Previous studies have found that during the differentiation of NG108-15 cells, dibutyryl cyclic AMP is a key factor in the culture medium, which can stimulate NG108-15 cells to present a morphological alterations (such as the increase in cell diameter, neurite length, and number of clear vesicles) and to develop the cholinergic neuronal properties including stimulus-dependent acetylcholine release and activities of ChAT and acetylcholinesterase
[2–4]. Our present study demonstrated that ChAT, a cholinergic neuron marker, was expressed in all differentiated NG108-15 cells (Figure
1). Based on these results, NG108-15 cell line is considered to be a suitable cell line for studying cholinergic neuronal function.
Although many studies focused on the measurement of ion channels (such as voltage-gated Na+, Ca++, and K+ channels) in NG108-15 cells
[5–10, 17–20], a few studies recorded the action potential and obtained inconsistent results
[21–23]. In Kowtha’s study, the differentiated-cell excitability is still lower under a very high current-stimulation (30 nA)
. Extracellularly added NH4Cl increased the cell excitability via an elevation in intracellular pH
. Doebler reported that a high current-stimulation (700 pA, 75 ms) induced the generation of action potential after NG108-15 cells were differentiated over 5 days
. Usually, action potential recording needs to be kept a long time for measuring the cell excitability (including action potential frequency and current threshold-inducing action potential) and investigating the effect of drugs. However, the high current-stimulation can induce the cell damage and shorten the cell recording time. Additionally, a current stimulation less than 300 pA is normally used for action potential recording in isolated primary neuron cells. In present study, therefore, we chose a low current stimulation (100 pA) to investigate the time course for differentiation-induced alteration of cell excitability in NG108-15 cells. We observed that short-time differentiation (9 days) didn’t change the cell excitability, compared with undifferentiated condition (Figure
2). Moreover, only about a half of cells generated action potentials after 3 week differentiation (Figure
2). A similar result was found in Ma’s study
. This research group considered there are two types of the cells after long-time differentiation: type 1 neuron-like cells with neuronal morphologies and excitable membrane properties, and type 2 cells with a proliferative property
It is well known that Na+ currents mainly mediate the upstroke of action potential
[15, 24]. However, it is unclear how much functional Na+ channel and Na+ current density are required for the generation of neuronal action potential in NG108-15 cells. We recorded the action potential and voltage-gated Na+ currents in the same cell. Using this technique, we found that differentiation-induced mild increase of Na+ currents didn’t trigger the generation of action potential (such as day 9 and day 21 without AP groups, Figure
3), and a significant increase of Na+ currents is needed for generating action potentials in differentiated NG108-15 cells (such as day 21 with AP group, Figure
3). The data from linear regression analysis (Figure
6) suggest that differentiation-enhanced Na+ current density should reach a high level (77 pA/pF) close to the current threshold for generating action potentials in NG108-15 cells, which is also supported by western blot and single-cell real-time PCR data (Figures
5). Similarly, a previous study has demonstrated that action potential generation requires a high Na+ channel density in the axon initial segment of cortical layer 5 pyramidal neurons
There is no direct evidence showing the involvement of Nav channels in acetylcholine release from cholinergic neurons. However, many studies have shown that Ca++ influx through the voltage-gated Ca++ channels is a key trigger for the release of neurotransmitters including acetylcholine
[25–31]. Voltage-gated Ca++ channels are activated and intracellular Ca++ level is increased when the cell membrane is depolarized by an action potential
[32–34]. Therefore, Na+ channel-initiated action potential might link to the acetylcholine release through triggering Ca++ influx.
Our recent study has shown that differentiation also induces the alteration of voltage-gated Ca++ channel mRNA, protein and current in NG108-15 cells
. Additionally, 0.1 mM Cd++ (a common voltage-gated Ca++ channel blocker) reduces the action potential frequency and increases the current threshold-inducing action potential in differentiated NG108-15 cells (data not shown). It is possible that enhanced expression of voltage-gated Ca++ channels also contributes to action potential generation in NG108-15 cells.
In addition to fasting activating and inactivating Na+ current, the persistent Na+ current, also known as non-inactivating Na+ current are recorded in many types of excitable neurons
[35, 36]. Wu, et al. reported that the non-inactivating Na+ current was characterized in differentiated NG108-15 cells, and this type of Na+ current might facilitate neuronal hyper-excitability
. However, the origin of the non-inactivating Na+ current is unclear. Three hypotheses have been proposed: first, the non-inactivating Na+ current originates from the window current; second, the non-inactivating Na+ current originates from the mutation in the inactivation properties of the same channels that generate the fasting activating and inactivating Na+ currents; third, the non-inactivating Na+ current originates from a special subtype of Na+ channels
[35, 36]. Based on these uncertainties, we did not address the correlation between non-inactivating Na+ current and action potential in the present study.
Although established neuronal cell lines including NG108-15 cells may provide some valuable data, extrapolation to the original neuronal cells should be cautiously used because an endless debate for the neuronal cell lines is still presented in respect of genetic, differentiated, biochemical, and physiological aspects.