Agradecemos ao Dr. D. Chen e ao Dr. C. Yen por compartilharem o script AFNI para configurar a fMRI em tempo real para PV 5 e à equipe AFNI pelo suporte de software. Esta pesquisa foi apoiada pelo financiamento da NIH Brain Initiative (RF1NS113278-01, R01 MH111438-01) e pela concessão do instrumento S10 (S10 RR023009-01) para o Martinos Center, a Fundação Alemã de Pesquisa (DFG) Yu215/3-1, BMBF 01GQ1702 e o financiamento interno da Max Planck Society.

Este estudo foi realizado de acordo com a Lei Alemã de Bem-Estar Animal (TierSchG) e a Portaria de Animais de Laboratório de Bem-Estar Animal (TierSchVersV). O protocolo experimental aqui descrito foi revisado pela comissão de ética (§15 TierSchG) e aprovado pela autoridade estadual (Regierungspräsidium, Tübingen, Baden-Württemberg, Alemanha).
1. Preparação da instalação experimental BOLD-fMRI para estudos em pequenos animais
<ol><li>Ligue o software do console para controlar os parâmetros de imagem e adquirir dados de ressonância magnética. NOTA: A configuração de fMRI em tempo real proposta é implementada utilizando macrofunções do software do console (versão 6) em paralelo com as funções de processamento de imagem do AFNI.</li>
	<li>Encontre sequências de RM (ou seja, Posição, Localizador, Aquisição Rápida com Aprimoramento de Relaxamento (RARE) e Imagem ecoplanar 3D (EPI) com o explorador de espaço de trabalho e, em seguida, arraste-as e anexe-as na lista de varredura. NOTA: As sequências de posição e localizador são usadas para identificar uma região de interesse (ROI) em um cérebro. Uma sequência RARE é usada para uma varredura de anatomia. Uma sequência EPI 3D é usada para medir respostas BOLD dinâmicas.</li>
	<li>Coloque os scripts de macro predefinidos, "Setup_rt3DEPI" e "Feed2AFNI_rt3DEPI" no caminho do script de macro (por exemplo, "/opt/(versão PV)/prog/curdir/(nome de usuário)/ParaVision/macros"). Ative as opções de reconstrução de EPI 3D, "Pre Image Series Activities" e "Execute Macro" no menu de interface do usuário "Data Reconstruction" e, em seguida, vincule o script de macro predefinido, "Setup_rt3DEPI", antes de clicar no botão "Scan". Observação : os scripts de macro estão incluídos nos arquivos suplementares.</li>
	<li>Instale o software AFNI para a análise e visualização BOLD-fMRI em tempo real.</li>
</ol>2. Cirurgia de cateterismo e ventilação
<ol><li>Instalar um ventilador e sistemas de monitoramento do estado fisiológico, como termômetro, registro da pressão arterial e da respiração, conforme mostrado na Figura 1. Defina uma frequência constante de 60 ± 1 respiração/min com o ventilador e uma temperatura de 37 °C usando uma almofada de aquecimento compatível com RM com um conjunto de controle de feedback.</li>
	<li>Anestesiar um rato Sprague-Dawley macho adulto (300-600 g) em uma câmara com isoflurano a 5% para indução e fornecer 2-2,5% de isoflurano para cirurgia a partir de um vaporizador. Verifique a profundidade da anestesia apertando a pata traseira e confirmando a falta de uma resposta de abstinência.</li>
	<li>Intubar o animal com cânula plástica de 14 G para ventilação (60 ± 1 respiração/min com uma mistura de 70% de ar e 30% de oxigênio). Ajustar o dióxido de carbono da maré final (CO2) para estar na faixa de 25 ± 5 mmHg29. NOTA: A intubação é fundamental para manter os níveis adequados de CO2 através de experimentos de fMRI.</li>
	<li>Coloque o animal em decúbito dorsal sobre uma mesa de cirurgia e raspe a coxa com uma navalha elétrica. E então, faça uma incisão na pele raspada com tesoura cirúrgica. NOTA: O comprimento da incisão é de cerca de 1-2 cm em uma direção longitudinal.</li>
	<li>Encontre uma artéria e veia femoral sob a região incisada para cateterismo e separe a artéria e a veia femoral individuais dos tecidos circundantes.</li>
	<li>Prenda um lado da artéria femoral separada com uma sutura cirúrgica e segure o outro lado com pinça micro buldogue. Em seguida, faça uma pequena incisão entre as regiões amarradas na artéria femoral.</li>
	<li>Insira um cateter na artéria femoral através da pequena incisão e amarre o cateter e a artéria juntamente com suturas cirúrgicas. Monitore a pressão arterial constantemente com o sistema de monitoramento fisiológico para estar na faixa de 80-120 mmHg e meça a gasometria arterial regularmente para manter o pO 2 de mínimo 90 mmHg e pCO2 de 30-45 mmHg durante a varredura. NOTA: Este cateterismo é fundamental para o monitoramento da pressão arterial durante experimentos de fMRI.</li>
	<li>Prender ambas as extremidades da veia femoral com suturas cirúrgicas trançadas de seda. Em seguida, faça uma pequena incisão entre as regiões amarradas na veia femoral. Use fórceps para realizar a sutura. NOTA: O tamanho da sutura é de cerca de 1-2 cm.</li>
	<li>Insira um cateter na veia femoral. Amarre o cateter e a veia juntamente com suturas cirúrgicas. NOTA: Este cateterismo é fundamental para administrar alfa-cloralose através da veia e ajustar os níveis anestésicos durante experimentos de fMRI. Se o animal não estiver bem anestesiado, ele começará a respirar espontaneamente. Neste caso, mais alfa-cloralose deve ser administrada para evitar artefatos de movimento respiratório.</li>
	<li>Suture a incisão cirúrgica na pele raspada. Uma vez concluídos os procedimentos cirúrgicos, manter o animal anestesiado por infusão de um bolo de alfa-cloralose com a dosagem de ~80 mg/kg através do cateter conectado à veia femoral e interromper a administração de isoflurano ao mesmo tempo.</li>
