Single units in the medial prefrontal cortex with anxiety-related firing patterns are preferentially influenced by ventral hippocampal activity

Abstract

The medial prefrontal cortex (mPFC) and ventral hippocampus (vHPC) functionally interact during innate anxiety tasks. To explore the consequences of this interaction, we examined task-related firing of single units from the mPFC of mice exploring standard and modified versions of the elevated plus maze (EPM), an innate anxiety paradigm. Hippocampal local field potentials (LFPs) were simultaneously monitored. The population of mPFC units distinguished between safe and aversive locations within the maze, regardless of the nature of the anxiogenic stimulus. Strikingly, mPFC units with stronger task-related activity were more strongly coupled to theta-frequency activity in the vHPC LFP. Lastly, task-related activity was inversely correlated with behavioral measures of anxiety. These results clarify the role of the vHPC-mPFC circuit in innate anxiety and underscore how specific inputs may be involved in the generation of behaviorally relevant neural activity within the mPFC.

Figure 1. mPFC single units have task-related firing patterns in the EPM

(A-C) Upper panels: Spatial distributions of spikes of representative single units that fired preferentially in the closed (A) or open (B) arms or the center (C) of the EPM. The behavior track is shown in grey and the location of occurrence of each spike is marked with a green circle. Lower Panel: Spatial firing rate maps of the same single units. Average normalized firing rates are color-coded (higher firing rates are indicated by warmer colors) for each pixel. See also Figure S4.

Figure 2. mPFC single units differentiate between open and closed arms in the EPM

(A) Depiction of the standard EPM. (B,C) Normalized firing rates (% difference from mean firing rate) from each of the arms for representative units with putative task-related (B) and task-unrelated (C) firing patterns. (D) Scatter plot of normalized firing rates (% difference form mean rate) across both closed arms for all recorded units with > 100 total spikes. Each point represents a single unit. Note that normalized rates in the closed arms are strongly positively correlated (r=0.38, p<0.0001, n=69 cells). (E) Same as (D), but for rates in the two open arms (r= 0.25, p<0.04, n=69 cells). (F) Correlation of normalized rates across closed and open arms. Note that firing rates are negatively correlated across arms of different type (r= −0.64, p<0.0001, n=69 cells). See also Figure S1.

Figure 3. mPFC firing patterns in the EPM are not due to novelty or locomotor differences

(A) Scatter plot of normalized firing rates for mPFC single units in both closed arms during a second ten-minute exposure to the EPM, a day after the original exposure. (B) Same as left panel, but for firing in both open arms. (C) Cumulative sum distribution of speed in the closed and open arms. Inset: Mean +/− s.e.m. speed across animals in the closed and open arms (p=0.56). (D) Cumulative sum distribution of acceleration. Shaded areas are ± s.e.m.

Figure 4. mPFC units with anxiety-related firing patterns are over-represented in the population

(A,B) Cells with positive EPM scores (black circles) have more marked task-related firing than those with negative EPM scores (grey circles), as measured by higher correlations between firing in the two closed (A) and two open (B) arms. Correlation values for grey and black circles are plotted in their respective colors. (C) Distribution of EPM scores for all recorded units with > 100 total spikes. The arrowheads and stars marks the median and mean of the subsamples of units with negative (grey) and positive (black) scores. The distribution differs significantly from that expected by chance (red line). See also Figure S6.

