Approaches to studying the brain functions of awake behaving mice
Recording neuronal activity in awake behaving mice is indispensable for studying sensory processing, cognition, or decision making. During an experiment, an optical or electrical probe must remain stable relative to the targeted brain region. This task is not trivial, taking into consideration that an awake mouse wants to move and explore its environment.
To achieve stability, neuroscientists can do one of two things. They can mount a miniaturized probe on the head of a freely moving mouse (“head-mounted” approach). Alternatively, they can immobilize the animal’s head in a stationary frame, thus securing stable access with full-size probes and advanced optical equipment, such as a two-photon microscope (“head-fixed” or “head-restrained” approach).
Head-mounted approaches to studying the rodent brain
Electrophysiologists have used the head-mounted solutions since the 1970s. More recently, miniaturized optical microscopes were developed (Ghosh et al./Schnitzer, 2011). Such microscopes have since become widely available both as a commercial product (nVista and nVoke from Inscopix Inc, www.inscopix.com, and Quartet from Neurescence Inc., www.neurescence.com) and as do-it-yourself (DIY) solutions (UCLA’s Miniscope, www.miniscope.org; University of Toronto’s CHEndoscope).
In the head-fixed camp, the research has progressed from anesthetized or fully-restrained mice to head-fixed awake and behaving mice. To minimize stress caused by immobility, researchers place awake head-fixed mice on a linear or circular treadmill, on an air-lifted ball, or in a floating flat-floored cage (Dombeck et al./Tank 2007, Royer et al./Buzsaki 2012, Kislin et al./Khiroug 2014). Solutions that combine head-fixation with body movement are available both as DIY (Dombeck and Tank, 2017; Nashaat et al./Larkum, 2016; Voigts et.al./Harnett, 2018) and as commercial products (e.g., treadmill from Luigs&Neumann GmbH www.luigs-neumann.com, JetBall from PhenoSys GmbH www.phenosys.com, Gramophone from Femtonics www.femtonics.eu, and Mobile HomeCage from Neurotar Oy Ltd www.neurotar.com).
Pros and cons of different approaches
Both head-mounted and head-fixed preparations have their advantages, but also limitations. Mice carrying a head-mounted device can move in a three-dimensional environment. However, their movement is constrained by the weight and length of the attached cables. Moreover, the performance of a head-mounted microscope is greatly compromised by miniaturization. This approach works best for studying neuronal networks (with relatively low resolution) within a limited field of view of a pre-implanted endoscopic probe.
In head-fixed mice, researchers can study brain activity at a cellular or subcellular level using high-resolution multi-color microscopy methods or single-cell electrophysiology. However, head-fixed mice move in only one or two dimensions, their natural head movements are restricted, and vestibular inputs are compromised (Thurley and Ayaz, 2016). A popular combination of an air-lifted Styrofoam ball with virtual reality (VR) allows tracking the animal’s movement, creating and controlling very complex virtual environments. Unfortunately, the sensory input is missing, VR systems tend to be expensive, and the ball is not easily compatible with most two-photon microscopes.
Compared to the ball, the floating flat-floored cage offers an environment rich in natural somatosensory stimuli and more efficient in reducing the stress experienced by animals under head-fixation conditions. The only commercial solution, the Mobile HomeCage, was originally developed for in vivo two-photon microscopy in awake mice. Nowadays, it is also used for electrophysiological recording (including patch-clamp), functional ultrasound (fUS) imaging, wide-field imaging, and optical coherent tomography (OCT). Besides that, the system is compact and suitable for the brain as well as the spinal cord imaging/recording. The recent addition of locomotion tracking capabilities allows integrating imaging/recording with behavioral monitoring.
Head-mounted and head-fixed approaches are not mutually exclusive
An increasing number of labs are starting to combine them to benefit from the strengths inherent in each approach. Recognizing this trend, Inscopix has recently joined forces with several multiphoton microscope manufacturers in launching a new Multimodal Image Registration and Analysis (MIRA) platform, which allows integrating one-photon and multiphoton imaging.
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