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

Summary

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.

Abstract

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.

Protocol

1. Preparation of the Auditory Stimuli 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 artif…

Representative Results

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 …

Discussion

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 …

Materials

Name of the reagent/equipment	Company	Catalogue number	Comments
Isoflurane anaesthetic	Isoflo	05260-05
PC-Sam hardware/software	SA-Instruments		http://www.i4sa.com
Monitoring and gating system		1025
MR-compatible small rodent heater system			Model 1025 compatible
Rectal temperature probe		RTP-102B	7”, 0.044”
7T MR scanner	Bruker Biospin	PHS 70/16
Paravision software		5.1
Gradient Insert		BGA9S	400 mT/m, 300A, 500V
Gradient Amplifiers	Copley Co., USA	C256
Transmit resonators			Inner diameter: 72 mm, transmit only, active decoupled
Receiver antenna – 20 mm quadrature Mouse Head			Receive only, active decoupled
WaveLab software	Steinberg
Praat software	Paul Boersma, University of Amsterdam		http://www.praat.org
Non-magnetic dynamic speakers	Visation, Germany	HK 150
Fiber optic microphone	Optoacoustics,	Optimic 1160
Sound amplifier	Phonic corporation	MM 1002a
Presentation software	Neurobehavioral Systems Inc.
MRIcro	Chris Rorden		http://www.cabiatl.com/mricro/mricro/
Statistical Parametric Mapping (SPM)	Welcome Trust Centre for Neuroimaging	8	http://www.fil.ion.ucl.ac.uk/spm/