microprism insertion for two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

The advent of in vivo two-photon fluorescent imaging1,2, combining the new technologies in optical systems and genetically modified fluorescence indicators, has emerged as a powerful technique in neuroscience to investigate the intricate structure, function, and plasticity in the living brain3,4. In particular, this imaging modality offers an unparalleled advantage over traditional electrophysiology by capturing both the morphology and dynamic activities of neurons, thereby facilitating long-term tracking of identified neurons5,6,7,8.
Despite its noteworthy strengths, the application of high-resolution fluorescence imaging often necessitates a static, head-fixed setup that constrains the animal&#39;s mobility9,10,11. Additionally, the use of a transparent glass surface for visualizing neurons restricts observations to one or more horizontal planes, limiting the exploration of the dynamics of vertical processes that extend across different cortical depths12.
Addressing these limitations, the present study outlines an innovative surgical procedure that integrates head plate fixation, microprism, and miniscope to create an imaging modality with multilayer and multimodal capabilities. The microprism allows the observation of the vertical processing along the cortical column13,14,15,16, which is critical in understanding how information is processed and transformed as it moves through different layers of the cortex and how the vertical processing is altered during plastic changes. Moreover, it permits imaging of the same neural populations in a head-fixed paradigm and in a freely moving setting, encompassing the versatile experimental settings17,18,19: for example, head-fixation is often required for well-controlled paradigms like sensory perception assessment and stable recordings under 2-photon paradigm, while freely moving offers a more natural, flexible environment for behavioral studies. Therefore, the ability to conduct a direct comparison in both modes is crucial to furthering our understanding of the microcircuits that enable flexible, functional responses.
In essence, the integration of head plate fixation, microprism, and miniscope in fluorescence imaging offers a promising platform for probing the intricacies of the brain&#39;s structure and functionality. Researchers can sample identified cell populations across various depths spanning all cortical layers, directly compare their responses in both well-controlled and natural paradigms, and monitor their long-term alterations over months20. This approach offers valuable insight into how these neural populations interact and change over time under different experimental conditions, providing a window into the dynamic nature of neural circuits.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

System neuroscience benefits greatly from advancement in imaging technology, especially combined with molecular and genetic approaches. Researchers are increasingly using high resolution, chronic recording, and cell type-specific targeting to dissect the brain's function at finer scale, longer time, and more position. One notable shift in experimental paradigms is moving away from the anesthetized recording towards awake or freely-moving states.This approach allows the characterization of brain activity in natural conditions, but also imposes challenges for precise control on stimulation and measurements. With this protocol, we can image the same neural populations in head-fixed and freely-moving animals. We can directly compare their activities in both well-controlled and natural paradigms.Our protocol enables the study of sensory information processing across cortical layers. It allows us to compare neural responses to controlled sensory stimuli in head-fixed states and its behavioral tasks during free movement, thus facilitating our understanding of sensory processing dynamics.

Microprism Insertion for Two-Photon Imaging in a Head-Fixed Position and One-Photon Imaging in a Freely Moving Paradigm

microprism-insertion-for-two-photon-imaging-head-fixed-position-one

Research

JoVE Journal

Neuroscience

154 Views.  University College London. Microprism Insertion for Two-Photon Imaging in a Head-Fixed Position and One-Photon Imaging in a Freely Moving Paradigm

Video: Microprism Insertion for Two-Photon Imaging in a Head-Fixed Position and One-Photon Imaging in a Freely Moving Paradigm

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...