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A morphofunctional investigation of the different neuronal subpopulations projecting through each of the nerves IV-VI emerging bilaterally from the terminal abdominal ganglion (TAG) was correlated with the octopaminergic activity in the ganglion that controls the ovipositor movements associated with calling behavior in the female gypsy moth Lymantria dispar. Tetramethylrodamine-dextran backfills from nerve stumps resulted in a relatively low number of TAG projections, ranging from 12 to 13 for nerve pair IV, 12 to 14 for nerve pair V, and 8 to 9 for nerve pair VI. Furthermore, as assessed by electrophysiological recordings, a number of fibers within each of these nerves displays spontaneous tonic activity, also when the ganglion is fully disconnected from the ventral nerve cord (VNC). Octopamine (OA) applications to the TAG strongly enhanced the activity of these nerves, either by increasing the firing rate of a number of spontaneously firing units or by recruiting new ones. This octopaminergic activity affected calling behavior, and specifically the muscle activity leading to cycling extensions of the intersegmental membrane (IM) between segments VIII and IX (ovipositor). Our results indicate that in the female gypsy moth the octopaminergic neural activity of the TAG is coupled with extensions and retractions of IM for the purpose of releasing pheromone, where motor units innervated by nerve pair IV appear antagonistic with respect to those innervated by nerve pair V.

Findings from studies using animal models expressing amyotrophic lateral sclerosis (ALS) mutations in RNA-binding proteins, such as Transactive Response DNA-binding protein-43 (TDP-43), indicate that this protein, which is involved in multiple functions, including transcriptional regulation and pre-mRNA splicing, represents a key candidate in ALS development. This study focuses on characterizing, in a Drosophila genetic model of ALS (TDP-43), the effects of Mucuna pruriens (Mpe) and Withania somnifera (Wse). Electrophysiological and behavioural data in TDP-43 mutant flies revealed anomalous locomotion (i.e. impaired climbing with unexpected hyperactivity) and sleep dysregulation. These features, in agreement with previous findings with a different ALS model, were at least partially, rescued by treatment with Mpe and Wse. In addition, electrophysiological recordings from dorsal longitudinal muscle fibers and behavioral observations of TDP-43 flies exposed to the volatile anaesthetics, diethyl ether or chloroform, showed paradoxical responses, which were normalized upon Mpe or Wse treatment. Hence, given the involvement of some potassium channels in the effects of anaesthetics, our results also hint toward a possible dysregulation of some potassium channels in the ALS-TDP-43 Drosophila model, that might shed new light on future therapeutic strategies pertaining to ALS.

Phote-HrTH (Phormia terraenovae hypertrehalosemic hormone) has been demonstrated in the Diptera to be involved in flight metabolism, reproduction, and diapause. Each of these events needs the hormone's action and requirement for carbohydrates is the common denominator. In Diptera, carbohydrates are taken up during feeding by action of the cibarial pump and are then stored in the crop. Using adult Phormia regina, both a bioassay and electrophysiological recordings show that Phote-HrTH slows down or inhibits the crop lobe muscles (P5) and, at the same time, stimulates the muscles of the pump 4 (P4) involved in pushing fluids out of the crop and up into the midgut for digestion. The EC50 for P4 was in the nanomolar range while the IC50 for P5 was 1.4-75.1 pM. The effect of Phote-HrTH on P4/5 suggests that the peptide is important in coordinating the two pumps, which are involved in moving carbohydrates up into the midgut for digestion. The adult crop organ is an essential storage organ for carbohydrates and now should be considered an important structure capable of delivering nutrients to the midgut for digestion.

