Vitronectin (VTN) is a blood protein produced mainly by the liver. We show that VTN leaks from the bloodstream into the injury site and neighboring subventricular zone (SVZ) following ischemic stroke (middle cerebral artery occlusion, MCAO) in adult mice. MCAO is known to increase neurogenesis after stroke. VTN inhibits this response in females, but not in males, as shown by ~70% more stroke-induced SVZ neurogenesis in female VTN−/− mice at 14 d. In female VTN−/− mice, stroke-induced expression of interleukin-6 (IL-6) at 24 h was reduced in the SVZ. The closely related leukemia inhibitory factor (LIF) or pro-neurogenic ciliary neurotrophic factor (CNTF) were not affected. The female-specific effect of VTN on IL-6 expression was not due to sex hormones, as shown by ovariectomy and castration. IL-6 injection next to the SVZ reversed the MCAO-induced increase in neurogenesis seen in VTN−/− mice. Our in vitro and vivo data suggest that plasma VTN activates focal adhesion kinase (FAK) in the SVZ following MCAO, which reduces IL-6 expression in astrocytes but increases it in other cells such as microglia/macrophages. Inducible conditional astrocytic FAK deletion increased MCAO-induced IL-6 expression in females at 24 h and blocked MCAO-induced neurogenesis at 14 d, confirming a key detrimental role of IL-6. Collectively, these data suggest that leakage of VTN into the SVZ reduces the neurogenic response to stroke in female mice by promoting IL-6 expression. Reducing VTN or VTN signaling may be an approach to promote neurogenesis for neuroprotection and cell replacement after stroke in females.

Pattern separation is the means by which the brain discriminates similar experiences. It enables retrieval of individuated memories without confusing them with other memories. It is the reason one remembers where one parked the car today and does not mix it up with where one parked it previously. Adult neurogenesis refers to the ongoing production of neurons in the mature brain. One of the likely roles of adult neurogenesis in the hippocampus is facilitating pattern separation. Induced reduction of adult neurogenesis in non-human animals is associated with depression- and anxiety-like behaviors. One possible explanation is that reduced neurogenesis leads to reduced pattern separation, further leading to overgeneralization of threat situations. Instead of perceiving threats where it should, the animal risks perceiving threats everywhere. Emotional resilience is the ability to recover from adversity with a minimum of lingering negative effects such as depression or anxiety. This thesis investigates whether pattern separation in the human hippocampus supports emotional resilience. I performed a systematic review of studies that used the Mnemonic Similarity Task – a memory task commonly used to measure human pattern separation – to investigate the relationship between pattern separation and anxiety. The results are inconclusive but suggest a possible interaction effect whereby pattern separation and high-arousal states like stress predict anxiety. Together with the evidence from the non-human animal studies, this suggests that reduced pattern separation as caused by reduced neurogenesis could make one vulnerable to developing anxiety disorders.

The ophthalmic trigeminal placode (opV) exclusively gives rise to sensory neurons. A number of signaling pathways including Wnt, PDGF, FGF, and Notch are all involved in the progression of an undifferentiated cell in the opV placode to a proneural cell in the condensing opV ganglion. However, the regulatory relationships between these signal transduction pathways are still unknown. To determine if FGF activation acts to modulate Notch signaling in the sensory neurogenesis pathway, a novel multifactorial approach was employed: FGF signaling was inhibited in individual cells and globally with simultaneous inactivation of Notch signaling in chick embryos to investigate if FGF activation downregulates Notch thereby driving neurogenesis. These experiments resulted in few differentiating opV cells in the mesenchymal region of future ganglion formation suggesting an alternate regulatory relationship between FGF and Notch where either reduced Notch activity allows for FGFR4 expression (leading to FGF signaling and neurogenesis), or a parallel relationship where FGF and Notch act independently of one another to induce neurogenesis. To distinguish between these two possibilities Notch signaling was inhibited with DAPT, a gamma-secretase inhibitor, and assayed for FGFR4 mRNA expression. These results indicated FGFR4 is not upregulated by reduced Notch activity, suggesting that FGF and Notch act in parallel to promote neurogenesis. During these experiments it was observed that Notch inhibition resulted in an undefined ectoderm in the opV placode region. To investigate this, FGF and Notch were inhibited by SU5402, an FGF antagonist, and DAPT, and later sectioned and stained for Laminin. In DAPT treated embryos the basement membrane became highly fragmented, a remarkable observation not yet reported. From these data a proposed mechanism was established where activation of FGF with parallel downregulation of Notch leads to disruption of extracellular matrix proteins in the basement membrane resulting in fragmentation and subsequent delamination of differentiating opV placode cells.

