This research was supported by grants from the Research Foundation - Flanders (FWO, project Nr G.0420.02 and G.0443.11N), the Hercules Foundation (grant Nr AUHA0012), Concerted Research Actions (GOA funding) from the University of Antwerp, and partly sponsored by EC - FP6 project DiMI, LSHB-CT-2005-512146 and EC - FP6 project EMIL LSHC-CT-2004-503569 to A.VdL. G.DG and C.P. are Postdoctoral Fellows of the Research Foundation - Flanders (FWO).

1. Preparation of the Auditory Stimuli
<ol>
	<li>First record the sound-stimuli while being played inside the bore of the 7T MR system. The bore is a confined space that can distort the auditory stimuli resulting in enhancement of certain auditory frequencies. Figure 1 shows the frequencies enhanced and suppressed as shown by our recordings of white noise made at the location of the bird's head within the magnet bore using a fiber-optic microphone (Optimic 1160, Optoacoustics). To compensate this artificial enhancement, an equalizer function is applied to each stimulus using WaveLab software. For our particular setup, the function consists of a Gaussian kernel with the following parameters: maximum amplitude: -20dB, centered on 3,750 Hz, width: 0.05 octaves (corresponding to the range 2,500-5,000 Hz for our system).</li>
	<li>The song stimuli are composed of several individual song motifs of each bird interleaved with periods of silence. The duration of these silent periods is adjusted to keep the total amount of sound and silence identical over all stimuli. This construction conserves the natural intra-individual and inter-individual variability of song length. The total length of each stimulus is 16 sec. The intensity of each song is normalized in terms of matched root-mean-square and high-pass filtered at 400 Hz before being integrated into the complete stimulus (song and silent periods). These manipulations are done using Praat software.</li>
	<li>The experiment consists of an ON/OFF block design alternating auditory stimulation periods (ON blocks) with resting periods (OFF blocks) (Figure 2). Each block (ON and OFF) lasts 16 sec, which corresponds to the acquisition time of 2 images (see below for acquisition). Each stimulus type is presented 25 times, resulting in the acquisition of 50 images per stimulus and per subject. The presentation order of the conditions should be randomized within and between subjects. This randomized order of the stimuli can be coded into Presentation software.</li>
</ol>
2. Subject Preparation
2.1 Subject and group size
Here we present a protocol specifically adapted to the use of (adult) zebra finches. The choice of the species depends on the scientific question. However, other considerations like bird robustness to anaesthesia may also be taken into account. Zebra finches (Taeniopygia guttata) should be housed in aviaries under a 12 hr light: 12 hr dark photoperiod and have access to food and water ad libitum throughout the study. The minimal number of individuals per experiment is 15. This number takes into account the sensitivity of spin-echo fMRI and the natural inter-individual variability of biological phenomena measured in the experiment.
2.2 Installation of setup and preparation of the animal
(For specification of the used equipment, we refer to the list of specific reagents and equipment at the end of this article)
<ol>
	<li>Install the beak mask on the MRI bed of a 7T MR system and connect it to the gas controller device with plastic tubes. Open both oxygen and nitrogen gas bottles and switch on the gas controller device (flow rate oxygen: 200 cc/min; nitrogen: 400 cc/min).</li>
</ol>
As mentioned above, a 7T MR system is used in the presented setup. Other MR systems with different field strengths are also possible, but at 7T a good compromise is reached between signal-to-noise ratio and degree of susceptibility artifacts (see discussion). At higher field strengths the signal-to-noise ratio will increase together with the degree of susceptibility artifacts.
<ol start="2">
	<li>Switch on the feedback controlled system and warm airflow device.</li>
	<li>Anaesthetize the zebra finch with 3% isoflurane in a mixture of oxygen and nitrogen by introducing its beak into the mask and holding the head down until the bird is fully anesthetised. This can be verified by pulling the foot softly: when the bird is fully sedated the foot will not be retracted by the bird. In addition, the eyes of the bird will be partly closed.</li>
	<li>Introduce the cloacal temperature probe to screen the body temperature and monitor the breathing rate by placing a pneumatic sensor underneath the zebra finch belly. Close the jacket to restrain the body of the bird (Figure 3).</li>
	<li>Maintain breathing rate within the range of 40 - 100 breaths per minute and keep body temperature constant within a narrow range of 40 &plusmn; 0.5 &deg;C. When the breathing range is too low/high, adjust the level of anaesthesia (% isoflurane) accordingly. When the problem persists, the experiment should be stopped and the animal removed from the setup in order to recover.</li>
	<li>Position the non-magnetic dynamic speakers on either side of the zebra finch head and connect them to the amplifier. Make sure that the wires of the speakers are led away from the temperature probe, because it can influence the temperature reading when too close.</li>
