This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.
The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.
The Drosophila visual system, comprised of the compound eye and underlying optic lobe, has been an excellent model for the study of neural circuit development and function. In recent years, the optic lobe in particular has emerged as a powerful system in which to study neurodevelopmental processes such as neurogenesis and circuit wiring1,2,3,4,5,6,7,8. It is made up of four neuropils: the lamina, medulla, lobula and lobula plate (the latter two comprise the lobula complex)1,2,3,4,5,6. Photoreceptors from the eye, target neurons of the lamina and medulla, which process visual inputs and relay them to the neuropils of the lobula complex1,2,3,4,5,6. Projection neurons in the lobula complex subsequently send visual information to the higher order processing centers in the central brain1,5,9. The complex organization of the optic lobe, necessitated by a need to maintain retinotopy and to process different types of visual stimuli, makes it an attractive system for studying how sophisticated neural circuits are assembled. Notably, the medulla shares striking similarities in both its organization and development with the neuroretina, which has long been a model for vertebrate neural circuit development3,8.
Optic lobe development begins during embryogenesis, with the specification of ~35 ectodermal cells that form the optic placode2,4,5,6,7,8. After larval hatching, the optic placode is subdivided into two distinct primordia: 1) the outer proliferation center (OPC), which generates the neurons of the lamina and outer medulla and 2) the inner proliferation center (IPC), which generates neurons of the inner medulla and lobula complex4,5,6,10. In the late second-instar larva, the neuroepithelial cells of the OPC and IPC begin to transform into neuroblasts that subsequently generate neurons via intermediate ganglion mother cells4,5,11,12. Optic lobe neuroblasts are patterned by spatially and temporally-restricted transcription factors, which act together to generate neural diversity in their progeny11,12,13,14. In the pupa, the circuits of the optic lobe neuropils are assembled via the coordination of several processes, including programmed cell death11,15, neuronal migration12,16, axonal/dendritic targeting10,17, synapse formation18,19 and neuropil rotations10,17.
Here, we describe the methodology by which larval and adult brains are dissected, immunostained and mounted for imaging the optic lobe. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Thus, we put special emphasis on how the orientation of mounting relates to the spatial organization of the optic lobe structures. We describe three mounting strategies for larval brains (anterior, posterior and lateral) and three for adult brains (anterior, posterior and horizontal), each of which provide an optimal angle for imaging a specific optic lobe progenitor population or neuropil.
In this protocol, we describe a method to immunostain larval and adult Drosophila brains and mount them in several orientations. While methods to stain larval and adult brains have been previously described22,23,24,27,28, mounting strategies for the optimal visualization of specific optic lobe structures have received less attention2…
The authors have nothing to disclose.
We would like to thank Claude Desplan for sharing with us an aliquot of the Bsh antibody. The DE-Cadherin, Dachshund, Eyes Absent, Seven-up and Bruchpilot monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was supported by an NSERC Discovery Grant awarded to T.E.. U.A. is supported by an NSERC Alexander Graham Bell Canada Graduate Scholarship. P.V. is supported by an Ontario Graduate Scholarship.
10x PBS | Bioshop | PBS405 | |
37% formaldehyde | Bioshop | FOR201 | |
Alexa Fluor 488 (goat) secondary | Invitrogen | A-11055 | use at 1:501 |
Alexa Fluor 555 (mouse) secondary | Invitrogen | A-31570 | use at 1:500 |
Alexa Fluor 647 (guinea pig) secondary | Invitrogen | A-21450 | use at 1:503 |
Alexa Fluor 647 (rat) secondary | Invitrogen | A-21247 | use at 1:502 |
Cover slips | VWR | 48366-067 | |
Dissecting forceps – #5 | Dumont | 11251-10 | |
Dissecting forceps – #55 | Dumont | 11295-51 | |
Dissection Dish | Corning | 722085 | |
Dry wipes | Kimbery Clark | 34155 | |
Goat anti-Bgal primary antibody | Biogenesis | use at 1:1000 | |
Guinea pig anti-Bsh primary antibody | Gift from Claude Desplan | use at 1:500 | |
Guinea pig anti-Vsx1 primary antibody | Erclik et al. 2008 | use at 1:1000 | |
Laboratory film | Parfilm | PM-996 | |
Microcentrifuge tubes | Sarstedt | 72.706.600 | |
Microscope slides | VWR | CA4823-180 | |
Mouse anti-dac primary antibody | Developmental Studies Hybridoma Bank (DSHB) | mabdac2-3 | use at 1:20 |
Mouse anti-eya primary antibody | DSHB | eya10H6 | use at 1:20 |
Mouse anti-nc82 primary antibody | DSHB | nc82 | use at 1:50 |
Mouse anti-svp primary antibody | DSHB | Seven-up 2D3 | use at 1:100 |
Polymer Clay | Any type of clay can be used | ||
Rabbit anti-GFP | Invitrogen | A-11122 | use at 1:1000 |
Rat anti-DE-Cadherin primary antibody | DSHB | DCAD2 | use at 1:20 |
Slowfade mounting medium | Invitrogen | S36967 | Vectashield mounting medium ( cat# H-1000) can also be used |
Triton-x-100 | Bioshop | TRX506 |