Whole-cell patch-clamp characterization of Neuro-2a cells

Abstract. Neuro-2a (N2a) cell line is a mouse derived neuroblastoma cell line. They can differentiate to neurons within a few days and have endogenous ion currents, but are not capable of firing action potentials. N2a cell line is widely used in electrophysiological research to study neuronal differentation, axonal growth and signaling pathways. Because of this it is important to know properties of the cells and how they typically behave. Cellular electrophysiology is based on different concentrations of ions and their movement in cellular environment powered by electrochemical gradients. Ions move across the cell membrane, a biological membrane that separates the cytoplasm from the extracellular environment and consists of a lipid bilayer and embedded proteins, via active and passive transport. Active transport moves ions against their electrochemical gradient and therefore requires energy to work. The energy is received from hydrolysis of the ATP or from transporting another solute along its electrochemical gradient. Ion channels are a form of passive transport. They are proteins that form pores in the cell membrane. These pores create a way for ions to pass through the membrane and they are usually highly selective for certain ion species. Some ion channels are gated, and they open and close in response to electrical, mechanical or chemical signals. Voltage-gated channels open and close in response to changes in membrane potential. Many of them are highly selective to be permeable to only one type of ion and, for example, generate and conduct an action potential, an electrical signal neurons conduct to synapse to send signal to an another cell. Ligand-gated channels are controlled by binding of ligands, known as agonists. They are important, for example, in chemical synapses. Mechanosensitive ion channels are gated by stretch in membrane formation or via tethers. They are important in sensory neurons. Not all ion channels have gating, ones that do not are leak channels. They participate in resting potential of the cell membrane. To study voltage-gated currents in N2a cells whole-cell voltage clamp was used. Voltage clamp is a patch clamp method where cell membrane is fixed to certain potential and current passing through it is recorded. In whole-cell configuration, a glass pipette, that has electrode inside of it and is filled with pipette solution, is brought in contact with the cell membrane. After contact a strong seal with the membrane is formed, after which the cell membrane is punctured using suction and a small zap. After a hole in the membrane has been formed, cells intracellular solution is gradually replaced with pipette solution. In whole-cell configuration, ohmic and capacitive “error” signals caused by series resistance and cell capacitance have to be compensated. In the recordings electrodes with median electrode resistance of 4,7 MΩ were used. The voltage pulse protocol used consisted of a holding potential of −70 mV, a prepulse of −100 mV and two series of voltage steps with a holding potential of 0 mV in between. The voltage step series went from −100 mV to 80 mV in 10 mV increments. For recordings, cells were chosen according to oval shape and smooth, well-defined membrane. Total of 11 cells were recorded, out of which ten were chosen for analysis. Passage of cells was from 6 to 9. Cells had median resting potential −38,5 mV, cell capacitance 23 pF, input resistance 3,3 GΩ and series resistance 18 MΩ before compensations. Resting potential of cells was determined to be formed by voltage-gated K+ currents and leak currents. Out of recorded currents K+ and Na+ currents could be clearly distinguished. K+ currents that could be distinguished are delayed rectifier currents. They are the major currents at steady-state reached at the end of the clamp step pulse. The median for activation and half-activation potentials were −50 mV and −7,75 mV, respectively. In N2a cells, there are also A-type and erg-mediated K+ currents, but these currents overlap with Na+ currents and leftover transients from incomplete compensations and are hard to distinguish. There are fast Na+ current in N2a cells and their peak currents were determined where possible for recorded cells. However, as these currents are fast, they got partly covered by recording artefacts and may not have passed low-pass filtering at all cases. They also overlap with A-type K+ currents which may change their peak locations and amplitudes. As currents overlap, if specific currents were of interest, should other currents be blocked by channel blockers. Because of this overlap no other voltage-gated currents than K+ and Na+ could be determined from the recordings. Mechanosensitive or ligand-gated currents of N2a cells could also not be studied because the research protocol was able to distinguish only voltage dependent currents. All of recorded cells were quite consistent, so their properties can be used as rough quality criteria for N2a cells in performing whole-cell patch-clamp. To make criteria more comprehensive larger sample size would be needed, with more diverse cells

