Nous remercions Mme Tess J. Lameyer, Mme Ruth Frank et le Dr Michael A. Muniak pour les modifications et les commentaires, et le Dr Ryohei Yasuda de Max Planck Florida pour le logiciel d'acquisition 2pFLIM. Ce travail a été soutenu par deux prix BRAIN Initiative U01NS94247 (H.Z. et T.M.) et R01NS104944 (H.Z. et T.M.), une subvention R01RS081071 (T.M.), et une subvention R21NS097856 (H.Z.). Tous les prix proviennent du National Institute of Neurological Disorders and Stroke, États-Unis.

<ol>
	<li>Greengard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Neurobiology+of+Slow+Synaptic+Transmission.">The Neurobiology of Slow Synaptic Transmission.</a> Science. 294 (5544), 1024-1030 (2001).</li><li>Petersen, S. E., Posner, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+attention+system+of+the+human+brain:+20+years+after.">The attention system of the human brain: 20 years after.</a> Annual Review of Neuroscience. 35 (2), 73-89 (2012).</li><li>Sun, Y., Hunt, S., Sah, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norepinephrine+and+Corticotropin-Releasing+Hormone:+Partners+in+the+Neural+Circuits+that+Underpin+Stress+and+Anxiety.">Norepinephrine and Corticotropin-Releasing Hormone: Partners in the Neural Circuits that Underpin Stress and Anxiety.</a> Neuron. 87 (3), 468-470 (2015).</li><li>Berke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+dopamine+mean?.">What does dopamine mean?.</a> Nature Neuroscience. 21 (6), 787-793 (2018).</li><li>Chen, 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=Endogenous+G&#945;q-Coupled+Neuromodulator+Receptors+Activate+Protein+Kinase+A.">Endogenous G&#945;q-Coupled Neuromodulator Receptors Activate Protein Kinase A.</a> Neuron. 96 (5), 1070-1083 (2017).</li><li>Madison, D. V., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+adenosine+3&#8217;,5&#8217;-monophosphate+mediates+beta-receptor+actions+of+noradrenaline+in+rat+hippocampal+pyramidal+cells.">Cyclic adenosine 3&#8217;,5&#8217;-monophosphate mediates beta-receptor actions of noradrenaline in rat hippocampal pyramidal cells.</a> The Journal of Physiology. 372 (1), 245-259 (1986).</li><li>Yasuda, H., Barth, A. L., Stellwagen, D., Malenka, 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=A+developmental+switch+in+the+signaling+cascades+for+LTP+induction.">A developmental switch in the signaling cascades for LTP induction.</a> Nature Neuroscience. 6 (1), 15-16 (2003).</li><li>Pedarzani, P., Storm, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKA+mediates+the+effects+of+monoamine+transmitters+on+the+K++current+underlying+the+slow+spike+frequency+adaptation+in+hippocampal+neurons.">PKA mediates the effects of monoamine transmitters on the K+ current underlying the slow spike frequency adaptation in hippocampal neurons.</a> Neuron. 11 (6), 1023-1035 (1993).</li><li>Brandon, E. P., Idzerda, R. L., McKnight, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKA+isoforms,+neural+pathways,+and+behaviour:+making+the+connection.">PKA isoforms, neural pathways, and behaviour: making the connection.</a> Current Opinion in Neurobiology. 7 (3), 397-403 (1997).</li><li>Radnikow, G., Feldmeyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-+and+Cell+Type-Specific+Modulation+of+Excitatory+Neuronal+Activity+in+the+Neocortex.">Layer- and Cell Type-Specific Modulation of Excitatory Neuronal Activity in the Neocortex.</a> Frontiers in Neuroanatomy. 12 (January), (2018).</li><li>Kennedy, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+trends+in+in+vivo+neurochemical+monitoring+by+microdialysis.">Emerging trends in in vivo neurochemical monitoring by microdialysis.</a> Current Opinion in Chemical Biology. 17 (5), 860-867 (2013).</li><li>Rodeberg, N. T., Sandberg, S. G., Johnson, J. A., Phillips, P. E. M., Wightman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hitchhiker&#8217;s+Guide+to+Voltammetry:+Acute+and+Chronic+Electrodes+for+In+Vivo+Fast-Scan+Cyclic+Voltammetry.">Hitchhiker&#8217;s Guide to Voltammetry: Acute and Chronic Electrodes for In Vivo Fast-Scan Cyclic Voltammetry.</a> ACS Chemical Neuroscience. 8 (2), 221-234 (2017).</li><li>Hamel, E. J. O., Grewe, B. F., Parker, J. G., Schnitzer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+level+brain+imaging+in+behaving+mammals:+An+engineering+approach.">Cellular level brain imaging in behaving mammals: An engineering approach.</a> Neuron. 86 (1), 140-159 (2015).</li><li>Allen, M. D., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+dynamics+of+protein+kinase+A+activity+visualized+by+FRET-based+reporters.">Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters.</a> Biochemical and Biophysical Research Communications. 348 (2), 716-721 (2006).</li><li>Zhang, J., Ma, Y., Taylor, S. S., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+reporters+of+protein+kinase+A+activity+reveal+impact+of+substrate+tethering.">Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (26), 14997-15002 (2001).</li><li>Chen, Y., Saulnier, J. L., Yellen, G., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PKA+activity+sensor+for+quantitative+analysis+of+endogenous+GPCR+signaling+via+2-photon+FRET-FLIM+imaging.">A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging.</a> Frontiers in Pharmacology. 5 (April), 1-12 (2014).</li><li>Ma, 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=A+Highly+Sensitive+A-Kinase+Activity+Reporter+for+Imaging+Neuromodulatory+Events+in+Awake+Mice.">A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice.</a> Neuron. 99 (4), 665-679 (2018).</li><li>Yellen, G., Mongeon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+two-photon+imaging+of+fluorescent+biosensors.">Quantitative two-photon imaging of fluorescent biosensors.</a> Current Opinion in Chemical Biology. 27, 24-30 (2015).</li><li>Tang, S., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+ERK+and+PKA+Activation+in+Single+Dendritic+Spines+during+Structural+Plasticity.">Imaging ERK and PKA Activation in Single Dendritic Spines during Structural Plasticity.</a> Neuron. 93 (6), 1315-1324 (2017).</li><li>Tillo, S. E., 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=Liberated+PKA+Catalytic+Subunits+Associate+with+the+Membrane+via+Myristoylation+to+Preferentially+Phosphorylate+Membrane+Substrates.">Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates.</a> Cell Reports. 19 (3), 617-629 (2017).</li><li>Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+spatiotemporal+dynamics+of+neuronal+signaling+using+fluorescence+resonance+energy+transfer+and+fluorescence+lifetime+imaging+microscopy.">Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy.</a> Current Opinion in Neurobiology. 16 (5), 551-561 (2006).</li><li>Theurkauf, W. E., Vallee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+cAMP-dependent+protein+kinase+bound+to+microtubule-associated+protein+2.">Molecular characterization of the cAMP-dependent protein kinase bound to microtubule-associated protein 2.</a> Journal of Biological Chemistry. 257 (6), 3284-3290 (1982).</li><li>Zhong, H., 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=Subcellular+dynamics+of+type+II+PKA+in+neurons.">Subcellular dynamics of type II PKA in neurons.</a> Neuron. 62 (3), 363-374 (2009).</li><li>Borrell, V., Yoshimura, Y., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+delivery+to+telencephalic+inhibitory+neurons+by+directional+in+utero+electroporation.">Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation.</a> Journal of Neuroscience Methods. 143 (2), 151-158 (2005).</li><li>Baumgart, J., Baumgart, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortex-,+Hippocampus-,+Thalamus-,+Hypothalamus-,+Lateral+Septal+Nucleus-+and+Striatum-specific+In+utero+Electroporation+in+the+C57BL/6+Mouse.">Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In utero Electroporation in the C57BL/6 Mouse.</a> Journal of Visualized Experiments. (107), e53303 (2016).</li><li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+Injection+of+Adeno-associated+Viral+Vectors.">Intracranial Injection of Adeno-associated Viral Vectors.</a> Journal of Visualized Experiments. (45), 1-4 (2010).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> Journal of Visualized Experiments. (12), 18-19 (2008).</li><li>Holtmaat, A., 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=Long-term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4 (8), 1128-1144 (2009).</li><li>Murakoshi, H., Lee, S. J., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+and+quantitative+FRET-FLIM+imaging+in+single+dendritic+spines+using+improved+non-radiative+YFP.">Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP.</a> Brain Cell Biology. 36 (1-4), 31-42 (2008).</li><li>Murakoshi, H., Shibata, A. C. E., Nakahata, Y., Nabekura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dark+green+fluorescent+protein+as+an+acceptor+for+measurement+of+F&#246;rster+resonance+energy+transfer.">A dark green fluorescent protein as an acceptor for measurement of F&#246;rster resonance energy transfer.</a> Scientific Reports. 5, 1-11 (2015).</li><li>Guo, Z. V., 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=Procedures+for+behavioral+experiments+in+head-fixed+mice.">Procedures for behavioral experiments in head-fixed mice.</a> PLoS ONE. 9 (2), (2014).</li><li>Yu, 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=The+central+amygdala+controls+learning+in+the+lateral+amygdala.">The central amygdala controls learning in the lateral amygdala.</a> Nature Neuroscience. 20 (12), 1680-1685 (2017).</li><li>Harvey, C. D., Collman, F., Dombeck, D. A., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+dynamics+of+hippocampal+place+cells+during+virtual+navigation.">Intracellular dynamics of hippocampal place cells during virtual navigation.</a> Nature. 461 (7266), 941-946 (2009).</li><li>Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+signaling+using+two-photon+fluorescent+lifetime+imaging+microscopy.">Imaging intracellular signaling using two-photon fluorescent lifetime imaging microscopy.</a> Cold Spring Harbor Protocols. 7 (11), 1121-1128 (2012).</li><li>Mehta, 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=Single-fluorophore+biosensors+for+sensitive+and+multiplexed+detection+of+signalling+activities.">Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities.</a> Nature Cell Biology. 20 (10), 1215-1225 (2018).</li></ol>

