Elevated dopamine signaling from ventral tegmental area to prefrontal cortical parvalbumin neurons drives conditioned inhibition Rongzhen 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 of Chemical 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 learned (dmPFC) subpopulation of neurons correlates with fear re- responses for optimal adaptation, but its underlying biological sponses after auditory conditioning, implicating their regulation mechanism is poorly understood. Here we used safety learning of freezing level in a bidirectional manner (19, 20). These (SL)/fear discrimination after fear conditioning as a conditioned results identify the PFC as a key brain region mediating inhibition model because it demonstrates the essential properties inhibitory control. of summation and retardation. Activity of the dorsomedial pre- Either reward or aversive stimuli causes the release of dopa- frontal cortex (dmPFC) parvalbumin (PV) neurons bidirectionally mine (21). Dopamine signaling is important in fear conditioning regulates spiking levels of dmPFC excitatory neurons and fear (FC), generalization, and discrimination (22). Importantly, ven- states. Responses to safety cues are increased in dopaminergic tral tegmental area (VTA) dopaminergic (DA) neurons con- (DA) neurons in the ventral tegmental area (VTA) and in PV tribute to the learning process in a projection-dependent manner neurons in dmPFC after SL. Plasticity in the VTA is implicated, since (23). Efferents of DA neurons to the PFC target both excitatory SL requires activation of N-methyl-D-aspartate receptors. Further- and inhibitory neurons and likely activate both D1 and more, in a posttraumatic stress disorder model, impaired SL is as- D2 subtype DA receptors (24, 25). Both in vitro and in vivo sociated with impaired potentiation of VTA DA neuron activity. studies have shown that DA inputs directly activate inhibitory Our results demonstrate a DA-dependent learning process that neurons, particularly fast-spiking interneurons, in the PFC, NEUROSCIENCE targets prefrontal inhibitory neurons for suppression of learned resulting in feed-forward inhibition of principal excitatory neu- responses, and have implications for the pathogenesis and treat- rons (24, 25). Thus, a DA-mediated increase in inhibition may ment of various psychiatric diseases. contribute or even mediate conditioned inhibition/fear discrim- ination. Given that altering DA receptor activity in the amygdala dopamine | prefrontal cortex | ventral tegmental area | parvalbumin affects conditioned inhibition or fear discrimination (26, 27), it neurons | safety learning will be of great interest to examine whether DA signaling in the PFC contributes to SL/conditioned inhibition by recruiting earning, especially emotional learning, is not and should not inhibitory neurons. Lbe precise in nature to allow optimal adaptation, since very In this study, we found that SL exhibits essential features of rarely the exact same circumstance occurs repeatedly in the real conditioned inhibition, namely summation and retardation. Fear world. Appropriate generalization of learned responses has suppression requires activation of dmPFC parvalbumin (PV) adaptive value (1, 2); for example, adequate generalization of neurons in an conditioned stimulus (CS)-dependent manner. fear memory may enable the expression of these responses under similar situations when a danger is eminent or predicted (3, 4). Significance On the other hand, discrimination between safe and dangerous circumstances/cues is critical to survival. An animal can learn to Suppression of a learned response is critical to survival and stop responding or to suppress learned responses in the presence adaptation and is impaired in various diseases. Conditioned of a safety cue, a form of learning termed conditioned inhibition inhibition is proposed to require learning and inhibitory pro- (1, 2, 5, 6). cesses, but its exact nature is poorly understood. Here we Conditioned inhibition has been recognized as an important studied safety cue-triggered fear suppression and found that biological process for survival and adaptation, as an animal its learning process requires plasticity in the ventral tegmental learns to take advantage of safety in its environment (7, 8), and area (VTA), leading to an enhanced dopaminergic (DA) neuron safety learning (SL) has antidepressant effects (9, 10) and activity by safety cue, and its inhibitory process requires VTA adaptive value for behavioral flexibility (2). Inability to suppress DA neuron inputs to parvalbumin