</ol>3. Colocar o animal dentro do scanner de ressonância magnética
<ol><li>Transfira o animal anestesiado para o scanner de ressonância magnética assim que o passo 2.10 for feito e prenda-o em um berço personalizado.</li>
	<li>Insira um termômetro retal de feedback em tempo real no animal para monitorar a temperatura do animal. Coloque uma almofada de aquecimento sob o tronco do animal para controlar a temperatura. Mantenha a temperatura corporal em 37,0 ± 0,5 °C durante exames de ressonância magnética.</li>
	<li>Administrar alfa-cloralose com ~25 mg/kg/h de solução numa mistura de pancurónio (~2 mg/kg/h), um relaxante muscular, mantendo o animal anestesiado e reduzindo os artefactos de movimento nas imagens de fMRI. Monitore a pressão arterial e a respiração, ajustando a quantidade de droga e a taxa de ventilação de acordo com o estado fisiológico.</li>
	<li>Administrar pomada oftálmica nos olhos do animal para evitar a secura durante os experimentos de fMRI. Fixe a cabeça do animal com segurança com duas barras auriculares para evitar artefatos de movimento da cabeça.</li>
	<li>Fixe uma bobina de superfície do transceptor na cabeça. Sintonize e combine a bobina com a frequência Larmor (por exemplo, 599 MHz em 14,1 T) na cabeça antes das medições de ressonância magnética. NOTA: Aqui, a bobina de 22 mm de diâmetro é usada para cobrir todo o cérebro de um rato.</li>
	<li>Insira um par de eletrodos de agulha na pele da pata dianteira entre os dígitos 1 e 4 e fixe-os com fita cirúrgica. E, em seguida, confirme se a estimulação funciona corretamente depois de conectar um cabo de entrada de estimulação a esses eletrodos30.</li>
	<li>Insira o animal no furo da ressonância magnética e coloque-o no isocentro aproximadamente.</li>
</ol>4. Medição de imagens anatômicas de RM
<ol><li>Clique no botão do menu de calibração na interface do usuário principal. Execute as calibrações do sistema de ressonância magnética clicando nos seguintes itens na interface do usuário da Plataforma de Ajuste (consulte o menu Ajuda no software do console): Encontre a frequência básica de ressonância, Calibre a potência de pulso de RF, Defina o ganho ideal do receptor, Meça o mapa B0 no animal para emenda, Execute calços lineares globais com base na integral de decaimento de indução livre (FID) não localizado. NOTA: Este passo demora menos de 2 minutos.</li>
	<li>Execute uma sequência de Posição clicando no botão "Scan" para encontrar a localização da cabeça do animal dentro do furo da ressonância magnética. Se a cabeça não estiver localizada no isocentro, ajuste a localização da cabeça enquanto move o berço para frente e para trás até que a cabeça esteja localizada no isocentro.</li>
	<li>Execute uma sequência do localizador clicando no botão "Scan" para identificar um ROI na cabeça. Selecione Map Shim e defina o ROI do volume do shim para cobrir todo o cérebro na imagem do localizador e, em seguida, execute um shimming de ordem alta (por exemplo, 2ª ou3ª ordem) usando a opção "Shim to" para reduzir as principais não homogeneidades do campo magnético (B0) no ROI. NOTA: O deslizamento de alta ordem é uma etapa crítica para melhorar a qualidade dos dados BOLD-fMRI quando as sequências EPI são usadas.</li>
	<li>Execute uma sequência RARE ponderada em T2 clicando no botão "Scan" para adquirir imagens anatômicas cobrindo todo o cérebro em uma visão coronal (por exemplo, os seguintes parâmetros de sequência são usados: tempo de repetição (TR) 4000 ms, tempo de eco efetivo (TE) 36,1 ms, matriz 128 x 128, campo de visão (FOV) 19,2x19,2 mm2, número de fatias 32, espessura da fatia 0,3 mm, Fator RARE 8). NOTA: Na seguinte etapa de visualização de fMRI em tempo real, as imagens anatômicas são usadas para registrar imagens EPI 3D como um modelo.</li>
</ol>5. Configuração do software de fMRI em tempo real e visualização da resposta de fMRI
<ol><li>Abra uma janela de terminal e vá para o caminho do plugin AFNI em tempo real usando o seguinte comando: cd /home/(nome de usuário)/rt_afni NOTA: O script do plug-in AFNI, "afni_rt" está incluído nos arquivos suplementares.</li>
	<li>Execute o software AFNI com o plugin em tempo real usando o comando e as opções abaixo. afni -rt -yestplugouts -DAFNI_REALTIME_MP_HOST_PORT=localhost:(número da porta) -DAFNI_REALTIME_Graph=Tempo real -DAFNI_FIM_IDEAL=(Paradigma) NOTA: No primeiro caso, o código permite que programas externos troquem dados com AFNI, enquanto no segundo caso o plug-in em tempo real tentará abrir um soquete TCP para o localhost e a porta definidos pelo usuário. No terceiro e quarto casos, os códigos plotarão o curso temporal dos dados de fMRI em tempo real e plotarão o curso de tempo do paradigma definido pelo usuário no curso de tempo de fMRI, respectivamente, quando os dados de fMRI em tempo real forem adquiridos. Para mais detalhes, consulte https://afni.nimh.nih.gov/pub/dist/doc/program_help/README.environment.html.</li>
	<li>Monitore os próximos arquivos AFNI BRIK definidos usando o comando "Dimon", como mostra a Figura 2 , com as seguintes opções: Dimon -tr (TR de EPI) -nt (NRepetições de EPI) -rt -sair -infile_pattern em tempo real*. BRIK -AFNI file_type NOTA: "Dimon" é um comando para monitorar a aquisição em tempo real de arquivos de imagem AFNI usando as seguintes opções: "-rt" que executa o plugin em tempo real e "-infile_pattern (nome dos dados). BRIK -file_type AFNI", que permite ao plugin ler os arquivos BRIK específicos e enviá-los para AFNI para exibição e formatação. Para mais detalhes, consulte https://afni.nimh.nih.gov/pub/dist/doc/program_help/Dimon.html.</li>
