Partially reinforced fear drives a hyperarousal phenotype in males

A, Schematic of behavior chambers and tone/shock presentations for fully- and partially reinforced fear conditioning and recall. B, Freezing during fear conditioning in males (Mixed-effects model, effect of tone F(2.482, 71.99)=16.76 p<.0001, effect of fear F(2,33)=27.24 p<.0001). C, last tone freezing during conditioning in males (one-way ANOVA F(2,29)=44.53 p<.0001, Tukey’s multiple comparisons test Full v. Part p=.0014, Full v. Ctrl p<.0001, Part v. Ctrl p<.0001). D, Freezing during fear recall in males (two-way RM ANOVA, main effect of tone: F(4.445, 146.7)=8.836 p<.0001, fear: F(2,33)=57.04 p<.0001, tone x fear: F(12,198)=3.121 p=.0004). E, Freezing during first tone of recall in males (one way ANOVA F(2,33)=28.49 p<.0001, Tukey’s multiple comparisons test Full v. Ctrl p<.0001, Part v. Ctrl p<.0001). F, Freezing during fear conditioning in females (Mixed-effects model, effect of tone F(2.024, 58.70)=20.85 p<.0001, effect of fear F(2,33)=27.28 p<.0001). G, last tone freezing during conditioning in females (one-way ANOVA F(2,33)=39.74 p<.0001, Tukey’s post-hoc Full v. Part p=.0122, Full v. Ctrl p<.0001, Part v. Ctrl p<.0001). H, Freezing during fear recall in females (two-way RM ANOVA, main effect of tone: F(4.499, 148.5)=18.16 p<.0001, fear: F(2,33)=49.20 p<.0001, tone x fear: F(12,198)=4.640 p<.0001). I, Freezing during first tone of recall in females (one way ANOVA F(2,33)=49.52 p<.0001, Tukey’s post-hoc Full v. Ctrl p<.0001, Part v. Ctrl p<.0001). J, behavioral timeline for anxiety-like behavior tests after fear. K, last tone freezing during fear conditioning in males (one-way ANOVA F(2,20)=42.71 p<.0001, Tukey’s multiple comparisons test: Ctrl v. Full p<.0001, Ctrl v. Part p<.0001). L, Latency to feed in the novelty-induced suppression of feeding task in males. M, food consumed during NSF refeed test in males. N, acoustic startle response in males (two-way RM ANOVA, main effect of fear (F(2,20)=11.61 p=.0005, main effect of stim intensity: F(1.808, 36.15)=166.9 p<.0001, interaction: F(4,40)=6.096 p=.0006, Tukey post-hoc 120 dB Full v. Part p=.0395). O, last tone freezing during fear conditioning in females (one way ANOVA, F(2, 33)=57.11, Tukey’s multiple comparisons test Full v. Ctrl p<.0001, Part v. Ctrl p<.0001, Full v. Part p=.0383). P, Latency to feed in the novelty-induced suppression of feeding task in females. Q, food consumed during NSF refeed test in females. R, acoustic startle response in females (two-way RM ANOVA, main effect of stim intensity F(2,66)=223.1 p<.0001). n=8-12/group.

BNST is dynamically engaged during fear conditioning and recall

A, surgical schematic for fiber photometry experiments. B,C schematic of tone and shock presentations during fear conditioning and recall. D, BNST representative images showing fiber placement. E, Fear Conditioning traces in males. F, tone response in males averaged from t=0-2 across all trials (paired two-tailed t-tests, Full v. BL t(6)=2.504 p=.0463, Part v. BL t(8)=3.935 p=.0043). G, comparison of first and last tone response in males (paired two-tailed t-test: F First v. F Last t(5)=3723 p=.0137). H, shock response in males averaged from t=28-30 across all trials (paired two-tailed t-tests, Full v. BL t(6)=9.423 p<.0001, Part v. BL t(8)=18.36 p<.0001). I, comparison of first and last shock response in males (paired two-tailed t-tests, F BNST v. BL. t(6)=2.587 p=.0414). J, Fear Conditioning traces in females. K, tone response in females averaged from t=0-2 across all trials (paired two-tailed t-tests, Part v. BL t(9)=3.218 p=.0105). L, comparison of first and last tone response in females. M, shock response in females averaged from t=28-30 across all trials (paired two-tailed t-tests, Full v. BL t(7)=18.04 p<.0001, Part v. BL t(9)=13.97 p<.0001). N, comparison of first and last shock response in females. O, Fear recall traces in males. P, tone onset response during fear recall in males (paired two-tailed t-tests, Part v. BL t(9)=3.111 p=.0125. Q, area under the curve during tone presentations in males from t=5-30 (two-way RM ANOVA, Part v. Full: main effect of fear F(1,14)=5.69 p=.0409). R, Fear recall traces in females. A, tone onset response during fear recall in females (paired two-tailed t-test, Full v. BL t(7)=3.361 p=.0121, Part v. BL t(9)=3.241 p=.0101). T, area under the curve during tone presentations in females from t=0-30. n=7-10/group.

CRF knockdown in the BNST potentiates partial but not fully reinforced fear in females

A, Surgical schematic and timeline CRF knockdown experiments. B,C, representative image of cre and GFP virus expression in BNST. D,E, freezing during full and partial fear conditioning in males. F,G, freezing during full and partial fear conditioning in females. H,I freezing during recall for full and partial fear groups in males. J, Full recall in females. K, Part recall in females (unpaired two-tailed t-test: baseline GFP v Cre t(17)=3.660 p=.0019, first tone GFP v. Cre t(17)=2.213 p=.0487).

