Toward a single‐cell‐based analysis of neuropeptide expression in Periplaneta americana antennal lobe neurons

Abstract

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.

Choline acetyltransferase‐like immunoreactivity in a physiologically distinct subtype of olfactory nonspiking local interneurons in the cockroach (periplaneta americana)

“…Additional LN types do exist that are neither GABAergic nor cholinergic, and the chemical identity of these subtypes remains to be uncovered. Previous immunocytochemical and mass spectrometric studies have shown that AL neurons can contain a variety of potential neurotransmitters and neuromodulators (Loesel and Homberg, ; Nässel and Winther, ; for reviews see Homberg, ; Homberg and Müller, ; Neupert et al, ). Overlapping expression patterns also suggested that classical transmitters might colocalize with potential neuromodulators such as biogenic amines (Dacks et al, , , ; Ignell, ; Sachse et al, ) and peptides (Berg et al, ; Nässel and Homberg, ; Nässel and Winther, ; Utz et al, ).…”

“…To understand precisely the physiological role of potential transmitters and neuromodulators in the AL, it is essential to match the different AL neuron types with their biochemical profiles by combining electrophysiological recordings with immunohistochemical studies and/or single‐cell mass spectrometry (Neupert et al, ). The marked physiological differences between LN types imply direct consequences for their computational capacity and synaptic output kinetics, and thus their functional role in the olfactory circuit.…”

The neuropeptide SIFamide in the brain of three cockroach species

“…The double‐label experiments allowed for discrimination of branches of individual SIFamide‐ir neurons among the meshwork of intermingled neurites originating from the different cell groups. With matrix‐assisted laser desorption/ionization–time of flight (MALDI‐TOF) mass spectrometry (MS), it was shown that the amino acid sequences of SIFamides from R. maderae and T. petiveriana are identical to SIFamide of P. americana , which confirmed the conserved nature of this neuropeptide in insects (Neupert et al, ), whereas behavioral data from R. maderae demonstrated a function as an input factor into the circadian clockwork.…”

Peptidomics for the discovery and characterization of neuropeptides and hormones

“…Detecting multiple predicted peptides by their molecular mass often is sufficient to assign peptides to a certain prohormone, in the same way as the peptide mass fingerprint method is widely used in proteomics for protein identification. This approach allows matching of the individual cell peptide profiles to electrophysiological activity [72], localization of molecular probes [73], or both [74, 75]. A unique two-step strategy hyphenates MS to targeted chemical analysis of immunocytochemical-selected peptidergic neurons containing selected biomarkers of interest [76].…”