Cellular mechanisms contributing to response variability of cortical neurons in vivo

Abstract

Cortical neurons recorded in vivo exhibit highly variable responses to the repeated presentation of the same stimulus. To further understand the cellular mechanisms underlying this phenomenon, we performed intracellular recordings from neurons in cat striate cortex in vivo and examined the relationships between spontaneous activity and visually evoked responses. Activity was assessed on a trial-by-trial basis by measuring the membrane potential (Vm) fluctuations and spike activity during brief epochs immediately before and after the onset of an evoked response. We found that the response magnitude, expressed as a change in Vm relative to baseline, was linearly correlated with the preceding spontaneous Vm. This correlation was enhanced when the cells were hyperpolarized to reduce the activation of voltage-gated conductances. The output of the cells, expressed as spike counts and latencies, was only moderately correlated with fluctuations in the preceding spontaneous Vm. Spike-triggered averaging of Vm revealed that visually evoked action potentials arise from transient depolarizations having a rise time of approximately 10 msec. Consistent with this, evoked spike count was found to be linearly correlated with the magnitude of Vm fluctuations in the gamma (20-70 Hz) frequency band. We also found that the threshold of visually evoked action potentials varied over a range of approximately 10 mV. Examination of simultaneously recorded intracellular and extracellular activity revealed a correlation between Vm depolarization and spike discharges in adjacent cells. Together these results demonstrate that response variability is attributable largely to coherent fluctuations in cortical activity preceding the onset of a stimulus, but also to variations in action potential threshold and the magnitude of high-frequency fluctuations evoked by the stimulus.

Fig. 1.

This figure illustrates the methodology we used to investigate the relationships between spontaneous and visually evoked activity from cortical neurons recorded intracellularly in vivo. The data are taken from a chattering cell and illustrate the response to a drifting sine wave grating (0.8 cycles/°, 2 °/sec, 10 cd/m²) presented to the left eye.A, These traces show the membrane potential (V_m) of the cell (top plot) and the time course of the stimulus (bottom plot) recorded during a single trial. The short horizontal bar on the left indicates the epoch chosen for the baseline V_m measurement, and the one shown during the onset of the stimulus indicates the epochs of activity chosen for analysis. The small arrow marks the onset of the visual response. B, Expanded traces ofV_m (1), the extracted spike train (2), and the median-filteredV_m (3) illustrating the signals just before and just after the onset of the response (arrow). The bracketed horizontal line marks the boundaries of the adjacent 64 msec windows of spontaneous (a) and visually evoked (b) activity sampled for analysis. When the relation between spontaneous V_m and evoked spike activity was analyzed, the window for the spike analysis was extended to the first 128 msec of the response. C, Expanded traces of V_m and the stimulus time course during a single trial while the cell was hyperpolarized below firing threshold. In this and all other measurements, the stimulus began moving at the moment of its appearance.

Fig. 2.

Illustration of the method for calculating action potential threshold. A, A brief epoch of data taken from a cell during the response to a visual stimulus. Three action potentials are shown that differ slightly in their threshold voltages (arrows). B, Expanded traces of the three action potentials shown in A illustrating the threshold voltage (lower arrows) and the peak rate of change (dV_m/dt_max) of the action potential upstroke (upper arrows).C, Expanded traces of the rate of change of voltage (dV_m/dt) for the same three action potentials as shown in B. Thetraces in B and C are plotted in temporal register so that the voltage values and their rates of change can be compared. The pair of vertical dashed lines in each trace illustrates the correspondence between the peak rate of change during the upstroke (right) and the threshold (left). The horizontal linesindicate a value of 0 fordV_m/dt.

Fig. 3.

