A multitransmitter, synaptic specification-segregated calyx-like synapse linking pontine pituitary adenylate cyclase-activating polypeptide neurons to forebrain-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 Kölliker-Fuse nucleus, and their terminals form calyx-like multirelease-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 vesicular glutamate transporter 1 (VGluT1), VGluT2, vesicular acetylcholine transport (VAChT), and the neuropeptides PACAP, calcitonin gene-related peptide, and neurotensin, together with calretinin in the presynaptic compartment. We identified a distinct neuronal subpopulation in the pontine Kölliker-Fuse region of the parabrachial complex that gives rise to these calyceal terminals, which engulf Protein kinase C delta (PKCδ+) / Glutamate delta receptor 1 (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 cluster 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.