Amygdala Intercalated Cells: Gate Keepers and Conveyors of Internal State to the Circuits of Emotion.

Generating adaptive behavioral responses to emotionally salient stimuli requires evaluation of complex associations between multiple sensations, the surrounding context, and current internal state. Neural circuits within the amygdala parse this emotional information, undergo synaptic plasticity to reflect learned associations, and evoke appropriate responses through their projections to the brain regions orchestrating these behaviors. Information flow within the amygdala is regulated by the intercalated cells (ITCs), which are densely packed clusters of GABAergic neurons that encircle the basolateral amygdala (BLA) and provide contextually relevant feedforward inhibition of amygdala nuclei, including the central and BLA. Emerging studies have begun to delineate the unique contribution of each ITC cluster and establish ITCs as key loci of plasticity in emotional learning. In this review, we summarize the known connectivity and function of individual ITC clusters and explore how different neuromodulators conveying internal state act via ITC gates to shape emotionally motivated behavior. We propose that the behavioral state-dependent function of ITCs, their unique genetic profile, and rich expression of neuromodulator receptors make them potential therapeutic targets for disorders, such as anxiety, schizophrenia spectrum, and addiction.

Deletion of ErbB4 Disrupts Synaptic Transmission and Long-Term Potentiation of Thalamic Input to Amygdalar Medial Paracapsular Intercalated Cells.

Identification of candidate risk genes and alteration in the expression of proteins involved in regulating inhibitory neuron function in various psychiatric disorders, support the notion that GABAergic neuron dysfunction plays an important role in disease etiology. Genetic variations in neuregulin and its receptor kinase ErbB4, expressed exclusively by GABAergic neurons in the CNS, have been linked with schizophrenia. In the amygdala, ErbB4 is highly expressed in GABAergic intercalated cell clusters (ITCs), which play a critical role in amygdala-dependent behaviors. It is however unknown whether ErbB4 deletion from ITCs affects their synaptic properties and function in amygdala circuitry. Here, we examined the impact of ErbB4 deletion on inhibitory and excitatory circuits recruiting medial paracapsular ITCs (mpITCs) using electrophysiological techniques. Ablation of ErbB4 in mpITCs suppressed NMDA receptor-mediated synaptic transmission at thalamo-mpITC synapses and enhanced thalamic driven GABAergic transmission onto mpITCs. Furthermore, long-term potentiation (LTP) at thalamo-mpITC synapses was compromised in ErbB4 mutant mice, indicating that ErbB4 activity is critical for LTP at these synapses. Together, our findings suggest that ErbB4 deletion from mpITCs disrupts excitation-inhibition balance and learning mechanisms in amygdala circuits.

Dysregulation of the mesoprefrontal dopamine circuit mediates an early-life stress-induced synaptic imbalance in the prefrontal cortex.

Stress adversely affects an array of cognitive functions. Although stress-related disorders are often addressed in adulthood, far less is known about how early-life stress (ELS) affects the developing brain in early postnatal periods. Here we show that ELS, induced by maternal separation, leads to synaptic alteration of layer 2/3 pyramidal neurons in the prefrontal cortex (PFC) of mice. We find that layer 2/3 neurons show increased excitatory synapse numbers following ELS and that this is accompanied by hyperexcitability of PFC-projecting dopamine (DA) neurons in the ventral tegmental area. Notably, excitatory synaptic change requires local signaling through DA D2 receptors. In vivo pharmacological treatment with a D2 receptor agonist in the PFC of control mice mimics the effects of ELS on synaptic alterations. Our findings reveal a neuromodulatory mechanism underlying ELS-induced PFC dysfunction, and this mechanism may facilitate a more comprehensive understanding of how ELS leads to mental disorders.

Apical intercalated cell cluster: A distinct sensory regulator in the amygdala.

GABAergic neurons regulate different aspects of information processing in the amygdala. Among these are clusters of intercalated cells (ITCs), which have been implicated in fear-related behaviors. Although a few of the ITC clusters have been studied, the functional role of apical ITCs (apITCs) is unknown. Here, we combine monosynaptic rabies tracing with optogenetics and demonstrate that apITCs receive synaptic input from medial geniculate nucleus (MGm), posterior intralaminar nucleus (PIN), and medial dorsal nucleus of the thalamus and from a diverse range of cortical areas including temporal association, entorhinal, insular, piriform, and somatosensory cortex. Upon fear learning, PIN/MGm inputs are strengthened, indicative of their involvement in fear behaviors. 3-D reconstruction of apITCs reveals local arborization and innervation of the dorsal striatum and lateral amygdala. We further show that apITCs provide sensory feedforward inhibition to LA principal cells, a putative mechanism for controlling plasticity during fear learning.

Deletion of NRXN1α impairs long-range and local connectivity in amygdala fear circuit.

Neurexins are a family of presynaptic cell adhesion proteins that regulate synaptic structure and maintain normal synaptic transmission. Mutations in the α-isoform of neurexin1-gene (NRXN1α) are linked with cognitive and emotional dysregulation, which are heavily dependent on the amygdala and medial prefrontal cortex (mPFC). It is however not known whether deletion of NRXN1α gene affect specific synaptic elements within the amygdala microcircuit and connectivity with mPFC. In this study, we show that NRXN1α deletion impairs synaptic transmission between the dorsal medial prefrontal cortex (dmPFC) and basal amygdala (BA) principal neurons. Stimulation of dmPFC fibers resulted in reduced paired pulse ratio (PPR) and AMPA/NMDA ratio at dmPFC to BA synapses in NRXN1α-knockout (KO) (NRXN1α KO) mice suggestive of pre- and postsynaptic deficits but there was no change at the lateral amygdala (LA) to BA synapses following LA stimulation. However, feedforward inhibition from either pathway was significantly reduced, suggestive of input-independent deficit in GABAergic transmission within BA. We further analyzed BA inhibitory network and found reduced connectivity between BA GABAergic and glutamatergic neurons in NRXN1α KO mice. As this circuit is tightly linked with fear regulation, we subjected NRXN1α KO and WT mice to discriminative fear conditioning and found a deficit in fear memory retrieval in NRXN1α KO mice compared with WT mice. Together, we provide novel evidence that deletion of NRNX1α disrupts amygdala fear circuit.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Sensory inputs to intercalated cells provide fear-learning modulated inhibition to the basolateral amygdala.

Increasing evidence suggests that parallel plastic processes in the amygdala involve inhibitory elements to control fear and extinction memory. GABAergic medial paracapsular intercalated cells (mpITCs) are thought to relay activity from basolateral nucleus (BLA) and prefrontal cortex to inhibit central amygdala output during suppression of fear. Recently, projection diversity and differential behavioral activation of mpITCs in distinct fear states suggest additional functions. Here, we show that mpITCs receive convergent sensory thalamic and cortical inputs that undergo fear learning-related changes and are dynamically modulated via presynaptic GABAB receptors recruited by GABA released from the mpITC network. Among mpITCs, we identify cells that inhibit but are also mutually activated by BLA principal neurons. Thus, mpITCs take part in fear learning-modulated feedforward and feedback inhibitory circuits to simultaneously control amygdala input and output nuclei. Our findings place mpITCs in a unique position to gate acquired amygdala-dependent behaviors via their direct sensory inputs.