Plasticity of amygdala intercalated cell microcircuits in feear learning

Anxiety spectrum disorders result from abnormal regulation of neural networks that support fear learning. A key brain region is the amygdala, which is composed of several nuclei with unique inputs and outputs. To study fear learning in the laboratory, Pavlovian fear conditioning is the most widely used model, where a neutral conditioned stimulus (CS) is associated with an aversive unconditioned stimulus (US). Conditioned fear responses are acquired through activity-dependent plasticity at glutamatergic sensory synapses on principal neurons of the basolateral amygdala. While increasing evidence also indicates a critical role for modulation and plasticity of inhibitory neurons, specific functions and plasticity mechanisms of different types of amygdala GABAergic neurons are still poorly understood. Recently, clusters of GABAergic projection neurons, the intercalated cells (ITC) received particular attention as crucial players in extinction learning. ITCs are ideally positioned to integrate information from the somatosensory system and neuromodulators such as dopamine, which has a critical role in acquisition and expression of fear. Our recent findings show that dorsal ITCs are also activated by fear learning, are a site of convergence for glutamatergic CS- and US-related sensory inputs, and send efferents to several local (amygdala) and extra-amygdala targets. Our key hypothesis is that salient sensory stimuli directly drive ITC neuronal plasticity and activity, which in turn affects multiple target areas to control conditioned fear behavior. Here, we propose an integrated approach to investigate fear learning-related synaptic plasticity in ITC networks, and to understand how ITC activity and outputs contribute to distinct acquired fear states in mice. First, we will elucidate functional and molecular mechanisms of sensory input plasticity onto dorsal ITCs following fear learning and memory retrieval using ex vivo electrophysiology and high-resolution imaging. Next, we will determine the role of physiologically released dopamine in modulating ITC plasticity by employing a specific optogenetic approach. Thirdly, for all recorded ITCs we will identify innervated brain regions and targeted cell types using anatomical techniques. As we hypothesize that subsets of dorsal ITCs with specific outputs are activated during distinct fear states, we will examine activation of ITCs with defined target regions upon high and low fear states by combining behavioral analysis with retrograde tracing and activity mapping. Lastly, to establish a causal link between ITC activity and distinct fear states, we will use a pharmacogenetic approach to attenuate ITC activity specifically during fear learning, memory retrieval, and extinction. Our data will provide important new insights into the layout, plasticity mechanisms, and role of these specialized GABAergic ITC microcircuits in the amygdala in distinct acquired fear states.

The amygdala plays a crucial role in attaching emotional significance to environmental cues. Its intercalated cells (ITC) are tight clusters of spiny GABAergic neurons, which are distributed around the basolateral amygdala complex (BLA). Distinct ITC clusters are involved in the acquisition and extinction of conditioned fear responses. Previously, we have shown that fear memory retrieval reduces the AMPA/NMDA ratio at thalamic afferents to ITC neurons within the dorsomedial (dm)-ITC cluster. We investigated the molecular mechanisms underlying the fear-mediated reduction in the AMPA/NMDA ratio at these synapses using the freeze-fracture replica immunolabeling technique and could demonstrate that changes in the synaptic density of AMPA receptors, particularly at spine synapses, underlie the observed change in AMPA/NMDA ratio. These findings directly link the regulation of AMPA receptor trafficking to memory processes in identified neuronal networks. Dopaminergic signaling plays an important role in associative learning including fear and extinction learning. Dopaminergic midbrain neurons encode prediction error-like signals when threats differ from expectations. Within the amygdala, ITC clusters receive the densest dopaminergic projections, but their physiological consequences were incompletely understood. In mice, we revealed two distinct novel mechanisms how mesencephalic dopaminergic afferents control ITCs. Firstly, they co-release GABA to mediate rapid, direct inhibition. Secondly, released dopamine (DA) directly hyperpolarizes ITCs, and suppresses inhibitory interactions between distinct ITC clusters via presynaptic D1-receptors. Early extinction training augments both GABA co-release onto dm-ITCs and DA-mediated suppression of the inhibition exerted by the dm onto the ventromedial ITC cluster. These findings provide novel insights into dopaminergic mechanisms shaping the activity balance between distinct ITC clusters that could support their opposing roles in fear behavior.