Figure 1.
Scanning Laser Optical Tomography setup.
Schematic drawing (A) and photograph (B) of the Scanning Laser Optical Tomograph. A laser beam is directed via two mirrors onto an x-y-galvanometer-scanning system. The system is placed in the back focal plane of the imaging lens, which produces a weakly focused beam in the imaging volume. Transmitted light collected with a collimating lens onto a photo diode. Scattered and fluorescent light is collimated with a lens at the bottom of the cuvette. A spectral filter suppresses the scattered light for fluorescence detection (indicated in red; in A) or the fluorescent light for scattered light detection (indicated in orange; in A). The filtered light is then collimated onto a photomultiplier tube (PMT). A capillary containing the sample is rotated within the cuvette which is filled with glycerol.
Figure 2.
Evaluation of size changes of the antennal lobe and mushroom body with Scanning Laser Optical Tomography (SLOTy).
A. Frontal view of the untreated locust brain (exclusive optic lobes) under reflected light. The mushroom body calyces are hardly visible (arrows). B,C. Raw data of 3D transmission-(B) and scattered light (C) projections of the locust brain. Brains were cleared with glycerol. Note that mushroom body calyces are clearly visible (arrows in B,C). D–G. Reconstructed 2D optical sections of transmission projections. D. Frontal section depicting left (untreated side) and right (ablated side) antennal lobe 7 days after ablation of the right antenna. Note that the right (deafferented) antennal lobe is smaller than the left (untreated) antennal lobe. E. Side view of brain with left antennal lobe. Antennal lobe size was approximated as an ellipsoidal volume V = π/6abc (D,E). F,G. Frontal section (F) and horizontal section (G) depicting mushroom body calyces. Calyx width was measured in frontal sections (red line in F). AL, antennal lobe; Ca, calyx. Scale bars = 400 µm in A–C; 200 µm in D–G.
Figure 3.
Changes of antennal lobe size after unilateral deafferentation via antennal ablation and nerve crush.
A. Relative size of right (treated) antennal lobe expressed as percentage of left (control) side. Datasets are based on estimated neuropil volume. Each bar represents the mean value of 5–10 preparations. Error bars indicate SEM. Levels of significance are indicated *p<0.05, **p<0.01, ***p<0.001. B–D. Reconstructed optical sections of antennal lobes after unilateral deafferentation. Asterisks indicate treated side. B. 21 days after ablation of right antenna. Size of right antennal lobe is reduced by ca. 60%. C. 7 days after nerve crush of right antenna. Size of right antennal lobe is significantly reduced compared to left (control) side. D. 21 days after nerve crush. Right antennal lobe has regained size almost back to normal. ns, not significant. Scale bar = 200 µm in D (applies to B–D).
Figure 4.
Changes of mushroom body size after unilateral deafferentation of the antennal lobe via antennal ablation and nerve crush.
A. Relative size of right mushroom body calyx (treated side) expressed as percentage of left (control) side. Each bar represents the mean value of 5–10 preparations. Error bars indicate SEM. Levels of significance are indicated *p<0.05, **p<0.01. B,C. Reconstructed optical sections of mushroom body calyces after unilateral deafferentation of the right antennal lobe. Asterisks indicate treated side. B. 21 days after ablation of right antenna. Size of right calyx is reduced by ca. 10%. C. 7 days after nerve crush of right antenna. Size of right calyx is significantly reduced compared to left (control) side. Scale bar = 200 µm in C (applies to B,C).
Figure 5.
Superimposed transmission and fluorescence projections of a locust midbrain (exclusive optic lobes) 14 days after crush of the right antenna.
Antennal lobes are labeled with Neurobiotin. Asterisks indicate treated side. No obvious differences in antennal lobe size are visible. A. Raw data of superimposed transmission-and emission projections. B–D. Reconstructed optical sections of antennal lobes. B. Transmission. C. Emission. D. Merged. Note that Neurobiotin labels distinct glomeruli in the left (control side; arrows in C,D) but not the right (treated side) antennal lobe. AL, antennal lobe. Scale bars = 200 µm (scale bar in D applies to B–D).