Pre- and Postsynaptic Role of Dopamine D2 Receptor DD2R in Drosophila Olfactory Associative Learning

Abstract

Dopaminergic neurons in Drosophila play critical roles in diverse brain functions such as motor control, arousal, learning, and memory. Using genetic and behavioral approaches, it has been firmly established that proper dopamine signaling is required for olfactory classical conditioning (e.g., aversive and appetitive learning). Dopamine mediates its functions through interaction with its receptors. There are two different types of dopamine receptors in Drosophila: D1-like (dDA1, DAMB) and D2-like receptors (DD2R). Currently, no study has attempted to characterize the role of DD2R in Drosophila learning and memory. Using a DD2R-RNAi transgenic line, we have examined the role of DD2R, expressed in dopamine neurons (i.e., the presynaptic DD2R autoreceptor), in larval olfactory learning. The function of postsynaptic DD2R expressed in mushroom body (MB) was also studied as MB is the center for Drosophila learning, with a function analogous to that of the mammalian hippocampus. Our results showed that suppression of presynaptic DD2R autoreceptors impairs both appetitive and aversive learning. Similarly, postsynaptic DD2R in MB neurons appears to be involved in both appetitive and aversive learning. The data confirm, for the first time, that DD2R plays an important role in Drosophila olfactory learning.

Figure 1

Larval olfactory learning assay. (Left) Training: third-instar larvae (92–96 h after egg laying) are trained on a 2.5% agar plate that is covered with 2 mL of either 1 M sucrose solution (SUC, for appetitive learning) or 0.1% quinine hemisulfate solution (QH, for aversive learning). Distilled water (DW) is used as a control. During training time, an odorant (Pentyl Acetate) is placed on a small piece of filter inside the lid. (Right) Test: Larvae are rinsed and transferred to the middle line of a new 2.5% agar plate after training. A small piece of filter paper with Pentyl Acetate (PA) is placed on one side of the plate, while the control is the other side. We then counted the number of larvae in the two semicircular areas and calculated the response index (R.I., see text for further detail) after 5 min.

Figure 2

Expression of DD2R-RNAi in dopaminergic neuron-impaired aversive olfactory learning in Drosophila larvae. A dopaminergic (DA)-specific driver, TH-Gal4, was used to drive expression of DD2R-RNAi in DA neurons (TH-DD2R-RNAi). Number (n) of separate experiments: wild type (WT) with water (DW) or QH (3), TH-Gal4 × UAS-DD2R-RNAi (TH-DD2R-RNAi) with DW or with QH (10). Student t-test, *** p < 0.001.

Figure 3

Expression of DD2R-RNAi in dopaminergic neuron-impaired appetitive olfactory learning in Drosophila larvae. Number (n) of separate experiments: wild type (WT) with water (DW) or SUC (3), TH-Gal4 × UAS-DD2R-RNAi (TH-DD2R-RNAi) with DW or with SUC (8). Student t-test, * p < 0.05, ** p < 0.01.

Figure 4

Expression of DD2R-RNAi in the mushroom body-impaired aversive olfactory learning in Drosophila larvae. DD2R-RNAi was expressed by using two mushroom body drivers, 201Y-Gal4 and 30Y-Gal4. The 201-Gal4 and UAS-DD2R-RNAi lines were used as controls for this experiment. Number (n) of separate experiments: 201-Gal4 with water (DW) or QH (4), UAS-DD2R-RNAi with DW or QH (3), 201Y-Gal4 × UAS-DD2R-RNAi (201Y-DD2R-RNAi) with DW or with QH (5), and 30Y-Gal4 × UAS-DD2R-RNAi (30Y-DD2R-RNAi) with DW or with QH (6). Student t-test, * p<0.05, *** p < 0.001.

Figure 5

Expression of DD2R-RNAi in the mushroom body-impaired appetitive olfactory learning in Drosophila larvae. 201Y-Gal4 and UAS-DD2R-RNAi lines were used as controls for this experiment. Number (n) of separate experiments: 201Y-Gal4 with water (DW) or QH (3), UAS-DD2R-RNAi with DW or QH (3), 201Y-Gal4 × UAS-DD2R-RNAi (201Y-DD2R-RNAi) with DW or with QH (5), and 30Y-Gal4 × UAS-DD2R-RNAi (30Y-DD2R-RNAi) with DW or with QH (6). Student t-test, ** p < 0.01.

Figure 6

Proposed neuronal and synaptic mechanisms underlying the role of Drosophila D2 receptor DD2R in larval olfactory associative learning. (A) The diagram shows neural circuits involved in Drosophila larval learning. The circuits include three components: (1) Olfactory sensory circuits for CS are comprised of olfactory sensory neurons (OSNs) in antennae, projection neurons (PNs) in antennal lobes, and mushroom body neurons (MBNs). (2) Gustatory sensory circuits for US are comprised of gustatory sensory neurons (GSN), subesophageal ganglion (SOG), dopaminergic (DA) neurons, and MBNs. (3) MBNs serve as coincidence detection of signals from OSNs and GSNs. Thus they can associate olfactory and gustatory signals and mediate olfactory learning in larvae. (B) Presynaptic mechanism underlying aversive and appetitive learning. Presynaptic DD2R autoreceptors suppress release of DA at the presynaptic terminals in the MB. Therefore, if presynaptic DD2R function is suppressed, then more DA is released in MB neurons. Increased DA tone in MB neurons impairs both aversive and appetitive learning behaviors. (C) Postsynaptic mechanism underlying aversive and appetitive learning. Postsynaptic DD2Rs in MBNs inhibits neuronal excitability as shown in Wiemerslage et al. [21]. Therefore, neural circuits associating CS with US maintain balanced excitability. However, DD2R-RNAi in MBNs suppresses postsynaptic DD2Rs and thus neural circuits responsible for learning are over-excited, resulting in impairment of olfactory learning. Our results strongly suggest that DA homeostasis is important for aversive and appetitive learning in Drosophila larvae.