Les auteurs n'ont rien à révéler.

Ce protocole démontre l'utilisation du capteur FRET-FLIM tAKARMD pour visualiser l'activité PKA déclenchée par la neuromodulation chez les souris ayant un comportement fixe à la tête. Cette application est basée sur des tests approfondis et des caractérisations de tAKARMD in vitro et in vivo pour démontrer que le signal FLIM obtenu est pertinent pour les événements neuromodulatoires physiologiques17. Ici, une application in vivo, l'activité PKA induite par la locomotion dans le cortex ...

La neuromodulation, également connue sous le nom de transmission synaptique lente, impose un contrôle fort sur la fonction cérébrale pendant différents états comportementaux, tels que le stress, l'excitation, l'attention, et la locomotion1,2,3, 4. Malgré son importance, l'étude du moment et de l'endroit où se déroulent les événements neuromodulatoires en est encore à ses balbutiements. Les neuromodulateurs, y compris l'acétylcholine, la dopamine, la noradrénaline, la sérotonine et de nombreux neuropeptides, activent les récepteurs couplés aux protéines G (GPCR), qui déclenchent à leur tour des voies intracellulaires de deuxième messager avec une large fenêtre d'échelles de temps allant de secondes à quelques heures. Tandis que chaque neuromodulateur déclenche un ensemble distinct d'événements de signalisation, la voie de kinase A de cAMP/protéine est une voie en aval commune pour beaucoup de neuromodulators1,5. La voie cAMP/PKA régule l'excitabilité neuronale, la transmission synaptique et la plasticité6,7,8,9, et donc, accorde la dynamique neuronale du réseau. Étant donné que différents neurones ou types neuronaux expriment différents types ou niveaux de récepteurs neuromodulateurs10, les effets intracellulaires du même neuromodulateur extracellulaire peuvent être hétérogènes à travers différents neurones, et doivent donc être avec résolution cellulaire. À ce jour, il reste difficile de surveiller les événements neuromodulatoires dans les neurones individuels in vivo pendant le comportement.
Pour étudier la dynamique spatiotemporale de la neuromodulation, une modalité appropriée d'enregistrement est exigée. La microdialyse et la voltamétrie cyclique à balayage rapide sont fréquemment utilisées pour étudier la libération des neuromodulateurs, mais ils n'ont pas la résolution spatiale pour surveiller les événements cellulaires11,12. Analogue à la dynamique du calcium utilisé comme un proxy pour l'activité électrique neuronale dans l'imagerie de la population13, pDJ peut être utilisé pour lire les événements neuromodulatoires à travers une population neuronale à la résolution cellulaire. Le protocole actuel décrit l'utilisation d'un journaliste d'activité A-kinase amélioré (AKAR) pour surveiller les activités De PKA in vivo pendant le comportement animal. La méthode décrite ici permet l'imagerie simultanée des populations neuronales à la résolution subcellulaire avec une résolution temporelle qui suit les événements neuromodulatoires physiologiques.
Les AKAR sont composés d'un donneur et d'un accepteur de protéines fluorescentes reliées par un peptide de substrat de phosphorylation PKA et d'un domaine associé à la tête de fourchette (FHA) qui se lie à la sérine phosphorylée ou à la thréonine du substrat14,15. Lors de l'activation de la voie PKA, le peptide de substrat d'AKAR est phosphorylé. En conséquence, le domaine FHA se lie au peptide phosphorylé de substrat, amenant de ce fait les deux fluorophores à proximité, dénommé l'état fermé d'AKAR. L'état fermé d'un AKAR phosphorylé entraîne une augmentation du transfert d'énergie par résonance (FRET) entre le donneur et les fluorophores accepteurs. Étant donné que la proportion d'AKAR phosphorylés est liée au niveau d'activité PKA16, la quantité de FRET dans un échantillon biologique peut être utilisée pour quantifier le niveau d'activité PKA16,17,18, 19,20.
Les premières versions des RLA ont été principalement conçues pour l'imagerie ratiométrique à deux couleurs14. Lors de l'imagerie plus profondément dans le tissu cérébral, la méthode ratiométrique souffre de distorsion du signal due à la diffusion de la lumière dépendante de la longueur d'onde17,18,21. Comme nous l'avons vu ci-dessous, la microscopie d'imagerie à vie par fluorescence (FLIM) élimine ce problème parce que la FLIM ne mesure que les photons émis par le fluorophore du donneur18,21. En conséquence, la quantification FLIM de FRET n'est pas affectée par la profondeur tissulaire17. En outre, une variante « foncée » (c.-à-d. faible rendement quantique [QY]) du fluorophore accepteur peut être utilisée. Cela libère un canal de couleur pour faciliter la mesure multiplexée des propriétés neuronales orthogonales par l'imagerie simultanée d'un deuxième capteur ou d'un marqueur morphologique17,19,20.