	<li>Use o comando "pvcmd" com as seguintes opções: pvcmd -a JMacroManager JMMExecuteMacro -category $USER -macro Feed2AFNI_rt3DEPI Observação : esse código existe no script de macro, "Setup_rt3DEPI", para executar o script de macro em segundo plano, "Feed2AFNI_rt3DEPI", logo após clicar no botão "Scan" para aquisição de EPI.</li>
	<li>Use o comando "exec pvcmd" com as seguintes opções para obter parâmetros de aquisição de EPI. exec pvcmd -a ParxServer -r ParamGetValue -psid $ParSpaceId -param (parâmetros PVM de EPI) -id 10 -args $AcqKey $ParSpaceId $ProcnoPath</li>
	<li>Use o comando "exec to3d" com as seguintes opções para converter dados brutos EPI em arquivos AFNI em tempo real no script de macro em segundo plano, "Feed2AFNI_rt3DEPI". exec to3d -omri -xFOV $FOV_X -yFOV $FOV_Y -zFOV $FOV_Z -prefixo $LastVolName $ImgFormat$Path2dseq</li>
	<li>Certifique-se de que as informações geométricas do EPI sejam consistentes com a orientação da anatomia. NOTA: O comando AFNI "to3d" será executado automaticamente com as informações geométricas, como o campo de visão (FOV) e o tamanho da matriz, para converter os dados brutos da fMRI em um dado AFNI BRIK sempre que cada dado de volume 3D for armazenado após cada TR, conforme mostrado na Figura 2. A orientação da imagem pode ser alterada com os parâmetros de informação geométrica de "to3d". Para mais detalhes, consulte https://afni.nimh.nih.gov/pub/dist/doc/program_help/to3d.html.</li>
	<li>Ligue um isolador de estímulo elétrico e realize estimulação elétrica da pata dianteira para um estudo de fMRI evocado (por exemplo, 3Hz, largura de pulso 4s 300us, 2,5mA) usando blocos de estimulação. NOTA: Aqui, o paradigma do design de blocos consiste em 10 varreduras de pré-estimulação, 3 varreduras de estimulação e 12 varreduras de interestimulação (15 varreduras por época).</li>
	<li>Execute uma sequência de EPI 3D ponderada em T2* clicando no botão "Scan" para o estudo BOLD-fMRI (por exemplo, os seguintes parâmetros são usados: TR/TE 1500/14 ms, matriz 64 x 64 x 32, FOV 19,2 x 19,2 x 9,6 mm 3 e resolução 300 x 300 x 300 μm3). NOTA: Assim que clicar no botão "Digitalizar", o monitoramento e o processamento de dados brutos serão feitos usando os scripts de macro predefinidos em tempo real. Uma vez que um conjunto de dados AFNI BRIK é convertido, os gráficos de curso de tempo voxel para imagens EPI 3D são exibidos no software AFNI e atualizados automaticamente para cada TR.</li>
	<li>Para sobrepor as imagens EPI sobre as imagens RARE anatômicas, converta as imagens RARE em um conjunto de dados AFNI BRIK usando o comando "to3d" como na etapa 5.6 e, em seguida, registre as imagens EPI nas imagens anatômicas usando o script AFNI "align_epi_anat.py" com as seguintes opções: align_epi_anat.py -anat anatomy_template_al+orig -epi epi.$(número de dados epi)+orig -epi_base 1 -sufixo _volreg -rat_align -custo lpa -epi2anat NOTA: Para obter mais detalhes, verifique https://afni.nimh.nih.gov/pub/dist/doc/program_help/ align_epi_anat.py.html.</li>
	<li>Para processar mapas funcionais das respostas BOLD, calcule a deconvolução do conjunto de dados 3D+time com uma série temporal de estímulo específica usando o comando "3dDeconvolve" com as seguintes opções: 3dDeconvolve -input (nome do arquivo de entrada)+orig. -nfirst 0 -polort 3 -num_stimts 1 -stim_times 1 (nome do arquivo do paradigma de estimulação) 'BLOCK(4,1)' -stim_label 1 forepaw -tout -fout -rout NOTA: As etapas de processamento de imagem, como suavização espacial ou filtragem temporal, foram incorporadas em um script de processamento de dados AFNI personalizado. Para mais detalhes, consulte https://afni.nimh.nih.gov/afni/doc/help/3dDeconvolve.html.</li>
	<li>Para visualizar mapas funcionais dos sinais BOLD, use um clustering interativo no software AFNI. Abra a opção "Definir sobreposição" e use a função "Clusters" no menu da interface do usuário AFNI.</li>
	<li>Após a última ressonância magnética funcional, retire o animal do scanner de ressonância magnética e eutanasie-o de acordo com os protocolos aprovados. NOTA: As funções de processamento de imagem do AFNI e as funções de macro no software de console mais recente foram usadas para processar os dados de fMRI em tempo real. Informações detalhadas e descrições de funções de macro podem ser encontradas no menu de ajuda no software do console. O software AFNI é um freeware, que pode ser baixado diretamente através do site NIMH-AFNI. Os scripts relacionados para construir a ligação entre o AFNI e o sistema de console são anexados.</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+animal+functional+magnetic+resonance+imaging+and+its+application+to+neurophamacological+studies.">Real-time animal functional magnetic resonance imaging and its application to neurophamacological studies.</a> Magnetic Resonance Imaging. 26 (9), 1266-1272 (2008).</li><li>Cox, R. W., Jesmanowicz, A., Hyde, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+functional+magnetic+resonance+imaging.">Real-time functional magnetic resonance imaging.</a> Magnetic Resonance Medicine. 33 (2), 230-236 (1995).</li><li>Lee, C. C., Jack, C. R., Rossman, P. J., Riederer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+reconstruction+and+high-speed+processing+in+functional+MR+imaging.">Real-time reconstruction and high-speed processing in functional MR imaging.