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Fibronectin-leucine-rich-repeat-transmembrane proteins (FLRTs) are a family of three single pass transmembrane proteins with extracellular leucine rich repeats and a short intracellular domain of largely unknown function. They are broadly expressed in the developing and adult nervous system as well as in other tissues. FLRTs have been implicated in a variety of different developmental processes mainly via two functions: as homophilic cell adhesion molecules, and as repulsive ligands for Unc5-positive cells. Furthermore, they can regulate cell adhesion via control of surface expression of C Cadherin and are involved in FGF signaling. Previously we found that all FLRTs are localized to synapses in the mouse brain and thus investigated a potential involvement of FLRTs in synapse formation. However, using different in vitro and in vivo approaches ranging from HEK293 cell-neuron coculture assays to ultrastructural analysis of synapse density in FLRT3 knock-out mouse brains, I did not find any evidence for an involvement of FLRTs in synapse development. This is in contrast to published results but can be explained by differences in the experimental approaches and timing of the experiments. In collaboration with structural biologists we solved the crystal structure of the FLRT and Unc5 extracellular domains and a complex of both, to gain insight into the structural basis of the adhesive and repulsive functions of FLRTs. We found that homophilic FLRT FLRT and heterophilic FLRT-Unc5 interactions both occur via the FLRT LRR domain but at distinct structural surfaces. Thus, the interactions can be uncoupled. Based on the structural results we developed FLRT and Unc5 glycosylation mutants that specifically inhibit FLRT-FLRT or FLRT-Unc5 interaction and validated them in vitro. I then used these mutants in in utero electroporation experiments to prove that the repulsive effect of Unc5D overexpression in radially migrating neurons that was discovered previously is indeed, at least partially, mediated by FLRT2. Furthermore I found that overexpression of FLRTs inhibits radial migration of cortical pyramidal neurons and this effect is dependent on FLRT-FLRT homophilic interaction and the FLRT intracellular domain but independent of FLRT-Unc5 binding. In summary, the work presented here provides new insights into adhesive and repulsive functions of the FLRT family of proteins in the regulation of cell migration during cortical development.

Before newly formed memories can last for the long-term, they must undergo a period of consolidation. It has been shown that sleep facilitates this process. One hypothesis about how this may occur is that learning-related neuronal activity is replayed during following sleep periods. Such a reactivation of neural activity patterns has been repeatedly shown in the hippocampal formation in animals. Hippocampally-induced reactivation can also be observed in other brain areas like the neocortex and basal ganglia. On the behavioral level, sleep has been found to benefit performance on a broad range of memory tasks that rely on different neural systems. Up to now, however, it is unclear whether the same mechanisms mediate effects of sleep on consolidation in different memory systems. In this thesis, we investigated both the effects and the mechanisms of sleep-dependent consolidation in multiple memory systems. We find that sleep benefits performance on a broad range of procedural and declarative memory tasks (studies 1 and 2). These beneficial effects of sleep go beyond a reduction of retroactive interference as effected by quiet wakeful meditation (study 1). In study 2, we demonstrate that the processes underlying these beneficial effects of sleep are different for different memory systems. We assessed performance on typical declarative and procedural memory tasks during one week after participants slept or were sleep deprived for one night after learning. Sleep-dependent consolidation of hippocampal and non-hippocampal memory follows different time-courses. Hippocampal memory shows a benefit of sleep only one day after learning. Performance after sleep deprivation recovers following the next night of sleep, so that no enduring effect of sleep can be observed. However, sleep deprivation before recall does not impair performance. For non-hippocampal memory, on the other hand, long-term benefits of sleep after learning can be observed even after four days. Here, delayed sleep cannot rescue performance. This indicates a dissociation between two sleep-related consolidation mechanisms, which rely on distinct neuronal processes. We studied the neuronal processes underlying sleep effects on declarative memory in study 3, where we investigate learning-related electrophysiological activity in the sleeping brain. With the help of multivariate pattern classification algorithms, we show that brain activity during sleep contains information about the kind of visual stimuli that were learned earlier. We thus find that learned material is actively reprocessed during sleep. In a next step, we examined whether procedural memory can also benefit from reactivation during sleep. We find that a procedural memory task that has been found to activate the hippocampus can be strengthened by externally cueing the reactivation process during sleep. Similar to study 2, this indicates that it is not the traditional distinction between declarative and procedural memory that determines how memories are consolidated during sleep. Rather, memory systems, and in particular hippocampal contribution, decide the sleep-dependent consolidation process. In the first four studies, we examined how sleep affects memory in different memory systems. In our last study, we went one step further and investigated whether multiple memory systems can also interact during consolidation in sleep. We devised a task during which both implicit and explicit memory develop during learning. Results show that sleep not only strengthens implicit and explicit memory individually, it also integrates these formerly separate representations of the learning task. Implicit and explicit memory are negatively correlated immediately after training. Sleep renders this association positive and allows cooperation between the two memory traces. We observe this change both in behavior, using structural equation modeling, and on the level of brain activity, measured by fMRI. After sleep, the hippocampus is more strongly activated during recall of implicit memory, whereas the caudate nucleus shows stronger activity during explicit memory recall. Moreover, both regions show correlated stimulus-induced responses in a task that allows memory systems cooperation. These results provide conclusive evidence that sleep not only strengthens memory, but also reorganizes the contributing neural circuits. In this way, sleep actually changes the quality of the memory representation.

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