GABA- and serotonin-expressing neurons take part in inhibitory as well as excitatory input pathways to the circadian clock of the Madeira cockroach Rhyparobia maderae.

In the Madeira cockroach, pigment-dispersing factor-immunoreactive (PDF-ir) neurons innervating the circadian clock, the accessory medulla (AME) in the brain's optic lobes, control circadian behaviour. Circadian activity rhythms are entrained to daily light-dark cycles only by compound eye photoreceptors terminating in the lamina and medulla. Still, it is unknown which neurons connect the photoreceptors to the clock to allow for light entrainment. Here, we characterized by multiple-label immunocytochemistry the serotonin (5-HT)-ir anterior fibre fan and GABA-ir pathways connecting the AME- and optic lobe neuropils. Colocalization of 5-HT with PDF was confirmed in PDF-ir lamina neurons (PDFLAs). Double-labelled fibres were traced to the AME originating from colabelled PDFLAs branching in accessory laminae and proximal lamina. The newly discovered GABA-ir medial layer fibre tract connected the AME to the medulla's medial layer fibre system, and the distal tract fibres connected the AME to the medulla. With Ca imaging on primary cell cultures of the AME and with loose-patch-clamp recordings in vivo, we showed that both neurotransmitters either excite or inhibit AME clock neurons. Because we found no colocalization of GABA and 5-HT in any optic lobe neuron, GABA- and 5-HT neurons form separate clock input circuits. Among others, both pathways converged also on AME neurons that coexpressed mostly inhibitory GABA- and excitatory 5-HT receptors. Our physiological and immunocytochemical studies demonstrate that GABA- and 5-HT-immunoreactive neurons constitute parallel excitatory or inhibitory pathways connecting the circadian clock either to the lamina or medulla where photic information from the compound eye is processed.