Dorsal Unpaired Median Neurons Research Articles

Flupyradifurone activates DUM neuron nicotinic acetylcholine receptors and stimulates an increase in intracellular calcium through the ryanodine receptors

Insect neuronal nicotinic acetylcholine receptors (nAChRs) are transmembrane receptors that play a key role in the development and synaptic plasticity of both vertebrates and invertebrates, and are considered to be major targets of several insecticides. We used dorsal unpaired median (DUM) neurons, which are insect neurosecretory cells, to explore what type of nAChRs are involved in flupyradifurone's (FLU) mode of action, and to study the role of calcium release from intracellular stores in this process. Using whole-cell patch-clamp and fura-2-AM calcium imaging techniques, we found that inhibition of IP3Rs through application of 2-APB reduced FLU inward currents, but did not affect the intracellular calcium release induced by FLU. In contrast, inhibition of RyRs using ryanodine, led to reduction of intracellular calcium increase following FLU pulse application. These results suggested that FLU inward currents are likely due to a combination of the direct effects of FLU on DUM neuron nAChRs and the subsequent calcium release from RyRs.

The complex neurochemistry of the cockroach antennal heart

The innervation of the antennal heart of the cockroach Periplaneta americana was studied with immunocytochemical techniques on both the light and electron microscopic levels. The antennal heart is innervated by two efferent systems, both using one biogenic amine in combination with neuropeptides. In one, we found co-localization of serotonin with proctolin and allatostatin. These fibers most likely originate from paired neurons located in the suboesophageal ganglion. In the second system, we found octopamine co-localized with the short neuropeptide F. The source of this second system is dorsal unpaired median (DUM) neurons, also located in the suboesophageal ganglion. The possible effects of these neuromediators on different targets are discussed.

The calcium-calmodulin-dependent protein kinase kinase inhibitor, STO-609, inhibits nicotine-induced currents and intracellular calcium increase in insect neurosecretory cells.

Insect neuronal nicotinic acetylcholine receptors (nAChRs) are transmembrane receptors that play a key role in the development and synaptic plasticity of both vertebrates and invertebrates and are considered to be major targets of neonicotinoid insecticides. We used dorsal unpaired median (DUM) neurons, which are insect neurosecretory cells, in order to explore the intracellular mechanisms leading to the regulation of insect neuronal nAChRs in more detail. Using whole-cell patch-clamp and fura-2AM calcium imaging techniques, we found that a novel CaMKK/AMPK pathway could be involved in the intracellular regulation of DUM neuron nAChRs. The CaMKK selective inhibitor, STO, reduced nicotinic current amplitudes, and strongly when co-applied with α-Bgt. Interestingly, intracellular application of the AMPK activator, A-76, prevented the reduction in nicotine-induced currents observed in the presence of the AMPK inhibitor, dorsomorphin. STO prevented the increase in intracellular calcium induced by nicotine, which was not dependent on α-Bgt. Currents induced by 1 mM LMA, a selective activator of nAChR2, were reduced under bath application of STO, and mecamylamine, which blocked nAChR2 subtype, inhibited the increase in intracellular calcium induced by LMA. These findings provide insight into potential complex mechanisms linked to the modulation of the DUM neuron nAChRs and CaMKK pathway.

Optical Regulation of GABA Receptor by Visible Light via Azobenzene-Phenylpyrazole.

Photopharmacology provides superior temporal and spatial resolution for the study of biological functions of γ-aminobutyric acid receptors (GABARs), and photopharmacology based on GABARs has significantly advanced the research of neurons and cells. Herein, we reported the azobenzene-phenylpyrazole (ABPs) for controlling the function of GABARs by light. The insecticidal activity of ABPs against Aedes albopictus larvae was different before and after light illumination. ABP3 (1 μM) could induce depolarization of dorsal unpaired median neurons and enable the real-time photoregulation of mosquito larval behavior. An electrophysiological experiment indicated that ABP3 had different inhibitory effects on GABARs before and after illumination. ABPs realized optical control of GABARs utilizing visible light, providing valuable supplements to the existing GABAR photocontrol tools.

