Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.
Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.
Functional Magnetic Resonance Imaging (fMRI) is a non-invasive method to measure the hemodynamic responses1,2,3,4,5,6,7,8,9, e.g., the blood-oxygen-level-dependent (BOLD), cerebral blood volume and flow signal, associated with neural activity in the brain. In animal studies, hemodynamic signals can be affected by anesthesia10, the stress level of awake animals11, as well as the potential non-physiological artifacts, e.g., cardiac pulsation and respiratory motions12,13,14,15. Although many post-processing methods have been developed to provide a retrospective analysis of the fMRI signal for the task-related and resting-state functional dynamics and connectivity mapping16,17,18,19, there are few techniques to provide a real-time brain function mapping solution and instantaneous readouts in the animal brain20 (most of which are mainly used for human brain mapping21,22,23,24,25,26,27). In particular, this kind of real-time fMRI mapping method is lacking in animal studies. It is necessary to set up an fMRI platform to enable the investigation of real-time brain state-dependent physiological stages and to provides real-time biofeedback stimulation paradigm for animal brain functional studies.
In the present work, we illustrate a real-time fMRI experimental set-up with the customized macro-functions of the MRI console software, demonstrating real-time monitoring of the evoked BOLD-fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of the anesthetized rats. This real-time set-up allows for the visualization of the ongoing brain activation in functional maps, as well as individual time courses in a voxel-wise manner, using the existing neuroimage analysis software, Analysis of Functional NeuroImages (AFNI)28. The preparation of the real-time fMRI experimental set-up for the animal study is described in the protocol. Besides the animal set-up, we provide detailed procedures to set up the visualization and analysis of the real-time fMRI signals using the latest console software in parallel with the image processing scripts. In summary, the proposed real-time fMRI set-up for animal studies is a powerful tool for monitoring the dynamic fMRI signals in the animal brain using the MRI console system.
This study was performed in accordance with the German Animal Welfare Act (TierSchG) and Animal Welfare Laboratory Animal Ordinance (TierSchVersV). The experimental protocol described here was reviewed by the ethics commission (§15 TierSchG) and approved by the state authority (Regierungspräsidium, Tübingen, Baden-Württemberg, Germany).
1. Preparing the BOLD-fMRI experimental set-up for small animal study
2. Catheterization and ventilation surgery
3. Placing the animal inside the MRI scanner
4. Measuring anatomical MR images
5. Real-Time fMRI software set-up and fMRI response visualization
Figure 3 and Figure 4 show a representative real-time voxel-wise BOLD-fMRI time course and functional maps with electrical forepaw stimulation (3 Hz, 4 s, pulse width 300 us, 2.5 mA). The fMRI design paradigm comprises 10 pre-stimulation scans, 3 stimulation scans, and 12 inter-stimulation scans with a total of 8 epochs (130 scans). The total scan time is 3 min 15 sec (195 sec). Figure 3 shows the voxel-wise time course (black line) of the contralateral FP-S1 corresponding to the block-design paradigm (red line) in the real-time acquisition format. Figure 4 shows the activated BOLD maps corresponding to the electrical forepaw stimulation. The activated regions are detected and displayed as the colored clusters (red and yellow colors). Experimenters can use the “Clusters” function in the AFNI software to interactively explore clustered volumes and display them as an overlaid color-coded image.
Figure 1: Real-time fMRI experimental set-up for forepaw stimulation. A simplified schematic of the real-time fMRI set-up and the flow (dashed lines) of the control parameters are shown. One computer (left) is used as a console for pulse sequence execution, stimulus isolator control, and data analysis with AFNI. The other computer (right) is used for monitoring physiological information (e.g., blood pressure, respiration, and chest movement, etc.). Please click here to view a larger version of this figure.
Figure 2: Diagram of the data processing during fMRI scanning. A simplified flow chart of data processing with the representative macro and AFNI functions in the real-time fMRI set-up is shown. Before starting fMRI scans, the “Pre Image Series Activities” and “Execute Macro” options are selected among the reconstruction options. The “Setup_rt3DEPI” script is executed by using those options when clicking the “Scan” button. With the “Dimon” command, the real-time AFNI files are monitored and sent into the AFNI plugin to display dynamic BOLD responses when the background macro script, “Feed2AFNI_rt3DEPI” converts the fMRI raw data to the AFNI files. Please click here to view a larger version of this figure.