Fluctuations in spontaneous and evokedV_m are linearly correlated. A, B, Raw data collected on two separate trials from the same cell as shown in Figure 1. The top plots display the entire duration of each of the two trials. The small arrowsmark the onset of the visual response, and thebracketed horizontal line indicates the epochs chosen for the correlation analysis. The middle plotshows the raw data at an expanded time scale. The action potentials have been truncated as indicated by the dashed lines. The bottom plots show the median-filtered data for the same epochs. The dotted line indicates the baseline membrane potential, the bracketed lines indicate the epochs chosen for analysis, and the bottom horizontal line indicates the stimulus time course. The visual response latencies are marked by the arrows. Note that the evoked activity was enhanced when the spontaneousV_m was depolarized (bottom trace in A) and reduced when the spontaneousV_m was hyperpolarized (bottom trace in B). C, Scatter plot of the mean V_m computed from two adjacent 64 msec epochs of spontaneous activity preceding the visual response on each trial. D, Scatter plot of the meanV_m of the evoked response and its immediately preceding spontaneous activity on each trial. InC and D the straight lineshows the linear regression fit to the data.

Fig. 4.

Time course of membrane potential correlation.A, The mean and SD of the correlation coefficient as a function of time lag for all the cells having a significant linear correlation. The value at time 0 displays the autocorrelation computed from the spontaneous V_m immediately preceding the response. Points lying to theleft and right display the correlation between this epoch and the preceding spontaneous and evoked activity, respectively. Each point in the plot is computed from a different fraction of the cells. B, The percentage of cells at each time lag showing a significant linear correlation.C, The mean and SD of the partial correlation coefficient as a function of time lag for all the cells in the sample. Note the rapid fall-off in correlation magnitude. D, Histogram of the decay time constant computed from the autocorrelation function from the entire sample of cells. The unfilledand filled bars show the values obtained from the spontaneous and stimulus-evoked data, respectively. Theinset shows the autocorrelation function computed from the spontaneous (thin line) and stimulus-evoked (thick line) activity in one cell. The autocorrelation functions and the data displayed in A andC were fit by the equation y =a + be^−x/c. The data in B were fit by the equation y =a + bx^c.

Fig. 5.

Correlation magnitude is increased by membrane hyperpolarization. A, Scatter plots of the spontaneous versus visually evoked V_m recorded across trials under control conditions (▵) and when the cell was hyperpolarized with −0.8 nA of current (▪). The thinand thick lines depict the linear regression fit to the data collected under control (r = 0.76) and hyperpolarized (r = 0.81) conditions, respectively.B, Mean and SD of the linear correlation coefficient for the same cell shown in A as a function of time lag for the control (▵) and hyperpolarized (▪) conditions, respectively. The filled square at time 0 displays the autocorrelation for both conditions. Note that the mean level of correlation at all time lags is higher when the cell is hyperpolarized below firing threshold. The data were fit by the equation y =a + bx^c.

Fig. 6.

The latency and number of visually evoked spikes are linearly correlated with the spontaneousV_m immediately preceding the visual response. A, B, Raw data (top plots) and corresponding spike trains (bottom plots) for the same two trials displayed in Figure 3A. Thearrows mark the response latency as determined from the change in V_m. C, Scatter plot of the number of spikes occurring in the first 128 msec of the response versus the mean spontaneous V_m in the 64 msec preceding the response onset. D, A similar scatter plot between latency to the first spike and the spontaneousV_m. Note that spike count increases and the latency of the first spike decreases in proportion to the spontaneousV_m.

Fig. 7.

Mean and SD of the correlation between evoked spike count and the preceding V_m as a function of time lag (n = 52). The filled circles depict the linear correlation coefficients and are fit by the equation y = 1/(a +bx³), and the open triangles depict the partial correlation coefficients and are fit by the equation y = a +be^−x/c. Note that the mean correlation between spontaneous V_m and evoked spike count is significantly lower than the correlation between spontaneous and evoked V_m. As with theV_m correlation depicted in Figure 4, the partial correlation decays much more rapidly with time lag.

Fig. 8.

Visually evoked changes inV_m are moderately correlated with the latency and number of evoked spikes. A, Scatter plot of the number of visually evoked spikes versus the mean evokedV_m in the 128 msec after the response onset.B, Similar scatter plot between latency to the first spike and the mean evoked V_m on each trial.

