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

7. Insect Physiol., 1975, Vol. 21, pp. 265 to 280. Pergamon Press. Printed in Great Britain

PERIPHERAL AND CENTRAL INTERACTIONS SUGAR, WATER, AND SALT RECEPTORS BLOWFLY, PHORMIA REGINA STEVEN

BETWEEN OF THE

M. FREDMAN

Department of Zoology, University of California, Berkeley, U.S.A. * (Received

8 April

1974;

revised

28 June

1974)

Abstract-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 set 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. * Present address : Worcester Massachusetts 01545, U.S.A.

Foundation 265

for Experimental

Biology,

Shrewsbury

266

STEVBN M. FREDMAN INTRODUCTION

THE GUSTATORY receptors of blowflies are located in sensilla on the tarsi and the margin of the labellum. MINNICH (1929, 1931) using Calliphwa found that these sensilla were sensitive to sugars. Similar results were reported for Phomia regina by DETHIER (1952). Stimulation of the tarsi or of a single labellar sensillum with sugars produced proboscis extension. Electrophysiological studies have demonstrated the presence of four chemoreceptors in each sensillum : a salt receptor which is sensitive to monovalent cations (HODGSONet al., 1955) and a monovalent anion receptor ( STEINHARDT,1965) in addition to the sugar receptor (HODGSON, 1957) and the water receptor (EVANSand MELLON, 1962). Each sensillum can thus be considered to be a single sensory channel for each individual receptor type. Behavioural studies on the water receptor have shown that stimulation of more than one labellar sensillum is usually necessary to elicit a response (DETHIER and EVANS,1961; EVANS,1961; DIXTHIERet al., 1965, 1968), although dehydration and loss of haemolymph can increase a fly’s responsiveness (EVANS, 1961). Other behavioural studies have indicated a possible central inhibitory role for the salt receptor (DETHIER, 1955; DETHIERet al., 1965, 1968). These studies propose that proboscis extension is determined by the summation of excitatory and inhibitory sensory information at some point in the central nervous system. Behavioural rejection of mixtures of sugar and hydrocarbons or sugar and dilute acids can be accounted for by direct inhibition of the sugar receptor itself by alcohols (STEINHARDTet al., 1966) and extremes of pH (SHIFWSHI and MORITA, 1969) without invoking a central inhibitory mechanism. FREDMAN and STEINHARDT(1973) found evidence that peripheral inhibition is also involved in rejection of sugar solutions containing high salt concentrations. Although REES(1970) found that the water receptor was inhibited by high salt concentrations, the possibility remains that the salt receptor is involved in a central inhibition of responsiveness to water, as indicated by the work of DETHIER et al. (1965, 1968). GETTING (1971), in an electrophysiological study of the blowfly’s responsiveness to sugar, found that stimulating two sugar receptors simultaneously gave a motor response that was larger than the sum of the responses to stimulation of each individual sensillum separately. He concluded that the effect was due to crosschannel summation and that there was a non-linear gain mechanism in the fly’s central nervous system. Other effects involving sensory summation have been reported. DETHIER et al. (1965, 1968) in a behavioural study found that even though a fly would not extend its proboscis to water stimulation of a single sensillum, it would respond to water if it was first stimulated with sugar on another labellar sensillum. They termed this phenomenon central excitatory state (CES). It was also reported that CES could be blocked by interposing a salt stimulus between the sugar and water stimulations. They concluded that the latter effect was due to central inhibition. The present study is concerned with the basic relationship between the sugar, water, and salt receptors, and also with how many water receptors must be stimulated to give a proboscis extension, and how this is affected by increased

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AND CENTRAL

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RECEPTORS

267

saIt concentration. Cross-channel summation and central excitation are also investigated electrophysiologically. Does simultaneous stimulation of the sugar and water receptors or the sugar and salt receptors give a greater motor response than that to sugar alone ? Does preceding a water receptor stimulation by a sugar receptor stimulation alter the responsiveness to water, and is this blocked by interposing a salt receptor stimulation ? To answer these questions, the technique described by GETTING (1971) of recording both the activity of the muscle responsible for proboscis extension and the sensory input is utilized. This permits the observation of motor activity that might be undetectable behaviourally. In addition, any changes in the programme of motor unit discharges can be analysed quantitatively.