This study showed that in adult Drosophila melanogaster, the type of sugar-either present within the crop lumen or in the bathing solution of the crop-had no effect on crop muscle contraction. What is important, however, is the volume within the crop lumen. Electrophysiological recordings demonstrated that exogenous applications of serotonin on crop muscles increases both the amplitude and the frequency of crop contraction rate, while adipokinetic hormone mainly enhances the crop contraction frequency. Conversely, octopamine virtually silenced the overall crop activity. The present study reports for the first time an analysis of serotonin effects along the gut-brain axis in adult D. melanogaster. Injection of serotonin into the brain between the interocellar area shows that brain applications of serotonin decrease the frequency of crop activity. Based on our results, we propose that there are two different, opposite pathways for crop motility control governed by serotonin: excitatory when added in the abdomen (i.e., directly bathing the crop) and inhibitory when supplied within the brain (i.e., by injection). Finally, our results point to a double brain-gut serotonergic circuitry suggesting that not only the brain can affect gut functions, but the gut can also affect the central nervous system. On the basis of our results, and data in the literature, a possible mechanism for these two discrete serotonergic functions is suggested.

Bioassays and electrophysiological recordings were conducted in the adult blowfly Phormia regina to provide new insights into the regulatory mechanisms governing the crop filling and emptying processes of the supercontractile crop muscles. The cibarial pump drives ingestion. Simultaneous multisite extracellular recordings show that crop lobe (P5) distension during ingestion of a 4.7 μl sugar meal does not require muscle activity by any of the other pumps of the system. Conversely, pumping of fluids toward the anterior of the crop system during crop emptying is brought about by active muscle contraction, in the form of a highly coordinated peristaltic wave starting from P5 and progressively propagating to P6, P4 and P3 pumps, with P5 contracting with a frequency about 3.4 times higher than the other pumps. The crop contraction rate is also modulated by hemolymph-borne factors such as sugars, through ligand recognition at a presumptive receptor site rather than by an osmotic effect, as assessed by both behavioural and electrophysiological experiments. In this respect, sugars of equal osmolarity produce different effects, glucose being inhibitory and mannose ineffective for crop muscles, while trehalose enhances crop activity. Finally, voltage and current clamp experiments show that the muscle action potentials (mAPs) at the P4 pump are sustained by a serotonin-sensitive calcium conductance. Serotonin enhances calcium entry into the muscle cells and this could lead, as an indirect modulatory effect, to activation of a Ca2+-activated K+ conductance (IK(Ca)), which sustains the following mAP repolarization phase in such a way that further mAPs can be generated early and the frequency consequently increased.

The present study was aimed at characterizing the effects of Withania somnifera (Wse) and Mucuna pruriens (Mpe) on a Drosophila melanogaster model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse- but not in Mpe-treated hSOD1 mutants. Hence, given the role of inflammation in the development of ALS, the high translational impact of the model, the known anti-inflammatory properties of these extracts, and the viability of their clinical use, these results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptoms.

Bioassays and electrophysiological recordings were conducted to determine the role of serotonin and calcium on the supercontractile pump muscles of the diverticulated crop of adult blowflies. Using in situ crop preparations, serotonin was found to significantly increase the rates of contractions of a specific pump in the crop wall, pump P4. The addition of the serotonin antagonist, mianserin, or calcium free saline, both significantly reduced the contraction rates of this pump. Recordings, using suction electrodes from pump P4, confirm the in situ bioassay data and show that serotonin promotes muscle activity in empty crops in which no pump activity is normally observed. Moreover, our data indicate the crucial role of extracellular calcium ions in crop pump contractile activity. These results provide new information on how the crop of adult dipterans is modulated and suggest that serotonin, possibly supplied by neurons in the thoracico-abdominal neural plexus, may be involved in modulating the pumping of crop contents into the midgut for digestion or triggering antiperistalsis from the foregut in the process known as regurgitation or 'bubbling'.

