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In order to select the appropriate behavior, it is important to choose the right behavior at the right time out of many options. It still remains unclear nowadays how exactly this is managed. To address this question, I expose flies (Drosophila melanogaster) to uncontrollable stress to study their behavior under restrictive circumstances by using the so-called shock box. Exposing animals to uncontrollable stress may have an impact on subsequent behavior and can last for some time. The animal learns that whatever it does, it cannot change the situation and therefore can develop something called learned helplessness. The term was first conceptualized by two American psychologists Maier and Seligman (1967), who discovered this phenomenon while doing experiments with dogs. They found out that dogs which are exposed to inescapable stress, later fail in a learning task (‘shuttle box’).
In this work the walking patterns of three different types of experimental flies, walking in a small dark chamber, were evaluated. Using the triadic design (Seligman and Maier, 1967), flies were either exposed to electric shock randomly (yoked), could turn it off by being active (master) or did not receive punishment at all (control). Master flies were shocked whenever they sat for more than 0.9 seconds. At the same time yoked flies received a shock as well independent of what they were doing, to ensure the same amount of shocks received and to create random punishment pattern for the yoked group. With this so-called no-idleness paradigm flies were conditioned either 10 minutes, which resulted in a short (3 minutes) after-effect, or 20 minutes that turned out to be more stable (10 minutes).
In a second part, the behavior during the 20 minute conditioning and a 10 minutes post-test was described in detail. Female flies of the yoked group developed lower activity levels, longer pauses and walked more slowly than master and control flies during conditioning. In the time after the shocks while still in the box, the yoked flies also reduced the frequency and duration of walking bouts as well as their walking speed. Additionally, they took more time to resume walking after the onset of an electric shock than master flies (escape latency) and turned out to make less pauses lasting between 1-1.5 seconds which supports the finding concerning the escape latency.
Male flies, tested under the same conditions, showed a slightly weaker after-effect regarding the difference between master and yoked during conditioning and post-test when compared to female flies.
When comparing the 20 minutes conditioning with subsequent 10 minutes test in the heat and the shock box in parallel, one finds the same effect: Flies which do not have control over the shocks, lower their activity, make less but longer pauses and walk more slowly than their respective master flies. Despite the similar effect of heat and shock on the flies, some differences between the devices occurred, which can partly be explained by different humidity conditions as well as by different surfaces within the chambers.
When the control over the shocks is given back to the yoked flies, it takes them about seven minutes to realize it. One could also show that dopamine levels in the brain were reduced in comparison to flies which did not receive shocks. Yoked flies also were impaired in a place learning task (place learning) and their reaction to light (exit from the box towards the light) directly after conditioning.
After characterizing the walking behavior in the chambers, the study deals with the question whether the effects observed in the chambers transfer to different environments.
In free walk they only differed from flies which did not receive electric shocks and no effect of uncontrollability was transferred to courtship behavior. Handling as the cause could be excluded. Since handling could be exclude to be the cause of losing the effect, I assumed that the behavior shown in the boxes are context depend.
Not only were the after-effects of inescapable shock subject of the current research also the impact of the rearing situation on the response to electric shock was investigated in the present study. Flies which grew up in a single-reared situation turned out to be less affected by inescapable stress in both sexes.
In the next part, the first steps to unravel the neuronal underpinning were taken. A mutant – fumin – which is defective in the dopamine re-uptake transporter showed less reaction to inescapable foot shocks, while a mutant for the gene which encodes an adenylate cyclase (rutabaga2080) resulted in a good score during conditioning, but showed no stable after-effect. Downregulating the expression of the adenylate cyclase gene (rutabaga) in different parts of the mushroom bodies showed, that rutabaga is necessary in the α’β’-lobes for expressing the differences between master and yoked flies in the no-idleness paradigm. The study further confirmed previous findings, that rutabaga is needed in operant but not in classical conditioning.
As a result, the study could show that not the stimulus itself causes the state of uncontrollability but the fact that the fly learned that it was not in control of the stimulus. This state turned out to be context and time dependent.
