The circadian pacemaker of the Madeira cockroach, Rhyparobia (Leucophaea) maderae, is located in the accessory medulla (AME). Ipsi- and contralateral histaminergic compound eyes are required for photic entrainment. Light pulses delay locomotor activity rhythm during the early night and advance it during the late night. Thus, different neuronal pathways might relay either light-dependent delays or advances to the clock. Injections of neuroactive substances combined with running-wheel assays suggested that GABA, pigment-dispersing factor, myoinhibitory peptides (MIPs), and orcokinins (ORCs) were part of both entrainment pathways, whereas allatotropin (AT) only delayed locomotor rhythms at the early night. To characterize photic entrainment further, histamine and corazonin were injected. Histamine injections resulted in light-like phase delays and advances, indicating that the neurotransmitter of the compound eyes participates in both entrainment pathways. Because injections of corazonin only advanced during the late subjective night, it was hypothesized that corazonin is only part of the advance pathway. Multiple-label immunocytochemistry in combination with neurobiotin backfills demonstrated that a single cell expressed corazonin in the optic lobes that belonged to the group of medial AME interneurons. It colocalized GABA and MIP but not AT or ORC immunoreactivity. Corazonin-immunoreactive (-ir) terminals overlapped with projections of putatively light-sensitive interneurons from the ipsi- and contralateral compound eye. Thus, we hypothesize that the corazonin-ir medial neuron integrates ipsi- and contralateral light information as part of the phase-advancing light entrainment pathway to the circadian clock. J. Comp. Neurol. 525:1250-1272, 2017. © 2016 Wiley Periodicals, Inc.
The release of neuromodulators by widely projecting neurons often allows sensory systems to alter how they process information based on the physiological state of an animal. Neuromodulators alter network function by changing the biophysical properties of individual neurons and the synaptic efficacy with which individual neurons communicate. However, most, if not all, sensory networks receive multiple neuromodulatory inputs, and the mechanisms by which sensory networks integrate multiple modulatory inputs are not well understood. Here we characterized the relative glomerular distribution of two extrinsic neuromodulators associated with distinct physiological states, serotonin (5-HT) and dopamine (DA), in the antennal lobe (AL) of the moth Manduca sexta. By using immunocytochemistry and mass dye fills, we characterized the innervation patterns of both 5-HT- and tyrosine hydroxylase-immunoreactive processes relative to each other, to olfactory receptor neurons (ORNs), to projection neurons (PNs), and to several subsets of local interneurons (LNs). 5-HT immunoreactivity had nearly complete overlap with PNs and LNs, yet no overlap with ORNs, suggesting that 5-HT may modulate PNs and LNs directly but not ORNs. TH immunoreactivity overlapped with PNs, LNs, and ORNs, suggesting that dopamine has the potential to modulate all three cell types. Furthermore, the branching density of each neuromodulator differed, with 5-HT exhibiting denser arborizations and TH-ir processes being sparser. Our results suggest that 5-HT and DA extrinsic neurons target partially overlapping glomerular regions, yet DA extends further into the region occupied by ORNs.
A multitude of potential neurotransmitters and neuromodulators, including peptides, have been detected in the antennal lobe (AL), the first synaptic relay of the central olfactory pathway in the insect brain. However, the functional role of neuropeptides in this system has yet to be revealed. An important prerequisite to understanding the role of neuropeptides is to match the functionally different cell types in the AL with their peptide profiles by using electrophysiological recordings combined with immunocytochemical studies and/or single-cell mass spectrometry. The olfactory system of Periplaneta americana is particularly well suited to accomplish this goal because several physiologically distinct neuron types can be unequivocally identified. With the aim to analyze the neuropeptide inventory of the P. americana AL, this study is an essential step in this direction. First, we systematically analyzed different parts of the AL by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry to obtain the complete set of neuropeptides present. Altogether, 56 ion signals could be assigned to products of 10 neuropeptide genes (allatostatins A, B, C, SIFamide, allatotropin, FMRFamide-related peptides [myosuppressin, short neuropeptides F, extended FMRFamides], crustacean cardioactive peptide, tachykinin-related peptides). In a second step, a combination of immunocytochemistry and mass spectrometric profiling of defined AL compartments was used to reveal the spatial distribution of neuropeptide-containing cells. Finally, we demonstrated the feasibility of MALDI-TOF mass spectrometric profiling of single AL neurons, which is an important precondition for combining electrophysiology with peptide profiling at the single-cell level.