Non-stationary fluctuation analysis


Non-stationary fluctuation analysis is a method by which information regarding the single channel currents that underlie a recorded macroscopic current can be extracted, under conditions where single channel properties cannot be measured directly. We have used this to investigate the properties of synaptic AMPA channels directly. What follows is a very brief introduction to non-stationary fluctuation analysis. For more detailed information, see Benke et al (1998).
Figure 1.The total recorded current measured is the sum of the individual currents generated by individual channels.


A recorded current is built up from the individual opening/closing cycles of activated channels For simplicity, consider that ion channels can either be open (ie passing current) or closed. A population of channels may be stimulated and open together, but will close in a random fashion. Thus the sum of the individual currents from individual channels gives rise to the commonly seen excitatory post synaptic current, EPSC (Fig. 1). Because the closing of channels is random, there is variation in the decay phase of the EPSC. Thus the shape of the decay phase of individual EPSCs varies from the mean decay of all the currents recorded when scaled to the same peak amplitude. Subtraction of the mean current from the individual currents leaves fluctuations. A plot of the variance in these fluctuations against current amplitude is parabolic, the initial slope of which gives the mean single channel conductance of the channel population. Thus changes in channel conductance can be diustinguished from changes in channel number and mean open time (Fig. 2). See Benke et al 2001 for a full description of the mathematical model describing the application of NSFA to synaptic AMPA receptors.
effect of changes in conductance and number on amplitude vs variance curveFigure 2. Increased current amplitude may be the result of increased channel number or an increase in single channel conductance.

Examples of Application

Different Mechanistic properties of LTP and LTD

NSFA has been used here at the MRC Centre to investigate the cellular mechanisms that underlie LTP and LTD. The first questions to be adressed was whether LTP was the result of a change in the conductance of existing receptors in the synapse or whether there was an increase in the number of receptors expressed Benke et al (1998). Stimulating and recording electrodes were placed very close to the dendrites of CA1 hippocampal neurons and minimal stimulation applied, to activate only a few synapses. It was shown that in 2 out of every 3 cells, LTP could be accounted for by an increase in the mean single channel conductance, i.e. and increase in the initial slope of the current-variance relationship; in 1 out of 3 cells, no change in single channel conductance was seen. These results indicated that there are at least two mechanisms underlying an apparently single celullar response - either a modification of existing receptors or an increase in the number of receptors expressed. In contrast, when the mechanism underlying LTD ( Luthi et al 1999) was studied, all cells undergoing LTD showed no change in single channel conductance, indicating that LTD is the result in a reduction in receptor number.