Development and Characterization of Primary Brain Cultures from Japanese Quail Embryos.

Cell cultures play a crucial role in neuroscience research, facilitating the elucidation of the complexities of cellular physiology and pathology. The relative simplicity in producing cultures and the accessibility to cells that the cultures provide, in contrast to in vivo settings, allow users to manipulate and monitor cells more easily at higher throughputs and lower costs. These are ideal for screening purposes and electrophysiological characterizations. Despite the prevalence of methodologies for producing brain cultures from various animal models, rodents in particular, approaches for culturing neurons (and glia) from birds are less established or completely absent as in the case of the Japanese quail model. Here, we present a unique culturing protocol for brain cells (e.g., neurons at different maturation levels, such as progenitor cells, excitatory and inhibitory neurons, microglia, and endothelial cells) from entire forebrains of Japanese quail embryos for high-throughput screening of viral vectors in vitro and other various purposes. Following dissection and digestion methods uniquely suited for avian brains, we tailored the growth media and culturing surface to allow the survival of quail brain cultures for more than three weeks in vitro. Key features • We introduce a detailed protocol for producing primary brain cultures from quail embryos' forebrains for up to 30 days. • We show that the cultures support in vitro viral transfections effectively. • We demonstrate the use of the cultures for rapid (days) screening for suitable viruses for quail brain cells, electrophysiological characterizations, and single mRNA sequencing.

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.