Name: functional magnetic resonance imaging (fmri) with auditory stimulation in songbirds
Uploaded: 2013-06-03T08:56:00
Description: Watch this Scientific Journal Video about Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds at JoVE.com

Questa ricerca è stata sostenuta da finanziamenti della Fondazione per la Ricerca - Fiandre (FWO, progetto Nr. G.0420.02 e G.0443.11N), l&#39;Ercole Foundation (Grant Nr. AUHA0012), concertate azioni di ricerca (finanziamento GOA) presso l&#39;Università di Anversa, e in parte sponsorizzato dalla CE - FP6 progetto Dimi, LSHB-CT-2005-512146 e CE - FP6 progetto EMIL LSHC-CT-2004-503569 per A.VdL. G.DG e CP sono borsisti post-dottorato della Fondazione di Ricerca - Fiandre (FWO).

1. Preparazione degli stimoli uditivi <ol><li> Prima di registrare il suono-stimolo, mentre in fase di riproduzione all&#39;interno del foro del sistema MR 7T. L&#39;alesaggio è uno spazio limitato che può distorcere gli stimoli uditivi con conseguente valorizzazione di determinate frequenze uditive. Figura 1 mostra le frequenze enfatizzate e repressa come dimostrano le nostre registrazioni di rumore bianco realizzato in corrispondenza della posizione della testa degli stessi all&#39;interno del mag...