Calmidazolium induces a decrease in nicotine-induced currents and intracellular calcium levels after pulse application of nicotine onto insect neurosecretory cells

Dorsal unpaired median (DUM) neurons, are a class of insect neurosecretory cells, which are involved in the control of several functions, such as excretion and reproduction, or the release of neurohormones. Previous studies demonstrated that they express different nicotinic acetylcholine receptor subtypes, in particular α-bungarotoxin-insensitive receptors, with nAChR1 and nAChR2 subtypes. Here, we demonstrated that pulse application of 1 mM nicotine (300 ms pulse duration) induced inward currents which were reduced under bath application of 15 µM calmidazolium, a calmodulin inhibitor. Bath application of 0.5 µM α-bungarotoxin had no effect on calmidazolium action, suggesting that it could have an indirect effect through α-bungarotoxin-insensitive receptors. Indeed, nicotine-evoked currents were reduced by 10 µM d-tubocurarine, and completely blocked by 5 µM mecamylamine, which affected nAChR1 and nAChR2 subtypes, respectively. Our results demonstrated that nAChR2 subtypes are involved in the indirect effect of calmidazolium. Moreover, we found that this calmidazolium effect was associated to a strong reduction in intracellular calcium levels after pulse application of 1 mM nicotine. Thus, compared to previous studies on mammalian cells, calmidazolium did not cause an increase in intracellular calcium levels in DUM neurons, suggesting that different calcium mechanisms are involved in the calmidazolium effect.

Azobenzene-Avermectin B1a Derivatives for Optical Modulation of Insect Behaviors.

Naturally occurring avermectins are allosteric modulators of glutamate-gated chloride channels (GluCls) and possess exceptionally potent anthelmintic, acaricidal, and insecticidal activities. Here, we develop photoswitchable azobenzene-avermectin (ABAVM) derivatives, which can be photoactivated upon ultraviolet irradiation. After illumination, the best compound p-AB4″AVM had a 1.88-fold and 2.74-fold insecticidal activity enhancement toward Culex pipiens pallens and Mythimna separata larvae, respectively. p-AB4″AVM allows for optical regulation of dorsal unpaired median neuron membrane potential with a 2.15-fold fluorescence intensity decrease after illumination. p-AB4″AVM and p-AB5AVM enable optical modulation of the behavioral response of Culex pipiens pallens larvae with 1- and 4-fold reduced mobility upon irradiation, respectively. The ABAVMs could be used to reversibly manipulate GluCls with light and may be useful for the mechanistic study of macrocyclic lactone insecticides.

Azobenzene-Semicarbazone Enables Optical Control of Insect Sodium Channels and Behavior.

Photopharmacology uses molecular photoswitches to establish control over the action of bioactive molecules. The application of photopharmacology in the research of invertebrate sodium channels has not been investigated. Here we report several photochromic ligands of metaflumizone. One ligand, termed ABM04, underwent reversible trans-cis isomerization under ultraviolet or blue light irradiation. cis-ABM04 had excellent larvicidal activity against mosquito larvae with an LC50 value of 4.39 μM and showed insecticidal activity against Mythimna separata with an LC50 value of 7.19 μM. However, trans-ABM04 was not found to have biological activity. ABM04 (10 μM) can induce depolarization of dorsal unpaired median neurons and enable the real-time photoregulation of mosquito larval behavior. The precise regulation of invertebrate sodium channels is realized for the first time, which provides a new strategy for the basic and accurate research of invertebrate sodium channels.

Azobenzene-isoxazoline as photopharmacological ligand for optical control of insect GABA receptor and behavior.

