Four individually identified paired dopamine neurons signal reward in larval Drosophila

Dopaminergic neurons serve multiple functions, including reinforcement processing during associative learning [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12]. It is thus warranted to understand which dopaminergic neurons mediate which function. We study larval Drosophila, in which only approximately 120 of a total of 10,000 neurons are dopaminergic, as judged by the expression of tyrosine hydroxylase (TH), the rate- limiting enzyme of dopamine biosynthesis [ 5 and 13]. Dopaminergic neurons mediating reinforcement in insect olfactory learning target the mushroom bodies, a higher-order “cortical” brain region [ 1, 2, 3, 4, 5, 11, 12, 14 and 15]. We discover four previously undescribed paired neurons, the primary protocerebral anterior medial (pPAM) neurons. These neurons are TH positive and subdivide the medial lobe of the mushroom body into four distinct subunits. These pPAM neurons are acutely necessary for odor-sugar reward learning and require intact TH function in this process. However, they are dispensable for aversive learning and innate behavior toward the odors and sugars employed. Optogenetical activation of pPAM neurons is sufficient as a reward. Thus, the pPAM neurons convey a likely dopaminergic reward signal. In contrast, DL1 cluster neurons convey a corresponding punishment signal [5], suggesting a cellular division of labor to convey dopaminergic reward and punishment signals. On the level of individually identified neurons, this uncovers an organizational principle shared with adult Drosophila and mammals [ 1, 2, 3, 4, 7, 9 and 10] (but see [6]). The numerical simplicity and connectomic tractability of the larval nervous system [ 16, 17, 18 and 19] now offers a prospect for studying circuit principles of dopamine function at unprecedented resolution

The serotonergic central nervous system of the Drosophila larva: anatomy and behavioral function.

The Drosophila larva has turned into a particularly simple model system for studying the neuronal basis of innate behaviors and higher brain functions. Neuronal networks involved in olfaction, gustation, vision and learning and memory have been described during the last decade, often up to the single-cell level. Thus, most of these sensory networks are substantially defined, from the sensory level up to third-order neurons. This is especially true for the olfactory system of the larva. Given the wealth of genetic tools in Drosophila it is now possible to address the question how modulatory systems interfere with sensory systems and affect learning and memory. Here we focus on the serotonergic system that was shown to be involved in mammalian and insect sensory perception as well as learning and memory. Larval studies suggested that the serotonergic system is involved in the modulation of olfaction, feeding, vision and heart rate regulation. In a dual anatomical and behavioral approach we describe the basic anatomy of the larval serotonergic system, down to the single-cell level. In parallel, by expressing apoptosis-inducing genes during embryonic and larval development, we ablate most of the serotonergic neurons within the larval central nervous system. When testing these animals for naïve odor, sugar, salt and light perception, no profound phenotype was detectable; even appetitive and aversive learning was normal. Our results provide the first comprehensive description of the neuronal network of the larval serotonergic system. Moreover, they suggest that serotonin per se is not necessary for any of the behaviors tested. However, our data do not exclude that this system may modulate or fine-tune a wide set of behaviors, similar to its reported function in other insect species or in mammals. Based on our observations and the availability of a wide variety of genetic tools, this issue can now be addressed

Biogenic amines in the Drosophila melanogaster larval central nervous system : an anatomical and behavioural function description

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Characterization of the octopaminergic and tyraminergic neurons in the central brain of Drosophila larvae

Drosophila larvae are able to evaluate sensory information based on prior experience, similar to adult flies, other insect species and vertebrates. Larvae and adult flies can be taught to associate odor stimuli with sugar reward and prior work has implicated both the octopaminergic and dopaminergic modulatory systems in reinforcement signaling. Here we use genetics to analyze the anatomy, up to the single-cell level, of the octopaminergic/tyraminergic system in the larval brain and suboesophageal ganglion. Genetic ablation of subsets of these neurons allowed us to determine their necessity for appetitive olfactory learning. These experiments reveal that a small subset of about 39 largely morphologically distinguishable octopaminergic/tyraminergic neurons is involved in signaling reward in the Drosophila larval brain. In addition to prior work on larval locomotion, these data functionally separate the octopaminergic/tyraminergic system into two sets of about 40 neurons. Those situated in the thoracic/abdominal ganglion are involved in larva locomotion, whereas the others in the suboesophageal ganglion and brain hemispheres mediate reward signaling

Anatomy and behavioral function of serotonin receptors in <i>Drosophila melanogaster</i> larvae

