Peripheral and central interactions between sugar,water, and salt receptors of the blowfly, Phormia regina

The peripheral and central nervous interactions between the sugar, water, and salt receptors of the blowfly were investigated electrophysiologically by simultaneously recording from the labellar chemoreceptors and the extensor muscle of the haustellum. Simultaneous stimulation of two water receptors with 10 mM LiCl resulted in a motor response even though stimulating the same two sensilla separately with 10 mM LiCl did not. There was a linear decrease in the motor response to two sensilla stimulation as the salt concentration in the stimulating solution increased. Although stimulating two sensilla simultaneously with 200 mM NaCl gave no motor response, simultaneously stimulating two sensilla with 10 mM LiCl and a third with 200 mM NaCl gave a greater response than did two sensilla stimulation with 10 mM LiCl alone, indicating cross-channel summation between the water and salt receptors. Similarly, simultaneously stimulating one sensillum with 100 mM sucrose and another with 10 mM LiCl or 500 mM NaCl gave a larger response than did 100 mM sucrose stimulation alone. The cross-channel summation between the sugar and water receptors was not affected by feeding the flies water. A central excitatory state (CES) which previously had been demonstrated behaviourally was investigated. A stimulation of one sensillum with 10 mM LiCl which previously had been ineffective gave a motor response if proceeded by a stimulation with 1 M sucrose on another sensillum. The effect of the time interval between the sugar and water stimuli was tested, but for intervals of 100 msec to 4 sec no definite correlation was found. In addition, CES with respect to the sugar receptor was demonstrated. The motor response to stimulation of a single sensillum with 100 mM sucrose was enhanced by preceding it with 1 M sucrose stimulation of another sensillum. The motor response to stimulation of two water receptors with 10 mM LiCl was partially inhibited by simultaneously stimulating a third sensillum with 4 M NaCl. Inhibition was also seen when a single sensillum was stimulated with a mixture of 10 mM LiCl and 10 mM sucrose and an adjacent sensillum was simultaneously stimulated with 1 M NaCl. Behavioural experiments showing inhibition of CES by salt were confirmed. Interposing a salt stimulus of 4 M NaCl between the 1 M sucrose and 10 mM LiCl stimuli reduced but did not totally eliminate the motor response to 10 mM LiCl. The basis for peripheral control of the relative activities of the water and salt receptors is discussed. A model is proposed to account for all the receptor interactions in the central nervous system.     

Sugar response differences related to sensillum type and location on the labella of Protophormia terraenovae: a contribution to spatial representation of the stimulus

The chemoreceptor spike activity in response to sucrose in the concentration range 1-500mM was recorded from each of the 11 Intermediate and 13 Large labellar sensilla in the blowfly Protophormia terraenovae. The results showed that: (1) three of the four cells present in each sensillum are activated by sucrose stimulation; (2) differences between the Large and Intermediate types exist in the dose-response profiles of one of these cells (the 'water' cell), possibly reflecting different sugar receptor site populations on the dendritic membranes of homologous cells in the two types; (3) sensilla of both types are differentially responsive to sucrose solutions according to their location on the labellum. These differences may provide elements for a spatial representation of the stimulus source within the sensory coding process.     

Stimulation of the salt receptor of the blowfly. I. NaCl

Application of NaCl solutions to the tip of a labellar sensillum of the blowfly elicited a repetitive neural response from the salt receptor. The response was examined with respect to reproducibility and adaptation. A threshold was observed for tests with dilute solutions. Above this, the response increased linearly with the logarithm of the molarity. The response was not significantly affected by the pH of stimulating solutions, buffered or not, between 3 and 10. Beyond this range, it was reversibly inhibited until, at greater extremes of pH, atypical stimulation independent of the presence of salt was seen. Receptor sensitivity increased with fly age. The results presented here may be due to effects at sites in the sensillum other than the receptor membrane.     

Stimulation of the salt receptor of the blowfly. III. The alkali halides

Application of solutions of each of the alkali halides to the tip of a labellar sensillum of the blowfly elicited a repetitive neural discharge from the salt receptor. The records were qualitatively similar to those for NaCl. For each of the alkali chlorides and sodium halides, the shapes of the curves of the response of the salt receptor as a function of concentration were similar to that for NaCl. The alkali halides exhibited a regular pattern of relative stimulating effectiveness for the salt receptor. The effectiveness of the anions increased monotonically with atomic number. The effectiveness of the cations was greatest for potassium and declined as the atomic number was increased or decreased. This hierarchy for stimulating effectiveness was maintained at all tested molarities. The response to a mixture of two salts appeared to be an average of those to the single salts at concentrations equal to the total concentration of the mixture. Cross-adaptation was observed between the alkali halides. The results indicate that an explanation of stimulation of the salt receptor must apply to all the salts tested and that both the anion and the cation affect a salt's stimulating effectiveness.     