Photopharmacology is a fast-growing photonics-based technology, which realizes the high-resolution regulation of drugs in time and space through light. The purpose of this research was to introduce photochromic groups into the isoxazoline structure to realize the regulation of γ-Aminobutyric acid receptors (GABARs) targeting insect behavior. Azobenzene-Fluralaner analogs ABF02, ABF03 and ABF04 have been proven to have larvicidal activity against mosquito larvae. Cis-ABF03 had excellent larvicidal activity against mosquito larvae with a median lethal concentration (LC50 ) value of 1.63, which was better than that of trans-ABF03 (LC50 =3.90). In particular, ABF03 also showed insecticidal activity against Mythimna separata. Further experiments showed that ABF03 (1μm) induced depolarization of dorsal unpaired median neurons after ultraviolet light irradiation, enhanced affinity to the receptor, and blocked ligand-gated chloride channels of GABARs. ABF03 (1μm) realized the real-time photoregulation of the behavior of mosquito larvae, which indicated that the synthesized ligand can complete the binding and off-target action of drugs and targets in vivo under the regulation of light. Azobenzene-Isoxazoline as photopharmacological ligand was synthesized and evaluated for optical control of insect GABARs and behavior for the first time. ABF03 completed the differential regulation of cockroach neurons and the real-time reversible regulation of insect behavior. The establishment of photochromic ligands provides a new strategy for basic and convenience-oriented research on GABARs in invertebrates. © 2021 Society of Chemical Industry.

Neuromodulation Can Be Simple: Myoinhibitory Peptide, Contained in Dedicated Regulatory Pathways, Is the Only Neurally-Mediated Peptide Modulator of Stick Insect Leg Muscle.

In the best studied cases (Aplysia feeding, crustacean stomatogastric system), peptidergic modulation is mediated by large numbers of peptides. Furthermore, in Aplysia, excitatory motor neurons release the peptides, obligatorily coupling target activation and modulator release. Vertebrate nervous systems typically contain about a hundred peptide modulators. These data have created a belief that modulation is, in general, complex. The stick insect leg is a well-studied locomotory model system, and the complete stick insect neuropeptide inventory was recently described. We used multiple techniques to comprehensively examine stick insect leg peptidergic modulation. Single-cell mass spectrometry (MS) and immunohistochemistry showed that myoinhibitory peptide (MIP) is the only neuronal (as opposed to hemolymph-borne) peptide modulator of all leg muscles. Leg muscle excitatory motor neurons contained no neuropeptides. Only the common inhibitor (CI) and dorsal unpaired median (DUM) neuron groups, each neuron of which innervates a group of functionally-related leg muscles, contained MIP. We described MIP transport to, and receptor presence in, one leg muscle, the extensor tibiae (ExtTi). MIP application reduced ExtTi slow fiber force and shortening by about half, increasing the muscle's ability to contract and relax rapidly. These data show neuromodulation does not need to be complex. Excitation and modulation do not need to be obligatorily coupled (Aplysia feeding). Modulation does not need to involve large numbers of peptides, with the attendant possibility of combinatorial explosion (stomatogastric system). Modulation can be simple, mediated by dedicated regulatory neurons, each innervating a single group of functionally-related targets, and all using the same neuropeptide.SIGNIFICANCE STATEMENT Vertebrate and invertebrate nervous systems contain large numbers (around a hundred in human brain) of peptide neurotransmitters. In prior work, neuropeptide modulation has been complex, either obligatorily coupling postsynaptic excitation and modulation, or large numbers of peptides modulating individual neural networks. The complete stick insect neuropeptide inventory was recently described. We comprehensively describe here peptidergic modulation in the stick insect leg. Surprisingly, out of the large number of potential peptide transmitters, only myoinhibitory peptide (MIP) was present in neurons innervating leg muscles. Furthermore, the peptide was present only in dedicated regulatory neurons, not in leg excitatory motor neurons. Peptidergic modulation can thus be simple, neither obligatorily coupling target activation and modulation nor involving so many peptides that combinatorial explosion can occur.

Azobenzene-diamides as Photopharmacological Ligands for Insect Ryanodine Receptor.

