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. 2011 Jun 29;31(26):9696-707.
doi: 10.1523/JNEUROSCI.6542-10.2011.

Presynapses in Kenyon Cell Dendrites in the Mushroom Body Calyx of Drosophila

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Free PMC article

Presynapses in Kenyon Cell Dendrites in the Mushroom Body Calyx of Drosophila

Frauke Christiansen et al. J Neurosci. .
Free PMC article

Abstract

Plastic changes at the presynaptic sites of the mushroom body (MB) principal neurons called Kenyon cells (KCs) are considered to represent a neuronal substrate underlying olfactory learning and memory. It is generally believed that presynaptic and postsynaptic sites of KCs are spatially segregated. In the MB calyx, KCs receive olfactory input from projection neurons (PNs) on their dendrites. Their presynaptic sites, however, are thought to be restricted to the axonal projections within the MB lobes. Here, we show that KCs also form presynapses along their calycal dendrites, by using novel transgenic tools for visualizing presynaptic active zones and postsynaptic densities. At these presynapses, vesicle release following stimulation could be observed. They reside at a distance from the PN input into the KC dendrites, suggesting that regions of presynaptic and postsynaptic differentiation are segregated along individual KC dendrites. KC presynapses are present in γ-type KCs that support short- and long-term memory in adult flies and larvae. They can also be observed in α/β-type KCs, which are involved in memory retrieval, but not in α'/β'-type KCs, which are implicated in memory acquisition and consolidation. We hypothesize that, as in mammals, recurrent activity loops might operate for memory retrieval in the fly olfactory system. The newly identified KC-derived presynapses in the calyx are, inter alia, candidate sites for the formation of memory traces during olfactory learning.