The effects of amiloride on the labellar taste receptor cells of the fleshfly Boettcherisca peregrina

Amiloride is known to inhibit the taste response of vertebrates to salt by blocking the amiloride-sensitive sodium channel. In this study, we investigated electrophysiologically the effect of amiloride on the taste response of the fleshfly Boettcherisca peregrina. When 0.5 mM amiloride was included in taste solutions, the response of the salt receptor cell (salt response) to sodium chloride (NaCl) was not depressed but those of the sugar receptor cell (sugar responses) to sucrose, glucose, fructose, l-valine (l-Val) and l-phenylalanine (l-Phe) were strongly depressed. An inhibitory effect of amiloride on the concentration-response relationship for both sucrose and l-Phe was clearly revealed, but not at high concentrations of sucrose. After pretreatment of a chemosensory seta with 0.15 mM amiloride for 10 min, the salt response to NaCl was not affected. On the other hand, the sugar responses to sucrose, fructose, l-Val and l-Phe were depressed just after amiloride pretreatment. The sugar response to adenosine 5’-diphosphate (ADP) mixed with 0.5 mM amiloride was not depressed, but the response to ADP alone was depressed after amiloride pretreatment. It was therefore observed that amiloride depressed the responses to all stimulants that react with each of the receptor sites of the sugar receptor cell.     

The Effects of pH on the Labellar Sugar Receptor of the Fleshfly

Reproducible results describing the effects of pH on the response of the labellar sugar receptor of the fleshfly, Boettcherisca peregrina, were obtained. The response to sucrose was independent over a wide range of pH (3.0 to 10.0 for sucrose stimulation), but was inhibited fairly sharply on both sides of this range. Similar results were obtained for monosaccharide stimulation. The receptor was excited on stimulation by water above pH 12.0. The effects of high pH, both inhibitory and excitatory, were affected by the presence of salts. In the presence of 0.5 molar NaCl, for example, the pH-inhibition curve was shifted toward lower pH's by about one pH unit. The effects of low pH, on the other hand, were not affected by salts. Following Dixon's theory, it was concluded that at least five ionizable groups (loosing positive charges above pH 10.5) were located at the receptor site.     

Apparent opposing effects of cyclic AMP and dibutyryl cyclic GMP on the neuronal firing of the blowfly chemo-receptors.

Cyclic AMP and its dibutyryl derivative inhibit neuronal firing of the labellar sugar sensitive receptor of the blowfly when applied in conjunction with the stimulant sucrose. Furthermore, simultaneous application of aminophylline (phosphodieterase inhibitor) and sucrose or in combination with cyclic AMP caused a similar depression of the sugar receptors response. In contrast, dibutyryl cyclic GMP elicited an increase in sugar receptor firing when applied with sucrose to sugar receptor. Either 5'-AMP or 5'-GMP in combination with sucrose had no discernable effect on the sugar receptors response. Different ratio combinations of cyclic AMP and dibutyryl cyyclic GMP showed the striking inhibitory effect of cyclic AMP upon the dibutyryl cyclic GMP elicited increases in receptor firing frequency. Therefore, it is suggested that these two nucleotides may be mediating different but complimentary aspects of sugar receptor function in a push-pull manner.     

Apparent opposing effects of cyclic AMP and dibutyryl-cyclic GMP on the neuronal firing of the blowfly chemoreceptors

Cyclic AMP and its dibutyryl derivative inhibit neuronal firing of the labellar sugar sensitive receptor of the blowfly when applied in conjunction with the stimulant sucrose. Furthermore, simultaneous application of aminophylline (phosphodiesterase inhibitor) and sucrose or in combination with cyclic AMP caused a similar depression of the sugar receptors response. In contrast, dibutyryl cyclic GMP elicited an increase in sugar receptor firing when applied with sucrose to the sugar receptor. Either 5′-AMP or 5′-GMP in combination with sucrose had no discernable effect on the sugar receptors response. Different ratio combinations of cyclic AMP and dibutyryl cyclic GMP showed the striking inhibitory effect of cyclic AMP upon the dibutyryl cyclic GMP elicited increases in receptor firing frequency. Therefore, it is suggested that these two nucleotides may be mediating different but complimentary aspects of sugar receptor function in a push-pull manner.     

