A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats

Article

A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats Highlights d

CRF neurons in the CeA are critical for discriminative fear

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CRF neurons in the CeA are not required for generalized fear

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CRF neurons project locally to enhance excitatory synaptic transmission

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CRF provides gain control for fear processing

Authors Christina A. Sanford, Marta E. Soden, Madison A. Baird, ..., Richard D. Palmiter, Michael Clark, Larry S. Zweifel

Correspondence [email protected]

In Brief Sanford et al. demonstrate that a unique population of CRF-producing neurons in the central nucleus of the amygdala (CeA) provides a modulatory gain control signal within the CeA that operates at low threat levels to facilitate learning through the release of CRF.

Sanford et al., 2017, Neuron 93, 1–15 January 4, 2017 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.neuron.2016.11.034

Please cite this article in press as: Sanford et al., A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats, Neuron (2016), http:// dx.doi.org/10.1016/j.neuron.2016.11.034

Neuron

Article A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats Christina A. Sanford,1 Marta E. Soden,1 Madison A. Baird,1 Samara M. Miller,1 Jay Schulkin,2,3,4 Richard D. Palmiter,5 Michael Clark,6 and Larry S. Zweifel1,6,7,* 1Department

of Pharmacology, University of Washington, Seattle, WA 98105, USA of Physiology and Biophysics 3Department of Neuroscience Georgetown University, Washington, DC 20057, USA 4Department of Obstetrics and Gynecology 5Department of Biochemistry 6Department of Psychiatry and Behavioral Sciences University of Washington, Seattle, WA 98105, USA 7Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2016.11.034 2Department

SUMMARY

Fear is a graded central motive state ranging from mild to intense. As threat intensity increases, fear transitions from discriminative to generalized. The circuit mechanisms that process threats of different intensity are not well resolved. Here, we isolate a unique population of locally projecting neurons in the central nucleus of the amygdala (CeA) that produce the neuropeptide corticotropin-releasing factor (CRF). CRF-producing neurons and CRF in the CeA are required for discriminative fear, but both are dispensable for generalized fear at high US intensities. Consistent with a role in discriminative fear, CRF neurons undergo plasticity following threat conditioning and selectively respond to threat-predictive cues. We further show that excitability of genetically isolated CRF-receptive (CRFR1) neurons in the CeA is potently enhanced by CRF and that CRFR1 signaling in the CeA is critical for discriminative fear. These findings demonstrate a novel CRF gain-control circuit and show separable pathways for graded fear processing.

INTRODUCTION Defensive behavioral responses to threatening stimuli involve both innate and adaptive processes (Anderson and Adolphs, 2014; Duvarci and Pare, 2014; Tovote et al., 2015). The amygdala is a key brain region that serves as an integration point for sensory and affective information from cortical and thalamic brain regions to promote defensive behavior and threat-related learning (LeDoux, 2000). Within the amygdala, information processing and behavioral output occur through highly interconnected subdivisions defined by distinct cell types (Janak and

Tye, 2015). While considerable progress has been made in establishing basic circuitry underlying fear-related behavior, a major unresolved question is how threats of different intensity are processed by this circuitry to give rise to adaptive discriminative fear in response to low-to-moderate threats and the maladaptive processes that lead to generalized fear in response to high-intensity threats. One possibility is that distinct neural circuit components or signaling pathways provide gain control to fine-tune temporal precision in discriminative processes, and that high-intensity signals bypass this gain control to promote aberrant learning and stimulus generalization. Neuropeptides are of particular interest in gain control due to their ability to modulate synaptic strength (Rosen and Schulkin, 1998; Keifer et al., 2015). Support for the graded dependency of neuropeptides in the modulation of behavioral responses to a noxious threat has been shown in spinal cord circuitry (Lichtman and Fanselow, 1991), but whether this occurs within the brain is not clear. The central nucleus of the amygdala (CeA) is rich in the expression of neuropeptides (Cassell et al., 1986), which include cholecystokinin (CCK) (Micevych et al., 1988), neuropeptide Y (NPY) (Gustafson et al., 1986), corticotropinreleasing factor (CRF) (Joseph and Knigge, 1983), dynorphin (Dyn) (Weber and Barchas, 1983), and enkephalin (Enk) (Cassell et al., 1986). Many of these peptides have been shown to modulate threat-related behaviors and are thought to allow for complex bidirectional control over a variety of defensive responses (Davis et al., 2010; Bowers et al., 2012). Of the neuropeptides expressed in the CeA, CRF is the most often associated with fear-related behaviors (Gafford and Ressler, 2015). It is interesting to note that previous studies designed to assess the function of CRF signaling in the brain for modulating defensive behavior yielded conflicting results, with one study demonstrating a key role for CRF (Swerdlow et al., 1989) and a second study using a similar strategy finding no effect (de Jongh et al., 2003). It was proposed that these differences may relate to the intensity of the unconditioned stimulus (US) (de Jongh et al., 2003), such that threat conditioning with a weaker US (Swerdlow et al., 1989) is more sensitive to Neuron 93, 1–15, January 4, 2017 ª 2016 Elsevier Inc. 1

