Webinar Q&A Report - InsideScientific

Webinar Q&A Report: Making Optical and Electrophysiological Measurements in the Brain of Head-Fixed, Freely-Moving Rodents

Q: Can you tell more about the animal training protocol for Mobile HomeCage? How long is the habituation process? (L. Khiroug): The animal training protocol consists of 4 days of training with a total of 8 training sessions, each lasting 2 hours. After the animal is ready for experiments. The training protocol can be found from the following open-access JoVE article: Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents Q: Once the animals have the head-apparatus (helicopter?) attached, how long can a subject be studied in the cage (what have the speakers experienced in the lab)? (L. Khiroug): We normally experiment for 2 hours whilst the animal is in the cage. Since the animal can fall asleep in Mobile HomeCage, the experiment can last longer as long as water and food is provided for the animal. Q: Chronic cranial window preparation, do you have any advice on that or we will need to use whichever method we can find? (L. Khiroug): At Neurotar we use published protocols for cranial window implantation. We can provide you with detailed protocols for implantation surgeries if needed.

Q: Would the mobile home cage be suitable for gerbils or even rats?? (L Khiroug): The current Mobile HomeCage version is designed for mice and other small rodents. Neurotar is developing a larger Mobile HomeCage (MHC-L) which will be suitable for rats. The estimated launch date for MHC-L is in summer 2015.

Q: Can you provide different head fixation mechanisms, e.g. to make it more suitable with a custom two photon microscope? The current system seems too big perhaps? (L. Khiroug): The Mobile HomeCage fits under most commonly used two-photon microscopes and the head-fixation system was designed to be optimally stable in order to support imaging and patch clamp electrophysiological experiments. If you have any questions regarding compatibility with your microscope, please contact Neurotar for more details. Custom parts are also available.

Q: Does the focal plane shift when you take a series of pictures? (L. Khiroug): Z-stacks can be taken during imaging in a stable manner. As the skull plates have a degree of elasticity around the sutures, any movement artefacts will spring back to the original position and do not require correction. Q: How would a researcher deliver sensory stimulation to a subject while in the homecage? (L. Khiroug): You can deliver sensory stimulation by for example attaching pictures, textures and odors to the inner wall or the floor of the carbon cage.

Q: What is the feasibility of implementing operant behavior paradigms in the mobile home cage? Is it possible to incorporate nose pokes, reward ports, levers, small video screens etc on the floor/walls? One concern is that the weight of these things and their cables would severely limit the mobility of the cage. (L. Khiroug): The vertical head-fixation bar could be used to mount various instruments such as reward ports. The carbon cage can support hundreds of grams of additional weight and therefore for example low weight touch screens with wireless connection could be mounted on the cage wall.

Q: Mobile Home Cage: is there any noise/vibration associated with the cage floating that may affect the behavior? (L. Khiroug): The Mobile HomeCage has been designed to be maximally vibration-free, using aircraftgrade aluminium alloy and carbon fibre. Airflow noise is no higher than quiet office background noise at around 45dB.

Q: What kind of Objectives do you recommend for the cranial window when using two-photon imaging? What is the max imaging depth in your experience? (L. Khiroug): We recommend a 25x high NA (e.g., 1.05) water immersion objective. The maximum imaging depth typically achievable is 1mm.

Q: Is there any limitation on which part of the brain can be exposed for imaging, in order to achieve best stability? For example, how anteriorly or laterally can the head plate be glued to image with the brain still stable? (L. Khiroug): The only real limit to imaging position for stability is which part of cortex you can physically access without the animal being in too unnatural a position. For imaging work you can place a window unilaterally over primary sensory cortex (S1), so the mice's heads are at an angle of about 20 degrees. For electrophysiology you can place window over the entire dorsal surface of the parietal bones, from V1 to S1, and teardrop-shaped windows over bregma for access to striatum and M1.

Q: Is there other evidence for the MHC not being very stressful other than sleep? (L. Khiroug): From our experience, we have not observed any overt signs of undue stress in our animals. After the first training session, there is a brief dip in body weight that is a good indicator of stress. This rebounds on the second day. During the trainings process, mouse movement exceeds that observed in a home cage with free access to a wheel. We interpret this as the mouse feeling progressively more comfortable. Combined with lack of tail erection, biting or freezing, we believe that stress levels upon completion of training are very low. We plan to collect more detailed physiological data in future.

Q: For the patch clamping experiment, how is the insertion done vertically? My previous experience had always been that the electrode would need to have an angle in order for the lens to be placed – please elaborate. (L. Khiroug): Patch clamping so far has been done blind, entering vertically or at angles of 15-30 degrees from vertical.

Q: Can you compare and contrast this device versus a virtual reality ball? (L. Khiroug): Compared to the virtual reality ball, the Mobile HomeCage has a much faster habituation time (4 days versus 10+ days), is less stressful for the animal, provides tactile contact with walls for the animal to touch with its whiskers & to smell, which provides a feeling of security. It is also more compact in size, is orders of magnitude quieter, and there is no danger of the mechanism sticking. Mobile HomeCage is not currently optimal for place cell research but Neurotar is working on this by creating a larger version (MHC-L).

If you have additional questions for Neurotar and our guest speakers regarding content from their webinar, or wish to receive additional information about the Mobile HomeCage please contact them by email:

Leo Khiroug, PhD – Neurotar Oy [email protected] Eero Castren, MD, PhD – University of Helsinki [email protected] Petri Hyytia, PhD – University of Helsinki [email protected] Roustem Khazipov, MD, PhD – U901 INSERM [email protected] Marat Minlebaev, MD, PhD – U901 INSERM [email protected]

Neurotar Ltd Viikinkaari 4 00790 Helsinki, Finland Tel: +358 9 428 344 56 [email protected]

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