The entorhinal cortex modulates trace fear memory formation and neuroplasticity in the mouse lateral amygdala via cholecystokinin

Although fear memory formation is essential for survival and fear-related mental disorders, the neural circuitry and mechanism are incompletely understood. Here, we utilized trace fear conditioning to study the formation of trace fear memory in mice. We identified the entorhinal cortex (EC) as a critical component of sensory signaling to the amygdala. We adopted both loss-of-function and gain-of-function experiments to demonstrate that release of the cholecystokinin (CCK) from the EC is required for trace fear memory formation. We discovered that CCK-positive neurons project from the EC to the lateral nuclei of the amygdala (LA), and inhibition of CCK-dependent signaling in the EC prevented long-term potentiation of the auditory response in the LA and formation of trace fear memory. In summary, high-frequency activation of EC neurons triggers the release of CCK in their projection terminals in the LA, potentiating auditory response in LA neurons. The neural plasticity in the LA leads to trace fear memory formation.

Measuring human trace fear conditioning

Trace fear conditioning is an important research paradigm to model aversive learning in biological or clinical scenarios, where predictors (conditioned stimuli, CS) and aversive outcomes (unconditioned stimuli, US) are separated in time. The optimal measurement of human trace fear conditioning, and in particular of memory recall after consolidation, is currently unclear. We conducted two identical experiments with a 15-s trace interval and a recall test 1 week after acquisition, while recording several psychophysiological observables. We explored learning and memory measures in the first experiment and confirmed the most sensitive measures in the second experiment. Retrodictive validity was used as a metric to estimate measurement error. We found that in the recall test without reinforcement, only fear-potentiated startle but not skin conductance, pupil size, heart period, or respiration amplitude, differentiated CS+ and CS-. During acquisition without startle probes, only skin conductance responses and pupil size responses but none of the other measures differentiated CS+ and CS-. We establish the optimal way of quantifying these conditioned responses. As a side finding, there was no evidence for extinction of fear-potentiated startle over 30 trials without reinforcement. These results may be useful to inform future substantive research using human trace fear conditioning protocols.

Behavioral phenotyping of Zip8 393T-KI mice for in vivo study of schizophrenia pathogenesis

Genetic studies have informed on the genetic landscape of schizophrenia, and the next challenge is to link the genetic associations to mechanistic studies. A common single nucleotide polymorphism in the zinc and manganese transporter ZIP8 (rs13107325; ZIP8 A391T) is a top candidate to prioritize for functional studies because it is a missense mutation that results in hypomorphic protein function. With this goal, we have established a mouse model (Zip8 393T-knock-in (KI)), and here, we report the results of brain necropsy and initial behavioral phenotyping experiments in the KI mice using open field testing, elevated plus maze, Y-maze, and trace fear conditioning. Overall, male, homozygous KI mice may exhibit subtle defects in cognition and spatial learning, otherwise the baseline testing supports minimal behavioral differences between wild-type and Zip8 393T-KI mice. There were no genotype-specific alterations of gross or microscopic neuroanatomy. These experiments are important to establish the baseline characteristics of the Zip8 393T-KI mice that may be perturbed in animal models of schizophrenia and position the Zip8 393T-KI mouse as an important model for translational studies of schizophrenia pathogenesis.

Entorhinal cortical Island cells regulate temporal association learning with long trace period

Temporal association learning (TAL) allows for the linkage of distinct, nonsynchronous events across a period of time. This function is driven by neural interactions in the entorhinal cortical–hippocampal network, especially the neural input from the pyramidal cells in layer III of medial entorhinal cortex (MECIII) to hippocampal CA1 is crucial for TAL. Successful TAL depends on the strength of event stimuli and the duration of the temporal gap between events. Whereas it has been demonstrated that the neural input from pyramidal cells in layer II of MEC, referred to as Island cells, to inhibitory neurons in dorsal hippocampal CA1 controls TAL when the strength of event stimuli is weak, it remains unknown whether Island cells regulate TAL with long trace periods as well. To understand the role of Island cells in regulating the duration of the learnable trace period in TAL, we used Pavlovian trace fear conditioning (TFC) with a 60-sec long trace period (long trace fear conditioning [L-TFC]) coupled with optogenetic and chemogenetic neural activity manipulations as well as cell type-specific neural ablation. We found that ablation of Island cells in MECII partially increases L-TFC performance. Chemogenetic manipulation of Island cells causes differential effectiveness in Island cell activity and leads to a circuit imbalance that disrupts L-TFC. However, optogenetic terminal inhibition of Island cell input to dorsal hippocampal CA1 during the temporal association period allows for long trace intervals to be learned in TFC. These results demonstrate that Island cells have a critical role in regulating the duration of time bridgeable between associated events in TAL.