Photoresponsive ligands are powerful tool compounds for studying receptor function with spatiotemporal resolution. However, to the best of our knowledge, such a ligand is not available for the ryanodine receptor (RyR). Herein, we present a photochromic ligand (PCL) for insect RyR by decorating chlorantraniliprole (CHL) with photoswitchable azobenzene (AB). We demonstrated that one potent ligand, named ABCHL13, shows light-induced reversible trans-cis isomerization and 3.5-fold insecticidal activity decrease toward oriental armyworm (Mythimna separata) after UV-light irradiation, that is, trans-ABCH13 has higher activity than the cis-ABCH13. ABCHL13 enables optical control over intracellular Ca2+ release in dorsal unpaired median (DUM) neurons of M. separata and American cockroach (Periplaneta americana) and cardiac function of P. americana. Our results provide a first photopharmacological toolkit that is applicable to light-dependent regulation of RyR and heart beating.

Auditory DUM neurons in a bush-cricket: inhibited inhibitors

Thoracic ganglia of many hearing insects house the first level of auditory processing. In bush-crickets, the largest population of local auditory neurons in the prothoracic processing centre are dorsal unpaired median (DUM) neurons. It has been suggested that DUM neurons are inhibitory using γ-aminobutyric acid (GABA) as transmitter. Immunohistochemistry reveals a population of about 35–50 GABA-positive somata in the posterior medial cluster of the prothoracic ganglion. Only very few small somata in this cluster remain unstained. At least 10 neurites from 10 neurons can be identified. Intracellularly stained auditory DUM neurons have their soma in the cluster of median GABA positive cells and most of them exhibit GABA-immunoreactivity. Responses of certain DUM neurons show obvious signs of inhibition. Application of picrotoxin (PTX), a chloride-channel blocker in insects, changes the responses of many DUM neurons. They become broader in frequency tuning and broader or narrower in temporal pattern tuning. Furthermore, inhibitory postsynaptic potentials (IPSPs) may be replaced by excitatory postsynaptic potentials. Loss of an IPSP in the rising graded potential after PTX-application leads to a significant reduction of first-spike latency. Therefore, auditory DUM neurons receive effective inhibition and are the best candidates for inhibition in DUM neurons and other auditory interneurons.

Descending octopaminergic neurons modulate sensory-evoked activity of thoracic motor neurons in stick insects.

Neuromodulatory neurons located in the brain can influence activity in locomotor networks residing in the spinal cord or ventral nerve cords of invertebrates. How inputs to and outputs of neuromodulatory descending neurons affect walking activity is largely unknown. With the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunohistochemistry, we show that a population of dorsal unpaired median (DUM) neurons descending from the gnathal ganglion to thoracic ganglia of the stick insect Carausius morosus contains the neuromodulatory amine octopamine. These neurons receive excitatory input coupled to the legs' stance phases during treadmill walking. Inputs did not result from connections with thoracic central pattern-generating networks, but, instead, most are derived from leg load sensors. In excitatory and inhibitory retractor coxae motor neurons, spike activity in the descending DUM (desDUM) neurons increased depolarizing reflexlike responses to stimulation of leg load sensors. In these motor neurons, descending octopaminergic neurons apparently functioned as components of a positive feedback network mainly driven by load-detecting sense organs. Reflexlike responses in excitatory extensor tibiae motor neurons evoked by stimulations of a femur-tibia movement sensor either are increased or decreased or were not affected by the activity of the descending neurons, indicating different functions of desDUM neurons. The increase in motor neuron activity is often accompanied by a reflex reversal, which is characteristic for actively moving animals. Our findings indicate that some descending octopaminergic neurons can facilitate motor activity during walking and support a sensory-motor state necessary for active leg movements.NEW & NOTEWORTHY We investigated the role of descending octopaminergic neurons in the gnathal ganglion of stick insects. The neurons become active during walking, mainly triggered by input from load sensors in the legs rather than pattern-generating networks. This report provides novel evidence that octopamine released by descending neurons on stimulation of leg sense organs contributes to the modulation of leg sensory-evoked activity in a leg motor control system.