<div><p>The biogenic amine serotonin (5-HT) is an important neuroactive molecule in the central nervous system of the majority of animal phyla. 5-HT binds to specific G protein-coupled and ligand-gated ion receptors to regulate particular aspects of animal behavior. In <i>Drosophila</i>, as in many other insects this includes the regulation of locomotion and feeding. Due to its genetic amenability and neuronal simplicity the <i>Drosophila</i> larva has turned into a useful model for studying the anatomical and molecular basis of chemosensory behaviors. This is particularly true for the olfactory system, which is mostly described down to the synaptic level over the first three orders of neuronal information processing. Here we focus on the 5-HT receptor system of the <i>Drosophila</i> larva. In a bipartite approach consisting of anatomical and behavioral experiments we describe the distribution and the implications of individual 5-HT receptors on naïve and acquired chemosensory behaviors. Our data suggest that <i>5-HT</i>_<i>1A</i>, <i>5-HT</i>_<i>1B</i>, and <i>5-HT</i>_<i>7</i> are dispensable for larval naïve olfactory and gustatory choice behaviors as well as for appetitive and aversive associative olfactory learning and memory. In contrast, we show that 5-HT/<i>5-HT</i>_<i>2A</i> signaling throughout development, but not as an acute neuronal function, affects associative olfactory learning and memory using high salt concentration as a negative unconditioned stimulus. These findings describe for the first time an involvement of 5-HT signaling in learning and memory in <i>Drosophila</i> larvae. In the longer run these results may uncover developmental, 5-HT dependent principles related to reinforcement processing possibly shared with adult <i>Drosophila</i> and other insects.</p></div

5-HT/<i>5-HT</i>_<i>2A</i> signaling throughout development specifically impairs odor-salt learning and memory.

<p>(A) The <i>TRH-Gal4</i> line was crossed with UAS-<i>mCD8</i>::<i>GFP</i> to visualize its expression pattern (green; anti-GFP staining) in addition to a reference labeling of the CNS (magenta; anti-ChAT/anti-FasII double-staining). (B) The <i>TRH-Gal4</i> line was crossed with UAS-<i>hid</i>,<i>rpr</i> to genetically induce apoptosis in 5-HT cells. In addition, the <i>Gal4</i> line and UAS-<i>hid</i>,<i>rpr</i> were crossed with <i>w</i>^<i>1118</i> to obtain heterozygous genetic control larvae. Above the panel a color scheme describes the three different groups used in the experiment. Ablation of 5-HT cells completely abolished aversive olfactory learning and memory reinforced by high salt concentration. (C) shows an overview on the experimental procedure that was used in larvae to test for odor-electric shock learning and memory. (D) Ablation of <i>5-HT</i>_<i>2A</i><i>-Gal4</i> positive cells via UAS-<i>hid</i>,<i>rpr</i> did not impair odor-electric shock learning and memory. Sample size (n = 15) is indicated at the bottom of each box plot. Differences against zero are given at the top of each box plot. Differences between the groups are shown at the bottom of the panel. Visualization of statistical evaluations: if only n.s. is shown the initial KWT did not suggest for a difference between the three groups (p > 0.05). When differences between each group are shown this provides the results of the BWRT as the initial KWT suggested for significance (p < 0.05). *** (p < 0.001), ** (p < 0.01), * (p < 0.05), n.s. (not significant p ≥ 0.05). Scale bar: 50 μm.</p

<i>Gal4</i> expression patterns of four potential 5-HT receptor lines.

<p><i>5-HT</i>_<i>1A</i>-, <i>5-HT</i>_<i>1B</i>-, <i>5-HT</i>_<i>7</i>-, and <i>5-HT</i>_<i>2A</i><i>-Gal4</i> positive cells are shown in A, B, C, and D, respectively. (A-C) <i>Gal4</i> lines were crossed with UAS-<i>mCD8</i>::<i>GFP</i> to analyze their expression patterns (green; anti-GFP staining) in addition to reference labeling of the central nervous system (CNS) (magenta; anti-ChAT/anti-FasII double-staining). In (D) <i>5-HT</i>_<i>2A</i><i>-Gal4</i> was crossed with UAS-<i>myr</i>::<i>tomato</i> to visualize its expression pattern (green; anti-dsRed staining) within the larval CNS (magenta; anti-ChAT/anti-FasII double-staining). For all four lines the first column shows a frontal view onto a z-projection of the entire CNS. In addition, for each line representative z-projections of close-ups of the ventral nerve cord (VNC), one hemisphere (HEMI), the suboesophageal ganglion (SOG), one antennal lobe (AL), the calyx (CA) of the mushroom body (MB), the lobes of the MB, and the larval optic neuropil (LON) are shown from left to right. Below each close-up only the GFP channel is shown as an inverted black and white image to visualize innervation patterns with higher contrast and no neuropil background. White arrows highlight aspects of the expression patterns that are further described in the results. Additional abbrevations: VL vertical lobe, ML medial lobe, PED peduncle; Scale bars: 50 μm (in A, B, C, D) and 20 μm (in all other panels).</p

Ablation of potential <i>5-HT</i>_<i>2A</i> receptor cells throughout development impairs aversive olfactory learning and memory.