A number of oxaspiropentane derivatives (OXPs) were tested as potential (+)-disparlure analogues, with the aim of identifying any possible interaction of these compounds, be it additive, synergetic, or inhibitory, with the pheromone response in the male gypsy moth Lymantria dispar. As assessed by male electroantennograms, 2 OXPs, 2-decyl-1-oxaspiro[2.2]pentane (OXP-01) and 4-(1-oxaspiro[2.2]pent-2-yl)butan-1-ol (OXP-04), were found to be effective. OXP-01 had no stimulatory effect but strongly decreased the response to (+)-disparlure in a blend in a 1:1 ratio. By contrast, OXP-04 proved to be more stimulating than (+)-disparlure and also had an additive effect in the blend. Single-cell recordings from the sensilla trichoidea showed the activity of 2 cells, one of which responded to (+)-disparlure. OXP-01 reduced the stimulating effectiveness of pheromone by silencing the pheromone-responding unit when the 2 compounds were presented in blend, whereas OXP-04 mimicked the pheromone response, evidenced by exciting the pheromone-responding neuron when tested alone. Behavioral observations are in agreement with electrophysiological results.

Blowflies respond to sugars, salts, and water through the activation of specific chemoreceptor neurons in the labellar taste chemosensilla. These insects also detect deterrent stimuli, but identification of a specific “deterrent” chemoreceptor within their sensilla has been elusive. Here electrophysiological evidence is provided that the so-called “fifth” cell in taste chemosensilla of blowflies responds to deterrent compounds, such as quinine, amiloride, nicotine, and caffeine, which are also known to be bitter tasting for vertebrates. Therefore, comparison of behavioral and electrophysiological data, including crossadaptation analysis, suggests that the blowfly can detect “bitter” stimuli by activation of the “fifth” cell. A possible chemoreception mechanism is discussed.

The feeding of Aedes aegypti (L.) on blood is induced by the presence of phagostimulants: adenine nucleotides. Three chemoreceptive cells in the labral apical sensilla can distinguish the presence of adenine nucleotides depending on the other stimulus components. This work aims at correlating the sensory information arising from the labral apical sensilla with the feeding behavior in response to the same stimuli. The saline stimulating solution, containing adenine nucleotides, is modulated by changing one of the following components: salt concentration, buffer or pH. Cell 3 that responds to NaCl in a dose dependent manner seems to have another unique modality. The response of this cell is unaffected by ATP when the stimulating solution is NaCl buffered by NaHCO3. It responds at a higher spike frequency to the presence of ATP in a NaCl solution without NaHCO3. Thus in the presence of ATP Cell 3 detects whether the NaCl solution is buffered by NaHCO3. Both the blood feeding response and the sensory information from Cell 2 (which responds at high spike frequencies to the presence of ATP) are modulated by pH in a similar way. Both responses present a bi-modal response, with a major peak at pH 4.0 and a moderate peak at the most alkaline pH value tested.

The phagostimulants from the cellular fraction of blood induce gorging of Aedes aegypti (L.), and this process is enhanced by some plasma components. This project examines the responses of the labral apical chemoreceptors to plasma components enhancing phagostimulation. From the electrophysiological responses of the labral apical chemoreceptors four cells were identified by the waveform of their action potentials. Three of the cells (Cell 2, Cell 3 and Cell 4) responded in a dose dependent manner to NaCl. The responses of Cell 2 and Cell 3 to NaCl concentrations from 1 to 500 mmol/l can be described by a logarithmic equation. The response of Cell 2 to 150 mmol/l NaCl is modulated when a buffer is added. The magnitude of the modulation of the response is determined by the nature of the buffer: NaHCO3 inhibits while Na2HPO4 enhances the response. High osmotic pressure inhibits the response of Cell 4, regardless of how it is achieved. Cell 4 responds with a high frequency to the presence of l -alanine, the C-terminal amino acid of albumin, but shows a reduced response to the same concentration of albumin. From these results it can be concluded that labral apical chemoreceptors of A. aegypti are capable of detecting the plasma components involved in blood recognition.

