JultikaUniversity of Oulu repository

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.