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Browsing by discipline "Neuroscience"

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  • Detection and quantification of GABAergic markers in histaminergic neurons 
    Kauhanen, Jenna (2018)
    Histamine is an important neurotransmitter in the central nervous system (CNS). It is involved e.g. in the sleep-wake cycle, endocrine and energy homeostasis as well as in synaptic plasticity and learning. It is produced from L-histidine by histidine decarboxylase (HDC). Almost all species have histamine in their body although the amount varies between species. Histaminergic neurons are located in the tuberomamillary nucleus (TMN) of the posterior hypothalamus. There are four different histamine receptors in mammals and they are all metabotropic GPCR receptors. The first three (Hrh1, Hrh2 and Hrh3) are located in the brain while Hrh1 and Hrh2 along with Hrh4 that is mainly found in mast cells, are found in the periphery. Receptors have different functions e.g. Hrh1 regulates wakefulness and alertness while Hrh2 is involved in learning and memory. It is established that histaminergic neurons contain GABA-producing enzyme GAD1 and GABA itself. In the present study we aimed to evaluate GABAergic phenotype of the hypothalamic histaminergic neurons with double fluorescent in situ hybridization. Specifically, we were interested in co-existence of VGAT, which is responsible for vesicular release of GABA, and HDC mRNA. The animals used in this study were mouse and zebrafish. The percentage of mouse HDC-neurons that expressed GAD1 was 99.65% and co-expression for VGAT was also high (94.53%). This coexistence was verified also in the zebrafish model. Our data suggest that histaminergic neurons containing VGAT mRNA and are potentially able to release GABA. If GABA is released in a paracrine manner like histamine, it causes tonic inhibition that counterbalances the effects of histamine during wakefulness. The fact that VGAT mRNA was also found in zebrafish histaminergic neurons indicates that histamine-GABA system is preserved among species.
  • The Role of Angiopoietin 1 During Embryonic Neurogenesis in the Zebrafish 
    de Aquino dos Santos Martins, Tomás (2017)
    Angiopoietin 1 (ANGPT1) is an endothelial growth factor and the ligand of the tyrosine endothelial kinase (TEK). The ANGPT-TEK system is known to mediate endothelial cell (EC) interactions and contributes to angiogenesis and vascular remodeling through angiocrine signaling. Although TEK is expressed in neural stem and progenitor cells, a vascular-independent role for ANGPT1 in neurogenesis is still unclear. This study focused on the embryonic expression of angiopoietins and their receptors in wild-type (WT) zebrafish (Danio rerio) and on further characterization of two zebrafish lines: the Angpt1sa14264 line, with a nonsense mutation in the angpt1 gene, and a transgenic line of angpt1 overexpression. Whole-mount in situ hybridization (WISH) and real-time quantitative polymerase chain reaction (RT-qPCR) showed a relatively high expression of angpt1 and tek throughout the first three days of WT development. Imaging of live Angpt1sa14264 and transgenic overexpression embryos revealed the bidirectional effect of angpt1 on the cardiovascular system, suggesting an essential role for normal embryonic development. In the absence of angpt1, gene expression analysis showed a dramatic disturbance in genes involved in neural patterning and neuronal development. The spatiotemporal expression of wingless-type MMTV integration site 1 (wnt1) implicated the midbrain-hindbrain boundary as a major site of Angpt1-mediated regulation of neuronal organization. The downregulated spatiotemporal expression of nestin (nes) indicated a decrease in neural progenitor-like cells throughout the central nervous system (CNS). In the context of angpt1 overexpression, the neurogenic locus notch homolog 1a (notch1a) was slightly increased despite the unchanged expression of all other neuronal markers and neural patterning genes analyzed. The spatiotemporal expression of notch1a was exacerbated in a large cranial vein, albeit detected in typical notch1a domains in the brain. These findings suggest that Angpt1 plays an important role in zebrafish embryogenesis and possibly regulates the organization of the zebrafish midbrain and hindbrain regions.

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