Title Promoter Affiliations Abstract "Single cell(-type) platform for biological applications." "Lut Arckens" "Animal Physiology and Neurobiology" "The recent soar in single cell and single cell-type analyses relies on breakthrough technologies to unveil tissue  heterogeneity. Until recently, researchers in biological and life sciences would work at the resolution of entire organs or tissues, for example by trying to establish how the brain displays plasticity and how that affects the entire organism. It is however no secret that organs and tissues consist of different cell types, which contribute to different extents to distinct processes.Recent technological advancements lead to robust, userfriendly Fluorescence Activated Cell Sorting (FACS) instruments, breaking open this niche field to a wide user base of researchers, now having the technology at hand for unmasking biological phenomena that would otherwise remain hidden in whole-tissue analyses. Thanks to accessible FACS technology, scientists can easily isolate single cells of interest to analyze individually, or grow clonal populations from them. In addition, sorting out tissues into their composite cell types can easily be done based on marker use.While FACS technology per se has been around for several years, the newest instruments are truly innovative in that they abolish the need of restricting access to specialist operators. It is clear that the future of biological research will be driven by findings of single cell(-type) studies, and via the acquisition of such a FACS instrument, our consortium aspires to remain a leading party in neurobiology research. " "Impact of vision loss on cellular processing in the visual cortex and its relation to multisensory processing and behavior" "Vincent Bonin" "Animal Physiology and Neurobiology" "The loss of senses such as seeing and hearing is one of the major sources of disability and is associated with high economic costs. With an aging population, there is an acute need for low-cost and effective therapies to help patients cope with and to compensate for sensory losses, as well as for new devices that can restore function. Key to these developments is an understanding of the physiological changes that occur in the brain upon loss of sensory inputs. In this research project, I will team up with two laboratories with extensive expertise in systems neuroscience and brain plasticity, combining the latest genetic, imaging and computational techniques, to address this question at the cellular level. I will use a mouse model to examine how the brain’s visual center, the visual cortex, responds to loss of eye inputs. I will examine how neurons in the visual cortex of mice adapt their processing to take on a new function, namely the ability to sense tactile stimuli. First, I will compare activity measured in sighted and blind animals at different locations in the cortex. Second, I will use a rodent behavioral assay to study the functional consequences of the physiological changes I have observed. It will provide all the critical data needed to validate an emerging animal model of adult plasticity. Finally, it will lead to valuable information to constrain the design of cortical prostheses and inspire new therapeutic approaches to facilitate recovery and regain function." "The molecular signature of neuropeptidergic control of long-term memory" "Liliane Schoofs" "Animal Physiology and Neurobiology" "Learning and memory processes are crucial for animals to adapt their choices and behavioral responses according to previous experiences to increase their chances of survival. Neuropeptides are essential signaling molecules in the brain and are emerging as important modulators of learned behaviors. For many neuropeptides, however, the central function in the nervous system remains largely unknown and their precise role is not well understood. In this study, we used the genetically tractable neurobiological model Caenorhabditis elegans to investigate the neuropeptidergic modulation of learning and memory.We hypothesized that the best candidate neuropeptides to study in learning and memory are those that are conserved across the Animal Kingdom and of which the cellular expression is largely observed in brain areas known to be involved in learning and memory. Such a neuropeptide system is the evolutionarily conserved myoinhibitory peptide (MIP) system, initially discovered in locusts and mainly studied in insects. Although MIP neuropeptides and their receptors have not been directly implicated in learning, we hypothesized that they may modulate learning circuits based on their expression in the insect mushroom body, an area of the central nervous system that is crucial for olfactory and gustatory conditioning in insects.C. elegans has three predicted MIP receptor orthologs: SPRR-1, SPRR-2, and SPRR-3 (sex peptide receptor related), belonging to the rhodopsin class of G protein coupled receptors (GPCRs) and named after their sequence similarity with the Drosophila MIP receptor which is also known as Sex Peptide Receptor. Previous studies in our lab showed that the SPRR-1 and SPRR-2 receptors can be activated by peptides encoded by the NLP-42 and NLP-38 precursors in vitro, respectively. In addition to activating the MIP-related receptors in vitro, these peptides exhibit the