Small animal brain surgery with neither a brain atlas nor a stereotaxic frame

Stereotaxic surgery is a cornerstone in brain research for the precise positioning of electrodes and probes, but its application is limited to species with available brain atlases and tailored stereotaxic frames. Addressing this limitation, we introduce an alternative technique for small animal brain surgery that requires neither an aligned brain atlas nor a standard stereotaxic frame. This method requires a high-contrast MRI scan of a specimen and access to a microCT scanner. The process involves attaching miniature markers to the skull, followed by CT scanning of the head. Subsequently, MRI and CT images are co-registered using standard image processing software and the targets for recordings in the brain are marked in the MRI image. During surgery, the animals head is stabilized in any convenient orientation, and the probes 3D position and angle are tracked using a multi-camera system. We have developed a software that utilizes the on-skull markers as fiducial points to align the CT/MRI 3D model with the surgical positioning system, and in turn instructs the surgeon how to move the probe to reach the targets within the brain. Our technique allows the execution of insertion tracks connecting two points in the brain. We successfully applied this method for neuropixels probe positioning in owls, quails, and mice, demonstrating its versatility and its potential to open new avenues for research in non-standard and novel animal models.