MATERIALS AND METHODS Blowflies, Phormiu regina Meig. 1 to 10 days after emergence were used throughout. The flies were raised at 27 to 30°C on beef liver through pupation. After emergence, the flies were kept in cages with free access to sugar and water. The flies used in experiments were taken directly from the cages without prior starvation, since it has been shown that the motor responses to labellar stimulations are not influenced by internal receptors in the gut (GETTING and STEINHARDT, 1972). To increase their responsiveness to water, some flies were bled by cutting off their legs and gently compressing the thorax while blotting the haemolymph with tissue (EVANS,1961). Electrophysiological recordings from the extensor muscle of the haustellum and from single chemosensory sensilla were made using a technique previously described in detail (GETTING, 1971). Briefly, the proboscis of a fly whose legs had been removed and the cut stumps covered with melted wax to prevent further loss of haemolymph was fixed in a semi-extended position by inserting the constriction between the haustellum and the rostrum into a slot in a silver chloride plate which served as the indifferent electrode. A glass-insulated tungsten electrode was inserted through the cuticle into the extensor of the haustellum, under the proximal end of the apodeme. Sensory activity from single labellar sensilla was recorded by placing a capillary over the tip of the sensillum. The capillary, whose movement was controlled by pulses from a square-wave generator, was blown out just prior to each stimulation to minimize the effect of evaporation at the tip. It was connected to the input of a high impedance amplifier. A second electronically controlled capillary was also used and a third was advanced by hand. As a water stimulus 10 mM LiCl was used. This concentration of LiCl was insufficient to stimulate the salt receptor, but was still able to activate the water receptor at close to the same frequency obtained with distilled water, while providing sufficient conductivity so the sensory impulses could be recorded (GILLARP, 1968b; REES, 1970). Solutions of 100 mM or 1 M sucrose contained 50 mM LiCl which provided electrical conductivity but was insufficient to stimulate the salt receptor (GILLARY, 1966b). Other solutions used contained 50 mM LiCl, 200 mM,

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500 mM, 1 M or 4 M NaCl. Sensory and motor signal were amplified by conventional means, displayed simultaneously on an oscilloscope, and filmed. Sensory and motor responses could be recorded for several hours.

RESULTS

At the start of each experiment, single long, posterior labellar sensilla were tested with 100 mM sucrose to ascertain whether they were capable of eliciting a motor response. After a wait of 10 to 15 min (30 min in later experiments) to permit recovery from habituation, the same sensilla were tested with 10 mM LiCl. Of 76 flies tested, 23 gave some motor response to single sensillum water stimulation, but only 5 gave a response greater than one or two small motor unit impulses. Many other flies were subsequently rejected when they failed to respond to multiple sensilla water stimulation. Following this, testing then proceeded at 10 to 15 min intervals (up to 30 min intervals in later experiments) with paired simultaneous stimulations with the same solution in each pipette. Initial experiments used ascending concentrations (10 mM LiCl, 50 mM NaCI, 100 mM NaCI, 200 mM NaCI). In later experiments, stimulation with 200 mM NaCl and then 100 mM NaCl followed those with 10 mM LiCl. This was to avoid a systematic artefact of testing the most concentrated solutions at the end of an experiment when the real sensitivity might be masked by long-term habituation. At the end of each experiment, individual sensilla were again tested with 10 mM LiCl.

IO

50

8--o

Large

e

Small motor unit

motor unit

100

Salt concentration

(mM)

FIG. 1. The effect of increased salt concentration on the motor response to simultaneous stimulation of two labellar water receptors. The mean motor response for both the large and small motor units decreases with increasing salt concentration (decreasing water receptor activity). Both units reach zero response at about 130 mM salt. The numbers above each point refer to the number of responses from 4 flies averaged to obtain that value.

