| NSF Org: |
CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems |
| Recipient: |
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| Initial Amendment Date: | May 20, 2016 |
| Latest Amendment Date: | August 27, 2021 |
| Award Number: | 1605679 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Rizia Bardhan
rbardhan@nsf.gov (703)292-2390 CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems ENG Directorate for Engineering |
| Start Date: | September 1, 2016 |
| End Date: | December 31, 2021 (Estimated) |
| Total Intended Award Amount: | $314,811.00 |
| Total Awarded Amount to Date: | $314,811.00 |
| Funds Obligated to Date: |
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| History of Investigator: |
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| Recipient Sponsored Research Office: |
100 INSTITUTE RD WORCESTER MA US 01609-2280 (508)831-5000 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
100 Institute Road Worcester MA US 01609-2247 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Engineering of Biomed Systems |
| Primary Program Source: |
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| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.041 |
ABSTRACT
PI: Albrecht, Dirk R.
Proposal #: 1605679
The focus of this 3 year proposal is developing a much needed microfluidic platform for long-term, high resolution optical imaging and recording of stimulated brain activity in living animals. Existing optical systems are limited by 1) requirements of relatively intense excitation light that causes photobleaching and phototoxicity that limits the duration of the experiment and hinders stimulating the neuronal circuit under investigation or 2) by the incompatability of optical systems that can operate at lower intensities with standard microfluidic stimulation methods. The central aim of this proposal is to develop microfluidic devices using optical index-matched materials compatible with a low intensity microscopy method, "selective plane illumination microscopy (SPIM)." Preliminary results show feasibility of hydrogel-based systems to record neural responses in C elegans for hours, as well as compatibility with simultaneous optogenetic (pulsed visible light) neural activation and readout. The proposed system will for the first time enable simultaneous chemical and optical stimulation and perturbation of brain circuits under investigation, with multiple neurons monitored for activity over several hours. The methods developed will impact the broader neuroscience community, in which neural imaging is a critical method for developmental, structural, and functional brain studies. Materials and hardware, including microfluidic systems, will be made accessible to the research and commercial community. Broader impact is also achieved through a comprehensive educational plan including innovative curricula in advanced biomedical imaging focused on neuroscience applications and outreach activities to increase involvement of STEM-underrepresented students and local communities, through exciting, hands-on modules for summer programs and scientific demonstrations.
Sensation, memory, and behaviors are encoded in dynamic electrochemical patterns within neurons of the brain. Recent advances in fast three-3D microscopy have enabled the optical imaging of activity in large numbers of neurons at once, in some cases nearly the entire brain of an organism. Such systems promise to revolutionize the study of neural circuit regulation, compared with sparse single-neuron recordings that do not capture neural dynamics elsewhere in the circuit. However, current confocal and structured illumination systems are limited by their requirements of relatively intense excitation light, causing photobleaching and phototoxicity that limits the duration of an experiment, and by difficulty in stimulating the neuronal circuit under investigation. While light sheet or selective plane illumination microscopy (SPIM) captures more emission light and therefore operates at lower excitation intensity, optical requirements are incompatible with standard microfluidic stimulation methods. Therefore, there exists an urgent need for SPIM-compatible microfluidic stimulation methods to enable long-term, high resolution recording of stimulated brain activity in living animals. The central aim of this proposal is to develop microfluidic devices using optical index-matched materials compatible with SPIM. Preliminary results show feasibility of hydrogel-based systems to record neural responses in C elegans for hours, as well as compatibility with simultaneous optogenetic neural activation and readout. Specific objectives are: 1) development of diSPIM-compatible sample immobilization and microfluidic stimulation, 2) multi-neuronal imaging of sensory-stimulated brain circuits to observe and study sensory feedback, and 3) multi-neuronal imaging of optogenetically-stimulated brain circuits to observe the effect of reversible circuit perturbations on ensemble neural activity in the same animal. Innovations of the propose include: 1) identification of hydrogel encapsulants compatible with diSPIM that immobilize living animals but maintain organism health and function; 2) microfluidic designs, including hydrogel-glass or hydrogel-silicone hybrids, that deliver precise chemical concentrations; 3) identification of sensory neurons detecting novel chemical stimuli; 4) identification of sensory feedback and study of its regulation via candidate genetic mutants; 5) simultaneous optogenetic and chemical stimulation while monitoring multiple sensory and interneurons, to observe neural responses during dynamic circuit perturbation. The end result will be a new configuration of the dual-view inverted (diSPIM) system and protocols suitable for the embedding of cells and small organisms to record high-resolution, isotropic, fluorescent 3-D volumetric images for long time periods during and after chemical and/or optogenetic stimulation. The studies planned will improve the understanding of circuit computation in C. elegans when stimulated with natural sensory stimuli (e.g., chemicals) and by arbitrary optogenetic stimulation within a compact and well-defined neural circuit. The methods developed will impact the broader neuroscience community, in which neural imaging is a critical method for developmental, structural, and functional brain studies. Materials and hardware, including microfluidic systems, will be made accessible to the research and commercial community. Integrated with the proposed research is a comprehensive educational program toward training and educating the next generation of interdisciplinary scientists, particularly biologist-engineers, including: 1) innovative curricula in advanced biomedical imaging with focus on neuroscience applications, 2) mentoring undergraduate and graduate students through research and engineering design projects, and 3) outreach to increase involvement of STEM-underrepresented students and local communities, through exciting, hands-on modules for summer programs and scientific demonstrations.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
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PROJECT OUTCOMES REPORT
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This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
In 2016, Worcester Polytechnic Insititute and PI Albrecht received a CBET award entitled “Long-term brain circuit imaging with chemical and optogenetic stimulation.” This project developed and tested several new methods aimed at improving microscope imaging quality and duration of living, healthy organisms. To study how biological processes change, techniques are needed to observe them over time, typically hours to days, and ideally in the same cells or animals to account for differences across the population. In this project, light-activated hydrogels and polymers with beneficial optical properties were used to safely surround cells and small organisms and hold them in place for hours to days. These methods are compatible with stimulation by chemicals, drugs, and light during microscope imaging for live viewing of dynamic biological responses, and initial applications studied neural circuit response changes by sleep, traumatic injury, drugs, and adaptation.
