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Allen Brain Observatory Neuropixels recording rig, from the paper in Nature Protocols. The label “f” indicates one of the six three-axis New Scale manipulators in the rig.

As the creator and supplier of the Multi-Probe Manipulator System (MPM), we often are asked to recommend support equipment and procedures to optimize the process of inserting multiple silicon probes in awake, head-fixed mice.

A team at the Allen Institute recently published (Nature Protocols) the step-by-step process they use to insert six probes into the visual cortex of a mouse. We developed the MPM system and the Virtual Coordinate System (VCS) software in collaboration with this Allen Institute team, particularly under the mentorship of Josh Siegle. Since they have successfully performed experiments with hundreds of mice, we think their protocol paper likely contains the answers to many of the questions we receive.

In case you missed it, the paper describing the Allen Institute multi-probe recording protocol, Acute head-fixed recordings in awake mice with multiple Neuropixels probes,  can be accessed at Nature.com.

The Allen Brain Observatory rig

It should be noted that these procedures and equipment have been optimized for a dense concentration of probes in the visual cortical region.  Therefore, some procedures and some support equipment are not universally applicable.  For example: Intrinsic Signal Imaging (ISI) can be used only in cortical regions, since light scattering prohibits its use in deeper brain regions.

The Allen Institute rig is also evolved from the MPM Multi-Probe Micromanipulator System, which is suitable for work in all parts of the brain.  The Allen Institute team tailored the design for studying the visual cortex. As they explain in the paper:

On the Allen Institute rigs, we use the most compact commercially available three-axis stages (New Scale Technologies) and mount each one at a fixed angle relative to the brain. This minimizes the space requirements but limits the targeting flexibility.

Working in Stereotactic Coordinate Space

The New Scale Pathfinder software’s Virtual Coordinate System (VCS) is key to insertion of multiple probes. We highlight the Allen Institute team’s VCS perspective here:

When inserting many probes simultaneously, some probes will require different entry angles to avoid collisions between the probes, probe holders and manipulators. This makes targeting stereotaxic coordinates non-trivial, because the manipulator axes are no longer aligned to the atlas axis. Similarly, precise targeting based on visible landmarks becomes a time-consuming trial-and-error process. In both cases, the solution is to precalibrate the three axis manipulators for each probe to a global coordinate space. Some commercial manipulator systems, such as New Scale Technologies’ Multi-Probe Manipulator, include software for performing this alignment based on graduations on the physical hardware.

The Nature Protocols paper includes detailed instructions for calibrating three-axis manipulators to a shared coordinate system. VCS software make this process even easier by automatically displaying probe locations relative to bregma, the most commonly used anatomical landmark.


Using Microscopes or Digital Cameras with the MPM System

We are often asked to recommend a microscope or digital camera, especially when our MPM Platform is being used with inverted manipulators.  Here is the Allen Institute configuration:

 Clearly visualizing the brain during probe insertion is essential. This is most commonly achieved with a stereomicroscope mounted on a flexible arm. In our case, we chose to mount a microscope a fixed distance from the probes and replace the eyepieces with universal serial bus (USB) cameras. This makes it possible to visualize the brain remotely while the subject is in an isolated chamber. In practice, we use only one camera at a time, so the stereomicroscope could be replaced with a more compact, single field of view (FOV) microscope (e.g., InfiniProbe, Infinity PhotoOptical). In either case, a polarizing filter will probably be needed for reducing glare from the silicone oil/insertion windows used during the experiment.

Other MPM customers have had success with a Dinolight digital microscope – basically a camera mounted on an articulating arm attached to the MPM ring to visualize the probe insertion.

 Inserting Multiple Probes Simultaneously

In a recent blog post, we highlighted the advisability of slow insertion, at speeds of 200 – 300 micrometers/minute, to minimize brain damage and optimize the success rate of recordings.  The ability of the MPM System to automatically insert multiple probes simultaneously is a great advantage. Josh Siegle explains:

 We move [six probes] to the brain surface one by one, and then insert them all simultaneously to the final depth. If [this was not possible], our 6-probe experiments would take 3 hours to set up! Instead, it takes 30 minutes to insert the probes, followed by ~2.5 hours of recording.

With these considerations in mind, we think this paper delivers many excellent recommendations that should result in higher productivity and better recordings.