Regulation of elementary exocytotic and endocytotic events in cultured astrocytes

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 citosolle-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.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

TYPOLOGY OF (CO)LIVING 65+ - Development of a model of dispersed communities for the elderly in the urban structure

Magistrska naloga se ukvarja z demografskim problemom večanja števila starega prebivalstva, prezasedenostjo kapacitet domov za ostarele, pomanjkanjem sodobnih tipologij bivanja in problemom odmikanja starih na obrobje mesta zunaj mestnega življenja, na obrobje družbe. Naloga skozi razumevanje razvoja bivanja starejših, analizo trenutnih tipologij in raziskovanjem možnih lokacij pride do zaključka, da je potrebna disperzna razporeditev manjših enot bivanja za starejše, s ciljem, da zmanjšamo občutek institucionalizacije bivanja starostnikov, hkrati pa tako omogočimo, da se starostnik postara v domačem okolju, ki mu je poznano, je nanj navajen in navezan. Toda kakšna je primerna tipologija bivanja za starostnika? Potrebno je načrtovanje tipologij bivanja za starejše, ki se prilagaja karakterju mesta in načinu bivanja, ki so ga imeli do sedaj. Z zasnovo treh konceptov bivanja, kot treh odgovorov na tri glavne karakterje mesta Ljubljana je vpeljana rešitev, ki se ne ukvarja zgolj z bivanjem, temveč s socialnimi interakcijami na ravni starostnik, bivalna enota, skupnost, objekt, mesto. Vpeljani novi modeli bivanja se ukvarjajo z ravnmi zasebnosti in ravnmi skupnega glede na karakter bivanja. Tako dobimo model bivanja z bolj individualnim karakterjem - model individualne skupnosti, ki na subtilen način omogoča interakcije med stanovalci. Model sobivanja manjše skupnosti po vzoru karakterja družine, kjer je v ospredju skupno življenje stanovalcev, ter model makro skupnosti, kot nekakšne bivanjske skupnosti starejših, kjer se neprestano prepletata karakterja zasebnega in skupnega življenja. Bivanjska rešitev starejših je tako v bivanje starejših z mestom in v mestu ali drugače bivanje starejših v družbi in z družbo.This master\u27s thesis addresses the demographic problem of an increasing elderly population, overcrowded eldercare facilities, the lack of modern living typologies, and the problem of pushing older people to the outskirts of cities and society. Through an understanding of the development of living arrangements for seniors, an analysis of current typologies, and research into possible locations, the thesis concludes that the dispersal of smaller senior living units is necessary to reduce the feeling of institutionalisation of eldercare and to enable seniors to age in a familiar environment to which they are accustomed and attached. However, what is the appropriate living typology for seniors? It is necessary to plan living typologies for seniors that adapt to the character of the city and their previous way of living. By designing three living concepts that respond to Ljubljana\u27s three main characteristics, the thesis proposes a solution that deals with living arrangements and focuses on social interactions at the level of the elderly, living unit, community, building, and city. The newly introduced living models deal with levels of privacy and communal living based on the character of living. The result is a living model with a more individualistic character - the model of an individual community that subtly allows interactions between residents. The model of living in a small community is based on the character of a family, where communal living is at the forefront of residents\u27 lives. The macro-community model is like a residential community of seniors, where the character of private and communal living continually intertwines. Therefore, the solution for senior living is to live with the city and in the city or to live with and in society

Astrocyte specific remodeling of plasmalemmal cholesterol composition by ketamine indicates a new mechanism of antidepressant action

Ketamine is an antidepressant with rapid therapeutic onset and long-lasting effect, although the underlying mechanism(s) remain unknown. Using FRET-based nanosensors we found that ketamine increases [cAMP]$_i$ in astrocytes. Membrane capacitance recordings, however, reveal fundamentally distinct mechanisms of effects of ketamine and [cAMP]$_i$ on vesicular secretion: a rise in [cAMP]$_i$ facilitated, whereas ketamine inhibited exocytosis. By directly monitoring cholesterol-rich membrane domains with a fluorescently tagged cholesterol-specific membrane binding domain (D4) of toxin perfringolysin O, we demonstrated that ketamine induced cholesterol redistribution in the plasmalemma in astrocytes, but neither in fibroblasts nor in PC 12 cells. This novel mechanism posits that ketamine affects density and distribution of cholesterol in the astrocytic plasmalemma, consequently modulating a host of processes that may contribute to ketamine\u27s rapid antidepressant action

Vesicle cholesterol controls exocytotic fusion pore

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca$^{2+}$-dependent exocytosis regulated by distinct Ca$^{2+}$ sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca$^{2+}$ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD

Vesicle cholesterol controls exocytotic fusion pore

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.</p