Neurologiset jänniteriippuvat natriumkanavataudit

Tiivistelmä. Jänniteriippuvat natriumkanavat ovat hermostossa merkittävässä roolissa aktiopotentiaalin tuottamisessa ja etenevän aktiopotentiaalin ylläpidossa. Ne muodostavat solukalvon signalointiproteiinien suurimman ryhmän. Jänniteriippuvat natriumkanavat jaotellaan niiden rakenteen mukaan yhdeksään alatyyppiin. Kullakin näistä on oma roolinsa ja paikkansa elimistössä ja ne eroavat toisistaan myös toiminnaltaan. Muutokset ionikanavien toiminnassa voivat aiheuttaa vakavia oireyhtymiä. Neurologisia natriumin jänniteriippuviin kanaviin liittyviä tauteja ovat ainakin jotkin epilepsiat, kipuoireyhtymät ja migreenit. Tässä tutkielmassa perehdytään hermostossa esiintyviin jänniteriippuviin natriumkanaviin sekä niihin liitettyihin sairauksiin. Kanavia tarkastellaan niiden rakenteen ja toiminnan kautta. Sen jälkeen käydään läpi tunnettuja neurologisia natriumkanavatauteja ja niiden toimintamekanismeja

Lakka kasvaa viljeltynäkin

Lakan viljelyä on kokeiltu Kainuussa kuuden vuoden ajan. Tulokset ovat olleet lupaavia. Tällä hetkellä Kainuun alueella on noin kolme hehtaaria lakkapeltoja, ja pienimuotoisia viljelykokeiluja on perustettu muuallekin Väli- ja Pohjois-Suomen alueelle.vo

Lakasta viljelykasvi?

Lakka (Rubus chamaemorus L.) on Pohjois-Suomen arvokkaimpia luonnonmarjoja. Vuosittaiset satovaihtelut ja poimijapula vaikeuttavat marjojen ostajien ja jatkojalostajien toimintaa. Lakan saaminen peltoviljelyyn varmistaisi marjojen tarjontaa, koska luonnossa satovaihtelua aiheuttavat riskitekijät pystytään peltoviljelyssä hallitsemaan paremmin. MTT Kainuun tutkimusasemalla Sotkamossa on tutkittu lakan viljelyn edellytyksiä kenttäkokeissa MTT:n ja ProAgria Kainuun yhteistyönä.Tässä esitellyt kolme koetta, kasvualustakoe, lannoituskoe sekä lajikekoe, perustettiin MTT Kainuun tutkimusasemalle elokuussa 1999. Vuosina 2001–2004 niistä seurattiin sadontuottoa ja versonkasvua. Kasvualustakokeessa verrattiin lakan kasvua neljässä erilaisessa maalajissa tai kasvualustassa, lannoituskokeessa tutkittiin erikseen N-, P- ja K-lannoituksen vaikutuksia ja lajikekokeessa verrattiin neljää norjalaista lajiketta.Parhaaksi kasvualustaksi osoittautui vaalean rahkaturpeen ja maatumattoman rahkasammalen sekoitus 1:1. Lakka kasvoi erittäin huonosti kivennäismaassa (LjHHt). Kaksi muuta kasvualustaa, edellä mainitun kivennäismaan ja vaalean rahkaturpeen sekoitus 1:1 sekä kivennäismaan ja maatumattoman rahkasammalen sekoitus 1:1 sijoittuivat tuloksissa näiden kahden käsittelyn välille.N-, P- tai K-lannoitus ei vaikuttanut lakan sadontuottoon. Vuoteen 2003 saakka lannoitus ei vaikuttanut myöskään lakan versonkasvuun. Vuonna 2004 kokeessa olleen parhaan versotiheyden, noin 600 kpl/m2, saavuttamiseen tarvittava kasvualustan lannoitusvuoden 2000 typpipitoisuus oli ≥600 mg/l, fosforipitoisuus ≥30 mg/l ja kaliumpitoisuus ≤200 mg/l. Kokeessa testattiin ravinteita vain erikseen. Ravinteiden yhdysvaikutuksia ei testattu.Lajikekokeessa verrattiin norjalaisia satoa tuottavia emilajikkeita Fjellgull ja Fjordgull sekä pölyttäviä hedelajikkeita Apollen ja Apolto. Emilajikkeista satoisammaksi osoittautui Fjellgull. Versonkasvussa ei ollut lajikkeiden välisiä eroja.Kokeissa luotiin tarpeellista pohjatietoa lakan viljelyä varten. Lakan viljelyvaatimukset tunnetaan kuitenkin edelleen huonosti ja tutkimustarve on suuri. Vuosina 2005–2007 MTT Kainuun tutkimusasemalla jatketaan edelleen uusissa kokeissa lakan kasvualusta- ja lajikekysymysten selvittämistä