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269

Single sensillum stimulations with 10 mM LiCl were usually ineffective ; when two sensilla were stimulated simultaneously, there was a vigorous but readily habituated motor response. Flies that failed to give a motor response when two sensilla were stimulated were discarded. Motor latencies measured from the onset of 80 stimulations averaged 47 f 15 msec and 60 + 19 msec for the small and large units respectively. These are in close agreement with the values GETTING (1971) obtained with sugar receptor stimulation latencies of 43 and 54 msec respectively. More vigorous responses tended to have slightly shorter latencies than did weaker responses. Increased salt concentrations gave a reduced motor response (Fig. 1). Stimulating two sensilla with 50 mM LiCl or 100 mM NaCl was effective, but 200 mM NaCl was not. The decline in responsiveness with increasing salt concentrations is linear. Since two sensilla stimulation with 200 mM NaCl never gave a response, its potential effectiveness was further examined by stimulating two sensilla with 10 mM LiCl and a third with 200 mM NaCl. This was done in two ways. The first technique was to bend two sensilla with a fine dissecting needle until their shafts were touching and their tips were in close proximity. When the stimulating pipette advanced, the tips of both sensilla were stimulated and impulses could be simultaneously recorded from each. The second method consisted of extruding a small drop of 10 mM LiCl from the pipette, such that when the pipette advanced, two sensilla made contact with the surface of the drop. Although sensory impulses could not be seen with this technique, the motor responses were more vigorous than with the two separate pipettes on the same two sensilla. This was probably due to the solution concentrating less from evaporation with the drop than at the tip of the pipette. It also suggests that the actual stimulating solutions, at the very tips of the pipettes, were more concentrated than the initial concentration in the proximal portion of the pipettes. When two sensilla were stimulated with 10 mM LiCl by either of these techniques and a third was simultaneously stimulated with 200 mM NaCl, there were more large motor unit impulses (Fig. 2) than with just 10 mM LiCl presented on the same two sensilla alone (Table 1). Since REES(1970) has shown that the water receptor is almost totally inhibited by salt concentrations in excess of 150 mM, this indicates that the salt receptor can be excitatory. However, 200 mM NaCl is much less effective than more dilute solutions in which the water receptor is active. The same technique was used to test for central inhibition due to the presence of salt on another sensory channel. In these experiments a single sensillum was stimulated with 4 M NaCl, 50 to 100 msec before the onset of the two sensilla 10 mM LiCl stimulation. The interval between the onset of the salt and water stimuli was to permit any inhibition to be established before the water stimulation began. Other temporal relationships such as simultaneous stimulation of the water and salt receptor were also tried. Since they did not seem to be more effective, the temporal sequence of stimuli described by FREDMANand STEINHARDT(1973) was used. The response to 10 mM LiCl on two sensilla was reduced by 4 M NaCl,

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STEVENM. FREDMAN

cM FIG. 2. Summation of the activities of the water and salt receptors. Electrophysiological recordings from two labellar sensilla (S,, S,) and the resultant motor activity (M). W, a water receptor spike; T, a salt receptor spike; L, large motor unit; S, small motor unit; P, tonic motor unit. (A) and (C) motor response to stimulation of a single sensillum with 10 mM LiCI. This fly was unusual in that it responded strongly to single sensillum water stimulation. (B) Motor response to simultaneous stimulation of the same sensillum with 10 mM LiCl and an adjacent sensillum with 200 mM NaCl. The motor response is significantly larger than that with 10 mM LiCl alone. There was no motor response to 200 mM NaCl alone. (A), (B), and (C) were consecutive stimulations. Time mark: 100 msec. which

drives the salt receptor close to its maximum frquency (Table 1). Only 1 fly out of 3 failed to show a significant reduction. The overall results indicate that 4 M NaCl on a separate sensory channel produced a central nervous inhibition which significantly reduced the motor response to water. To further define the range of concentrations over which salt was inhibitory, the following experiment was performed. A single long posterior labellar sensillum was stimulated with a mixture of 10 mM LiCl and 10 mM sucrose. That sugar concentration is very close to the absolute threshold of the sugar receptor (OMAND and DETHIER, 1969). This mixture, combined with bleeding the flies, permitted obtaining motor responses to single sensillum stimulation, which were still largely due to the activity of the water receptor, and were not so vigorous that they could not be inhibited by salt stimulation on another channel (FREDMANand STEINHARDT,1973). The motor responses to the mixture alone were compared to those preceded by 50 to 100 msec with 1 M NaCl on an adjacent sensillum. All three flies tested in this way showed significant inhibition by salt on a separate channel (Table 1). Th’ 1s a g rees with observations of inhibition of the motor response when two sensilla were stimulated with 10 mM LiCl and a third with 1 M KNO,.

