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Ciliary neurotrophic factor (Cntf) acts as a differentiation and survival factor for different types of neurons and glial cells. It is expressed by peripheral Schwann cells and astrocytes in the central nervous system and mediates its effects via a receptor complex involving CntfRα, LifRß and gp130, leading to downstream activation of Stat3. Recent studies by our group have shown that Cntf modulates neuronal microtubule dynamics via Stat3/stathmin interaction. In a mouse model for motor neuron disease, i.e. pmn, Cntf is able to rescue axonal degeneration through Stat3/stathmin signaling. While these findings suggest a role of Cntf in controlling axonal functions in the neuromuscular system, additional data indicate that Cntf might also play a role in synaptic plasticity in the hippocampus. Electrophysiological recordings in hippocampal organotypic cultures and acute slices revealed a deficit in long-term potentiation (LTP) in Cntf -/- mice. This deficit was rescued by 24 h stimulation with Cntf, combined with an acute application of Cntf during LTP-measurements indicating that Cntf is both necessary and sufficient for hippocampal LTP, and possibly synaptic plasticity. Therefore, Cntf knockout mice were investigated to elucidate this possible role of Cntf in hippocampal LTP and synaptic plasticity.
First, we validated the presence of Cntf in the target tissue: in the hippocampus, Cntf was localized in Gfap-positive astrocytes surrounding small blood vessels in the fissure and in meningeal areas close to the dentate gyrus. Laser micro-dissection and qPCR analysis showed a similar distribution of Cntf-coding mRNA validating the obtained immunofluorescent results. Despite the strong LTP deficit in organotypic cultures, in vivo behavior of Cntf -/- mice regarding hippocampus-dependent learning and anxiety-related paradigms was largely inconspicuous. However, western blot analysis of hippocampal organotypic cultures revealed a significant reduction of pStat3 levels in Cntf -/- cultures under baseline conditions, which in turn were elevated upon Cntf stimulation. In order to resolve and examine synaptic structures we turned to in vitro analysis of cultured hippocampal neurons which indicated that pStat3 is predominantly located in the presynapse. In line with these findings, presynapses of Cntf -/- cultures were reduced in size and when in contact to astrocytes, contained less pStat3 immunoreactivity compared to presynapses in wildtype cultures.
In conclusion, our findings hypothesize that despite of a largely inconspicuous behavioral phenotype of Cntf -/- mice, Cntf appears to have an influence on pStat3 levels at hippocampal synapses. In a next step these two key questions need to be addressed experimentally: 1) is there a compensatory mechanism by members of the Cntf family, possibly downstream of pStat3, which explains the in vivo behavioral results of Cntf -/- mice and can likewise account for the largely inconspicuous phenotype in CNTF-deficient humans? 2) How exactly does Cntf influence LTP through Stat3 signaling? To unravel the underlying mechanism further experiments should therefore investigate whether microtubule dynamics downstream of Stat3 and stathmin signaling are involved in the Cntf-induced modulation of hippocampal synaptic plasticity, similar to as it was shown in motoneurons.
Behavioral adaptation to environmental changes is crucial for animals’ survival. The prediction of the outcome of one owns action, like finding reward or avoiding punishment, requires recollection of past experiences and comparison with current situation, and adjustment of behavioral responses. The process of memory acquisition is called learning, and the Drosophila larva came up to be an excellent model organism for studying the neural mechanisms of memory formation. In Drosophila, associative memories are formed, stored and expressed in the mushroom bodies. In the last years, great progress has been made in uncovering the anatomical architecture of these brain structures, however there is still a lack of knowledge about the functional connectivity.
Dopamine plays essential roles in learning processes, as dopaminergic neurons mediate information about the presence of rewarding and punishing stimuli to the mushroom bodies. In the following work, the function of a newly identified anatomical connection from the mushroom bodies to rewarding dopaminergic neurons was dissected. A recurrent feedback signaling within the neuronal network was analyzed by simultaneous genetic manipulation of the mushroom body Kenyon cells and dopaminergic neurons from the primary protocerebral anterior (pPAM) cluster, and learning assays were performed in order to unravel the impact of the Kenyon cells-to-pPAM neurons feedback loop on larval memory formation.
In a substitution learning assay, simultaneous odor exposure paired with optogenetic activation of Kenyon cells in fruit fly larvae in absence of a rewarding stimulus resulted in formation of an appetitive memory, whereas no learning behavior was observed when pPAM neurons were ablated in addition to the KC activation. I argue that the activation of Kenyon cells may induce an internal signal that mimics reward exposure by feedback activation of the rewarding dopaminergic neurons. My data further suggests that the Kenyon cells-to-pPAM communication relies on peptidergic signaling via short neuropeptide F and underlies memory stabilization.
In this thesis I studied psychological aspects in the behaviour of Drosophila, and especially Drosophila larvae. After an introduction where I present the general scientific context and describe the mechanisms of olfactory perception as well as of classical and operant conditioning, I present the different experiments that I realised during my PhD. Perception The second chapter deals with the way adult Drosophila generalise between single odours and binary mixtures of odours. I found that flies perceive a mixture of two odours as equally similar to the two elements composing it; and that the intensity as well as the physico-chemical nature of the elements composing a mixture affect the degree of generalisation between this mixture and one of its elements. These findings now call for further investigation on the physiological level, using functional imaging. Memory The third chapter presents a series of experiments in Drosophila larvae in order to define some characteristics of a new protocol for classical aversive learning which involves associating odours with mechanical disturbance as a punishment. The protocol and the first results should open new doors for the study of classical conditioning in Drosophila larvae, by allowing the comparison between two types of aversive memory (gustatory vs. mechanical reinforcement), including a comparison of their neurogenetic bases. It will also allow enquiries into the question whether these respective memories are specific for the kind of reinforcer used. Agency The fourth chapter documents our attempts to establish operant memory in Drosophila larvae. By analysing the first moments of the test, I could reveal that the larvae modified their behaviour according to their previous operant training. However, this memory seems to be quickly extinguished during the course of the test. We now aim at repeating these results and improving the protocol, in order to be able to systematically study the mechanisms allowing and underlying operant learning in Drosophila larvae. In the fifth chapter, I use the methods developed in chapter four for an analysis of larval locomotion. I determine whether larval locomotion in terms of speed or angular speed is affected by a treatment with the “cognitive enhancer” Rhodiola rosea, or by mutations in the Synapsin or SAP47 genes which are involved in the formation of olfactory memory. I also characterize the modifications induced by the presence of gustatory stimuli in the substrate on which the larvae are crawling. This thesis thus brings new elements to the current knowledge of Drosophila