Roger Marek

The amygdala and medial prefrontal cortex: partners in the fear circuit

Abstract  Fear conditioning and fear extinction are Pavlovian conditioning paradigms extensively used to study the mechanisms that underlie learning and memory formation. The neural circuits that mediate this learning are evolutionarily conserved, and seen in virtually all species from flies to humans. In mammals, the amygdala and medial prefrontal cortex are two structures that play a key role in the acquisition, consolidation and retrieval of fear memory, as well extinction of fear. These two regions have extensive bidirectional connections, and in recent years, the neural circuits that mediate fear learning and fear extinction are beginning to be elucidated. In this review, we provide an overview of our current understanding of the neural architecture within the amygdala and medial prefrontal cortex. We describe how sensory information is processed in these two structures and the neural circuits between them thought to mediate different aspects of fear learning. Finally, we discuss how changes in circuits within these structures may mediate fear responses following fear conditioning and extinction.

p300/CBP-associated factor selectively regulates the extinction of conditioned fear

It is well established that the activity of chromatin-modifying enzymes is crucial for regulating gene expression associated with hippocampal-dependent memories. However, very little is known about how these epigenetic mechanisms influence the formation of cortically dependent memory, particularly when there is competition between opposing memory traces, such as that which occurs during the acquisition and extinction of conditioned fear. Here we demonstrate, in C57BL/6 mice, that the activity of p300/CBP-associated factor (PCAF) within the infralimbic prefrontal cortex is required for long-term potentiation and is necessary for the formation of memory associated with fear extinction, but not for fear acquisition. Further, systemic administration of the PCAF activator SPV106 enhances memory for fear extinction and prevents fear renewal. The selective influence of PCAF on fear extinction is mediated, in part, by a transient recruitment of the repressive transcription factor ATF4 to the promoter of the immediate early gene zif268, which competitively inhibits its expression. Thus, within the context of fear extinction, PCAF functions as a transcriptional coactivator, which may facilitate the formation of memory for fear extinction by interfering with reconsolidation of the original memory trace.

Paradoxical enhancement of fear extinction memory and synaptic plasticity by inhibition of the histone acetyltransferase p300

It is well established that the coordinated regulation of activity-dependent gene expression by the histone acetyltransferase (HAT) family of transcriptional coactivators is crucial for the formation of contextual fear and spatial memory, and for hippocampal synaptic plasticity. However, no studies have examined the role of this epigenetic mechanism within the infralimbic prefrontal cortex (ILPFC), an area of the brain that is essential for the formation and consolidation of fear extinction memory. Here we report that a postextinction training infusion of a combined p300/CBP inhibitor (Lys-CoA-Tat), directly into the ILPFC, enhances fear extinction memory in mice. Our results also demonstrate that the HAT p300 is highly expressed within pyramidal neurons of the ILPFC and that the small-molecule p300-specific inhibitor (C646) infused into the ILPFC immediately after weak extinction training enhances the consolidation of fear extinction memory. C646 infused 6 h after extinction had no effect on fear extinction memory, nor did an immediate postextinction training infusion into the prelimbic prefrontal cortex. Consistent with the behavioral findings, inhibition of p300 activity within the ILPFC facilitated long-term potentiation (LTP) under stimulation conditions that do not evoke long-lasting LTP. These data suggest that one function of p300 activity within the ILPFC is to constrain synaptic plasticity, and that a reduction in the function of this HAT is required for the formation of fear extinction memory.

Prefrontal and Auditory Input to Intercalated Neurons of the Amygdala

The basolateral amygdala (BLA) and prefrontal cortex (PFC) are partners in fear learning and extinction. Intercalated (ITC) cells are inhibitory neurons that surround the BLA. Lateral ITC (lITC) neurons provide feed-forward inhibition to BLA principal neurons, whereas medial ITC (mITC) neurons form an inhibitory interface between the BLA and central amygdala (CeA). Notably, infralimbic prefrontal (IL) input to mITC neurons is thought to play a key role in fear extinction. Here, using targeted optogenetic stimulation, we show that lITC neurons receive auditory input from cortical and thalamic regions. IL inputs innervate principal neurons in the BLA but not mITC neurons. These results suggest that (1) these neurons may play a more central role in fear learning as both lITCs and mITCs receive auditory input and that (2) mITC neurons cannot be driven directly by the IL, and their role in fear extinction is likely mediated via the BLA.

Balanced Interhemispheric Cortical Activity Is Required for Correct Targeting of the Corpus Callosum

Bilateral integration of sensory and associative brain processing is achieved by precise connections between homologous regions in the two hemispheres via the corpus callosum. These connections form postnatally, and unilateral deprivation of sensory or spontaneous cortical activity during a critical period severely disrupts callosal wiring. However, little is known about how this early activity affects precise circuit formation. Here, using in utero electroporation of reporter genes, optogenetic constructs, and direct disruption of activity in callosal neurons combined with whisker ablations, we show that balanced interhemispheric activity, and not simply intact cortical activity in either hemisphere, is required for functional callosal targeting. Moreover, bilateral ablation of whiskers in symmetric or asymmetric configurations shows that spatially symmetric interhemispheric activity is required for appropriate callosal targeting. Our findings reveal a principle governing axon targeting, where spatially balanced activity between regions is required to establish their appropriate connectivity.

