aumente gradualmente a intensidade da EMT em incrementos de 2% até que as PEmáx sejam observadas.</li>
		<li>Uma vez identificada a localização ótima, na qual os MEPs podem ser provocados de forma confiável na maioria (~75%) dos pulsos na menor intensidade possível do estimulador, determine o limiar motor de repouso (RMT) encontrando o nível de intensidade que produz MEPs com uma amplitude pico-a-pico de >50 μV em cinco de 10 pulsos.</li>
		<li>Marque a posição colocando suavemente tiras finas de fita refletiva na cabeça do participante ao longo do perímetro da bobina. Mantenha o posicionamento da bobina segurando manualmente a bobina ou usando um suporte para apoiá-la.</li>
	</ol></li>
</ol>8. Alcançando a configuração da tarefa
<ol><li>Coloque uma luva Velcro na mão direita do participante para facilitar uma postura de aperto de poder relaxada.</li>
	<li>Prenda a caneta à luva e aconselhe o sujeito a manter a mão relaxada entre os movimentos de alcance.</li>
	<li>Comunique as instruções da tarefa, que são as seguintes: Guie o cursor para a posição inicial na parte inferior da tela. Você verá uma sugestão em um dos dois locais de destino. Quando o alvo preencher com cor, alcance o alvo o mais rápido e com a maior precisão possível. Em seguida, retorne à posição inicial. Indique os locais das posições residenciais, pistas e alvos (Figura 1A).</li>
	<li>Treine o participante para cortar alvos com a caneta o mais rápido e com a maior precisão possível. Desligue as luzes da sala de tarefas para obscurecer a visão do participante dos movimentos dos braços e melhorar a visibilidade do monitor de tarefas.</li>
</ol>9. Design de tarefas
<ol><li>Controle a apresentação de estímulos visuais com o Psychtoolbox 3.0 no Matlab 2018 (Arquivo de Codificação Suplementar 1).</li>
	<li>Use os seguintes parâmetros para corresponder aos dados atuais: 20 ensaios práticos; 270 ensaios de teste; TMS em 4/5 dos ensaios de teste; A EMT coincide com o início da sugestão preparatória (EMT basal) ou 100 ms antes da sugestão imperativa (EMT atrasada) com igual frequência; 1/10 do total de ensaios são ensaios de captura, nos quais a sugestão imperativa não aparece; a posição inicial é um círculo com um raio de 2 cm posicionado no centro inferior do espaço de trabalho; dois alvos circulares com 1 cm de raio são posicionados a 15 cm da posição inicial a +45° e -45° de distância da linha média.</li>
	<li>Defina a ordem e a duração do evento da seguinte forma: sugestão preparatória a 900 ms e sugestão imperativa a 900 ms.</li>
</ol>10. Administração do TMS
<ol><li>A caixa de ferramentas VETA administra simultaneamente o TMS e registra o EMG https://github.com/greenhouselab/Veta.</li>
	<li>Controle o tempo dos pulsos TMS com a caixa de ferramentas VETA para coincidir com os eventos comportamentais escolhidos (ou seja, o início da pista preparatória ou 100 ms antes do início do alvo).</li>
	<li>Fornecer TMS com frequência suficiente para garantir um número suficiente de eurodeputados para análise. NOTA: Tal como escrito, o código de tarefa fornecerá um pulso TMS em 4/5 do total de ensaios, quer no início da sugestão preparatória - para provocar eurodeputados de linha de base - ou 100 ms antes da sugestão imperativa - para provocar eurodeputados atrasados. Os parâmetros podem ser ajustados no código de acordo com as necessidades do usuário. Ensaios sem EMT podem ser usados para avaliar o desempenho comportamental na ausência de EMT. Isso é útil para determinar qualquer possível influência do TMS no desempenho.</li>
</ol>

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Todos os autores declaram que não há conflitos de interesse

Os métodos descritos acima oferecem uma nova abordagem para estudar a preparação motora no contexto de alcançar comportamentos. Embora o alcance represente uma tarefa de modelo popular no estudo do controle motor e da aprendizagem, há uma necessidade de avaliar com precisão a dinâmica da SC associada ao comportamento de alcance. O TMS oferece um método não invasivo e temporalmente preciso de capturar a atividade de CS em pontos de tempo discretos durante o alcance. A abordagem aqui descrita comb...