Fig. 9.

STA of V_m reveals the time course of depolarization leading to action potentials.A, This plot illustrates an epoch of raw data sampled during the response to a visual stimulus in a chattering cell.B, STA of V_m for all the action potentials recorded on 20 consecutive trials during spontaneous (thin line, 232 spikes) and stimulus-evoked (thick line, 1214 spikes) activity. The filled circles (spontaneous) and filled diamonds(evoked) mark the points at which the mean voltage crosses a predefined baseline. The time elapsed between these points is defined as the width of the peak in the STA, and the time between the leading point and time point 0 is defined as the rise time of the peak in the STA. C, D, These plots show the distribution of peak widths (C) and rise times (D) and associated Gaussian fits for the spontaneous and visually evoked data across the entire sample of cells (n = 37).

Fig. 10.

The number of visually evoked spikes is linearly correlated with the γ-band power in the V_mand more weakly correlated to the mean V_m.A, Raw data collected on two separate trials (a, b) from the same cell.B, Fourier power spectra of the corresponding median-filtered V_m traces inA, and the extracted spike train (top traces). C, Scatter plot of the number of spikes occurring in the first 1024 msec of the response versus the normalized γ power (20–70 Hz) in the median-filteredV_m. D, Scatter plot of the number of spikes versus the mean evoked responseV_m for the same 1024 msec epoch of data. Thecalibration bar in B represents the percentage of power with respect to the peak value at the DC level. In these examples, firing rate is enhanced when both the γ-band power and the mean V_m are enhanced.

Fig. 11.

Visual cortical neurons exhibit dynamic variations in the threshold of spike initiation. The plots inA and B show examples of the raw data collected from two cells that exhibit different ranges of variation in action potential threshold. The top plots show the data collected during the visual response on a single trial for each cell. The middle and bottom plots display brief epochs of this data at medium and fast time scales, respectively. Action potential threshold is marked by a filled diamondin the top and middle plots and byarrows in the bottom plots. Thecontinuous horizontal lines in the middle plots are used as a reference for comparison of different spike thresholds. The dashed lines indicate truncated spikes.

Fig. 12.

A, B, Distributions ofV_m (unfilled bars) and spike threshold (filled bars) accumulated from the visual responses across all trials for the same two cells shown in Figure 11. The distributions of V_m were sampled during the periods when the cells were not firing an action potential. Note that the distributions overlap substantially, indicating that V_m can often exceed the minimum threshold without the cell firing a spike.

Fig. 13.

The correlation between spontaneous and evoked activity is dependent on response strength. A, Peristimulus time histograms of spike activity in response to five different spatial frequencies of a drifting square-wave grating (0.4–1.2 cycles/° in steps of 0.2 cycles/°). Thecalibration bar at the bottom rightindicates the number of spikes/bin. B, Normalized tuning curve for the cell computed from the mean firing rate during the first second of the visual response. C, Scatter plot of the normalized strength (■, thick line) and regression slope (▴, thin line) of the correlation between the spontaneous and evoked V_m and the normalized response strength to gratings of different spatial frequencies.D, Scatter plot of the normalized strength (■,thick line) and regression slope (▴, thin line) of the correlation between the spontaneousV_m and evoked spike count and the normalized response strength to gratings of different spatial frequencies.

Fig. 14.

Extracellular spike activity is correlated with fluctuations in V_m. Two examples of simultaneously recorded extracellular and intracellular activity taken from the same electrode penetrations. The recording electrodes were ∼500 μm apart. A, The top trace shows the V_m of an intracellularly impaled cell during spontaneous and evoked activity. The bottom trace shows the extracellular multiunit activity recorded at the same time. B, The same extracellular units as in A, but a different intracellularly impaled cell. The epoch marked with an asterisk is shown at an expanded time scale in the bottom panels. Note that the spontaneous spike activity of the extracellularly recorded units is correlated with brief periods of depolarization in the intracellularly recorded units.