1. A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats
Christina A. Sanford, Marta E. Soden, Madison A. Baird, Samara M. Miller, Jay Schulkin, Richard D. Palmiter, Michael Clark, Larry S. Zweifel 
Neuron 
Volume 93, Issue 1, Pages (January 2017)
DOI: /j.neuron
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2. Figure 1 CRF Neurons Represent a Distinct Population within the CeAL
(A) Representative rostral-caudal images of CRF neurons from CrhIREs-Cre;Ai14TdTmto reporter mouse with co-staining against PKCδ (left), somatostatin (Sst, middle), and neurotensin (Nts, right).
(B) Quantification of PKCδ and CRF neurons along rostral-caudal axis (n = 3 sections from 3 mice per region, two-way ANOVA with Bonferroni multiple comparisons, F(4,12) = 21.89, ∗∗p < 0.01, ∗∗∗∗p < ).
(C) qPCR of translating RNA isolated from CRF neurons (n = 3 groups of tissue collected from 5–7 mice per group; ordinary one-way ANOVA with multiple comparisons, F(5) = 6.6, ∗p < 0.05, ∗∗p < 0.01 relative to Crh).
Error bars are mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2
CRF Neurons Exhibit Synaptic Plasticity and Cue Selectivity following Fear Conditioning
(A) Representative image of CRF neurons in the CeA from CrhIRES-Cre;Ai14TdTmto reporter mouse.
(B) Example traces of AMPAR- and NMDAR-mediated currents under control (top) and shock (bottom) conditions.
(C) Ratios of peak AMPAR- to NMDAR-mediated currents (n = 10 cells total per group, 2–3 mice per group; unpaired t test, t = 3.76, ∗∗p = ).
(D) Postsynaptic currents following bath application of AMPA (n = 9–10 cells from 2 mice per condition; two-way repeated-measures ANOVA, time × treatment interaction, F(29,493) = 3.63, ∗∗∗∗p < ).
(E) Postsynaptic currents following bath application of NMDA (n = 9–10 cells from 2 mice per condition; two-way ANOVA, time × treatment interaction, F(29,493) = 0.78, p = 0.78).
(F) LTP occlusion in shock-conditioned mice following high-frequency stimulation (n = 11–12 cells from 3–4 mice per condition; two-way repeated-measures ANOVA, time versus treatment interaction, F(104,2184) = 1.77, ∗∗∗∗p < ).
(G) Quantification of percent change versus baseline (one-sample t test relative to 100%, tshock = 2.81, ∗p = 0.02; tcontrol = 0.815, p = 0.43).
(H) Targeting schematic of fiber-optic guide cannula placement.
(I) Representative image of GCaMP6 expression in CRF neurons.
(J) Average of fluorescence traces during CS+ (left) and CS− (right) presentations. Inset: quantification of the area under the curve for each cue (n = 32 cells from 4 mice; paired t test, t = 3.59, ∗∗p < 0.01).
(K) Heatplot of the ΔF/F for each cell during the CS+ (left) and CS− (right).
Error bars are mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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4. Figure 3
Fear Learning about Weak Threats Is Dependent on CRF Produced by the CeAL
(A) Schematic of targeted genetic inactivation of Crh (left) and representative image of AAV-CreGFP expression (right).
(B) Behavioral strategy.
(C) Freezing behavior on conditioning days to 0.3 mA foot shocks (n = 12 control, 14 Crh:CeA KO; two-way repeated-measures ANOVA, trial × genotype interaction to CS+, F(9,216) = 2.0, ∗p = 0.04).
(D) Freezing behavior during baseline and test sessions following 0.3 mA foot shock conditioning (n = 13 control, 15 Crh:CeA KO, two-way repeated-measures ANOVA with post hoc Tukey multiple comparisons, F(3,52) = 3.38, p < 0.02, ∗p ≤ 0.05, ∗∗p < 0.01).
(E) Distribution of mice displaying fear to 0.3 mA foot shock conditioning (chi-square test, X2 = 5.05, ∗p = 0.02).
