Fig. 6: Sinusoidal 8-Hz stimulation of the RE attenuates the renewal of extinguished fear.

A A representative image showing viral expression and optic fiber placement targeting the RE. Similar photomicrographs were taken to confirm placements for all animals. B Experimental timeline for fear renewal procedure. C Schematic of the optogenetic stimulation protocol by which RE neurons were stimulated with an 8-Hz sine wave pattern beginning five seconds before CS onset and stopping five seconds after CS termination. D Spectrograms showing that blue-laser stimulation using 8-Hz sine waves induces 8-Hz rhythms in the RE LFPs of ChR2-expressing rats, (E) but not control mCherry-expressing rats. F Red-laser stimulation was also insufficient to induce 8-Hz rhythms in ChR2 rats. G Average laser-evoked theta power was greatest in ChR2 under blue-laser stimulation, relative to red-laser (n = 5/group, p = 0.0014, two-way ANOVA with Bonferroni correction). H Raster plot of one example single unit phase-locked to blue-laser stimulation in a ChR2-expressing rat. I 8-Hz sinusoidal stimulation entrained 7/8 RE units in ChR2-expressing rats, compared to 0/8 units in mCherry-expressing rats (n = 8 units/group, p = 0.0014, Fisher’s exact test). J Average firing rate in relation to the phase of laser stimulation of the same example neuron in H. K 8-Hz sinusoidal stimulation strongly entrained the firing of RE neurons (n = 8 units/group, p = 0.0061, Unpaired t-test), (L) despite not uniformly increasing their firing rates (p = 0.374, Unpaired t-test). M Average freezing data during conditioning and extinction sessions. N Freezing behavior during fear renewal trials shows that sinusoidal 8-Hz stimulation of RE attenuates fear renewal (p = 0.006; Three-way ANOVA, Laser x Group interaction) in rats expressing ChR2 (n = 11; p = 0.0493, Bonferroni correction) and not mCherry (n = 11; p = 0.0835, Bonferroni correction). O This is further confirmed by comparing the average freezing across the five CS trials (p = 0.004; Two-way ANOVA, Laser × Group interaction). RE nucleus reuniens of the thalamus, MRL mean resultant length. Line plots represent mean ± s.e.m.s; boxplots represent mean plus minima and maxima with lower and upper quantiles. *p < 0.05; **p < 0.01. Source data are provided as a Source Data file.