Identification of transcriptional and metabolic programs related to mammalian cell size

SummaryBackgroundRegulation of cell size requires coordination of growth and proliferation. Conditional loss of cyclin-dependent kinase 1 in mice permits hepatocyte growth without cell division, allowing us to study cell size in vivo using transcriptomics and metabolomics.ResultsLarger cells displayed increased expression of cytoskeletal genes but unexpectedly repressed expression of many genes involved in mitochondrial functions. This effect appears to be cell autonomous because cultured Drosophila cells induced to increase cell size displayed a similar gene-expression pattern. Larger hepatocytes also displayed a reduction in the expression of lipogenic transcription factors, especially sterol-regulatory element binding proteins. Inhibition of mitochondrial functions and lipid biosynthesis, which is dependent on mitochondrial metabolism, increased the cell size with reciprocal effects on cell proliferation in several cell lines.ConclusionsWe uncover that large cell-size increase is accompanied by downregulation of mitochondrial gene expression, similar to that observed in diabetic individuals. Mitochondrial metabolism and lipid synthesis are used to couple cell size and cell proliferation. This regulatory mechanism may provide a possible mechanism for sensing metazoan cell size

Towards a more comprehensive understanding of lacustrine greenhouse gas dynamics — two-year measurements of concentrations and fluxes of CO2, CH4 and N2O in a typical boreal lake surrounded by managed forests

Peer reviewe

Co-culture of human induced pluripotent stem cell-derived retinal pigment epithelial cells and endothelial cells on double collagen-coated honeycomb films

