Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging
February 24th, 2021
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This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.
Overview
This protocol outlines the use of functional ultrasound (fUS) for quantifying hemodynamic variations in the mouse brain. It showcases 3D functional activation mapping following sensory stimulation and resting-state functional connectivity in both anesthetized and awake mice.
Key Study Components
Area of Science
- Neuroimaging
- Cerebral hemodynamics
- Mouse models
Background
- Functional ultrasound allows mapping of hemodynamic changes in live rodent brains.
- It offers unmatched spatial and temporal resolution compared to traditional methods.
- FUS is accessible for the larger neuroscience community with the availability of commercial scanners.
- Facilitates studies in awake, freely moving subjects, reducing anesthesia-related biases.
Purpose of Study
- To demonstrate a protocol for transcranial functional ultrasound imaging in mice.
- To illustrate methods for capturing brain hemodynamic responses during sensory activation.
- To establish procedures for resting-state functional connectivity analysis.
Methods Used
- Functional ultrasound imaging system, utilizing ultra-fast plane wave imaging.
- Experimental model includes both anesthetized and awake mice for imaging sessions.
- Focus on preparation including eye ointment application, head shaving, and surgical head fixation.
- Detailed steps for optimizing imaging parameters and data acquisition processes.
- Includes techniques for data analysis and visualization in the Allen Mouse Common Coordinates Framework.
Main Results
- Exhibits the capability to perform high-resolution imaging of cerebral blood volume.
- Details the effectiveness of fUS in studying hemodynamic responses without anesthesia.
- Validates its application for both sensory stimulation and monitoring resting-state connectivity.
- Highlights acute post-operative care and recovery protocols for awake animal studies.
Conclusions
- This study illustrates a powerful imaging technique that enhances understanding of cerebral dynamics.
- It opens new avenues in behavioral studies through non-invasive imaging of awake subjects.
- Reveals the potential for fUS to facilitate understanding of neuronal mechanisms and functional brain connectivity.
Frequently Asked Questions
What advantages does functional ultrasound have over traditional imaging techniques?
Functional ultrasound provides high portability, ease of use, and allows imaging of awake, freely moving subjects, minimizing anesthesia effects.
How are awake mice prepared for functional ultrasound imaging?
Awake mice require head fixation through surgical procedures to allow imaging without the confounding effects of anesthesia.
What types of hemodynamic data can be obtained using this method?
Functional ultrasound measures cerebral blood volume changes and can capture both sensory-evoked responses and resting-state connectivity aspects.
Can this protocol be adapted for other animal models?
While this protocol is optimized for mice, similar techniques can potentially be adapted to larger animal models with appropriate adjustments.
What are some limitations of functional ultrasound?
Functional ultrasound may have limitations in depth resolution and may require prior surgical interventions, which could affect experimental design.
What is the significance of using the Allen Mouse Common Coordinates Framework?
This framework allows precise alignment of functional data to anatomical structures, enhancing the accuracy of brain mapping studies.
Executive Industry Relevance
Functional ultrasound (fUS) imaging enables non-invasive, high-resolution mapping of cerebral hemodynamic responses in awake and behaving mice, reducing anesthesia-related confounds in preclinical neuroscience studies. This capability supports target validation and mechanistic de-risking by linking neuronal activity to vascular responses in disease-relevant systems. The method enhances predictive confidence in early discovery by providing reproducible, quantifiable readouts of brain activation and functional connectivity.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through sensory-evoked activation mapping in defined brain regions.
- Operational Value: Provides reproducible 3D functional connectivity patterns for network identification and target engagement assessment.
- Strategic Value: Supports biological de-risking by validating target modulation effects on brain-wide hemodynamic responses.
Screening & Assay Development
- Scientific Value: Delivers quantitative cerebral blood volume (CBV) measurements as a hemodynamic proxy for neuronal activity.
- Operational Value: Enables standardized, whole-brain 3D activation mapping with high spatiotemporal resolution for assay readiness.
- Strategic Value: Facilitates scalable screening of pharmacological compounds on functional brain networks in awake animals.
Translational & Preclinical Research
- Scientific Value: Captures resting-state functional connectivity in disease-relevant brain networks, such as hippocampal-cortical circuits.
- Operational Value: Enables longitudinal monitoring of functional connectivity changes in preclinical models.
- Strategic Value: Advances translational biomarker alignment by linking hemodynamic signatures to neuronal network function.
Pipeline & Workflow Integration
fUS imaging integrates into the discovery continuum from hypothesis testing in early discovery to lead optimization through quantifiable brain activation and connectivity readouts.
- Discovery Biology: Supports hypothesis testing via sensory stimulation paradigms and GLM-based activation mapping.
- Screening: Delivers assay-ready, reproducible hemodynamic responses with defined stimulation and recovery timelines.
- Analytics: Provides voxel-wise time courses and correlation matrices for statistical analysis of brain-wide responses.
- Translational Research: Connects hemodynamic changes to anatomical structures via Allen Mouse Common Coordinates Framework registration.
- Enterprise Reuse: Establishes a reusable platform for cross-functional teams to assess target modulation in awake, behaving animals.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation through hemodynamic response quantification.
- Operational Value: Standardization and reproducibility across anesthetized and awake experimental conditions.
- Strategic Value: Reduced late-stage biological risk via early functional connectivity profiling.
- Portfolio Impact: Informed go/no-go decisions based on brain network engagement and modulation.
Implementation Considerations
- Expertise in neuroscience and ultrasound-based imaging techniques.
- Instrumentation including motorized linear transducer and fUS acquisition software.
- Cross-team standardization of head fixation, anesthesia levels, and stimulation protocols.
- Adaptation considerations for different mouse strains and disease models.
- Practical limitations including surgical preparation for awake imaging and signal depth constraints.
Biopharma FAQs
Why does GLM-based activation mapping matter for target validation?
General linear model (GLM) analysis of voxel-wise time courses enables significant activation detection, such as 15-20% CBV increase in S1BF, providing quantifiable hemodynamic readouts for assessing target engagement in sensory pathways.
How does independent variable isolation fit the discovery pipeline?
Predefined stimulation sequences with controlled timing, duration, and repetition allow isolation of sensory-evoked responses, enabling reliable comparison of hemodynamic changes across experimental conditions in target validation studies.
What quantitative dependent variable measurements enable mechanistic de-risking?
Cerebral blood volume (CBV) fluctuations serve as a quantitative hemodynamic proxy for neuronal activity, allowing measurement of activation magnitude and functional connectivity strength between regions like hippocampus and cortex.
Why do replication requirements matter for cross-functional collaboration?
Reproducible 3D functional connectivity patterns, such as interhemispheric hippocampal correlations, ensure consistent data across teams and sites, supporting reliable target validation and assay transfer in discovery programs.
What statistical analysis capabilities are required before implementation?
Functional connectivity analysis requires seed-based correlation mapping and bandwidth filter adjustment in ICO studio software to export correlation matrices for statistical evaluation of network-level hemodynamic changes.
Chapters in this video
0:05
Introduction
1:22
Animal Preparation
3:58
Probe Positioning
4:34
Angiographic Scan and Atlas Registration
5:49
Brain Positioning System (BPS)
7:14
Task-Evoked Experiment: Whisker Stimulation
8:14
4D Functional Connectivity
9:08
Results: Transcranial Activation Maps and rCBV Time Course Following Whiskers Stimulation in an Anesthetized and Awake Behaving Mouse
11:08
Conclusion
