Neuropharmacological Properties of the Histaminergic System in the Zebrafish

Biogenic amine neurotransmitters are important modulators of the central nervous system. Studies on the roles of the histaminergic system have revealed that it regulates many key brain functions. It has also been implicated to be involved in many brain disorders. Modulation of histamine signalling may thus provide an option for the treatment of these conditions. Due to its relatively recent discovery, many unanswered questions about the histaminergic system remain. In this thesis, I studied the neuropharmacology of the histaminergic system using the zebrafish as a model of the vertebrate brain. In the first study, the effect of acute ethanol on the histaminergic and dopaminergic systems was analysed. The mRNAs of enzymes synthesizing dopamine and histamine were upregulated by a short ethanol exposure in a dose-dependent manner. The morphology of the histaminergic and dopaminergic systems was unaltered by ethanol treatment, suggesting that the changes observed were localized to cells that produce these transmitters under normal conditions. No significant changes were seen in histamine levels following ethanol treatment. Ethanol treatment resulted in a rapid, dose-dependent increase in locomotion of larvae. The behavioural and transcriptional changes occurred within a similar time frame, suggesting that histamine and dopamine may play a role in the locomotor effects of ethanol. In the second study, pharmacological inhibition of the vesicular monoamine transporter 2 (VMAT2) was shown to result in an almost total loss of histamine immunoreactivity in neuronal fibres and a decreased number of histamine neurons. Additionally, levels of histamine and other amine neurotransmitters were decreased. Depletion of histamine did not upregulate its synthesis, although enzymes synthesizing the other aminergic transmitters were upregulated. Behaviourally, VMAT2 inhibition resulted in a decreased histamine-dependent dark-induced flash response of zebrafish larvae, a brief spike in locomotor activity during the first second of sudden onset of darkness, which has been described as histamine dependent. In the third study, we characterized the expression of the zebrafish histamine receptor hrh3 and generated a hrh3 knockout strain using the CRISPR-Cas9 method of targeted genome editing. Double fluorescent in situ hybridization revealed that the majority of hrh3 expressing neurons in the telencephalon were glutamatergic, suggesting that hrh3 regulates glutamatergic transmission in the zebrafish. We saw that hrh3 knockout larval zebrafish adapted faster to sudden onset of darkness, indicating that hrh3 can regulate adaptation to sudden changes in the environment. Although basic behavioural parameters of hrh3 knockout larvae were unaltered, adult hrh3 knockout fish showed decreased locomotor activity, suggesting that hrh3 signalling may have different roles at different ages. No changes were seen in the morphology and transcriptional profile of other aminergic networks. However, zebrafish larvae had decreased levels of dopamine and serotonin, supporting a role for hrh3 in the regulation of these systems. In total, these results provide some new answers to open questions in the field of histamine neuropharmacology, specifically concerning the effects of alcohol on the histaminergic system, the mechanisms of vesicular storage of histamine and interactions between histamine and other aminergic systems. This information will be valuable when planning further studies to fully elucidate the pharmacological potential of this system.Aivojen aminergiset järjestelmät ovat keskeisiä aivotoimintojen säätelijöitä. Aminergisia välittäjäaineita ovat esimerkiksi dopamiini ja serotoniini, ja merkittävä osa keskushermoston sairauksien hoidossa käytetyistä lääkkeistä vaikuttaa näitä välittäjäaineita säätelemällä. Histamiini on verrattain myöhään löydetty aminerginen välittäjäaine, jonka toiminta on puutteellisesti tunnettua. Histamiinin tiedetään liittyvän moniin aivojen toimintoihin ja aivosairauksiin. Histamiinijärjestelmään vaikuttavilla lääkkeillä voi tulevaisuudessa olla merkitystä aivosairauksien hoidossa. Lääkkeiden kehitys edellyttää välittäjäainejärjestelmän ominaisuuksien perusteellista tuntemista. Tässä tutkimuksessa käsitellään histamiinijärjestelmän toimintaa käyttäen seeprakalaa mallieläimenä. Ensimmäisessä osatyössä tutkittiin alkoholin akuutteja vaikutuksia histamiini- ja dopamiinijärjestelmiin seeprakalan poikasilla. Lyhyt alkoholialtistus aktivoi näiden välittäjäaineiden tuotantokoneistoa geenitasolla. Sama altistus aiheutti myös annosriippuvaisen liikeaktiivisuuden kiihtymisen poikasilla. Alkoholi ei vaikuttanut näiden välittäjäaineverkostojen hienorakenteeseen. Löydökset viittaavat siihen, että histamiini, kuten myös dopamiini, on olennainen alkoholin aivovaikutuksien välittäjä. Toisessa osatyössä kuvailtiin mekanismi, jolla histamiinia kuljetetaan välittäjäainerakkuloihin. Koska välittäjäaineen toiminta on riippuvainen aineen pakkautumisesta rakkuloihin, on tämän mekanismin tunteminen olennaista. Estämällä vesikulaarisen monoamiinitransportteri 2:n toimintaa histamiinin määrä histamiiniaivosolujen tumissa ja ulokkeissa pieneni merkittävästi. Lisäksi kalapoikasten äkillinen liikevaste pimeydelle oli heikentynyt. Kyseisen vasteen tiedetään olevan histamiinin säätelemä. Tulokset osoittavat, että histamiinin kuljetus tapahtuu kyseisen proteiinin välityksellä. Toisin kuin muiden amiinien kohdalla, histamiinitasojen lasku ei johtanut histamiinin tuotantokoneiston aktivaatioon. Löydös viittaa siihen, että histamiinituotannon säätelyn mekanismit eroavat muista amiinivälittäjäaineista. Kolmannessa osatyössä tuotettiin poistogeeninen seeprakalakanta, jolta puuttuu yksi histamiinin vaikutuksia välittävistä reseptoreista, hrh3. Poistogeenisten kalanpoikasten dopamiini- ja serotoniinitasot olivat verrokkeja matalammat, viitaten siihen, että hrh3:n puuttuminen muuttaa näiden välittäjäaineverkostojen toimintaa. Lisäksi poistogeenisten kalanpoikasten liikevaste äkilliselle valojen sammumiselle palautuu verrokkeja nopeammin perustasolle. Tämän perusteella hrh3 näyttäisi säätelevän eläimen mukautumista muutoksiin ympäristössä. Poistogeenisten kalojen havaittiin uivan hitaammin kuin verrokit, vaikka poikasissa hrh3-poistogeenisyys ei vaikuttanut nopeuteen. On siis mahdollista, että hrh3:lla on erilaisia toimintoja eri kehitysvaiheissa, mikä on lääkekehityksen näkökulmasta olennaista. Lisäksi todettiin, että merkittävä osa etuaivojen glutamaattia välittäjäaineena käyttävistä hermosoluista ilmensivät hrh3-reseptoria. Glutamaatti on nykytiedon perusteella keskeisimpiä keskushermoston välittäjäaineita, joka on olennainen mm. muistitoimintojen säätelyssä. Histamiinin merkitys glutamaattivälitteisen hermovälityksen toiminnassa on toistaiseksi puutteellisesti tunnettu. Tätä yhteyttä olisi mielekästä selvittää jatkotutkimuksin. Väitöskirjan löydökset tuovat esille uutta tietoa histamiinin toiminnasta välittäjäaineena suhteessa käyttäytymiseen, lääkkeisiin ja muihin välittäjäainejärjestelmiin, josta on hyötyä histamiinin neurofarmakologisen potentiaalin selvittämisessä