Figure 5. Changes in unit activity precede transitions across compartments in the EPM

(A,B) Representative transitions (black) from the closed arm to the open arm (A) and from the open arm to the closed arm (B), superimposed on the behavioral trace from the entire session (grey). Arrows indicate the direction of movement and the green bars indicates the boundary between closed arm and center. (C,D) Peri-event time histograms averaged across all closed-to-center transitions (C) and center-to-closed transitions (D) for all closed arm-preferring units (blue), open arm-preferring units (red), and for units without task-related firing, as defined by negative EPM scores (grey). Firing rates were converted to Z-scores in 0.5 s bins. Arrows indicate the time point at which significant changes in firing rate begin to occur, as calculated by the change point method (p<0.01, see Experimental Procedures). Note that all significant changes in mPFC unit activity occur 1–3 s prior to the animal leaving (C) or entering (D) the closed arm. No significant change points were identified for the units with negative EPM scores. Units recorded in the standard EPM at 200 and 0–5 Lux were pooled for this analysis (n=69 units from the standard EPM at 200 Lux and n=122 units from the standard EPM in the dark). (E) Principal components analysis of firing rates during transitions. Upper panel: first principal component of the entire population of units is shown for closed to center transitions. Note that the curve in has a time-course similar to the firing patterns of closed arm units (C, blue bars). Lower panel: Mean scores of the first principal component (PC1) of closed arm-preferring units, open arm-preferring units and units with negative EPM scores. (F) Same as (E), but for center to closed arm transitions (n=69 units from the standard EPM at 200 Lux and n=122 units from the standard EPM in the dark, pooled). *p<0.01, Wilcoxon rank sum test. Error bars are ± S.E.M.s.

Figure 6. Paradigm-related firing patterns do not depend on the geometric arrangement of the arms

(A) 18 units were recorded during exposure to both the standard EPM (upper panel) and an altered EPM (lower panel) in which arms of the same type were adjacent to each other rather than across from each other. (B, C) Upper panels: Firing rate maps (warmer colors represent higher firing rates) for a units that fired preferentially in the closed (A) and open (C) arms of the altered EPM. Lower panel: Scatter plots of normalized rates (% difference from mean firing rate) for all 18 units across the two closed (B) and open (C) arms in the altered EPM. (D,E) Correlation between firing rates in closed arms (D) and open arms (E) across the two mazes. Firing rates were significantly positively correlated across arms of the same type even across mazes. The correlation in (E) is significant even if the point on the upper right corner is excluded (r=0.46, p=0.04).

Figure 7. mPFC single units respond similarly to different aversive stimuli

(A) Standard EPM in the dark. Light level was <5 lux. (B) EPM with four closed arms. Two of the arms were illuminated with bright (600 lux) light. Light in the other arms remained < 5 lux. (C) Upper panel: spatial firing rate map of a representative single unit recorded in the standard EPM in the dark. Lower panel: Behavior track of the session from which this recording was obtained. Safe (closed) and aversive (open) arms are depicted in blue and red, respectively. (D) Upper panel: spatial firing map of the same unit shown in (C), but recorded in dark/bright maze. Note that the example unit fires preferentially in the aversive arms of both mazes. Lower panel: Behavior track of the session of the recording shown in the upper panel. Safe (dark) and aversive (bright) arms are depicted in blue and red, respectively. (E) Scatter plot showing that firing rates across closed and open arms were negatively correlated (maze shown in (A), n=105 units). (F) Scatter plot showing that normalized firing rates in the dark and bright arms were negatively correlated (maze shown in (B), n=105 units). (G–H) Correlations of normalized firing rates across the two mazes for safe arms (G) and aversive arms (n = 105 units) (H). Note that rates for arms of the same type were positively correlated even across mazes in which the aversive stimuli were different. (I) Bar graph showing the % time spent in the aversive arms in the standard and modified EPMs. In all configurations of the EPM naive mice spent less than 50% time in the aversive arm (*p<0.05, Wilcoxon’s signrank test). See also Figure S5.