NPY Released From GABA Neurons of the Dentate Gyrus Specially Reduces Contextual Fear Without Affecting Cued or Trace Fear

Disproportionate, maladapted, and generalized fear are essential hallmarks of posttraumatic stress disorder (PTSD), which develops upon severe trauma in a subset of exposed individuals. Among the brain areas that are processing fear memories, the hippocampal formation exerts a central role linking emotional-affective with cognitive aspects. In the hippocampus, neuronal excitability is constrained by multiple GABAergic interneurons with highly specialized functions and an extensive repertoire of co-released neuromodulators. Neuropeptide Y (NPY) is one of these co-transmitters that significantly affects hippocampal signaling, with ample evidence supporting its fundamental role in emotional, cognitive, and metabolic circuitries. Here we investigated the role of NPY in relation to GABA, both released from the same interneurons of the dorsal dentate gyrus (DG), in different aspects of fear conditioning. We demonstrated that activation of dentate GABA neurons specifically during fear recall reduced cue-related as well as trace-related freezing behavior, whereas inhibition of the same neurons had no significant effects. Interestingly, concomitant overexpression of NPY in these neurons did not further modify fear recall, neither under baseline conditions nor upon chemogenetic stimulation. However, potentially increased co-release of NPY substantially reduced contextual fear, promoted extinction learning, and long-term suppression of fear in a foreground context–conditioning paradigm. Importantly, NPY in the dorsal DG was not only expressed in somatostatin neurons, but also in parvalbumin-positive basket cells and axoaxonic cells, indicating intense feedback and feedforward modulation of hippocampal signaling and precise curtailing of neuronal engrams. Thus, these findings suggest that co-release of NPY from specific interneuron populations of the dorsal DG modifies dedicated aspects of hippocampal processing by sharpening the activation of neural engrams and the consecutive fear response. Since inappropriate and generalized fear is the major impediment in the treatment of PTSD patients, the dentate NPY system may be a suitable access point to ameliorate PTSD symptoms and improve the inherent disease course.

The entorhinal cortex modulates trace fear memory formation and neuroplasticity in the lateral amygdala via cholecystokinin

Although the neural circuitry underlying fear memory formation is important in fear-related mental disorders, it is incompletely understood. Here, we utilized trace fear conditioning to study the formation of trace fear memory. We identified the entorhinal cortex (EC) as a critical component of sensory signaling to the amygdala. Moreover, we used the loss of function and rescue experiments to demonstrate that release of the neuropeptide cholecystokinin (CCK) from the EC is required for trace fear memory formation. We discovered that CCK-positive neurons extend from the EC to the lateral nuclei of the amygdala (LA), and inhibition of CCK21 dependent signaling in the EC prevented long-term potentiation of sensory signals to the LA and formation of trace fear memory. Altogether, we suggest a model where sensory stimuli trigger the release of CCK from EC neurons, which potentiates sensory signals to the LA, ultimately influencing neural plasticity and trace fear memory formation.

miR-142 downregulation alleviates rat PTSD-like behaviors, reduces the level of inflammatory cytokine expression and apoptosis in hippocampus, and upregulates the expression of fragile X mental retardation protein

Background FMRP is a selective mRNA-binding protein that regulates protein synthesis at synapses, and its loss may lead to the impairment of trace fear memory. Previously, we found that FMRP levels in the hippocampus of rats with post-traumatic stress disorder (PTSD) were decreased. However, the mechanism underlying these changes remains unclear. Methods Forty-eight male Sprague-Dawley rats were randomly divided into four groups. The experimental groups were treated with the single-prolonged stress (SPS) procedure and injected with a lentivirus-mediated inhibitor of miR-142-5p. Behavior test as well as morphology and molecular biology experiments were performed to detect the effect of miR-142 downregulation on PTSD, which was further verified by in vitro experiments. Results We found that silence of miRNA-142 (miR-142), an upstream regulator of FMRP, could alleviate PTSD-like behaviors of rats exposed to the SPS paradigm. MiR-142 silence not only decreased the levels of proinflammatory mediators, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α, but also increased the expressive levels of synaptic proteins including PSD95 and synapsin I in the hippocampus, which was one of the key brain regions associated with PTSD. We further detected that miR-142 silence also downregulated the transportation of nuclear factor kappa-B (NF-κB) into the nuclei of neurons and might further affect the morphology of neurons. Conclusions The results revealed miR-142 downregulation could alleviate PTSD-like behaviors through attenuating neuroinflammation in the hippocampus of SPS rats by binding to FMRP.