PERIPHERAL AND CENTRAL TABLEI-THE

INTERACTIONS

OF BLOWFLY

EFFECT OF SEPARATE SENSILLA STIMULATION MOTOR

WITH

271

RECEPTORS DIFFERENT SOLUTIONS

ON

OUTPUT

Mean response of large motor unit

N

P (by the t-test)

1084 + 782

13

0.10

10 mM LiCl 10 mM LiCl/4 M NaCl*

8.83 + 2.26 3.85 + 2.02

7

0.01

10 mM LiCl + 10 mM sucrose 10 mM LiCl + 10 mM sucrose/l M NaCl*

8.76 + 5.65 1.61 f 1.73

13

0.001

27

0.001 NS

Stimulating solution 10mM

LiCl

5.16+5.99

10 mM LiClj200 mM NaCl*

100 mM sucrose 100 mM sucrose/l0 mM LiCl* 100mM sucrose/lOmM LiCl* ingestion)

(after Ha0

5.78 rf:6.44 15.70 f 10.70 11.82f8.12

100 mM sucrose 100 mM sucrose/500 mM NaCl*

14.36 + 6.51 20.81 f 6.69

11

0.05

100 mM sucrose 1 M sucrose/100 mM sucrose* (500 m set IBS)

8.63 + 7.69 16.78 + 9.25

33

0.001

1 M sucrose/l0 mM LiCl* 1 M sucrose/4 M NaCI/lO mM LiCl* (1600 msec IBS) 1 M sucrose 1 M sucrose/4 M NaCl* (1600 msec IBS)

12.75 + 8.69 6.75 f 7.00

32

0.01

21.65 f 12.00 22.25 _+10.50

32

NS

* Solutions applied to separate sensilia. NS, Difference of the means not significant.

To test to see if there was cross-channel summation between the sugar and water receptors, flies were kept at 20 to 30°C and given access to dry sucrose but no water 18 to 24 hr prior to each experiment. Tests consisted of alternating single, long posterior labellar stimulation with 100 mM sucrose and simultaneous stimulation with 100 mM sucrose on the same sensillum and 10 mM LiCl on an adjacent sensillum. Stimulations were 500 msec long and were performed at 30 min intervals. After several pairs of stimulations, the flies were fed 10 mM LiCl from the stimulating pipette, which was inserted between the labella, thus avoiding contact with the sensilla themselves. The pipette was withdrawn as soon as the flies stopped sucking. The volume of solution fed each fly was not monitored. After a 30 min interval alternating stimulations were resumed. As seen in Table 1, simultaneous stimulation with 100 mM sucrose on one sensillum and 10 mM LiCl on another gave a larger motor response than did 100 mM sucrose alone. In all experiments the mean response was always greater when water was simultaneously presented on a separate sensillum. However,

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STEVENM. FRKDMAN

in only 7 out of 9 experiments was the difference statistically significant. To check the overall effect, the results from all experiments were pooled. The motor response when the water receptor was stimulated was significantly larger than that with sugar alone (Table 1). There was no significant difference between the summated responses before and after being fed 10 mM LiCl except in 2 flies. For the data treated as a whole, the difference was not significant before and after water ingestion. DJZY~HIER and EVANS(1961) found behavioural evidence for excitatory effects with salt solutions. Flies drank the same volume of 500 mM NaCl as they did water over a 3-day period. To test for cross-channel summation between the sugar and salt receptors, fresh flies were stimulated with 100 mM sucrose on one labellar sensillum and simultaneously with 500 mM NaCl on an adjacent sensillum. As shown in Table 1, the magnitude of the motor response to 100 mM sucrose was slightly but significantly increased when 500 mM NaCl was presented on a separate sensory channel. While this is indicative of cross-channel summation between the receptor types, only in 1 fly out of 3 was the difference in the mean response large enough to be significantly different statistically. If the activities of the sugar and salt or water receptors can summate, can the activity of one receptor influence the effectiveness of another when they are not simultaneous 7 Could the central excitatory state (CES) found in behavioural tests by DETHIER et al. (1965) be demonstrated physiologically ? To test this, flies that had been taken fresh from their cages had the water responsiveness of single long, posterior labellar sensilla tested with 10 mM LiCl. While 12 of 41 flies gave some response, only in 5 was it greater than one or two small motor unit impulses. A single sensillum was then stimulated with 1 M sucrose for 500 msec. This was followed by a 500 msec stimulation with 10 mM LiCl on an adjacent sensillum 100 to 4000 msec after the sucrose stimulation. An interval between stimuli (IBS) of approximately 500 msec was used as a standard. Testing proceeded with 30 min rests between stimulations to avoid habituation (GETTING, 1971). With a 500 msec IBS flies responded regularly to the following water stimulus. Tests with 10 mM LiCl alone at various times during each experiment usually were negative, or gave a small motor unit response only. On some occasions, however, the motor response was vigorous enough to include the large motor unit, indicating possible long-term effects from the pairing of the two stimuli. In some individuals, increasing the IBS reduced the motor response to the water stimulation; for the data as a whole, however, there was so much variability, especially when comparing different animals, that no overall trends could readily be distinguished (Table 2). Because the variation might be due to differences in the responses to sucrose, responses from all experiments were broken down into three arbitrary groups: those whose motor response to 1 M sucrose was 20 or more large motor unit impulses, those whose response was 10 to 19 large motor unit impulses, and those whose response was 0 to 9 large motor unit impulses. The response to 10 mM LiCl at all IBS’s were compared for each group. Comparison was also made for each IBS tested. Motor responses to 10 mM LiCl for the 20+ group were