	<li>Place the surface RF coil on top of the zebra finch head and position the zebra finch in the center of the magnet (and automatically the center of the transmit coil which is situated in the middle of the magnet).</li>
	<li>Reduce the anaesthesia level to 1.5% isoflurane mixed with oxygen and nitrogen.</li>
</ol>
3. Data Acquisition
<ol>
	<li>Acquire a set of 1 sagittal, 1 horizontal and 1 coronal gradient-echo (GE) scout image (tri-pilot sequence) and sets of horizontal, coronal and sagittal multi-slice images (piloting T2-weighted rapid acquisition relaxation-enhanced (RARE) SE sequence) to determine the position of the brain in the magnet (Figure 4).</li>
	<li>Decrease the noise of the gradients by increasing their ramp times to 1,000 &mu;s.</li>
	<li>Prepare the fMRI sequence: RARE T2-weighted sequence, effective TE: 60 msec, TR: 2,000 msec, RARE factor: 8, FOV: 16 mm, matrix size: 64 x 32, orientation: sagittal, slice thickness: 0.75 mm, Inter-slice gap thickness: 0.05 mm, 15 slices covering nearly the whole brain (Figure 4).</li>
	<li>Select the auditory protocol (auditory stimuli and timing of stimulus delivery) in the presentation software. This protocol consists of a sequence of commands - for the initiation of specific auditory stimuli - which are executed at a specific scan-number. At every repetition within the fMRI sequence, the scanner software will send a trigger to the auditory presentation software which in turn registers the scan number and executes the corresponding command.</li>
	<li>To ensure that the auditory presentation software does not miss any trigger from the scanner, the auditory protocol is initiated first. Once the protocol is fully loaded, the fMRI sequence is started.</li>
	<li>Each fMRI experiment is preceded by the acquisition of 12 dummy images to allow the signal attributed to the scanner noise to reach a steady state before starting auditory stimulation.</li>
	<li>After acquisition zero-fill the data to 64 x 64.</li>
	<li>Take a first (preliminary) look at the results using the Functional Tool of Paravision (option Processing/Functional Imaging). Calculate the differential BOLD response between all ON blocks and the baseline (OFF blocks). This analysis gives a first indication of the quality of the experiment. If no activation is seen in the primary auditory areas at this stage, the bird did probably not hear/processed the auditory stimuli due to technical problems with the stimulus presentation, anaesthesia level, etc. The setup should be verified and the measurement repeated.</li>
	<li>Run an anatomical 3D RARE T2-weighted sequence in the same orientation as the previous fMRI scans and with effective TE: 60 msec, TR: 2,000 msec, RARE factor: 8, FOV: 16 mm, matrix size: 256 x 128 x 64.</li>
	<li>Zero-fill the data to 256 x 256 x 256.</li>
	<li>Take the zebra finch from the MRI bed and let it recover from anaesthesia in a cage under a red lamp. Normally, the recovery of a zebra finch after isoflurane anaesthesia goes relatively fast (maximal 5 min). After only a few minutes, the birds will try to stand up and once the bird is fully recovered, it will perch on a branch instead of sitting on the bottom of the cage. The duration of anaesthesia is about 2 hr for the present experiment. The maximum time of isoflurane anaesthesia applied to zebra finches in our lab is 6 hr, after which the birds also recovered within 5 min.</li>
</ol>
4. Data Processing
<ol>
	<li>Convert the MR-data into Analyze or Nifti format.</li>
	<li>Because SPM has been developed to process fMRI data acquired in humans, that is for voxels of around 2 mm. Numerous SPM settings are adapted to this approximate voxel size. If one does not want to change all these settings, the simplest way to proceed is to artificially increase the voxel size of bird fMRI data. Adjust the voxel size in the header by multiplying the real voxel size by 10 using MRIcro. It should be noted, that such adjustment does not influence the data in itself, no resampling or any other modifications to the data is applied.</li>
</ol>
An alternative to this is the use of 'SPMMouse' which is a toolbox allowing SPM to open and analyze files of any voxel dimension. The tool allows SPM 'glass brains' to be created from any image, and automatically adjusts defaults length scales based on the headers of image files or user entered data. Hence, this toolbox works in the opposite way than what we propose. Instead of changing the voxel size of the images to fit in SPM, the default settings of SPM are changed to use images with different voxel sizes.
<ol start="3">
	<li>Realign the fMRI data. Co-register the anatomical 3D dataset to the fMRI time series. Normalize the 3D data (and the co-registered fMRI time series) to the zebra finch brain MRI atlas. Apply the transformation matrix to the fMRI dataset. This can all be done using Statistical Parametric Mapping (SPM) 8 software.</li>
	<li>Smooth the data with a 0.5-mm width Gaussian kernel using SPM8.</li>
	<li>Carry out statistical voxel-based analyses using SPM8. Model the data as a box-car (no hemodynamic response function). Estimate model parameters with the classical Restricted Maximum Likelihood algorithm. Compute the mean effect of each auditory stimulus in each subject (fixed-effect analysis) and then compute statistics as wished for group analyses (mixed-effect analyses).</li>
	<li>Project the statistical parametric map onto the zebra finch atlas (Figure 5) 9 in SPM8 to localize the functional activations (Figure 6).</li>
</ol>

