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

BACKGROUND: 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 stereotaxic frame. NEW METHOD: The new method requires an ex-vivo high-contrast MRI brain scan of one specimen and access to a micro-CT 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 brain recordings are marked in the MRI image. During surgery, the animal's head is stabilized in any convenient orientation, and the probe's 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. RESULTS: 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. COMPARISON WITH EXISTING METHODS: We present an alternative to traditional stereotaxic brain surgeries that does not require established stereotaxic tools. Thus, this method is especially of advantage for research in non-standard and novel animal models.

A custom-made AAV1 variant (AAV1-T593K) enables efficient transduction of Japanese quail neurons in vitro and in vivo.

The widespread use of rodents in neuroscience has prompted the development of optimized viral variants for transduction of brain cells, in vivo. However, many of the viruses developed are less efficient in other model organisms, with birds being among the most resistant to transduction by current viral tools. Resultantly, the use of genetically-encoded tools and methods in avian species is markedly lower than in rodents; likely holding the field back. We sought to bridge this gap by developing custom viruses towards the transduction of brain cells of the Japanese quail. We first develop a protocol for culturing primary neurons and glia from quail embryos, followed by characterization of cultures via immunostaining, single cell mRNA sequencing, patch clamp electrophysiology and calcium imaging. We then leveraged the cultures for the rapid screening of various viruses, only to find that all yielded poor to no infection of cells in vitro. However, few infected neurons were obtained by AAV1 and AAV2. Scrutiny of the sequence of the AAV receptor found in quails led us to rationally design a custom-made AAV variant (AAV1-T593K; AAV1*) that exhibits improved transduction efficiencies in vitro and in vivo (14- and five-fold, respectively). Together, we present unique culturing method, transcriptomic profiles of quail's brain cells and a custom-tailored AAV1 for transduction of quail neurons in vitro and in vivo.

Directional tuning in the hippocampal formation of birds.

Birds strongly rely on spatial memory and navigation. Therefore, it is of utmost interest to reveal how space is represented in the avian brain. Here we used tetrodes to record neurons from the hippocampal formation of Japanese quails-a ground-dwelling species-while the quails roamed in an open-field arena. Whereas spatially modulated cells (place cells, grid cells, border cells) were generally not encountered, the firing rate of about 12% of the neurons was unimodally and significantly modulated by the head azimuth-i.e., these were head-direction cells (HD cells). Typically, HD cells were maximally active at one preferred direction and minimally at the opposite null direction, with preferred directions spanning all 360° across the population. The preferred direction was independent of the animal's position and speed and was stable during the recording session. The HD tuning was broader compared to that of HD cells in rodents, and most cells had non-zero baseline firing in all directions. However, similar to findings in rodents, the HD tuning usually rotated with the rotation of a salient visual cue in the arena. Thus, these findings support the existence of an allocentric HD representation in the quail hippocampal formation and provide the first demonstration of HD cells in birds.