Figures

Figure 1.
Figure 1.
Evidence for KC-derived presynapses within the MB calyx of Drosophila larvae. A–G, Expression of the BRP fragment BRP-shortGFP under control of the KC enhancer mb247 reveals a strong signal in the MB lobes (A, maximum-intensity projection) and the calyx of the larva (C, F). Costaining with BRPNc82, an antibody against the presynaptic AZ protein BRP (A, mushroom body; B, E, calyx), showed a clear overlap with the KC-derived BRP-shortGFP signal in the calyx (D, G, arrows), suggesting the existence of a KC-derived population of AZs. E–G, Cutout of the calyx shown in B–D in a higher magnification. H–Q, UAS-brp-RNAi expressed in KCs (ok107-GAL4) provoked a strong reduction of the BRPNc82 label in both MB lobes and calyx. H, I, Larval brain overview. In the control brain stained with αBRPNc82, major neuropils are clearly visible, including the mushroom body lobes (H, arrows). UAS-brp-RNAi expressed in KCs resulted in a strong reduction of the MB lobe label (I, arrows). J, K, Colabeling of αBRPNc82 together with an antibody against the AZ protein DSyd1 showed a specific effect of the expression of UAS-brp-RNAi in KCs on the BRP label in the calyx. Reduction of BRP was confined to the regions in the center of the calyx (K, dotted circle) but did not occur in areas where KCs are postsynaptic to PN presynaptic terminals, the macroglomeruli (K, arrows). L–Q, Higher magnification views of calycal regions shown in J and K, marked in J and K by white rectangles. While the BRP label is clearly reduced (O, Q, below the dotted line) compared with the control (L, N), the DSyd1 label remains unaffected (M, P). The macroglomeruli also show no reduction in BRP label (Q, arrows). ML, Medial lobe; P, peduncle; MP, medial protocerebrum; EF, esophageal foramen; VLP, ventrolateral protocerebrum; SEG, subesophageal ganglion; MB, mushroom body. All images show single optical slices, except when stated differently. Slice thickness, 200 nm. Scale bars: A, H, I, 50 μm; B–D, J, K, 10 μm; E–G, 5 μm; L–Q, 2 μm.
Figure 2.
Figure 2.
Evidence for KC-derived presynapses within the MB calyx of Drosophila adults. A–G, Expression of the BRP fragment BRP-shortGFP under control of the KC enhancer mb247 reveals a strong signal in MB lobes (A, maximum-intensity projection) and calyces of adult flies (C; higher magnification in F). The costaining of the presynaptic AZ protein BRP (B; high magnification in E) showed a clear overlap with the KC-derived BRP-shortGFP signal (D; higher magnification in G; arrows), suggesting the existence of a KC-derived population of AZs. H–Q, UAS-brp-RNAi expressed in KCs (ok107-GAL4) provoked a strong reduction of the BRPNc82 label in both MB lobes (H, I, arrows) and calyces (J, K, arrows; higher magnification in L and O). In the calyx, the reduction of BRP was confined to the regions outside of microglomeruli (K, arrows). For a higher magnification, compare L–Q. Since αDSyd-1 localization at AZs was shown to be independent of BRP (Owald et al., 2010), we used this marker as an independent reference. DSyd-1 labeling remained unaffected from the brp-RNAi expression in KCs (M, P). MB, Mushroom body; AL, antennal lobe. All images show single optical slices, except when stated differently. Slice thickness, 200 nm. Scale bars: A, H, I, 50 μm; B–D, K, 10 μm; E–G, 5 μm; L–Q, 2 μm.
Figure 3.
Figure 3.
Segmentation and quantification of BRPNc82 signal within the calyx, with and without expression of UAS-brp-RNAi in KCs. A–F, Visualization of the segmentation and 3D reconstruction of the calyx used for quantification. A–C, Top view. D–F, Side view. A, D, Segmented calyx (green) within the BRPNc82 staining labeling all neuropils (magenta). B, E, Computed 3D surface covering the complete BRPNc82-positive signal in the calyx (gray), superimposed on the staining shown in A and D. C, F, Three-dimensional surfaces of individual BRPNc82-positive spots (gray), superimposed on the staining shown in A and D. The surfaces used for the detection of the number of AZs. For additional detailed images of such a 3D reconstruction of the BRPNc82 signal, also see Kremer et al. (2010). G, H, Results of the quantification. Blue bar, Median; green box, interquartile range; red whiskers, min/max values. The Mann–Whitney U test was used to determine two-sided exact p values. G, Larval calyces, n = 6 animals for both genotypes. Ratio of number of spots is the ratio of BRPNc82-positive spots versus DSyd-1-positive spots, p = 0.002. Density is the number of spots per calyx area. DSyd-1 density, p = 0.485; BRPNc82 density, p = 0.002. BRP intensity/DSyd-1 intensity is the ratio of the BRPNc82 signal intensity versus the DSyd-1 signal intensity within a surface covering the complete calyx, p = 0.002. H, Adult calyces, n = 10 animals for brp-RNAi and n = 9 for controls. Ratio of number of spots, p < 0.001. DSyd-1 density, p = 0.968; BRPNc82 density, p = 0.001. Ratio of signal intensity, p = 0.002.
Figure 4.
Figure 4.