<ol>
	<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>

No conflicts of interest declared.

In this report, we describe an optimized protocol for the detailed in vivo characterization of neural substrates of auditory stimulation in anaesthetized zebra finches.
In line with the presented protocol, the majority of functional brain activation studies in animals using BOLD fMRI, anaesthetize the animals during the acquisition. Training animals to accustom them to the magnet environment and the scanner noise during the study periods is also possible but rather time-consuming and ...

<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

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5 (for a review, see 6). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8 . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

We here visually presented an optimized sequence of procedures for successful imaging of neural substrates of auditory stimuli in the zebra finch brain. Firstly, the described procedure for preparation of the auditory stimuli results in stimuli that can be incorporated into an ON/OFF block paradigm (Figure 2) and that are normalized to eliminate potential differences in sound pressure level that could evoke a differential response in the brain. After preparing the zebra finch for MRI scanning and ...

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Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

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

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and ...

functional-magnetic-resonance-imaging-fmri-with-auditory-stimulation

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18.0K Views.  University of Antwerp. This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

Video: Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

Best Current Practice for Obtaining High Quality EEG Data During Simultaneous fMRI

Simultaneous EEG-fMRI allows the excellent temporal resolution of EEG to be combined with the high spatial accuracy of fMRI. The data from these two modalities can be combined in a number of ways, but all rely on the acquisition of high quality EEG and fMRI data. EEG data acquired during simultaneous fMRI are affected by several artifacts, including the gradient artefact (due to the changing magnetic field gradients required for fMRI), the pulse artefact (linked to the cardiac cycle) and movement artifacts (resulting from movements in the strong magnetic field of the scanner, and muscle activity). Post-processing methods for successfully correcting the gradient and pulse artifacts require a number of criteria to be satisfied during data acquisition. Minimizing head motion during EEG-fMRI is also imperative for limiting the generation of artifacts.
Interactions between the radio frequency (RF) pulses required for MRI and the EEG hardware may occur and can cause heating. This is only a significant risk if safety guidelines are not satisfied. Hardware design and set-up, as well as careful selection of which MR sequences are run with the EEG hardware present must therefore be considered.
The above issues highlight the importance of the choice of the experimental protocol employed when performing a simultaneous EEG-fMRI experiment. Based on previous research we describe an optimal experimental set-up. This provides high quality EEG data during simultaneous fMRI when using commercial EEG and fMRI systems, with safety risks to the subject minimized. We demonstrate this set-up in an EEG-fMRI experiment using a simple visual stimulus. However, much more complex stimuli can be used. Here we show the EEG-fMRI set-up using a Brain Products GmbH (Gilching, Germany) MRplus, 32 channel EEG system in conjunction with a Philips Achieva (Best, Netherlands) 3T MR scanner, although many of the techniques are transferable to other systems.

Simultaneous electroencephalography (EEG) and functional Magnetic Resonance imaging (fMRI) is a powerful neuroimaging tool. However, the inside of an MRI scanner forms a difficult environment for EEG data recording and safety must be considered whenever operating EEG equipment inside a scanner. Here, we present an optimised EEG-fMRI data acquisition protocol.