Song production in songbirds is controlled by parts of the brain known as the song control regions (SCRs). During spring, gonads increase in size, males sing to attract mates, and SCRs become larger. This neuroplasticity is controlled by the change in day length and increased plasma testosterone (T) levels. Plasma T can be reduced by stress through the production of corticosterone (CORT), through the production of beta-endorphin, or through direct effects on the testes via the nervous system. We determined the T, estradiol, and CORT hormonal profiles of wild House Finches by capturing and sampling blood from the finches every season for two years. To track SCR neuroplasticity in the wild, we also measured the volume of two specific SCRs, the HVC and RA, every season. We then examined the effects of stress on the finch endocrine system in the wild by performing a 30-minute restraint stress protocol once every season and took blood samples before and after the restraint. To determine whether stress and/or CORT affect neuroplasticity in SCRs, we captured male house finches during winter and brought them into captivity. They were allowed to acclimate to captivity for one month on short days (8L:16D) before we transferred them to long days (16L:8D) and restraint stressed half the birds. We measured their gonads, plasma T and CORT levels, volumes of the HVC and RA, and the number of new neurons in the HVC. HVC volumes were smaller in stressed than non-stressed birds, while RA volumes did not differ. There was no difference in number of new neurons or estimated total number of neurons in the HVC between control and restrained birds. Because the HVC is involved in song production, it is possible that stress negatively impacts singing behavior and reproductive success in House Finches. Future work should address how natural stressors may affect neuroplasticity in birds.

Neuronal Nuclei (NeuN) and Doublecortin (DCX) are neuron specific proteins that are used in histological studies of brain structure in a variety of vertebrate taxa.Antibodies against NeuN (anti-NeuN) bind to the Fox-3 protein, an RNA binding protein common in mature neurons. Anti-DCX labels a microtubule-associated protein expressed in actively dividing neural progenitor cells and migrating neurons. The DCX gene encodes a protein that is well conserved across mammalian, avian, and a few reptilian species, therefore anti-DCX staining has been used successfully across a range of vertebrate taxa. Successful neuronal staining using anti-NeuN has been demonstrated in mammals, birds, and the Testudines order (turtles). However, herpetologists who study neurobiology in squamates have had limited success with anti-NeuN and anti-DCX binding to their respective antigens. All commercially available anti-NeuN and anti-DCX antiserums were designed to mammalian antigens, and significant differences in tertiary structure divergence at the epitope where these antibodies bind may explain the failure of anti-NeuN and anti-DCX immunohistochemistry in many squamate species. This study aims to characterize evolutionary differences in gene and protein structure between two species of reptiles (Crotalus oreganus and Sceloporus occidentalis) and mammals. We sequenced the Fox-3 and DCX coding sequences using polymerase chain reaction (PCR) and Sanger sequencing, which allowed us to build phylogenetic trees comparing Fox-3 and DCX deduced protein structures. By identifying structural differences linked to evolutionary variation, new polyclonal antibodies specifically targeting Fox-3 and DCX in reptile brains can be developed to facilitate future investigations of neurogenesis and brain structure in squamate reptiles.

