Elucidating projection patterns and postsynaptic partners of medial paracapsular intercalated cells in the mouse amygdala

Abstract

The amygdala plays a crucial role in the processing of fear and anxiety and in related disorders. It is situated in the temporal lobe and consists of several distinct nuclei that receive sensory inputs and project to downstream regions that mediate behavioral responses associated with fear. Whereas the role of glutamatergic projection neurons has been thoroughly investigated, the function of amygdala GABAergic networks is incompletely understood. GABAergic neurons are thought to modulate fear processing via inhibition and disinhibition. Part of the inhibitory network is, composed of distinct clusters of small GABAergic neurons, the intercalated cells (ITC), situated along fiber tracts surrounding the basolateral complex. Current evidence suggest that medial paracapsular ITCs (mpITCs) modulate fear processing, as they are active in fear and extinction learning, and their ablation impairs extinction memory retrieval. In addition, recent findings suggest that these cells exhibit heterogeneous projection patterns. Therefore, investigation of their anatomical projection patterns and postsynaptic targets may allow a better understanding of fear and extinction memory processes. Here I started to address the following questions: 1) To which intra- and extraamgygdaloid regions do mpITCs project in adult mice? 2) How is the communication infrastructure of mpITCs organized? 3) Which putative postsynaptic partners are targeted by mpITC axons? I addressed these questions using fluorescent and confocal imaging, and axonal reconstruction of labeled mpITCs in brain slices. I obtained qualitative data that show mpITC axonal projection patterns in accordance with published cell types in juvenile animals. I also observed putative new intra-amygdala projection patterns to the basolateral complex and extraamygdala projections to the caudate putamen and along amygdalo-striatal transition zone (Astr) to the internal capsule. In a subset of cells, we obtained 3D-reconstructions of axons using the Neurolucida software and determined quantitative parameters such as axonal length, branchpoints, and the number of presynaptic terminal boutons in specific target regions. Among these, axonal length in a specific region seems to be a main parameter that determines innervation efficacy. Lastly, I used confocal microscopy and immunohistochemical staining for markers of distinct neuron types in CEA and the Astr, to identify possible contact sites with mpITCs axons. Our findings suggest that mpITCs could make contacts with FearONcells (SOM+cells) and FearOFFcells (PKC d +cells) in CEA, but appear to pass cholinergic cells in the Astria. Together, our results reveal an unprecedented complexity of axonal projection patterns and give first quantitative insights into the wiring and postsynaptic partners of mpITCs in adult animals.