O alcance direcionado a metas é um comportamento motor fundamental que permite que os seres humanos interajam e manipulem o ambiente externo. O estudo do alcance nos campos da fisiologia motora, psicologia e neurociência produziu uma literatura rica e extensa que inclui uma variedade de metodologias. Os primeiros estudos de alcance utilizaram registros neurais diretos em primatas não humanos para investigar a atividade neural no nível de neurônios individuais 1,2. Estudos mais recentes têm investigado o alcance utilizando paradigmas comportamentais que empregam a adaptação sensório-motora para explorar a natureza da aprendizagem motora e do controle 3,4,5. Tais tarefas comportamentais combinadas com ressonância magnética funcional e eletroencefalografia podem medir toda a atividade cerebral durante o alcance em humanos 6,7. Outros estudos aplicaram a EMT on-line para investigar várias características da preparação e execução do alcance 8,9,10,11,12,13,14. No entanto, continua a haver uma necessidade de uma abordagem flexível e de código aberto que combine a avaliação comportamental do alcance com o TMS. Embora a utilidade de combinar TMS com protocolos comportamentais esteja muito bem estabelecida15, aqui, examinamos especificamente a aplicação da TMS dentro do contexto de alcançar usando uma abordagem de código aberto. Isso é novo na medida em que outros grupos que publicaram usando essa combinação de métodos não disponibilizaram suas ferramentas prontamente, proibindo a replicação direta. Essa abordagem de código aberto facilita a replicação, o compartilhamento de dados e a possibilidade de estudos em vários locais. Além disso, se outros desejarem buscar novas questões de pesquisa com ferramentas semelhantes, o código-fonte aberto pode atuar como uma plataforma de lançamento para a inovação, pois é prontamente adaptável.
A EMT oferece um meio não invasivo de sondar o sistema motor em pontos de tempo precisamente controlados16. Quando aplicada sobre o córtex motor primário (M1), a EMT pode provocar uma deflexão mensurável no eletromiograma de um músculo alvo. A amplitude dessa onda de voltagem, conhecida como potencial evocado motor (PEmáx), fornece um índice do estado de excitabilidade momentânea da via corticoespinhal (SC) - um análogo resultante de todas as influências excitatórias e inibitórias na via CS17. Além de fornecer uma medição confiável dentro do sujeito da excitabilidade intrínseca da CS, a EMT pode ser combinada com outras métricas comportamentais ou cinemáticas para investigar as relações entre a atividade e o comportamento da CS de maneira temporalmente precisa. Muitos estudos têm utilizado uma combinação de EMT e eletromiografia (EMG) para abordar uma variedade de questões sobre o sistema motor, particularmente porque essa combinação de métodos torna possível investigar PEmáx sob uma vasta gama de condições comportamentais15. Uma área em que isso tem se mostrado particularmente útil é no estudo da preparação da ação, na maioria das vezes através do estudo de movimentos uniarticulares18. No entanto, há comparativamente menos estudos de EMT de movimentos naturalistas multi-articulares, como o alcance.
O objetivo atual era projetar uma tarefa de resposta atrasada que incluísse cinemática comportamental, administração on-line de EMS de pulso único e gravação EMG simultânea de vários músculos. A tarefa inclui um paradigma bidimensional de alcance ponto-a-ponto com feedback visual on-line usando um monitor orientado horizontalmente de modo que o feedback visual corresponda às trajetórias de alcance (ou seja, uma relação 1: 1 durante o feedback verídico e nenhuma transformação entre feedback visual e movimento). O projeto atual também inclui um conjunto de ensaios com uma perturbação visuomotora. No exemplo fornecido, este é um deslocamento rotacional de 20° no feedback do cursor. Estudos anteriores utilizaram um paradigma de alcance semelhante para abordar questões sobre os mecanismos e cálculos associados à adaptação sensório-motora 19,20,21,22,23,24,25. Além disso, essa abordagem permite avaliar a dinâmica da excitabilidade do sistema motor em pontos de tempo precisos durante o aprendizado motor on-line.
Como o alcance provou ser um comportamento frutífero para investigar a aprendizagem/adaptação, avaliar a excitabilidade da SC no contexto desse comportamento tem um enorme potencial para lançar luz sobre os substratos neurais envolvidos nesses comportamentos. Estes podem incluir influências inibitórias locais, mudanças nas propriedades de ajuste, o momento dos eventos neurais, etc., como foi estabelecido em pesquisas com primatas não humanos. No entanto, essas características têm sido mais difíceis de quantificar em humanos e populações clínicas. A dinâmica neural também pode ser investigada na ausência de movimento evidente em humanos usando a abordagem combinada de EMT e EMG (ou seja, durante a preparação do movimento ou em repouso).
As ferramentas apresentadas são de código aberto e o código é facilmente adaptável. Este novo paradigma produzirá insights importantes sobre os mecanismos envolvidos na preparação, execução, término e adaptação dos movimentos de alcance. Além disso, essa combinação de métodos tem o potencial de descobrir relações entre a eletrofisiologia e o comportamento de alcance em humanos.