L'imagerie FLIM quantifie le temps qu'un fluorophore passe dans l'état excité, c'est-à-dire la durée de vie de la fluorescence18. Le retour d'un fluorophore à l'état de sol, donc la fin de l'état excité, se concomite souvent avec l'émission d'un photon. Bien que l'émission d'un photon pour une molécule excitée individuelle soit stochastique, dans une population la durée de vie moyenne de fluorescence est une caractéristique de ce fluorophore particulier. Quand une population pure de fluorophores sont excitées simultanément, la fluorescence résultante suivra une seule décomposition exponentielle. La constante temporelle de cette carie exponentielle correspond à la durée de vie moyenne de la fluorescence, qui varie généralement de une à quatre nanosecondes pour les protéines fluorescentes. Le retour d'un fluorophore donneur excité à l'état du sol peut également se produire par FRET. En présence de FRET, la durée de vie de fluorescence du fluorophore donneur est réduite. Les AKAR non phosphorylés présentent une durée de vie relativement plus longue de fluorescence des donneurs. Lors de la phosphorylation par PKA, le capteur montre une durée de vie plus courte parce que le donneur et les fluorophores accepteurs sont amenés près de l'autre et FRET est augmenté. La quantification de la durée de vie de la fluorescence dans une population d'AKAR représente donc le niveau d'activité de la PKA.
Les premières versions des RCR n'ont pas été utilisées avec succès pour l'imagerie in vivo à résolution monocellulaire. Cela est principalement dû à l'amplitude du signal faible des capteurs AKAR aux activations physiologiques17. Récemment, en comparant systématiquement les capteurs AKAR disponibles pour la microscopie d'imagerie à vie à deux photons (2pFLIM), un capteur appelé FLIM-AKAR a été trouvé pour surpasser les capteurs alternatifs. En outre, une série de variantes FLIM-AKAR appelées AKAR ciblées (tAKARs) ont été développées pour visualiser l'activité de La PKA à des endroits sous-cellulaires spécifiques : microtubules (tAKARMD), cytosol (tAKARMD), actine (tAKARMD), actine filamentuse (tAKARMD), membrane (tAKARMD), et la densité postsynaptique (tAKAR- ). Parmi les tAKAR, le tAKARMD a multiplié par 2,7 l'amplitude du signal obtenue par la noradrénaline. Ceci est compatible avec la connaissance que la majorité de PKA dans les neurones sont ancrés aux microtubules à l'état de repos22,23. tAKAR a été le meilleur performer parmi les AKAR existants pour 2pFLIM. En outre, tAKARMD a détecté l'activité physiologiquement pertinente de PKA obtenue par les neuromodulators multiples, et l'expression de tAKAR mD n'a pas modifié des fonctions neuronales17.
Récemment, tAKAR a été utilisé avec succès pour visualiser les activités PKA chez les souris de comportement fixes à la tête17. Il a été démontré que la locomotion forcée déclenchait l'activité de la PKA dans le soma des neurones de couche superficielle (couche 1 à 3, jusqu'à une profondeur de 300 m de pia) dans le moteur, le baril et les cortices visuels. L'activité de PKA locomotion-déclenchée a été en partie dépendante de la signalisation par l'intermédiaire des récepteurs adrénergiques et des récepteurs de dopamine d'A1, mais n'a pas été affectée par un antagoniste de récepteur de dopamine de D2. Ce travail illustre la capacité des tAKARs à suivre les événements de neuromodulation in vivo à l'aide de 2pFLIM.
Dans le protocole actuel, la méthode entière pour l'imagerie d'activité de PKA dans les souris éveillées tête-fixes pendant un paradigme forcé de locomotion est décrite en six étapes. Tout d'abord, l'ajout de capacités 2pFLIM à un microscope conventionnel à deux photons (figure 1). Deuxièmement, la construction d'un tapis roulant motorisé (figure 2). Troisièmement, l'expression du capteur tAKARMD dans le cortex de la souris par électroporation in utero (IUE) des plasmides d'ADN, ou injection stéréotaxique du virus adéno-associé (AAV). D'excellents protocoles pour les chirurgies pour IUE24,25 et l'injection stéréotaxique de particules virales26 ont été précédemment publiés. Les paramètres clés que nous avons utilisés sont décrits ci-dessous. Forth, l'installation d'une fenêtre crânienne. D'excellents protocoles ont déjà été publiés pour la chirurgie de fenêtre crânienne27,28. Plusieurs étapes qui ont été modifiées à partir des protocoles standard sont décrites. Cinquième, effectuer in vivo 2pFLIM. Sixièmement, les analyses des images 2pFLIM (figure3 et figure 4). Cette approche devrait être facilement applicable à beaucoup d'autres paradigmes comportementaux et zones cérébrales fixés à la tête.