neurons in the dorsomedial fear by safety cues results in excessive generalization of fear re- prefrontal cortex (dmPFC) to reduce dmPFC activity and fear sponses to harmless stimuli, which has been proposed as a core responses. This DA-dependent learning process is impaired in a symptom of anxiety disorders (4, 11, 12). Although suppression posttraumatic stress disorder model. Thus, conditioned in- of learned responses has been modeled as a conditioned in- hibition requires complex interactions between DA and hibitor (6), the underlying biological process remains poorly GABAergic signaling to suppress learned responses. understood. The core feature of conditioned inhibition is suppression of Author contributions: R.Y. and Q.Z. designed research; R.Y., T.W., and Q.Z. performed learned behavior through learning. SL can be a good model research; R.Y., T.W., and Q.Z. analyzed data; and R.Y. and Q.Z. wrote the paper. system for studying conditioned inhibition due to its robust na- The authors declare no conflict of interest. ture and clear relevance to various psychiatric disorders. Oppo- This article is a PNAS Direct Submission. site changes in neuronal spiking and dendritic spine size have Published under the PNAS license. been reported after fear conditioning (FC) and SL (13, 14). 1To whom correspondence may be addressed. Email: [email protected]. Evidence both for and against the contribution of the medial This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. prefrontal cortex (mPFC) to SL/fear discrimination have been 1073/pnas.1901902116/-/DCSupplemental. reported (15–18). Spiking of a dorsomedial prefrontal cortex Published online June 10, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1901902116 PNAS | June 25, 2019 | vol. 116 | no. 26 | 13077–13086 Downloaded by guest on September 25, 2021 This association between safety cue and fear suppression is inative fear responses to generalized responses by using a greater mediated by increased activity of VTA DA neurons, requires N- foot shock current (SI Appendix, Fig. S3; but see ref. 13). Taken methyl-D-aspartate receptor (NMDAR) activity during learning, together, our SL protocol establishes an adequate conditioned and is impaired in a mouse model of posttraumatic stress dis- inhibition model and allows us to explore the cellular basis of order (PTSD). The finding that conditioned inhibition requires conditioned inhibition. interactions between DA and GABAergic signaling sheds light on the mechanisms underlying emotional regulation and its Dopamine Signaling from the VTA to dmPFC Is Required for SL. Do- dysregulation in diseases. pamine signaling has been implicated in SL/fear generalization (27, 31, 32). Consistent with the importance of DA for SL, we Results found that i.p. injection of D1 receptor antagonist SCH 23390 Spiking Activity in dmPFC Neurons Discriminates Between Reinforced (0.5 mg/kg) resulted in similar dmPFC spike changes during CS+ CS (CS+) and Nonreinforced CS (CS−) After SL. Spiking in a sub- and CS− after SL (Fig. 3A). Freezing levels were not different population of dmPFC neurons was increased during reinforced between CS+ and CS− (SI Appendix, Fig. S4A), likely due to the CS (CS+; danger cue) and low during nonreinforced CS (CS−; increased freezing response after SCH 23390 injection (SI Ap- safety cue) after FC (20). We simultaneously measured spike pendix, Fig. S4B). To circumvent this problem, we infused SCH rates of dmPFC neurons and fear responses (freezing percent) in 23390 locally into dmPFC (0.5 μg/per side, bilateral). In these the same behaving mouse. Our recording region included the experiments, we used SL protocol consisted of six pairings of prelimbic cortex and cingulate cortex area 1. To best understand CS+/CS−. We found that differential fear responses disappeared changes in neuronal activity, we monitored spiking activity from after SCH 23390 infusion and reappeared after saline infusion the same neurons under different experimental conditions over (Fig. 3B). In similar experiments, local infusion of D2 receptor several days. The recorded spikes were distinct and stable, as antagonist raclopride had no effect (SI Appendix, Fig. S4C). shown by spike waveforms (SI Appendix, Figs. S1 A–C) and Thus, DA D1 receptor-coupled signaling in the dmPFC is re- principal component space cylinder (SI Appendix, Fig. S1D). quired for SL. After FC (Fig. 1A), freezing levels were high during both CS+ To understand whether dynamic modulation of DA release in and CS− [unconditioned CS at this time], with no significant the dmPFC