Figure 1.
During adaptation animals (n = 13) were exposed to six CS- only and the entire session was repeated six hours later. Conditioning took place on the following two days (days -1 and 0): The CS+ was presented three times during each conditioning session, every time co-terminating with an electric footshock. Memory was tested on days 1 and 2. On day 1, six consecutive retrieval sessions were carried out (R1 through R6), with 30 minutes between sessions. Extinction memory was recalled on day 2 in two sessions (E1 and E2), again separated by 30 minutes. All sessions on days 1 and 2 were identical (see inset), and contained four CS- and four CS+ presentations. CS-evoked conditioned freezing (B). The fraction of time spent freezing during presentation of the CS+ declined over extinction sessions (R1-6) and remained low during extinction memory recall (E1&2). Note the very low levels of conditioned freezing during CS- presentations. Freezing in response to the CS+ was compared across sessions, revealing that, from session R3 onwards, the observed freezing values significantly differed from R1. Values are mean ± SEM; asterisks indicate the significance level: *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 2.
Verification of recording sites.
(A) Schematic representation of electrode locations in the medial prefrontal cortex (mPFC), hippocampal CA1, and lateral amygdala (LA). Black dots mark field potential and unit activity recordings sites; grey dots represent sites of electrical microstimulation. (B) Representative Nissl stained coronal sections showing electrode positions in the mPFC, CA1 and LA. Arrows indicate the electrode tip positions. PrL, prelimbic region of mPFC; IL, infralimbic region of mPFC; BLA, basolateral amygdala.
Figure 3.
Representative data from one animal in all three channels (CA1, LA, mPFC) during CS+ presentation at R1 (top), R6 (middle) and E1 (bottom).
For each session, LFP waveforms (left) as raw (light colored) and 2–12 Hz filtered (dark colored) traces, cross-correlograms (middle) and time-frequency representations (right) are shown. Note the coupled theta activity, evident as periodic patterns in the cross-correlograms, among all three channels during R1. This effect is absent at R6, while at E1 synchronous activity emerges again, predominantly in the CA1-mPFC and the mPFC-LA pathways. Characters indicate animal behavior (f: freezing; w: risk-assessment).
Figure 4.
Group averages from recordings in 13 animals of cross-correlation between LA, CA1 and mPFC during extinction (R1-R6) and extinction recall (E1 & E2).
(A) Magnitude of cross-correlation waveforms at different latencies (±π, ±2π) during presentation of CS+ (top) and CS- (bottom). Note the decrease in cross-correlation magnitude evident in all channel combinations as extinction progresses. (B) Mean cross-correlation values (averages of ±π, ±2π), summing up the panels presented in (A). Coupling clearly decreases over the course of extinction in all channel pairs. (C) Mean cross-correlation values as shown in (B), however, considering only periods during which the animal actually displayed freezing while exposed to the CS+. Sessions R3-5 are binned, due to the little freezing observed from R3 onwards (no freezing periods survived artifact rejection for R6). Likewise, freezing was rarely observed in response to CS- and is therefore not quantified. Note the similar dynamics over sessions in comparison to (B). Values are mean ± SEM. in all plots; asterisks indicate the significance level: *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 5.
Effects of theta burst stimulation on CS+ - evoked freezing.
Trains of stimuli were delivered locally to CA1 and LA at the same phase (“in phase”; n = 7 animals tested) or at 180° phase offset (“anti phase”; n = 8), after each CS+ presentation in each retrieval session (R1-R6) during extinction learning. Note the delay in extinction of conditioned freezing and maintained fear responsiveness during extinction recall in the “in phase” group (n = 7) as compared to “sham” controls (n = 6). Asterisks indicate significant differences “in phase” vs. “anti phase” (p<0.05). Values are mean ± SEM.