<table>
	<tbody>
		<tr>
			<td>0.2 &#956;m cellulose acetate syringe filter</td>
			<td>Nalgene</td>
			<td>190-2520</td>
			<td>Step 3.2.2.</td>
		</tr>
		<tr>
			<td>16x 0.8 NA water-immersion objective</td>
			<td>Nikon</td>
			<td>MRP07220</td>
			<td>Step 5.5.</td>
		</tr>
		<tr>
			<td>3-pin cable</td>
			<td>US digital</td>
			<td>CA-MIC3-SH-NC</td>
			<td>Step 2.5. To connect rotation sensor to the DAQ input of the microscope</td>
		</tr>
		<tr>
			<td>Aluminum bread board</td>
			<td>Thorlabs</td>
			<td>MB1012</td>
			<td>Step 2.5.</td>
		</tr>
		<tr>
			<td>AnimalTracker MATLAB software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 2.5 and sections 5 - 6. Will be provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Band-pass barrier filter</td>
			<td>Chroma</td>
			<td>ET500-40m</td>
			<td>Step 1.4.</td>
		</tr>
		<tr>
			<td>Cage plate</td>
			<td>Thorlabs</td>
			<td>CP01</td>
			<td>Step 2.4. Used as mount for rotation sensor</td>
		</tr>
		<tr>
			<td>Carbon steel burrs for micro drill, 0.5 mm tip diameter</td>
			<td>FST</td>
			<td>19007-05</td>
			<td>Steps 3.2.3. and 4.4.</td>
		</tr>
		<tr>
			<td>Circular coverslip (5mm diameter)</td>
			<td>VWR</td>
			<td>101413-528</td>
			<td>Step 4.5.</td>
		</tr>
		<tr>
			<td>Custom-made injection needle holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 3.2.4. Technical details provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Dental acrylic</td>
			<td>Yates Motloid</td>
			<td>44114</td>
			<td>Steps 4.3. and 4.5.</td>
		</tr>
		<tr>
			<td>Dental drill; Microtorque ii</td>
			<td>Ram products</td>
			<td>66699</td>
			<td>Steps 3.2.3. and 4.4.</td>
		</tr>
		<tr>
			<td>Dowsil transparent polymer</td>
			<td>The Dow Chemical Company</td>
			<td>3-4680</td>
			<td>Step 4.5. Artificial dura</td>
		</tr>
		<tr>
			<td>Electroporation electrode</td>
			<td>Bex</td>
			<td>LF650P5</td>
			<td>Step 3.1.4.</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bex</td>
			<td>CUY21</td>
			<td>Step 3.1.4.</td>
		</tr>
		<tr>
			<td>Fast green FCF</td>
			<td>Sigma-aldrich</td>
			<td>F7258-25G</td>
			<td>Step 3.1.1.</td>
		</tr>
		<tr>
			<td>FLIMimage MATLAB software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Section 5. Kindly provided by Dr. Ryohei Yasuda, Max Planck Florida</td>
		</tr>
		<tr>
			<td>FLIMview MATLAB software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Sections 5. and 6. Will be provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Foam-compatible glue (Gorilla White Glue)</td>
			<td>Gorilla</td>
			<td>5201204</td>
			<td>Step 2.3.</td>
		</tr>
		<tr>
			<td>Headplate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 4.3. Technical details provided upon request to the lead author</td>
		</tr>
		<tr>
			<td>Headplate holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Step 2.6. Technical details provided upon request lead author, used in combination with mounting post bracket and right-angled bracket</td>
		</tr>
		<tr>
			<td>Hydraulic micromanipulator</td>
			<td>Narishige</td>
			<td>MO-10</td>
			<td>Step 3.2.4.</td>
		</tr>
		<tr>
			<td>Krazy glue</td>
			<td>Krazy glue</td>
			<td>KG82648R</td>
			<td>Step 4.3. Cyanoacrylate-based glue</td>
		</tr>
		<tr>
			<td>Low-noise fast photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422PA-40 or H10769PA-40</td>
			<td>Step 1.3.</td>
		</tr>
		<tr>
			<td>MATLAB 2012b</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>Steps 2.6, and sections 5, and 6. Used to run microscope acquisition and data analysis software</td>
		</tr>
		<tr>
			<td>Motor</td>
			<td>Zhengke</td>
			<td>ZGA37RG</td>
			<td>Step 2.4.</td>
		</tr>
		<tr>
			<td>Motor speed controller</td>
			<td>Elenker</td>