Light-sheet-compatible sample immobilization and microfluidic stimulation: Advances in microscopy and optogenetics over the last decade enabled direct viewing of brain activity, but initial methods were limited to short experiment times and limited stimulation. In this project, we first developed and optimized methods for mounting live samples in hydrogels, improving both imaging quality and sample health (Burnett et al., Comms Bio 2018). We later expanded to fluoropolymer materials for even better deep imaging quality (Han et al., Lab Chip 2021). In both methods, neuronal activity was demonstrated over 24 hours during microfluidic chemical and optical stimulation, in neurons and whole brains of the nematode C. elegans, and cellular structures in several small organisms. These and other open-access reports shared sample preparation and microscopy protocols.
To validate these methods, we measured how neural responses to sensory stimuli changed over hours to days after sleep, stimulation, drugs, and mechanical injury:
Neural circuit changes during sleep: We first applied these imaging and stimulation methods to study how sensory and interneuron responses change between sleep and wake states, causing a change in arousal threshold. By monitoring each animal over 12 hours, identifying sleep transitions, and stimulating equally in each sleep or wake state, we identified that neural responses to harmful sensation were identical in sensory neurons but different downstream in the neural circuits (Lawler et al., J Neurosci 2021). Further study of the mechanisms by which sensory information is suppressed during sleep are ongoing.
Random variability in neural responses: Changes in neural responsiveness allow an organism to shift attention to the most important sensory information. Randomness in neural signals can be beneficial to test a broader range of outcomes. We identified different odor stimuli that elicit either consistent or variable responses that changed within minutes. Comparing them provides a framework to study how attention is focused at the neural level.
Adaptation and habituation behavior: When repeatedly presented with the same stimulus, there is often a decline in both neural responses (adaptation) and behavioral responses (habituation)-- but not always. Sometimes, neural adaptation does not change beahvior. To understand why, we built and validated a computational model that correctly predicted adaptation and habituation to different stimulus patterns. The model predicted conditions that would produce “paradoxical” behavior, such as avoidance of a normally attractive food cue. Indeed, we observed this unexpected and irrational behavior, which suggests a flexible positive-negative signal balance that may relate to an excitation-inhibition imbalance underlying some neuropsychiatric disorders.
Pharmaceutical modulation of neural responses: We also used hydrogel encapsulation methods to develop a live organism microwell plate screening system for testing the in vivo effects of drugs on neural function. This system could be used to identify new chemical tools to study brain function or lead to more selective drug therapies. In a validation experiment, we screened 1,280 FDA-approved compounds for drugs that affected direct neural activation, discovering several hits and validating an expected class of voltage-gated calcium channel blockers. We published the method and analysis software (Lagoy et al., 2021), and future applications will assess synaptic modulators and screen genes and environments that increase drug effectiveness.
Broader impacts: The methods of this project will benefit future projects in many areas, including our current study of traumatic brain injury (TBI) by sonication or blast waves. Overall, the project resulted in 2 book chapters, 6 peer-reviewed research articles, 1 feature article, 8 conference presentations, and 1 issued patent. It supported 3 PhD dissertations, 4 graduate students, a lab technician, and 7 undergraduates with summer and academic year research stipends.
Summary: This project successfully developed immobilization techniques for long-term 3D microscopy of live organisms, with applications demonstrated to the study of dynamic, responsive changes to neural activity in the brain.
Last Modified: 01/09/2025
Modified by: Dirk R Albrecht
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