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	<li>Van Meir, 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=Spatiotemporal+properties+of+the+BOLD+response+in+the+songbirds'+auditory+circuit+during+a+variety+of+listening+tasks.">Spatiotemporal properties of the BOLD response in the songbirds' auditory circuit during a variety of listening tasks.</a> Neuroimage. 25, 1242-1255 (2005).</li><li>Boumans, T., Theunissen, F. E., Poirier, C., Van Der Linden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+representation+of+spectral+and+temporal+features+of+song+in+the+auditory+forebrain+of+zebra+finches+as+revealed+by+functional+MRI.">Neural representation of spectral and temporal features of song in the auditory forebrain of zebra finches as revealed by functional MRI.</a> The European Journal of Neuroscience. 26, 2613-2626 (2007).</li><li>Boumans, T., 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=Functional+magnetic+resonance+imaging+in+zebra+finch+discerns+the+neural+substrate+involved+in+segregation+of+conspecific+song+from+background+noise.">Functional magnetic resonance imaging in zebra finch discerns the neural substrate involved in segregation of conspecific song from background noise.</a> Journal of Neurophysiology. 99, 931-938 (2008).</li><li>Boumans, T., 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=Functional+MRI+of+auditory+responses+in+the+zebra+finch+forebrain+reveals+a+hierarchical+organisation+based+on+signal+strength+but+not+selectivity.">Functional MRI of auditory responses in the zebra finch forebrain reveals a hierarchical organisation based on signal strength but not selectivity.</a> PloS ONE. 3, e3184 (2008).</li><li>Vignal, C., 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=Measuring+brain+hemodynamic+changes+in+a+songbird:+responses+to+hypercapnia+measured+with+functional+MRI+and+near-infrared+spectroscopy.">Measuring brain hemodynamic changes in a songbird: responses to hypercapnia measured with functional MRI and near-infrared spectroscopy.</a> Physics in Medicine and Biology. 53, 2457-2470 (2008).</li><li>Van der Linden, A., Van Meir, V., Boumans, T., Poirier, C., Balthazart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+in+small+brains+displaying+extensive+plasticity.">MRI in small brains displaying extensive plasticity.</a> Trends in Neurosciences. 32, 257-266 (2009).</li><li>Poirier, C., Van der Linden, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin+echo+BOLD+fMRI+on+songbirds.">Spin echo BOLD fMRI on songbirds.</a> Methods Mol. Biol. 771, 569-576 (2011).</li><li>Poirier, C., Verhoye, M., Boumans, T., Van der Linden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+spin-echo+blood+oxygen+level-dependent+(BOLD)+functional+MRI+in+birds.">Implementation of spin-echo blood oxygen level-dependent (BOLD) functional MRI in birds.</a> NMR in Biomedicine. 23, 1027-1032 (2010).</li><li>Poirier, C., 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+three-dimensional+MRI+atlas+of+the+zebra+finch+brain+in+stereotaxic+coordinates.">A three-dimensional MRI atlas of the zebra finch brain in stereotaxic coordinates.</a> Neuroimage. 41, 1-6 (2008).</li><li>Zhao, F., 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=Cortical+depth-dependent+gradient-echo+and+spin-echo+BOLD+fMRI+at+9.4T.">Cortical depth-dependent gradient-echo and spin-echo BOLD fMRI at 9.4T.</a> Magnetic Resonance in Medicine: Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 51, 518-524 (2004).</li><li>Harel, N., Lin, J., Moeller, S., Ugurbil, K., Yacoub, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+imaging-histological+study+of+cortical+laminar+specificity+of+fMRI+signals.">Combined imaging-histological study of cortical laminar specificity of fMRI signals.</a> NeuroImage. 29, 879-887 (2006).</li><li>Duong, T. Q., 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=Microvascular+BOLD+contribution+at+4+and+7+T+in+the+human+brain:+gradient-echo+and+spin-echo+fMRI+with+suppression+of+blood+effects.">Microvascular BOLD contribution at 4 and 7 T in the human brain: gradient-echo and spin-echo fMRI with suppression of blood effects.</a> Magnetic Resonance in Medicine: Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 49, 1019-1027 (2003).</li><li>Lee, S. P., Silva, A. C., Ugurbil, K., 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=Diffusion-weighted+spin-echo+fMRI+at+9.4+T:+microvascular/tissue+contribution+to+BOLD+signal+changes.">Diffusion-weighted spin-echo fMRI at 9.4 T: microvascular/tissue contribution to BOLD signal changes.</a> Magnetic Resonance in Medicine: Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 42, 919-928 (1999).</li><li>Uludag, K., Muller-Bierl, B., Ugurbil, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrative+model+for+neuronal+activity-induced+signal+changes+for+gradient+and+spin+echo+functional+imaging.">An integrative model for neuronal activity-induced signal changes for gradient and spin echo functional imaging.</a> NeuroImage. 48, 150-165 (2009).</li><li>Yacoub, 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=Spin-echo+fMRI+in+humans+using+high+spatial+resolutions+and+high+magnetic+fields.">Spin-echo fMRI in humans using high spatial resolutions and high magnetic fields.</a> Magnetic Resonance in Medicine: Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 49, 655-664 (2003).</li><li>Keilholz, S. D., Silva, A. C., Raman, M., Merkle, H., Koretsky, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+MRI+of+the+rodent+somatosensory+pathway+using+multislice+echo+planar+imaging.">Functional MRI of the rodent somatosensory pathway using multislice echo planar imaging.</a> Magnetic Resonance in Medicine: Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 55, 316-324 (2006).</li><li>Keilholz, S. D., Silva, A. C., Raman, M., Merkle, H., Koretsky, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+MRI+of+the+rodent+somatosensory+pathway+using+multislice+echo+planar+imaging.">Functional MRI of the rodent somatosensory pathway using multislice echo planar imaging.</a> Magnetic Resonance in Medicine: Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine. 52, 89-99 (2004).</li><li>Goloshevsky, A. G., Silva, A. C., Dodd, S. J., Koretsky, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BOLD+fMRI+and+somatosensory+evoked+potentials+are+well+correlated+over+a+broad+range+of+frequency+content+of+somatosensory+stimulation+of+the+rat+forepaw.">BOLD fMRI and somatosensory evoked potentials are well correlated over a broad range of frequency content of somatosensory stimulation of the rat forepaw.</a> Brain Research. 1195, 67-76 (2008).</li><li>Kida, I., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus+frequency+dependence+of+blood+oxygenation+level-dependent+functional+magnetic+resonance+imaging+signals+in+the+somatosensory+cortex+of+rats.">Stimulus frequency dependence of blood oxygenation level-dependent functional magnetic resonance imaging signals in the somatosensory cortex of rats.</a> Neuroscience Research. 62, 25-31 (2008).</li><li>Poirier, C., Boumans, T., Verhoye, M., Balthazart, J., Van der Linden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Own-song+recognition+in+the+songbird+auditory+pathway:+selectivity+and+lateralization.">Own-song recognition in the songbird auditory pathway: selectivity and lateralization.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 29, 2252-2258 (2009).</li><li>Poirier, C., 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=Own+song+selectivity+in+the+songbird+auditory+pathway:+suppression+by+norepinephrine.">Own song selectivity in the songbird auditory pathway: suppression by norepinephrine.</a> PloS ONE. 6, e20131 (2011).</li><li>Logothetis, N. K., Pauls, J., Augath, M., Trinath, T., Oeltermann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurophysiological+investigation+of+the+basis+of+the+fMRI+signal.">Neurophysiological investigation of the basis of the fMRI signal.</a> Nature. 412, 150-157 (2001).</li></ol>