characteristic protostomian C-terminal MIP motif, which is why we suggest to rename the NLP-38 precursor as MIP-1 and NLP-42 as MIP-2. Here, we attempted to find the ligand(s) of the C. elegans SPRR-3 receptor as well as the putative human MIPRs GPR139 and GPR142. However, none of the ~350 currently identified and predicted C. elegans neuropeptides could activate these receptors in our in vitro GPCR activation assay.We next analyzed the in vivo expression patterns of SPRR-1, SPRR-2, SPRR-3, NLP-38, and NLP-42 using fluorescent reporter genes. Expression was revealed in the C. elegans nervous system including in chemosensory and interneurons that function in gustatory and olfactory learning, thereby pointing to a role for this system in gustatory and/or olfactory learning.Guided by our in vivo expression analysis and the previously reported MIP expression analysis in insects we compared the behavior of mutants with impaired MIP signaling with wild-type behavior in gustatory associative learning assays in C. elegans. We found that MIP-1 signaling through activation of the SPRR-2 receptor modulates two types of aversive, but not appetitive, gustatory associative learning. On the one hand, MIP-1/SPRR-2 signaling modulates short-term gustatory plasticity, wherein worms learn to avoid normally attractive NaCl concentrations when salt is paired with short-term food withdrawal. On the other hand, MIP-1/SPRR-2 as well as SPRR-3 signaling modulates aversion towards normally attractive NaCl concentrations when paired with a long, 6 hours, starvation period. We additionally showed that the memory formed upon this salt avoidance learning shows hallmarks of long-term memory. By contrast, an appetitive association between the presence of food and NaCl is not modulated by MIP signaling and does not display hallmarks of long-term memory.Taken together, our findings expand our current knowledge on the neuropeptidergic modulation of learning and memory. Our results suggest that MIP signaling promotes learning and memory and appears to specifically modulate aversive gustatory learning in C. elegans. Moreover, MIP signaling regulates both short-term memory and memory displaying characteristics of long-term memory. Given that the putative deuterostomian MIPRs are expressed in brain regions related to learning and memory but otherwise remain largely unexplored, these findings lay a foundation for further research into the involvement of MIP receptor signaling in avoidance learning in other organisms, including humans. With the recent increased interest in avoidance learning from psychology literature, this study contributes to our understanding of how aversive events in life become anchored in memory and contribute to normal as well as disordered psychological functioning." "Detection of new targets for the treatment of obesity using the cholecystokinin signal system in C. elegans." "Liliane Schoofs" "Animal Physiology and Neurobiology" "In summary, we will contribute with a present project proposal to a solution to one of the most comprehensive medical problems of modern western society" "Matrix metalloproteinases in optic nerve regeneration: functions and substrate identification." "Lieve Moons" "Animal Physiology and Neurobiology" "Dysfunction of the central nervous system (CNS) after injury or in neurodegenerative diseases increasingly impairs life quality in our aging society. Also optic neuropathies, like glaucoma, are becoming increasingly prevalent in our elderly population, affecting over 60 million people worldwide. They are characterized by retinal ganglion cell (RGC) axonal degeneration and death, ultimately resulting in irreversible blindness because adult mammals lack the capacity to repair/regrow damaged axons in the CNS. Although combinatorial approaches indicate that it might be possible to repair the injured mammalian optic nerve (ON), they have not yet induced substantial visual recovery, nor are they, up to now, suitable for clinical applications. Therefore, comparative studies focus on the spontaneously regenerating adult CNS of zebrafish, which proves to be a valuable research model to investigate regenerative strategies of damaged neurons and to identify novel pro-regenerative molecules. Notably, a recent transcriptome profiling study performed on regenerating adult zebrafish eyes at various time-points after optic nerve crush (ONC), pinpointed several potential pro-regenerative molecules, under which four matrix metalloproteinases (mmp-2, -9, -13a, -14). While MMPs have been implicated in axonal outgrowth during CNS development and are known to be upregulated during vertebrate CNS repair, their impact on in vivo CNS regeneration remains undocumented. Within this context, this PhD project then also intended to unravel the role of Mmp-2, -9, -13a and -14 in ON regeneration in adult zebrafish subjected to ONC.The first part of this thesis was dedicated to an in-depth characterization of RGC survival, axonal regrowth/extension and primary visual recovery in the zebrafish ONC model.Next, we obtained first in vivo insights in a contributory role for MMPs in RGC axonal regrowth as retinal broad-spectrum MMP inhibition seriously attenuated optic tectum reinnervation by regenerating RGC axons without influencing RGC survival. Moreover, combined use of Western Blotting and