Fear discrimination is the ability to distinguish between different sources or types of fear-inducing stimuli or situations. Accurate threat discrimination through fear learning is essential for survival, whereas overgeneralized fear is a characteristic feature of anxiety disorders, including posttraumatic stress disorder (PTSD). The contextual differential fear conditioning paradigm using a mouse model, in which individuals are required to differentiate between safe and threatening contexts, provides a valuable model for investigating fear discrimination. The prelimbic (PL) subregion of the ventromedial prefrontal cortex (vmPFC) is believed to play a pivotal role in regulating these processes. Previous research has explored the importance of an amygdala-hippocampal-prefrontal circuit in fear learning, yet the specific roles of the PL substructure remain unclear. Prior studies have emphasized the role of long-term memory formation in the vmPFC for contextual differential fear learning. However, how the local PL network, consisting of hundreds of neurons, functions concerning fear discrimination has not been fully explored due to the complexity of the recordings and the length of behavioral design. This study investigates the role of network population dynamics within PL during a fear-safety learning task discriminating between ambiguous aversive and safe environments, using large-scale population recordings with calcium imaging during a contextual fear discrimination learning paradigm. Our behavioral results indicate that PL memory consolidation is not required for fear acquisition or generalization but is critical for distinguishing between similar yet distinct safe and dangerous contexts during the later stages of discrimination. Since PL has been shown to be involved with fear expression, an initial hypothesis is that we will observe similar population dynamics during fear generalization and differential dynamics after successful discrimination. An alternative hypothesis is that PL's function is to integrate contextual information and guide behavior in situations of contextual ambiguity. Using calcium imaging in mice and advanced unsupervised analysis techniques, this research reveals specific groups of neurons that respond to safe and dangerous cues throughout the learning paradigm. Blocking memory consolidation impairs fear discrimination and the development of neurons relevant to fear discrimination. This highlights the PL's role in processing contextual information, particularly in ambiguous environments.

Developmental factors involved in schizophrenic pathology are known to exist, but complex chemical interactions during critical stages of brain development are unclear. Environmental and genetic factors contribute to the development of cortical brain circuits and disruption during specific critical times in development could have lifelong consequences. N-methyl-D-aspartate receptors (NMDARs) are of critical importance for synaptic formation as well as synaptic plasticity throughout the lifespan. Hypofunctioning NMDARs have been implicated in schizophrenic patients and have been extensively used as the basis of animal models to study schizophrenia in a lab environment. Here we use a developmental model induced by drugs that block NMDARs during early stages of postnatal development in order to model schizophrenia in mice. These mice have shown permanent reductions in endocannabinoid (eCB) signaling in the medial prefrontal cortex (mPFC). These findings emphasize a possible involvement of eCB signaling in the development of schizophrenia. eCBs are unusual in that they act retrogradely at the synapse and are released on demand under high levels of post synaptic activation. Since the overall effects of eCBs at the synapse are inhibitory, it has been suggested that eCBs may play an important role in maintaining a nominal level of neuronal activation in the cortex. This also led us to investigate a well-known risk factor of schizophrenia, adolescent cannabis abuse. Cannabis contains Δ9-tetrahydrocannabinol (THC) which directly activates cannabinoid receptors in the brain. Prolonged exposure to cannabis during adolescence has been speculated to have long-term effects in some people lasting into adulthood. An adolescent cannabis abuse model induced by a specific agonist of the primary cannabinoid receptor (CB1R) in the brain shows permanent reduction in the eCB system signaling including impaired eCB-dependent synaptic plasticity and CB1R function. In addition, the cannabis abuse model shows deficiency in a function of another presynaptic receptor involved in control of presynaptic release, metabotropic glutamate receptor 2/3 (mGluR2/3). CB1Rs and mGluR2/3 receptors both share the same intracellular signaling pathways which suggest that the critical maladaptation underlying the etiology of psychosis may involve signaling mediated by presynaptic metabotropic receptors or downstream processes. These data open a new avenue for understanding and treatments of schizophrenia taking into account important developmental factors.

Several mental disorders present with symptoms related to inaccurate fear memory discrimination, including post-traumatic stress disorder (PTSD), in which a non-aversive stimulus resembling a previously experienced aversive situation triggers a fear response. The neural mechanisms underlying the attainment of fear memory accuracy for appropriate discriminative responses to aversive and non-aversive stimuli are unclear. While much research has involved the hippocampus in these processes, more recent studies implicate the medial prefrontal cortex (mPFC). By using a combination of novel behavioral paradigms and viral-mediated genetic manipulation, we were able to disrupt specific processes within the mPFC and measure their effect on fear memory accuracy. We found that several elements important to the cellular mechanisms underlying memory consolidation within the mPFC are required for appropriate discrimination of aversive and non-aversive stimuli with overlapping qualities of similarity. These include cyclic AMP response element binding protein (CREB), CREB-binding protein (CBP) histone acetyltransferase (HAT) activity, and N-methyl-D-aspartate receptors (NMDARs). This work contributes to the growing body of literature on prefrontal contributions to memory systems at the basic level, and can be applied to our understanding of deficient memory specificity found in disorders like PTSD.