In questo rapporto, descriviamo un protocollo ottimizzato per la dettagliata caratterizzazione in vivo di substrati neurali della stimolazione uditiva in anestetizzati diamante mandarino. In linea con il protocollo presentato, la maggior parte degli studi di attivazione funzionale del cervello in animali utilizzando fMRI BOLD, anestetizzare gli animali durante l&#39;acquisizione. Animali di addestramento per abituarli all&#39;ambiente magnete e il rumore dello scanner durante i peri...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Isoflurane anaesthetic</td>
 <td>Isoflo</td>
 <td>05260-05</td>
 <td></td>
 </tr>
 <tr>
 <td>PC-Sam hardware/software</td>
 <td>SA-Instruments</td>
 <td></td>
 <td><a href="http://www.i4sa.com" target="_blank">http://www.i4sa.com</a></td>
 </tr>
 <tr>
 <td>Monitoring and gating system</td>
 <td></td>
 <td>1025</td>
 <td></td>
 </tr>
 <tr>
 <td>MR-compatible small rodent heater system</td>
 <td></td>
 <td></td>
 <td>Model 1025 compatible</td>
 </tr>
 <tr>
 <td>Rectal temperature probe</td>
 <td></td>
 <td>RTP-102B</td>
 <td>7'', 0.044'' </td>
 </tr>
 <tr>
 <td>7T MR scanner</td>
 <td>Bruker Biospin</td>
 <td>PHS 70/16 </td>
 <td></td>
 </tr>
 <tr>
 <td>Paravision software</td>
 <td></td>
 <td>5.1</td>
 <td></td>
 </tr>
 <tr>
 <td>Gradient Insert</td>
 <td></td>
 <td>BGA9S</td>
 <td>400 mT/m, 300A, 500V</td>
 </tr>
 <tr>
 <td>Gradient Amplifiers</td>
 <td>Copley Co., USA</td>
 <td>C256</td>
 <td></td>
 </tr>
 <tr>
 <td>Transmit resonators</td>
 <td></td>
 <td></td>
 <td>Inner diameter: 72 mm, transmit only, active decoupled</td>
 </tr>
 <tr>
 <td>Receiver antenna - 20 mm quadrature Mouse Head</td>
 <td></td>
 <td></td>
 <td>Receive only, active decoupled</td>
 </tr>
 <tr>
 <td>WaveLab software</td>
 <td>Steinberg</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Praat software</td>
 <td>Paul Boersma, University of Amsterdam</td>
 <td></td>
 <td><a href="http://www.praat.org" target="_blank">http://www.praat.org</a></td>
 </tr>
 <tr>
 <td>Non-magnetic dynamic speakers</td>
 <td>Visation, Germany</td>
 <td>HK 150</td>
 <td></td>
 </tr>
 <tr>
 <td>Fiber optic microphone</td>
 <td>Optoacoustics, </td>
 <td>Optimic 1160</td>
 <td></td>
 </tr>
 <tr>
 <td>Sound amplifier</td>
 <td>Phonic corporation</td>
 <td>MM 1002a</td>
 <td></td>
 </tr>
 <tr>
 <td>Presentation software</td>
 <td>Neurobehavioral Systems Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>MRIcro</td>
 <td>Chris Rorden</td>
 <td></td>
 <td><a href="http://www.cabiatl.com/mricro/mricro/" target="_blank">http://www.cabiatl.com/mricro/mricro/</a></td>
 </tr>
 <tr>
 <td>Statistical Parametric Mapping (SPM)</td>
 <td>Welcome Trust Centre for Neuroimaging</td>
 <td>8</td>
 <td><a href="http://www.fil.ion.ucl.ac.uk/spm/" target="_blank">http://www.fil.ion.ucl.ac.uk/spm/</a></td>
 </tr>
 </tbody>
</table>

