Long term ex vivo culturing of Drosophila brain as a method to live image pupal brains: insights into the cellular mechanisms of neuronal remodeling

Abstract

Holometabolous insects, including Drosophila melanogaster, undergo complete metamorphosis that includes a pupal stage. During metamorphosis, the Drosophila nervous system undergoes massive remodeling and growth, that include cell death and large-scale axon and synapse elimination as well as neurogenesis, developmental axon regrowth, and formation of new connections. Neuronal remodeling is an essential step in the development of vertebrate and invertebrate nervous systems. Research on the stereotypic remodeling of Drosophila mushroom body (MB) γ neurons has contributed to our knowledge of the molecular mechanisms of remodeling but our knowledge of the cellular mechanisms remain poorly understood. A major hurdle in understanding various dynamic processes that occur during metamorphosis is the lack of time-lapse resolution. The pupal case and opaque fat bodies that enwrap the central nervous system (CNS) make live-imaging of the central brain in-vivo impossible. We have established an ex vivo long-term brain culture system that supports the development and neuronal remodeling of pupal brains. By optimizing culture conditions and dissection protocols, we have observed development in culture at kinetics similar to what occurs in vivo. Using this new method, we have obtained the first time-lapse sequence of MB γ neurons undergoing remodeling in up to a single cell resolution. We found that axon pruning is initiated by blebbing, followed by one-two nicks that seem to initiate a more widely spread axon fragmentation. As such, we have set up some of the tools and methodologies needed for further exploration of the cellular mechanisms of neuronal remodeling, not limited to the MB. The long-term ex vivo brain culture system that we report here could be used to study dynamic aspects of neurodevelopment of any Drosophila neuron.

Filopodial dynamics and growth cone stabilization in Drosophila visual circuit development

“…Our key goal was to follow subcellular dynamics at the resolution limit of conventional light microscopy with fast enough time-lapse to quantitatively describe subcellular dynamic properties in developing brains over many hr. Important advances in Drosophila ex vivo brain imaging have recently established high-resolution imaging in short developmental time windows ( Medioni et al, 2015 ; Zschätzsch, et al, 2014) and over long periods at low resolution and with slow time lapse ( Rabinovich et al, 2015 ). We identify phototoxicity and drift as key problems to obtain high spatial and temporal resolution 3D dynamics data over long developmental time periods, for which the imaging system presented here provides a successful approach.…”

“…However, intravital imaging has reduced resolution in deeper brain regions and is limited to early pupal stages. Previous imaging in cultured brains established high-resolution imaging in short developmental time windows ( Medioni et al, 2015 ; Zschätzsch, et al, 2014) and over long periods at low resolution and with slow time lapse ( Rabinovich et al, 2015 ), thus preventing in depth analysis of the role of filopodial dynamics during an entire neural circuit assembly process. Here we present the development of an ex vivo imaging method for Drosophila eye-brain development using 2-photon microscopy that allows widely applicable continuous, fast, high-resolution 4D live imaging anywhere in the fly brain throughout pupal development.…”

CytoCensus: mapping cell identity and division in tissues and organs using machine learning

“…Elucidating the molecular basis of such developmental processes is not only essential for understanding basic neuroscience, but is also important for discovering new treatments for neurological diseases and cancer. Modern imaging approaches have proven indispensable in studying development in intact zebrafish (Danio rario) and Drosophila tissues (Barbosa & Ninkovic, 2016;Dray et al, 2015;Medioni et al, 2015;Rabinovich et al, 2015;Cabernard & Doe., 2013;Graeden & Sive, 2009). Tissue imaging approaches have also been combined with functional genetic screens, for example to discover neuroblast (NB) behaviour underlying defects in brain size or tumour formation (Berger et al, 2012;Homem & Knoblich, 2012;Neumüller et al, 2011).…”

Ataxin-7 and Non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization

“…To determine whether Non-stop regulates SCAR entry into a proteasomal degradation pathway 3 rd instar larval brains were cultured ex vivo to permit pharmacological inhibition of the proteasome in defined genetic backgrounds (83,84). Wandering 3 rd instar larval brains were isolated from non-stop homozygous mutant larvae and cultured for 24 hours in the presence of MG132 proteasome inhibitor.…”

“…Ex vivo culturing of larval brains was performed as described (83,84). Third instar brains were dissected in 1xPBS and quickly placed into culture media (Schneider's insect media supplemented with 10% FBS, 1% pen/strp, 1:10,000 insulin, and 2 µg/ml ecdysone) in 24 well dish.…”