PERIPHERAL ANDCWTRALINTJIRACTIONS OF BLOWFLYRECEPTORS TABLE ~-THE

Motor response to 1 M sucrose O-19 large motor unit impulses 20+ large motor unit impulses

O-19 large motor unit impulses 20+ large motor unit impulses

273

EFFECTOF THE INTERVALBETWEENSTIMULION CENTRALEXCITATION

100 (N)

200 (N)

5.50 k 8.75 (6)

4.62 f 3.67 (8)

1300 (N) 8.50 + 8.07 (16)

1400 (N) 3.81 It.6.56 (11)

Mean response to 10 mM LiCl* IBS (msec) 300 400 450 500 6.50 (N) (N) (N) (N) (N) 1.53 2.94 + 2.23 k4.50 (13) (17) 13.12 7.88 + 9.76 + 8.72 (8) (9) 1500 (N)

1900 (N)

16.75 35.50 f 16.61 * 8.50 (4) (2)

4.77 rt 3.93 (9) 8.12 + 9.53 (8)

1.25 + 1.29 (4) 1.75 rt:1.08 (4)

2100 (N) 3.00 f 2.54 (4)

3700 (N) 6.00

800 (N)

900 (N)

0.50 11.50 6.33 + 0.50 + 6.72 + 10.32 (2) (6) (4) 25.00 11.85 19.50 + 15.00 f 12.26 k 14.16 (2) (7) (7)

All (N) 3*9-s+ + 4.69 (1) (40) 40.00 12.06 f 0.00 + 12.82 (49) (2)

* Taken from 14 flies. t Applies to the 10 to 19 group; mean for the 0 to 9 group was 240 f 5~20.

significantly greater than those of the other two groups. The 0 to 9 and the 10 to 19 groups were not significantly different. This is one source of the variation observed. Flies in the 20+ group often had very vigorous responses (20 or more large motor unit spikes) even at long IBS’s (Table 2). It was often impossible to test these flies at short (100 to 500 msec) IBS’s due to the magnitude of the response to 1 M sucrose. On the other hand, flies in the other two groups (which were more typical) would not respond to 10 mM LiCl at long (1000 to 4000 msec) IBS’s. Since a CES could clearly be demonstrated with the water receptor, could it be demonstrated with another sugar receptor ? Could the activity of one sugar receptor enhance the response to stimulation of another sugar receptor when they were not simultaneous ? To test this, a similar procedure was adopted, using 100 mM sucrose instead of 10 mM LiCl. A 500 msec IBS was used throughout. Experiments consisted of alternating 100 mM sucrose stimulation of one sensillum and 100 mM sucrose preceeded by 1 M sucrose on an adjacent sensillum. CES can act on the sugar receptor. Responses to 100 mM sucrose following stimulation with 1 M sucrose on an adjacent sensillum were significantly larger than with 100 mM sucrose alone (Table 1). Of 6 flies tested, only 1 fly failed to show an enhanced motor response.

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STEVENM. FREDMAN

DETHIER et al. (1965) reported that CES could be blocked by interposing a salt stimulation between the sugar stimulus and the water stimulus. This was tested in the following way. Sucrose (1 M) and LiCl (10 mM) were used as previously described. In addition, a third pipette, containing 4 M NaCl was advanced by hand after the 1 M sucrose stimulation had ended and before the onset of the 10 mM LiCl test. An IBS of 1600 msec was used. Flies that failed to respond to 10 mM LiCl at this IBS were discarded. The duration of the salt stimulation, while not controlled, was typically 500 to 700 msec. Trials with 4 M NaCl interposed alternated at 30 min intervals with those in which 4 M NaCl was not presented.

