The role of dopamine in Drosophila larval classical olfactory conditioning.
Bottom Line: Single cell analysis suggests that dopaminergic neurons do not directly connect gustatory input in the larval suboesophageal ganglion to olfactory information in the mushroom bodies.We found that dopamine receptors are highly enriched in the mushroom bodies and that aversive and appetitive olfactory learning is strongly impaired in dopamine receptor mutants.Genetically interfering with dopaminergic signaling supports this finding, although our data do not exclude on naïve odor and sugar preferences of the larvae.
Affiliation: Department of Biology, University of Fribourg, Fribourg, Switzerland.
Learning and memory is not an attribute of higher animals. Even Drosophila larvae are able to form and recall an association of a given odor with an aversive or appetitive gustatory reinforcer. As the Drosophila larva has turned into a particularly simple model for studying odor processing, a detailed neuronal and functional map of the olfactory pathway is available up to the third order neurons in the mushroom bodies. At this point, a convergence of olfactory processing and gustatory reinforcement is suggested to underlie associative memory formation. The dopaminergic system was shown to be involved in mammalian and insect olfactory conditioning. To analyze the anatomy and function of the larval dopaminergic system, we first characterize dopaminergic neurons immunohistochemically up to the single cell level and subsequent test for the effects of distortions in the dopamine system upon aversive (odor-salt) as well as appetitive (odor-sugar) associative learning. Single cell analysis suggests that dopaminergic neurons do not directly connect gustatory input in the larval suboesophageal ganglion to olfactory information in the mushroom bodies. However, a number of dopaminergic neurons innervate different regions of the brain, including protocerebra, mushroom bodies and suboesophageal ganglion. We found that dopamine receptors are highly enriched in the mushroom bodies and that aversive and appetitive olfactory learning is strongly impaired in dopamine receptor mutants. Genetically interfering with dopaminergic signaling supports this finding, although our data do not exclude on naïve odor and sugar preferences of the larvae. Our data suggest that dopaminergic neurons provide input to different brain regions including protocerebra, suboesophageal ganglion and mushroom bodies by more than one route. We therefore propose that different types of dopaminergic neurons might be involved in different types of signaling necessary for aversive and appetitive olfactory memory formation respectively, or for the retrieval of these memory traces. Future studies of the dopaminergic system need to take into account such cellular dissociations in function in order to be meaningful.
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Mentions: Approximately 70 putative DA neurons have been described in the cns of third instar larvae by catecholamine histofluorescence  (see also Table 1) and by immunoreactivity to DA, TH  and Dopa decarboxylase (DDC) , , . Apart from three bilaterally symmetrical clusters of DA neurons in the brain called DL1, DL2 and DM , DA cell bodies were reported from the sog and the thoracic and abdominal neuromeres (Table 1) . For analyzing the gross anatomy of the larval DA system with respect to the published data –, , , we used the TH-GAL4 driver line  to express either UAS-mCD8::GFP (data not shown)  or UAS-Cameleon2.1 . A significantly stronger signal was obtained with UAS-Cameleon2.1 compared to UAS-mCD8::GFP, the former providing two anti-GFP binding sites, it allowed us to identify cells with low GAL4 expression levels. By double-labeling with anti-GFP and anti-TH antibodies we were able to visualize the DL1, DL2 and DM clusters in TH-GAL4 (Figure 2). In DL1, seven to eight cell bodies were labeled (Table 1), although the TH-GAL4 line labeled one neuron that was not TH-positive. DL2 consisted of about six cell bodies per hemisphere in TH-GAL4 (Table 1), all of which were TH-positive. In the DM cluster, only eight DA cells were strongly labeled in all brains; in the remaining cell bodies, staining intensity varied, depending on the applied effectors and antibodies (Table 1, Figure 2H and data not shown). Concerning the sog, previous studies categorized the DA neurons as paired and unpaired types , . Based on our single cell labelings we doubt such a distinction. Rather we prefer the more neutral terms lateral and medial, describing exclusively the position of the cell body. The same nomenclature was applied for thoracic and abdominal neuromeres. In the sog we were able to distinguish two anteriomedial clusters, SM1 and SM2, and a more lateral cluster SL (Figure 