The bullies are the leaders of the next generation : Inherited aminergic neurotransmitter system changes in socially dominant zebrafish, Danio rerio

We studied the social hierarchy in zebrafish and assessed differences in neurotransmitters and behavior in the F1 generation offspring of dominant and subordinate zebrafish (Danio rerio). We used behavioral assays to study locomotion, ability to complete cognitive tasks, social interaction and aggression. To study the neurochemical changes, we applied quantitative polymerase chain reaction, high pressure liquid chromatography and immunohistochemistry. Social hierarchies were formed both by males and females when animals were kept in same sex pairs in the dyadic dominant-subordinate hierarchy test. The offspring of dominant animals were the leaders in social interactions, however aggression in the mirror-test was not altered in any group. Serotonin and noradrenaline levels were lower in the F1 generation subordinate animals when compared with dominant animals, but not compared with animals that were naive to social hierarchy. The mRNA level of the rate-limiting enzyme in histamine synthesis, histidine decarboxylase, was significantly lower in dominant and subordinate larval zebrafish when compared with control animals. In the dominant adult zebrafish tyrosine hydroxylase 1 mRNA level was lower compared with control animals, whereas tyrosine hydroxylase 2 mRNA was not different. The result was verified with immunohistochemistry. There were gender specific differences between the dominant and subordinate animals, where the dominant females performed better in cognitive tasks such as the T-maze than subordinate females. This was not observed in males, as the behavior of the dominant and subordinate males did not differ. These results add to the understanding of the plastic nature of the central nervous system and show that neurochemical features in aminergic neurotransmitter systems are associated with social leadership and dominance.Peer reviewe

Storage of neural histamine and histaminergic neurotransmission is VMAT2 dependent in the zebrafish

