Footshock stress induces associative fear, SEFR, and SEFL.

(A) Mice received a single session of footshock stress (Stress group) or were exposed to the same context and received no footshocks (NoStress group). Beginning 24 hours after stress, mice underwent the SEFR tone test, fear retrieval in the stress context, and the two-day SEFL procedure. (B) In the stress session, freezing behavior increased as a function of the 4 shock presentations. (C) In the SEFR tone test, freezing behavior was evaluated before (Pre-Tone) and during the tone and inter-tone periods (Tone). All mice exhibited low levels of pre-tone freezing. However, during the tone period, stressed mice displayed increased levels of freezing relative to the NoStress group. (D) Stressed animals displayed robust context-elicited freezing behavior to the stress chamber. (E) During the SEFL conditioning session, all mice displayed little to no freezing prior to the onset of footshock. (F) In the SEFL test session, stressed mice exhibited increased levels of context-elicited freezing relative to the NoStress group. Data presented as mean +/-SEM. N=23: NoStress group (6 males, 6 females), Stress group (6 males, 5 females). βˆ— 𝑝 < 0.05, βˆ—βˆ— 𝑝 < 0.01, βˆ—βˆ—βˆ— 𝑝 < 0.001.

Varying the footshock stress intensity reveals dissociations among associative fear, SEFR, and SEFL.

(A) Animals received either no footshocks (NoStress group) or footshocks at intensities of 0.25 mA, 0.5 mA, or 1 mA (Stress groups). In the stress session, all footshock stress-exposed groups exhibited elevated levels of freezing relative to the NoStress group. (B) In the SEFR tone test, the 0.5mA-Stress and 1mA-Stress groups showed significantly higher freezing than the NoStress group. (C) In the stress context test, the 0.25mA-Stress, 0.5mA-Stress, and 1mA-Stress groups displayed elevated freezing compared to the NoStress group. (D) During SEFL conditioning, no group differences in freezing were observed prior to the footshock. Following footshock, the 0.5mA-Stress and 1mA-Stress groups exhibited increased freezing relative to the NoStress group. (E) In the SEFL test, the 0.5mA-Stress and 1mA-Stress groups exhibited elevated freezing relative to the NoStress group. (F) Correlation of individual subjects’ percent freezing between the stress context test, SEFR tone test, and SEFL test, calculated among animals in the 0.25mA-Stress, 0.5mA-Stress, and 1mA-Stress groups. Data presented as mean +/-SEM. N=27: NoStress (7 males), 0.25mA-Stress (7 males), 0.5mA-Stress (6 males), 1mA-Stress (7 males). βˆ— 𝑝 < 0.05, βˆ—βˆ— 𝑝 < 0.01, βˆ—βˆ—βˆ— 𝑝 < 0.001.

Footshock stress potentiates c-Fos expression in the paraventricular thalamus in response to the SEFR tone test.

(A) Quantification of c-Fos expression across brain regions: BLA, basolateral amygdala; CeA, central amygdala; lPBN, lateral parabrachial nucleus; dlPAG, dorsolateral periaqueductal gray; lPAG, lateral periaqueductal gray; PVT, paraventricular thalamus. (B) Representative images of c-Fos expression in the NoStress-ToneTest and Stress-ToneTest groups. (C) Correlation between c-Fos expression and freezing levels during the tone period of the SEFR tone test. Black circles represent subjects in the NoStress-ToneTest group; red circles represent subjects in the Stress-ToneTest group. Data presented as mean +/-SEM. N=33: NoStress-HomeCage (8 males), Stress-HomeCage (8 males), NoStress-ToneTest (8 males), Stress-ToneTest (9 males) βˆ— 𝑝 < 0.05, βˆ—βˆ— 𝑝 < 0.01, βˆ—βˆ—βˆ— 𝑝 < 0.001.

Mice previously exposed to footshock stress exhibit elevated pPVT activity in response to the novel tone used to elicit SEFR but not to the footshock used to test SEFL.

(A) Representative image GCamP8s expression and fiber tract in the pPVT. (B) Left; Ca2+ signals of pPVT averaged across the three tone presentations in the SEFR tone test for the Stress and NoStress groups. Right; Quantification of AUC during the 30-s tone presentation compared between the NoStress and Stress groups. (C) Left; Ca2+ signals in pPVT during the SEFL conditioning session for the Stress and NoStress groups. Right; Quantification of AUC during the 2-s footshock presentation compared between the NoStress and Stress groups. Data presented as mean +/-SEM. For (B) N=16: NoStress group (6 males, 2 females), Stress group (6 males, 2 females). For (C) N=11: NoStress group (3 males, 2 females), Stress group (4 males, 2 females). βˆ— 𝑝 < 0.05, βˆ—βˆ— 𝑝 < 0.01, βˆ—βˆ—βˆ— 𝑝 < 0.001.

