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>

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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 ...

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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.