Vpliv ketamina na znotrajcelično dinamiko kalijevega kanalčka Kir4.1 v astrocitih v kulturi

Gajić, Arijana (author), Stenovec, Matjaž (mentor)

Astrociti specifično izražajo kalijeve kanalčke Kir4.1, s katerimi prerazporejajo kalijeve ione v možganovini in vplivajo na vzdražnost nevronov. Prekomerno izražanje astrocitnih kanalčkov Kir4.1 lahko povzroči hiperpolarizacijo nevronov v možganskem predelu imenovanem lateralna habenula (LHb) in izzove atipični vzorec proženja akcijskih potencialov, ki ga povezujemo s pojavom depresivnega vedenja pri glodalcih. Ketamin, splošni anestetik, ki izzove hiter, močan in dolgotrajen antidepresivni učinek, blokira atipično električno aktivnost nevronov LHb in zmanjša depresivno vedenje. V nalogi smo raziskali vpliv ketamina na mobilnost in izražanje kalijevega kanalčka Kir4.1 na površini astrocitov v kulturi. V astrocite smo vnesli plazmidno DNA z zapisom za fluorescentno označen kalijev kanalček Kir4.1 (pKir4.1 EGFP) in mikroskopsko raziskali mobilnost Kir4.1 pozitivnih mešičkov. V časovnih nizih konfokalnih mikrografij smo izmerili mobilnost in njeno spremembo po kratkoročnem (30 min) tretiranju astrocitov s subanestetičnim odmerkom ketamina (2,5 in 25 µM). Tretiranje celic s ketaminom je izzvalo zmanjšanje (P<0,05) vseh parametrov mobilnosti (prepotovana pot (TL), največji odmik na poti (MD), indeks usmerjenosti (DI) in hitrost) Kir4.1 pozitivnih mešičkov glede na mobilnost v kontrolnih, ne tretiranih, astrocitih. Kratkoročno tretiranje s ketaminom je zmanjšalo površinsko gostoto imunocitokemično označenih kanalčkov Kir4.1 v plazmalemi astrocitov. Poleg kratkoročnega vpliva ketamina na mobilnost mešičkov smo raziskali še dolgoročni vpliv povečane koncentracije zunajceličnih K+ ([K+]o, 15 mM) in dbcAMP (dibutiril ciklični adenozin 5' monofosfat, 1 mM), s katerima smo simulirali vnetni odziv. Oba načina tretiranja celic (15 mM K+ in 1 mM dbcAMP) sta značilno zmanjšala (P<0,05) mobilnost Kir4.1 pozitivnih mešičkov. Zaključimo lahko, da vsi uporabljeni načini tretiranja celic zavrejo znotrajcelično mobilnost kanalčkov Kir4.1 ter da subanestetični odmerki ketamina učinkovito zavirajo dostavo Kir4.1 pozitivnih mešičkov proti perifernim predelom celice in zmanjšajo površinsko gostoto kanalčkov v plazmalemi. Zmanjšana površinska gostota kanalčkov Kir4.1 lahko zavre atipični vzorec proženja akcijskih potencialov nevronov LHb in vivo in olajša depresivno občutje.

• Language: Slovenian
• Keywords: astrociti, kalijev kanalček, Kir4.1, mobilnost mešičkov, ketamin, dbcAMP, K+
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [A. Gajić]
• Year: 2020
• PID: 20.500.12556/RUL-118208
• UDC: 601.2:575.853:576.342:606:61(043.2)
• COBISS.SI-ID: 26837251
• Publication date in RUL: 27.08.2020

Secondary language

• Language: English
• Title: Influence of ketamine on the intracellular dynamics of potassium channel Kir4.1 in cultured astrocytes

Abstract

Potassium channel Kir4.1 is specifically expressed in astrocytes and regulates neuronal excitability by mediating spatial potassium buffering. Overexpression of astrocyte Kir4.1 may contribute to hyperpolarization of neurons in brain region of lateral habenula (LHb) and increases the atypical action potential firing that augments depression like behavior in rodents. Ketamine, a general anaesthetic, that exerts rapid, potent and long lasting antidepressant effect, blocks the atypical electrical activity of LHb neurons and alleviates depressive behavior. In this study we evaluated the impact of ketamine on mobility and surface expression of Kir4.1 channel in cultured astrocytes. We transfected astrocytes with the plasmid encoding potassium channel Kir4.1 tagged with enhanced green fluorescent protein (pKir4.1 EGFP) and microscopically examined the intracellular dynamic of mobile vesicles carrying Kir4.1. We examined the spontaneous mobility of Kir4.1 positive vesicles and analyzed changes in mobility after a short term (30 min) treatment with subanesthetic doses of ketamine (2.5 in 25 µM). Ketamine treatment significantly reduced (P<0.05) all mobility parameters (track length (TL), maximal displacement (MD), directionality index (DI) and speed) when compared to non treated controls. Short term treatment also reduced the surface density of immunolabeled Kir4.1 in the astrocyte plasmalemma. In addition to the effects mediated by ketamine, we also examined the impact of long term elevation in extracellular concentration of K+ ([K+]o, 15 mM) and dbcAMP (dibutyryl cyclic adenosine 5′ monophosphate, 1 mM), that mimics an inflammatory response. Both treatments (15 mM K+ and 1 mM dbcAMP) statistically significantly attenuated (P<0.05) mobility of Kir4.1 positive vesicles. We conclude that the listed treatments significantly attenuate the mobility of Kir4.1 positive vesicles and that the subanesthetic doses of ketamine inhibit the delivery of Kir4.1 towards the astrocyte plasmalemma and reduce the surface density of Kir4.1. Reduced surface expression of astrocyte Kir4.1 may reduce atypical firing of action potentials by LHb neurons in vivo and contribute to rapid alleviation of depressive behavior.

• Keywords: astrocyte, potassium channel, Kir4.1, intracellular dynamic, ketamine, dbcAMP, K+