FIG. 3. Inhibition of CES by salt. Electrophysiological recordings from three labellar sensilla (S,, Sa, .‘&a) and the resultant motor activity (M). R, a sugar receptor spike; W, a water receptor spike; T, a salt receptor spike; L, large motor unit. (A) CES motor response to 10 mM LiCl stimulation of a single sensillum following stimulation of an adjacent sensillum with 1 M sucrose. (B) Inhibition of the CES motor response to 10 mM LiCl by stimulation with 4 M NaCl of a third sensillum and recorded through the same amplifier as the 10 mM LiCl stimulation. The motor response is significantly reduced. Time mark: 250 msec.

Fig. 3 shows a reduced motor response to 10 mM LiCl when 4 M NaCl is presented during the IBS. Since many flies would not respond to 10 mM LiCl presented at an IBS of 1600 msec, only those trials where there was a control motor response to 10 mM LiCl (without 4 M NaCl) immediately following the test with salt were counted. As a further control, the responses to the 1 M sucrose ‘conditioning’ stimulus were also compared and not found to be significantly different (Table 1). DISCUSSION

The relative effectiveness of sensory information provided by the labellar sugar water and salt receptors of the blowfly is controlled at two levels. The first is peripheral and acts on the sensitivity of the receptors themselves. The second is central, where the activity of the receptors converge and are processed by the fly’s brain. The result of this two-layered control system is both excitatory and inhibitory interactions between the receptor types. This has been demonstrated by

PERIPHERAL

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stimulating specific receptors in separate sensilia (and thus separate sensory channels). Peripherally there are inhibitory effects of salt solutions on the sugar and water receptors. Centrally the activities of separate water receptors and sugar and water receptors can summate to give an enhanced motor response, as can the activities of the sugar and water receptors and the low frequency firing of a salt receptor on another channel. The high frequency firing of a salt receptor can also inhibit the motor response to water stimulation on a separate channel. Concentrated sugar can enhance the motor response to sugar or water inputs on separate channels that follow it, and this effect can be blocked by high frequency firing by a salt receptor on a third channel. Peripherally, the effect of increased salt concentration is to inhibit the water receptor (EVANS and MELLON, 1962; REES, 1970). In pure water the firing of the water receptor is at its maximum (PEES, 1970). As the salt concentration increases, the water receptor’s activity declines. As the concentration increases further, it reaches the threshold of the salt receptor; higher salt concentrations progressively stimulate the salt receptor (GILLARY, 1966a). Thus when stimulated by a solution containing monovalent cations, the firing frequencies of both the water and salt are determined by how the salt concentration affects the receptors themselves. When two Iabellar water receptors are simultaneously stimulated, the receptor activities summate to produce a motor response, even though none was elicited by the activity of each receptor acting separately. The magnitude of the response is determined by the frequency at which the water receptors are firing. Thus 10 mM LiCl is more effective in eliciting a motor response than in 50 mM LiCl. This correlation is due strictly to the effect of the salt concentration of the stimulating solution on the water receptor alone, since 50 mM LiCl is below the threshold of the salt receptor (GILLARY, 1966b). Even after the threshold of the salt receptor has been reached, the linear decline in the motor output when two labellar sensilla are stimulated is consistent with the decline in the activity of the water receptors. When further increases in the salt concentration reduce the firing of the water receptor close to zero, two sensilla stimulation is no longer effective. Thus 100 mM NaCl is a slightly effective stimulus, but 200 mM NaCl is not. GJXTING (1971) found that two separate sugar sensory channels could summate. He failed to obtain this effect in water-satiated flies when 50 mM LiCl was used instead of one of the sugar inputs. When 10 mM LiCl is used and the water receptor activity is greater, summation between the sugar and water receptors is possible. There are two basic reasons for this discrepancy. First, the water receptor is only weakly excitatory compared to the sugar receptor. Second, PEES (1970) found that the water receptor is 80 per cent inhibited by 50 mM LiCI, but is almost 100 per cent active when 10 mM LiCl is used. Getting’s failure to find summation between the sugar and water receptors is thus probably due to insufficient water receptor input. The summation process appears to be independent of the state of thirst of the flies. Feeding thirsty flies water did not have a significant effect on the summation of the receptors’ activity. This may mean that the control of the responsiveness to water may have its effect after the point at which the sensory