Simultaneous  EEG-fMRI allows the excellent temporal resolution of EEG to be combined with the  high spatial accuracy of fMRI. The data  from these two ...

Quantitative Assessment of Cortical Auditory-tactile Processing in Children with Disabilities

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology to quantitatively assess children&#39;s cortical processing of light touch, speech sounds and the multisensory processing of the 2 stimuli, without requiring active subject participation or causing children discomfort. To accomplish this we developed a dual channel, time and strength calibrated air puff stimulator that allows both tactile stimulation and sham control. We combined this with the use of event-related potential methodology to allow for high temporal resolution of signals from the primary and secondary somatosensory cortices as well as higher order processing. This methodology also allowed us to measure a multisensory response to auditory-tactile stimulation.

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology to quantitatively assess infants and children&#39;s cortical processing of light touch, speech sounds, and the multisensory processing of the 2 stimuli, without requiring active subject participation or causing discomfort in vulnerable patients.

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology ...

Stimulating the Lip Motor Cortex with Transcranial Magnetic Stimulation

Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception. Researchers have used single-pulse and repetitive TMS to stimulate the lip representation in the motor cortex. The excitability of the lip motor representation can be investigated by applying single TMS pulses over this cortical area and recording TMS-induced motor evoked potentials (MEPs) via electrodes attached to the lip muscles (electromyography; EMG). Larger MEPs reflect increased cortical excitability. Studies have shown that excitability increases during listening to speech as well as during&#160;viewing speech-related movements. TMS can be used also to disrupt the lip motor representation. A 15-min train of low-frequency sub-threshold repetitive stimulation has been shown to suppress motor excitability for a further 15-20 min. This TMS-induced disruption of the motor lip representation impairs subsequent performance in demanding speech perception tasks and modulates auditory-cortex responses to speech sounds. These findings are consistent with the suggestion that the motor cortex contributes to speech perception. This article describes how to localize the lip representation in the motor cortex and how to define the appropriate stimulation intensity for carrying out both single-pulse and repetitive TMS experiments.

Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception.&#160; This article describes how to record motor evoked potentials (MEPs) from the lip muscles and how to disrupt the motor lip representation using repetitive TMS.

Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception. ...

Transcranial Direct Current Stimulation and Simultaneous  Functional Magnetic Resonance Imaging

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses weak electrical currents administered to the scalp to manipulate cortical excitability and, consequently, behavior and brain function. In the last decade, numerous studies have addressed short-term and long-term effects of tDCS on different measures of behavioral performance during motor and cognitive tasks, both in healthy individuals and in a number of different patient populations. So far, however, little is known about the neural underpinnings of tDCS-action in humans with regard to large-scale brain networks. This issue can be addressed by combining tDCS with functional brain imaging techniques like functional magnetic resonance imaging (fMRI) or electroencephalography (EEG).
In particular, fMRI is the most widely used brain imaging technique to investigate the neural mechanisms underlying cognition and motor functions. Application of tDCS during fMRI allows analysis of the neural mechanisms underlying behavioral tDCS effects with high spatial resolution across the entire brain. Recent studies using this technique identified stimulation induced changes in task-related functional brain activity at the stimulation site&#160;and also in more distant brain regions, which were associated with behavioral improvement. In addition, tDCS administered during resting-state fMRI allowed identification of widespread changes in whole brain functional connectivity.
Future studies using this combined protocol should yield new insights into the mechanisms of tDCS action in health and disease and new options for more targeted application of tDCS in research and clinical settings. The present manuscript describes this novel technique in a step-by-step fashion, with a focus on technical aspects of tDCS administered during fMRI.

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique. It has successfully been used in basic research and clinical settings to modulate brain function in humans. This article describes the implementation of tDCS and simultaneous functional magnetic resonance imaging (fMRI), to investigate the neural basis of tDCS effects.

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses weak electrical currents administered to the scalp ...