Labellar lobe spreading in the blowfly: Regulation by taste and satiety

Summary Lobe spreading behavior was studied by recording electromyograms from the muscles which spread the labellar lobes, the retractors of the furca (RF) inPhormia regina. RF responses and lobe spreading could be elicited by stimulating labellar, but not tarsal, taste hairs with sucrose (Fig. 3). RF activity was important to spread the lobes at the beginning of a meal, but was not necessary for continued feeding (Fig. 4).Temporal summation between sugar receptor spikes was necessary to elicit RF responses. Central response decrement occurs independently for different labellar hairs and may participate in the termination of motor responses.RF responses were more probable and more intense when either the sucrose concentration of the stimulus or the number of hairs stimulated was increased (Fig. 7). Stimulation with NaCl had no effect on the response to simultaneous sucrose stimulation of other hairs (Table 1).Feeding caused decreases in the probability and intensity of motor responses, but did not alter chemosensory responses (Figs. 8 and 9). Section of either the recurrent or median abdominal nerves prevented this postingestional inhibition of lobe spreading (Fig. 9).These results are discussed with regard to the possible role that regulation of lobe spreading may play in the control of food intake.This work was supported by United States Public Health Service Training Grant 5T01 GM 00457-13S2 and by a grant from the National Science Foundation to Dr. Vincent G. Dethier. I wish to thank Dr. Dethier for his support and encouragement.     

Biochemical and physiological evidence that calmodulin is involved in the taste response of the sugar receptor cells of the blowfly, Phormia regina

The gustatory system is essential for almost all animals. However, the signal transduction mechanisms have not yet been fully elucidated. We isolated labellar chemosensilla from blowfly, Phormia regina, and purified calcium binding proteins from the water soluble fraction. The most abundant calcium-binding protein was calmodulin. To investigate the role of calmodulin in taste transduction, electrophysiological responses were recorded with the calmodulin inhibitor, W-7. When we stimulated the labellar chemosensillum with sucrose plus W-7, a dose-dependent decrease of impulse frequency was observed when the concentration was <50 microM. In addition, when W-7 at 50 microM or higher concentration was added, an initial short-term impulse generation from the sugar receptor cell was observed, but this was followed by a silent period. When the sensillum was stimulated with W-7 plus a membrane-permeable cGMP analog, dibtyryl-cGMP or 8-bromo-cGMP, impulses of the sugar receptor cell were induced but the frequency was decreased. By the sidewall-recording method, we observed that the receptor potential induced by sucrose stimulation was decreased by W-7 in the sugar receptor cell, and corresponded with a disappearance of impulses. These data strongly suggest that the cGMP-gated channel generating receptor potential in the sugar receptor cell requires calmodulin for its gating.     

Clonidine effects on protein and carbohydrate electrophysiological responses of labellar and tarsal sensilla in Phormia regina

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.     

Chromosaponin I stimulates the sugar taste receptor cells of the blowfly, Phormia regina

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, stimulates the growth of roots in a variety of plants. In the present work, we introduce CSI as a sugar taste substance for the blowfly, Phormia regina. The blowfly has taste chemosensilla on the labellum. The sensory receptor cells in the chemosensillum are highly specialized for the tastes of sugar, salt and water, respectively. Application of CSI induced the feeding response of blowflies including full proboscis extension. CSI also induced impulses of the sugar taste receptor cell in the LL-type sensillum. The optimum concentration of CSI in these responses was 0.1 mM which is much lower than that of sucrose. Based on the comparison of dose-response relationships, CSI is 100 times more effective than sucrose in stimulating the sugar taste receptor cells. CSI-induced impulses appeared after a significant latency compared with sucrose. As far as we know, this is the first report describing that a natural saponin induces sugar responses in insects. CSI is a unique saponin because of its bifunctional property in plants and insects.     

Ion dependence of the tarsal sugar receptor of the blowfly Phormia regina.

The activity of the tarsal sugar receptor is greatly reduced following prolonged water exposure. The animal's behavior, which characteristícally reflects receptor input, also shows decreased acceptance of sucrose solutions following prolonged tarsal immersion in deionized water. Long exposure of the tarsi to Bodenstein's saline instead of water does not produce as large a decrement in the acceptance response as does water exposure.Recovery of the behavioral response occurs spontaneously after a few hours. The original response level can also be restored immediately if a moderate concentration (0.05 to 0.2 M) of KCl or NaCl is added to the sucrose stimulus. The effect of LiCl is ambiguous: it inhibits the normal sucrose response, thereby tending to mask any restorative effects. The electrophysiological data show that the cellular response level is also restored when Na⁺ or K⁺ ions are present in the stimulus.The above data are interpreted to mean that the effect of tarsal water exposure is to slowly leach out ions in the effective extracellular fluid surrounding the receptor membrane, thus lowering the membrane potential and deceasing the receptor potential upon stimulation. The fact that Na⁺ and K⁺ when supplied in the stimulating solution temporarily restore the original response level suggests that these extrinsically added ions can be used as current carrying ions to depolarize the cell. The data suggest that the sensillum contains three functional compartments interconnected by partial diffusion barriers: (1) a ‘receptor compartment’ (2) an axial cylinder which contains the dendrites and functions as the immediate extracellular ion source, and (3) a larger axial cylinder which serves as an ion reservoir.A method for statistically analyzing behavioral acceptance data is presented.     

