Structure and function of ITC

The intercalated cell masses (ITC) of the amygdala are composed primarily of medium sized GABAergic neurons and represent an important inhibitory gate between the basolateral and central nuclei of the amygdala, under cortical control. These clusters are critically involved in the extinction of conditioned fear and probably also in the storage of extinction memory. Despite the anatomical structure of the different ITC clusters and their intrinsic and extrinsic connectivity have been only superficially investigated, they are often regarded as structurally and functionally homogeneous. Recently we have obtained a large body of preliminary data indicating that the different ITC clusters may have distinctive cytoarchitectonical, neurochemical and functional properties. In the present proposal, we aim to define the structure and function of the different ITC cell clusters. A highly detailed 3- dimensional nanostructure of the dorsomedial cluster will be analyzed by means of serial block-face scanning electron microscopy (SBF-SEM) and pre-embedding immuno-electron microscopy. The data obtained with these approaches will provide large datasets of the intrinsic and extrinsic connections of identified neurons in an ITC cluster. In addition, morphological and physiological features of ITC neurons recorded and filled in vivo will be analyzed to elucidate the full axonal projections of these neurons and to understand how the activity of distinct neuronal populations of the ITC contribute to network oscillations in the amygdala during specific brain states. The intrasynaptic arrangement of signaling proteins, such as AMPA and NMDA receptors at thalamic-ITC and BLA- ITC synapses, will also be investigated at the EM level using the freeze-fracture replica labeling and pre- embedding techniques. This study will reveal whether these receptors are distinctly organized in an input- dependent manner. In order to explore the function of ITC circuits in fear extinction, we will investigate whether pharmacological manipulation of signaling molecules present in these circuits, such as mGlu7 receptors, is effective in reversing/improving the behavioural phenotype of a mouse model (129S1/SvImJ strain) of impaired fear extinction and characterized by aberrant activity in the ITCs.

In recent years, the intercalated cell masses (ITC) of the amygdala, which are clusters of medium sized GABAergic projection neurons surrounding the basolateral amygdaloid complex (BLA), received particular attention as possible important players in the extinction of fear. So far, however, ITC neurons have been refractory to a detailed functional and anatomical characterization. This project, using a multidisciplinary approach, has allowed us to provide the first anatomical, neurochemical and functional characterization of several neuronal subtypes forming ITC complexes in rodents. We mostly focused our work on a cluster localized dorsomedially between the BLA, the primary input station for sensory inputs in the amygdala, and the central nucleus, the main output structure responsible for the fear response. We could identify that medium spiny neurons within this cluster inhibit, but are also mutually activated by, BLA principal neurons, and are also an important site of convergence for glutamatergic sensory inputs. This finding has major relevance for our understanding of the formation of fear memories. The encoding of fear memories is often studied both in rodents and in humans using classical Pavlovian fear conditioning. In fear conditioning, a neutral conditioned stimulus (CS, e.g. a tone) is paired with an aversive unconditioned stimulus (US, e.g. a mild footshock). Training leads to a CS-US association and to a conditioned fear response upon presentation of the CS alone. If the CS is repetitively presented in the absence of the US, a progressive decrease in the conditioned fear response is observed, a phenomenon, which is generally referred to as extinction learning. Extinction thus has great clinical relevance in the context of behavioural therapy for human anxiety disorders. Our results indicate that the ITC take part in fear learning-modulated feed-forward and feedback inhibitory circuits to simultaneously control amygdala input and output nuclei. Moroever, we have shown that large neurons encircling the medium spiny cells are part of ITC microcircuits. They also receive sensory inputs from the thalamus and respond in vivo to noxious stimuli. These neurons were shown to be GABAergic and to selectively innervate BLA interneurons. Taken together, our findings reveal novel wiring and function of ITCs that significantly extend the current view on parallel inhibitory and disinhibitory processes in amygdala circuits. They also support the idea that the reciprocal interaction between ITCs and BLA may be part of a sensory feedback circuit engaged in time- and stimulus strength-dependent regulation of fear expression.