</a> American Journal of Neuroradiology. 19 (7), 1297-1300 (1998).</li><li>Voyvodic, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+fMRI+paradigm+control,+physiology,+and+behavior+combined+with+near+real-time+statistical+analysis.">Real-time fMRI paradigm control, physiology, and behavior combined with near real-time statistical analysis.</a> Neuroimage. 10 (2), 91-106 (1999).</li><li>Cohen, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+functional+magnetic+resonance+imaging.">Real-time functional magnetic resonance imaging.</a> Methods. 25 (2), 201-220 (2001).</li><li>Posse, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+measure+single-event+related+brain+activity+using+real-time+fMRI:+Feasibility+of+sensory,+motor,+and+higher+cognitive+tasks.">A new approach to measure single-event related brain activity using real-time fMRI: Feasibility of sensory, motor, and higher cognitive tasks.</a> Human Brain Mapping. 12 (1), 25-41 (2001).</li><li>Decharms, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reading+and+controlling+human+brain+activation+using+real-time+functional+magnetic+resonance+imaging.">Reading and controlling human brain activation using real-time functional magnetic resonance imaging.</a> Trends in Cognitive Sciences. 11 (11), 473-481 (2007).</li><li>Bruhl, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+Sense+of+Real-Time+Functional+Magnetic+Resonance+Imaging+(rtfMRI)+and+rtfMRI+Neurofeedback.">Making Sense of Real-Time Functional Magnetic Resonance Imaging (rtfMRI) and rtfMRI Neurofeedback.</a> International Journal of Neuropsychopharmacology. 18 (6), (2015).</li><li>Cox, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFNI:+Software+for+analysis+and+visualization+of+functional+magnetic+resonance+neuroimages.">AFNI: Software for analysis and visualization of functional magnetic resonance neuroimages.</a> Computers and Biomedical Research. 29 (3), 162-173 (1996).</li><li>Liou, W. W., Goshgarian, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+the+effect+of+chronic+phrenicotomy+on+the+induction+of+the+crossed+phrenic+phenomenon.">Quantitative assessment of the effect of chronic phrenicotomy on the induction of the crossed phrenic phenomenon.</a> Experimental Neurology. 127 (1), 145-153 (1994).</li><li>Shih, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra+high-resolution+fMRI+and+electrophysiology+of+the+rat+primary+somatosensory+cortex.">Ultra high-resolution fMRI and electrophysiology of the rat primary somatosensory cortex.</a> Neuroimage. 73, 113-120 (2013).</li><li>Masamoto, K., Kim, T., Fukuda, M., Wang, P., Kim, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+neural,+vascular,+and+BOLD+signals+in+isoflurane-anesthetized+rat+somatosensory+cortex.">Relationship between neural, vascular, and BOLD signals in isoflurane-anesthetized rat somatosensory cortex.</a> Cerebral Cortex. 17 (4), 942-950 (2007).</li><li>van Alst, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+differentially+modulates+neuronal+and+vascular+contributions+to+the+BOLD+signal.">Anesthesia differentially modulates neuronal and vascular contributions to the BOLD signal.</a> Neuroimage. 195, 89-103 (2019).</li><li>Wu, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+isoflurane+anesthesia+on+resting-state+fMRI+signals+and+functional+connectivity+within+primary+somatosensory+cortex+of+monkeys.">Effects of isoflurane anesthesia on resting-state fMRI signals and functional connectivity within primary somatosensory cortex of monkeys.</a> Brain and Behavior. 6 (12), 00591 (2016).</li><li>Liu, X., Zhu, X. H., Zhang, Y., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+change+of+functional+connectivity+specificity+in+rats+under+various+anesthesia+levels+and+its+neural+origin.">The change of functional connectivity specificity in rats under various anesthesia levels and its neural origin.</a> Brain Topography. 26 (3), 363-377 (2013).</li><li>Liu, X. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphasic+modification+of+intrinsic+functional+connectivity+of+the+rat+brain+during+increasing+levels+of+propofol.">Multiphasic modification of intrinsic functional connectivity of the rat brain during increasing levels of propofol.</a> Neuroimage. 83, 581-592 (2013).</li><li>Hutchison, R. M., Hutchison, M., Manning, K. Y., Menon, R. S., Everling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induces+dose-dependent+alterations+in+the+cortical+connectivity+profiles+and+dynamic+properties+of+the+brain's+functional+architecture.">Isoflurane induces dose-dependent alterations in the cortical connectivity profiles and dynamic properties of the brain's functional architecture.</a> Human Brain Mapping. 35 (12), 5754-5775 (2014).</li><li>He, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-Slow+Single-Vessel+BOLD+and+CBV-Based+fMRI+Spatiotemporal+Dynamics+and+Their+Correlation+with+Neuronal+Intracellular+Calcium+Signals.">Ultra-Slow Single-Vessel BOLD and CBV-Based fMRI Spatiotemporal Dynamics and Their Correlation with Neuronal Intracellular Calcium Signals.</a> Neuron. 97 (4), 925-939 (2018).</li><li>Yoshida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+effects+of+a+habituation+procedure+for+functional+MRI+in+awake+mice+using+a+cryogenic+radiofrequency+probe.">Physiological effects of a habituation procedure for functional MRI in awake mice using a cryogenic radiofrequency probe.</a> Journal of Neuroscience Methods. 274, 38-48 (2016).</li></ol>