immunostainings revealed a phase-dependent expression pattern for each MMP in the retina after ONC, primarily implying Mmp-2 and -13a in RGC axonal regrowth.As such, via utilization of several loss-of-function approaches to interfere with the functioning of one specific MMP, e.g. pharmaceutical MMP inhibition, morpholino and genome editing technology, we were able to identify Mmp-2 and -13a as in vivo regulators of RGC axonal regeneration in adult zebrafish after ONC. Notably, since zebrafish lack an inhibitory environment, these data suggest a novel, neuron intrinsic, function for multiple MMPs in RGC axonal regrowth in addition to their role in breaking down environmental barriers as deduced from mammalian studies. The future identification of the underlying MMP substrates by means of quantitative proteomics and subsequent validation of promising candidate molecules in fish and mice might then also unravel new pro-regenerative molecules for the injured mammalian CNS.While being an important part of the neuronal circuitry, the response of dendrites upon induction of axonal regeneration in mammals is almost unattended. Notably, the few publications that do discuss this issue, indicate that dendritic morphology is seriously influenced by the pro-regenerative molecule used to trigger axonal regrowth, ranging from a reduction to an increase in size and complexity. As the eventual recovery of sight after ON injury will equally depend on the proper restoration of dendritic structure, we then also characterized the inherent response of dendrites during successful RGC axonal regeneration in adult zebrafish. Remarkably, both temporal expression analyses of synaptic and dendritic markers, as well as morphometric analysis of inner plexiform layer thickness, indicated that regeneration-competent injured neurons repeat the developmental order of neurite growth, where axogenesis is primary to dendritogenesis.Finally, a last part of this work focused on the elucidation of a potential role of MMPs in retinal dendritic remodeling in adult zebrafish after ONC. Both retinal broad- and narrow-spectrum Mmp(-2) inhibition seemingly prevented RGC dendrites from shrinking upon ON injury. Intriguingly, in both conditions, a disturbance of dendritic retraction occurred concurrently with a reduced RGC axonal regrowth, thus indicative of a potential antagonistic interplay between dendritic remodeling and axonal regrowth after ONC. In addition, due the consecutive progress of axonal and dendritic growth during development and regeneration, and based on a publication discussing TRAK1/2 driven polarized mitochondrial transport in neurons respectively regulating axonal and dendritic outgrowth, we hypothesized that an energetic trade-off might lie at the base of this apparent inter-dependency. As such, future experiments should interfere with mitochondrial trafficking in zebrafish RGC dendrites and analyze the effect on RGC axonal regeneration. If these data would result in a proof-of-concept, i.e. dendritic remodeling serving as fuel for axonal regeneration, we envision a major shift in the research focus of the neuroregenerative research field and the potential uncovering of various novel therapeutic targets. " "Glaucoma and the brain: a novel optogenetic approach towards retinal ganglion cell protection." "Lieve Moons" "Animal Physiology and Neurobiology" "Summary PhD thesis Eline DekeysterGlaucoma refers to a group of optic neuropathies, all characterized by progressive degeneration of retinal ganglion cells (RGCs) - the cells that send visual information from the eye to the brain - resulting in a gradual loss of vision. Ocular hypertension (OHT) is considered one of the major risk factors for development and progression of glaucoma, and thus lowering eye pressure through topical application of hypotensive eye drops or surgery, is the cornerstone of glaucoma therapy today. Unfortunately, a number of patients do not benefit from such treatments, and although it generally slows down disease progression, controlling eye pressure does not really halt RGC degeneration. This stresses the need for development of new therapies aimed at long‑term retinal neuroprotection and preservation of vision. Within this context, this PhD project intends to contribute to the knowledge on the pathological mechanisms underlying glaucomatous RGC death and to highlight possible novel strategies towards RGC protection.The first part of this thesis was dedicated to the optimization and characterization of two mouse models of glaucoma: the OHT model and the normotensive optic nerve crush (ONC) model, each focusing on different aspects of the disease.Next, a closer look was taken at the link between glaucoma and the brain. Although long considered purely an eye disease, glaucoma is increasingly becoming recognized as a disease of the entire visual system. Using the OHT model, the effect of elevated eye pressure on the central visual brain areas was evaluated. Reduced RGC synapse density, retinotopically correlated to degeneration of RGC soma, and intensive but transient astroglial reactivity were observed in the main subcortical RGC projection area, which for mice is the superior colliculus (SC). Furthermore, diminished neuronal activity in the primary visual cortex extending to specific extrastriate areas was seen