Morphology and synaptic connections of pigment-dispersing factor-immunoreactive neurons projecting to the lateral protocerebrum in the large black chafer, Holotrichia parallela

Pigment-dispersing factor (PDF) is a well-known output neuropeptide modulator of circadian pacemakers in insects. Here, we investigated PDF-immunoreactive (ir) neurons in the brain of the large black chafer Holotrichia parallela to search for circadian neural components, which are potentially involved in its circabidian rhythm. PDF-ir cells were exclusively detected near the accessory medulla (AME) as a cluster of similar to 100 cells with almost homogeneous size. No other cells exhibited immunoreactivity. The PDF-ir cells send beaded fibers into the proximal half of the AME and ventral elongation in an anterior region between the medulla (ME) and lobula (LO). Neither the lamina, ME, LO, nor lobula plate receives PDF-ir fibers. Primary axons derived from the PDF-ir cells extend toward the contralateral hemisphere through the dorsolateral protocerebrum anterior to the calyx to connect the bilateral AME. The axons form varicose outgrowths exclusively in the lateral protocerebrum. Double labeling with antisynapsin revealed partial overlaps between PDF-ir varicosities and synapsin-ir puncta. Thus, it was assumed that the PDF-ir fibers form output synapses there. To verify this, we investigated the ultrastructure of the PDF-ir varicosities in the lateral protocerebrum by preembedding immunoelectron microscopy. The PDF-ir profiles contain small clear synaptic vesicles as well as both PDF-positive and PDF-negative dense-core vesicles, and the profiles form output synapses upon unknown profiles and receive synapses from other PDF-ir profiles. PDF neurons near the AME are considered to be prominent circadian pacemakers in the cockroach and flies. Their possible function in the circabidian rhythm was discussed based on these anatomical insights.