			<td>EK-G00015A1-1</td>
			<td>Step 2.5.</td>
		</tr>
		<tr>
			<td>Motorized micromanipulator</td>
			<td>Sutter</td>
			<td>MP-285</td>
			<td>Step 3.2.4.</td>
		</tr>
		<tr>
			<td>Mounting base</td>
			<td>Thorlabs</td>
			<td>BA1S</td>
			<td>Step 2.5. Used for posts for motor and sensor in combination with PH4 and TR2</td>
		</tr>
		<tr>
			<td>Mounting post</td>
			<td>Thorlabs</td>
			<td>P14</td>
			<td>Step 2.6. Used for headplate holder post in combination with PB2</td>
		</tr>
		<tr>
			<td>Mounting post base</td>
			<td>Thorlabs</td>
			<td>PB2</td>
			<td>Step 2.6. Used for headplate holder post in combination with P14</td>
		</tr>
		<tr>
			<td>Mounting post bracket</td>
			<td>Thorlabs</td>
			<td>C1515</td>
			<td>Step 2.6. Used in combination with right-angle bracket and headplate holder</td>
		</tr>
		<tr>
			<td>Optical post</td>
			<td>Thorlabs</td>
			<td>TR2</td>
			<td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and PH4</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>&#925;/&#913;</td>
			<td>&#925;/&#913;</td>
			<td>Step 3.2.2. Protocol: Cold Spring Harbor Protocols 2006, doi: 10.1101/pbd.rec8247</td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Thorlabs</td>
			<td>FDS010</td>
			<td>Step 1.2.</td>
		</tr>
		<tr>
			<td>Photon timing counting module</td>
			<td>Becker and Hickl</td>
			<td>SPC-150</td>
			<td>Step 1.1.</td>
		</tr>
		<tr>
			<td>Plasmid: tAKAR&#945; (CAG-tAKAR&#945;-WPRE)</td>
			<td>Addgene</td>
			<td>119913</td>
			<td>Step 3.1.3.</td>
		</tr>
		<tr>
			<td>Post holder</td>
			<td>Thorlabs</td>
			<td>PH4</td>
			<td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and TR2</td>
		</tr>
		<tr>
			<td>Right-angle bracket</td>
			<td>Thorlabs</td>
			<td>AB90</td>
			<td>Step 2.6 Used in combination with mounting post bracket and headplate holder</td>
		</tr>
		<tr>
			<td>Rotation sensor</td>
			<td>US digital</td>
			<td>MA3-A10-250-N</td>
			<td>Step 2.4.</td>
		</tr>
		<tr>
			<td>Rubber mat</td>
			<td>Rubber-Cal</td>
			<td>B01DCR5LUG</td>
			<td>Step 2.1.</td>
		</tr>
		<tr>
			<td>Shaft coupling (1/4 inch x 1/4 inch)</td>
			<td>McMaster</td>
			<td>6208K433</td>
			<td>Steps 2.3. and 2.4.</td>
		</tr>
		<tr>
			<td>ScanImage 3.6</td>
			<td>Svoboda Lab/Vidrio Technology</td>
			<td>N/A</td>
			<td>Steps 5.9. and 6.1.</td>
		</tr>
		<tr>
			<td>Signal splitter</td>
			<td>Becker and Hickl</td>
			<td>HPM-CON-02</td>
			<td>Step 1.3.1.</td>
		</tr>
		<tr>
			<td>Stainless steel axle (diameter 1/4 inch, L = 12 inch)</td>
			<td>McMaster</td>
			<td>1327K66</td>
			<td>Step 2.3.</td>
		</tr>
		<tr>
			<td>Stereotaxic alignment systsem</td>
			<td>David kopf</td>
			<td>1900</td>
			<td>Steps 3.2. and 4.1. modified; Sutter micromanipulator, custom-made injection needle holder, hydraulic micromanipulator</td>
		</tr>
		<tr>
			<td>Two-photon microscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Section 5. Built based on Modular in vivo multiphoton microscopy system (MIMMS) from HHMI Janelia Research Campus (https://www.janelia.org/open-science/mimms)</td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>14006</td>
			<td>Step 3.2.6.</td>
		</tr>
		<tr>
			<td>Virus: tAKAR&#945; (AAV2/1 hSyn-tAKAR&#945;-WPRE)</td>
			<td>Addgene</td>
			<td>119921</td>
			<td>Step 3.2.2.</td>
		</tr>
		<tr>
			<td>White PE foam roller (8 x 12 inch)</td>
			<td>Fabrication enterprises INC.</td>
			<td>30-2261</td>
			<td>Step 2.1.1.</td>
		</tr>
		<tr>
			<td>White polystyrene fom ball halves</td>
			<td>GrahamSweet</td>
			<td>200mm diameter 2 hollow halves</td>
			<td>Step 2.1.1.</td>
		</tr>
		<tr>
			<td>Zipkicker</td>
			<td>PACER</td>
			<td>PT29</td>
			<td>Step 4.3. Hardening accelerator</td>
		</tr>
	</tbody>
</table>