Transcranial Magnetic Stimulation for Investigating Causal Brain-behavioral Relationships and their Time Course

Transcranial magnetic stimulation (TMS) is a safe, non-invasive brain stimulation technique that uses a strong electromagnet in order to temporarily disrupt information processing in a brain region, generating a short-lived &#8220;virtual lesion.&#8221; Stimulation that interferes with task performance indicates that the affected brain region is necessary to perform the task normally. In other words, unlike neuroimaging methods such as functional magnetic resonance imaging (fMRI) that indicate correlations between brain and behavior, TMS can be used to demonstrate causal brain-behavior relations. Furthermore, by varying the duration and onset of the virtual lesion, TMS can also reveal the time course of normal processing. As a result, TMS has become an important tool in cognitive neuroscience. Advantages of the technique over lesion-deficit studies include better spatial-temporal precision of the disruption effect, the ability to use participants as their own control subjects, and the accessibility of participants. Limitations include concurrent auditory and somatosensory stimulation that may influence task performance, limited access to structures more than a few centimeters from the surface of the scalp, and the relatively large space of free parameters that need to be optimized in order for the experiment to work. Experimental designs that give careful consideration to appropriate control conditions help to address these concerns. This article illustrates these issues with TMS results that investigate the spatial and temporal contributions of the left supramarginal gyrus (SMG) to reading.

Transcranial magnetic stimulation (TMS) is a technique for non-invasively disrupting neural information processing and measuring its effect on behavior. When TMS interferes with a task, it indicates that the stimulated brain region is necessary for normal task performance, allowing one to systematically relate brain regions to cognitive functions.

Transcranial magnetic stimulation (TMS) is a safe, non-invasive brain stimulation technique that uses a strong electromagnet in order to temporarily ...

Transferring Cognitive Tasks Between Brain Imaging Modalities: Implications for Task Design and Results Interpretation in fMRI Studies

As cognitive neuroscience methods develop, established experimental tasks are used with emerging brain imaging modalities. Here transferring a paradigm (the visual oddball task) with a long history of behavioral and electroencephalography (EEG) experiments to a functional magnetic resonance imaging (fMRI) experiment is considered. The aims of this paper are to briefly describe fMRI and when its use is appropriate in cognitive neuroscience; illustrate how task design can influence the results of an fMRI experiment, particularly when that task is borrowed from another imaging modality; explain the practical aspects of performing an fMRI experiment. It is demonstrated that manipulating the task demands in the visual oddball task results in different patterns of blood oxygen level dependent (BOLD) activation. The nature of the fMRI BOLD measure means that many brain regions are found to be active in a particular task. Determining the functions of these areas of activation is very much dependent on task design and analysis. The complex nature of many fMRI tasks means that the details of the task and its requirements need careful consideration when interpreting data. The data show that this is particularly important in those tasks relying on a motor response as well as cognitive elements and that covert and overt responses should be considered where possible. Furthermore, the data show that transferring an EEG paradigm to an fMRI experiment needs careful consideration and it cannot be assumed that the same paradigm will work equally well across imaging modalities. It is therefore recommended that the design of an fMRI study is pilot tested behaviorally to establish the effects of interest and then pilot tested in the fMRI environment to ensure appropriate design, implementation and analysis for the effects of interest.

Transferring a paradigm with a history of use in EEG experiments to an fMRI experiment is considered. It is demonstrated that manipulating the task demands in the visual oddball task resulted in different patterns of BOLD activation and illustrated how task design is crucial in fMRI experiments.

As cognitive neuroscience methods develop, established experimental tasks are used with emerging brain imaging modalities. Here transferring a paradigm ...

Design and Implementation of an fMRI Study Examining Thought Suppression in Young Women with, and At-risk, for Depression

Ruminative brooding is associated with increased vulnerability to major depression. Individuals who regularly ruminate will often try to reduce the frequency of their negative thoughts by actively suppressing them. We aim to identify the neural correlates underlying thought suppression in at-risk and depressed individuals. Three groups of women were studied; a major depressive disorder group, an at-risk group (having a first degree relative with depression) and controls. Participants performed a mixed block-event fMRI paradigm involving thought suppression, free thought and motor control periods. Participants identified the re-emergence of &#8220;to-be-suppressed&#8221; thoughts (&#8220;popping&#8221; back into conscious awareness) with a button press. During thought suppression the control group showed the greatest activation of the dorsolateral prefrontal cortex, followed by the at-risk, then depressed group. During the re-emergence of intrusive thoughts compared to successful re-suppression of those thoughts, the control group showed the greatest activation of the anterior cingulate cortices, followed by the at-risk, then depressed group. At-risk participants displayed anomalies in the neural regulation of thought suppression resembling the dysregulation found in depressed individuals. The predictive value of these changes in the onset of depression remains to be determined.