The vertebrate inner ear is a remarkable sensory organ, harboring two different senses: the auditory system, responsible for hearing, and the vestibular system, responsible for balance. Even though the anatomical structure of the vertebrate inner ear is very complex, only three different cell types are mainly involved on a cellular level in the perception of sound as well as balance and movement: sensory hair cells that are surrounded by supporting cells receive the stimulus and transfer it via sensory neurons to the brain. Worldwide, millions of people suffer from sensorineural hearing loss, caused by the loss of sensory hair cells and/or their innervating neurons within the inner ear. In mammals, including humans, both cell types are only produced during fetal stages making loss of these cells and the resulting consequences irreversible. In contrast, it is known that zebrafish produce sensory hair cells throughout life and additionally possess the remarkable capacity to regenerate them upon lesion. However, it is unknown whether new sensory neurons are also formed throughout life in the zebrafish statoacoustic ganglion (SAG), which transduces signals from the inner ear to the brain. Moreover, it is unknown whether sensory neurons are replaced upon loss. Hence, the first aim of this study was to investigate whether new sensory neurons are produced beyond larval stages. To this end, analysis of different transgenic lines combined with immunohistochemistry against known markers for neuronal stem and progenitor cells, neurons, glia and myelinating cells as well as markers for proliferation were used to identify distinct cell populations and anatomical landmarks in the juvenile and adult SAG. In the juvenile SAG, a pool of highly proliferating Neurod/Nestin-positive neuronal progenitors produces large amounts of new sensory neurons. In contrast, at adult stages this neurogenic niche transitions to a quiescent state, in which Neurod/Nestin-positive neuronal progenitor cells are no longer proliferating and the neurogenesis rate is very low. Moreover, BrdU pulse chase experiments revealed the existence of a proliferative but otherwise marker-negative cell population that replenishes the Neurod/Nestinpositive progenitor pool throughout life, indicating a neural stem cell-like cell population upstream of the neuronal progenitor cell pool. Additionally, expression of glia markers and a switch in the myelination pattern was found to mark the peripheral and central nervous system transitional zone (PCTZ) as a prominent landmark of the SAG. To further study the nature of the proliferating but otherwise unknown stem cell-like cell population replenishing the Neurod/Nestin-positive neuronal progenitor pool, the transcriptome of proliferating cells and their progeny of the juvenile and adult SAG was analyzed via single cell RNA-sequencing using the Smart-Seq2 technology. Therefore, a pipeline including preparation of the SAG as well as cell dissociation followed by fluorescence-activated cell sorting was established to obtain single cells from the SAG. The fluorescent reporters Tg(pcna:GFP) and Tg(nestin:mCherry-CreERT2) were used to label proliferating cells (GFP-only positive), proliferating progenitors (GFP/mCherry-double positive as well as nonproliferating progenitor cells (mCherry-positive). Additionally, based on the perdurance of the fluorophores in the progeny of the cells expressing the reporter constructs, this sorting strategy also enables to sort the progeny of proliferating cells differentiating into neuronal progenitor cells (GFP/mCherry-double positive but not expressing pcna) to trace back the putative stem cell-like cell population replenishing the Neurod/Nestin-positive progenitor population. Similar, the sorting strategy also included newborn neurons as the progeny of neuronal progenitors (mCherry-positive but not expressing nestin). In the transcriptome data obtained from the juvenile SAG, the majority of the analyzed cells could be assigned to the neuronal lineage, reflecting the neuronal differentiation trajectory from neuronal progenitor cells transitioning to newborn neurons and even further differentiating into mature neurons. Additionally, two different putative neuronal stem cell-like cell clusters were identified which are currently under validation. In contrast, in the adult transcriptome data the majority of cells were identified as cells from the sensory lineage, including cells expressing markers specific for hair cells and the sensory epithelium. Only a minority of cells came from the neuronal lineage, with the group of newborn and differentiating neurons clustering together in one cluster. Very few cells were identified as neuronal progenitor cells and did not cluster together, whereas both putative stem cell-like cell populations could be identified as distinct cluster. However, validation of the putative stem cell population remains subject to further studies. The second aim of this thesis was to investigate the regenerative capacity of the adult SAG and to study whether the neurogenic progenitor cell niche can be reactivated and to give rise to new sensory neurons upon damage. Therefore, a lesion paradigm using unilateral injections into the otic capsule was established. Upon lesion, mature SAG neurons undergo apoptosis and a massive infiltration with immune cells was found. Importantly, the Neurod-positive progenitor cells reentered the cell cycle displaying a peak in proliferation at 8 days post lesion before they returned to homeostatic levels at 57 days post lesion. In parallel to reactive proliferation, an increase in neurogenesis from the Neurod-positive progenitor pool was observed. Reactive neurogenesis started at around 4 days post lesion, peaked at 8 days post lesion decreased again to low homeostatic levels at 57 days post lesion. The administration of the thymidine analog BrdU to label proliferating cells and their progeny revealed the generation of new sensory neurons from proliferating neuronal progenitor cells within 19 days post lesion. Interestingly, reactive proliferation as well as an increased neurogenesis rate were also detected in the unlesioned SAG, revealing a systemic effect of the unilateral lesions. Taken together, this study is the first to show that neurogenesis in the zebrafish SAG persists way beyond larval stages. New neurons descend from a population of Neurod/Nestin-positive neuronal progenitor cells that is highly proliferative during juvenile stages but turn quiescent at adulthood. Nevertheless, this neuronal progenitor cell pool is replenished throughout life by a currently unknown neuronal stem cell-like cell population. Additional this study reveals the regenerative capacity of the adult SAG: upon lesion Neurod/Nestin-positive progenitor cells are reactivated to re-enter the cell cycle, proliferate and give rise to new neurons leading to an increased neurogenesis rate to replace lost mature neurons. Studying the underlying genes and pathways in zebrafish compared to mammalian species will hopefully provide valuable insights that will help developing cures for auditory and vestibular neuropathies in the future.