visualizing protein kinase a activity in head-fixed behaving mice using in vivo two-photon fluorescence lifetime imaging microscopy

La neuromodulation exerce un contrôle puissant sur le fonctionnement du cerveau. La dysfonction des systèmes neuromodulatoires entraîne des troubles neurologiques et psychiatriques. Malgré leur importance, les technologies de suivi des événements neuromodulatoires avec résolution cellulaire commencent à peine à émerger. Les neuromodulateurs, tels que la dopamine, la noradrénaline, l'acétylcholine et la sérotonine, déclenchent des événements de signalisation intracellulaire s'il y a des signaux intracellulaires par l'intermédiaire de leurs récepteurs couplés aux protéines G respectifs afin de moduler l'excitabilité neuronale, les communications synaptiques et d'autres fonctions, régulant ainsi le traitement de l'information dans le réseau neuronal. Les neuromodulateurs mentionnés ci-dessus convergent vers la voie cAMP/protéine kinase A (PKA). Par conséquent, la formation image in vivo de PKA avec la résolution unicellulaire a été développée comme readout pour des événements neuromodulatoires d'une manière analogue à l'imagerie de calcium pour des activités électriques neuronales. Ici, une méthode est présentée pour visualiser l'activité de PKA au niveau des neurones individuels dans le cortex des souris se comportant head-fixed. Pour ce faire, un journaliste d'activité A-kinase amélioré (AKAR), appelé tAKARMD, est utilisé, qui est basé sur le transfert d'énergie de résonance de Fârster (FRET). Ce capteur PKA génétiquement codé est introduit dans le cortex moteur par électroporation in utero (IUE) des plasmides d'ADN, ou injection stéréotaxique du virus adéno-associé (AAV). Les changements de FRET sont représentés à l'aide de la microscopie d'imagerie à vie à deux photons (2pFLIM), qui offre des avantages par rapport aux mesures fret ratiométriques pour quantifier le signal FRET dans le tissu cérébral qui disperse la lumière. Pour étudier les activités de La PKA pendant la locomotion forcée, tAKAREstEst est représenté à travers une fenêtre crânienne chronique au-dessus du cortex des souris éveillées, fixées à la tête, qui courent ou se reposent sur un tapis roulant motorisé à commande de vitesse. Cette approche d'imagerie s'appliquera à de nombreuses autres régions du cerveau pour étudier les activités Correspondantes de LTP induites par le comportement et à d'autres capteurs FLIM pour l'imagerie in vivo.

