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Developing 2-photon optical imaging for neural-network studies in medial entorhinal cortex of freely moving mice

Periodic Reporting for period 1 - ANAT-MEC (Developing 2-photon optical imaging for neural-network studies in medial entorhinal cortex of freely moving mice)

Reporting period: 2019-05-01 to 2021-04-30

New generations of portable microscopes for calcium imaging enable observation of simultaneous activity in hundreds-to-thousands of distinguishable neurons while animals perform experimental tasks, allowing researchers to identify the underlying neural dynamics and computational algorithms, goals that would be out of reach if cells were recorded one by one or a few at the time. Among these new miniature microscopy techniques, the miniature two-photon microscopes -2P miniscopes- is of particular interest for applications that require high anatomical resolution and the ability to distinguish between recorded cells based on their genetic expression, somatic location, or axonal projection pattern. Early versions of 2P miniscopes did not catch on, however, because they lacked the capacity to image sensitive calcium indicators such as GCaMP6, because their scanning speed was slow, because the miniscopes were heavy and difficult to carry for rodents, or because optical cables were stiff and inflexible. These constraints motivated the development of a new generation of 2P miniscopes with benchtop-comparable 2P resolution, fast speed, Z-scanning capability, and a large field of view (FOV). However, while these 2P miniscopes represented major advances in imaging quality, their applicability in tasks that require movement is still limited, due to heavyweight, stiff optical cable connections, low cell yield, and high system complexity. As of today, calcium imaging technology is thus not suitable for recording activity at the scale of hundreds to thousands of neurons, and at the high spatial and temporal resolution, during spatially dispersed behavior in freely-moving animals. Therefore, the aim of the ANAT-MEC project is to develop a new generation of 2P miniscope that enables the study of neural activity, at high sampling rates and with high spatial resolution, in thousands of individually identifiable cells in cortices with different architectures and locations, while mice move freely in the environment. The new 2P miniscope developed in this project will open many new doors to the neuroscientists to image large-scale cellular and subcellular structures in actively moving animals and will bring us closer than ever before to understanding the fundamental principles underlying complex behaviors.

The overall objectives include 1) developing a new 2P miniscope that enables the study of neural activity, at high sampling rates and with high spatial resolution, while mice move freely in the environment, 2) demonstrating the feasibility of large-scale imaging through prisms implanted along the elongated dorsoventrally surface of MEC to study the anatomical organization of grid cells, and 3) providing the open-source materials for building this 2P miniscope system so it can be easily duplicated in any biology lab with basic optics and electronic experience.
The main aim of this project is to develop improved optical imaging methods that can be used to explain if and how distinct functional cell types are anatomically organized in MEC and how this organization enables mapping of local space. During this project, the researcher introduced and adapted the miniature two-photon technology to the host lab, further developed the miniature two-photon technology, successfully recorded hundreds of neurons in MEC of freely moving mice, and identified clusters of hundred grid cells, head-direction cells, border cells and object-vector cells in MEC. The new technique developed in this project -MINI2P- can be constructed in biology labs with basic optics and electronic experience. A list of key components such as objectives, μTlens, hollow-core photonic crystal fiber, TFB, with ordering numbers, 3D models, and drawings of the customized components is provided for open access. These documents also contain building instructions and an operation manual. Behavioral recording software, aberration-correction software, and processing code are also provided. The complete data-processing and animal behavior analysis pipeline is depicted, with output in a standardized format that can be shared and analyzed by multiple programming languages.
The new 2P miniscope (MINI2P) developed in this project enables the study of neural activity, at high sampling rates and with high spatial resolution, in thousands of individually identifiable cells in brain regions with different architectures and locations, at the same time as mice move freely in the environment. By retaining the optical sectioning capability afforded by hardware-demanding 2P excitation, MINI2P can be used for high-signal-to-noise calcium imaging, irrespective of somatic packing density, the density of neuropil, or neuronal activity level, in brain regions where imaging is often precluded with current 1P miniscopes. With the present reduction in headpiece weight and the increase in flexibility caused by tapering of the optical cable connection, MINI2P allows for behavioral flexibility similar to that of 1P miniscopes, despite the added 2P hardware. The microscope can be carried on the head of mice uninterruptedly for half an hour or more, while the animals roam freely in an open environment, at speeds, directions, and turning rates comparable to those of untethered animals without miniscopes. The miniscope can be carried for successive recording sessions, without bleaching of imaged neurons or change in the quality of signal or the animal’s behavior. Taken together, these features of MINI2P allow for high-resolution imaging of unprecedented numbers of neurons during spatially dispersed behaviors that have not been accessible to optical imaging until now.

By open-sourcing protocols and software for assembly and use of the new miniscope, we hope that a growing number of investigators will be involved in the further development of large-scale high-resolution imaging in freely behaving rodents.
A mouse with the new miniature two-photon microscope developed in this project on the head