Sascha Köhler é funcionário da Bruker BioSpin MRI GmbH.

O monitoramento em tempo real do sinal de fMRI ajuda os experimentadores a ajustar a fisiologia dos animais para otimizar o mapeamento funcional. Artefatos de movimento em animais acordados, bem como o efeito anestésico, são os principais fatores que medeiam a variabilidade dos sinais de fMRI, confundindo a interpretação biológica do sinal por si só 31,32,33,34,35,36,37,38 <sup...

A Ressonância Magnética Funcional (RMf) é um método não invasivo para medir as respostas hemodinâmicas 1,2,3,4,5,6,7,8,9, por exemplo, dependentes do nível de oxigênio no sangue (BOLD), volume sanguíneo cerebral e sinal de fluxo, associados à atividade neural no cérebro. Em estudos com animais, os sinais hemodinâmicos podem ser afetados pela anestesia10, pelo nível de estresse de animais acordados 11, bem como pelos potenciais artefatos não fisiológicos, por exemplo, pulsação cardíaca e movimentos respiratórios12,13,14,15. Embora muitos métodos de pós-processamento tenham sido desenvolvidos para fornecer uma análise retrospectiva do sinal de fMRI para a dinâmica funcional relacionada à tarefa e ao estado de repouso e mapeamento de conectividade16,17,18,19, existem poucas técnicas para fornecer uma solução de mapeamento da função cerebral em tempo real e leituras instantâneas no cérebro animal 20 (a maioria das quais é usada principalmente para mapeamento cerebral humano21, 22,23,24,25,26,27). Em particular, esse tipo de método de mapeamento de fMRI em tempo real está faltando em estudos com animais. É necessário criar uma plataforma de fMRI para permitir a investigação de estágios fisiológicos dependentes do estado cerebral em tempo real e fornecer paradigma de estimulação de biofeedback em tempo real para estudos funcionais do cérebro animal.
No presente trabalho, ilustramos uma configuração experimental de fMRI em tempo real com as macrofunções personalizadas do software de console de ressonância magnética, demonstrando o monitoramento em tempo real das respostas evocadas de BOLD-fMRI no córtex somatossensorial primário da pata dianteira (FP-S1) dos ratos anestesiados. Essa configuração em tempo real permite a visualização da ativação cerebral em curso em mapas funcionais, bem como cursos de tempo individuais de forma voxel-wise, utilizando o software de análise de neuroimagem existente, Analysis of Functional NeuroImages (AFNI)28. A preparação da configuração experimental de fMRI em tempo real para o estudo em animais é descrita no protocolo. Além da configuração animal, fornecemos procedimentos detalhados para configurar a visualização e análise dos sinais de fMRI em tempo real usando o software de console mais recente em paralelo com os scripts de processamento de imagem. Em resumo, a configuração de fMRI em tempo real proposta para estudos em animais é uma ferramenta poderosa para monitorar os sinais dinâmicos de fMRI no cérebro animal usando o sistema de console de ressonância magnética.

