Circadian plasticity in the neuromuscular junction of Drosophila melanogaster

University dissertation from Stockholm : Zoologiska institutionen

Abstract: A biological clock exists in many organisms and controls rhythmic behaviors such as the activity pattern of locomotion. At the cellular level, it has been shown that circadian rhythms in the morphology of neurons exist in the fruit fly Drosophila melanogaster. The studies presented in this thesis contribute to an increased understanding of this novel aspect of neuronal plasticity and its regulation.We demonstrated the existence of a circadian rhythm in the morphology of neuromuscular terminals that innervate two identified flight muscles. Synaptic boutons are larger during the day than during the night under light:dark conditions (LD) as well as in constant darkness (DD). However, this rhythm is abolished in normal flies older than 30 days and in arrhythmic ones with null mutations in the clock genes period (per) or timeless (tim). Furthermore, these clock mutants show a completely different branching pattern indicating that the proteins PER and TIM not only function in well-described mechanisms in the circadian clock but also on neuronal morphology.We found that boutons grow during a period of six hours around light´s on and decrease again during six hours around light´s off. In order to test a possible relationship between synaptic activity and bouton rhythm, we manipulated synaptic activity of the fly during critical periods of morning and evening times when the flies show an increased activity pattern. We also decapitated the animals, which results in flies that do not move. Bouton size is largely independent of synaptic activity and seems to be driven by a peripheral pacemaker.We found the expression of tim in glial cells and focused on their possible role as a peripheral pacemaker. Rescue and knock down experiments showed slight effects on the rhythm of bouton size indicating that the expression of tim in glial cells could be part of the mechanism governing the rhythmic change of motor terminals.Furthermore, we investigated the question of whether bouton rhythm also involves changes in synapses. After paralyzing flies for a twelve-hour interval, we investigated the number of boutons and synapses as well as the distribution of synapses per bouton. Synapse numbers did not change but the other parameters were affected by paralysis, which might suggest that synaptic activity and/or endocytosis are involved in the formation of boutons and synapses. Furthermore, we noticed that the majority of boutons either contain none or only a small number of synapses.

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