Presence of SVs in KCACs. A–H, Expression of a GFP-fusion of the SV marker Synaptotagmin (sytGFP) with the KC driver ok107-GAL4 in MB lobes and calyces of larvae (A, MB lobes, maximum-intensity projection; B–G, calyx) and adult flies (H, MB lobes, maximum-intensity projection; I–N, calyx). Costaining with BRPNc82 revealed that sytGFP expressed in KCs locates between microglomerular and macroglomerular complexes (B–D, arrows; I–K, arrows). For a higher magnification in larvae, compare E–G (arrows), and for adults, L–N (arrows). All images show single optical slices, except when stated differently. Slice thickness, 200 nm. MB, Mushroom body. Scale bars: A, B–D, H–K, 10 μm; E–G, L–N, 1 μm.
Figure 5.
Figure 5.
Evidence that KCACs are part of active synapses. Optical imaging of SV release at KCs analyzed using Synapto-pHluorin. A, Experimental setup during functional imaging. B, Expression of Synapto-pHluorin in KCs. Note that the calyx region can easily be distinguished from the lobe regions in the in vivo preparation. C, Relative change in fluorescence emission after stimulation with KCl in the calycal region, indicated by the red line shown in the inserted image of the calyx. D, Average of change in fluorescence in five flies. The trace indicates mean values; the gray shadows represent SEMs. The increase in fluorescence emission after stimulation with 100 mm KCl solution (superimposed on the decrease in emission caused by photobleaching) demonstrates that the KC dendrites in the calyx are capable of releasing SVs. Scale bars: B, 100 μm; C, 10 μm.
Figure 6.
Figure 6.
Presynaptic and postsynaptic domains segregate within KC calyx dendrites. A–H, Analysis of synaptic elements in the calyx of adult flies. KCACs cluster distant from PN::KC synapses. A–D, Visualization of cholinergic PSDs of KCs (mb247-dα7GFP) (A) together with PN-derived AZs (gh146-GAL4 driving UAS-brp-shortmCherry) (B). A higher magnification of both merged labels shows that PN AZs locate at the inner edge of the microglomeruli, juxtaposed to KC PSDs (C, arrows; single microglomerulus in D). E–H, In contrast, KC-expressed BRP-short (mb247::brp-shortGFP) (E) clustered distant from PN AZ populations (marked by gh146::brp-shortmCherry) (F), as the merge of both labels at a higher magnification demonstrates (G, arrows; H, single microglomerulus). I–L, Visualization of single KCs in the calyx using the MARCM technique. I, J, Single KC (maximum-intensity projection) expressing Dα7mCherry together with mCD8::GFP. A preferential localization of ACh receptors at the KC claw-like endings is visible (I, arrows; J, single claw). K, L, Single KC (maximum-intensity projection) expressing BRP-shortmCherry together with mCD8::GFP. BRP signal is distributed along the KC dendrites (K, arrows), mostly separated from the KC claw-like endings (K, arrows; L, single claw). M, Schematic drawing of synaptic input and output regions on a single KC within the calyx. All images show single optical slices, except when stated differently. Slice thickness, 200 nm. Scale bars: A, B, E, F, 10 μm; C, G, I, K, 5 μm; D, H, 1 μm.
Figure 7.
Figure 7.
Coexpression of presynaptic (BRP-short) and postsynaptic (Dα7) markers identifies distinct regions within KC dendrites of adult calyces. A–L show cutouts of maximum-intensity projections of calyces. A–C, ok107-GAL4 driving expression of both UAS-dα7GFP (A) and UAS-brp-shortmCherry (B) within KC dendrites. The merged image shows that presynaptic and postsynaptic regions appeared largely segregated (C, arrows). D–F, α′β′ KCs (c305a-GAL4) expressing both UAS-dα7GFP (D) and UAS-brp-shortmCherry (E). While a strong Dα7 signal was present (D), no BRP-short signal could be detected within the calyx (E, F). G–I, γ KC (h24-GAL4) expressing both UAS-dα7GFP (G) and UAS-brp-shortmCherry (H). Both signals were present and clearly separated from each other (I). Dα7 was distributed in microglomerular structures, while the BRP-short signal localized in between (I, arrows). J–O, αβ KCs (c739-GAL4) expressing both UAS-dα7GFP (J, M) and UAS-brp-shortmCherry (K, N). M–O show the whole calyx (maximum-intensity projection). The dendrites showed a similar segregation of presynaptic and postsynaptic regions (L, O). Both signals were arranged into four distinct patches clearly separated from each other (O, asterisks). Here, BRP-short was located at the more peripheral part of the calyx (K, N), whereas Dα7 showed a stronger signal toward the center of the neuropil (J, M). P, Horizontal section through the calyx of an adult fly. Visualization of calycal microglomeruli by UAS-brp-shortmCherry expressed in PNs (gh146-GAL4) and Dα7 expressed in KCs (mb247::dα7GFP). A αBRPNc82 label of all presynapses is shown in blue. The calyx is divided into five subunits, each harboring microglomeruli in the center, that are surrounded by other synapses. Four subunits protrude to the posterior part and one further subunit is located between them and the iACT. Q, Schematic drawing of the distribution of presynaptic and postsynaptic regions of the different KC subtypes within the MB calyx. Postsynapses are shown in green, and presynapses are shown in blue. All images show single optical slices, except when stated differently. Slice thickness, 200 nm. iACT, Inner antennocerebral tract. Scale bars: C, F, I, L, 5 μm; M–P, 10 μm. BRP-short can form agglomerations within the somata of the cells it is expressed in. As the KC somata are located very close to the calyx neuropil, we manually removed the somata from images M–O for clarity.

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