El Poder como mecanismo de control o liberación

To test whether dendritic spines could be genetically modifiable in this system, the level of a factor known to affect spine morphology and density in vertebrates were manipulated. dRac1 was chosen because the effects of Rac1 on spines in vertebrates are particularly well characterized (Govek et al., 2005). Full length dRac1 (FL: rac1.L; (Luo et al., 1996; Nakayama et al., 2000) as well as dominant negative (DN: rac1.N17; (Luo et al., 1994) and constitutively active (CA: rac1.V12; (Luo et al., 1994) versions of dRac1 were expressed specifically in the LPTCs under the control of db331-GAL4, and coexpressed GMA was used to visualize the dendritic trees. Since overexpression of CA dRac1 led to lethality at pupal stages only the effects of full length and dominant negative dRac1 over-expression could be analyzed (Figure 4.7). The overall dendritic architecture in both genotypes appeared to be similar to the wild type condition: neither position nor branching patterns of primary and secondary order dendrites were obviously affected. This is consistent with previous evidence that alteration of Rac activity does not affect the dendrite structure of pyramidal neurons or of cerebellar Purkinje neurons (Luo et al., 1996; Nakayama et al., 2000). However, a difference in spine morphology and an increase in spine density was

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noticed (compare Figure 4.7B and Figure 4.7A). To quantify these observations spine density analysis was quantified as described above (Figure 4.7G).Spine density was found to be increased by around 30% upon overexpression of either full length dRac1 (Figure 4.7G; 1.54 spines/μm; n= 5; p= 0.0064 by t-test) or dominant negative dRac1 (1.51 spines/μm; n=5; p= 0.0051), in comparison to the control expressing myr-mRFP (1.15 spines/μm; n=5). Moreover, spines appeared shorter and less well defined. Although opposite effects might be in principle expected upon overexpression of full length and dominant negative proteins, both genotypes appeared indistinguishable and yielded similar results in the quantification (see Discussion). Next, it was addressed whether the processes present upon Rac1 over-expression share characteristics of spines as described above. Actin and Dα7 distribution were therefore analyzed (Figure 4.7B, C, E, F). Actin was found to be enriched in the Rac1-induced spines 2–7 times more than in the dendrite shaft in comparison to cytoplasmic mRFP (VS1 fragments from 5 animals quantified; Figure 4.7B, E; Supplemental Figure 6.2). Dα7-GFP was also present on these processes (Figure 4.7F). Because of low signal level in these experiments, reliable quantifications could not be done. Rac1-induced spines seem thus to share two distinct characteristics of LPTC spines.

Figure 4.6 | Dα7 is localized at dendritic spines

(A) Projection of a confocal stack (spanning approx. 3 μm) through a representative fragment of VS1 expressing myr-mRFP (red) and Dα7-GFP (green) under the control of

db331-GAL4. (B) Magnified view of the branchlet boxed in A, showing the single channels and the merge, as indicated. >90% of all spines contain the ectopically expressed ACh- receptor subunit Dα7-GFP. Note also the specificity of localization to the spines: 70% of the Da7-GFP positive puncta localize to spines. (C) View of a branchlet of VS1, expressing

mCD8-GFP under the control of db331-GAL4 and immunolabelled with anti-Dα7 antibodies. The single channels and the merge are shown. Arrows point to single spines. Scale bars: 10 μm.

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Figure 4.7 | Spine density is modulated by Rac1

(A-F) Similar dendritic fragments of VS1 are shown for animals carrying db331-GAL4 and that were heterozygous for UAS-GMA (used for imaging) and either UAS-myr-mRFP

(control, A) or UAS-rac1.L (D); UAS-actin-GFP and UAS-mRed with (E) or without (B)

UAS-rac1.L or UAS-Dα7-GFP and UAS-mCD8-cherry with (F) or without (C) UAS-rac1.L

respectively. (G) Quantification of spine density. >500 spines from 5 animals (as shown in A and G) were analyzed per data point. T-test p< 0.007. Scale bars: 5 μm.

Taken together, alterations of Rac1 levels appear to be capable of modulating spine density in Drosophila as previously reported for vertebrates.

In summary, Ewa Koper and I characterized the morphology and cytoskeletal organization of Lobula Plate Tangential cell (LPTC) dendrites and demonstrate that they bear small protrusions that closely resemble dendritic spines of vertebrates. LPTCs were considered to be an interesting model system to study dendrite and spine morphogenesis genetically. The first step towards this goal was the identification of Rac1 as a modulator of spine morphology in flies as previously described for vertebrates. Rac1 was identified as an interesting candidate molecule in initial attempts to screen for factors involved in dendritogenesis. These experiments will be introduced in more detail after the presentation of anatomical work describing Kenyon cell dendrites and their synaptic partners because the attempts to identify genes involved in dendrite morphogenesis were based on the anatomical knowledge of both LPTCs and Kenyon cells.

4.10 Microglomerular complexes in the mushroom body calyx

Kenyon cells are no ideal cellular system to study dendritic morphology because of their poorly defined morphology and the lack of a GAL4 line allowing the labelling of individual Kenyon cells. The morphology of single Kenyon cells can only be studied using MARCM (Lee and Luo, 1999). Kenyon cell dendrites form several branches throughout

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the calyx which often end with characteristic claw-like structures. Because of the high number of Kenyon cells (around 2000 per hemisphere) and the lack of specific markers it is an open question if they have an individually stereotyped morphology – but electrophysiology combined with theoretic considerations suggest that this might not be the case (Murthy et al., 2008). Although Kenyon cell dendrites seem to share some common features they are not very stereotyped between different cells. These constraints make cellular studies with the resolution presented for the LPTCs impossible. A number of advantages seemed to outweigh these technical shortcomings. First, Kenyon cells are known to play an important role in learning and memory. Second, Kenyon cells dendrites are the intrinsic elements of the mushroom body calyx and it has been shown that the volume of the calyx is sensitive to alterations of sensory environment in a number of insect species including the fly. Third, they are part of the well-characterized olfactory system. It is possible to manipulate the activity of the presynaptic partners of Kenyon cells with both odour stimulation and molecular tools. While the first two points make it plausible that Kenyon cell dendrites might undergo morphological rearrangements in an activity-dependent manner the third opens interesting opportunities for ultimately investigating the relation between neuronal activity and structural plasticity. For these reasons I decided to investigate if the mushroom body calyx (with the Kenyon cells as the intrinsic elements) could be a suitable model system for structural plasticity in the fly. I will describe the anatomy of microglomerular complexes in the mushroom body calyx before presenting my attempts to exploit this anatomical information for studies on structural plasticity.

4.11 Actin-rich microglomerular structures are present throughout the calyx