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			<td>2-Port Native PCI Express&#160;</td>
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			<td>Computer monitor for EMG/TMS</td>
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			<td>Dell</td>
			<td>xps 8930</td>
			<td>RAM: 16 GB, Storage: 1TB, Graphics: 1060 6GB&#160;</td>
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			<td>EMG electrodes</td>
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			<td>Handle of paint roller</td>
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			<td>to be used as stylus handle, hollowed out center must be large enough for stylus to sit securely inside&#160;</td>
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			<td>Medical tape</td>
			<td>any</td>
			<td></td>
			<td>To secure EMG electrodes</td>
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			<td>PCI-6220 card DAQ</td>
			<td>National Instruments</td>
			<td></td>
			<td>To interface EMG system</td>
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			<td>Photodiode Sensor</td>
			<td>Vishay</td>
			<td>BPW21R</td>
			<td>To record timing of task events into EMG trace.</td>
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			<td>Rear TMS port</td>
			<td>Magstim</td>
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			<td>Right-handed polyethylene glove</td>
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			<td>Sensory Adhesive Interface, 2-slot</td>
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			<td>Stylus</td>
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			<td>Intuos-4 grip pen</td>
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			<td>Tablet-to-Computer USB cable&#160;</td>
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			<td>Task Monitor</td>
			<td>Asus</td>
			<td>VG248</td>
			<td></td>
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			<td>TMS coil</td>
			<td>Magstim</td>
			<td>D70 Remote Coil</td>
			<td>7cm diameter, figure-of-eight coil</td>
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			<td>TMS machine</td>
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assessing corticospinal excitability during goal-directed reaching behavior

Alcançar é um comportamento amplamente estudado em fisiologia motora e pesquisa em neurociências. Embora o alcance tenha sido examinado usando uma variedade de manipulações comportamentais, ainda existem lacunas significativas na compreensão dos processos neurais envolvidos no planejamento, execução e controle do alcance. A nova abordagem descrita aqui combina uma tarefa de alcance bidimensional com estimulação magnética transcraniana (EMT) e eletromiografia simultânea (EMG) gravação de múltiplos músculos. Este método permite a detecção não invasiva da atividade corticoespinhal em pontos de tempo precisos durante o desdobramento dos movimentos de alcance. O código de tarefa de exemplo inclui uma tarefa de resposta atrasada com dois possíveis destinos exibidos ± 45° fora da linha média. A EMT de pulso único é entregue na maioria dos ensaios de tarefa, seja no início da sugestão preparatória (linha de base) ou 100 ms antes da sugestão imperativa (atraso). Este desenho amostral é adequado para investigar alterações na excitabilidade corticoespinhal durante a preparação do alcance. O código de amostra também inclui uma perturbação visuomotora (ou seja, rotação do cursor de ± 20°) para investigar os efeitos da adaptação na excitabilidade corticoespinhal durante a preparação do alcance. Os parâmetros da tarefa e a entrega da EMT podem ser ajustados para abordar hipóteses específicas sobre o estado do sistema motor durante o comportamento de alcance. Na implementação inicial, os potenciais evocados motores (PEmáx) foram obtidos com sucesso em 83% dos ensaios de EMT, e as trajetórias de alcance foram registradas em todos os ensaios.

A execução bem-sucedida dos métodos descritos inclui o registro de dados do tablet, traços EMG e elicitação confiável de eurodeputados. Um experimento foi concluído que incluiu 270 ensaios de teste com TMS entregues em 4/5 dos ensaios (216 ensaios).
Os dados foram coletados de 16 participantes (oito mulheres; oito homens) com idades entre 25 ± 10 anos, todos autodeclarados destros. Avaliamos a eficácia da perturbação visual no desempenho comportamental derivando uma função de apr...

Watch this Scientific Journal Video about Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior at JoVE.com

Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior

Alcançar é uma habilidade fundamental que permite que os seres humanos interajam com o ambiente. Diversos estudos têm como objetivo caracterizar o comportamento de alcance utilizando uma variedade de metodologias. Este artigo oferece uma aplicação de código aberto de estimulação magnética transcraniana para avaliar o estado de excitabilidade corticoespinhal em humanos durante o desempenho da tarefa.

Avaliando a excitabilidade corticoespinhal durante o comportamento de alcance direcionado a objetivos

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral ...

assessing-corticospinal-excitability-during-goal-directed-reaching

Research

JoVE Journal

Neuroscience

1.6K visualizações.  University of Oregon. Alcançar é uma habilidade fundamental que permite que os seres humanos interajam com o ambiente. Diversos estudos têm como objetivo caracterizar o comportamento de alcance utilizando uma variedade de metodologias. Este artigo oferece uma aplicação de código aberto de estimulação magnética transcraniana para avaliar o estado de excitabilidade corticoespinhal em humanos durante o desempenho da tarefa.