Temporal processing properties of auditory DUM neurons in a bush-cricket

Insects with ears process sounds and respond to conspecific signals or predator cues. Axons of auditory sensory cells terminate in mechanosensory neuropils from which auditory interneurons project into (brain-) areas to prepare response behaviors. In the prothoracic ganglion of a bush-cricket, a cluster of local DUM (dorsal unpaired median) neurons has recently been described and constitutes a filter bank for carrier frequency. Here, we demonstrate that these neurons also constitute a filter bank for temporal patterns. The majority of DUM neurons showed pronounced phasic-tonic responses. The transitions from phasic to tonic activation had different time constants in different DUM neurons. Time constants of the membrane potential were shorter in most DUM neurons than in auditory sensory neurons. Patterned stimuli with known behavioral relevance evoked a broad range of responses in DUM neurons: low-pass, band-pass, and high-pass characteristics were encountered. Temporal and carrier frequency processing were not correlated. Those DUM neurons producing action potentials showed divergent processing of temporal patterns when the graded potential or the spiking was analyzed separately. The extent of membrane potential fluctuations mimicking the patterned stimuli was different between otherwise similarly responding neurons. Different kinds of inhibition were apparent and their relevance for temporal processing is discussed.

Mode of action of sulfoxaflor on α-bungarotoxin-insensitive nAChR1 and nAChR2 subtypes: Inhibitory effect of imidacloprid

Cockroach neurosecretory cells, dorsal unpaired median (DUM) neurons, express two distinct α-bungarotoxin-insensitive nicotinic acetylcholine receptor subtypes, nAChR1 and nAChR2 which are differently sensitive to the neonicotinoid insecticides and intracellular calcium pathways. The aim of this study is to determine whether sulfoxaflor acts as an agonist of nAChR1 and nAChR2 subtypes. We demonstrated that 1 mM sulfoxaflor induced high current amplitudes, compared to acetylcholine, suggesting that it was a full agonist of DUM neuron nAChR subtypes. Sulfoxaflor evoked currents were not inhibited by the nicotinic acetylcholine receptor antagonist d-tubocurarine (dTC) which reduced nAChR1. But, sulfoxaflor evoked currents were reduced in the presence of 5 μM mecamylamine which is known to reduce nAChR2 subtype. Interestingly, when 1 μM imidacloprid was added in the extracellular solution, sulfoxaflor-induced currents were significantly suppressed. Moreover, when extracellular calcium concentration was increased, bath application of 1 μM imidacloprid partially reduced sulfoxaflor activated currents when nAChR1 was inhibited with 20 μM dTC and completely suppressed sulfoxaflor currents when nAChR2 was inhibited with 5 μM mecamylamine. Our data demonstrated therefore that sulfoxaflor activates both nAChR1 and nAChR2 subtypes.

Identification and functional study of three nAChR regulators, ubiquilin-1, PICK1, and CRELD2, in Locusta migratoria manilensis dorsal unpaired median neurons.

Insect nicotinic acetylcholine receptors (nAChRs) are not only important neurotransmitter receptors but also effective insecticide targets. The regulation of nAChRs has been mainly studied in vertebrates, especially in mammals. Here, two types of nAChRs were found present in the locust Locusta migratoria manilensis dorsal unpaired median (DUM) neurons, α-bungarotoxin (α-Bgt)-sensitive nAChRs and α-Bgt-resistant nAChRs, responding to acetylcholine (ACh) at different concentrations. The homologs to three mammalian nAChR regulators, ubiquilin-1, CRELD2 (cysteine-rich with EFG-like domain 2), and PICK1 (protein interacting with PRKCA 1), were characterized in L.migratoria, and their functions on regulating native nAChRs were investigated via RNAi followed by membrane potential measurement with DiBAC4 (3) and agonist-evoked macroscopic current recording in cultured L.migratoria DUM neurons. Ubiquilin-1 and PICK1 negatively regulated nAChRs because silencing of ubiquilin-1 and PICK1 both resulted in increased membrane potential and increased inward currents in DUM neurons, while CRELD2 positively regulated nAChRs as decreased membrane potential and inward currents were observed in DUM neurons. In addition, ubiquilin-1 regulated both α-Bgt-sensitive and α-Bgt-resistant types of nAChRs whereas PICK1 and CRELD2 regulated only the α-Bgt-resistant nAChRs. The present study broadened our understanding on the regulation of insect nAChRs and will benefit pest management given the important role of nAChRs in insect neurons and insecticide science. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Permethrin enhances the agonist activity of dinotefuran on insect cholinergic synaptic transmission and isolated neurons