Electrophysiological responses to NaCl in the concentration range 1–1000 mM were obtained from each of the 11 intermediate and 13 large labellar chemosensilla in the blowfly Protophormia terraenovae . Up to three of the four chemoreceptor cells associated with each chemosensillum responded to NaCl in both sensillum types; the spike discharges of two of these cells were analysed and cross-compared. Differences in chemoreceptor sensitivity and response pattern resulted between intermediate and large sensilla. Sensitivity differences were found, by means of the cluster analysis, within each sensillum type in relation to sensillar topology and are discussed in the light of a possible spatial contribution to the sensory coding process.

Electrophysiological recordings show that the hair-like sensilla on the tip of the labrum in Culex pipiens L. have neurones that respond to adenine nucleotides. Adenine nucleotides show the following descending order of stimulating effectiveness: ADP > ATP=AMP-PNP=AMP. Sensillar sensitivity to these adenine nucleotides were tested at the concentration range of 10 −5 –10 −3 M. These electrophysiological data are in good accordance with data reported from behavioural studies of this species.

The results of the present study demonstrate that series of gustatory stimulations can induce shortening of the cardiac cycle in the blowfly, Protopbormia terraenovae. This effect is due to a decrease in duration of one of the two phases which compose the cardiac cycle, that is the Fast‐phase. No changes in action potential frequency of either phase were evoked by stimulation. The data are discussed in terms of sensory‐induced changes in hemolymph circulation, which may function to optimize the insect's performance with respect to a state of alertness.

The electrophysiological response of labellar and tarsal chemosensilla in the blowfly Phormia regina was studied in response to a complex stimulus naturally encountered by flies such as sheep faeces, and to beef liver, a proteinaceous feeding source. Responses were investigated both before or after injection of clonidine, an octopamine agonist previously shown to enhance sucrose ingestion, while decreasing that of proteins. As assessed by single sensillum recordings, the four different chemosensory - "salt", "sugar", "deterrent" and "water" - cells were all activated by both stimuli, regardless of sex and sensillum type, the "sugar" one being in all cases the most sensitive to beef liver before clonidine injection. Clonidine treatment affected neither labellar nor tarsal sensitivity to sucrose. Conversely, clonidine-injected flies showed a significant increase in the activity of the "deterrent" cell to beef liver, thus accounting for a decrease in protein ingestion. This study for the first time provides evidence of a key role of a clonidine-sensitive peripheral taste sensitivity in down-regulation of protein ingestion in blowflies. Correlation between peripheral sensitivity and behavioural output is discussed.

The feeding of Aedes aegypti (L.) on blood and nectar is induced by phagostimulants: adenine nucleotides and sugars respectively. This work examines the responses of the four chemoreceptor cells in the labral apical sensilla to these phagostimulants. The apical chemoreceptors can detect the presence of adenine nucleotides. This part of the response is in good agreement with the gorging behavior. The output of the chemoreceptors cannot distinguish among different adenine nucleotides or among their concentrations (0.01–1 mmol/l), whereas gorging behavior is affected by the identity of adenine nucleotides and by their concentrations. Hence the gorging behavior cannot be driven by the output of these chemoreceptors alone. To the presence of adenine nucleotides Cell 2 was the only cell that responded with high frequencies, while the response of Cell 4 was almost abolished. The response of Cell 2 to ATP depended on the mosquito's physiological state. This dependence accorded well with the gorging behavior; Cell 2 responded with a higher frequency to ATP in the gorging state, than when not in a gorging state. The responses to sucrose and fructose constituted the only case recorded in which all these chemoreceptors failed to respond. This depression of response implies that other chemoreceptors must be present as sugar detectors.

In the attempt to gain more information on the mechanisms underlying bitter and/or sweet taste reception, we have investigated the responses of labellar chemosensilla in the blowfly Protophormia terraenovae to Na-saccharin, as compared to sweet stimuli (sucrose or fructose) and bitter stimuli (denatonium benzoate or amiloride). Electrophysiological and behavioral results indicate that the sweetener Na-saccharin inhibits the "sugar" cell in the labellar taste sensilla of the blowfly P. terraenovae. In multichoice preference tests, flies ingested more of the solutions containing sugar to those with sugar+Na-saccharin. This finding is in good agreement with the spike frequency reduction observed for the "sugar" cell activity. Analysis of the spike discharges also shows a positive dose-response for the "deterrent" cell following stimulation with Na-saccharin and denatonium benzoate. Flies drank any of the Na-saccharin solutions, regardless of their concentration, less than water, thus indicating a weak deterring effect on water drinking. The prevailing activation of the "deterrent" cell by stimulation with Na-saccharin is not directly coupled with a coherent behavioral output. Cross adaptation was found to occur between responses to Na-saccharin and denatonium benzoate or amiloride regardless of the order of adapting stimuli. In the case of sweet stimuli, cross adaptation occurred when the adapting stimulus was Na-saccharin, but it did not when the adapting stimuli were sucrose or fructose. Addition of Na-saccharin to both sugars significantly depressed the spike firing frequency, while an increase was observed with denatonium benzoate or amiloride.

The fruit fly Drosophila melanogaster (Dm) mutant for PTEN-induced putative kinase 1 (PINK1B9) gene is a powerful tool to investigate physiopathology of Parkinson's disease (PD). Using PINK1B9 mutant Dm we sought to explore the effects of Mucuna pruriens methanolic extract (Mpe), a L-Dopa-containing herbal remedy of PD. The effects of Mpe on PINK1B9 mutants, supplied with standard diet to larvae and adults, were assayed on 3-6 (I), 10-15 (II) and 20-25 (III) days old flies. Mpe 0.1% significantly extended lifespan of PINK1B9 and fully rescued olfactory response to 1-hexanol and improved climbing behavior of PINK1B9 of all ages; in contrast, L-Dopa (0.01%, percentage at which it is present in Mpe 0.1%) ameliorated climbing of only PINK1B9 flies of age step II. Transmission electron microscopy analysis of antennal lobes and thoracic ganglia of PINK1B9 revealed that Mpe restored to wild type (WT) levels both T-bars and damaged mitochondria. Western blot analysis of whole brain showed that Mpe, but not L-Dopa on its own, restored bruchpilot (BRP) and tyrosine hydroxylase (TH) expression to age-matched WT control levels. These results highlight multiple sites of action of Mpe, suggesting that its effects cannot only depend upon its L-Dopa content and support the clinical observation of Mpe as an effective medication with intrinsic ability of delaying the onset of chronic L-Dopa-induced long-term motor complications. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the properties of Mucuna pruriens for PD treatment.

Using a modified “grease electrode”, we recorded extracellular spike activity from either member of each of the six pairs of peripheral nerves of the terminal abdominal ganglion in the female gypsy moth. Our results show that the three most caudally located nerve pairs (nerve pairs IV, V and VI) carry most of the spike activity related to the ovipositor movements associated with calling. The spike trains from nerve pair VI presented a temporal pattern which correlated most closely to the extension and retraction of the ovipositor during calling. Transection of the ventral nerve cord anterior to the terminal ganglion stopped neither the spike activity nor the calling behaviour, but some slight changes in both the spike frequency patterns and the duration of ovipositor extension and retraction were detected. We conclude that the terminal abdominal ganglion exhibits a considerable degree of autonomy in its control of the calling behaviour in the female gypsy moth.

The effectiveness of labellar taste input in evoking the feeding response of labellar lobe spreading in Protophormia was greatly increased by injecting flies with clonidine or dl -octopamine. This effect was suppressed when either drug was administered together with yohimbine; when injected alone, the latter determined a strong decrease in the feeding responses. Drug treatment did not determine variations in labellar taste input or evoke spontaneous activity in the denervated muscle. These results suggest that the injected substances lower the feeding-related reflex threshold at the CNS level.