Monoaminergic neurotransmission is greatly dependent on the function of the vesicular monoamine transporter VMAT2, which is responsible for loading monoamines into secretory vesicles. The role of VMAT2 in histaminergic neurotransmission is poorly understood. We studied the structure and function of the histaminergic system in larval zebrafish following inhibition of VMAT2 function by reserpine. We found that reserpine treatment greatly reduced histamine immunoreactivity in neurons and an almost total disappearance of histamine-containing nerve fibers in the dorsal telencephalon and habenula, the most densely innervated targets of the hypothalamic histamine neurons. The reserpine treated larvae had an impaired histamine-dependent dark-induced flash response seen during the first second after onset of darkness, implying that function of the histaminergic network is VMAT2 dependent. Levels of histamine and other monoamines were decreased in reserpine treated animals. This study provides conclusive evidence of the relevance of VMAT2 in histaminergic neurotransmission, further implying that the storage and release mechanism of neural histamine is comparable to that of other monoamines. Our results also reveal potential new insights about the roles of monoaminergic neurotransmitters in the regulation of locomotion increase during adaptation to darkness.Peer reviewe

Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish

Aim We aimed at identifying potential roles of vesicular monoamine transporter 2, also known as Solute Carrier protein 18 A2 (SLC18A2) (hereafter, Vmat2), in brain monoamine regulation, their turnover, behaviour and brain development using a novel zebrafish model. Methods A zebrafish strain lacking functional Vmat2 was generated with the CRISPR/Cas9 system. Larval behaviour and heart rate were monitored. Monoamines and their metabolites were analysed with high-pressure liquid chromatography. Amine synthesising and degrading enzymes, and genes essential for brain development, were analysed with quantitative PCR, in situ hybridisation and immunocytochemistry. Results The 5-bp deletion in exon 3 caused an early frameshift and was lethal within 2 weeks post-fertilisation. Homozygous mutants (hereafter, mutants) displayed normal low locomotor activity during night-time but aberrant response to illumination changes. In mutants dopamine, noradrenaline, 5-hydroxytryptamine and histamine levels were reduced, whereas levels of dopamine and 5-hydroxytryptamine metabolites were increased, implying elevated monoamine turnover. Consistently, there were fewer histamine, 5-hydroxytryptamine and dopamine immunoreactive cells. Cellular dopamine immunostaining, in wild-type larvae more prominent in tyrosine hydroxylase 1 (Th1)-expressing than in Th2-expressing neurons, was absent in mutants. Despite reduced dopamine levels, mutants presented upregulated dopamine-synthesising enzymes. Further, in mutants the number of histidine decarboxylase-expressing neurons was increased, notch1a and pax2a were downregulated in brain proliferative zones. Conclusion Lack of Vmat2 increases monoamine turnover and upregulates genes encoding amine-synthesising enzymes, including histidine decarboxylase. Notch1a and pax2a, genes implicated in stem cell development, are downregulated in mutants. The zebrafish vmat2 mutant strain may be a useful model to study how monoamine transport affects brain development and function, and for use in drug screening.Peer reviewe

Distribution of Lewy-related pathology in the brain, spinal cord, and periphery : the population-based Vantaa 85+study

Evolving evidence has supported the existence of two anatomically distinct Lewy-related pathology (LRP) types. Investigation of spinal cord and peripheral LRP can elucidate mechanisms of Lewy body disorders and origins of synuclein accumulation. Still, very few unselected studies have focused on LRP in these regions. Here we analysed LRP in spinal cord, dorsal root ganglion, and adrenal gland in the population-based Vantaa 85 + study, including every ≥ 85 years old citizen living in the city of Vantaa in 1991 (n = 601). Samples from spinal cord (C6-7, TH3-4, L3-4, S1-2) were available from 303, lumbar dorsal root ganglion from 219, and adrenal gland from 164 subjects. Semiquantitative scores of LRP were determined from immunohistochemically stained sections (anti-alpha-synuclein antibody 5G4). LRP in the ventral and dorsal horns of spinal cord, thoracic intermediolateral column, dorsal root ganglion and adrenal gland were compared with brain LRP, previously determined according to DLB Consortium criteria and by caudo-rostral versus amygdala-based LRP classification. Spinal LRP was found in 28% of the total population and in 61% of those who had LRP in the brain. Spinal cord LRP was found only in those subjects with LRP in the brain, and the quantity of spinal cord LRP was associated with the severity of brain LRP (p Peer reviewe

Content analysis: What are they talking about?