Insect resistance mechanisms against pesticides lead to the development and the search of new pesticide combinations in order to delay the resistance. The combination of neonicotinoids with pyrethroids was currently proposed but the mode of action of these compounds at synaptic and extrasynaptic levels needs to be further explored. In the present study, we evaluated the effect of the combination of two insecticides, permethrin and dinotefuran, on cockroach cholinergic synaptic transmission and on isolated cell bodies. We first found that combination of 5 μM permethrin and dinotefuran enhances depolarization of the sixth abdominal ganglion compared to dinotefuran alone, without an inhibition of the spontaneous activity. However, a pretreatment with 1 μM dinotefuran or permethrin before bath application of the mixture inhibits the ganglionic depolarization. Compared to permethrin, 1 μM dinotefuran induces a persistent enhancement of spontaneous activity. Interestingly, at extrasynaptic level, using dorsal unpaired median neurons and Kenyon cells, we found that combination of both 1 μM dinotefuran and permethrin resulted in an increase of the mixture-induced current amplitudes. Pretreatment with 1 μM dinotefuran strongly decreases the currents whereas permethrin induces a time-dependent inhibition. These data demonstrate that the combination of dinotefuran and permethrin enhances the effect of dinotefuran.

Purification and Characterization of a Novel Insecticidal Toxin, μ-sparatoxin-Hv2, from the Venom of the Spider Heteropoda venatoria.

The venom of the spider Heteropoda venatoria produced lethal effect to cockroaches as reported in our previous study, and could be a resource for naturally-occurring insecticides. The present study characterized a novel cockroach voltage-gated sodium channels (NaVs) antagonist, μ-sparatoxin-Hv2 (μ-SPRTX-Hv2 for short), from this venom. μ-SPRTX-Hv2 is composed of 37 amino acids and contains six conserved cysteines. We synthesized the toxin by using the chemical synthesis method. The toxin was lethal to cockroaches when intraperitoneally injected, with a LD50 value of 2.8 nmol/g of body weight. Electrophysiological data showed that the toxin potently blocked NaVs in cockroach dorsal unpaired median (DUM) neurons, with an IC50 of 833.7 ± 132.2 nM, but it hardly affected the DUM voltage-gated potassium channels (KVs) and the DUM high-voltage-activated calcium channels (HVA CaVs). The toxin also did not affect NaVs, HVA CaVs, and Kvs in rat dorsal root ganglion (DRG) neurons, as well as NaV subtypes NaV1.3–1.5, NaV1.7, and NaV1.8. No envenomation symptoms were observed when μ-SPRTX-Hv2 was intraperitoneally injected into mouse at the dose of 7.0 μg/g. In summary, μ-SPRTX-Hv2 is a novel insecticidal toxin from H. venatoria venom. It might exhibit its effect by blocking the insect NaVs and is a candidate for developing bioinsecticide.

Auditory DUM neurons in a bush-cricket: A filter bank for carrier frequency.

In bush-crickets the first stage of central auditory processing occurs in the prothoracic ganglion. About 15 to 50 different auditory dorsal unpaired median neurons (DUM neurons) exist but they have not been studied in any detail. These DUM neurons may be classified into seven different morphological types, although, there is only limited correlation between morphology and physiological responses. Ninety seven percent of the stained neurons were local, 3% were intersegmental. About 90% project nearly exclusively into the auditory neuropile, and 45% into restricted areas therein. Lateral extensions overlap with the axons of primary auditory sensory neurons close to their branching point. DUM neurons are typically tuned to frequencies covering the range between 2 and 50 kHz and thereby may establish a filter bank for carrier frequency. Less than 10% of DUM neurons have their branches in adjacent and more posterior regions of the auditory neuropile and are mostly tuned to low frequencies, less sensitive than the other types and respond to vibration. Thirty five percent of DUM show indications of inhibition, either through reduced responses at higher intensities, or by hyperpolarizing responses to sound. Most DUM neurons produce phasic spike responses preferably at higher intensities. Spikes may be elicited by intracellular current injection. Preliminary data suggest that auditory DUM neurons have GABA as transmitter and therefore may inhibit other auditory interneurons. From all known local auditory neurons, only DUM neurons have frequency specific responses which appear suited for local processing relevant for acoustic communication in bush crickets.

Octopaminergic innervation and a neurohaemal release site in the antennal heart of the locust Schistocerca gregaria.

A detailed account is given by the octopaminergic innervation of the antennal heart in Schistocerca gregaria using various immunohistochemical methods. Anterograde axonal filling illustrates the unilateral innervation on the medial ventral surface of the pumping muscle of the antennal heart via the paired corpora cardiaca nerve III. In addition, antibody staining revealed that ascending axons of this nerve terminate at the ampullae of the antennal heart forming synaptoid structures and extensive neurohaemal release sites. Due to the innervation by two dorsal unpaired median neurons, the presence of the biogenic amines octopamine and tyramine could be visualized by immunocytochemistry in an insect antennal heart for the first time. The data suggest that tyramine acts as a precursor and not purely as an independent transmitter. While the octopaminergic fibers innervating the pumping muscle of the antennal heart indicate a cardioregulatory role, we conclude that octopamine released from the neurohaemal area is pumped into the antennae and an involvement in the modulation of the antennal sensory sensitivity is discussed.

Subchronic exposure to sublethal dose of imidacloprid changes electrophysiological properties and expression pattern of nicotinic acetylcholine receptor subtypes in insect neurosecretory cells

Neonicotinoids are the most important class of insecticides used in agriculture over the last decade. They act as selective agonists of insect nicotinic acetylcholine receptors (nAChRs). The emergence of insect resistance to these insecticides is one of the major problems, which limit the use of neonicotinoids. The aim of our study is to better understand physiological changes appearing after subchronic exposure to sublethal doses of insecticide using complementary approaches that include toxicology, electrophysiology, molecular biology and calcium imaging. We used cockroach neurosecretory cells identified as dorsal unpaired median (DUM) neurons, known to express two α-bungarotoxin-insensitive (α-bgt-insensitive) nAChR subtypes, nAChR1 and nAChR2, which differ in their sensitivity to imidacloprid. Although nAChR1 is sensitive to imidacloprid, nAChR2 is insensitive to this insecticide. In this study, we demonstrate that subchronic exposure to sublethal dose of imidacloprid differentially changes physiological and molecular properties of nAChR1 and nAChR2. Our findings reported that this treatment decreased the sensitivity of nAChR1 to imidacloprid, reduced current density flowing through this nAChR subtype but did not affect its subunit composition (α3, α8 and β1). Subchronic exposure to sublethal dose of imidacloprid also affected nAChR2 functions. However, these effects were different from those reported on nAChR1. We observed changes in nAChR2 conformational state, which could be related to modification of the subunit composition (α1, α2 and β1). Finally, the subchronic exposure affecting both nAChR1 and nAChR2 seemed to be linked to the elevation of the steady-state resting intracellular calcium level. In conclusion, under subchronic exposure to sublethal dose of imidacloprid, cockroaches are capable of triggering adaptive mechanisms by reducing the participation of imidacloprid-sensitive nAChR1 and by optimizing functional properties of nAChR2, which is insensitive to this insecticide.