<table>
	<tbody>
		<tr>
			<td>14.1T Bruker MRI system</td>
			<td>Bruker BioSpin MRI GmbH</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>A365 Stimulus Isolator</td>
			<td>World Precision Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>AcqKnowledge Software</td>
			<td>Biopac</td>
			<td>RRID:SCR_014279, http://www.biopac.com/product/acqknowledge-software/</td>
			<td></td>
		</tr>
		<tr>
			<td>AFNI</td>
			<td>Cox, 1996</td>
			<td>RRID:SCR_005927, http://afni.nimh.nih.gov</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2SMO (ETCO2/SpO2 Monitor), Model 7100</td>
			<td>Novametrix Medical Systems Inc</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>CP-Pharma</td>
			<td>Cat# 1214</td>
			<td></td>
		</tr>
		<tr>
			<td>Master-9</td>
			<td>A.M.P.I</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoliter Injector</td>
			<td>World Precision Instruments</td>
			<td>Cat# NANOFIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pancuronium Bromide</td>
			<td>Inresa Arzneimittel</td>
			<td>Cat# 34409.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>ParaVision 6</td>
			<td>Bruker BioSpin MRI GmbH</td>
			<td>RRID:SCR_001964</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>Cat# 10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat: Sprague Dawley rat</td>
			<td>Charles River Laboratories</td>
			<td>Crl:CD(SD)</td>
			<td></td>
		</tr>
		<tr>
			<td>SAR-830/AP Ventilator</td>
			<td>CWE</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-chloralose</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# C0128-25G;RRID</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time fmri brain mapping in animals

As respostas dinâmicas de fMRI variam em grande parte de acordo com as condições fisiológicas dos animais, sob anestesia ou em estados acordados. Desenvolvemos uma plataforma de fMRI em tempo real para orientar os experimentadores a monitorar as respostas de fMRI instantaneamente durante a aquisição, que pode ser usada para modificar a fisiologia de animais para alcançar as respostas hemodinâmicas desejadas em cérebros de animais. A configuração de fMRI em tempo real é baseada em um sistema de ressonância magnética pré-clínica de 14,1T, permitindo o mapeamento em tempo real das respostas dinâmicas de fMRI no córtex somatossensorial primário da pata dianteira (FP-S1) de ratos anestesiados. Em vez de uma análise retrospectiva para investigar fontes de confusão que levam à variabilidade dos sinais de fMRI, a plataforma de fMRI em tempo real fornece um esquema mais eficaz para identificar respostas dinâmicas de fMRI usando macrofunções personalizadas e um software comum de análise de neuroimagem no sistema de ressonância magnética. Além disso, fornece viabilidade imediata de solução de problemas e um paradigma de estimulação de biofeedback em tempo real para estudos funcionais cerebrais em animais.