The mental state of fear together with its related behavioral, physiological, and perceptual responses to threatening stimuli, is a highly conserved response in the animal kingdom that serves important functions for an animal’s survival. One of the challenges in modern neuroscience is to understand the mechanisms underlying how brain networks resolve ambiguous contextual information for appropriate valence determination and, subsequently, guide appropriate behavior responses. Fear based behaviors have been extensively studied over a hundred years using classical Pavlovian fear conditioning, in which a conditioned stimulus (CS) such as context is paired with an inherently aversive stimulus (US), like a mild foot shock. This form of associative learning has been used extensively to study the neural correlates of fear including the circuit-level, cellular-level, and molecular-level mechanisms of learning and memory. While it is well established that fear conditioning is encoded primarily in basolateral amygdala, the subsequent modulation of fear behavior involves a broader fear modulation circuit. There is strong evidence for the involvement of infralimbic (IL) and prelimbic (PL) subdivisions of prefrontal cortex (PFC) in fear memory modulation; however, the circuit-level mechanisms guiding appropriate fear responses are unclear. Current studies reveal that neurons in both IL and PL are active across fear discrimination learning. For example, fear conditioning triggers neuronal representation for CS+ specifically in PL but not in IL. In addition, differential fear conditioning triggers subtle and distinctive temporal patterns of neuronal representations of CS- in both PL and IL. Viral targeting of prefrontal cortex subdivisions with a specific inhibitor of memory consolidation demonstrated that both PL and IL are separately coding critical information during fear discrimination learning, which is consistent with prior data. In addition, neurons in PL projecting to BLA are also highly involved in coding information critical to learning appropriate balancing of fear responses. These data suggest that while there is functional division between IL and PL as previously suggested, both regions are necessary during differential fear discrimination learning. Furthermore, IL cannot compensate for impaired PL function, and PL cannot compensate for impaired IL function under the condition that both regions remain engaged during learning.

Survival relies on the ability to discriminate between danger and safety. Fear discrimination is crucial for appropriate adaptive responses while fear generalization is important for recalling and avoiding dangerous situations. Previous research suggests that fear discrimination learning is mediated by the prefrontal cortex (PFC). It has been demonstrated that disruption in either cAMP element response binding protein (CREB), its co-activator, CREB binding protein (CBP), or N-methyl D-apsarate (NMDA) receptor function specifically within the PFC causes deficits in fear discrimination. Mice exhibiting disruption of these signalling pathways show normal fear responses to aversive stimuli but inappropriate fear responses to similar, yet distinct, non-aversive stimuli. Recent evidence suggests that the prelimbic (PL) and infralimbic (IL) subregions of the PFC provide excitatory and inhibitory effects on the fear circuit, respectively, but the subregion-specific mechanisms driving fear discrimination are unknown. To get insight into the circuit mechanisms underlying fear discrimination, we investigated prefrontal neuronal ensembles representing distinct experiences associated with learning to disambiguate between dangerous and similar, yet distinct, harmless stimuli. We show distinct quantitative activation differences in response to conditioned and generalized fear responses, as well as modulation of neuronal ensembles associated with successful acquisition of contextual fear. Prefrontal neuronal ensembles of fear memories trace functional context-danger and context-safety associations. The PL subdivision of the PFC monitors context-danger associations to conditioned fear, whereas fear discrimination learning engages memory ensembles associated with the inhibition of generalized fear in both PL and IL subdivisions of the PFC. Our data suggests that fear discrimination learning is associated with the modulation of prefrontal memory representations in a subregion- and experience-specific manner while learning appropriate responses to conditioned and generalized fear experiences is driven by updating and rebalancing these prefrontal memory ensembles. These findings provide novel insight into prefrontal mechanisms that underlie fear generalization, a common symptom in patients with posttraumatic stress disorder, phobia and generalized anxiety disorder.

Determining the appropriate circumstances in which to express fear-related behavior is a difficult yet important cognitive challenge for behaving animals; insufficient fear increases vulnerability to predation and other threats, but excessive fear may lead to lost opportunities and foregone resources. Despite considerable research, the neural circuitry that enables the mammalian brain to successfully associate danger and safety cues with appropriate behavioral responses is not yet fully understood. However, recent studies have identified a pivotal role of the medial prefrontal cortex (mPFC), particularly for complex tasks requiring an appropriate balancing of fear versus safety learning and/or involving complex or ambiguous danger-associated cues. In the present work, we investigated the involvement of the prelimbic (PL) and infralimbic (IL) subdivisions of mPFC in the acquisition of contextual fear discrimination learning, a task in which subjects are challenged to maintain appropriate expression of fear to an aversive context (CS+) while suppressing the inappropriate expression of fear to a similar yet harmless context (CS-). We tested the role of PL and IL by selectively transforming these subregions with CBP∆HAT, a dominant-negative mutation of CBP (CREB Binding Protein) with eliminated intrinsic histone acetyltransferase activity but intact transcriptional facilitation, which induces hypofunction of local memory storage by impairing neuronal capacity for activity-dependent histone acetylation. We found that transforming either PL or IL with CBP∆HAT impairs the acquisition of contextual fear discrimination. Furthermore, we found that selectively transforming the subpopulation of PL neurons which projects to the basolateral amygdala (BLA) is sufficient to recapitulate the impairment produced by indiscriminately transforming the entire PL. These results show that both PL and IL are important loci for the storage of contextual fear discrimination learning, and that the PL→BLA pathway is a key component of PL’s role as such. These findings further build and clarify our understanding of what neural perturbations can upset the delicate balance between fear and safety, and suggest new insights into the possible etiology of anxiety disorders such as Post-Traumatic Stress Disorder.