doktorska disertacija
Eva Lasič (Author), Matjaž Stenovec (Mentor), Robert Zorec (Co-mentor)

Abstract

Astrociti so celice nevroglije v živčnem sistemu, ki sodelujejo v medcelični komunikaciji z ekso-/endocitozo mešičkov. Z namenom, da bi bolje razumeli interakcije posameznih mešičkov s plazmalemo, smo uporabili elektrofiziološko metodo vpete napetosti membrane celice v konfiguraciji pripete celice in super-ločljivostno mikroskopijo s strukturirano osvetlitvijo ter raziskali vpliv različnih farmakoloških učinkovin na ekso-/endocitozo in fuzijsko poro mešičkov v podganjih astrocitih v kulturi. Ketamin, lipofilni anestetik z antidepresivnim delovanjem, je inhibiral izzvano eksocitotsko izločanje BDNF ter popolno in prehodno zlivanje mešičkov s plazmalemo. Dodatno je ketamin inhibiral endocitozo, saj je izzval utripanje fuzijske pore mešičkov pripetih na plazmalemo, ki se niso odcepili v citosol. Podobno kot ketamin, sta inhibitorja aktivnosti dinamina Dynole-34-2 in Dyngo-4a povzročila utripanje fuzijske pore z zmanjšano prevodnostjo. S pomočjo fluorescentnih dekstranov in lipofilnega barvila DiD smo pokazali, da inhibicija dinamina prepreči internalizacijo mešičkov v citosol; le-ti verjetno ostanejo povezani s plazmalemo z utripajočo fuzijsko poro. Aktivator dinamina Ryngo-1-23 je zmanjšal utripajočo aktivnost fuzijske pore, favoriziral zaprto stanje pore in povečal endocitotsko internalizacijo mešičkov v citosol. Rezultati kažejo, da je dinamin vpleten tudi v zlivanje VAMP2- in Syt4-pozitivnih eksocitotskih mešičkov. Povečana koncentracija sekundarnega prenašalca cAMP pa je povečala prevodnost fuzijske pore, favorizirala odprto stanje pore ter spodbudila popolno zlivanje večjih mešičkov s plazmalemo. Naši rezultati kažejo, da ketamin zmanjša, medtem ko cAMP poveča verjetnost za izločanje snovi iz mešičkov. Novo odkriti farmakološki vplivi na procese ekso-/endocitoze v astrocitih prispevajo k razumevanju medcelične komunikacije na ravni posameznega mešička v nevrofiziologiji.

Keywords

astrociti;eksocitoza;endocitoza;fuzijska pora;elektrofiziologija;mikroskopija SIM;disertacije;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL MF - Faculty of Medicine
Publisher: [E. Lasič]
UDC: 61:537.533.35:576.6(043.3)=163.6
COBISS: 921463 Link will open in a new window
Views: 1235
Downloads: 595
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Other data

Secondary language: English
Secondary title: Regulation of elementary exocytotic and endocytotic events in cultured astrocytes
Secondary abstract: Astrocytes are neuroglial cells in the nervous system that participate in cellular communication via vesicle exo-/endocytosis. To better understand single vesicle interactions with the plasmalemma, we employed electrophysiological cell-attached capacitance measurements and super-resolution structured illumination microscopy, and examined the effects of different pharmacological agents on exo-/endocytosis and the fusion pore in cultured rat astrocytes. Ketamine, a lipophilic anesthetic with antidepressant action, inhibited evoked exocytotic BDNF secretion as well as full and transient vesicle fusions with the plasmalemma. Ketamine also inhibited endocytosis by evoking fusion pore flickering of vesicles that remained attached to the plasmalemma and were unable to undergo endocytotic fission. Similar to ketamine, the dynamin inhibitors Dynole-34-2 and Dyngo-4a caused flickering of fusion pores with decreased conductances. Using fluorescent dextrans and the lipophilic dye DiD, we demonstrated that dynamin inhibition prevents internalization of vesicles that instead likely remained attached to the plasmalemma via a flickering fusion pore. On the other hand, the dynamin activator Ryngo-1-23 decreased fusion pore flickering, favored a closed fusion pore configuration, and increased vesicle internalization. Dynamin is also involved in fusion of exocytotic VAMP2- and Syt4-positive vesicles. In contrast, the secondary messenger cAMP increased fusion pore conductance, favored an open fusion pore configuration, and caused larger vesicles to transit to full fusion. Whereas ketamine decreased secretion, cAMP increased the probability of gliotransmitter secretion. These newly discovered pharmacological effects on the fusion pore and exo-/endocytosis at the single vesicle level contribute to a better understanding of gliotransmission in neurophysiology.
Secondary keywords: astrocytes;exocytosis;endocytosis;fusion pore;electrophysiology;SIM microscopy;dissertations;
Type (COBISS): Doctoral dissertation
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak.
Pages: X, 84 str.
ID: 10932987