This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.
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Method Article
Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila
In This Article
Summary
Abstract
Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.
Introduction
Dendrites, which are the branched projections of a neuron, cover the field that encompasses the neuron's sensory and synaptic inputs from other neurons1,2. Dendrites are an important component of synapse formation and play a critical role in integrating synaptic inputs, as well as propagating the electrochemical stimulation in a neuron. Dendritic arborization (da) is a process by which neurons form new dendritic trees and branches to create new synapses. The development and morphology of da, such as branch density and grouping patterns, result from multi-step biological processes and are highly correlated ....
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Protocol
1. Experimental Preparation
- Prepare the following reagents: Dulbecco's phosphate buffered saline (PBS); Triton X-100; 0.2% PBST (PBS + 0.2% Triton X-100); 32% paraformaldehyde (PFA), diluted into 4% before use; silicone elastomer base and curing agent; antifade mounting medium (e.g., ProLong Gold); and fingernail polish.
- Prepare the following equipment: dissection microscope, two sharp forceps and a pair of scissors for microdissection, a number of pins for microdissection, a Petri dish for making the dissection dish, microscope slides and coverslips, a confocal microscope, and a computer with Fiji ImageJ software installed.
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Results
The dendrites of da neurons were labeled by co-overexpressing GFP (UAS-GFP; ppk-GAL4) in the da neural soma and dendritic arbors for GFP fluorescence imaging analysis. The morphology of da neuron dendrites was imaged by an inverted confocal microscope (Figure 2).
The dendrites of da neurons were traced using Fiji ImageJ software. The file was used to estimate the dendrite length (
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Discussion
Dendrites that innervate the epidermis are the input regions of neurons, and their morphologies determine how information is received and processed by individual neurons. Development dendrite morphology reflects gene modulation of dendrite organization. The Drosophila larval da neuron of the peripheral nervous system is an important model for studying dendrite development because of: 1) the functional similarity with mammals11,12; 2) four class distincti.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank William A. Eimer for imaging technical assistance. This work was supported by the Cure Alzheimer's Fund [to R.E.T], the National Institute of Health [R01AG014713 and R01MH60009 to R.E.T; R03AR063271 and R15EB019704 to A.L.], and the National Science Foundation [NSF1455613 to A.L.].
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Phosphate buffered saline(PBS) | Gibco Life Sciences | 10010-023 | |
| TritonX-100 | Fisher Scientific | 9002-93-1 | |
| Paraformaldehyde(PFA) | Electron Microscopy Sciences | 15714-S | |
| Sylgard 184 silicone elastomer base and curing agent | Dow Corning Corportation | 3097366-0516;3097358-1004 | |
| ProLong Gold Antifade Mountant | Thermo Fisher Scientific | P36931 | |
| Fingernail polish | CVS | 72180 | |
| Stereo microscope | Nikon | SMZ800 | |
| Confocal microscope | Nikon | Eclipse Ti-E | |
| Petri dish | Falcon | 353001 | |
| Forceps | Dumont | 11255-20 | |
| Scissors | Roboz Surgical Instrument Co | RS-5611 | |
| Insect Pins | Roboz Surgical Instrument Co | RS-6082-25 | |
| Microscope slides and cover slips | Fisher Scientific | 15-188-52 |
References
- Wassle, H., Boycott, B. B. Functional architecture of the mammalian retina. Physiol Rev. 71 (2), 447-480 (1991).
- MacNeil, M. A., Masland, R. H. Extreme diversity among amacrine cells: implications for function. Neuron. 20 (5), 971-982 (1998).
- Losonczy, A., Makara, J. K.,....
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