Frontiers In Cellular Neuroscience Template

Abstract
Abstract
Octopamine (OA) and its precursor tyramine (TA) are neurotransmitters operating in many different neuronal and physiological processes. We investigated the role of those two transmitters in Drosophila sugar responsiveness. Tyrosine-\(\(\beta\)\)-hydroxylase (t\(\(\beta\)\)h ) mutants are unable to convert TA into OA. Starved mutants show a reduced sugar response and their hemolymph sugar concentration is elevated compared to control flies. When starved to death, they survive longer. Temporally controlled rescue experiments revealed an action of the OA/TA-system during the sugar response, while spatially controlled rescue experiments suggest actions also outside of the nervous system. Additionally, the analysis of four OA- and four TA-receptor mutants suggests an involvement of both receptor types in the animals' physiological and neuronal response to starvation. These results complement the pharmacological investigations in Apis mellifera described in our companion paper (Buckemheimer et al.).

Methods


Results

tßhnM18 mutants respond less to sucrose.
First, we explored the involvement of OA and TA in the modulation of sugar responsiveness. We used tßhnM18 mutant flies that lack OA and accumulate TA (Monastirioti et al., 1996), tethered them to a hook glued between head and thorax and tested their proboscis extension response to a serial dilution of sucrose after 20 h of starvation. The proportion of flies extending the proboscis increased with increasing sucrose concentration to reach almost 90% (Fig. 1A). tßhnM18 mutant flies responded almost 40% less than their control (Fig. 1A). The sum of all positive responses over the 7 sucrose presentations was significantly different (Wilcoxon rank sum test, p = 2*10-12, Fig. 1B). These results suggest that tßh-expressing and consequently that OA and TA play a crucial role in controlling sugar responses under starvation conditions.

Physiological response to starvation is weaker in tßhnM18 compared to wild type flies.
In order to determine whether tßhnM18 mutants might be less affected by starvation, we compared carbohydrate content (trehalose plus glucose) in the hemolymph of starved and fed flies. To this end, the hemolymph was extracted and all glucose and trehalose was enzymatically converted into spectrometrically measurable glucose. Trehalose is the “blood-sugar” in insects (Thompson, 2003) and is degraded under starvation conditions (Isabel et al., 2005; Meunier et al., 2007). Starvation treatment reduced the carbohydrate content in both, mutants and wild type flies (ANOVA: starvation p = 4*10-6, F = 27.449, genotype p = 0.14, F = 2.261, starvation x genotype p = 0.189, F = 1.777, Fig. 1A). The change in carbohydrate level after starvation was significantly smaller in tßh mutants compared to wild type controls (Welch Two Sample t-Test, p = 0.0497, Fig. 1B). As a second measure of starvation resistance, we recorded survival rate under starvation conditions with ad libitum access to water. As expected from their increased sugar content, tßhnM18 mutants survived longer than wild type controls (Wilcoxon rank sum test, p = 0.039, Fig. 1). The prolonged survival cannot be due to remaining eggs in the ovaries of tßh mutants (Partridge et al., 1987; Monastirioti, 2003) since we found the same survival effect on male tßh mutants (Fig. 1) and tßh-expression in mutant background did rescue egg-laying but not the survival phenotype (unpublished observations). Our experiments show that tßhnM18 mutants are less affected by starvation compared to wild type.

Acute tßh induction rescues the sugar response phenotype.
In order to rescue the tßhnM18 mutant sugar response phenotype, we induced ubiquitous tßh expression in the mutant background at different time points by means of the heat-induced construct hsp-tßh. With heat shock-induced tßh expression 3 h before testing, we found an increase in the mutants’ sugar response (paired Wilcoxon rank sum test, ap = 6*10-5, bp = 0, cp = 4*10-12, Fig. 1A). With heat shock-induced tßh expression exclusively during the starvation period, we did not find a rescue of the tßh mutant phenotype (paired Wilcoxon rank sum test with bonferroni correction, ap = 0.008, bp = 0, hsp-tßh x tßhp = 1, Fig. 1B). Those data indicate an acute role of the OA/TA-system during sugar response.

Sensitivity of taste receptor neurons is lower in tßhnM18 mutants.
OA is known to modulate different kinds of receptors in insects (Kass et al., 1988; Ramirez and Orchard, 1990; Pophof, 2000). In order to test a potential role of OA on gustatory receptor sensitivity we recorded the response of labellar sensilla to 100 mM sucrose of fed and starved flies by the tip-recording method (Hodgson et al., 1955; Hiroi et al., 2002). We found a decreased sensillar response to sucrose stimulation after starvation in tßhnM18 mutants, compared to wild type controls (Fig. 1A, Wilcoxon rank sum test, p = 0.008). The increase of spiking rate after starvation seems to underlie great genetic variation since different wild types exhibit different phenotypes (Fig. 1B, Wilcoxon rank sum test, w1118p = 0.039, CantonSp = 0.001; (Meunier et al., 2007; Inagaki et al., 2012; Nishimura et al., 2012).
Those data indicate a role of the OA/TA-system in the starvation-dependent modulation of receptor potentials.

Differential outcome of OA/TA-receptor manipulations on survival and sugar responsiveness
Because the tßh mutation leads to increased TA and decreased OA levels (Monastirioti et al., 1996), we performed additional experiments to disentangle the relative importance of each amine in the regulation of survival and sugar response. We tested published and novel mutants for several OA- and TA-receptors in our PER and survival under starvation conditions assays (Fig. 1, Table 1).
TyrRf05682 was generated by piggyback transposition (Thibault et al., 2004), real-time PCR revealed a 100-fold decrease of mRNA transcript (Zhang and Blumenthal, submitted). TyrRII29 and the double mutant TyrRII-TyrR124 are deletion mutants of 13567316-13576610 and the entire region 13567316-13579400, respectively. TyrRII has low mRNA transcript levels such that quantification of mutant levels is not possible (Zhang and Blumenthal, submitted). Octß2R3.22 and Octß2R4.3 are deletion mutants of 17,539 bp and 33,489 bp, respectively. Deletions were confirmed by genomic PCR. oamb and honoka were described elsewhere (Han et al., 1998; Kutsukake et al., 2000).
The two TA-receptor mutants TyrRf05682 and honoka showed a decreased sugar response (Wilcoxon rank sum test with correction for multiple measurements, TyrRf05682p = 0.003, honokap = 0.012) and an increased survival (TyrRf05682p = 0.002, honokap = 0.009) comparable to tßhnM18 mutants. Interestingly, the two phenotypes could be decorrelated indicated by the differential behavioral outcome of the tested receptors: Octß2R and the double mutant TyrII-TyrR124 show an increase in survival (Octβ2R3.22p = 0.001, Octß2R4.3p = 0.021, TyrRII-CG7431124p = 0.015) and a normal sugar response (Octß2R3.22p = 0.825, Octß2R4.3p = 0.06), while TyrRII29 shows normal survival (p = 0.354) and a decrease in sugar response (p = 0.007). Finally, the oamb mutants showed no phenotype at all (survival: oamb286p = 0.397, oamb584p = 0.867; sugar response: oamb286p = 0.506, oamb584p = 0.388), in contrast to a previously published report (Erion et al., 2012). The receptor mutant data suggest that flies can exhibit a wild type survival simultaneously with a lower sugar response (TyrRII24), or a higher survival simultaneously with a wild type sugar response (Octß2R3.22 and Octß2R4.3). That indicates that starvation affects sugar responsiveness and survival via different pathways.

Neuronal and non-neuronal tßh expression rescues PER
OA and TA act both inside and outside of the nervous system, functioning as either a neurotransmitter or -hormone in insects (Cole et al., 2005). Thus, we explored whether the sugar response phenotype of tßh mutants was a result of alterations in neurons inside or outside of the brain or in non-neuronal cells. For this experiment, we expressed UAS-tßh in tßhnM18 mutant males driven by different GAL4-lines. We found a significant increase in sugar response compared to the respective mutant control when we used the ubiquitous Actin-promoter (Wilcoxon rank sum test, p = 0.016, Fig. 1), the pan-neuronal nSyb-promoter (p = 0.013, Fig. 1), or the non-neuronal Tdc1-GAL4 driver (Cole et al., 2005) (p = 0.028, Fig. 1). In contrast, tßh expression in subsets of OA/TA-neurons in thoracic nerve cord and the brain by using either Tdc2- or NP7088-GAL4 did not significantly affect the mutants’ response (Tdc2p = 0.098, NP7088p = 0.58, Fig. 1) in contrast to a previous report (NP7088-Gal4, Scheiner et al., 2014). These results indicate that tßh expression induced in neurons in the central nervous system as well as in non-neuronal cells is sufficient to enhance the sugar responsiveness of tßhnM18 mutant flies.