Mechanism of bilateral communication in the suprachiasmatic nucleus

The central circadian pacemaker of the suprachiasmatic nuclei (SCN) is a bilaterally symmetrical structure. Little is known about the physiological mechanisms underlying communication between the left and right SCN and yet the degree of synchronization between SCN neurons can have a critical impact on the properties of the circadian system. In this study, we used electrophysiological tools and calcium (Ca2+) imaging to examine the mechanisms underlying bilateral signaling in mouse SCN. Electrical stimulation of one SCN produced responses in the contralateral SCN with a short delay (approximately 5 ms) and Ca2+‐dependence that are consistent with action potential‐mediated chemical synaptic transmission. Patch‐clamp recordings of stimulated cells revealed excitatory postsynaptic inward‐currents (EPSCs), which were sufficient in magnitude to elicit action potentials. Electrical stimulation evoked tetrodotoxin‐dependent Ca2+ transients in about 30% of all contralateral SCN neurons recorded. The responding neurons were widely distributed within the SCN with a highest density in the posterior SCN. EPSCs and Ca2+ responses were significantly reduced after application of a glutamate receptor antagonist. Application of antagonists for receptors of other candidate transmitters inhibited the Ca2+ responses in some of the cells but overall the impact of these antagonists was variable. In a functional assay, electrical stimulation of the SCN produced phase shifts in the circadian rhythm in the frequency of multiunit activity rhythm in the contralateral SCN. These phase shifts were blocked by a glutamate receptor antagonist. Taken together, these results implicate glutamate as a transmitter required for communication between the left and right SCN.

Hippocampus-driven feed-forward inhibition of the prefrontal cortex mediates relapse of extinguished fear

The medial prefrontal cortex (mPFC) has been implicated in the extinction of emotional memories, including conditioned fear. We found that ventral hippocampal (vHPC) projections to the infralimbic (IL) cortex recruited parvalbumin-expressing interneurons to counter the expression of extinguished fear and promote fear relapse. Whole-cell recordings ex vivo revealed that optogenetic activation of vHPC input to amygdala-projecting pyramidal neurons in the IL was dominated by feed-forward inhibition. Selectively silencing parvalbumin-expressing, but not somatostatin-expressing, interneurons in the IL eliminated vHPC-mediated inhibition. In behaving rats, pharmacogenetic activation of vHPC→IL projections impaired extinction recall, whereas silencing IL projectors diminished fear renewal. Intra-IL infusion of GABA receptor agonists or antagonists, respectively, reproduced these effects. Together, our findings describe a previously unknown circuit mechanism for the contextual control of fear, and indicate that vHPC-mediated inhibition of IL is an essential neural substrate for fear relapse.Neurons in the ventral hippocampus project to parvalbumin inhibitory interneurons in the infralimbic (IL) region of medial prefrontal cortex. Activation of this projection produces feed-forward inhibition of IL and causes relapse of extinguished fear.

Excitatory connections between the prelimbic and infralimbic medial prefrontal cortex show a role for the prelimbic cortex in fear extinction

We elucidated the intrinsic circuitry of the medial prefrontal cortex and its role in regulating fear extinction, using neuronal tracing and optogenetic stimulation in vitro and in vivo. We show that pyramidal neurons in layer 5/6 of the prelimbic medial prefrontal cortex project to pyramidal cells in layer 5/6 of the infralimbic cortex. Activation of this connection enhances fear extinction, redefining the role of the prelimbic cortex in extinction learning.Prelimbic (PL) and infralimbic (IL) mPFC are thought to mediate fear expression and fear extinction, respectively. The authors show that PL projects to IL and innervates projections to amygdala and that this connection is engaged in fear extinction.

Neural circuits for a top-down control of fear and extinction

Fear learning and extinction are controlled by the activity of three interconnected regions: the amygdala, hippocampus, and prefrontal cortex. Of these, the medial prefrontal cortex modulates specific aspects in fear and extinction via a top-down regulation. In recent years, extensive progress has been made in our understanding of the neural circuits that mediate fear-related behaviors and their modulation by ascending systems. The development of new experimental techniques is now revealing the details of the intrinsic circuits within these structures as well as the connections between them. Here, we highlight recent advances in our understanding of how the prefrontal cortex may mediate such a top-down regulation.

Neural Circuits Mediating Fear Learning and Extinction

The activity of neural circuits that underpin particular behaviours are one of the most interesting questions in neurobiology today. This understanding will not only lead to a detailed understanding of learning and memory formation, but also provides a platform for the development of novel therapeutic approaches to a range of neurological disorders that afflict humans. Among the different behavioural paradigms, Pavlovian fear conditioning and its extinction are two of the most extensively used to study acquisition, consolidation and retrieval of fear-related memories. The amygdala, medial prefrontal cortex (mPFC) and hippocampus are three regions with extensive bidirectional connections, and play key roles in fear processing. In this chapter, we summarise our current understanding of the structure and physiological role of these three regions in fear learning and extinction.