Vpliv pogojev gojenja podganjih nevronov jedra Locus coeruleus v primarni kulturi na izražanje adrenergičnih receptorjev α1A in β2

Horvat, Paula (author), Haque Chowdhury, Helena (mentor)

Jedro Locus Coeruleus (LC) je skupek nevronov v možganskem deblu, ki predstavlja glavni vir noradrenalina (NA) v osrednjem živčnem sistemu (OŽS). Prevladujoči tip celic v možganih pa so celice glija, med katerimi so najštevilčnejši astrociti, ki imajo strukturno, regulatorno in presnovno vlogo v OŽS. Astrociti energijsko zalagajo nevrone LC (LCn) v procesu, ki se začne z aktivacijo LCn in sproščanjem NA, kar je energetsko zelo zahtevno. NA se veže na adrenergične receptorje (AR), kar v astrocitih sproži nastajanje L-laktata (LL), ki prehaja iz astrocitov v LCn. AR so sicer prisotni tudi na membrani nevronov. Spadajo v skupino receptorjev, sklopljenih z G proteini, ki po vezavi NA prožijo znotrajcelično signalno pot. Razlikujemo dve glavni skupini, α-AR in β-AR, ki se delita še na 9 podtipov. Nepravilna komunikacija med nevroni in astrociti je eden izmed vzrokov za razvoj nevrodegenerativnih bolezni. Zato so poskusi, izvedeni na celičnih ko-kulturah, pomembni za boljši približek celičnih interakcij, ki se pojavljajo in vivo. Poskusi so pokazali, da je nevronska aktivnost funkcija temperature. Ni pa jasno, kako temperatura vpliva na izražanje ključnih receptorjev, kot so AR. Zanimalo nas je, kako znižana temperatura gojenja (34 °C) vpliva na izražanje α1A-AR in β2-AR na LCn, ter kako prisotnosti drugih celic v kulturi vpliva na izražanje α1A-AR in β2-AR na LCn in na izražanje α1A-AR na astrocitih. Problema smo se lotili z imunocitokemijo. S fluorescenčno označenimi protitelesi smo označili AR in s konfokalnim mikroskopom opazovali fluorescenco posameznih celic. Rezultati so pokazali, da je gojenje LCn pri nižji temperaturi zvišalo izražanje β2-AR, ni pa vplivalo na izražanje α1A-AR, kar je lahko pomembna informacija pri interpretaciji rezultatov poskusov na LCn, ki se izvajajo pri nižji od optimalne temperature 37 °C. Gojenje astrocitov in nevronov v ko-kulturi je znižalo izražanje α1A-AR pri obeh tipih celic, kar kaže na pomen interakcij med obema tipoma celic. Vendar so za natančnejšo oceno vpliva temperature in prisotnosti drugega tipa celic v kulturi na izražanje AR potrebni nadaljnji poskusi.

• Language: Slovenian
• Keywords: nevroni Locus coeruleus, astrociti, temperatura gojenja, ko-kultura, adrenergični receptorji
• Work type: Master's thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2024
• PID: 20.500.12556/RUL-164460
• Publication date in RUL: 25.10.2024

Secondary language

• Language: English
• Title: The effect of culture conditions of rat Locus coeruleus neurons in primary culture on the expression of α1A and β2 adrenergic receptors

Abstract

The nucleus Locus Coeruleus (LC), a cluster of neurons in the brainstem, represents the main source of noradrenaline (NA) in the central nervous system (CNS). However, the predominant cell type in the brain are glial cells, with astrocytes being the most numerous. Astrocytes play a structural, regulatory and metabolic role in the CNS. Astrocytes supply LC neurons (LCn) with energy in a process that begins with LCn activation and NA release, which is energetically demanding. NA binds to adrenergic receptors (AR), triggering the production of L-lactate (LL) in astrocytes, which then passes to LCn. ARs are also present on the neuronal membrane. They belong to the group of G-protein-coupled receptors (GPCRs), which initiate intracellular signalling pathways upon NA binding. The two main groups of ARs: α-AR and β-AR, are further divided into nine subtypes. Impaired communication between neurons and astrocytes is one of the causes of neurodegenerative diseases. Therefore, experiments conducted on cell co-cultures are important for better approximation of the cell interactions that occur in vivo. Experiments have shown that neuronal activity is a function of temperature. However, it remains unclear how the temperature affects the expression of key receptors such as ARs. We were interested in how a reduced temperature (34 °C) affects the expression of α1A-AR and β2-AR on LCn, and how the presence of other cells in the culture influences the expression of α1A-AR and β2-AR on LCn and the expression of α1A-AR on astrocytes. We tackled the problem with immunocytochemistry. ARs were labelled with fluorescently tagged antibodies and the fluorescence of individual cells was observed using confocal microscopy. The results showed that culturing LCn at 34 °C increased the expression of β2-AR, but did not affect the expression of α1A-AR, which may be important information when interpreting results from experiments on LCn conducted at suboptimal temperatures. Culturing astrocytes and neurons in co-culture reduced the expression of α1A-AR in both cell types, highlighting the importance of interactions between the two cell types. However, further experiments are needed to more precisely assess the effects of temperature and the presence of other cell types in culture on AR expression.

• Keywords: Locus coeruleus neurons, astrocytes, cultivation temperature, co-culture, adrenergic receptors