No description available.

Adult hippocampal neurogenesis is orchestrated by neural stem cell (NSC) activity. Some associations exist between autophagy and neurogenesis, yet much remains unknown about autophagic regulation of adult neurogenesis. This thesis interrogates the requirement and role of Beclin1 and Atg5, two regulators of autophagy, in the formation of adult hippocampal neurons. To examine adult brain NSCs, the experiments presented in the first objective of this thesis test the ability to isolate adult NSCs using flow cytometry and a DNA-binding dye, DyeCycleViolet. While adult NSCs could not be isolated from the adult neurogenic niches using this methodology, it was effective in isolating endothelial cells. This provided valuable insight on the use of DNA-binding dyes and a new method for isolation of brain endothelial cells. The next objective determines the role of Beclin1 in adult NSCs and their progeny using an inducible model. Beclin1 loss in Nestin-expressing hippocampal NSCs resulted in reduced proliferation, autophagy, and adult neurogenesis within one month. Single-cell RNA sequencing and other methods illuminated that loss of Beclin1 resulted in mitosis reduction, disrupted mitotic regulation of chromatin maintenance, and induction of DNA damage. The final objective first tests whether Beclin1 loss results in similar deficits within GLAST-expressing NSCs and progeny. This model mirrored neurogenesis deficits and requirement of Beclin1 in mitosis and DNA maintenance. Next, to test whether this phenotype occurs with other autophagy proteins, Atg5 was removed from GLAST NSCs. This resulted in reduced autophagy and a transient decrease in neurons in the absence of any effect on NSC proliferation. Thus, proliferation deficits are unique to Beclin1 loss and do not underlie reduced adult hippocampal neurogenesis after Atg5 removal. This work demonstrates a novel discovery of mitosis regulation in adult NSCs by Beclin1, and individual roles of Beclin1 and Atg5 in neurogenesis.

In contrast to mammals, adult zebrafish undergo successful neural regeneration following spinal cord injury (SCI). Radial glia (RG) lining the zebrafish central canal undergo injury-induced proliferation and subsequent neuronal differentiation to replace damaged cells and restore motor function. However, the molecular mechanisms that underlie these processes remain elusive. Here, we demonstrate that signaling through the evolutionarily conserved purinergic P2X7 receptor is involved. Within the zebrafish spinal cord, P2X7 receptors have widespread distribution with specific localization to neurons and radial glia. At the protein level, the predominant P2X7 receptor isoforms in zebrafish did not include the full-length variant expressed throughout the murine central nervous system, but two truncated splice variants. In response to SCI, protein expression of the 50 kDa isoform became downregulated at 7 dpi and returned to basal levels of expression at 14 and 21 dpi when compared to naïve controls. Meanwhile, expression of the 37 kDa isoform did not change following injury. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi, while P2X7 inhibition appeared to have no effect. At 14 dpi, these treatments did not have a significant effect on the number of neurons within the injured spinal cord. This data indicates that P2X7 receptor activation is sufficient to induce cellular proliferation, but not a necessary mediator of either proliferation or neurogenesis following SCI in adult zebrafish. Our findings suggest that unlike in humans, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish. / Thesis / Master of Science (MSc) / Spinal cord injury in mammals causes widespread neuronal cell death and paralysis. In comparison, zebrafish regenerate damaged neurons and restore motor function. Radial glial cells within the zebrafish spinal cord maintain stem-cell properties. Following injury, these cells divide and replace motor neurons. Since mammals have similar cell-types within the spinal cord, understanding the molecular cues driving this adaptive response is of great interest. Here, we examined the evolutionarily conserved purinergic signaling system and found that the expression of the P2X7 receptor varies significantly from mammals and promotes radial glia division following injury.

No description available.