In vitro cell culture models representing the physiological and pathological features of the outer retina are urgently needed. Artificial tissue replacements for patients suffering from degenerative retinal diseases are similarly in great demand. Here, we developed a co-culture system based solely on the use of human induced pluripotent stem cell (hiPSC)-derived cells. For the first time, hiPSC-derived retinal pigment epithelium (RPE) and endothelial cells (EC) were cultured on opposite sides of porous polylactide substrates prepared by breath figures (BF), where both surfaces had been collagen-coated by Langmuir–Schaefer (LS) technology. Small modifications of casting conditions during material preparation allowed the production of free-standing materials with distinct porosity, wettability and ion diffusion capacity. Complete pore coverage was achieved by the collagen coating procedure, resulting in a detectable nanoscale topography. Primary retinal endothelial cells (ACBRI181) and umbilical cord vein endothelial cells (hUVEC) were utilised as EC references. Mono-cultures of all ECs were prepared for comparison. All tested materials supported cell attachment and growth. In mono-culture, properties of the materials had a major effect on the growth of all ECs. In co-culture, the presence of hiPSC-RPE affected the primary ECs more significantly than hiPSC-EC. In consistency, hiPSC-RPE were also less affected by hiPSC-EC than by the primary ECs. Finally, our results show that the modulation of the porosity of the materials can promote or prevent EC migration. In short, we showed that the behaviour of the cells is highly dependent on the three main variables of the study: the presence of a second cell type in co-culture, the source of endothelial cells and the biomaterial properties. The combination of BF and LS methodologies is a powerful strategy to develop thin but stable materials enabling cell growth and modulation of cell-cell contact. Statement of significance: Artificial blood-retinal barriers (BRB), mimicking the interface at the back of the eye, are urgently needed as physiological and disease models, and for tissue transplantation targeting patients suffering from degenerative retinal diseases. Here, we developed a new co-culture model based on thin, biodegradable porous films, coated on both sides with collagen, one of the main components of the natural BRB, and cultivated endothelial and retinal pigment epithelial cells on opposite sides of the films, forming a three-layer structure. Importantly, our hiPSC-EC and hiPSC-RPE co-culture model is the first to exclusively use human induced pluripotent stem cells as cell source, which have been widely regarded as an practical candidate for therapeutic applications in regenerative medicine.publishedVersionPeer reviewe

LAKE 2.0: A model for temperature, methane, carbon dioxide and oxygen dynamics in lakes

A one-dimensional (1-D) model for an enclosed basin (lake) is presented, which reproduces temperature, horizontal velocities, oxygen, carbon dioxide and methane in the basin. All prognostic variables are treated in a unified manner via a generic 1-D transport equation for horizontally averaged property. A water body interacts with underlying sediments. These sediments are represented by a set of vertical columns with heat, moisture and CH4 transport inside. The model is validated vs. a comprehensive observational data set gathered at Kuivajarvi Lake (southern Finland), demonstrating a fair agreement. The value of a key calibration constant, regulating the magnitude of methane production in sediments, corresponded well to that obtained from another two lakes. We demonstrated via surface seiche parameterization that the near-bottom turbulence induced by surface seiches is likely to significantly affect CH4 accumulation there. Furthermore, our results suggest that a gas transfer through thermocline under intense internal seiche motions is a bottleneck in quantifying greenhouse gas dynamics in dimictic lakes, which calls for further research.Peer reviewe

Applicability and consequences of the integration of alternative models for CO2 transfer velocity into a process-based lake model

Freshwater lakes are important in carbon cycling, especially in the boreal zone where many lakes are supersaturated with the greenhouse gas carbon dioxide (CO2) and emit it to the atmosphere, thus ventilating carbon originally fixed by the terrestrial system. The exchange of CO2 between water and the atmosphere is commonly estimated using simple wind-based parameterizations or models of gas transfer velocity (k). More complex surface renewal models, however, have been shown to yield more correct estimates of k in comparison with direct CO2 flux measurements. We incorporated four gas exchange models with different complexity into a vertical process-based physico-biochemical lake model, MyLake C, and assessed the performance and applicability of the alternative lake model versions to simulate air-water CO2 fluxes over a small boreal lake. None of the incorporated gas exchange models significantly outperformed the other models in the simulations in comparison to the measured near-surface CO2 concentrations or respective air-water CO2 fluxes calculated directly with the gas exchange models using measurement data as input. The use of more complex gas exchange models in the simulation, on the contrary, led to difficulties in obtaining a sufficient gain of CO2 in the water column and thus resulted in lower CO2 fluxes and water column CO2 concentrations compared to the respective measurement-based values. The inclusion of sophisticated and more correct models for air-water CO2 exchange in process-based lake models is crucial in efforts to properly assess lacustrine carbon budgets through model simulations in both single lakes and on a larger scale. However, finding higher estimates for both the internal and external sources of inorganic carbon in boreal lakes is important if improved knowledge of the magnitude of CO2 evasion from lakes is included in future studies on lake carbon budgets.peerReviewe