Inhibition of pPVT during footshock stress impaired the induction of SEFR but not SEFL.

(A) Schematic of the AAV viral strategy and representative image of pPVT viral expression. Animals received AAVs encoding the inhibitory designer receptor Hm4Di or a control mCherry fluorescent reporter. Scale bar indicates 100 Β΅m. (B) Experimental timeline. (C) Inhibition of pPVT had no effect on freezing behavior during footshock stress. (D) In the SEFR tone test, prior inhibition of pPVT during footshock stress decreased tone-elicited freezing. (E) The distance traveled in the open field test was comparable between the hM4Di and mCherry groups. (F) Associative fear of the stress context was not affected by pPVT inhibition during footshock stress. (G) In the SEFL conditioning session, mice displayed low levels of freezing prior to the presentation of footshock. (H) In the SEFL test session, all groups displayed similar levels of freezing to the context. Data presented as mean +/-SEM. N=29: Hm4Di group (8 males, 7 females), mCherry group (7 males, 7 females). βˆ— 𝑝 < 0.05, βˆ—βˆ— 𝑝 < 0.01, βˆ—βˆ—βˆ— 𝑝 < 0.001..

Inhibition of pPVT attenuates the expression of sensitization of unlearned fear but not sensitization of fear learning.

(A) Schematic of the AAV viral strategy and representative image of viral expression in the pPVT. Mice received AAVs encoding the inhibitory Hm4Di receptor or the mCherry fluorescent reporter. Scale bar indicates 100 Β΅m. (B) Experimental timeline. (C) In the footshock stress session, freezing behavior was comparable between the hM4Di and mCherry groups. (D) In the SEFR tone test, the hM4Di group displayed significantly decreased levels of freezing compared to the mCherry group, specifically during the tone period. (E) In the open field test, total distance traveled did not differ between the groups. (F) In the stress context test, both the hM4Di and mCherry groups showed similar levels of freezing. (G) In the SEFL conditioning session, mice displayed low levels of freezing prior to the footshock presentation. (H) In the SEFL test session, freezing behavior did not differ between the hM4Di and mCherry groups. Data presented as mean +/-SEM. N=30: Hm4Di group (6 males, 10 females), mCherry group (7 males, 7 females). βˆ— 𝑝 < 0.05, βˆ—βˆ— 𝑝 < 0.01, βˆ—βˆ—βˆ— 𝑝 < 0.001.

Activation of pPVT in stress-naΓ―ve mice potentiates freezing to the novel tone but does not enhance fear conditioning.

(A) Schematic of the AAV viral strategy and representative image of viral expression in pPVT. Animals received AAVs encoding the excitatory Hm3Dq receptor or the mCherry fluorescent reporter. Scale bar indicates 100 Β΅m. (B) Experimental timeline. (C) In the SEFR tone test, the Hm3Dq group displayed significantly higher levels of freezing compared to the mCherry group during the tone period. (D) In the open field, no significant group differences were observed in total distance traveled. (E) In the SEFL conditioning session, pre-Shock freezing levels did not differ between groups. However, the Hm3Dq group displayed increased levels of post-shock freezing relative to the mCherry group. (F) In the SEFL test session, the hM3dq and mCherry groups showed similar levels of freezing. Data presented as mean +/-SEM. N=35: Hm3Dq (9 males, 8 females), mCherry (9 males, 9 females). βˆ— 𝑝 < 0.05, βˆ—βˆ— 𝑝 < 0.01, βˆ—βˆ—βˆ— 𝑝 < 0.001.

Inhibition of pPVT in stress-naΓ―ve mice does not attenuate freezing responses to a 115-dB tone.

(A) Schematic of the AAV viral strategy and representative image of viral expression in pPVT. Animals received an AAV encoding the inhibitory hM4Di receptor or a fluorescent mCherry reporter. Scale bar indicates 100 Β΅m. (B) In the SEFR tone test, no significant group differences in freezing behavior was observed. Data presented as mean +/-SEM. N=19: Hm4Di (5 males, 4 females), mCherry (5 males, 5 females). βˆ— 𝑝 < 0.05, βˆ—βˆ— 𝑝 < 0.01, βˆ—βˆ—βˆ— 𝑝 < 0.001.