Novel multi-transmitter calyx-like giant synapse revealed in the extended amygdala

We have discovered a highly specialized innervation of the forebrain by pituitary adenylate cyclase activating polypeptide (PACAP) immunohistochemistry originating from the brain stem that uses glutamate, acetylcholine and PACAP and other peptides as neurotransmitters. The parent neurons of the axons are in the Kolliker-Fuse nucleus and their terminals form calyx-like multi-release site synapses in the rodent forebrain extended amygdala similar to the calyx of Held in the auditory brain stem. The latter is a giant, excitatory, cup-like axo-somatic high-fidelity synapse. The PACAP-positive terminals also form enveloping axo-somatic specialization with mixed glutamatergic and cholinergic molecular identities, co-expressing VGluT1, VGluT2, VAChT, and the neuropeptides PACAP, CGRP, and neurotensin, together with calretinin in the presynaptic compartment. We identified a distinct neuronal subpopulation in the pontine Kolliker-Fuse region of the parabrachial complex that gives rise to these calyceal terminals, which engulf PKC/GluD1 somata in the capsular central amygdala and oval bed nucleus of the stria terminalis. Strikingly, GluD1 immunolabeling is concentrated at axo-somatic contact zones apposed to VAChT presynaptic vesicle clusters zones but is absent from postsynaptic densities of conventional type I synapses within the same terminals. The results demonstrate a previously unrecognized multimodal calyx-like synapse in the forebrain, with parallel fast ionotropic and modulatory peptidergic neurotransmission mechanisms, which is a substrate for high-fidelity signal transmission within viscerosensory -emotional circuits. Significance StatementA high-fidelity axo-somatic glutamatergic synapse is exemplified by the calyx of Held in the auditory brainstem, but is unknown in the forebrain. Here we reveal a multimodal calyceal synapse in the extended amygdala formed by glutamatergic afferents of the pontine Kolliker-Fuse nucleus that contain also several neuropeptides. These giant terminals co-package glutamate, acetylcholine, PACAP, CGRP and neurotensin. They envelop select PKC/GluD1 neurons in the capsular central amygdala and oval BNST. These unique forebrain synapses link viscerosensory input from the hindbrain to limbic autonomic centers. Such high-fidelity multimodal transmission is likely to underlie the precise synchronization of emotional and homeostatic control circuits.