Scientists at the Max Planck Florida Neuroscience Institute (MPFI), in collaboration with ZEISS and MetaCell, have developed a powerful new imaging pipeline called Neuroplex. Published in e-lifeThe technology allows the simultaneous monitoring of the activity of up to nine different populations of neurons in freely moving mice, dramatically accelerating the pace of scientific exploration of how the brain controls behavior.
challenge
Neuroscientists have long faced fundamental limitations in linking brain activity to behavior. Miniscopes are small, head-mounted microscopes used to observe neural activity in behaving animals. Although they can capture neural activity, they cannot reliably distinguish between more than two different types of brain cells at once.
To understand the brain, we need to link the activity patterns of specific neurons to behavior. Although it is easy to color-code different neuronal populations using labels, it was not possible to distinguish between two or more of these populations when correlating neural activity and behavior using the miniscope. This has made it difficult to compare activity across multiple cell types and circuits to understand how specific circuits control their behavior. ”
Dr. Mary Phillips, first author
To get around this, the researchers had to test one type of cell at a time, repeating the same behavioral experiment and labeling a different neuron type each time. However, this iterative process was time-consuming and costly. It was also not possible to directly compare different neuron types within the same animal, and differences between individual animals clouded the conclusions. Alternatively, the scientists depicted different neuron types by removing and slicing brain tissue after behavioral experiments, color-coding different neuron types, and imaging the processed brain tissue using a microscope that can distinguish multiple colors. However, matching cells imaged with a miniscope in a living animal to cells in brain tissue processed post-mortem is difficult and results in significant data loss due to low throughput. Additionally, this approach destroyed the ability to track the activity of identified cell types over time to determine how their activity changes during learning, aging, or disease progression.
Solution: Neuroplex
To overcome these challenges, the MPFI team collaborated with collaborators at ZEISS and MetaCell to develop Neuroplex, an imaging pipeline that combines two complementary imaging approaches in the same live animal. Researchers first use a spectrum of different colored fluorescent tags to label up to nine different neural circuits or cell types. They then record neural activity across a labeled population of freely moving, behaving mice using a small lens and a miniscope attached to their heads. Although we were unable to distinguish the fluorescent tags with miniscope imaging, we gently removed the miniscope and placed the mouse under a confocal microscope, which can distinguish many different colors.
In this case, the scientists used a ZEISS LSM 980, a confocal microscope with spectral detection, to distinguish between different color tags. With confocal microscopy, the same neurons visualized with the miniscope are imaged through the same lens, but this time color-coded tags are visualized to identify which neurons belong to which specific type. Finally, images from the miniscope and confocal are aligned to each other using anatomical landmarks and a custom Python-based alignment tool developed by the scientists at MetaCell. As a result, the team was able to map each neuron’s color identity directly to its functional activity recordings.
“As part of MetaCell’s contribution to this project, we helped turn the complex data collected into practical computational workflows that enable imaging, alignment, and analysis with greater accuracy, reproducibility, and reliability. Neuroplex shows how carefully designed computational tools can help researchers understand complex biological imaging data and study multiple neuronal populations at once and over time,” said Zhe, co-author and data scientist at MetaCell. Dr. Dong said.
As a proof of principle, the researchers retrogradely targeted nine brain regions that receive projections from the medial prefrontal cortex, a brain region important for decision-making. This allowed them to use distinct fluorescent markers to distinguish neurons that project from the prefrontal cortex to nine other brain regions. They simultaneously recorded the activity of neurons across all nine circuits as the animals interacted socially by sniffing, approaching, and chasing.
“Neuroplex allows us to directly compare patterns of neural activity across cellular circuits during social behavior, overcoming long-standing challenges in miniscope recording and dramatically increasing the efficiency and reproducibility of data collection,” explains lead author Dr. Ryohei Yasuda.
The researchers found that about 75% of active neurons were assigned to one of nine specific cell types, and that the automatic program they built to assign neurons to specific groups performed with 90% accuracy and few false positives.
“Because Neuroplex runs entirely within a living animal through the same implanted lens, scientists can measure how different populations of neurons change their activity over time. Researchers can identify cell populations prior to behavior and monitor the same neurons over weeks or months, making it possible to study learning, aging, and disease progression over time,” Dr. Phillips explained.
what happens next
The team is already working on further improvements to the technology to improve the accuracy of color code identification. Additionally, we want to make Neuroplex accessible to all laboratories, including those without access to high-end spectral confocal systems. Their goal is to widely disseminate this approach to the neuroscience community using standard filter-based wide-field microscopy, bringing the core benefits of this approach to the entire research community.
“Improved data collection efficiency for cell-type or circuit-specific functional data will accelerate our understanding of the neural computations underlying behavior,” said Phillips. “Beyond basic research, we expect this approach to facilitate the understanding of circuit-specific functional changes in disease models, particularly neurodevelopmental or neurodegenerative disease models, which would benefit from longitudinal studies examining disease progression.”
To popularize this approach, the team has also developed a tutorial for scientists who want to use Neuroplex in their research. In addition, this approach will be presented in a ZEISS webinar on July 14th with lead author Dr. Mary Philips, who will share the technique and resources with the scientific community. Learn more and register here.
sauce:
Max Planck Florida Neuroscience Institute
Reference magazines:
Phillips, M.L.; Others. (2026). Functional imaging of nine different neuronal populations under the miniscope in freely behaving animals. e-life. DOI: 10.7554/elife.110277. https://elifesciences.org/articles/110277
