Applicability of Peak-Scaled Nonstationary Fluctuation Analysis to the Study of Inhibitory Synaptic Transmission in Hippocampal Cultures

Abstract

At present, there are no direct methods to determine the number of synaptic receptor-related channels activated in the course of synaptic transmission (N) or a value of the single-channel conductance (γ). Peak-scaled nonstationary fluctuation analysis (PS NSFA) should be considered the most well-developed indirect approach used for estimating these parameters. Despite the relatively wide using of this approach for the analysis of various synaptic currents, some aspects of possible errors that can occur in the course of data acquisition or their subsequent processing have not been studied. We examined in detail the problem of applicability of PS NSFA in the study of spontaneous and evoked GABA-ergic inhibitory postsynaptic currents (IPSCs). IPSCs were recorded using a dual patch-clamp technique from hippocampal neurons growing in low-density cultures. Parameters of the recorded IPSCs and values for different components of GABA-ergic synaptic transmission reported earlier were used for simulations and PS-NSFA analysis. In Monte Carlo computer simulations of evoked IPSCs, the influence of series resistance, background noise, asynchronicity of transmitter release, GABA A channel properties, dendritic attenuation, and instrumental filtering on γ estimates obtained by PS NSFA was examined. We concluded that the γ and, consequently, N values may be satisfactorily estimated by the suggested approach using spontaneous and evoked IPSCs recorded in inhibitory synaptic connections in hippocampal cultures within a wide range of experimental conditions. We also estimated the mean of the single-channel conductance of synaptic GABA A receptors in neurons from primary hippocampal cultures and found that this value (29 ± 5 pS) agrees well with the high conductance of single synaptic GABA A receptors observed in acute hippocampal slices. This indicates that dissociated cultures are an adequate model for studying the properties of synaptic GABA A receptors.

Studying properties of neurotransmitter receptors by non-stationary noise analysis of spontaneous postsynaptic currents and agonist-evoked responses in outside-out patches

“…Several studies have employed whole-cell recordings of spPSCs and non-stationary noise analysis (typically peak-scaled) to estimate the single-channel conductance of synaptic receptor channels (e.g., see refs. 26,31,42,45,46,48,52,53,[74][75][76][77]. In cases where it has been possible to compare the conductance estimates obtained from non-stationary noise analysis with those obtained from directly resolved single-channel openings in the spPSCs, a reasonably good correspondence between the estimates can be observed 26,31 .…”

“…Rather, the degree of skewness depends on the temporal structure of the fluctuations around the mean during the decay phase after the peak response and can be investigated with covariance analysis of the ensemble of PSCs 50 (see below). Skewed variance versus mean relationships have been observed both for physiologically recorded PSCs and for simulated responses based on Markov-style kinetic schemes 45,[50][51][52][53] .…”

“…Although the series resistance should be as low as possible to minimize low-pass filtering of recorded events 6,53,62 (see ref. 50 for detailed examples of distortion of single-channel conductance estimates from non-stationary noise analysis), changes in the series resistance can also compromise the results.…”

Maximum likelihood estimation of biophysical parameters of synaptic receptors from macroscopic currents

“…The number of currents necessary for a particular algorithm to secure a given accuracy of unitary current estimate is an important practical issue. With many hundreds or even thousands of simulated macroscopic currents accuracy of PS NSFA in estimating the unitary current is fairly good (Markova et al, 2005 ; Hartveit and Veruki, 2006 ). At the same time it is hard to collect more than about 100 of evoked postsynaptic currents in steady-state conditions in routine electrophysiological experiments.…”