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CRF manipulation than conditioning with a higher-intensity US (de Jongh et al., 2003). These findings indirectly implicate a threshold dependence and putative gain control function of CRF, but whether CRF actually operates in this manner is not known. Moreover, because these experiments used intracerebroventricular and not site-specific antagonist delivery, which cells are responsible for the production of CRF in this context, and where in the brain CRF is required, is not discernable. Here we establish the identity of CRF-producing neurons in the CeA and the role of CRF in this region for conditioning to different threat levels. We find that CRF-producing neurons are largely distinct from previously characterized somatostatin (Sst) and PKCd neurons in the CeA. CRF neurons form local inhibitory connections, undergo plasticity following threat conditioning, and selectively respond to threat-predictive stimuli. To establish the role of CeA-derived CRF, we generated a mouse line that allows for the conditional inactivation of the CRF gene (Crhlox/lox). Consistent with a gain control function for CRF, we find that CRF facilitates conditioned fear acquisition at low, but not high, US intensities. Surprisingly, we also find that inhibitory synaptic transmission from CRF neurons is also nonessential for fear at high US intensities, demonstrating a functional specificity for these cells. Using a newly generated Cre-driver mouse line, Crhr1IRES-Cre, we demonstrate that CRF-receptive neurons are broadly localized within the central CeA (CeAC), lateral CeA (CeAL), and medial CeA (CeAM), partially overlapping with Sst neurons in the caudal CeAL. We further show that CRF potently enhances glutamatergic synaptic transmission onto CRFR1 neurons, and CRFR1 signaling in the CeA is critical for discriminative fear. Collectively, these data define a novel modulatory gain control circuit within the CeA that specifically operates at low threat levels to facilitate learning through the release of CRF. RESULTS CRF Neurons Are Largely Distinct from Other Populations within the CeAL Electrophysiological recordings in the CeAL during presentations of threat-associated stimuli have revealed two primary response profiles: ‘‘fear-on’’ and ‘‘fear-off’’ neurons (Haubensak et al., 2010). Genetic and functional analyses of cells within the CeAL have demonstrated that ‘‘fear-on’’ neurons principally express the neuropeptide Sst (Li et al., 2013), whereas ‘‘fear-off’’ neurons are predominantly found to express the delta isoform of a calcium-activated protein kinase (PKCd) (Haubensak et al., 2010). To determine whether CRF-producing neurons are selectively encapsulated by one of these two major cell types in the CeAL, we performed immunostaining for PKCd and Sst, as well as the neuropeptide neurotensin (Nts), which labels the CeAC, in tissue sections obtained from a CrhIRES-Cre mouse line (Taniguchi et al., 2011) crossed with a reporter (CrhIRES-Cre;Ai14TdTomato; Figure 1A). Quantification of PKCd immunoreactivity, which robustly labeled cell bodies, and tdTomato across the rostralcaudal axis revealed very little overlap between PKCd and CRF neuronal populations (Figure 1B). Sst also did not significantly overlap with the distribution of CRF neurons (Figure S1, available online) and NTS, which predominantly labeled nerve terminals, showed little co-localization with tdTomato. Results from

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all three immunostaining experiments revealed that CRF neurons are predominantly localized to the rostral regions of the CeAL, diminishing significantly within the caudal-most aspect. PKCd, Sst, and Nts localized prominently within the caudal aspect of the CeAL, with a small number of CRF neurons showing partial overlap with these markers (Figures 1A and 1B). To confirm the findings of our immunohistochemistry analysis, we utilized the RiboTag (Sanz et al., 2009) strategy to selectively isolate actively translated mRNA from genetically defined CRF neurons. This approach allows for the identification of genetic markers with enriched expression within specific cell populations. Selective expression of the HA-tagged ribosomal protein L22 (Rpl22-HA) in CRF neurons was achieved by injecting an adeno-associated viral vector encoding a Cre-dependent Rpl22HA (AAV1-FLEX-Rpl22-HA) into the CeAL of mice expressing Cre-recombinase from the endogenous Crh locus encoding CRF (CrhIRES-Cre; Figure S1B). HA-tagged polyribosomes were immunoprecipitated (IP) from homogenates of tissue punches collected from injected mice (Figure S1C). RNA isolated from the IP fraction was screened for enrichment relative to the total RNA (input) for specific genes via qPCR. The genes included those encoding CRF (Crh), Sst (Sst), PKCd (Prkcd), as well as other neuropeptides known to be expressed in the CeAL such as neurotensin (Nts) and dynorphin (Pdyn) (Cassell et al., 1986). Analysis of the IP versus the input RNA revealed a significant enrichment of Crh message relative to all other markers (Figure 1C). Enrichment of other markers was not significantly different than the negative control oligodendrocyte marker Cnp. Neurons within the CeAL have been shown to project locally within the CeA as well as to distant structures (Keifer et al., 2015). To determine whether CRF neurons show similar projection patterns to previously identified cell groups, we performed a semi-quantitative analysis of projection data from the Allen Brain Institute Mouse Connectivity Atlas (Oh et al., 2014), in which the projection patterns of genetically identified cell types within the CeA, including PKCd neurons, have been established. The weighted density of fibers from CRF neurons is highest within the CeA, suggestive of strong local projections (Figure S1D). In line with our molecular and histological findings, the few long-range projections of CRF neurons differ from those of PKCd neurons (Figure S1D). Conditional viral-mediated expression of the synaptic marker synaptophysin fused to GFP (AAV1-FLEX-SynGFP) in CRF neurons of the CeA (Figures S1E and S1F) confirmed the projection data from the connectivity atlas. Collectively, these results indicate that CRF neurons are largely distinct from other known populations in the CeAL. CRF Neurons Undergo Synaptic Plasticity and Respond to Threat-Predictive Cues Neuronal cell types within the CeAL, such as those producing Sst, have been shown to undergo plasticity following fear conditioning that contributes to conditioned threat learning (Li et al., 2013; Penzo et al., 2014, 2015). To determine whether CRF neurons also undergo synaptic plasticity at excitatory synapses, we measured the ratio of AMPA/NMDA receptor (AMPAR/ NMDAR) currents (Malinow and Malenka, 2002) following conditioning in a differential delayed cue fear conditioning paradigm. To allow for visual identification of CRF neurons in an ex vivo

Please cite this article in press as: Sanford et al., A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats, Neuron (2016), http:// dx.doi.org/10.1016/j.neuron.2016.11.034