functional magnetic resonance imaging (fmri) with auditory stimulation in songbirds

La neurobiologia del canto degli uccelli, come modello per il linguaggio umano, è una zona marcata di ricerca in neuroscienze comportamentali. Considerando che l&#39;elettrofisiologia e molecolare approcci permettono l&#39;indagine sia stimoli differenti su alcuni neuroni, o uno stimolo in grandi parti del cervello, il livello di ossigenazione del sangue dipendente (BOLD), risonanza magnetica funzionale (fMRI) permette di combinare entrambi i vantaggi, cioè confrontare l&#39;attivazione neurale indotta da stimoli differenti in tutto il cervello in una volta. fMRI in uccelli canori è impegnativo a causa delle piccole dimensioni del loro cervello e perché le loro ossa e soprattutto il loro cranio comprendono numerose cavità d&#39;aria, inducendo importanti artefatti di suscettibilità. Gradient-echo (GE) fMRI BOLD è stato applicato con successo per uccelli canori 1-5 (per una rassegna, vedi 6). Questi studi si sono concentrati sulle aree cerebrali uditive primarie e secondarie, che sono le regioni libere di artefatti di suscettibilità. Tuttavia, poiché proccessi di interesse possono verificarsi al di là di queste regioni, è necessaria tutta la fMRI BOLD cervello utilizzando una sequenza MRI meno suscettibile a questi manufatti. Ciò può essere ottenuto utilizzando spin-echo (SE) BOLD fMRI 7,8. In questo articolo, si descrive come utilizzare questa tecnica in diamanti mandarini (Taeniopygia guttata), che sono piccoli uccelli canori, con un peso di 15-25 g ampiamente studiato in neuroscienze comportamentali del canto degli uccelli. Il tema principale di studi fMRI su uccelli canori è percezione canzone e canzone apprendimento. La natura degli stimoli uditivi in ​​combinazione con i deboli sensibilità BOLD di SE (rispetto a GE) sequenze fMRI basati rende l&#39;implementazione di questa tecnica molto impegnativo.

<html> Siamo qui presentate visivamente una sequenza ottimizzata di procedure per l&#39;imaging di successo di substrati neurali di stimoli uditivi nel cervello fringuello zebra. In primo luogo, la procedura descritta per la preparazione dei stimoli uditivi risultati in stimoli che può essere incorporato in un ON / OFF blocco paradigma (Figura 2) e che sono normalizzati per eliminare potenziali differenze nel livello di pressione sonora che potrebbe evocare una risposta differenziale nel cervello . Dopo...

Watch this Scientific Journal Video about Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds at JoVE.com

Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

Questo articolo mostra una procedura ottimizzata per l&#39;imaging dei substrati neurali della stimolazione uditiva nel cervello Songbird utilizzando la risonanza magnetica funzionale (fMRI). Esso descrive la preparazione degli stimoli sonori, il posizionamento del soggetto e l&#39;acquisizione e la successiva analisi dei dati fMRI.

Risonanza magnetica funzionale (fMRI) con Auditory Stimulation in uccelli canori

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Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

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