Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

Published: April 28, 2021

doi:

Urfa Arain, Priscilla Valentino, Ishrat Maliha Islam, Ted Erclik

¹Departments of Biology and Cell & Systems Biology,University of Toronto – Mississauga

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Preparing larval brains for confocal imaging Dissections NOTE: Before starting the dissection, prepare the fix (4% formaldehyde in phosphate buffered saline (PBS)) and PBT (0.1-0.3% Triton in PBS) solutions. The fix solution should be placed on ice during the dissection. Although paraformaldehyde (PFA) fixative is used in this protocol, alternative fixing strategies (using PLP20 or PEM21) have been described for specific epitopes. For …

Representative Results

Confocal images of larval and adult optic lobes mounted in the orientations described in the protocol are presented in Figure 1 and Figure 2. Figure 1 shows schematics and representative confocal slices of larval brains positioned in the anterior, posterior and lateral orientations. In the anterior mounting orientation, the OPC epithelium (DE-Cadherin), medulla neuroblasts (deadpan>βgal)…

Discussion

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 attention^2…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Citazione di questo articolo

Arain, U., Valentino, P., Islam, I. M., Erclik, T. Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization. J. Vis. Exp. (170), e61273, doi:10.3791/61273 (2021).