Sleepers, awake!

An overview of approaches for studying the brain functions of non-anesthetized and behaving mice.

This is a must read white paper for the scientists contemplating a transition form in vitro to in vivo preparations, trying to understand the pros and cons of different approaches and to identify commercial and DIY solutions.  

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 is not trivial taking into consideration that an awake mouse wants to move and explore its environment.

To achieve the 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” approach).

Alternative solutions for research in awake mice

The head-mounted approach has been used by electrophysiologists 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, 2014; Nashaat et al./Larkum, 2016) and as commercial products (e.g., Treadmill from Luigs&Neumann GmbH www.luigs-neumann.com, JetBall from PhenoSys GmbH www.phenosys.com, and Mobile HomeCage from Neurotar Oy Ltd www.neurotar.com).

Both head-mounted and head-fixed preparations have their advantages, but also limitations. Mice carrying a head-mounted device can move freely in a three-dimensional environment; however, their movement is limited by the weight and length of the attached cables, and the performance of a head-mounted microscope is greatly compromised by miniaturization. This approach is best suited for studying neuronal networks (with relatively low resolution) within a limited field of view of a pre-implanted endoscopic probe.

In head-fixed mice, brain activity can be studied at a cellular or subcellular level with 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 a virtual reality allows tracking the animal’s movement, creating and controlling very complex virtual environments. However, the sensory input is missing, virtual reality systems tend to be expensive, and the ball is not easily compatible with most commercial two-photon microscopes. Compared to the ball, the floating flat-floored cage offers a more natural 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 optimized for in vivo microscopy. It is compact and was recently enhanced with motion tracking capabilities.

Head-mounted and head-fixed approaches are not mutually exclusive. In fact, increasing number of labs are starting to combine them to benefit from the strength inherent in each individual approach.

Contact us using a form below for a free consultation on how to combine various approaches or selecting the one that best suits your experimental needs. Please also tell us if we have missed something in this article.

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Selected literature:

Dombeck DA, Khabbaz AN, Collman F, Adelman TL, Tank DW. 2007 Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron. 56(1):43-57.

Dombeck D, Tank D. 2017 Two-photon imaging of neural activity in awake mobile mice. Cold Spring Harb Protoc. 2014(7):726-36

Ghosh KK, Burns LD, Cocker ED, Nimmerjahn A, Ziv Y, Gamal AE, Schnitzer MJ. 2012 Miniaturized integration of a fluorescence microscope. Nat Methods. 8(10):871-8.

Kislin M, Mugantseva E, Molotkov D, Kulesskaya N, Khirug S, Kirilkin I, Pryazhnikov E, Kolikova J, Toptunov D, Yuryev M, Giniatullin R, Voikar V, Rivera C, Rauvala H, Khiroug L. 2014 Flat-floored air-lifted platform: a new method for combining behavior with microscopy or electrophysiology on awake freely moving rodents. J Vis Exp. (88):e51869.

Nashaat MA, Oraby H, Sachdev RN, Winter Y, Larkum ME.  2016 Air-Track: a real-world floating environment for active sensing in head-fixed mice. J Neurophysiol. 116(4):1542-1553.

Royer S, Zemelman BV, Losonczy A, Kim J, Chance F, Magee JC, Buzsáki G. 2012 Control of timing, rate and bursts of hippocampal place cells by dendritic and somatic inhibition. Nat Neurosci. 15(5):769-75

Thurley K. and Ayaz A. 2016 Virtual reality systems for rodents. Current Zoology 1–11 doi: 10.1093/cz/zow070