Eine der größten Herausforderungen in der Neurobiologie ist es, die neuronalen Prozesse zu verstehen, die Lernen und Gedächtnis zugrundeliegen. Welche biochemischen Pfade liegen z.B. der Koinzidenzdetektion von Reizen (klassische Konditionierung) oder einer Handlung und ihren Konsequenzen (operante Konditionierung) zugrunde? In welchen neuronalen Unterstrukturen werden diese Informationen gespeichert? Wie ähnlich sind die Stoffwechselwege, die diese beiden Arten des assoziativen Lernens vermitteln und auf welchem Niveau divergieren sie? Drosophila melanogaster ist wegen der Verfügbarkeit von Lern-Paradigmen und neurogenetischen Werkzeugen ein geeigneter Modell-Organismus, zum diese Fragen zu adressieren. Er ermöglicht eine umfangreiche Studie der Funktion des Gens S6KII, das in der Taufliege in klassischer und operanter Konditionierung unterschiedlich involviert ist (Bertolucci, 2002; Putz et al., 2004). Rettungsexperimenten zeigen, dass die olfaktorische Konditionierung in der Tully Maschine (ein klassisches, Pawlow’sches Konditionierungsparadigma) von dem Vorhandensein eines intakten S6KII Gens abhängt. Die Rettung war sowohl mit einer vollständigen, als auch einer partiellen Deletion erfolgreich und dies zeigt, dass der Verlust der phosphorylierenden Untereinheit der Kinase die Hauptursache des Funktionsdefektes war. Das GAL4/UAS System wurde benutzt, um die S6KII Expression zeitlich und räumlich zu steuern. Es wurde gezeigt, dass die Expression der Kinase während des adulten Stadiums für die Rettung hinreichend war. Dieser Befund schließt eine Entwicklungsstörung als Ursache für den mutanten Phänotyp aus. Außerdem zeigte die gezielte räumliche Rettung von S6KII die Notwendigkeit der Pilzkörper und schloss Strukturen wie das mediane Bündel, die Antennalloben und den Zentralkomplex aus. Dieses Muster ist dem vorher mit der rutabaga Mutation identifizierten sehr ähnlich (Zars et al., 2000). Experimente mit der Doppelmutante rut, ign58-1 deuten an, dass rutabaga und S6KII im gleichen Signalweg aktiv sind. Vorhergehende Studien hatten bereits gezeigt, dass die unterschiedlichen Ergebnisse bei operanter und klassischer Konditionierung auf verschiedenen Rollen für S6KII in den zwei Arten des Lernens hindeuten (Bertolucci, 2002; Putz, 2002). Diese Schlussfolgerung wurde durch den mutanten Phänotyp der transgenen Linien in der Positionskonditionierung und ihr wildtypisches Verhalten in der klassischen Konditionierung zusätzlich bekräftigt. Eine neue Art von Lern-Experiment, genannt „Idle Experiment“, wurde entworfen. Es basiert auf der Konditionierung der Laufaktivität, stellt eine operante Aufgabenstellung dar und überwindet einige der Limitationen des „Standard“ Heat-Box Experimentes. Die neue Art des Idle Experimentes erlaubt es, „gelernte Hilflosigkeit“ in Fliegen zu erforschen, dabei zeigte sich eine erstaunliche Ähnlichkeit zu den Vorgängen in komplizierteren Organismen wie Ratten, Mäusen oder Menschen. Gelernte Hilflosigkeit in der Taufliege wurde nur in den Weibchen beobachtet und wird von Antidepressiva beeinflusst.
The learned helplessness phenomenon is a specific animal behavior induced by prior exposure to uncontrollable aversive stimuli. It was first found by Seligman and Maier (1967) in dogs and then has been reported in many other species, e.g. in rats (Vollmayr and Henn, 2001), in goldfishes (Padilla, 1970), in cockroaches (Brown, 1988) and also in fruit flies (Brown, 1996; Bertolucci, 2008). However, the learned helplessness effect in fruit flies (Drosophila melanogaster) has not been studied in detail. Thus, in this doctoral study, we investigated systematically learned helplessness behavior of Drosophila for the first time.
Three groups of flies were tested in heatbox. Control group was in the chambers experiencing constant, mild temperature. Second group, master flies were punished in their chambers by being heated if they stopped walking for 0.9s. The heat pulses ended as soon as they resumed walking again. A third group, the yoked fly, was in their chambers at the same time. However, their behavior didn’t affect anything: yoked flies were heated whenever master flies did, with same timing and durations. After certain amount of heating events, yoked flies associated their own behavior with the uncontrollability of the environment. They suppressed their innate responses such as reducing their walking time and walking speed; making longer escape latencies and less turning around behavior under heat pulses. Even after the conditioning phase, yoked flies showed lower activity level than master and control flies. Interestingly, we have also observed sex dimorphisms in flies. Male flies expressed learned helplessness not like female flies. Differences between master and yoked flies were smaller in male than in female flies. Another interesting finding was that prolonged or even repetition of training phases didn’t enhance learned helplessness effect in flies.
Furthermore, we investigated serotonergic and dopaminergic nervous systems in learned helplessness. Using genetic and pharmacological manipulations, we altered the levels of serotonin and dopamine in flies’ central nervous system. Female flies with reduced serotonin concentration didn’t show helpless behavior, while the learned helplessness effect in male flies seems not to be affected by a reduction of serotonin. Flies with lower dopamine level do not display the learned helplessness effect in the test phase, suggesting that with low dopamine the motivational change in learned helplessness in Drosophila may decline faster than with a normal dopamine level.