Anatomy of two populations of local interneurons labelled by NP1227- and NP2426-GAL4by NP1227- and NP2426-GAL4

A first step in the investigation of the inhibitory local interneurons included an anatomical analysis of the distribution of neurons targeted by the two GAL4-enhancer-trap lines. Therefore, images were taken at a confocal microscope together with Dr. T. Riemensperger. Both lines were crossed with flies carrying two different GFP constructs under UAS control. The coupling of GFP with mCD8 results in a localization of GFP to the neuronal membranes whereas coupling with synaptobrevin targeted GFP to the synapses. Both constructs were expressed in the fly line (w⁻;U AS −mCD8 : GF P, U AS −syb : GF P; +) used to visualize the cells targeted by NP1227-GAL4 and NP2426-GAL4 respectively. Using immunos-tainings, the signal of GFP was enhanced with a GFP antibody and brain neuropils were stained with the nc82-antibody against the presynaptic protein Bruchpilot [Hofbauer, 1991; Wagh et al., 2006]. Both types of interneurons innervate the whole antennal lobe and synapse within the glomeruli. However, the expression patterns differed substantially.

Anatomy of NP1227-GAL4 positive neurons (type I local interneurons, LN1) The expression pattern of the NP1227 GAL4-enhancer-trap line targeting type I in-hibitory local interneurons (LN1) shows a strong labeling of the antennal lobes in the central nervous system as previously described [Sachse et al., 2007]. Apart from the antennal lobe, only a few cells were expressing GFP in the fly brain. In the peripheral nervous system, the LN1 line labels a few glia cells around the thoracic ganglion, but no neuronal structures were stained with GFP (Figure 3.11 A). A magnified view of the antennal lobe shows the labeled cell bodies of the inhibitory local interneurons in the surrounding of the lobe. Within the antennal lobe, the interneurons innervate a large amount of glomeruli. The neuronal arborizations are very distinct and sparsely target the core regions of the glomeruli (Figure 3.11 B).

A ^B

Figure 3.11. Anatomy of LN1 in-terneurons.

(A) The neurons expressing GFP (green) are targeted by the NP1227 GAL4-enhancer-trap line. In the tho-racic ganglion, GFP expression could be found in cells surrounding the neu-ropils stained in magenta. However, no expression in neurons could be ob-served. In the central nervous system, the LN1 line targets mainly the anten-nal lobe structures. The picture was merged from two separate scans in the same brain; gray dotted lines represent the borders of the two images.(B)A more detailed picture of the expression pattern within the antennal lobe reveals cell bodies surrounding the lobe structure. Neuronal pro-cesses extend inside the antennal lobe and arborize on the different glomeruli with distinct processes. green: GFP; magenta: nc82; Scale bars: 25µm

3. Results

Anatomy of NP2426-GAL4 positive neurons (type II local interneurons, LN2) The NP2426 GAL4-enhancer-trap line targets type II inhibitory local interneurons. In the central nervous system, the GFP expression is mainly targeted to the antennal lobes as previously described [Sachse et al., 2007]. However, previous studies did not report the expression pattern of NP2426 in the peripheral nervous system. Here, the thoracic ganglion is also labeled by the NP2426 line (Figure 3.12 A). Several neuronal structures and cell bodies are targeted by NP2426 expression. In addition, a co-expression of GFP and the neuropil marker nc82 could be observed. The cell bodies labeled with GFP are most likely motorneurons that extend their arboriza-tions into the legs [Baek & Mann, 2009]. Expression of shibire in these neurons would block synaptic transmission and might therefore result in an impairment of locomotion.

The magnification of the antennal lobe shows a more detailed view of the neurons within the lobe that are targeted by NP2426-GAL4 (Figure 3.12 B). The cell bodies of the type II inhibitory local interneurons (LN2) are located outside of the antennal lobe and send their processes to the different glomeruli. As previously reported by Sachse et al. [2007], the innervation of the glomeruli is very dense in contrast to the type I inhibitory local interneurons (LN1). Moreover, the arborizations are targeting the core and the surface of the glomeruli and seem to be much thinner and more distinct than the ones from LN1 interneurons.

In conclusion, the targeting of both GAL4-enhancer-trap lines NP1227 and NP2426 to the antennal lobes could be confirmed. However, the labeling of motorneurons in the thoracic ganglion by the NP2426-line is a novel finding and might cause prob-lems during olfactory conditioning with the Tully-machine. As a read-out during the test-phase of the paradigm includes the active avoidance or approach of one side of the T-maze, the line might not be suitable. Without normal locomotor activity, it is impossible to draw conclusion about the flies’ behavior after absolute or differ-ential training. Hence, several behavioral experiments were performed in order to investigate the general behavior of flies with a blocked synaptic transmission in both GAL4-enhancer-trap lines.

A ^B

Figure 3.12. Anatomy of LN2 in-terneurons.

(A) The neurons expressing GFP (green) are targeted by the NP2426 GAL4-enhancer-trap line. GFP ex-pression in the thoracic ganglion in-cludes several neuronal structures.

The cell bodies are located outside the neuropils and are likely to be mo-torneurons [Baek & Mann, 2009]. Be-sides the expression in the peripheral nervous system, the LN2 line predom-inantly targets the antennal lobe in the central nervous system. The picture was merged from two separate scans in the same brain; gray dotted lines represent the borders of the two images.(B)A magnified view of the antennal lobes shows the cell bodies of local interneurons around the lobe structures. The neurons arborize within the lobe and broadly innervate the distinct glomeruli.

green: GFP; magenta: nc82; Scale bars: 25µm

3.7.2. General behavioral performance of flies with blocked