Conditioned freezing in response to presentation of the first CS+ during R1, R6 and E.

<p>The fraction of time spent freezing during presentation of the first CS+ significantly declined comparing R1 with R6 and E. Values are mean±SEM; asterisks indicate the significance level: ***,p<0.001 [One-way ANOVA (F_2,36 = 16.03, p<0.001), followed by Tukey’s post hoc test for multiple comparison], n = 13.</p

Summary of directional unit phase-locking analysis.

<p>Each identified unit (CA1, LA, IL-PFC) was assigned a theta phase of LFPs recorded in the same and the other two brain areas (unit CA1 - LFP CA1, LA and IL-PFC, unit LA - LFP LA, CA1 and IL-PFC, unit IL-PFC – LFP IL-PFC, CA1 and LA), and lag or lead of the respective unit was determined by the peak MRL value. N [unit/animal] describes the total number of identified phase-locked units and the number of animals in which these units were identified. Results obtained at freezing during retrieval of fear memory (R1), and non-freezing during recall of fear extinction (E). A negative mean [ms] describes a preceding (lead) of the unit, phase-locked to the respective LFP, a positive mean circumscribes the opposite (lag). The p value determines the significance of directionality during R1 and E by use of paired Wilcoxon’s signed rank non-parametric test.</p

Directional Theta Coherence in Prefrontal Cortical to Amygdalo-Hippocampal Pathways Signals Fear Extinction

<div><p>Theta oscillations are considered crucial mechanisms in neuronal communication across brain areas, required for consolidation and retrieval of fear memories. One form of inhibitory learning allowing adaptive control of fear memory is extinction, a deficit of which leads to maladaptive fear expression potentially leading to anxiety disorders. Behavioral responses after extinction training are thought to reflect a balance of recall from extinction memory and initial fear memory traces. Therefore, we hypothesized that the initial fear memory circuits impact behavioral fear after extinction, and more specifically, that the dynamics of theta synchrony in these pathways signal the individual fear response. Simultaneous multi-channel local field and unit recordings were obtained from the infralimbic prefrontal cortex, the hippocampal CA1 and the lateral amygdala in mice. Data revealed that the pattern of theta coherence and directionality within and across regions correlated with individual behavioral responses. Upon conditioned freezing, units were phase-locked to synchronized theta oscillations in these pathways, characterizing states of fear memory retrieval. When the conditioned stimulus evoked no fear during extinction recall, theta interactions were directional with prefrontal cortical spike firing leading hippocampal and amygdalar theta oscillations. These results indicate that the directional dynamics of theta-entrained activity across these areas guide changes in appraisal of threatening stimuli during fear memory and extinction retrieval. Given that exposure therapy involves procedures and pathways similar to those during extinction of conditioned fear, one therapeutical extension might be useful that imposes artificial theta activity to prefrontal cortical-amygdalo-hippocampal pathways that mimics the directionality signaling successful extinction recall.</p></div

Phase-locking of single unit activity to LFP theta <i>across</i> regions.

<p>(A) Summary of phase-locking analysis during freezing states in R1 and non-freezing states in E. Each identified unit in CA1 (upper), LA (middle), and IL-PFC (bottom) was assigned a LFP theta phase recorded simultaneously in the other two brain areas (grey, blue, green diagrams indicating LFP in CA1, LA, IL-PFC, respectively). Plotted are mean time shifts between unit activity and the LFP theta phase calculated from MRLs in each area. Note the significant lead of unit activity in IL-PFC related to the LFP theta in CA1 and LA during non-freezing in E (highlighted by red lines). (B) Mean MRL of all identified theta phase-locked IL-PFC units during freezing in R1 and non-freezing in E related to LFP theta oscillations recorded simultaneously in CA1 (upper diagram) and LA (lower diagram). Note the peak MRL (indicated by red arrows) signaling a lead of unit activity in IL-PFC related to LFP theta in CA1 and LA during non-freezing in E. (C) Schematic representation of phase-locking of single unit activity to LFP theta across regions, during freezing at R1 and non-freezing at E. Black lines with two arrowheads indicate high theta synchrony (with no significant time lead or lag), red lines indicate theta directionality. Values are mean±SEM; asterisks indicate the significance level revealed by Wilcoxon Signed Rank test : *,p<0.05. MRL, mean resultant length vector; f, freezing; nf, non-freezing.</p

Phase-locking of single unit activity to LFP theta <i>within</i> regions.

<p>(A) Representative example of directional unit phase-locking analysis of an identified CA1 unit (same unit as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077707#pone-0077707-g003" target="_blank">Figure 3B</a>). Mean resultant length vector (MRL), computed by assigning each spike of the unit to the simultaneously recorded LFP theta phase, calculated for 50 different temporal offsets (4 ms bin, from −100 ms to +100 ms), indicate phase lead of the unit by approximately −50 ms (marked by red arrow). (B) Summary of phase-locking analysis of units and LFP theta in CA1, LA and IL-PFC, during freezing in R1 and non-freezing in E. Plotted are mean time shifts between unit activity and the LFP theta phase calculated from MRLs in each area. Note phase locking of units to LFP theta within each region during both freezing and non-freezing, except for phase lead of CA1 units (−33.9±10.7 ms during R1 freezing). Values are mean±SEM; asterisks indicate the significance level revealed by Wilcoxon Signed Rank test : **,p<0.01.</p

Verification of recording sites and isolation of single units.

<p>(A) Schematic representation of electrode locations in the infralimbic prefrontal cortex (IL-PFC), hippocampal CA1, and lateral amygdala (LA). Black dots mark recordings sites. (B) Representative examples of extracellular waveform sorting in IL-PFC, CA1 and LA. The top images show superimposed waveforms of two simultaneously recorded neurons and cluster analysis, formed in three-dimensional space after applying principal component analysis. The bottom images display the sorted unit (yellow) of the continuous signal below. PrL, prelimbic region of mPFC; IL, infralimbic region of mPFC; BLA, basolateral amygdala; LA lateral amygdala.</p

Directional phase locking of LFP theta.

<p>Directional phase locking of LFP theta at first CS+ presentation (A), freezing (B) and non-freezing (C) stages during fear memory retrieval (R1; open diagrams), extinction training (R6; grey diagrams), and recall of fear extinction (E; black diagrams). Diagrams indicate time shifts of correlation peaks of LFP theta amplitudes obtained between all possible pairs of recorded regions (CA1/LA, upper row; CA1/IL-PFC, middle; LA/IL-PFC, bottom) as an indicator of the directionality (lagging or leading) between oscillating signals. Positive values indicate that interactions are driven by one region, and negative values indicate that the interaction is driven by the respective other. Note lack of theta directionality during entire CS+ at R1 and R6, and during freezing at R1, R6 and E. Whereas IL-PFC leads to CA1 during CS+ and IL-PFC leads to CA1 and LA during non-freezing at E (highlighted by red lines in A and C). Further single unit phase-locking analyses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077707#pone-0077707-g004" target="_blank">Figure 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077707#pone-0077707-g005" target="_blank">5</a>) focused on R1 freezing (B) and non-freezing in E (C) representing the most prominent changes of fear behavior expression as well as LFP based shifts in directionality (grey background). (D) Schematic representation of LFP theta directionality in CA1, LA, and IL-PFC during entire CS+ presentation, freezing and non-freezing at R1, R6 and E. Black and grey lines with two arrowheads indicate high theta synchrony (with no significant time lead or lag), red lines indicate theta directionality. Values are mean±SEM; asterisks indicate the significance level revealed by Wilcoxon Signed Rank test : *,p<0.05; **,p<0.01.</p