1.

Introduction

As a model system in neurobiological studies, Drosophila has the advantage of utilizing simple brain circuits for diverse behaviors. Moreover, with the vast array of genomics, proteomics, powerful genetic tools, and large collection of available mutants, Drosophila has become an immensely popular experimental animal model.¹ Thus, understanding the brain-wide neuroanatomical architecture of Drosophila is essential for formulating hypotheses of neural information flow and deciphering neural mechanisms of brain function and dysfunction.²

Currently, two techniques are commonly used for neuroanatomy of the Drosophila brain: (1) staining with metal impregnation by Golgi methods and (2) labeling neurons with fluorescent reporters by genetic methods.³ The Golgi method randomly stains a small population of neurons; early studies have provided an indispensable first step in visualizing the cellular composition of the fly brain. However, using this method, our ability to bridge cell morphology to functional properties is hampered due to its lack of specificity and reproducibility. In contrast, using genetic approaches to target genetic reporters, such as fluorescent protein, tells us the distribution and morphology of specific types of neurons.

Two alternatives exist for detecting expressions of the reporter genes: immunofluorescence (IF) and immunoenzyme staining. Although widely used, IF still suffers from drawbacks, including photobleaching, photofading, and autofluorescence. In contrast, the immunoenzyme staining technique provides colored (absorption-based, opposite to fluorescence), permanent precipitates. Through immunoenzyme staining based on horseradish peroxidase/diaminobenzidine (HRP/DAB), Kimura et al. have observed sexual dimorphism of neurons for investigating the function of the fruitless gene.⁴ However, DAB penetrates poorly in thick tissue specimens, giving inconsistent results in deeper-lying cells.^5,6 Meanwhile, the colored reaction product of HRP/DAB cannot be axially resolved by bright-field microscopy and, subsequently, this leads to the lack of three-dimensional (3-D) information to distinguish adjacent neurons or neurites. These two issues restrict the application of HRP/DAB staining to the detection of neurons located in the surface layer.

Here, we set out to develop an immunoenzyme staining method to visualize the 3-D structure of the entire Drosophila neural network. In order to accomplish this, we modified the immunoenzymatic neuron labeling technique to attain a uniform staining effect over the whole fly brain. By means of the micro-optical sectioning tomography (MOST) system,^7,8 which combines histological ultrathin sectioning and bright-field line-scan imaging, we accomplished invariable axial resolution and unlimited imaging depth. Thus, by combining the improved HRP/DAB staining methodology and the MOST technique, we have developed a fast, automatic, and high spatial resolution method to image specific neurons in the Drosophila brain.

2.

Materials and Methods

3.

Results

To attain uniform staining through the whole fly brain, we modified the immunoenzyme staining protocol. We found that incubation conditions of the HRP-conjugated secondary antibody and the DAB solution were the most important factors determining staining penetration. The maximal projection of a stack of coronal sections (thickness: $35\mu \mathrm{m}$) in the center part of the fly brain was used to assess stain penetration (Fig. 1). As shown in Fig. 1(a), the shorter time the brains were incubated in the secondary antibody and the DAB solution, the less the brain was stained. Prolonging the incubation time of either step promoted stain penetration; however, the signal in the center of the brains remained weak [Figs. 1(b) and 1(c)]. We found that the best condition to obtain uniform brain-wide staining was to incubate for 24 h in secondary antibodies and 30 min in DAB solution [Fig. 1(d)]. In addition, for smoothing the high background caused by prolonging the reaction time, a 10-min immersion in 0.3% ${\mathrm{H}}_2{\mathrm{O}}_2$ was used to quench endogenous peroxidase.

Fig. 1

Projection images ($\text{thickness}=35\mu \mathrm{m}$) of fly brains stained under different immunoenzyme staining conditions. The incubation conditions of the second antibody were 4 h at room temperature (a), 12 h at 4°C (b), and 24 h at 4°C [(c) and (d)], respectively. The developing times in the diaminobenzidine (DAB) solution were 15 min [(a) and (c)] and 30 min [(b) and (d)]. $\text{Scale bar}=50\mu \mathrm{m}$.

To verify the invariable axial resolution and unlimited imaging depth of the MOST system, we compared immunoenzyme images from the MOST system [Figs. 2(a) to 2(c)] and IF images from the confocal microscope [Figs. 2(d) to 2(f)]. To distinguish between methods, a local brain region at the same location was zoomed-in. As can be seen from Figs. 2(b) and 2(e), the resolution in the x–y plane is similar for both types of imaging. The difference, however, could be observed when the same local brain region was rotated along the $y$ axis [Figs. 2(c) and 2(f)]; the neural fibers acquired by the MOST system were continuous and uniform in three dimensions [Fig. 2(c)], while they became spread out and fuzzy along the $z$ axis in the confocal system [Fig. 2(f)]. These results confirmed that the MOST system ensured consistent axial resolution and image quality in depth, and also verified the stronger signaling and contrast of the modified immunoenzyme staining method.