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 PKCd (left), somatostatin (Sst, middle), and neurotensin (Nts, right). (B) Quantification of PKCd 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 < 0.0001). (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.

slice preparation, we utilized CrhIRES-Cre;Ai14TdTomato reporter mice (Figure 2A). Mice were conditioned for 2 consecutive days to a predictive conditioned stimulus (ten presentations of a 10 s auditory cue, CS+) that co-terminated with a 0.5 s,

0.3 mA unconditioned foot shock stimulus (US). CS+ presentations were interleaved with ten presentations of a distinct auditory cue (CS
) not paired with the US. Approximately 24 hr after the last conditioning session, we isolated AMPAR- and NMDAR-mediated excitatory postsynaptic currents (EPSCs) in CeAL CRF neurons evoked by stimulation of the lateral amygdala (LA) in acute brain slices (Figure 2B). Shock-conditioned mice showed a significantly increased ratio of AMPA/ NMDA EPSCs relative to control mice that received cue presentations but no shock (Figure 2C). To confirm that the observed increase in AMPA/NMDA ratio is due to a selective enhancement of AMPAR-mediated currents, and not a decrease in the NMDAR-mediated current, we quantified currents evoked by bath application of either AMPA (1 mM) or NMDA (10 mM). AMPA-evoked currents were significantly enhanced in shocked mice relative to controls (Figure 2D), while NMDA-evoked currents remained equivalent (Figure 2E). To further establish that fear conditioning evokes plasticity in CRF neurons, we quantified long-term potentiation (LTP) induction in fluorescently identified neurons from shocked and non-shocked control mice (Figures 2F and 2G). Control mice that received only cue presentations displayed robust LTP in CRF neurons of the CeAL following high-frequency stimulation of the LA (Fu et al., 2007). The same stimulation pattern failed to induce LTP in shock-conditioned mice, consistent with the occlusion of LTP induction.

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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 = 0.0014). (D) Postsynaptic currents following bath application of AMPA (n = 9–10 cells from 2 mice per condition; two-way repeated-measures ANOVA, time 3 treatment interaction, F(29,493) = 3.63, ****p < 0.0001). (E) Postsynaptic currents following bath application of NMDA (n = 9–10 cells from 2 mice per condition; two-way ANOVA, time 3 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 < 0.0001). (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 DF/F for each cell during the CS+ (left) and CS
(right). Error bars are mean ± SEM.

Plasticity in CRF CeAL neurons suggests that these cells may differentially respond to conditioned versus non-conditioned stimuli. It has been previously demonstrated that subpopulations of neurons in the CeA selectively respond to cues predicting

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threatening outcomes (CSON or ‘‘fear-on’’), whereas as other neurons, specifically those expressing PKCd, are inhibited by predictive cues (CSOFF or ‘‘fear-off’’) (Ciocchi et al., 2010; Haubensak et al., 2010). To establish whether CRF CeAL neurons

Please cite this article in press as: Sanford et al., A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats, Neuron (2016), http:// dx.doi.org/10.1016/j.neuron.2016.11.034

respond to fear-predictive stimuli, we directly imaged calcium dynamics in the CeAL using fiber-optic confocal microscopy (Vincent et al., 2006; Gore et al., 2014) to detect the genetically encoded calcium indicator GCaMP6m (Chen et al., 2013) selectively expressed in CRF neurons. CrhIRES-Cre mice were injected with a conditional adeno-associated viral vector containing GCaMP6m (AAV1-DIO-GCaMP6) and implanted with a guide cannula targeting the CeAL (Figures 2H, 2I, and S2A–S2C). Two weeks following surgery, mice were fear conditioned for 2 days in a discriminative delayed cue conditioning paradigm, as described above. On the third day, a fiber-optic objective was lowered into the CeAL until fluorescence from CRF neurons was observed (Figure S2D). Mice were returned to the conditioning chamber, and calcium-associated fluorescence signals were imaged during CS+ and CS
presentations. Putative CRF neurons were visually identified and analyzed for changes in fluorescence intensity during CS+ and CS
presentations. Fluorescence signals were significantly increased in CRF neurons following CS+ deliveries relative to CS
(Figures 2J, 2K, and S2E). Consistent with the activity-dependent nature of increased calcium signals in vivo, calcium dynamics detected in CRF neurons in an acute slice preparation following stimulation of the LA at stimulus frequencies consistent with firing rates previously reported for CeAL neurons (Veinante and Freund-Mercier, 1998) demonstrated fluorescent changes similar in amplitude and duration to those observed in vivo (Figures S2F–S2I). CRF in CeAL Neurons Regulates Conditioned Threat Responses at Low US Intensities Our results thus far demonstrate that CRF neurons are largely distinct from previously defined populations. Furthermore, activity-dependent plasticity following threat conditioning implies that these neurons are engaged during fear processing. To determine the role of CRF for the regulation of defensive behaviors and establish the threshold dependence of this neuropeptide, we genetically inactivated the gene encoding CRF, Crh, in the CeAL (Crh:CeA KO [knockout]) by generating a mouse with a conditional floxed allele (Crhlox/lox; Figures S3A and S3B). Crh inactivation was anatomically restricted by site-specific injection of AAV1-CreGFP into the CeA of Crhlox/lox mice (Figure 3A). An adeno-associated virus containing an expression cassette for a Cre-dead enzyme (AAV1-CreDGFP) was used as a control. Immunohistochemical analysis of CRF following AAV1-CreGFP or AAV1-CreDGFP confirmed inactivation (Figure S3C). Different groups of Crh:CeA KO and control mice were conditioned with either a 0.3, 0.4, or 0.5 mA US using a discriminative delayed cued fear conditioning paradigm (Figure 3B). Baseline responses to two distinct auditory stimuli were established by three interleaved presentations of the cues. Mice were then conditioned to CS+ presentations (10 s auditory cue) co-terminating with the US and CS
presentations (distinct 10 s auditory cue that did not co-terminate with a US) on 2 consecutive days. Ten presentations of each cue were used for 0.3 and 0.4 mA, while five presentations were used for 0.5 mA (see below). Twenty-four hours after conditioning, mice were probed for discriminative threat responding by monitoring freezing in response to three interleaved presentations of the CS+ and CS
.

In mice that received the 0.3 mA US, we observed a significant interaction between groups (Crh:CeA KO versus control) and conditioning trial bin, with Crh:CeA KO mice exhibiting significantly less freezing relative to controls during acquisition (Figure 3C). During probe trials, Crh:CeA KO mice displayed less freezing to the CS+ relative to control mice (Figure 3D). To establish whether reduced freezing behavior represents a general lack of fear, we quantified the distribution of mice that showed no fear (cue-induced freezing below the 95th percentile of baseline freezing), low fear (cue-induced freezing one to two times greater than the 95th percentile of baseline), and high fear (>two times the 95th percentile of baseline). Mice with Crh genetic inactivation in the CeA showed a significant difference in the fear distribution compared to control mice (Figure 3E). Similar to 0.3 mA US intensity, at 0.4 mA we observed a significant difference between Crh:CeA KO mice and control mice during acquisition (Figure 3F), though the difference was less than that observed with the 0.3 mA stimulus. We also observed a significant difference between Crh:CeA KO mice and controls during testing, with Crh:CeA KO mice displaying reduced freezing (Figure 3G). Analysis of the distribution of mice displaying conditioned fear revealed a significant difference between groups, with fewer Crh:CeA KO mice displaying fear greater than the 95th percentile of baseline responses, although more Crh:CeA KO mice showed significant fear at 0.4 mA than was observed with the 0.3 mA US (Figure 3H). To assess the role of CRF at higher US intensity, we conditioned mice to a 0.5 mA foot shock. Due to the intense nature of the US, mice were conditioned with five CS-US pairings per day instead of ten (see Supplemental Experimental Procedures for additional details). Analysis of conditioned threat behavior did not reveal significant differences between Crh:CeA KO mice and control mice during acquisition or testing (Figures 3I and 3J). The distribution of mice displaying conditioned fear also did not differ significantly, though there were a slightly smaller number of control mice displaying low fear responses. However, both groups displayed predominantly high fear responses (Figure 3K). Interestingly, both control and Crh:CeA KO mice showed generalized threat responses, consistent with previous reports of behavioral phenotypes observed in response to increasing US strength (Baldi et al., 2004; Ghosh and Chattarji, 2015). It is possible that the differences observed in threat conditioning between Crh:CeA KO mice and controls in response to conditioning at lower US intensities is a reflection of reduced responsiveness to the US or to alterations in basal affective state, such as anxiety. To test this, we quantified changes in the velocity (cm/s) of mice in response to delivery of different US intensities. We did not observe any differences in US responsiveness of control and Crh:CeA KO mice (Figures S4A–S4F). Differences in fear conditioning also do not appear to be related to gross changes in affective state or motor deficits following inactivation of Crh in the CeA, as basal anxiety measures and locomotor activity were not different between groups (Figures S4G–S4J). CRF Neurons Operate at Low Threat Levels Our data are consistent with a role for CRF derived from the CeA in threat conditioning in response to low to moderate US

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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 AAVCreGFP 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 3 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 3 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 3 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 repeatedmeasures 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.

intensities. However, we have yet to directly establish local connectivity of CRF neurons. Moreover, we do not know whether other neurotransmitters are released from CRF neurons and the extent to which this synaptic transmission influences graded threat processing. CRF neurons have been shown to co-express the GABA-synthesizing enzyme GAD1 (Veinante et al., 1997); thus, GABA release from these neurons may play a key role at higher US intensities. To establish whether CRF neurons release GABA and project locally, we expressed the light-activated ion channel ChR2 in the CeA of CrhIRES-Cre/+ mice (AAV1-FLEX-ChR2mCherry). Wholecell patch-clamp recording of non-CRF neurons (non-ChR2 expressing) revealed light-evoked inhibitory postsynaptic currents (IPSCs) in the majority of neurons recorded (8 of 11); these IPSCs were sensitive to GABAA receptor antagonist pricotoxin, but not

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glutamate receptor antagonist CNQX (Figures 4A–4C), consistent with direct synaptic connectivity. To test whether inactivation of all synaptic transmission from CRF neurons leads to differential behavioral effects compared to loss of CRF alone, we selectively silenced CRF neurons through conditional expression of the light chain of tetanus toxin fused to GFP (AAV1-FLEX-GFP-TeTx), which allows for cell-specific inactivation of synaptic transmission (Han et al., 2015). Consistent with previous data (Han et al., 2015), co-expression of GFP-TeTx with ChR2 blocked synaptic transmission by preventing light-evoked IPSCs in postsynaptic neurons (0 of 10 cells; Figures 4B and 4C). Analysis of conditioned threat responses during acquisition revealed that Crh:CeA:TeTx mice had significantly reduced freezing in response to CS presentations relative to control

Please cite this article in press as: Sanford et al., A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats, Neuron (2016), http:// dx.doi.org/10.1016/j.neuron.2016.11.034

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 ChR2mCherry 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 3 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). (legend continued on next page)

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mice (Figure 4D). Analogous to reduced freezing during acquisition, Crh:CeA:TeTx mice had reduced freezing in response to CS+ presentations relative to control mice during test trials (Figure 4E). We also observed a significant difference in the proportion of control versus Crh:CeA:TeTx mice showing high, low, or no fear (Figure 4F). Similar to Crh inactivation, we did not observe any differences in US responsiveness between control and Crh:CeA:TeTx mice (Figures S5A–S5E), nor did we detect gross changes in anxiety following GFP-TeTx expression (Figures S5F–S5I). Complete silencing of CRF neurons did not affect conditioned fear responses following conditioning with a high US intensity (Figures 4G–4I), similar to inactivation of Crh. Thus, generalized fear at high US intensities is independent of both CRF and release of other transmitters from CRF neurons. The observation that inactivation of Crh in the CeA and inactivation of all synaptic release from CRF neurons result in nearly identical phenotypes suggests that CRF plays a major role in CRF neuron function. One potential function of CRF could be to regulate plasticity in CRF-producing neurons during fear conditioning in an autocrine-type fashion. To test this hypothesis, we measured AMPAR/NMDAR ratios in CRF neurons in non-shocked control and shocked mice both expressing GFPTeTx (Crh:CeA:TeTx). Similar to our observations described above, we observed a significant increase in AMPAR/NMDAR ratios in foot shock-conditioned Crh:CeA:TeTx mice relative to non-shocked controls (Figures S5J–S5L); thus, CRF neurons do not appear to regulate their own plasticity. Locally Derived CRF Is the Principal Mediator of CRF Function in the CeA In addition to locally projecting CRF neurons described here, the CeA receives input from other brain regions known to contain CRF-producing neurons, including the paraventricular hypothalamus (PVH) (Herna´ndez et al., 2015) and bed nucleus of the stria terminalis (BNST) (Gungor et al., 2015), that may contribute to threat conditioning at higher US intensities. To determine the extent of input to the CeA from CRF neurons in the PVH and BNST, we quantified projections from these neurons using the Allen Brain Institute Mouse Connectivity Atlas (Oh et al., 2014). Although CRF neurons in these regions did project to the CeA, they were relatively sparse (Figure S6A). Consistent with these projection data, analysis of CRF projections by retrograde labeling of inputs with retrobeads injected into the CeA of CrhIRES-Cre;Ai14TdTomato mice revealed projections from the BNST, PVH, and a region just dorsal to the PVH, the paraxiphoid nucleus of the thalamus (PaXi), but these cells showed little overlap TdTomato (Figure S6B). To confirm that the small number of TdTomato-labeled putative CRF neurons are actively expressing Cre from the endogenous Crh locus, we injected a Cre-dependent retrograde CAV2 vector, CAV2-DIO-ZsGreen, into the CeA of CrhIRES-Cre mice (Figure S6C). Consistent with our retro-

bead analysis, we observed sparse labeling of neurons in the BNST and PaXi (Figure S6D). Although inputs to the CeA from external CRF projection neurons are sparse, that does not preclude a role for these projections in threat processing at high US intensities. To address this, we inactivated Crh from all inputs to the CeA by injecting Crhlox/lox;Ai14TdTmto mice with a retrograde-transducing CAV2 containing a Cre expression cassette (CAV2-Cre) (Figure 5A). Analysis of tdTomato reporter expression revealed transduction of neurons within the CeA as well as in the BNST, the PVH, and the PaXi (Figure 5A), as well as other structures known to project to the CeA (data not shown). Similar to injection of AAV1-CreGFP into the CeA, CeA:Crh CAV2-Cre KO mice showed impaired fear at low (0.3 mA foot shock), but not high (0.5 mA foot shock), US intensity (Figures 5B, 5C, S6E, and S6F). Importantly, inactivation of Crh in the CeA and inputs to the CeA did not alter stress responsiveness as measured by corticosterone (Figure S6G). Collectively, these data demonstrate that CRF derived from the CeA promotes fear learning at low-to-moderate US intensities, but it is unclear whether CRF facilitates cue discrimination. Analysis of discriminative responses is consistent with impaired discrimination in CeA:Crh KO mice at low and high US intensities (Figure 5D), but because individual CeA:Crh KO groups from these different experiments display overall reduced fear levels, this is difficult to parse. However, if we pool all experimental CeA:Crh KO mice across groups (AAV1-Cre-GFP mice at 0.3 and 0.4 mA and CAV2-Cre at 0.3 mA), we find a reasonable number (11 of 37 mice) that demonstrated conditioned threat responses greater than the 95th percentile of baseline freezing (as compared to 29 of 38 control mice). Analysis of these pooled groups confirmed excellent discrimination by control animals at low thresholds (Figures 5E and S6H), but poor discrimination at higher US intensity (Figure 5F). In contrast, CeA:Crh KO mice that showed measurable fear at low US intensities failed to discriminate (Figure 5E). These findings support a general role for CRF in conditioned threat learning and discrimination. CRF Neurons Facilitate Fear Acquisition, but Not Expression We find that silencing CRF neurons impairs the acquisition of conditioned fear and recall during test sessions, but this does not allow us to disambiguate whether impaired fear expression during test trials is a consequence of impaired acquisition. It has been previously demonstrated that inactivation of Sst neurons in the CeAL influences both acquisition and expression of threat memory (Li et al., 2013), indicating that neurons within the CeAL can regulate both processes. To test whether CRF neurons in the CeAL influence the acquisition and expression of conditioned threat memory, we utilized the same strategy previously described for assessing the role of Sst neurons in these processes (Li et al., 2013). CrhIRES-Cre mice were injected with a Cre-conditional adenoassociated virus containing the designer receptor exclusively

(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 3 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).

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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.

and TeTx-mediated neuronal silencing, mice expressing HM4Di demonstrate significantly reduced freezing relative to controls during acquisition (Figure 6C) and during expression 24 hr later when no CNO was administered (Figure 6D). To assess the role of CRF neurons in threat memory expression, we conditioned mice as described above and only administered CNO prior to a test session. Inhibition of CRF neurons with CNO did not alter the expression of conditioned threat, as freezing following CNO treatment or saline treatment was indistinguishable (Figures 6E and 6F). Thus, unlike Sst neurons (Li et al., 2013), CRF neurons appear to be specialized for fear acquisition.

activated by designer drug (DREADD) HM4Di fused to YFP (AAV1-DIO-HM4Di-YFP) into the CeA (Figure 6A). As reported for numerous other cell types (Sternson and Roth, 2014), application of the HM4Di ligand clozapine-N-oxide (CNO, 5 mM) to CRF neurons in slice hyperpolarized HM4Di-YFP-expressing neurons and reduced excitability (Figure 6B). To examine the role of CRF neurons in threat memory acquisition, we conditioned mice as described previously, except mice were injected with CNO prior to conditioning sessions. As with Crh inactivation

CRFR1 Receptor Signaling in the CeA Is Critical for Learning about Weak Threats Altogether, our mapping experiments and functional analysis strongly suggest that CRF produced within the CeA specifically operates at low threat levels to facilitate fear learning. Postynaptically, CRF binds and activates G protein-coupled receptors, CRFR1 and CRFR2, with CRFR1 serving as the high-affinity receptor subtype that is broadly expressed in the CNS (Bonfiglio et al., 2011). To gain a better understanding of CRF circuitry within the CeA, we sought to establish the distribution and identity of CRFR1-expressing neurons. To achieve this, we generated a CRFR1 receptor Cre-driver line in which an IRESCre is knocked into the 30 UTR of the endogenous CRFR1 locus (Crhr1IRES-Cre; Figures S7A–S7C). Like CrhIRES-Cre, Crhr1IRES-Cre

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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.

does not alter expression from the endogenous locus (Figures S7D–S7I) or stress responsiveness (Figure S7J). Crossing Crhr1IRES-Cre with the Ai14 reporter line revealed a broad distribution of CRFR1 neurons in the brain (Figure S8A), including the cortex, dorsal striatum (DStr), BNST, hippocampal CA1 and dentate gyrus (DG), ventral pontine reticular formation (PnV), and the cerebellar cortex (CCtx), that is consistent with earlier reports (Grigoriadis et al., 1996; Aguilera et al., 2004). Consistent with previous reports of CRFR1 expression (Van Pett et al., 2000; Justice et al., 2008) and binding (Weathington and Cooke, 2012) in the CeA, we observed CRFR1-expressing neurons to be broadly distributed, including the CeAL, CeAM, and, to a lesser extent, CeAC, and that these neurons show continued Cre expression in the CeA from the Crfr1 locus (Figures S8B–S8D). Histological analysis of tdTomato-expressing CRFR1 neurons together with immunostaining for either PKCd, Sst, or NTS revealed a large number of CRFR1 neurons in the rostral CeA where PKCd, Sst, or NTS is not expressed; in more medial and caudal regions, CRFR1 showed a higher degree of

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overlap with Sst than with PKCd (Figures 7D–7G). Consistent with heterogeneity of CRFR1 neurons, analysis of action potential firing following depolarizing current injections revealed distinct firing patterns with 54% showing delayed spiking, 31% showing non-delayed spiking, and 15% showing non-delayed fast spiking (Figure S8D). In contrast, CRF neurons were more homogeneous, with 79% showing delayed spiking and 21% showing non-delayed spiking (Figure S8E). CRF has been demonstrated to enhance excitatory synaptic transmission from the LA onto neurons of the CeA in a CRFR1dependent manner (Pollandt et al., 2006). To confirm that genetically defined CRFR1 neurons respond to CRF and to provide greater insight into how CRF influences fear circuitry, we monitored evoked EPSCs onto CRFR1 neurons in the CeA by stimulating inputs from the LA. Bath application of CRF (100 nM) significantly enhanced AMPAR-mediated EPSCs, but not NMDAR EPSCs, resulting in a potent enhancement of AMPAR/NMDAR ratios that is consistent with functional CRFR1 signaling with the CeA (Figures 8A, 8B, and S8F). These findings demonstrate

Please cite this article in press as: Sanford et al., A Central Amygdala CRF Circuit Facilitates Learning about Weak Threats, Neuron (2016), http:// dx.doi.org/10.1016/j.neuron.2016.11.034

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 PKCd (left), somatostatin (Sst, middle), and neurotensin (Nts, right). (B) Quantification of PKCd 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 PKCd or Sst neurons. Error bars are mean ± SEM.

that CRFR1 signaling in the CeA is a potent modulator of excitatory transmission from the LA, an important circuit connection implicated in both fear acquisition and expression (Li et al., 2013). Our data are consistent with a gain-control model in which CRF neurons in the CeA form a local connection onto CRFR1-expressing neurons to facilitate conditioned fear acquisition in response to low-to-moderate threat levels by modulating excitatory transmission (Figure 8C). To confirm that CRF signaling through CRFR1 receptors in the CeA is critical to the function of CRF derived from the CeA, we tested the effects of locally infused CRFR1 antagonist antalarmin bilaterally into the CeA (0.5 mg/side) on conditioned fear at low threat levels (Figures 8D–8F). Consistent with inactivation of Crh and CRF-producing neurons, antalarmin impaired conditioned fear acquisition and expression when it was delivered during the acquisition phase (Figures 8E and 8F). DISCUSSION The findings of this study provide novel insight into the graded processing of fear by demonstrating a neuronal cell type dedi-

cated to facilitating cued fear discrimination at low US intensities, but one that is dispensable for threat generalization in response to high US intensities. Such a specialized cell type within the CeA has not been described and illustrates how graded fear processing occurs, at least in part, through the recruitment and exclusion of specific signaling pathways and neuronal cell types. Placing our findings in the context of existing data on fear circuitry, we propose a model in which CeA-CRF neurons in the rostral CeA receive input from the LA and provide feedforward inhibition onto CRFR1 neurons in the CeA, a portion of which are Sst neurons. During fear learning, LTP at excitatory synapses occurs in the LA (McKernan and Shinnick-Gallagher, 1997) that promotes selective responding to predictive versus non-predictive cues (Collins and Pare´, 2000). Within the CeA, plasticity at LA inputs to Sst neurons (Li et al., 2013) and CRF neurons (shown here) also occurs, and we propose that this is critical for establishing temporally precise control of fear-related responses and cue discrimination learning. Consistent with this model, it has been demonstrated that LTP in feedforward

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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.

inhibitory neurons of the hippocampus is critical for temporal fidelity and input discrimination (Lamsa et al., 2005). In contrast, when threat intensities are high, aberrant plasticity in LA neurons leads to non-discriminative coding and generalized fear responses (Ghosh and Chattarji, 2015). Thus, during intense generalized fear, gain control is mute and temporal fidelity and discriminative coding are lost through CRF-independent mechanisms. What are the circuit mechanisms in the CeA that underlie generalized fear? It has recently been demonstrated that extra-

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synaptic GABA release onto PKCd neurons in the CeAL protects against fear generalization (Botta et al., 2015). It has also been shown that silencing PKCd neurons enhances conditioned freezing during recall (Haubensak et al., 2010). Thus, it is likely that aberrant plasticity and hyperactivity in the LA associated with fear generalization disrupt inhibitory connectivity in the CeAL, reducing extrasynaptic GABA release onto PKCd neurons. Since CRF enhances excitatory transmission onto CRFR1 neurons, a portion of which are Sst positive, when hyperactivation of the LA occurs, a ceiling effect of excitation onto Sst neurons

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prevents further modulation by CRF signaling, thus resulting in CRF-independent processing. Why do CRF neurons in the CeA undergo synaptic plasticity and selectively respond to fear-predictive stimuli, but not regulate conditioned fear expression? One potential explanation for this observation is that CRF neurons facilitate acquisition through the modulation of Sst neurons, and likely other cell types in the CeA; once this occurs, plasticity in these neurons is sufficient to promote fear expression (Li et al., 2013). Plasticity and conditioned responding of CRF neurons may also facilitate higher-order conditioning (Gewirtz and Davis, 1998). Consistent with this latter hypothesis, previous assessment of the role of NMDAR-dependent plasticity in CRF neurons revealed that inactivation of Grin1 (the gene encoding the essential NR1 subunit of the NMDAR) specifically in CRF neurons results in homeostatic synaptic scaling of AMPARs, increasing neural activity in these cells (Gafford et al., 2014). In these ‘‘pre-potentiated’’ animals, conditioned fear was found to be increased; thus, cue-dependent elevations in CRF release following conditioning may enhance subsequent fear-related learning. An alternative, nonmutually exclusive explanation is that these neurons influence other aspects of affective state in response to threat. In support of this hypothesis, short hairpin RNA (shRNA)-mediated knockdown of Crh mRNA in the CeA reduces anxiety following stress (Regev et al., 2012) that has been shown to be dependent on CRF-CeA projections to the locus coeruleus (LC) (McCall et al., 2015). Thus, cue-evoked activation of CeA CRF neurons is likely to engage circuitry required for anxiety-related behavior following a threatening stimulus. In addition to co-localizing to a subset of Sst neurons in the caudal CeAL, we find a large number of CRFR1 neurons in the CeAM. Using a similar HM4Di strategy that we employed here, and in the inhibition of Sst neurons used elsewhere (Li et al., 2013), it was demonstrated that attenuating activity of Tac2expressing neurons in the CeAM resulted in a reduction in threat memory consolidation (Andero et al., 2014). In all three cases, CNO was administered prior to conditioning, and in the latter case acquisition was not affected (Andero et al., 2014). We observe a reduction in acquisition and expression when CRF neurons are inhibited during the acquisition phase and when these neurons are silenced throughout training, or when Crh is similarly inactivated throughout training. Because of the nature of these manipulations, we cannot exclude the possibility that CRF influences both acquisition and consolidation. Given the number of CRFR1 neurons located in the CeAM, it is likely both processes are modulated by CRF. Consistent with this hypothesis, it has been demonstrated that CeA-CRF inactivation with anti-sense oligonucleotides attenuates contextual fear memory consolidation (Pitts et al., 2009). Aside from CRFR1-positive Sst neurons in the caudal CeAL and CRFR1-positive neurons in the CeAM, we observed a large number of CRFR1 neurons in the rostral CeAL and CeAC where Sst and PKCd expression is low. The identity of these neurons is yet to be established, but it is interesting to note that analysis of the Allen Institute Mouse Expression Atlas (Lein et al., 2007) reveals differential expression patterns of numerous other neuropeptides, including Dyn, Enk, and NPY, in the rostral CeA. The extent to which peptidergic neurons in the CeA overlap, the

manner in which they contribute to graded fear processing, and the role of their respective neuropeptides in these processes will be important to establish. EXPERIMENTAL PROCEDURES Mice Crhlox/lox, CrhIRES-Cre, CrhIRES-Cre;Ai14, and Crhr1IRES-Cre;Ai14 mice (males and females) were housed on a 12 hr light/dark cycle with ad libitum access to standard rodent chow and water. All experiments were conducted during the light cycle. All procedures were approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Washington. For detailed generation of mouse lines, surgical, and viral injection procedures, see Supplemental Experimental Procedures. Viral Vectors AAV1-DIO-GFP-TeTx, AAV1-DIO-GCaMP6m, AAV1-DIO-Rpl22-HA, AAV1Cre-GFP, AAV1-DCre-GFP, AAV1-DIO-HM4Di-YFP, CAV2-Cre, and CAV2DIO-ZsGreen were produced in house with titers of 1–3 3 1012 particles per mL as described (Gore et al., 2013; Ibanes and Kremer, 2013). RiboTag Gene Expression CrhIRES-Cre mice were injected with AAV1-DIO-Rpl22-HA. Following 4 weeks to allow for sufficient HA-tagged Rpl22 incorporation, brain tissue was collected. Punches of the CeA were homogenized and immunoprecipitation was performed as described previously (Sanz et al., 2009). RNA purified from the IP sample was converted to cDNA and analyzed via qRT-PCR. For qRT-PCR analysis, TaqMan (Applied Biosystems) primers were used to detect gene expression levels. Relative expression values were obtained using the comparative CT method and normalized to Actb levels. Fold enrichment was calculated as the IP versus input ratio and represented the amount of the transcript in the targeted cell type (IP) when compared to equal amounts of RNA from the input. Slice Electrophysiology CRF neurons were identified by fluorescence. For electrophysiology solutions and additional information, see Supplemental Experimental Procedures. AMPA/NMDA Ratios CRF+ neurons were identified by fluorescence and were held at +40 mV while a concentric bipolar electrode placed in the LA delivered 1 ms stimuli at 0.1 Hz to elicit an EPSC containing both AMPA and NMDA components. In total, 15 traces were averaged per cell, followed by bath application of APV (100 mM) to isolate the AMPA component. In total, 15 AMPA EPSC traces were averaged and digitally subtracted from the initial recording in order to generate the NMDA trace. For AMPA/NMDA ratios following bath CRF application, see Supplemental Experimental Procedures. Bath AMPA and NMDA For AMPA currents, neurons were held at
60 mV and 50 mM cyclothiazide was perfused onto the slice for 30 s, followed by 1 mM AMPA (with cyclothiazide) for 30 s. For NMDA currents, neurons were held at +40 mV and 10 mM NMDA was perfused onto the slice for 30 s. Picrotoxin (100 mM) and tetrodotoxin (500 nM) were included in the bath. LTP Occlusion For LTP experiments, picrotoxin (100 mM) was included in the bath and neurons were held at
60 mV. EPSCs were evoked as described above at 0.5 Hz for a 10 min baseline period. LTP was induced with a high-frequency stimulation protocol consisting of 1 ms stimuli at 100 Hz for 1 s, repeated 5 times at 3 min intervals (Fu et al., 2007). Calcium Imaging For in vivo imaging in awake-behaving mice, a 300 nm diameter fiber-optic probe (NeuroPak fiber-optic, Mauna Kea Technologies) was lowered into the CeA of CrhIRES-Cre mice injected with AAV1-DIO-GCaMP6m until fluorescence was detected. GCaMP6m signals were recorded using a CellVizio 488 imaging system (Mauna Kea Technologies). Stable images were acquired from a total of four mice of ten that were implanted. For each region of interest

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(ROI), CS+ and CS
trials were segregated and the signal was averaged across all trials. Change in fluorescence (%DF/F) was calculated by subtracting the average normalized fluorescence during the baseline 2 s prior to CS onset from each time point during the imaging session (20 s total) and dividing by the fluorescence intensity at each point. Fitted curves were subtracted from both CS+ and CS
trials to generate normalized data. Data were smoothed using a three-point sliding average. Fear Conditioning Behavioral sessions were performed in a standard operant chamber (Med Associates Inc.) equipped with a house light and tone generator. All sessions were video recorded and analyzed with Ethovision (Noldus) tracking software. Conditioning Sessions Auditory cues included a 10 kHz pulsatile tone and a 20 kHz continuous tone, each 10 s in duration. Assignment of the tones as the CS+ and CS
was counterbalanced across groups. Daily sessions repeated over 2 consecutive days consisted of ten presentations of the CS+ co-terminating with a 0.3 or 0.4 mA foot shock alternating pseudo-randomly with ten presentations of the CS
on a 60 s intertrial interval. Conditioning sessions to the 0.5 mA footshock consisted of five CS+ and five CS
presentations. Test Sessions Test sessions were conducted in a context separate from the conditioning, consisting of solid white walls and flat white floor with acetic acid olfactory cues. Three presentations each of the CS+ and CS
were delivered at a 60 s interval in the absence of the US. hM4Di-Mediated Neuronal Silencing Animals were trained as described above. CNO (1 mg/kg) or saline was administered 3 hr prior to conditioning or test sessions as noted during the experimental period. Statistical Analyses All statistical tests indicated were performed using Prism (GraphPad) software. Normality assessments were performed on data where a normal distribution was assumed, followed by the appropriate parametric test and post hoc multiple comparisons with adjusted p values. Variance and subject matching are tested for each analysis. All t tests performed are two tailed, and unpaired t tests where the SD is not assumed to be equal between populations were performed with Welch’s correction. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and eight figures and can be found with this article online at http://dx.doi. org/10.1016/j.neuron.2016.11.034. AUTHOR CONTRIBUTIONS Conceptualization, L.S.Z. and C.A.S.; Methodology, C.A.S., M.E.S., R.D.P., M.C., and L.S.Z.; Investigation, C.A.S., M.E.S., M.A.B., S.M.M., and L.S.Z.; Writing – Original Draft, C.A.S. and L.S.Z.; Writing – Review & Editing, C.A.S., M.E.S., and L.S.Z.; Funding Acquisition, C.A.S. and L.S.Z.; Resources, M.C., R.D.P., J.S., and L.S.Z.; Supervision, L.S.Z. ACKNOWLEDGMENTS We thank members of the Allen Institute for Brain Science for data analysis assistance. We thank Dr. Eric Kremer for CAV2 plasmids and DK-Sce cells for generation of CAV2. We acknowledge support from the NIH (R01MH094536 and R21-MH098177, L.S.Z.) and the National Science Foundation (DGE-0718124, C.A.S.). Received: March 30, 2016 Revised: August 25, 2016 Accepted: November 11, 2016 Published: December 22, 2016

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