early after OHT induction. Complete recovery of cortical activity over time demonstrated the capacity of the adult visual cortex to functionally reorganize in an attempt to adapt to glaucomatous RGC degeneration. Our results uncovered for the first time effects on visual cortex activity patterns in a murine OHT model and, yet again, highlight the importance of including the brain in glaucoma research.For their survival, RGCs depend on neurotrophic factors, e.g. neurotrophins, produced locally in the eye as well as in the projection areas in the brain, from where they are retrogradely transported along the RGC axons towards the cell bodies in the retina. According to the neurotrophin deprivation hypothesis, diminished retrograde delivery of neurotrophic support during an early stage of glaucoma pathogenesis is one of the main triggers that induce apoptotic signaling in RGCs. Therefore, interfering with neurotrophic signaling seems an attractive tool to achieve neuroprotection. In this PhD project, the well-known neurotrophin brain-derived neurotrophic factor (BDNF) was chosen as the lead molecule to study the role of neurotrophic factors in glaucoma. The levels of BDNF and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), were examined in the retina and SC of mice subjected to OHT or ONC. Both models differentially influenced BDNF and TrkB levels. Defining a specific role for BDNF signaling within glaucoma pathology remains difficult as various studies using a variety of animal models of glaucoma have yielded unique results, including those presented within this manuscript.In line with the hypothesis of deprived neurotrophic support being an important contributor to glaucomatous RGC death, exogenous neurotrophin administration to the eye has been shown to reduce loss of RGCs; however, the neuroprotective effect was mostly transient and insufficient for sustained RGC survival. Therefore, we hypothesized that treatment at the level of extraretinal neurotrophin sources in the brain might be beneficial, as target-derived neurotrophins are likely to induce signaling pathways that diverge from local neurotrophin signaling. Brain-directed treatment was approached in two ways: 1) viral vector-mediated upregulation of BDNF in the SC was used to boost retrograde delivery of BDNF to the retina; 2) a more broad strategy to optogenetically increase neuronal activity in the SC was implemented to enhance production of a whole spectrum of endogenous neurotrophic factors. In light of these hypotheses, viral vector technology was optimized for use in the mouse SC.Although the previously reported temporary neuroprotective effect of intravitreally-delivered recombinant BDNF was confirmed, viral vector-induced overexpression of BDNF in the SC did not lead to protection of the RGCs in our glaucoma models. This unfortunate result most likely resulted from decreased neurotrophin responsiveness upon vector mediated BDNF overexpression. Regarding the second strategy employing SC manipulation to aim for RGC protection, optogenetic methods were introduced and validated. The basic principle underlying optogenetics is the introduction of genes, e.g. through viral vector technology, encoding light-sensitive ion channels or opsins into cells, rendering them responsive to light of a specific wavelength. For the first time, the application of an opsin with specifically slow kinetic properties, the stabilized step-function opsin (SSFO), was validated for use in the mouse SC. A setup for chronic optogenetic stimulation in awake, freely moving animals, was developed, and shown to effectively stimulate collicular activity. Two consecutive behavioral responses were observed upon unilateral SC stimulation, a short escape response, followed by prolonged pursuit-like behavior.In conclusion, a large component of this thesis was dedicated to method optimization, such as the two mouse models of glaucoma, viral vector technology, and a setup for in vivo optogenetic stimulation. These methods will importantly contribute to future research within the research group. Furthermore, new findings on the role of higher order visual brain centers in glaucoma pathogenesis were discovered. Although we were not able to deliver a proof-of-concept for the neurotrophin deprivation hypothesis, important insights concerning the complexity of neurotrophic factor treatments were highlighted." "The regulation of food uptake and digestion as a target for alternative pest control." "Jozef Vanden Broeck" "Animal Physiology and Neurobiology" "The fundamental processes of feeding and digestion are necessary to acquire sufficient building blocks and energy to fuel anabolic processes, such as growth, development and reproduction. Animals require the ability to respond to both environmental and internal cues and relate these to their nutritional state. A rigid regulatory system is necessary in all animals to ensure that feeding and digestion are dynamically controlled. This regulation of food uptake and digestion is coordinated by the nervous and endocrine system. Neuropeptides are ubiquitous and essential messengers that can serve as neurotransmitters, neuromodulators or circulating neurohormones and intervene in many processes that are influenced by the nervous and endocrine system. One of the many neuropeptides involved in the regulation of feeding and digestion in insects is sulfakinin (SK). SK is a sulfated neuropeptide that is homologous to vertebrate cholecystokinin and gastrin.SK acts as an inhibitor of food uptake and regulator of intestinal contractions in various insects. However, multiple effects of SK on the regulation of feeding and digestion have only been reported in the fruit fly, Drosophila melanogaster, so far. To expand the knowledge of the sulfakinin signaling system and its control of feeding and digestion, this study utilized two complementary insect models. The red flour beetle, Tribolium castaneum, provided complete and annotated sequence information that could be used to clone and characterize the different components of the SK signaling pathway. In the migratory locust, Locusta migratoria, the SK peptide has been isolated and can be used to assess multiple in vivo actions. The relative size of L. migratoria allows for straightforward physiological testing by means of peptide injections and microdissection. In addition, both models are categorized as important pest insects with a huge economical and humanitarian impact on cultivated and stored human food sources.An important step in elucidating the function of a neuropeptidergic system is the identification of its (G protein-coupled) receptor and peptide precursor and evidencing their functional coupling. In the current study, we managed to clone two putative SK receptors fromT. castaneum and were able to activate both of them using the endogenous SKs as ligands in an in vitro cell-based screening system. Stimulation of either receptor demonstrated positive coupling to cAMP and Ca2+ second messenger pathways in heterologous cell systems. Furthermore, the sulfated tyrosine residue and the C-terminal HMRFamide tetrapeptide were identified as the essential ligand core necessary for high-affinity receptor activation.A qRT-PCR study in T. castaneum mainly localized SK receptor transcripts in the brain and optic lobes, with little expression in the peripheral tissues. In L. migratoria, high expression of SK receptor was apparent in the brain and along the alimentary tract. Relative transcript levels of the components of the SK signaling system were influenced by starvation: inT. castaneum, significant upregulation was quantified upon starvation, while SK receptor transcripts were depleted in starved L. migratoria.Multiple experiments were performed to gain insight into the regulation of feeding and digestion by SK in L. migratoria. Injection of SK provided confirmation that it can inhibit food uptake. Furthermore, SK injections caused a significant downregulation of digestive enzyme activity in midgut and gastric caeca secretions and were able to mediate the removal of partially digested material and digestive enzymes from the gastric caeca. The sulfated tyrosine residue was revealed as an indispensable structural requirement for biological activity of SK in L. migratoria. SK-induced effects on digestive enzyme secretion and gastric caeca emptying were abolished by RNAi-mediated knockdown of the putative SK receptor inL. migratoria.In conclusion, this study describes the first in-depth characterization of SK receptor pharmacology and signaling properties in insects. The SK signaling system is clearly influenced by starvation in both L. migratoria and T. castaneum. Furthermore, SK negatively influences food uptake and digestive enzyme secretion and mediates the transport of partially digested food through the gut in L. migratoria. These observations greatly contribute to the understanding of the SK signalling system and its functions in insects.  " "Combined forward mutagenesis and proteomics to study vitellogenesis and steroidogenesis in C. elegans." "Liliane Schoofs" "Animal Physiology and Neurobiology" "In vertebrates like ourselves, reproduction is under control of gonadotropin releasing hormone (GnRH), which is produced in the hypothalamus and triggers the synthesis and release of gonadotropins (FSH and LH) from the pituitary. They bind to their specific receptors in the gonads leading to the production of sex hormones and the stimulation of gamete production. Very recently, we have identified the GnRH/AKH-like peptide and obtained evidence for its involvement in regulating reproduction in C. elegans. Because of its transparent body, C. elegans is ideally suited to identify other orthologs of this hypothalamo-hypophysial-gonadal axis. In order to do this, we will look for putative upstream genes that positively or negatively regulate the process of vitellogenesis and steroidogenesis, which is controlled by the sex hormones. We will persform a mutagenesis screen using GFP under control of the vitellogenin promoter as a readout. Meanwhile, we will also carry out an RNAi screen of candidate genes. All relevant mutations will be mapped. Phenotyping will be done using a range of physiological assays and by means of quantitative proteomics (mutant vs wildtype) to gather additional information on the pathways involved." "Hierarchical heterogeneous swarm (H2SWARM)." "Tom Wenseleers" "Ecology, Evolution and Biodiversity Conservation" "Deformed wing virus: a contributory factor of colony collapse disorder in honeybees?" "Tom Wenseleers" "Ecology, Evolution and Biodiversity Conservation, Animal Physiology and Neurobiology"