Figure 8. mPFC units phase-locked to vHPC theta oscillations have stronger task-related firing

(A,B) Left Panel: Distribution of the phases of firing relative to vHPC theta oscillations for an example mPFC single unit. This unit is significantly phase-locked to vHPC theta oscillations (p<0.05, Rayleigh’s test for circular uniformity). Right panel: Spatial firing rate map for the same unit. Note that this unit is preferentially active in the open arms. (B) Same as (A), but for a unit that is not significantly phase-locked to vHPC theta (left panel) and that does not distinguish robustly closed arms from open arms (right panel). (C) Left panel: bar graph showing mean EPM scores for units that were (black bars) and were not (grey bars) significantly phase-locked to vHPC theta oscillations. Phase locked units had on average higher EPM scores than other units (mean score = 0.31 ± 0.07 and 0.17 ± 0.04, for phase locked and other units, respectively, p<0.05, Wilcoxon’s test, n = 69 units) Right panel: normalized cumulative sum distributions of the EPM scores shown averaged in the left panel. Note that the difference seen in the left panel between the EPM score distributions of vHPC phase locked units and other units is not due to a change in the size of the right tail of the distribution. (D) and (E) Same as (C), but for phase locking to dHPC and mPFC theta oscillations. Phase-locking to dHPC or mPFC theta oscillations did not have significant effects on EPM scores (p = 0.31 and 0.07, respectively). See also Figure S2.

Figure 9. mPFC units that follow vHPC theta oscillations have more robust task-related firing patterns

(A) Shifting the spike train (black bars) backwards in time (lower panel) relative to the theta-filtered vHPC local field potential (grey lines) reveals stronger phase-locking. (B) and (C) Effect of shifting the spike train of two representative mPFC single units on the strength of phase-locking (MRL) to vHPC theta oscillations. The unit in (B) follows vHPC theta, as the maximal MRL value is observed at a negative lag (−12 ms), while the unit in (C) leads vHPC theta, with a peak at a positive lag (+54 ms). A star marks the position of the maximum MRL. A dashed line was plotted at zero lag for reference. (D) Left panel: bar graph showing mean +/− EPM scores for units with negative lags relative to hippocampal theta (grey) and all other units (black). Units that followed vHPC theta had significantly higher EPM scores (mean score = 0.24 ± 0.047 and 0.07 ± 0.05 for units that follow vHPC theta and other units, respectively. *p<0.05, Wilcoxon’s test, n = 69 units). Right panel: normalized cumulative sum distributions of the EPM scores shown averaged in the left panel. (E) and (F) Same as (D), but for dHPC and mPFC theta oscillations. Units that followed mPFC or dHPC theta oscillations did not have higher EPM scores than other units, p=0.08 and 0.51, respectively).

Figure 10. EPM scores and mPFC single unit activity are correlated with anxiety-related behavioral measures in the EPM

(A) Scatter plot of mean EPM score against % path in the open arms of the EPM for all animals with at least 3 simultaneously recorded single units with more than 100 spikes. EPM scores and open arm exploration are strongly positively correlated (r=+0.65, p<0.01). Animals were divided into two groups: those that were below (avoidant, n=9) and above (non-avoidant, n=8) 50 % path in the open arms. (B) Mean EPM scores for non-avoidant WT (mean score=0.171 ± 0.0051, n=61), avoidant WT (0.072 ± 0.0031, n= 95) and 5-HT1A receptor knockout mice (0.032 ± 0.011, n=20). (A–B) Units from WT mice recorded in the standard EPM at 200 and 0–5 Lux were pooled (n=39 units from the standard EPM at 200 Lux and n=117 units from the standard EPM in the dark). (C) EPM score normalized cumulative sum distributions for all WT single and all 5-HT1A receptor knockout single units. The two distributions are significantly different (p<0.05, Wilcoxon’s test). (D) Bar graph showing mean rate in the EPM for non-avoidant WT, avoidant WT and 5HT-1A receptor knockout mice. Rates are plotted as fold increase from the familiar environment. No change (fold increase of 1) is plotted as a dotted line. Avoidant WT and 5-HT1A receptor knockout mice had significant increases in firing rate relative to the familiar environment in the EPM (*p<0.05). (C–D) As 5-HT1A knockout mice were only exposed to the EPM at 200 Lux, only WT sessions used at this illumination were used to make comparisons across genotypes (n=69 and 24 units for WT and knockout mice, respectively). See also Figure S3.