A Figura 3 e a Figura 4 mostram um curso de tempo representativo em tempo real do BOLD-fMRI em tempo real e mapas funcionais com estimulação elétrica da pata dianteira (3 Hz, 4 s, largura de pulso 300 us, 2,5 mA). O paradigma de design da fMRI compreende 10 exames de pré-estimulação, 3 exames de estimulação e 12 exames de interestimulação com um total de 8 épocas (130 exames). O tempo total de varredura é de 3 min 15 seg (195 seg). <strong class="xfi...

Watch this Scientific Journal Video about Real-Time fMRI Brain Mapping in Animals at JoVE.com

Real-Time fMRI Brain Mapping in Animals

O mapeamento funcional do cérebro animal pode se beneficiar da configuração experimental de ressonância magnética funcional (fMRI) em tempo real. Usando o mais recente software implementado no sistema de ressonância magnética animal, estabelecemos uma plataforma de monitoramento em tempo real para fMRI de pequenos animais.

Mapeamento cerebral por fMRI em tempo real em animais

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Research

JoVE Journal

Neuroscience

3.4K visualizações.  Max Planck Institute for Biological Cybernetics. O mapeamento funcional do cérebro animal pode se beneficiar da configuração experimental de ressonância magnética funcional (fMRI) em tempo real. Usando o mais recente software implementado no sistema de ressonância magnética animal, estabelecemos uma plataforma de monitoramento em tempo real para fMRI de pequenos animais.

Vídeo: Real-Time fMRI Brain Mapping in Animals

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

FMRI simultânea e Eletrofisiologia no cérebro de roedores

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Neurociência

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: 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.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

Combinando Estimulação Magnética Transcraniana e fMRI para o Exame da rede de modo padrão