<p><i>5-HT</i>_<i>1A</i>-, <i>5-HT</i>_<i>1B</i>-, <i>5-HT</i>_<i>7</i>-, and <i>5-HT</i>_<i>2A</i><i>-Gal4</i> lines were crossed with UAS-<i>hid</i>,<i>rpr</i> to genetically induce apoptosis in potential 5-HT receptor cells. In addition, <i>Gal4</i> lines and UAS-<i>hid</i>,<i>rpr</i> were crossed with <i>w</i>^<i>1118</i> to obtain heterozygous genetic control larvae. (A) provides a color scheme for the three different groups used in each experiment. Appetitive olfactory learning and memory using fructose reinforcement is shown at the top (B-E). Aversive olfactory learning and memory is shown at the bottom (F-I). In most cases, experimental larvae and genetic control groups behaved similar. However, ablation of <i>5-HT</i>_<i>2A</i><i>-Gal4</i> positive cells specifically impaired aversive olfactory learning and memory (I), while leaving appetitive olfactory learning and memory intact (E). Sample size (n = 10–14) is indicated at the bottom of each box plot. Differences against zero are given at the top of each box plot. Differences between all three groups or individual groups are shown at the bottom of the panel. Visualization of statistical evaluations: if only n.s. is shown the initial Kruskal-Wallis test (KWT) did not suggest for a difference between the three groups (p > 0.05). When differences between each group are shown this provides the results of the Wilcoxon rank-sum tests with Bonferroni corrections (BWRT) as the initial KWT suggested for singnificance (p < 0.05). *** (p < 0.001), ** (p < 0.01), * (p < 0.05), n.s. (not significant p ≥ 0.05).</p

Impaired <i>5-HT</i>_<i>2A</i> receptor function throughout development impairs aversive olfactory learning and memory.

<p>Homozygous <i>5-HT</i>_<i>2A</i> receptor gene mutants and wild-type control larvae (<i>WT CS</i>) were used to analyze aversive (B) and appetitive (C) olfactory learning and memory, olfactory amyl acetate (AM, in D) and benzaldehyde (BA in E) preferences and gustatory sodium chloride (SALT, in F) and fructose (FRU, in G) preferences. (A) provides a color scheme for the two different groups used in each experiment. Whereas mutant larvae showed olfactory and gustatory preferences as well as appetitive olfactory learning and memory comparable to WT CS larvae, aversive olfactory learning and memory was significantly reduced. Sample size (n = 11–17) is indicated at the bottom of each box plot. Differences against zero are given at the top of each box plot. Differences between mutant and wild type larvae are shown at the bottom of the panel. *** (p < 0.001), ** (p < 0.01), * (p < 0.05), n.s. (not significant p ≥ 0.05).</p

Acute blockage of synaptic output of 5-HT cells and potential <i>5-HT</i>_<i>2A</i> receptor cells does not affect chemosensory behavior.

<p>The <i>5-HT</i>_<i>2A</i><i>-Gal4</i> and <i>TRH-Gal4</i> lines were crossed with UAS-<i>shi</i>^<i>ts</i> to genetically interfere with synaptic transmission only during training and testing but not during development. In addition, the <i>Gal4</i> lines and UAS-<i>hid</i>,<i>rpr</i> were crossed with <i>w</i>^<i>1118</i> to obtain heterozygous genetic control larvae. (A) provides a color scheme for the different groups used in each experiment. (B) shows the temperature regime that was applied to block synaptic output at the restrictive temperature of 35°C specifically during training (30 min) and testing (5 min). Immediately before the experiment, larvae were incubated (2 min) in a water-bath at 37°C. Aversive olfactory learning and memory reinforced by high salt concentration (C and D), olfactory preferences for AM and BA (E–H) and gustatory preferences for SALT (I and J) were analyzed. In none of the cases experimental larvae behaved significantly different compared to both genetic control groups. We thus reason that blockage of synaptic output of 5-HT cells and <i>5-HT</i>_<i>2A</i><i>-Gal4</i> positive cells does not impair all tested chemosensory behaviors. Sample size (n = 12–18) is indicated at the bottom of each box plot. Differences against zero are given at the top of each box plot. Differences between all three groups or individual groups are shown at the bottom of the panel, except for SALT, where it is placed above the box plots. Visualization of statistical evaluations: if only n.s. is shown the initial KWT did not suggest for a difference between the three groups (p > 0.05). When differences between each group are shown this provides the results of the BWRT as the initial KWT suggested for singnificance (p < 0.05). *** (p < 0.001), ** (p < 0.01), * (p < 0.05), n.s. (not significant p ≥ 0.05).</p