El Tatuaje y su Relación con el Grupo Delincuencial

Calycal microglomeruli were identified by electron microscopy in several insect species, including Drosophila, and were described to contain a single large presynaptic projection neuron bouton surrounded by numerous small post-synaptic profiles putatively from Kenyon cells and by few GABAergic profiles (Ganeshina and Menzel, 2001; Yasuyama et al., 2002). With the current work I introduce important additions to these early data. I demonstrate first that those small post-synaptic profiles are formed by the claw-like endings of Kenyon cell dendrites and by small spine-like structures protruding from them (Figure 4.9). Second, I show that each claw-like ending enwraps a single projection neuron bouton (Figure 4.10). Third, I identify each microglomerulus as a discrete unit, the boundaries of which are defined by the actin-enriched rim formed by the claw-like endings of several Kenyon cells contacting the projection neuron bouton. The implications of each of these findings are discussed below.

It is important to note that the microglomerular organization of the adult calyx, as described here, appears different than what reported for the larval mushroom body calyx (Masuda-Nakagawa et al., 2005; Ramaekers et al., 2005). In fact, unpublished observations (Supplemental Figure 6.6) suggest that each of the glomeruli in the larva comprises several microglomeruli and thus that the glomeruli in the larval calyx are a different structure than the microglomeruli in the adult calyx.

D i s c u s s i o n 84

5.7 Functional considerations

Electrophysiological recordings in several insect species have shown that the responses of Kenyon cells to odours are sparse, leading to the suggestion that Kenyon cells function as coincidence detectors, responding to coordinate input from projection neurons (Laurent, 2002; Perez-Orive et al., 2002; Szyszka et al., 2005). My morphological and connectivity data are consistent with this possibility.

Around 150-200 projection neurons provide olfactory input to the calyx (Stocker et al., 1990) forming an average of 5 boutons each (Marin et al., 2002). I counted around 1000 microglomeruli in 24 hours old flies. Given technical limitations in my counting methods these counts are approximate. Nevertheless, they are not inconsistent with the previous data, assuming that each microglomerulus contains one, and only one, projection neuron bouton, as our confocal microscopy data indicate. Importantly, they are also supported by 3D reconstructions of calycal microglomeruli obtained from serial-section electron microscopy. This data was obtained by Nancy Butcher and Claudia Groh in Ian Meinertzhagens laboratory as part of a collaboration and is presented in the supplemental information (Supplemental Figure 6.9 and Supplemental Figure 6.10).

A Kenyon cell has an average of five to seven claw-like endings (Lee et al., 1999; Zhu et al., 2003; F.L and G.T unpublished). Here, it is demonstrated that each of the claw-like endings contacts a single projection neuron bouton. Although additional input sites cannot be excluded, the data indicate that each Kenyon cell thus receives major input from a very limited number of projection neuron boutons, namely one per claw-like ending. If these boutons originate from different projection neurons, the morphology and connectivity of the Kenyon cells that were described would predict that they could act as detectors of coincident activity in several of their presynaptic partners, and that the number of their presynaptic partners is small compared with the locust (Jortner et al., 2007). Alternatively, all boutons presynaptic to a Kenyon cell could originate from a single projection neuron or from a functionally related set of projection neurons. In that case the functional task of Kenyon cells might be to improve the signal-to-noise ratio in the system.

The synaptic input from projection neurons to any one Kenyon cell is, as suggested from electron microscopy observations, provided at twenty or more active zones. If these sites of synaptic input were distributed evenly among 5-7 claw-shaped endings, each ending would receive three to six sites from any one projection neuron bouton. This number is possibly only what is sufficient to guarantee a reliable postsynaptic response to each incoming presynaptic potential. Based on the anatomical data it can be assumed that each clawed-shaped Kenyon cell ending contacts a different bouton. Thus, an average Kenyon cell could receive input from 5-7 projection neurons, assuming that all of these boutons were from different projection neurons. This estimate matches estimations of the PN:KC convergence ratio based on electrophysiological recordings (Turner et al., 2008). On the other hand, the widespread GABAergic input to a large fraction of microglomeruli revealed by my data (Figure 4.12) and by electron microscopy (Yasuyama et al., 2002, Supplemental Figure 6.9 and Supplemental Figure 6.10) could represent an instrument to keep the single claws silenced until inhibition is relieved. Laurent speculates that a short temporal integration window of Kenyon cells is critical to their specificity and thus to the sparseness of odour representations in the mushroom bodies. He suggests that GABAergic interneurons may provide a periodic reset, preventing temporal integration

85 D i s c u s s i o n

over successive oscillation cycles of projection neuron activity (Laurent, 2006). I believe that the widespread abundance of GABAergic presynaptic profiles supports these considerations. In honey bee, recurrent GABAergic interneurons innervating the lobes and the calyx (Mobbs, 1982) were suggested to be involved in olfactory memory formation (Grunewald, 1999). It is thus possible that GABAergic neurons in Drosophila also have important functions in odour coding and memory formation (Laurent and Naraghi, 1994; Yamazaki et al., 1998). Additional studies will be required to resolve the role of this GABAergic innervation into the calyx and must await more detailed reports on these pathways and further analysis of the physiology of the Kenyon cells and of their dendritic compartments.