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channels converge. It is also possible, judging from the two flies that did show a significant change following water ingestion, that different results might have been obtained if the flies had a more severe fluid loss, such as that caused by bleeding (EVANS, 1961). The activity of the water receptor is insufficient to account for the fly’s ability to respond to low concentrations of salt (DETHIERand EVANS,1961). Simultaneous stimulation of two sensilla with 10 mM LiCl and a third with 200 mM NaCl gave a larger motor response than did stimulation of the same two water receptors alone. Summation is also possible between the sugar and salt receptors. Usually, a large number of water receptor impulses from two or more channels must summate to produce a motor response. The salt receptor is moderately stimulated and the water receptor almost totally inhibited by 200 to 500 mM NaCl (EVANS and MELLON, 1962; REES, 1970). It is unlikely that the enhanced motor responses observed were caused by cross-channel summation with the one or two water receptor impulses that might have been present (but were not seen) during 200 and 500 mM NaCl stimulations on a separate sensory channel. From both behavioural and physiological evidence it would appear that salt at low concentrations is weakly excitatory and that the activity of the salt receptor can summate with the activity of the other receptors. GETTING (1971) found that it was the interval between sugar receptor impulses that determined how effective a stimulus was in eliciting a motor response. GILLARY (1966a, b) indicates that the impulse frequency of the salt receptor is low in dilute solutions. Therefore, several salt receptors would have to be stimulated simultaneously before the cross-channel interspike interval was sufficiently small to permit effective summation and a motor or behavioural response. The summation with the sugar receptor using 500 mM NaCl was not very strong. This indicates that 500 mM NaCl is probably close to the upper limit for the salt receptor to have an excitatory effect. When the salt concentration of a stimulating solution is greater and the salt receptor is firing at a high frequency, it is clearly inhibitory. The inhibition produced by single channel salt stimulation is not absolute, however. Stimulation of one sensillum with 4 M NaCl and two others with 10 mM LiCl caused a reduction, but not a total inhibition of the motor response. Since separate sensory channels are involved, this is a central inhibitory effect. It is possible that the same bleeding which increased the effectiveness of the water stimulation also decreased the inhibition produced by the salt receptor, as DETHIER and EVANS(1961) found that thirst increased flies responsiveness to concentrated salt. While FREDMANand STEINHARDT(1973) found that high frequency firing of the salt receptor from 2 and 4 M NaCl had no effect on the motor response to 100 mM sucrose on a separate sensory channel, stimulation with 1 M NaCl can cause central inhibition in the presence of dilute sugar. When 1 M NaCl was presented on one sensillum the motor response to a mixture of 10 mM LiCl and 10 mM sucrose was effectively reduced on another. This raises the possibility that the reason FREDMANand STEINHARDT(1973) failed to observe central inhibition by salt with respect to the activity produced by the sugar receptor was that the excitation

PERIPHERAL

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produced by 100 mM sucrose was too great to be significantly affected by the maximum amount of inhibitory input capable of being provided by a single salt channel. It is also possible that the inhibition produced by the salt receptor acts only with respect to the excitation produced by the water receptor. The inhibition observed might thus be due to the elimination of all or part of the excitation produced by the water receptor component of the sugar-water mixture. It might be possible to test these alternatives by stimulating several receptors with 4 M NaCl and a single labellar sugar receptor with a solution containing less than 100 mM sucrose. The water receptor activity of a dilute solution could be almost totally eliminated by raising its osmotic pressure (EVANS and MELLON, 1962). One possible explanation of the dual nature of effects produced by the salt receptor is based on the discovery of a dual-action synapse in the mollusc Aplysk