(F) Freezing behavior on conditioning days to 0.4 mA foot shocks (n = 10 control, 8 Crh:CeA KO; two-way repeated-measures ANOVA, trial × genotype interaction, F(27,288) = 2.12, ∗p = 0.03).
(G) Freezing behavior during baseline and test sessions following 0.4 mA foot shocks (n = 10 control, 8 Crh:CeA KO; two-way repeated-measures ANOVA with post hoc Tukey multiple comparisons, F(3,44) = 3.74, p < 0.02, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
(H) Distribution of mice displaying fear to CS+ with 0.4 mA foot shocks (chi-square test, X2 = 4.29, ∗p = 0.04).
(I) Freezing behavior on conditioning days to 0.5 mA foot shocks (n = 8 control, 8 Crh:CeA KO; two-way repeated-measures ANOVA, trial × genotype interaction, F(27,252) = 0.73, p = 0.70).
(J) Freezing behavior during baseline and test sessions following 0.5 mA foot shock conditioning (n = 8 control, 8 Crh:CeA KO; two-way repeated-measures ANOVA, F(3,32) = 0.60, p < 0.62).
(K) Distribution of mice displaying fear to CS+ with 0.5 mA foot shocks (chi-square test, X2 = 0.71, p = 0.41).
Error bars are mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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5. Figure 4
Silencing CeAL CRF Neurons Is Phenotypically Similar to Crh Inactivation
(A) Schematic of patch-clamp recordings in the CeA. Blue light stimulation evoked synaptic vesicle release from CRF neurons expressing ChR2-mCherry. Light-evoked IPSCs were recorded from non-CRF neurons.
(B) Example traces (average of 15 sweeps) showing light-evoked IPSC that could be blocked by PTX, but not CNQX. When CRF neurons co-expressed ChR2-mCherry and TeTx, no IPSCs were detected.
(C) Quantification of light-evoked IPSC amplitude (n = 11 control, 10 TeTx; unpaired t test, ∗p < 0.05).
(D) Freezing behavior on conditioning days to 0.3 mA foot shocks (n = 13 control, 10 Crh:CeA TeTx; two-way repeated-measures ANOVA, trial × genotype interaction, F(9,189) = 2.20, ∗p = 0.02).
(E) Freezing behavior during baseline and test sessions following 0.3 mA foot shock conditioning (two-way repeated-measures ANOVA with post hoc Tukey multiple comparisons, F(3,42) = 2.99, p < 0.05, ∗∗p ≤ 0.01).
(F) Distribution of mice displaying varying levels of fear to 0.3 mA foot shock conditioning (chi-square test, X2 = 5.57, ∗p = 0.02).
(G) Freezing behavior on conditioning days to 0.5 mA foot shocks (n = 12 control, 10 Crh:CeA TeTx; two-way repeated-measures ANOVA, trial × genotype interaction, F(9,180) = 1.78, p = 0.08).
(H) Freezing behavior during baseline and test sessions following 0.5 mA foot shock conditioning (two-way repeated-measures ANOVA with post hoc Tukey multiple comparisons, F(3,58) = 1.25, p < 0.30).
(I) Distribution of mice displaying varying levels of fear to 0.5 mA foot shock conditioning (chi-square test, X2 = 0.03, p = 0.86).
Neuron  , DOI: ( /j.neuron )
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6. Figure 5
Inactivation of Crh from Multiple CeA Inputs Does Not Prevent Threat Generalization
(A) CAV-Cre targeting schematic (left) and images from CeA afferents (right).
(B) Freezing behavior during baseline and test sessions following 0.3 mA foot shock conditioning (n = 13 control, 8 Crh:CeA CAV-Cre; two-way repeated-measures ANOVA with post hoc Tukey multiple comparisons, F(3,37) = 3.51, p = 0.02, ∗p < 0.05, ∗∗p < 0.01).
(C) Freezing behavior during baseline and test sessions following 0.5 mA foot shock conditioning (n = 8 control, 11 Crh:CeA CAV-Cre; two-way repeated-measures ANOVA, F(3,32) = 1.78, p < 0.2).