Les capteurs FRET-FLIM permettent la visualisation de nombreuses voies de signalisation différentes, y compris la voie cAMP/PKA impliquée dans la neuromodulation. Le protocole actuel utilise le capteur tAKARMD récemment développé en combinaison avec 2pFLIM pour visualiser les activités de PKA chez les souris ayant un comportement fixe à la tête. La plupart des microscopes à deux photons existants peuvent être améliorés avec des capacités 2pFLIM en ajoutant trois à quatre com...

Watch this Scientific Journal Video about Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy at JoVE.com

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Une procédure est présentée pour visualiser les activités de kinase a de protéine dans les souris head-fixed, se comportant. Un journaliste amélioré d'activité de A-kinase, tAKARMD, est exprimé dans les neurones corticaux et rendu accessible pour la formation image par une fenêtre crânienne. La microscopie d'imagerie à vie à deux photons est utilisée pour visualiser les activités de LPJ in vivo pendant la locomotion forcée.

Visualisation de la protéine Kinase Une activité chez des souris à behaving à l'aide de la microscopie d'imagerie à deux photons in vivo fluorescence à vie

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

Notre méthode utilise une nouvelle protéine kinase Un capteur appelé cible A kinase reporter d’activité alpha, ou tAKAR

visualizing-protein-kinase-activity-head-fixed-behaving-mice-using

Research

JoVE Journal

Neuroscience

8.3K vues.  Oregon Health & Science University. Notre méthode utilise une nouvelle protéine kinase Un capteur appelé cible A kinase reporter d’activité alpha, ou tAKAR

Vidéo: Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Dans l&#39;imagerie in vivo de la moelle épinière de souris utilisant microscopie à deux photons

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Recherche

Neurosciences

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Utilisation de neuro-imagerie fonctionnelle par ultrasons rupture de la barrière hémato-encéphalique et le manganèse-IRM

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

In Vivo microscopie à deux photons de terminaisons nerveuses dans la peau simples

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Deux-Photon<em&gt; In vivo</em&gt; Imagerie des épines dendritiques dans le cortex de souris utilisant un crâne-Diluée Préparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging de neurones corticaux en souris néonatales

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillaire Imagerie in vivo à deux photons de la rétine de souris

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.

Évaluation et manipulation de l’activité neuronale à l’aide de la microscopie holographique à deux photons

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

In vivo Imagerie calcique à grand champ et à deux photons d’une souris utilisant une grande fenêtre crânienne

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Suivi du comportement et modèle d’activité neuronale dans le cerveau antérieur du poisson-zèbre

Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.

Pleins feux sur l’auteur : Progrès de la recherche sur le cerveau du poisson-zèbre adulte 

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

Imagerie calcique à deux photons de l’activité du cerveau antérieur chez le poisson-zèbre adulte

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used ...