Figure 3: Real-time voxel-wise fMRI responses. An activated single voxel time course graph (black line) from the primary forepaw somatosensory (FP-S1) cortex is shown during the block-design stimulation paradigm. The repetitive fMRI design paradigm (red line) was defined by the “afni -rt -DAFNI_FIM_IDEAL=(Paradigm)”. The graph demonstrates that clear and stable BOLD responses follow electrical stimulation in real-time. Please click here to view a larger version of this figure.
Figure 4: Functional maps of BOLD responses to electrical stimulation in contralateral FP-S1 regions. The voxel clusters activated in the FP-S1 regions (yellow and red colors) were identified and significantly synchronized with the repetitive stimulation paradigm, overlaid on the T2-weighted anatomical images. Please click here to view a larger version of this figure.
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Real-time monitoring of the fMRI signal helps experimenters adjust the physiology of animals to optimize functional mapping. Motion artifacts in awake animals, as well as the anesthetic effect, are major factors that mediate the variability of fMRI signals, confounding the biological interpretation of the signal by itself31,32,33,34,35,36,37,38. The real-time fMRI platform offers instantaneous information to assist the optimization of scanning parameters and anesthetic administration schemes. Also, real-time brain hemodynamic responses can be used to provide fMRI-based biofeedback controlling signals for novel stimulation paradigms in multi-modal brain functional studies.
A remaining concern about the proposed real-time fMRI set-up is the technical dependency on the vendor-specific console software. In this protocol, the real-time fMRI analysis scripts implement a series of macro-functions using a console software (see Table of Materials) version 6 or higher. The workflow of the MR scan in the previous console software (e.g., PV version 5 or lower) is different from the latest version due to the upgraded user interface and new parameter definition. Using the previous version of the console system (PV version 3), Lu et al. (2008) have shown that the real-time fMRI set-up enabled the monitoring of the drug-induced hemodynamic signal changes in the rat brain to study the cocaine’s effect on the central nervous system20. However, those set-ups cannot be readily applied to the new console software with state-of-the-art electronic devices. In the latest console software, it is a critical step to run the predefined macro scripts and monitor fMRI raw data right after starting to scan by selecting the “Pre Image Series Activities” and “Execute Macro” options of the “Data Reconstruction”.
For further image processing, customized AFNI functions can be readily incorporated into the real-time image processing scripts. In particular, it will be valuable to provide real-time analysis using motion-related traces, e.g., electromyography (EMG) signal for awake animal fMRI38, and incorporate multi-modal dynamic brain signal, e.g., GCaMP-mediated Ca2+, to specify whole-brain hemodynamic correlation37. Furthermore, this real-time fMRI set-up can be extended to animal neurofeedback studies to investigate self-regulating brain and behavior similar to previous human studies27.
The authors have nothing to disclose.
We thank Dr. D. Chen and Dr. C. Yen for sharing the AFNI script to set up the real-time fMRI for PV 5 and the AFNI team for the software support. This research was supported by NIH Brain Initiative funding (RF1NS113278-01, R01 MH111438-01), and the S10 instrument grant (S10 RR023009-01) to Martinos Center, German Research Foundation (DFG) Yu215/3-1, BMBF 01GQ1702, and the internal funding from Max Planck Society.
14.1T Bruker MRI system | Bruker BioSpin MRI GmbH | N/A | |
A365 Stimulus Isolator | World Precision Instruments | N/A | |
AcqKnowledge Software | Biopac | RRID:SCR_014279, http://www.biopac.com/product/acqknowledge-software/ | |
AFNI | Cox, 1996 | RRID:SCR_005927, http://afni.nimh.nih.gov | |
CO2SMO (ETCO2/SpO2 Monitor), Model 7100 | Novametrix Medical Systems Inc | N/A | |
Isoflurane | CP-Pharma | Cat# 1214 | |
Master-9 | A.M.P.I | N/A | |
Nanoliter Injector | World Precision Instruments | Cat# NANOFIL | |
Pancuronium Bromide | Inresa Arzneimittel | Cat# 34409.00.00 | |
ParaVision 6 | Bruker BioSpin MRI GmbH | RRID:SCR_001964 | |
Phosphate Buffered Saline (PBS) | Gibco | Cat# 10010-023 | |
Rat: Sprague Dawley rat | Charles River Laboratories | Crl:CD(SD) | |
SAR-830/AP Ventilator | CWE | N/A | |
α-chloralose | Sigma-Aldrich | Cat# C0128-25G;RRID |