Detection of alkaloids and carbohydrates by taste receptor cells of the galea of gypsy moth larvae, Lymantria dispar (L.)

Gypsy moth larvae are polyphagous feeders. The electrophysiological responses of the medial and lateral styloconic sensilla to four secondary compounds (e.g., alkaloids), two carbohydrates, and one inorganic salt were examined using an extracellular tip-recording method. In the medial sensillum, one taste receptor cell responded to the alkaloids, strychnine, caffeine, nicotine, and aristolochic acid (i.e., deterrent-sensitive cell), while another, responded to the sugar alcohol and inositol (inositol-sensitive cell). In both medial and lateral sensilla, two taste receptor cells in each sensillum responded minimally and sporadically to 30?mM potassium chloride (KCl) (i.e., KCl-sensitive cells); one cell produced much larger amplitude action potentials than the other. In the medial sensillum, only the large-amplitude KCl-sensitive cell exhibited an increased firing rate with increasing salt concentration. When binary mixture experiments were conducted, it was confirmed that the large-amplitude KCl-sensitive cell and the deterrent-sensitive cell in the medial sensillum were one in the same cell. Only a single cell in the lateral sensillum responded to the sugar, sucrose (sucrose-sensitive cell). The temporal dynamics of responses of the deterrent-sensitive, sucrose-sensitive, and inositol-sensitive cells were compared. Concentration?Cresponse data were obtained for the deterrent-sensitive cell to various alkaloids, as well as to KCl.     

The peripheral inhibition of the tarsal sugar receptor by sodium chloride in the proboscis extension response of the blowfly,Phormia regina M.

Summary The proboscis extension reponse of the blowfly during stimulation of the tarsal sugar receptors was inhibited by the presence of NaCl. Acceptance thresholds for sucrose in various concentrations of NaCl were measured. The median acceptance thresholds for sucrose in mixtures of 0.01, 0.25, 0.5 and 1.0 M NaCl were 1.8 × 10^–3, 6.0 × 10^–3, 1.2 × 10^–2, and 2.0 × 10^–2 M, respectively. Concentration-response curves for sucrose in the tarsal D-type sugar receptor shifted to the right under the existence of high concentration of NaCl. Number of impulses per D-type sugar receptor at the median acceptance thresholds described above were 7.5, 8.4, 6.8 and 10.4 for the first 0.1 s of stimulation, respectively. The average number was 8.2 impulses per 0.1 s. Comparisons were made between the behavioral acceptance thresholds (1) on one leg exposed to sucrose mixed with 0.01 M NaCl and (2) on two contralateral legs, one of which was exposed to sucrose in 0.01 M NaCl and the other to 0.5 M NaCl alone. The acceptance thresholds from two experiments agreed with each other. The median threshold value was 1.7 × 10^–2 M sucrose. Behavioral inhibition by NaCl in mixtures with sucrose can be explained by its peripheral inhibition of sugar receptors.This research was supported in part by ITO foundation and Scientific Research Fund from the Ministry of Education of Japan.     

Variation of acceptance thresholds in the blowfly by increasing sugar concentrations in the food

Summary Aduld blowflies,Phormia regina M., were raised on different concentrations of sucrose. The thresholds of the behavioral responses to tarsal stimulation were elevated in blowflies raised on high concentrations of sucrose. The relationship between the median acceptance thresholds and the raising concentration of sucrose was logarithmically linear. Two groups of experimental flies were prepared: (1) coated flies, in which only D-type chemosensory hairs could respond physiologically, and (2) treated flies, in which all chemosensory hairs except D-type hairs functioned physiologically. Proboscis extension responses were ascertained in both groups. Median acceptance thresholds for the coated and treated flies, respectively, were presumed to be logarithmically linear in relation to the raising concentration of sucrose. It was supposed that D-type sugar receptor impulses initiate mainly the proboscis extension responses under the 0.01 M sucrose threshold and that B-type sugar receptor impulses initiate the responses above the 0.01 M sucrose threshold. Median acceptance thresholds for whole labellar stimulation were elevated to 0.026 M sucrose in blowflies raised on 1.0 M sucrose. Median acceptance thresholds were again lowered in blowflies raised on sucrose of more than 1.0 M.This research was supported in part by ITO foundation and Scientific Research Fund from the Ministry of Education of Japan.     

Multiple receptor sites for nucleotide reception in the labellar taste receptor cells of the fleshfly Boettcherisca peregrina

Nucleotides applied to the labellar chemosensory hair of the fleshfly, Boettcherisca peregrina, stimulated the taste receptor cells. Adenosine 5'-diphosphate (ADP) evoked a large response of the sugar receptor cell (sugar response) and guanosine 5'-diphosphate (GDP) evoked a large response of the salt receptor cell (salt response), but the salt response to ADP and the sugar response to GDP were relatively small. While the sugar response to ADP was independent over a wide range, pH5-9, the salt responses to GDP and ADP were inhibited at neutral and alkaline pH's, even though they elicited a marked salt response between pH's of about 5 and 6. Only adenine nucleotides (ADP, AMP, ATP) could stimulate the sugar receptor cell, with an order of stimulating effectiveness of ADP>AMP>/=ATP. However, the salt receptor cell could respond significantly not only to GDP but various nucleoside 5'-diphosphates, nucleoside 5'-monophosphates, cyclic nucleotides and thiamine diphosphate. These results clearly suggest that the specificity of the receptor site reacting with nucleotide in the sugar receptor cell is very different from that in the salt receptor cell.     

Different sensitivities of the sugar receptor of the lateral styloconic sensillum in fourth- and sixth-instar larvae of the spruce budworm Choristoneura fumiferana

Female fourth- and sixth-instar larvae, Choristoneura fumiferana, were tested individually for the response of the sugar cell on the lateral styloconic sensillum to 25 mM/l concentrations of 12 carbohydrates. The spruce budworm showed an age-related change in responsiveness of the sugar cell. The order of stimulating effectiveness for fourth-instars was melibiose > sucrose > raffinose.These storage di- and trisaccharides are present in the host plant at the beginning of budbreak. Sixth-instars responded to sucrose > fructose> m-inositol. These findings are in accordance with those of a previous behavioural study on feeding preferences of sixth-instars. The response for both melibiose and raffinose does not change from fourth- to sixth-instars; however, it does for sucrose, fructose and m- inositol.     

Effects of alkaloids on feeding by Phormia reginaconfirm the critical role of sensory inhibition

Abstract. Inhibition of peripheral chemoreceptors by micromolar concentrations of alkaloids may account for feeding deterrence. Earlier work showed a reduction in both the proboscis extension response and sensory activity recorded from sugar-sensitive cells in tarsal D and labellar 'largest' hairs when the stimulating solution was sucrose mixed with alkaloids. In the present work a similar behavioural effect was also seen when alkaloids were mixed with pyranose and furanose sugars as well as with water and deuterium oxide. Behavioural deterrence continued after the stimulus was removed. Complete recovery occurred after 120 s. To check against the possibility of a central inhibitory state (CIS) having been established two sets of experiments were run in which quinine was applied to a single tarsus followed by a sucrose post-test to the contralateral tarsus. In one half of the tests sugar was applied first (S₁Q₂), and in the other half quinine first (Q₁S₂). Comparable tests were run with contralateral labellar hairs. No CIS was set up by Q₁; however, a central excitatory state (CES) was set up by S₁. In order to test (a) whether quinine had different effects on other known or unknown receptors and (b) post-ingestive effects, volumes imbibed in single drinks of sucrose with and without quinine were compared. Volumes of the mixture imbibed were not significantly different from what was predicted based on the known reduction in sensory acitivty.     

Multiple receptor proteins for sweet taste inDrosophila discriminated by papain treatment

Summary Treatment of the labellar chemosensory setae of the fruit fly,Drosophila melanogaster, with 0.1 % papain for 3 min induced a complete elimination of the taste nerve response to fructose (Fig. 1). Responses to other sugars examined were not affected (Table 1). Responses to 20 mM LiCl and 0.1 M NaCl also remained unchanged by the treatment. The experiment on the time-dependency of the papain treatment showed a clear difference in the proteasesensitivity between the response to fructose and to glucose and sucrose (Fig. 2). The treatment with 0.005% trypsin for 3 min produced the same results. The response to fructose which was eliminated with the papain treatment, was restored after 3 hrs. These findings reveal the presence of a specific receptor site for fructose and its protease-sensitive nature and suggest the involvement of multiple receptor proteins in the sugar receptor ofDrosophila.We thank Dr. A. Shiraishi for valuable suggestions for electrophysiological recordings. This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.