2; Table 1). TH-GAL4 labeled about four cells in SM1 (Table 1) but only one of them was labeled by the anti-TH antibody, suggesting additional non-DA expression in three neurons. The SM2 cluster contained approximately three cells, which were all TH-positive (Table 1). The SL cluster of TH-GAL4 comprised about five cells per side; only three of them were double labeled and are therefore TH-positive. However, TH-GAL4 did not label three additional TH-positive cells (Table 1). Details about thoracic and abdominal DA clusters are provided in Table 1 and Figure 2. Taken together, TH-GAL4 labels a comprehensive set of DA neurons in the larval DA system and was therefore used in our behavioral approach (Figure 3). The expression pattern, however, is not complete and also includes a few TH-negative neurons [see also 36]. We next analyzed the cellular anatomy of TH-GAL4-positive cells in the larval brain. Due to their widespread arborization patterns, the anatomy of single DA neurons was difficult to untangle. Essentially, TH-GAL4 positive neurons innervated the protocerebra, the mbs, the sog as well as thoracic and abdominal ganglia (Figure 2). However, in insects DA is not only used as a neurotransmitter, but also as a neuromodulator [reviewed in 63], –. For example, Greer and colleagues have shown that the vesicular monoamine transporter mediates the transport of DA into secretory vesicles . Therefore, if DA acts as a neuromodulator, these types of neurons would not make direct synaptic connections and show diffuse anatomical projections. Yet, due to the limited resolution of the confocal microscope, our data did not allow to distinguish between these possibilities. The al was weakly labeled by TH-GAL4 driven UAS-Cameleon2.1, but not by the anti-TH antibody (Figure S2). Therefore, it is unlikely, although not formally excluded, that the al is innervated by DA neurons. Focusing on the mbs, we noticed that the TH-GAL4 driven Cameleon2.1 did not reveal any innervation of the main branch of its medial lobes (Figure 2C). In contrast, the larval-specific medial and lateral appendices (see above), as well as the vertical lobes, the spurs and the calyces were all innervated (Figure 2B–2E). Interestingly, about four DA neurons per hemisphere, having their cell bodies anterior to the dorsal part of the vertical lobe, densely innervated the medial lobe, as shown by anti-TH staining (Figure 2I). Therefore, the main branch of the medial lobes is innervated by DA neurons that are not included in the TH-GAL4 expression pattern. We further analysed the DA system by expressing post- and presynaptic effectors via TH-GAL4, reflecting potential input and output regions of the DA neurons respectively. From the available postsynaptic effectors, UAS-RDL::HA (resistence to dieldrin)  preferentially accumulated in the cell bodies, whereas UAS-PAK::GFP (p21/rac1-activated kinase)  and UAS-S97-DLG::GFP (Discs large)  labeled the whole neuron including axons (for all effectors data not shown). Thus, our data were limited to the dendrite-specific Drosophila Down Syndrome Cell Adhesion Molecule conjugated to GFP (Dscam[17.1]::GFP) . Similar to the adult fly , TH-GAL4/UAS-Dscam[17.1]::GFP larvae showed reduced staining in various parts of the brain including the mbs and sog compared to Cameleon2.1 (Figure 2L and 2M). In contrast, innervation was detected in the lateral horns, in the dorso- and basomedial protocerebra (Figure 2L and 2M) as well as in thoracic and abdominal neuromeres. To test the potential output regions of the DA system, we expressed – via the TH-GAL4 line – the presynaptic reporter genes n-synaptobrevin::GFP, synaptotagmin::HA and synaptotagmin::GFP ,  which yielded similar results (data not shown). Most brain regions, as well as the sog, were labeled with the same intensity, suggesting that there are no spatially separated cellular outputs in these regions (Figure 2J and 2K). In contrast, the mbs showed a defined dense innervation at the vertical lobes, the spurs and the pedunculi (Figure 2J and 2K) suggesting that these mb regions are presynaptic sites of the DA system. Analyzing the expression patterns of two DA receptors dDA1 and DAMB further supported this interpretation (Figure 4E–4J). Both dDA1 and DAMB showed strong expression in the mb lobes and the pedunculi, apart from some expression in the vnc (Figure 4F, 4H and 4J). Nevertheless, due to the limitationsof our immunohistochemical approach, we could not exclude low level receptor expression in other brain areas, as suggested by the presynaptic reporter expression (Figure 2J and 2K). To test whether the larval DA system is indeed involved in aversive and/or appetitive olfactory learning, we performed a set of conditioning experiments.
Affiliation: Department of Biology, University of Fribourg, Fribourg, Switzerland.