We aim to identify the neural correlates underlying sustained and transient thought suppression, and thought re-emergence in controls, at-risk and depressed individuals. Activation was greatest for controls compared to the at-risk and the depressed group in the dorsolateral prefrontal cortex during thought suppression and anterior cingulate cortex during thought re-emergence.

Ruminative brooding is associated with increased vulnerability to major depression. Individuals who regularly ruminate will often try to reduce the ...

Functional Near Infrared Spectroscopy of the Sensory and Motor Brain Regions with Simultaneous Kinematic and EMG Monitoring During Motor Tasks

There are several advantages that functional near-infrared spectroscopy (fNIRS) presents in the study of the neural control of human movement. It is relatively flexible with respect to participant positioning and allows for some head movements during tasks. Additionally, it is inexpensive, light weight, and portable, with very few contraindications to its use. This presents a unique opportunity to study functional brain activity during motor tasks in individuals who are typically developing, as well as those with movement disorders, such as cerebral palsy. An additional consideration when studying movement disorders, however, is the quality of actual movements performed and the potential for additional, unintended movements. Therefore, concurrent monitoring of both blood flow changes in the brain and actual movements of the body during testing is required for appropriate interpretation of fNIRS results. Here, we show a protocol for the combination of fNIRS with muscle and kinematic monitoring during motor tasks. We explore gait, a unilateral multi-joint movement (cycling), and two unilateral single-joint movements (isolated ankle dorsiflexion, and isolated hand squeezing). The techniques presented can be useful in studying both typical and atypical motor control, and can be modified to investigate a broad range of tasks and scientific questions.

Monitoring brain activity during upright motor tasks is of great value when investigating the neural source of movement disorders. Here, we demonstrate a protocol that combines functional near infrared spectroscopy with continuous monitoring of muscle and kinematic activity during 4 types of motor tasks.

There are several advantages that functional near-infrared spectroscopy (fNIRS) presents in the study of the neural control of human movement. It is ...

Neuronavigation-guided Repetitive Transcranial Magnetic Stimulation for Aphasia

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential therapeutic effects. Brain excitability is non-invasively modulated by rTMS, and rTMS to the language areas has proved its potential effects on treatment of aphasia. In our protocol, we aim to artificially induce virtual aphasia in healthy subjects by inhibiting Brodmann area 44 and 45 using neuronavigational TMS (nTMS), and F3 of the International 10-20 EEG system for conventional TMS (cTMS). To measure the degree of aphasia, changes in reaction time to a picture naming task pre- and post-stimulation are measured and compare the delay in reaction time between nTMS and cTMS. Accuracy of the two TMS stimulation methods is compared by averaging the Talairach coordinates of the target and the actual stimulation. Consistency of stimulation is demonstrated by the error range from the target. The purpose of this study is to demonstrate use of nTMS and to describe the benefits and limitations of the nTMS compared to those of cTMS.

This study is designed to test the hypothesis that neuronavigational system-guided transcranial magnetic stimulation has higher accuracy for targeting the intended target as demonstrated by eliciting a greater degree of virtual aphasia in healthy subjects, measured by delay in reaction time to picture naming.

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential ...

Treating Clinical Depression with Repetitive Deep Transcranial Magnetic Stimulation Using the Brainsway H1-coil

Repetitive transcranial magnetic stimulation (rTMS) is an emerging non-pharmacological approach to treating many brain-based disorders. rTMS uses electromagnetic coils to stimulate areas of the brain non-invasively. Deep transcranial magnetic stimulation (dTMS) with the Brainsway H1-coil system specifically is a type of rTMS indicated for treating patients with major depressive disorder (MDD) who are resistant to medication. The unique H1-coil design of this device is able to stimulate neuronal pathways that lie deeper in the targeted brain areas than those reached by conventional rTMS coils. dTMS is considered to be low-risk and well tolerated, making it a viable treatment option for people who have not responded to medication or psychotherapy trials for their depression.
Randomized, sham-control studies have demonstrated that dTMS produces significantly greater improvement in depressive symptoms than sham dTMS treatment in patients with major depression that has not responded to antidepressant medication. In this paper, we will review the methodology for treating major depression with dTMS using an H1-coil.

Here we present a protocol outlining the methodology for treatment of Major Depressive Disorder using the deep Transcranial Magnetic Stimulation (dTMS) system.