Fig. 2

Images reconstructed from a stack of coronal sections (thickness: $56\mu \mathrm{m}$) acquired by the micro-optical sectioning tomography system (a) and confocal microscopy (d). (b) and (e) Enlarged views of local brain regions (volume size: $50\times 45\times 55{\mu \text{m}}^3$) marked in (a) and (d). (c) and (f) The same local brain region in (b) and (e) rotated 30 deg around the $y$ axis. $\text{Scale bar}=50\mu \mathrm{m}$ [(a) and (d)] and $5\mu \mathrm{m}$ [(b), (c), (e), and (f)].

Finally, we acquired 3-D datasets of serotonin-specific neural circuits driven by the TPH-Gal4 transgenic line in the whole fly brain at a submicron voxel resolution. Taking advantages of the fast imaging speed and automated data collection of the MOST system, the average imaging time of one whole fly brain was $\sim 10\min$, much faster than that of traditional confocal microscopy. Meanwhile, no additional registration was needed because of the accurate spatial positioning of the obtained images.

To show the brain-wide distribution of the serotonin-specific neural processes, a series of coronal projection images of the entire fly brain are presented in Fig. 3. As shown, serotonin-containing neurons are widely distributed in most brain areas, but are comparatively sparse in the fan-shaped body, mushroom body pedunculus, lateral accessory lobes, and optic glomeruluses. According to the location of the cell bodies, serotonin neurons driven by the TPH-Gal4 transgenic line were classified into several distinct clusters: anterior lateral protocerebrum (ALP), lateral protocerebrum (LP), subesophageal (SE), posterior lateral protocerebrum (PLP), and posterior medial protocerebrum (PMP). A large portion of the serotonergic neurons could be observed in our results and their distributions are consistent with previous studies.^9–11 Furthermore, our results provide some unique neuronal structure. As shown in an enlarged view of local brain region, the neural process (marked by an arrow) can be resolved clearly in Fig. 3(g) rather than being faintly visible as in Fig. 3(h) (corresponding immunofluorescent results). To our knowledge, this is the first 3-D immunoenzyme-stained Drosophila brain dataset to offer a high-contrast, comprehensive outlook on the fly brain and could, thus, be a valuable comparison tool for more specific, targeted studies.

Fig. 3

[(a) to (f)] Coronal projection images of one immunostained Drosophila brain {thickness: 21 μm [(a) to (d)] and $35\mu \mathrm{m}$ [(e) and (f)]}. (g) Enlarged views of the region marked in (d). (h) Region corresponding to (g) imaged by confocal microscopy. Arrows mark neural processes. The nomenclature for naming the cell clusters is according to the previous studies.^9–11 LP1, cells between the lobula and the protocerebrum, posterior lateral protocerebrum; LP2, cells between the medulla/central neuropil; ALP, anterior cell body rind, lateral to midline; PLP, posterior lateral protocerebrum; PMP, posterior cell body rind, medial to the calyx, running dorso-ventral; SE1: anterior subesophageal neurons; SE2, posterior to SE1; Dor, dorsal; fb, fan-shaped body; lal, lateral accessory lobes; ped, mushroom body pedunculus; og, optic glomerulus. $\text{Scale bar}=50\mu \mathrm{m}$ [(a) to (f)] and $10\mu \mathrm{m}$[(g) and (h)].

4.

Summary

In summary, by combining the improved immunoenzyme staining method and MOST technology, we demonstrated fast, automatic, and high spatial resolution imaging of specific neurons in the Drosophila brain. By optimizing the experimental conditions of HRP-conjugated secondary antibodies and DAB developing, the penetration of the immunoenzyme stain could be extended to the whole fly brain. Furthermore, using thin-section imaging via the MOST system, we were able to achieve a 3-D volume rendering of the HRP/DAB-stained fly brain. Compared with conventional histological methods, automation of data collection in the present study greatly improved the imaging speed of anatomical studies, avoided the burdensome manual operation, and ensured the coherence of the acquisition conditions such that the datasets were more standardized. High axial resolution and better image uniformity would provide great conveniences for subsequent image analyses. Furthermore, in combination with advanced genetic tools, such as the mosaic analysis with a repressible cell marker (MARCM) system and flippase (FLP)-out, this method offers the potential to provide a finer atlas of the Drosophila nervous system.