Vídeo: Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Criopreservação de tecido cortical Blocos de Geração de culturas altamente enriquecido Neuronal

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Neurociência

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-maze Alternância forçado e Tarefas esquerda-direita para a Avaliação da Discriminação de Trabalho e Memória de Referência em Ratos

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.

Avaliação do nervo Excitabilidade em induzida por quimioterapia Neurotoxicidade

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Um Processo de Implantação de uma Câmara Spinal para Longitudinal<em&gt; In Vivo</em&gt; Criação de Imagens do Cordão do rato Spinal

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

Ensaios para detectar estruturas de reflexão de UV e determinar a sua importância na preferência do companheiro usando o Sailfin Molly<em&gt; Poecilia latipinna</em

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

Na Vivo Medida eletrofisiológica do nervo Ulnar rato com teste de excitabilidade Axonal

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their consequences. This process is integral to the normal human behavior and has been shown to be disrupted by neurological and psychiatric disorders such as addiction, schizophrenia, and depression. Rodents have long been used to uncover the neurobiology of human cognition. To this end, several behavioral tasks have been developed; however, most are non-automated and are labor-intensive. The recent development of the open-source microcontroller has enabled researchers to automate operant-based tasks for assessing a variety of cognitive tasks, standardizing the stimulus presentation, improving the data recording and consequently, improving the research output. Here, we describe an automated delay-based reinforcement-guided decision-making task, using an operant T-maze controlled by custom-written software programs. Using these decision-making tasks, we show the changes in the local field potential activities in the anterior cingulate cortex of a rat whilst it performs a delay-based cost-and-benefit decision-making task.

A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them.

Operante protocolos para avaliar o custo-benefício durante a tomada de decisão reforçada por roedores

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

Geração de hidrogéis com base em colágeno 3-D para analisar o crescimento e o comportamento axonal durante o desenvolvimento do sistema nervoso

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less vulnerable to interference. The neural mechanisms underlying the benefit of sleep for memory can be investigated using polysomnography (PSG). PSG is a combination of physiological recordings including signals from the brain (EEG), eyes (EOG), and muscles (EMG) that are used to classify sleep stages. In this protocol, we describe how PSG can be used in conjunction with behavioral memory assessments, actigraphy, and parent-report to examine sleep-dependent memory consolidation. The focus of this protocol is on early childhood, a period of significance as children transition from biphasic sleep (consisting of a nap and overnight sleep) to monophasic sleep (overnight sleep only). The effects of sleep on memory performance are measured using a visuospatial memory assessment across periods of sleep and wakeful-rest. A combination of actigraphy and parent report is used to assess sleep rhythms (i.e., characterizing children as habitual or non-habitual nappers). Finally, PSG is used to characterize sleep stages and qualities of those stages (such as frequencies and the presence of spindles) during naps. The advantage of using PSG is that it is the only tool currently available to assess sleep quality and sleep architecture, pointing to the relevant brain state that supports memory consolidation. The main limitations of PSG are the length of time it takes to prepare the recording montage and that recordings are typically taken over one sleep bought. These limitations can be overcome by engaging young participants in distracting tasks during application and combining PSG with actigraphy and self/parent-report measures to characterize sleep cycles. Together, this unique combination of methods allows for investigations into how naps support learning in preschool children.

This protocol describes methods used to examine neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood. It includes procedures for examining the effect of sleep on behavioral memory performance, as well as the application and recording of both polysomnography and actigraphy.

Medição de mecanismos neurais subjacentes à consolidação da memória dependente do sono durante cochilos na primeira infância

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less ...

Determining the Functional Status of the Corticospinal Tract Within One Week of Stroke

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential for recovery based on clinical assessments alone. The functional integrity of the corticospinal tract is an important prognostic biomarker for recovery of UL function, particularly for those with severe initial UL impairment. This article presents a protocol for evaluating corticospinal tract function within 1 week of stroke. This protocol can be used to select and stratify patients in trials of interventions designed to improve UL motor recovery and outcomes after stroke. The protocol also forms part of the PREP2 algorithm, which predicts UL function for individual patients 3 months poststroke. The algorithm sequentially combines a UL strength assessment, age, transcranial magnetic stimulation, and stroke severity, within a few days of the stroke. The benefits of using PREP2 in clinical practice are described elsewhere. This article focuses on the use of a UL strength assessment and transcranial magnetic stimulation to evaluate corticospinal tract function.

This protocol is for evaluating corticospinal tract function within 1 week of stroke. It can be used to select and stratify patients in trials of interventions designed to improve upper limb motor recovery and outcomes and in clinical practice for predicting upper limb functional outcomes 3 months after stroke.

Determinando o Status Funcional do Trato Corticorrindiano dentro de uma semana de Derrame

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential ...