Octopamine less flies survive longer
on Tuesday, March 13th, 2012 8:22 | by Christine Damrau
Octopamine is a biogenic amine involved in insect physiology and behavioral control. In Drosophila, it was suggested to be necessary for appetitive olfactory learning (Schwaerzel et al., 2003). The available fly mutant tßh cannot convert tyramine into octopamine. We have found that the preference of these mutant flies for sugar (tested in a T-maze) as well as the responsiveness to a serial dilution of sugar (tested in a proboscis extension assay) is decreased compared to their genetic control “w+” (unpublished). We wondered whether the flies have different physiological state and hence different motivation, or if octopamine is involved in the neuronal coding of motivation per se. We therefore want to examine the physiological state of these flies.
In a first approach, we starved flies to death in bottles containing only a cotton pad moisturized with water. Dead flies were counted periodically and kept in the bottles. The survival curve shows that the tßh mutant flies die later (Fig.1a): The time point for 50 % of the flies to die is significantly higher in tßh mutant flies than the one of the control w+. Gender had no effect (ANOVA, gender: p=0.13, genotype: p<0.04). Data from male and female were thus pooled in the figures. These results show that starvation affects differently control and mutant flies. Obviously tßh mutants are more resistant to starvation.
The different physiological state could be due to different activity levels and consequently less energy requirements in tßh flies. To test for this hypothesis, we will use a second approach and measure trehalose content in the hemolymph of the flies. It has been shown that trehalose level decreases with starvation (Meunier et al., 2007). Furthermore there is a correlation found between trehalose level and survival (Isabel et al., 2004). A different metabolic rate in tßh mutants may explain a different trehalose content and the higher survival rate after the same starvation time.
In the future, we hope to be able to titrate the physiological state of the fly using the measure of the trehalose content in their hemolymph, in order to test the sugar responsiveness of mutant and control flies with adapted physiological state. This will allow us to separate the role of octopamine on the fly physiology and its role on the fly motivation.
Category: Biogenic Amines
Have you tested activity levels just by looking at general behavior – walking , preening etc
Also, I guess I don’t quite understand the line of reasoning that connects sugar response to physiological state to then resistance to starvation in these octopamine mutants.
It would seem that if you block an important amine like this throughout the brain, it would have multiple effects that don’t have to be linked.
Walking behavior was tested in Buridan’s paradigm. Flies with cut wings are sat into a round water surrounded arena. They are supposed to walk back and forth to two opposite black stripes on the wall of the arena. Several behavioral aspects can be measured in that kind of assay.
In general activity there was no difference found but the walking speed is decreased in tßh mutant flies compared to the control. That gives a hint that the mutants may be less exhausted after the same time of movement. That could be related to the observation in the survival. Flies that are moving slower abuse less energy resources and may survive longer.
Starvation and consecutively physiological state are directly determining the sugar response of a fly. That coherence was shown by many publications. The longer a fly is starved the stronger is the sugar response (among others Colomb et al., 2009; Menieur eta l., 2007).
Starvation resistance is one option to measure physiological state. The longer the animal survives the less starved it probably is although the time for starvation was the same. It would mean that not the length but the strength of starvation is important for sugar response.
The correlation of starvation and sugar response makes it very reasonable to look for other aspects of physiological state like survival.
Hey Christine,
This is quite curious. So am I correct in stating your current hypothesis is that 1. The flies don’t move as much (and possibly don’t do very much at all) and save energy 2.They don’t use their limited resources to make octopamine. Hence they live longer?
It’s interesting that neuromodulator production is such a physiological burden but surely that would mask any role octopaminergic neurons have on physiological state by vastly reducing the metabolic requirements of the fly’s day-to-day life.
I guess I’m not sure if the goal here is to control for that (using trehalose experiments) or to examine it.
If the latter perhaps you could skirt around these overall metabolic rate issues using a localised RNAi/spatiotemporal knockdown experiment you could see which neurons are involved in motivation/metabolic rate control (if at all) etc.
However, I don’t feel this represents a significant upheaval in terms of appeditive memory as perhaps you hint at in the first paragraph. Did you starve the flies before performing the T-maze experiments?
Also I am wondering can you explain the lack of difference in the 2003 paper? (if you starved them I guess)
Finally, I should mention Schroll et al. 2006 which shows Octopaminergic neuron activation was sufficient for appeditive learning. This seems to support a role for Octopaminergic signalling in providing the US for appeditive learning.
Very cool of you guys to publish data on the net! I wish this was more common.
Mike
Octopamine is also active outside the nervous system so that the tßh mutants may survive longer either due to slower movements (we quantified that in Buridan’s paradigm) which could be a neuronal effect, or due to a difference in the metabolic rate, which would be a physiological effect, but not neuronal.
(We do not think that the energy savings are that great when no octopamine has to be synthesized.)
We want to quantify the potential difference in starvation level by measuring the trehalose content in the hemolymph. If there is a difference between octopamine less and control flies we will adjust the state of the flies and measure whether there is still a difference in performance.
Future rescue experiments (but not RNAi) will tell us if the effect is neuronal and if the starvation effect can be dissociated from activity differences. If the survival effect is neuronal we want to narrow down the subsets of octopaminergic neurons responsible for those mechanisms.
In a T-maze there is no preference or learning detectable without starvation. If the starvation level may be different between tßh mutants and w+ control, a different performance in the T-maze would appear so that we think that the investigation of starvation levels in those mutants is important for interpretation of learning experiments. The Schroll paper shows sufficiency of octopaminergic neurons but the question of necessity stays unclear especially since tßh mutants sometimes show appetitive memory (unpublished).
Which paper do you mean? And what lack of difference? If you mean Schwaerzel et al., 2003: This assay is already shown to be not sensitive enough to show a difference (compare Colomb et al., 2009 and Krashes et al., 2008).