Background

Bird species offer intriguing prospects for neuroscience research due to their sophisticated cognitive abilities and specialized behaviors [1–6]. Despite differences in neuroarchitecture and neuronal densities in avian brains when compared with mammals [4,7], birds demonstrate remarkable and distinctive behavioral performances, including long-distance navigation [8], imprinting, homing, food-caching, and song-learning [9,10], to name a few. Consequently, these provide unique opportunities for comparative studies aiming to elucidate the cellular mechanisms underlying these capabilities [6]. Indeed, these realizations are reflected by the growing interest in avian neuroscience [1,3,9,11–18]. Among the different avian species explored, our focus centers on the domestic Japanese quail (Coturnix japonica). The quail offers several advantages over other birds due to its small size, rapid sexual maturation, and frequent egg-laying; advantages that make this species highly suitable for routine experimentations [17]. Importantly, the ground-dwelling nature of quails makes them ideal for studying birds’ spatial navigation by reducing dimensionalities (from 3D to 2D) [19]. In spite of these advantages and the extensive use of quails in the field of developmental biology [17,20], this species remains relatively underexplored in neuroscience [5,19]. This is partly attributed to the absence of methods and tools to manipulate and examine quail neurons [21], in particular, the lack of efficient viral vectors for neuronal transduction [5,17]. To screen for suitable viral vectors, we choose not to pursue in vivo exploration, as is common practice, due to the considerably large number of animals and time this task necessitates (to overcome animal-to-animal variability, variable injection performances, and other technical limitations). Instead, we envisioned that primary brain cultures would present a more rapid and cost-effective approach for examining multiple viruses in parallel [22].

Primary brain cell cultures constitute a valuable tool in neuroscience [23,24]. Brain cultures faithfully preserve many complex cellular behaviors, electrophysiological properties, and phenotypic traits, as observed in vivo, despite the loss of the precise network architecture and organization in the tissue, as is exclusively found in vivo [23-28]. Nevertheless, the alteration in tissue architecture, which includes the removal of the blood-brain barrier (BBB) and extracellular matrix, proves to be advantageous as it enhances cellular accessibility and reduces time to expression. Indeed, cultures are ideal for high-throughput screening of pharmacology, which should likely translate toward the screening of different viruses [22].

We have initially attempted to produce primary cultures based on standard protocols for culturing cells from the brains of neonatal rodents (such as rats and mice, e.g., [29]) or chicken embryos (e.g., [30,31]). However, these attempts were unsuccessful. Consequently, we decided to optimize the procedure by experimenting with embryos collected from different days in ovo (DIO), modifying the culturing media, and assessing cell growth directly on plastic dishes instead of glass coverslips. After successfully establishing suitable conditions for maintaining viable cultures for several weeks (up to 30 days), we leveraged them for screening several commonly employed viruses in neuroscience research, including adeno-associated viruses (AAV), lentiviruses, and baculovirus. We further harnessed the cultures for the rational engineering of a novel AAV variant, specifically tailored for Japanese quail neurons (denoted AAV1*) [22]. Lastly, we characterized the diverse cellular populations in the cultures through use of electrophysiology, calcium imaging, and single-cell mRNA sequencing [22].