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), magnetoencephalography or functional magnetic-resonance imaging. These techniques have either inherently low temporal or low spatial resolution, and suffer from low signal-to-noise ratio and/or poor high-frequency sensitivity. Thus, they are suboptimal for exploring the short-lived spatio-temporal dynamics of many of the underlying brain processes. In contrast, the invasive technique of electrocorticography (ECoG) provides brain signals that have an exceptionally high signal-to-noise ratio, less susceptibility to artifacts than EEG, and a high spatial and temporal resolution (i.e., &lt;1 cm/&lt;1 millisecond, respectively). ECoG involves measurement of electrical brain signals using electrodes that are implanted subdurally on the surface of the brain. Recent studies have shown that ECoG amplitudes in certain frequency bands carry substantial information about task-related activity, such as motor execution and planning1, auditory processing2 and visual-spatial attention3. Most of this information is captured in the high gamma range (around 70-110 Hz). Thus, gamma activity has been proposed as a robust and general indicator of local cortical function1-5. ECoG can also reveal functional connectivity and resolve finer task-related spatial-temporal dynamics, thereby advancing our understanding of large-scale cortical processes. It has especially proven useful for advancing brain-computer interfacing (BCI) technology for decoding a user's intentions to enhance or improve communication6 and control7. Nevertheless, human ECoG data are often hard to obtain because of the risks and limitations of the invasive procedures involved, and the need to record within the constraints of clinical settings. Still, clinical monitoring to localize epileptic foci offers a unique and valuable opportunity to collect human ECoG data. We describe our methods for collecting recording ECoG, and demonstrate how to use these signals for important real-time applications such as clinical mapping and brain-computer interfacing. Our example uses the BCI2000 software platform8,9 and the SIGFRIED10 method, an application for real-time mapping of brain functions. This procedure yields information that clinicians can subsequently use to guide the complex and laborious process of functional mapping by electrical stimulation.
Prerequisites and Planning:
Patients with drug-resistant partial epilepsy may be candidates for resective surgery of an epileptic focus to minimize the frequency of seizures. Prior to resection, the patients undergo monitoring using subdural electrodes for two purposes: first, to localize the epileptic focus, and second, to identify nearby critical brain areas (i.e., eloquent cortex) where resection could result in long-term functional deficits. To implant electrodes, a craniotomy is performed to open the skull. Then, electrode grids and/or strips are placed on the cortex, usually beneath the dura. A typical grid has a set of 8 x 8 platinum-iridium electrodes of 4 mm diameter (2.3 mm exposed surface) embedded in silicon with an inter-electrode distance of 1cm. A strip typically contains 4 or 6 such electrodes in a single line. The locations for these grids/strips are planned by a team of neurologists and neurosurgeons, and are based on previous EEG monitoring, on a structural MRI of the patient's brain, and on relevant factors of the patient's history. Continuous recording over a period of 5-12 days serves to localize epileptic foci, and electrical stimulation via the implanted electrodes allows clinicians to map eloquent cortex. At the end of the monitoring period, explantation of the electrodes and therapeutic resection are performed together in one procedure.
In addition to its primary clinical purpose, invasive monitoring also provides a unique opportunity to acquire human ECoG data for neuroscientific research. The decision to include a prospective patient in the research is based on the planned location of their electrodes, on the patient's performance scores on neuropsychological assessments, and on their informed consent, which is predicated on their understanding that participation in research is optional and is not related to their treatment. As with all research involving human subjects, the research protocol must be approved by the hospital's institutional review board. The decision to perform individual experimental tasks is made day-by-day, and is contingent on the patient's endurance and willingness to participate. Some or all of the experiments may be prevented by problems with the clinical state of the patient, such as post-operative facial swelling, temporary aphasia, frequent seizures, post-ictal fatigue and confusion, and more general pain or discomfort.
At the Epilepsy Monitoring Unit at Albany Medical Center in Albany, New York, clinical monitoring is implemented around the clock using a 192-channel Nihon-Kohden Neurofax monitoring system. Research recordings are made in collaboration with the Wadsworth Center of the New York State Department of Health in Albany. Signals from the ECoG electrodes are fed simultaneously to the research and the clinical systems via splitter connectors. To ensure that the clinical and research systems do not interfere with each other, the two systems typically use separate grounds. In fact, an epidural strip of electrodes is sometimes implanted to provide a ground for the clinical system. Whether research or clinical recording system, the grounding electrode is chosen to be distant from the predicted epileptic focus and from cortical areas of interest for the research. Our research system consists of eight synchronized 16-channel g.USBamp amplifier/digitizer units (g.tec, Graz, Austria). These were chosen because they are safety-rated and FDA-approved for invasive recordings, they have a very low noise-floor in the high-frequency range in which the signals of interest are found, and they come with an SDK that allows them to be integrated with custom-written research software. In order to capture the high-gamma signal accurately, we acquire signals at 1200Hz sampling rate-considerably higher than that of the typical EEG experiment or that of many clinical monitoring systems. A built-in low-pass filter automatically prevents aliasing of signals higher than the digitizer can capture. The patient's eye gaze is tracked using a monitor with a built-in Tobii T-60 eye-tracking system (Tobii Tech., Stockholm, Sweden). Additional accessories such as joystick, bluetooth Wiimote (Nintendo Co.), data-glove (5th Dimension Technologies), keyboard, microphone, headphones, or video camera are connected depending on the requirements of the particular experiment.
Data collection, stimulus presentation, synchronization with the different input/output accessories, and real-time analysis and visualization are accomplished using our BCI2000 software8,9. BCI2000 is a freely available general-purpose software system for real-time biosignal data acquisition, processing and feedback. It includes an array of pre-built modules that can be flexibly configured for many different purposes, and that can be extended by researchers' own code in C++, MATLAB or Python. BCI2000 consists of four modules that communicate with each other via a network-capable protocol: a Source module that handles the acquisition of brain signals from one of 19 different hardware systems from different manufacturers; a Signal Processing module that extracts relevant ECoG features and translates them into output signals; an Application module that delivers stimuli and feedback to the subject; and the Operator module that provides a graphical interface to the investigator.
A number of different experiments may be conducted with any given patient. The priority of experiments will be determined by the location of the particular patient's electrodes. However, we usually begin our experimentation using the SIGFRIED (SIGnal modeling For Realtime Identification and Event Detection) mapping method, which detects and displays significant task-related activity in real time. The resulting functional map allows us to further tailor subsequent experimental protocols and may also prove as a useful starting point for traditional mapping by electrocortical stimulation (ECS).
Although ECS mapping remains the gold standard for predicting the clinical outcome of resection, the process of ECS mapping is time consuming and also has other problems, such as after-discharges or seizures. Thus, a passive functional mapping technique may prove valuable in providing an initial estimate of the locus of eloquent cortex, which may then be confirmed and refined by ECS. The results from our passive SIGFRIED mapping technique have been shown to exhibit substantial concurrence with the results derived using ECS mapping10.
The protocol described in this paper establishes a general methodology for gathering human ECoG data, before proceeding to illustrate how experiments can be initiated using the BCI2000 software platform. Finally, as a specific example, we describe how to perform passive functional mapping using the BCI2000-based SIGFRIED system.

Recording Human Electrocorticographic ECoG Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

Gravação Humanos eletrocorticográfica (ECOG) Sinais para pesquisa neurocientífica e mapeamento em tempo real Funcional Cortical

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

Concepção e montagem de um Microdrive Ultra-leve motorizada para doenças crônicas Recordings Neurais em Pequenos Animais

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Imagem Funcional do Cortex Auditivo em gatos adultos usando de alta campo fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

Estimulação Cerebral Profunda com fMRI simultâneo em Roedores

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Um<em&gt; Ex Vivo</em&gt; Induzida por laser Lesão Medular modelo para avaliar mecanismos de axonal Degeneration em tempo real

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Um protocolo para a administração do real-time fMRI treinamento de Neurofeedback

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

Itoforese em tempo real com tetrametilamônio para quantificar fração de volume e tortuosidade do espaço extracelular cerebral

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

Viver-pilha medição de odorante Receptor de ativação usando um ensaio de campo em tempo real

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...