CRF knockdown in the BNST modulates anxiety-like behavior after fear

A, Surgical schematic. B, behavioral timeline. C, Latency to feed in the novelty-induced suppression of feeding test in males (two-way ANOVA, main effect of fear: F(2,46)=15.24 p<.0001, Fear x virus interaction F(2,46)=2.972 p<.0611, Bonferroni post-hoc GFP part v. Cre part p=.0266). D, amount consumed during refeed test in males. E, Latency to feed in the novelty-induced suppression of feeding test in females (two-way ANOVA, main effect of fear F(2,41)=4.627 p<.0154). F, amount consumed during refeed test in females. G, acoustic startle response in control males. H, acoustic startle response in fear males (three way anova, main effect of virus: F(1,30)=5.101 p=.0313, fear x virus interaction: F(1,30)=5.700 p=.0235,Tukey post-hoc: 105 dB Full Cre v. 105 dB Full GFP p=.0357). I, acoustic startle response in control females. J, acoustic startle response in fear females. K, acoustic startle response at 105dB in males (two-way ANOVA, main effect of virus: F(1,45)=5.335 p=.0256, main effect of fear: F(2,45)= 5.759 p=.0059. interaction: F(2,45)=.0119, Tukey post-hoc: Full GFP v. Full cre p=.0045, Ctrl Cre v. full cre p=.0029). L, acoustic startle response at 105dB in females. N=7-10/group. All data are shown as mean + SEM. # denotes significant main effect or interaction, * denotes significant post-hoc comparison.

CRF neurons differentially encode partial fear in males and females

A, Viral strategy schematic. B, representative raw FOV. C, representative processed FOV. D, representative image showing GCaMP expression and GRIN lens placement. E, example traces of different responses to footshock. F, Pie charts showing the proportion of cells excited and inhibited in response to the first footshock in males, fear groups combined. G, Spike frequency during conditioning baseline and trial end (last 2 min of session) in males (two-way RM ANOVA main effect of epoch F(1,76)=99.61 p<.0001). Full: n=39 cells from 6 mice. Part: n=39 cells from 5 mice. H, Comparison of trial end spike frequency as a change from BL between full and part fear in males. I, spike frequency during first and last tones for Full fear in males. J, spike frequency during first and last tones for Part fear in males (paired two-tailed t-test t(40)=2.270 p=.0096. K, Pie charts showing the proportion of cells excited and inhibited in response to the first footshock in females. L, Spike frequency during conditioning baseline and trial end (last 2 min of session) in females (two way RM ANOVA main effect of epoch F(1,82)=163.1 p<.0001). Full: n= 33 cells from 5 mice. Part: n=51 cells from 5 mice. M, Comparison of trial end spike frequency as a change from BL frequency between full and part fear in females. N, spike frequency during first and last tones for Full fear in females (paired two-tailed t-test t(32)=2.832 p=.0079). O, spike frequency during first and last tones for Part fear in females (paired two-tailed t-test t(51)=6.385 p=.0001). P, example traces during baseline and trial end in fear recall. Q, Spike frequency during baseline and trial end during recall in males (two way RM ANOVA: main effect of epoch F(1,87)=47.88 p<.0001, epoch x fear interaction F(1,87)=4.677 p=.0333). Full: n= 45 cells from 6 mice. Part: n=44 cells from 5 mice. R, Comparison of spike frequency during the last 2 minutes of the recall trial as a change from BL between full and part fear in males (unpaired t-test: t(86)=2.048 p=.0436). S, Spike frequency during first and last blocks of tone presentations during Full fear recall in males. T, Spike frequency during first and last blocks of tone presentations during Part fear recall in males (paired two-tailed t-test t(44)=3.729 p=.0005). U, Spike frequency during baseline and trial end during recall in females (two way RM ANOVA: main effect of epoch F(1,76)=35.69 p<.0001, epoch x fear interaction F(1,76)=4.908 p=.0297). Full: n=33 cells from 5 mice. Part: n=45 cells from 5 mice. V, Comparison of trial end spike frequency as a change from BL during recall between full and part fear in females (unpaired t-test: t(76)=2.215 p=.0297). W, Spike frequency during first and last blocks of tone presentations during Full fear recall in females. X, Spike frequency during first and last blocks of tone presentations during Part fear recall in females (paired two-tailed t-test t(44)=3.729 p=.0005).

BNST activity negatively correlate with freezing

A schematic. B. spike freeze. C-D, freezing males. E. spikes freeze main effect of mobility F(1,15)=72.38 p<.0001, mobility x fear interaction F(1,15)=5.358 p<.0352. F spike freeze recall. Main effect mobility F(1,16)=53.70 p<.0001. G-H. female freezing. I. spikes freeze main effect of mobility F(1,16)=54.49 p<.0001. J. F spike freeze recall. Main effect mobility F(1,15)=17.79 p<.0007, mobility x fear F(1,15)=6.095 p=.0261

Sex differences in BNSTCRF response to shock omission

A, experimental design schematic for 1-photon recordings. B,D, representative traces showing a response to shock and shock omission trials for partial fear only in males and females. C, Spike frequency during the 10 s window post-shock or post-omission during partial fear in males (two- way RM ANOVA: main effect of shock F(1,40)=5.790 p=.0208). E, Spike frequency during the 10 s window post-shock or post-omission during partial fear in females (two-way RM ANOVA: main effect of shock F(1,51)=8.022 p=.0066, main effect of trial F(2,102)=5.950 p=.0036.