(D) Fear discrimination plot of freezing during CS− versus freezing during CS+ for all control and Crh:CeA KO animals described above.
(E and F) Discrimination index (freezing during CS−/freezing during CS+) following 0.3 or 0.4 mA shock conditioning (E; t = 2.856, p < 0.01) or 0.5 mA shock conditioning (F).
Error bars are mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

7. Figure 6
CeAL CRF Neurons Are Required for the Acquisition of Fear, but Not Expression
(A) Targeting schematic for Cre-dependent expression of hM4Di-YFP (left) and representative image in CRF neurons (right).
(B) Example traces showing action potential firing induced by current injection (bottom) before and after CNO application to Crh:CeA hM4Di neurons.
(C) Freezing behavior on conditioning days to 0.3 mA foot shocks following CNO administration (n = 8 control, 9 Crh:CeA hM4Di; two-way repeated-measures ANOVA, treatment factor, F(1,15) = 5.85, ∗p = 0.03).
(D) Freezing behavior during baseline and test sessions following 0.3 mA foot shock conditioning and CNO administration prior to conditioning sessions (n = 10 control, 10 Crh:CeA hM4Di; two-way repeated-measures ANOVA with post hoc Tukey multiple comparisons, F(3,67) = 8.17, p < 0.01, ∗p < 0.05, ∗∗∗p < 0.001).
(E) Freezing behavior on conditioning days to 0.3 mA foot shocks following saline administration (n = 12 control, 9 Crh:CeA hM4Di; two-way repeated-measures ANOVA with post hoc Tukey multiple comparisons, F(9,171) = 0.84, p < 0.50).
(F) Freezing behavior during baseline and test sessions following 0.3 mA foot shock conditioning and CNO administration prior to final test session (n = 12 control, 9 Crh:CeA hM4Di; two-way repeated-measures ANOVA with post hoc Tukey multiple comparisons, F(2,38) = 0.55, p < 0.60).
Error bars are mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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8. Figure 7 CRF Receptive Neurons Are Broadly Expressed in the CeA
(A) Representative rostral-caudal images of CRFR1 neurons from Crhr1IRES-Cre;Ai14TdTmto reporter mouse with co-staining against PKCδ (left), somatostatin (Sst, middle), and neurotensin (Nts, right).
(B) Quantification of PKCδ and CRFR1 neurons along rostral-caudal axis (n = 3 sections from 3 mice per region).
(C) Quantification of Sst and CRFR1 neurons along rostral-caudal axis (n = 3 sections from 3 mice per region).
(D) Quantification of overlap between CRFR1 neurons and PKCδ or Sst neurons.
Error bars are mean ± SEM.
Neuron  , DOI: ( /j.neuron )
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9. Figure 8
CRFR1 Receptors Are Required for Low-to-Moderate Fear Learning
(A) Example traces showing enhanced AMPAR-mediated EPSCs in CRFR1 neurons following bath application of CRF.
(B) Quantification of AMPA/NMDA ratios before and after bath application of ACSF (control) or CRF (n = 7 cells/group; two-way repeated-measures ANOVA, F(1,12) = 6.01, ∗∗p < 0.01).
(C) Diagram of proposed circuit for CRF-dependent fear learning at low-to-moderate threat intensities.
(D) Schematic of local infusion of CRFR1 antagonist antalarmin (Ant) in the CeA.
(E) Freezing behavior on conditioning days to 0.3 mA foot shocks following Ant or vehicle administration (n = 15 vehicle, 13 Ant; two-way repeated-measures ANOVA, effect of drug, F(1,27) = 5.59, p < 0.03).
(F) Freezing behavior during baseline and test sessions following 0.3 mA foot shock conditioning (n = 15 vehicle, 13 Ant; two-way repeated-measures ANOVA with post hoc Tukey multiple comparisons, F(3,54) = 3.10, p < 0.05, ∗p < 0.05). Ant or vehicle was administered only prior to conditioning sessions.
Error bars are mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions