Elevated dopamine signaling from ventral tegmentalarea to prefrontal cortical parvalbumin neuronsdrives conditioned inhibitionRongzhen Yana, Tianyu Wanga, and Qiang Zhoua,b,1

aSchool of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, 518055 Shenzhen, China; and bState Key Laboratory ofChemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, 518055 Shenzhen, China

Edited by Robert Malenka, Stanford University School of Medicine, Stanford, CA, and approved May 16, 2019 (received for review January 31, 2019)

Conditioned inhibition is an important process to suppress learnedresponses for optimal adaptation, but its underlying biologicalmechanism is poorly understood. Here we used safety learning(SL)/fear discrimination after fear conditioning as a conditionedinhibition model because it demonstrates the essential propertiesof summation and retardation. Activity of the dorsomedial pre-frontal cortex (dmPFC) parvalbumin (PV) neurons bidirectionallyregulates spiking levels of dmPFC excitatory neurons and fearstates. Responses to safety cues are increased in dopaminergic(DA) neurons in the ventral tegmental area (VTA) and in PVneurons in dmPFC after SL. Plasticity in the VTA is implicated, sinceSL requires activation of N-methyl-D-aspartate receptors. Further-more, in a posttraumatic stress disorder model, impaired SL is as-sociated with impaired potentiation of VTA DA neuron activity.Our results demonstrate a DA-dependent learning process thattargets prefrontal inhibitory neurons for suppression of learnedresponses, and have implications for the pathogenesis and treat-ment of various psychiatric diseases.

dopamine | prefrontal cortex | ventral tegmental area | parvalbuminneurons | safety learning

Learning, especially emotional learning, is not and should notbe precise in nature to allow optimal adaptation, since very

rarely the exact same circumstance occurs repeatedly in the realworld. Appropriate generalization of learned responses hasadaptive value (1, 2); for example, adequate generalization offear memory may enable the expression of these responses undersimilar situations when a danger is eminent or predicted (3, 4).On the other hand, discrimination between safe and dangerouscircumstances/cues is critical to survival. An animal can learn tostop responding or to suppress learned responses in the presenceof a safety cue, a form of learning termed conditioned inhibition(1, 2, 5, 6).Conditioned inhibition has been recognized as an important

biological process for survival and adaptation, as an animallearns to take advantage of safety in its environment (7, 8), andsafety learning (SL) has antidepressant effects (9, 10) andadaptive value for behavioral flexibility (2). Inability to suppressfear by safety cues results in excessive generalization of fear re-sponses to harmless stimuli, which has been proposed as a coresymptom of anxiety disorders (4, 11, 12). Although suppressionof learned responses has been modeled as a conditioned in-hibitor (6), the underlying biological process remains poorlyunderstood.The core feature of conditioned inhibition is suppression of

learned behavior through learning. SL can be a good modelsystem for studying conditioned inhibition due to its robust na-ture and clear relevance to various psychiatric disorders. Oppo-site changes in neuronal spiking and dendritic spine size havebeen reported after fear conditioning (FC) and SL (13, 14).Evidence both for and against the contribution of the medialprefrontal cortex (mPFC) to SL/fear discrimination have beenreported (15–18). Spiking of a dorsomedial prefrontal cortex

(dmPFC) subpopulation of neurons correlates with fear re-sponses after auditory conditioning, implicating their regulationof freezing level in a bidirectional manner (19, 20). Theseresults identify the PFC as a key brain region mediatinginhibitory control.Either reward or aversive stimuli causes the release of dopa-

mine (21). Dopamine signaling is important in fear conditioning(FC), generalization, and discrimination (22). Importantly, ven-tral tegmental area (VTA) dopaminergic (DA) neurons con-tribute to the learning process in a projection-dependent manner(23). Efferents of DA neurons to the PFC target both excitatoryand inhibitory neurons and likely activate both D1 andD2 subtype DA receptors (24, 25). Both in vitro and in vivostudies have shown that DA inputs directly activate inhibitoryneurons, particularly fast-spiking interneurons, in the PFC,resulting in feed-forward inhibition of principal excitatory neu-rons (24, 25). Thus, a DA-mediated increase in inhibition maycontribute or even mediate conditioned inhibition/fear discrim-ination. Given that altering DA receptor activity in the amygdalaaffects conditioned inhibition or fear discrimination (26, 27), itwill be of great interest to examine whether DA signaling in thePFC contributes to SL/conditioned inhibition by recruitinginhibitory neurons.In this study, we found that SL exhibits essential features of

conditioned inhibition, namely summation and retardation. Fearsuppression requires activation of dmPFC parvalbumin (PV)neurons in an conditioned stimulus (CS)-dependent manner.

Significance

Suppression of a learned response is critical to survival andadaptation and is impaired in various diseases. Conditionedinhibition is proposed to require learning and inhibitory pro-cesses, but its exact nature is poorly understood. Here westudied safety cue-triggered fear suppression and found thatits learning process requires plasticity in the ventral tegmentalarea (VTA), leading to an enhanced dopaminergic (DA) neuronactivity by safety cue, and its inhibitory process requires VTADA neuron inputs to parvalbumin neurons in the dorsomedialprefrontal cortex (dmPFC) to reduce dmPFC activity and fearresponses. This DA-dependent learning process is impaired in aposttraumatic stress disorder model. Thus, conditioned in-hibition requires complex interactions between DA andGABAergic signaling to suppress learned responses.

Author contributions: R.Y. and Q.Z. designed research; R.Y., T.W., and Q.Z. performedresearch; R.Y., T.W., and Q.Z. analyzed data; and R.Y. and Q.Z. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: zhouqiang@pkusz.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1901902116/-/DCSupplemental.

Published online June 10, 2019.

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This association between safety cue and fear suppression ismediated by increased activity of VTA DA neurons, requires N-methyl-D-aspartate receptor (NMDAR) activity during learning,and is impaired in a mouse model of posttraumatic stress dis-order (PTSD). The finding that conditioned inhibition requiresinteractions between DA and GABAergic signaling sheds lighton the mechanisms underlying emotional regulation and itsdysregulation in diseases.

ResultsSpiking Activity in dmPFC Neurons Discriminates Between ReinforcedCS (CS+) and Nonreinforced CS (CS−) After SL. Spiking in a sub-population of dmPFC neurons was increased during reinforcedCS (CS+; danger cue) and low during nonreinforced CS (CS−;safety cue) after FC (20). We simultaneously measured spikerates of dmPFC neurons and fear responses (freezing percent) inthe same behaving mouse. Our recording region included theprelimbic cortex and cingulate cortex area 1. To best understandchanges in neuronal activity, we monitored spiking activity fromthe same neurons under different experimental conditions overseveral days. The recorded spikes were distinct and stable, asshown by spike waveforms (SI Appendix, Figs. S1 A–C) andprincipal component space cylinder (SI Appendix, Fig. S1D).After FC (Fig. 1A), freezing levels were high during both CS+and CS− [unconditioned CS at this time], with no significantdifference between them (Fig. 1B). This state of fear general-ization can also be quantified as a low discrimination index (Fig.1C). In contrast, SL (i.e., discriminative learning) led to signifi-cantly lower freezing during CS− (Fig. 1 A and B) and a higherdiscrimination index (Fig. 1C). In the same mice, dmPFC neu-rons (∼25% of all recorded neurons) showed similar spike rateincreases during CS+ and CS− after FC (Fig. 1 D and E) butmuch lower spike increases during CS− than during CS+ afterSL (Fig. 1 D and E). Thus, there is a good correlation betweenchanges in dmPFC spike rate and freezing level. After SL, re-sponses to a third CS (CSn, to which mice had not been exposed)were similar to the responses to CS+ but significantly higher thanthose to CS− in terms of both freezing level (Fig. 1F) and dmPFCspike rate (Fig. 1G), demonstrating the selectivity of SL to CS−.Although the foregoing results are consistent with dmPFC

neurons driving fear behavior (28, 29), the observed dmPFCspike changes could be responding to elevated freezing. Thus, westimulated dmPFC neurons during CS− after SL via theimplanted stimulating electrodes and found elevated dmPFCspike rates (Fig. 1H) and freezing levels (Fig. 1I), while stimu-lation in the absence of CS did not change freezing levels (SIAppendix, Fig. S2).

SL Exhibits Essential Properties of Conditioned Inhibition. Condi-tioned inhibition has two fundamental properties: (i) summa-tion, reduced fear response when CS+ and CS− are presentedsimultaneously (5, 6), and (ii) retardation, lower fear responseswhen safety cue is paired to the same US (6, 30). We testedwhether these two properties are met at both behavioral andneuronal activity levels after SL. First, when both CS+ and CS−were presented simultaneously, freezing level was between thelevels seen during either CS+ or CS− alone (Fig. 2A), indicatingsuppression of CS+-induced fear response by CS−. In the samemice, changes in the spike rate of dmPFC neurons were of asimilar pattern, that is, the responses to CS+/CS− were betweenthe responses to CS+ or CS− alone (Fig. 2B).Retardation was tested by pairing CS− with foot shock using

the same protocol as for the CS+/US pairing. The freezing levelafter the CS−/US pairing was significantly lower than thatachieved with CS as a neutral cue during pairing [i.e., SL (CS+)in Fig. 2C]. In the same mice, the increase in dmPFC neuronalspiking after US pairing was lower than that seen with CS as aneutral cue (Fig. 2 D and E). We failed to reverse the discrim-

inative fear responses to generalized responses by using a greaterfoot shock current (SI Appendix, Fig. S3; but see ref. 13). Takentogether, our SL protocol establishes an adequate conditionedinhibition model and allows us to explore the cellular basis ofconditioned inhibition.

Dopamine Signaling from the VTA to dmPFC Is Required for SL. Do-pamine signaling has been implicated in SL/fear generalization(27, 31, 32). Consistent with the importance of DA for SL, wefound that i.p. injection of D1 receptor antagonist SCH 23390(0.5 mg/kg) resulted in similar dmPFC spike changes during CS+and CS− after SL (Fig. 3A). Freezing levels were not differentbetween CS+ and CS− (SI Appendix, Fig. S4A), likely due to theincreased freezing response after SCH 23390 injection (SI Ap-pendix, Fig. S4B). To circumvent this problem, we infused SCH23390 locally into dmPFC (0.5 μg/per side, bilateral). In theseexperiments, we used SL protocol consisted of six pairings ofCS+/CS−. We found that differential fear responses disappearedafter SCH 23390 infusion and reappeared after saline infusion(Fig. 3B). In similar experiments, local infusion of D2 receptorantagonist raclopride had no effect (SI Appendix, Fig. S4C).Thus, DA D1 receptor-coupled signaling in the dmPFC is re-quired for SL.To understand whether dynamic modulation of DA release in

the dmPFC can affect SL, we injected AAV2/9-TH-NLS-cre andAAV2/9-Ef1α-DIO-eNpHR3.0 viruses in the VTA and usedyellow laser to suppress dmPFC DA axonal terminals from theVTA (Fig. 3C and SI Appendix, Fig. S5A). The reason forselecting TH-cre over DA transporter (DAT)-cre is the obser-vation that PFC-projecting VTA DA neurons express signifi-cantly lower levels of DAT (33). After SL, freezing during CS−was significantly elevated with laser on compared with laser off(Fig. 3D). Freezing levels did not differ between laser on andlaser off in mice injected with eYFP virus (Fig. 3D), and yellowlaser stimulation without CS did not alter freezing levels (SIAppendix, Fig. S5B). Thus, suppression of fear responses by CS−requires VTA DA release in the dmPFC. In the converseexperiment, AAV2/9-TH-NLS-cre and AAV2/9-Ef1α-DIO-hChR2 viruses were injected in the VTA (Fig. 3E), and the ef-ficacy of optogenetic manipulation was confirmed by slice elec-trophysiology (SI Appendix, Fig. S5C). Blue laser stimulation inthe dmPFC significantly reduced freezing level during CS+ afterSL (Fig. 3F), while no difference in freezing level was seen be-tween laser on and off in eYFP-injected mice (Fig. 3F), and laserstimulation without CS did not alter freezing level (SI Appendix,Fig. S5D).

VTA DA Neurons Show Enhanced Activation by CS− After SL. Theresults presented so far suggest that DA release from VTA DAneurons is required for SL, but do not distinguish whether this isdue to constitutive, activity-independent release or to DA neu-ronal activity-dependent release. A subgroup of VTA neurons(∼28% of all recorded neurons) showed minimal responsesduring CS+ or CS− after FC (Fig. 4A), with responses increasingsignificantly only during CS− after SL (Fig. 4A). These changesin spiking were correlated with a larger discrimination index (SIAppendix, Fig. S6A). This subtype of VTA neurons had a rela-tively high firing frequency (28.74 ± 5.75 Hz), which is un-characteristic of typical dopamine neuron spiking frequency (34)but is consistent with the characteristics of PFC-projecting VTADA neurons (33, 35).To further verify the activation of DA neurons, we recorded

population activity in VTA neurons infected with AAV2/9-TH-NLS-Cre and AAV2/9-Ef1α-DIO-GCaMP6s viruses, using Ca2+

imaging. Small Ca2+ increases were seen during CS− before SL,with larger increases seen after SL (Fig. 4B; freezing levels in SIAppendix, Fig. S6B). In contrast, no significant changes in Ca2+

response (SI Appendix, Fig. S6C) or fear response during CS+

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(SI Appendix, Fig. S6D) were observed after SL. In addition, weinjected AAV2-Retro-hsyn-Cre virus in the dmPFC and AAV2/9-Ef1α-DIO-GCaMP6s virus in the VTA to measure Ca2+ re-sponses of dmPFC-projecting VTA neurons (Fig. 4C). IncreasedCa2+ responses during CS− were seen after SL, but not beforeSL (Fig. 4D), corresponding to the reduced freezing level afterSL (SI Appendix, Fig. S6E). Calcium responses and freezinglevels remained unchanged during CS+ (SI Appendix, Fig. S6 Fand G).

If elevated VTA DA neuronal activity is required for dis-criminative fear responses to emerge, inhibiting VTA DA neu-ron activity should block SL. To test this, we injected AAV2/9-TH-NLS-Cre and AAV2/9-Ef1α-DIO-hM4D (Gi) viruses in theVTA. Injection of clozapine-N-oxide (CNO; 3 mg/kg, i.p.) at30 min before fear recall led to significantly higher freezingduring CS− compared with levels seen before or 48 h after CNOinjection (Fig. 4E and SI Appendix, Fig. S6H). No significantchange was seen during CS− in mice injected with mCherry virus

Fig. 1. Differential fear responses and dmPFC neuronal activity associated with fear generalization and SL. (A) Experimental procedure for FC and SL. (B)Freezing levels during CS+ and CS− after FC and after SL [two-way repeated-measures (RM) ANOVA; interaction, F(1,8) = 12.62, P < 0.01; stimulus, F(1,8) = 16.37,P < 0.01; training, F(1,8) = 2.746, P = 0.136; n = 9 mice; post-FC, CS− vs. CS+, P = 0.15, post-SL, CS− vs. CS+, P < 0.001, Bonferroni’s posttest]. (C) Discriminationindex post-FC and post-SL (two-tailed paired t test, t = 3.608, df = 8; n = 9 mice, post-FC vs. post-SL, P < 0.01). (D) Sample recording of dmPFC neuronal spikespost-FC (Upper) and post-SL (Lower). (E) Population data on dmPFC neuronal spike change post-FC (Upper) and post-SL (Lower), with corresponding averagez-scores (two-tailed paired t test; post-FC, t = 1.856, df = 49; post-SL, t = 10.95, df = 49; n = 50 units/11 mice; post-FC, CS− vs. CS+, P = 0.07; post-SL, CS− vs. CS+,P < 0.0001). In this and subsequent figures, thick lines represent mean and thin lines represent SEM. (F) Fear suppression was selective to CS− after SL [one-way RM ANOVA, F(2, 20) = 40.18, P < 0.0001; n = 11 mice, CS− vs. CSn, P < 0.0001; CS− vs. CS+, P < 0.0001; CSn vs. CS+, P = 0.82, Bonferroni’s posttest]. (G)Smaller dmPFC neuronal spike change occurred only during CS− after SL [one-way RM ANOVA, F(2, 72) = 46.52, P < 0.0001; n = 37 units/7 mice; CS− vs. CSn, P <0.0001; CS− vs. CS+, P < 0.0001; CSn vs. CS+, P = 0.28, Bonferroni’s posttest]. (H) Electrical stimulation in the dmPFC elevated dmPFC spiking (n = 30 units/7 mice). (I) Elevated freezing level during electrical stimulation in dmPFC (two-tailed paired t test, t = 2.57, df = 8; n = 9 mice, no stim vs. stim, P < 0.05). *P <0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data represent mean ± SEM.

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(SI Appendix, Fig. S6I). Furthermore, similar spike changes inthe dmPFC were seen during CS+ and CS− at 30 min after CNOinjection (Fig. 4F), in contrast to the low spike changes seenduring CS− before or 48 h after CNO injection (Fig. 4F and SIAppendix, Fig. S6J). Thus, VTA DA neuronal activity is requiredfor the expression of differential freezing responses and dmPFCactivity after SL. Viral expression was largely restricted to theVTA DA neurons (SI Appendix, Fig. S6K). The efficacy of CNOon spiking in VTA DA neurons was also verified in slices (SIAppendix, Fig. S7 A and B).In further experiments, we found that inhibiting dmPFC-

projecting VTA neurons using pharmacogenetic approachessignificantly increased freezing levels during CS− (Fig. 5 A andB). In optogenetic experiments in which dmPFC-projecting VTAneurons were inhibited, increased freezing levels during CS−were also observed (Fig. 5 C and D). In addition, optogeneticexcitation of dmPFC-projecting VTA neurons decreased freez-ing levels during CS+ (Fig. 5 E and F), suggesting that high ac-tivity in dmPFC-projecting VTA neurons is sufficient toreduce fear.We next tested whether activation of dmPFC-projecting VTA

DA neurons is required for SL. We injected AAV2-Retro-TH-NLS-Cre virus into the dmPFC and AAV2/9-Ef1α-DIO-hM4D(Gi)-mCherry virus into the VTA for pharmacogenetic inhibitionof dmPFC-projecting VTA DA neurons (Fig. 5G), and thismanipulation significantly increased freezing level during CS−(Fig. 5H).The selectively enhanced responses to CS− after SL in

dmPFC-projecting VTA DA neurons suggest that CS− leads toelevated VTA DA neuronal spiking after SL. One possible

mechanism for such enhancement is the NMDAR-dependentplasticity in the VTA (21, 36). To test this possibility, the com-petitive NMDAR antagonist D-APV (1.0 μg/per side, bilateral)was infused into the VTA at 15 min before SL (Fig. 5I). Nosignificant difference in freezing levels between CS− and CS+was seen in the D-APV–infused mice (Fig. 5J), corresponding toa low discrimination index (Fig. 5K). In contrast, subsequentinfusion of saline in the same mice at 48 h after APV infusionresulted in a significant decrease in freezing level (Fig. 5J) and alarge discrimination index (Fig. 5K). Thus, an NMDAR-dependent plasticity process in the VTA likely occurs duringSL, which may underlie the selective elevation of VTA DAneuron activity by CS− (37).

PV-Positive Neurons in the dmPFC Mediate SL. What are the targetsof VTA DA neurons in the dmPFC? The dmPFC neuronsshowing spiking reduction appear to be excitatory neurons based

Fig. 3. DA in the dmPFC is required for differential fear response anddmPFC spiking. (A) Spike change during CS+ and CS− before and afterSCH23390 i.p. injection (two-tailed paired t test, before SCH, t = 8.359, df =30 SCH, t = 0.547, df = 30; n = 31 units/9 mice; CS− vs. CS+, P < 0.0001 beforeSCH; CS−/SCH vs. CS+/SCH, P = 0.59). (B) Freezing levels after localSCH23390 or saline infusion [two-way RM ANOVA; interaction, F(2,18) =10.40, P = 0.001; treatment, F(2,18) = 58.91, P < 0.0001; stimulus, F(1,9) = 36.26,P < 0.001; n = 10 mice, CS− vs. CS+, P < 0.0001 (before SCH); CS− vs. CS+, P =0.07 (SCH); CS− vs. CS+, P < 0.0001 (Sal), Bonferroni’s posttest]. (C) Schematicillustration of optogenetic inhibition of VTA DA projections in the dmPFC.(D) Optogenetic inhibition after SL elevated freezing levels during CS− (two-tailed paired t test; eNpHR3.0, t = 4.913, df = 4; eYFP, t = 1.997, df = 5; Left,eNpHR3.0, n = 5 mice, laser off vs. laser on, P < 0.01; Right, eYFP, n = 6 mice;laser off vs. laser on, P = 0.10). (E) Schematic illustration of optogeneticexcitation of VTA DA projections in the dmPFC. (F) Optogenetic excitationafter SL reduced freezing levels during CS+ (two-tailed paired t test; hChR2,t = 6.770, df = 5; eYFP, t = 0.5894, df = 5; Left, hChR2, n = 6 mice, laser off vs.laser on, P < 0.01; Right, eYFP, n = 6 mice, laser off vs. laser on, P = 0.58).

Fig. 2. SL meets the criteria for conditioned inhibition. (A) Summation ef-fect at the behavioral level after SL [one-way RM ANOVA, F(2, 38) = 131.6, P <0.0001; n = 20 mice, CS− vs. CS+, P < 0.0001; CS− vs. CS+/−, P < 0.0001; CS+ vs.CS+/−, P < 0.0001, Bonferroni’s posttest]. (B) Summation effect at neuronalactivity level after SL [one-way RM ANOVA, F(2, 106) = 36.53, P < 0.0001; n =54 units/12 mice; CS− vs. CS+, P < 0.0001; CS− vs. CS+/−, P < 0.001; CS+ vs.CS+/−, P < 0.001, Bonferroni’s posttest]. (C) Retardation effect at behaviorallevel after pairing of CS− with US [one-way RM ANOVA, F(2, 18) = 41.94, P <0.0001; n = 10 mice, SL (CS+) vs. CS−/US, P < 0.01, Bonferroni’s posttest]. (D)Sample recording in the dmPFC after pairing CS− with US. (E) Retardationeffect at the neuronal activity level after pairing of CS− with US [one-wayRMANOVA, F(2, 72) = 18.95, P < 0.0001; n = 37 units/10mice; SL (CS+) vs. CS−/US,P < 0.001, Bonferroni’s posttest].

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on their spike properties; thus, a likely candidate for causing thisreduction is inhibitory GABAergic neurons in the PFC, espe-cially those containing PV, which has a powerful perisomaticaction that suppresses the spiking activity of excitatory neurons(38). We encountered a few dmPFC neurons with waveformsand basal spiking frequency resembling those of inhibitory neu-rons (SI Appendix, Fig. S8A). They showed lower spike changesto CS− with CNO than without CNO (Fig. 6A, neurons fromexperiments in Fig. 4). These results prompted us to examine PVneurons in the dmPFC.To further verify the dmPFC PV neurons, we monitored Ca2+

responses in PV neurons in the dmPFC (GCaMP6s virus injectedin the dmPFC of PV-cre mice; SI Appendix, Fig. S8 B and C).The selectivity of GCaMP6s expression in PV neurons wasconfirmed using immunohistochemistry (SI Appendix, Fig. S8D).Responses were small during CS− before SL and became sig-nificantly larger after SL (Fig. 6B), corresponding to decreasedfreezing level (SI Appendix, Fig. S9A). In contrast, Ca2+ re-sponses during CS+ were not significantly altered by SL in thesame set of mice (Fig. 6B), nor was freezing level (SI Appendix,Fig. S9B). These results suggest that SL selectively elevates PVneuron activity in the dmPFC during CS−.We next tested whether ongoing PV neuron activity is re-

quired for the expression of SL. By injecting DIO-eNpHR3.0-eYFP virus into PV-cre mice, we inhibited PV neurons withyellow laser during CS− (Fig. 6C). Freezing level during CS− wassignificantly higher with the laser on than with it off (Fig. 6D)after SL, in contrast to no difference between laser on and off inmice injected with eYFP virus (Fig. 6D). Yellow laser stimulation

without CS did not alter freezing level (SI Appendix, Fig. S9C).Thus, fear level is likely modulated bidirectionally by the activityof dmPFC PV neurons.

Elevated dmPFC PV Neuron Activity Mimics the Effects of SL. Al-though the foregoing results indicate that PV neuron activity isrequired for SL expression, they do not show whether elevatedPV neuron activity alone is sufficient to mimic the effect of SL.To test this, we injected DIO-hChR2-eYFP/mCherry virus intodmPFC of PV-cre mice after FC. The locations of virus ex-pression and implanted optic fiber were verified after experi-ments (SI Appendix, Fig. S10A).When PV neurons were excitedduring CS−, freezing level was significantly lower with laser on,in contrast to no difference between laser on and laser off inmCherry-injected mice (Fig. 6E and SI Appendix, Fig. S10B),indicating that increasing PV neuron activity is sufficient to re-duce fear expression. We obtained a similar result when thesame experiment was repeated with CS+ (Fig. 6F and SI Ap-pendix, Fig. S10B). Furthermore, laser stimulation of VTA-dmPFC DA terminals in mice that only received FC also ledto reduced freezing (Fig. 6G). Taken together, these results in-dicate that fear responses associated with a given CS are de-termined by DA release in the dmPFC and the resulted activityof PV neurons. Since increasing the activity of dmPFC DAterminals or dmPFC PV neurons reduces fear in a similar way asSL, these results suggest that this portion of the circuitry isconstitutively active and unlikely affected by SL.Since retardation effect is a key feature of SL, we next ex-

amined the role of PV neuronal activity in retardation. Twopossible mechanisms could underlie the retardation effect:

Fig. 4. VTA DA neurons are selectively activated by CS− after SL. (A) Population spike change in VTA neurons after FC (Left) and after SL (Right) (two-tailedpaired t test; post-FC, t = 1.004, df = 32; post-SL, t = 7.857, df = 32; n = 33 units/5 mice; post-FC, CS− vs. CS+, P = 0.32; post-SL, CS− vs. CS+, P < 0.0001). (B) Peri-eventplot of averaged Ca2+ responses during CS− post-FC and post-SL (two-tailed paired t test, t = 3.554, df = 11; n = 12 mice; post-FC vs. post-SL, P < 0.01). (C)Schematic illustration of recording Ca2+ responses in dmPFC-projecting VTA neurons. (D) Averaged Ca2+ responses during CS− post-FC and post-SL (two-tailedpaired t test, t = 5.211, df = 6; n = 7 mice; post-FC vs. post -SL, P < 0.01). (E) Freezing levels during CS− after CNO injection [two-way RM ANOVA; interaction,F(2,14) = 13.53, P < 0.001; stimulus, F(1,7) = 387.1, P < 0.0001; treatment, F(2,14) = 39.11, P < 0.0001; n = 8 mice, CS− (pre CNO) vs. CS− (CNO 30 min), P < 0.0001;CS− (CNO 30 min) vs. CS− (CNO 48 h), P < 0.0001, Bonferroni’s posttest]. (F) Spike change of dmPFC neurons before (Left) and 30 min after (Right) CNOinjection [two-tailed paired t test; pre-CNO, t = 7.373, df = 20; CNO (30 min), t = 2.534, df = 20; n = 21 units/5 mice; pre-CNO, CS− vs. CS+, P < 0.0001; CNO(30 min), CS− vs. CS+, P < 0.05].

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reduced association between CS− and US during CS−/US pairing,or reduced fear expression due to suppression by CS− (i.e., itfunctions as a safety cue). To distinguish between these twopossibilities, we first compared the Ca2+ response of PV neuronsduring CS− after SL and after pairing of CS− with US in thesame mice. We found no significant difference (Fig. 6H), whichsuggests that PV neuronal responses are likely not altered bypairing. Thus, we asked whether inhibiting PV neurons couldabolish the retardation effect. PV neurons in the dmPFC wereinhibited in mice injected with DIO-hM4D (Gi)-mCherry viruswith CNO injection before CS− recall. The efficacy of CNO ininhibiting PV neuron spiking was confirmed in slices (SI Ap-pendix, Fig. S10C). This manipulation resulted in a significantincrease in freezing level, which was similar to the freezing levelachieved by CS+/US pairing (Fig. 6I). In contrast, there was nosignificant change in freezing level in mice injected with mCherry(SI Appendix, Fig. S10D). Thus, retardation is mediated by areduced fear expression via activation of dmPFC PV neurons.

This result thus indicates that CS− still functions as a safety cueafter pairing with US.

Deficits in Elevated VTA DA Signaling Underlies the Impaired SL in aPTSD Model.Clinical evidence suggests that fear discrimination orSL is impaired in PTSD patients (11, 39), but the underlyingmechanisms are poorly understood. In a single prolonged stress(SPS) model of PTSD (Fig. 7A) (40), we found a significant in-crease in freezing level during CS− (Fig. 7B) and a low dis-crimination index (Fig. 7C), compared with control mice afterSL. Thus, similar to human PTSD patients, SPS mice alsoexhibited deficits in SL. Since elevated VTA DA and dmPFC PVneuronal activities are required for SL, we examined which sig-naling may be altered in SPS mice. In SPS mice after SL, sig-nificantly lower Ca2+ responses in VTA DA neurons were seenduring CS−, while responses during CS+ were not altered (Fig.7D; freezing levels in SI Appendix, Fig. S11). Activating VTA DAprojection terminals in the dmPFC with blue laser excitation

Fig. 5. Activation of dmPFC-projecting VTA neuronsis required for fear suppression after SL, and NMDARin VTA is required for SL. (A) Schematic illustration ofpharmacogenetic inhibition of dmPFC-projectingVTA neurons. (B) Freezing levels during CS+ andCS− before, 30 min after, and 48 h after CNO in-jection in hM4D (Gi)- and mCherry-injected mice[two-way RM ANOVA; Left: interaction, F(2,24) =2.524, P = 0.101; stimulus, F(1,12) = 130.1, P < 0.0001;treatment, F(2,24) = 57.33, P < 0.0001; Right: in-teraction, F(2,10) = 3.52, P = 0.0696; stimulus, F(1,5) =309.9, P < 0.0001; treatment, F(2,10) = 1.992, P =0.187; Left: hM4D (Gi), n = 13 mice; CS−, pre-CNO vs.CNO (30 min), P < 0.001; Right: mCherry, n = 6 mice;CS−, pre-CNO vs. CNO (30 min), P = 0.40, Bonferroni’sposttest]. (C) Schematic drawing of optogenetic in-hibition of dmPFC-projecting VTA neurons. (D)Freezing levels during yellow laser stimulation andCS− in eNpHR3.0-injected mice (two-tailed pairedt test, t = 4.555, df = 4; n = 5 mice; laser off vs. laseron, P < 0.05). (E) Schematic drawing of optogeneticexcitation of dmPFC-projecting VTA neurons. (F)Freezing levels during blue laser stimulation and CS+in hChR2-injected mice (two-tailed paired t test, t =2.896, df = 4; n = 5 mice; laser off vs. laser on, P <0.05). (G) Schematic illustration of pharmacogeneticinhibition of dmPFC-projecting VTA DA neurons. (H)Freezing levels during CS+ and CS− before, 30 minafter, and 48 h after CNO injection in hM4D (Gi)- andmCherry-injected mice [two-way RM ANOVA; Left:interaction, F(2,10) = 7.165, P = 0.0117; stimulus,F(1,5) = 85.14, P = 0.0003; treatment, F(2,10) = 19.32,P = 0.0004; Right: interaction, F(2,10) = 1.441, P =0.2818; stimulus, F(1,5) = 129.5, P < 0.0001; treatment,F(2,10) = 3.085, P = 0.0905; Left: hM4D (Gi), n = 6 mice;CS−, pre-CNO vs. CNO (30 min), P < 0.001; Right:mCherry, n = 6 mice; CS−, pre-CNO vs. CNO (30 min),P = 0.98, Bonferroni’s posttest]. (I) Experimentalprocedure for APV and saline infusion in the VTA. (J)Infusion of APV blocked SL [two-way RM ANOVA;interaction, F(1,11) = 60.46, P < 0.0001; stimulus,F(1,11) = 100.4, P < 0.0001; treatment, F(1,11) = 10.09,P = 0.0088; n = 12 mice; CS−, APV vs. saline, P <0.0001; CS+, APV vs. saline, P = 0.08, Bonferroni’sposttest]. (K) Discrimination index after SL with APVand saline infusion (two-tailed paired t test, t =6.481, df = 11; n = 12 mice; APV vs. saline, P <0.0001).

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during CS− reduced the freezing level in SPS mice to a levelsimilar to that in control mice (Fig. 7E), suggesting that theseoutputs are largely unaltered and thus PV neuronal functionsremain largely intact in SPS mice. These findings indicate thatthe SL-associated increase in VTA DA neuronal activity is se-lectively impaired in SPS mice, which may underlie the impairedSL in these mice.

DiscussionIn this study, we found that conditioned inhibition in the form ofsuppression of fear responses by safety cues is acquired through alearning process that results in elevated DA neuron activity inthe VTA and enhanced parvalbumin neuron activity in thedmPFC. This enhanced inhibition reduces spiking of excitatory(principal) neurons in the dmPFC to suppress fear expression(Fig. 7F). This mechanism confirms the long-held belief that

conditioned inhibition is inhibitory in nature, and further em-phasizes the critical contribution of DA signaling during learn-ing. Interestingly, this DA contribution is impaired in a PTSDmodel, which may underlie the impaired SL in this model. Theseinsights reveal the complex nature of conditioned inhibition,which requires interactions between DA and GABAergic sys-tems. These findings may also provide new thinking and thera-peutic targets for treating various psychiatric diseases, especiallythose with altered anxiety and/or fear.Based on our findings, we propose the following model for

how conditioned inhibition mediates fear discrimination (Fig.7F): (i) the activity of dmPFC excitatory neurons bidirectionallydetermines fear responses, and this activity is modulated by thelocal GABAerigc (especially PV-containing) neurons; (ii) whenfear is generalized, CS+ or CS− leads to high-level dmPFCspiking and high fear; (iii) SL selectively increases CS− -induced

Fig. 6. Contribution of dmPFC PV neurons to theexpression of SL and the retardation effect. (A) Pu-tative dmPFC inhibitory neurons showed reducedchanges in spike change during CS− after CNO in-jection to inhibit DA neuron activity (two-tailedpaired t test; CS−, t = 2.870, df = 24; CS+, t =0.7844, df = 24; n = 25 units/10 mice; CS−, no CNO vs.with CNO, P < 0.01; CS+, no CNO vs. with CNO, P =0.44). (B) Averaged Ca2+ responses during CS− orCS+ post-FC and post-SL (two-tailed paired t test;CS−, t = 3.734, df = 14; CS+, t = 0.5759, df = 14; CS−,n = 15 mice, post-FC vs. post-SL, P < 0.01; CS+, n =15 mice, post-FC vs. post-SL, P = 0.57). (C) Schematicillustration of optogenetic inhibition of PV neuronsin the dmPFC. (D) In PV-cre mice injected witheNpHR3.0, freezing levels during CS− was elevatedby yellow laser illumination (two-tailed paired t test;eNpHR3.0, t = 8.919, df = 7; eYFP, t = 1.401, df = 4;Left: eNpHR3.0, n = 8 mice, laser off vs. laser on, P <0.0001; Right: eYFP, n = 5 mice, laser off vs. laser on,P = 0.23). (E) In PV-cre mice injected with hChR2,freezing levels during CS− post-FC (two-tailed pairedt test, t = 9.941, df = 4; n = 5 mice; laser off vs. laseron, P < 0.001). (F) In PV-cre mice injected withhChR2, freezing levels during CS+ post-FC (two-tailed paired t test, t = 5.199, df = 4; n = 5 mice;laser off vs. laser on, P < 0.01). (G) Optogenetic ex-citation of VTA DA terminals in the dmPFC duringCS− in hChR2- and mCherry-injected mice post-FC(two-tailed paired t test; hChR2, t = 5.276, df = 5;mCherry, t = 1.861, df = 5; Left: hChR2, n = 6 mice,laser off vs. laser on, P < 0.01; Right: mCherry, n =6 mice, laser off vs. laser on, P = 0.12). (H) AveragedCa2+ responses in dmPFC PV neurons post-SL andpost-CS−/US (foot shock) pairing (retardation) (two-tailed paired t test, t = 0.849, df = 4; n = 5 mice; post-SL vs. CS−/US, P = 0.44). (I) A retardation effect wasabsent with pharmacogenetic inhibition of dmPFCPV neurons (two-tailed paired t test, t = 3.45, df = 5;n = 6 mice, CS−/US, pre-CNO vs. CNO 30 min, P <0.05; two-tailed paired t test, t = 4.635, df = 5, CS−/US,pre-CNO vs. CS+/US, P < 0.01).

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VTA DA neuron activity and spiking of PV neurons; (iv) acti-vation of PV neurons reduces spiking of dmPFC excitatoryneurons and results in the suppression of fear responses duringCS− (discrimination) or simultaneous presentation of CS+ andCS− (summation); (v) plasticity in the VTA, via an NMDAR-dependent mechanism, is required for SL; (ix) a retardation ef-fect is mediated by fear suppression as CS− functions as bothdanger and safety cues, and summation of these two cues de-termines fear responses. This model emphasizes the associativenature of conditioned inhibition and interactions among multipleimportant neurotransmitter/neuromodulator systems.

Conditioned Inhibition and Fear Discrimination. It is critical toconsider the similarities and differences between these twoprocesses, as these differences could explain the differencesbetween our findings and those reported by others. Fear dis-crimination occurs with a sufficiently great difference betweenresponses to CS+ and CS−, and was tested by giving separateCS+ and CS− in most studies. Conditioned inhibition or SL usesCS− as a safety cue to reduce responses to CS+ and was testedwith simultaneous presentation of CS+ and CS− (summation).Thus, interaction between CS+ and CS− is an essential property

of conditioned inhibition but might not be necessary for dis-crimination. For example, Jo et al. (31) suggested that discrim-inative training is effective in preventing the occurrence of feargeneralization, but whether CS− inhibits CS+ response is un-clear. Thus, it is possible that different conditioning/trainingprotocols may result in either discrimination or conditioned in-hibition with distinct underlying mechanisms.

Contributions of PV-Containing GABAergic Neurons. Parvalbumin-neurons are known to provide powerful, perisomatic inhibitionto effectively suppress the spiking activity of their targets (38).Both D1 and D2 DA receptors are expressed on both excitatoryand inhibitory neurons, with D1 receptors the most abundantlyexpressed (41). Inputs from VTA DA neurons directly activate/excite PFC PV neurons in vivo, activate D1 receptors, and resultin enhanced inhibition in the PFC excitatory neurons in vitro (24,25), consistent with our present findings. Since enhancement ofVTA DA-dmPFC connections switched high fear to low fearregardless of whether SL occurred (Fig. 6G), it is likely that thesection of inhibitory circuitry functions in a default mode tomediate conditioned inhibition. Marek et al. (42) recentlyreported that ventral hippocampal projections to infralimbic

Fig. 7. Impaired SL and reduced increase in VTA DA signaling in a single prolonged stress model of PTSD. (A) Procedure for establishing the SPS/PTSD model.(B) Freezing levels during CS− [two-way RM ANOVA; interaction, F(1,17) = 4.148, P = 0.0576; stimulus, F(1,17) = 130.1, P < 0.0001; group, F(1,17) = 21.83, P < 0.001;n = 9 mice for control (Ctrl), 10 mice for SPS; CS−, Ctrl vs. SPS, P < 0.0001; CS+, Ctrl vs. SPS, P = 0.15. Bonferroni’s posttest]. (C) Discrimination index in Ctrl andSPS mice (two-tailed unpaired t test, t = 3.470, df = 17; Ctrl vs. SPS, P < 0.01; same set of mice as in B). (D) VTA DA Ca2+ responses during CS− (Left) or CS+(Right) post-SL in SPS and Ctrl mice [two-tailed unpaired t test; CS−, t = 2.319, df = 10; CS+, t = 0.0158, df = 10; n = 7 mice (Ctrl), 5 mice (SPS); CS−, Ctrl vs. SPS,P < 0.05; CS+, Ctrl vs. SPS, P = 0.99]. Heat maps showed corresponding Ca2+ responses during CS−. (E) Reduced freezing levels during CS− by excitation of VTADA terminals in the dmPFC after SL in SPS mice (two-tailed paired t test, t = 10.90, df = 9; n = 10 mice; laser off vs. laser on, P < 0.0001). (F) Model ofconditioned inhibition. (Left) CS+ (danger signal) leads to high activity in principal neurons (PN) in the dmPFC and high fear via outputs to subcortical regions,such as the basolateral amygdala, (BLA). (Right) After SL, CS− (safety cue) leads to stronger activation of VTA DA neurons, which results in stronger PV neuronactivity and reduced PN activity, with low fear expression, in the presence of CS+. The main consequence of SL is enhanced VTA DA neurons from the CS−input, which leads to enhanced transmission from VTA DA neurons to PV neurons and reduced PN neuronal activity (illustrated by thicker lines).

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PFC contribute to fear extinction by driving local PV neurons,suggesting that a similar inhibitory process may mediate fearsuppression after extinction training. Different from the reducedresponses in PV neurons during CS reported by Courtin et al.(20), we found enhanced PV neuron activity after SL. Nonetheless,both studies found higher PV neuron activity during CS− thanduring CS+.Fear responses can be suppressed at multiple levels. Increased

inhibition of fear output neurons of the central amygdala wasreported after fear extinction training via potentiation of syn-aptic inputs to the inhibitory GABAergic intercalated neurons inthe amygdala (43). Some studies implicated mPFC in SL/feardiscrimination (16–18), while others did not (15, 44). Our find-ings indicate that computation and decision to allow or suppressfear expression may occur in the PFC, with this decision thenrelayed to the subcortical regions for execution. Outputs ofdmPFC excitatory neurons can mediate fear responses in a bi-directional manner, and dmPFC-driven oscillatory events inamygdala neurons may mediate fear expression (20, 45).Whether and how circuits at cortical and subcortical levels maycoordinate or interact to generate coherent and appropriateresponses is an interesting and important topic for furtherexploration.It is also interesting and important to note the similarities

between conditioned inhibition and fear extinction. First, bothare suppressive in nature, mediated by enhanced inhibition, al-though the exact location and/or cell types mediating this in-hibition might differ (i.e., PV neurons in dmPFC for conditionedinhibition, GABAergic neurons in the hippocampus and/or in-tercalated amygdala for fear extinction) (42, 46–48). In the ab-sence of this inhibition, fear reappears/returns. Second, thesuppression of fear is selective to the specific condition/cue/context, with a safety cue for conditioned inhibition and extinc-tion context for fear extinction (47, 49, 50).

Contributions of DA Signaling. Previous studies have demon-strated a critical role of DA in FC (37, 51). Ng et al. (27)provided evidence that DA signaling in the amygdala influencesthe effectiveness of safety cues. As for reward learning, re-sponses of DA neurons were reduced by reward omission,which functions as a conditioned inhibitor (52). The omissionof an aversive stimulus (foot shock) indicated by safety cue canbe considered a reward signal, and thus the contribution of DAsignaling may be of a similar nature for both reward andaversive stimuli. In addition, our findings indicate that DAsignaling is required for the expression of SL, and future work isneeded to examine whether a plasticity process underlies thepotentiation of CS− inputs to VTA DA neurons. Previousstudies have shown that knockout of NMDARs on DAT-expressing DA neurons led to poor fear discrimination (32),while activation of DAT-DA neurons prevented fear general-ization (31). The foregoing results appear to be different fromours, but the differences may be explained by the following: (i)different groups of DA neurons being targeted: we targetedTH-positive, PFC-projecting DA neurons while Jones et al.(32) and Jo et al. (31) targeted DAT-positive neurons whichlikely innervate subcortical regions. (ii) Differences in theconditioning/learning protocols: we used high US (shock) in-tensity to induce fear generalization and SL started from ageneralization state, while Jones et al. (32) and Jo et al. (31)used low US intensity to avoid generalization. Different DApopulations might contribute to different aspects of fear gen-eralization/discrimination, with activation of DAT-DA neuronspreventing fear generalization at a state of fear discriminationand activation of TH-positive neurons leading to fear discrim-ination at a state of fear generalization. This distinction of DAsubgroups could also be subcortical vs. cortical, which should beexamined in future studies.

Implications for Psychiatric Disorders. Deficits in SL/fear discrimi-nating have been found in certain psychiatric disorders, espe-cially anxiety disorders, and has been proposed as a biomarker(11, 53, 54). Defective DA signaling has been implicated inPTSD, including deficits in the reward and reinforcement cir-cuits, decreased reward anticipation and reduced hedonic re-sponses, reduced DA metabolites in cerebral spinal fluidfollowing traumatic reminders, altered expression of striatal DAtransporter, and polymorphisms in genes encoding DAT and DAreceptors in PTSD patients (40). A reduced basal spiking rate ofVTA DA neurons (55) and altered DA level (56) were reportedin PTSD models. We have provided clear evidence of impairedfear suppression and the absence of potentiated activation ofVTA DA neurons after SL, while DA-dmPFC connections ap-pear to be largely intact.One important implication from our study is that multiple dis-

tinct causes can result in the observed deficits in conditioned in-hibition. Impaired DA signaling, PV signaling, or both, can occur,each with distinct pathological implications and treatment strate-gies. The required participation of both DA and GABAergicsystems also provides an explanation for the diversity of impairedfunctions in PTSD patients, including motivation, reward, andfear discrimination, and raises the possibility that deficits inseemingly distinct domains of neural functions are actuallydriven by deficits in different sections of the same circuitry. Thus,identifying the exact cause/deficit may enable more precise andeffective therapy. For example, treating impaired DA functionsmay require targeting the VTA, while ameliorating reduced in-hibitory functions may require enhancing GABAergic function.

Major Unresolved Questions. Although we have provided strongevidence for the involvement of DA and GABA signalingin conditioned inhibition, several key questions need to beaddressed in future studies. First, how are DA neurons activatedduring SL and become associated with the safety cue? What isthe role of CS+/US in SL? Second, are the DA projections to PVneurons a separate pathway distinct from DA projections to theexcitatory neurons? Third, is there any contribution from thesubcortical (such as amygdala) circuitry to SL and conditionedinhibition? If so, how is this circuitry coordinated with the PFCcircuitry examined in this study?

ConclusionsIn summary, our findings reveal the biological basis of condi-tioned inhibition and how this important biological process ismalfunctioning in relevant diseases. The fact that conditionedinhibition involves two of the most important players in neuralfunction, plasticity, and disease—namely dopamine and GABA—isclearly consistent with their critical contributions to both adaptivephysiological functions and pathogenesis of various psychiatricdisorders. The insights gained from this study may also help usdesign better treatments based on a more precise understandingof the underlying pathology. All animal experiments wereperformed in accordance with the ARRIVE (Animal Research:Reporting of In Vivo Experiments) guidelines on the Care andUse of Experimental Animals, approved by the Peking UniversityShenzhen Graduate School Animal Care and Use Committee.

Materials and MethodsComplete descriptions of the study methodology are provided in SIAppendix.

ACKNOWLEDGMENTS. We thank Dr. Meifang Ma for excellent technicalsupport and help with in vivo recording and data analysis, Xiaoyan Ma forhelp with slice recording, Dr. Weidong Yao for comments and suggestions,Yangmei Huang for help with immunofluorescence staining, and Dr. MiaoHe (Fudan University) for the PV-cre mice. This work was supportedby the Shenzhen Science and Technology Innovation Fund (GrantsKQTD2015032709315529 and JCYJ20170412150845848).

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