Quantitative content analysis is increasingly used to surpass surface level analyses in Computer-Supported Collaborative Learning (e.g., counting messages), but critical reflection on accepted practice has generally not been reported. A review of CSCL conference proceedings revealed a general vagueness in definitions of units of analysis. In general, arguments for choosing a unit were lacking and decisions made while developing the content analysis procedures were not made explicit. In this article, it will be illustrated that the currently accepted practices concerning the ‘unit of meaning’ are not generally applicable to quantitative content analysis of electronic communication. Such analysis is affected by ‘unit boundary overlap’ and contextual constraints having to do with the technology used. The analysis of e-mail communication required a different unit of analysis and segmentation procedure. This procedure proved to be reliable, and the subsequent coding of these units for quantitative analysis yielded satisfactory reliabilities. These findings have implications and recommendations for current content analysis practice in CSCL research

APOE epsilon 4 associates with increased risk of severe COVID-19, cerebral microhaemorrhages and post-COVID mental fatigue : a Finnish biobank, autopsy and clinical study

Apolipoprotein E epsilon 4 allele (APOE4) has been shown to associate with increased susceptibility to SARS-CoV-2 infection and COVID-19 mortality in some previous genetic studies, but information on the role of APOE4 on the underlying pathology and parallel clinical manifestations is scarce. Here we studied the genetic association between APOE and COVID-19 in Finnish biobank, autopsy and prospective clinical cohort datasets. In line with previous work, our data on 2611 cases showed that APOE4 carriership associates with severe COVID-19 in intensive care patients compared with non-infected population controls after matching for age, sex and cardiovascular disease status. Histopathological examination of brain autopsy material of 21 COVID-19 cases provided evidence that perivascular microhaemorrhages are more prevalent in APOE4 carriers. Finally, our analysis of post-COVID fatigue in a prospective clinical cohort of 156 subjects revealed that APOE4 carriership independently associates with higher mental fatigue compared to non-carriers at six months after initial illness. In conclusion, the present data on Finns suggests that APOE4 is a risk factor for severe COVID-19 and post-COVID mental fatigue and provides the first indication that some of this effect could be mediated via increased cerebrovascular damage. Further studies in larger cohorts and animal models are warranted.Peer reviewe

Trends and challenges in the development of bio-based barrier coating materials for paper/cardboard food packaging; a review

Currently, petroleum-based synthetic plastics are used as a key barrier material in the paper-based packaging of several food and nonfood goods. This widespread usage of plastic as a barrier lining is not only harmful to human and marine health, but it is also polluting the ecosystem. Researchers and food manufacturers are focused on biobased alternatives because of its numerous advantages, including biodegradability, biocompatibility, non-toxicity, and structural flexibility. When used alone or in composites/multilayers, these biobased alternatives provide strong barrier qualities against grease, oxygen, microbes, air, and water. According to the most recent literature reports, biobased polymers for barrier coatings are having difficulty breaking into the business. Technological breakthroughs in the field of bioplastic production and application are rapidly evolving, proffering new options for academics and industry to collaborate and develop sustainable packaging solutions. Existing techniques, such as multilayer coating of nanocomposites, can be improved further by designing them in a more systematic manner to attain the best barrier qualities. Modified nanocellulose, lignin nanoparticles, and bio-polyester are among the most promising future candidates for nanocomposite-based packaging films with high barrier qualities. In this review, the state-of-art and research advancements made in biobased polymeric alternatives such as paper and board barrier coating are summarized. Finally, the existing limitations and potential future development prospects for these biobased polymers as barrier materials are reviewed.Peer reviewe

Temporal Dynamics of Gene Expression During Endothelial Cell Differentiation From Human iPS Cells: A Comparison Study of Signalling Factors and Small Molecules

Endothelial cell (EC) therapy may promote vascular growth or reendothelization in a variety of disease conditions. However, the production of a cell therapy preparation containing differentiated, dividing cells presenting typical EC phenotype, functional properties and chemokine profile is challenging. We focused on comparative analysis of seven small molecule-mediated differentiation protocols of ECs from human induced pluripotent stem cells. Differentiated cells showed a typical surface antigen pattern of ECs as characterized with flow cytometry analysis, functional properties, such as tube formation and ability to uptake acetylated LDL. Gene expression analysis by RNA sequencing revealed an efficient silencing of pluripotency genes and upregulation of genes related to cellular adhesion during differentiation. In addition, distinct patterns of transcription factor expression were identified during cellular reprogramming providing targets for more effective differentiation protocols in the future. Altogether, our results suggest that the most optimal EC differentiation protocol includes early inhibition of Rho-associated coiled-coil kinase and activation of cyclic AMP signaling, and inhibition of transforming growth factor beta signaling after mesodermal stage. These findings provide the first systematic characterization of the most potent signalling factors and small molecules used to generate ECs from human induced pluripotent stem cells and, consequently, this work improves the existing EC differentiation protocols and opens up new avenues for controlling cell fate for regenerative EC therapy