by WACHTEL and KANDEL (1967, 1971). This synapse is excitatory at low frequencies and inhibitory at high frequencies. The excitatory component is antifacilitating and blocks at high frequencies. Assuming a similar relationship, at low frequencies of firing the salt receptor would excite its sensory interneuron. At high frequencies the interneuron would be inhibited. Alternatively, if such a system could be demonstrated in the central nervous system of flies, the same results could be achieved by an inhibitory interneuron with a high threshold which might act at some point in the excitatory pathway of the water receptor. The activities of the water and salt receptors is determined primarily by the monovalent cation concentrating in a stimulating solution. Depending on their relative activities, both excitatory and inhibitory interactions in the central nervous system are possible. The point at which the excitation caused by the salt receptor ceases and inhibition begins probably varies not only from fly to fly but in an individual fly as well depending on its nutritional state. DETHIER and EVANS (1961) found that flies became progressively more responsive to 1 M NaCl with prolonged dehydration while OUTLAND (1971) indicates that there may be feedback from the nutritional state of the fly which influences the impulse frequencies of the receptors themselves. DETHIERet aZ. (1965) found that by stimulating a single sugar receptor, a subsequent single water receptor stimulation, which previously had been ineffective, now resulted in proboscis extension. This has been confirmed physiologically. A direct comparison of their results and those described above is not possible In their behavioural experiments, due to differences in stimulus parameters. both the sugar and water stimulation lasted for several seconds, and the shortest IBS tested was 15 sec. It was not possible, given the limitations of the parameters that we used, to duplicate the decay of the CES process which they observed, except in some individual flies which did show such a decay. Given the duration and interval parameters of the stimuli used by DETHIERet al. (1965), it seems likely that their results were obtained with what would correspond to our ‘20+ large motor unit’ flies, which did not show any discernible decline over short IBS’S. That there was a difference in the responsiveness of the ‘O-19’ and ‘20 + ’ flies indicates that the important factor may be the ‘efficiency’ with which the sugar receptor excites those neurons upon which the sugar and water sensory pathways converge. 9

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GETTING(1971) f ound no evidence of alterations in response to sucrose stimulation of one sensillum following sucrose stimulation of another sensillum. The above results indicate that if the first sugar stimulation is strong (i.e. 1 M sucrose) it does alter the motor response of a subsequent sugar stimulation on another sensillum. The effectiveness of central excitation appears to be at least in part dependent on how effective the first stimulation is in eliciting a motor response. Probably Getting failed to observe CES due to not using a strong enough ‘conditioning’ stimulus, and due to habituation, since his stimulations lasted 1 set and were performed only 10 min apart. The blocking of CES which DETHIER et al. (1965) demonstrated by immersing one of the fly’s legs in a salt solution has been confirmed using labellar stimulation. The inhibition of CES by a single salt receptor was not absolute; the motor response to water was reduced but not eliminated. This is in keeping with results showing that the inhibitory effects of high frequency firing by the salt receptor on the excitation produced by two water receptors also were not total. It is worth noting that in two experiments, the salt receptor fired in doublets, indicating an injury discharge. In these experiments the motor response to 10 mM LiCl appeared to be enhanced compared to controls rather than inhibited. This is indicative that it is the impulse frequency which determines whether the salt receptor has an excitatory or inhibitory effect. The blocking of CES by the activity of a salt receptor also provides further evidence for convergence of the sensory information provided by three of the four chemoreceptor modalities.

I

r-

t

’ OtherMoior I Centers ; L____r___-l

Extensor

M!Ed?

FIG.~. A minimal neuronal model to account for the motor activity of the extensor muscle in response to labellar stimulation of the sugar, .water, and salt receptors (modified from GETTING,1971). The labellar sugar (R), water (W), and salt (T) receptors from each sensillum synapse on interneuron A. The sugar and water receptors are both excitatory. The salt receptor has both excitatory and inhibitory effects, and send branches to other cells which control other motor activities, such as proboscis withdrawal. Interneuron A excites interneuron B. Interneuron B also receives both excitatory and inhibitory input from other motor areas, and in turn drives the large, small and tonic motor inputs recorded in the extensor muscle. The excitatory and inhibitory input that interneuron B receives can account for spontaneous changes, habituation, and motor activity in the absence of labellar stimulation. This minimal model accounts for all the known functional relationships. See text for discussion. Open triangles indicate excitation. Partly filled triangles indicate excitation and inhibition.

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GETTING (1971) described a minimal model for the neuronal circuitry in the fly’s brain which would account for the summation and habituation he observed with sugar receptor stimulation. This model postulates that all the sugar receptors synapse on a sensory interneuron (A) which then drives a pre-motor interneuron (B). This in turn triggers the motor neurons which excite the extensor muscles. To account for the above results, the sugar, water, and salt receptors would all synapse on interneuron A (Fig. 4). The sugar and water receptors are strongly and weakly excitatory, respectively. The salt receptor is both excitatory and inhibitory. This would account for all the direct summation and inhibition effects observed during simultaneous stimulations. Furthermore, if the membrane potential of interneuron A was slow in returning to its resting level following excitatory input, then subsequent excitatory input would be much closer to the cell’s firing threshold. Inhibitory input during this period would reduce the elevated potential and tend to keep the added component from summating with depolarizations by subsequent excitatory input. Excitatory input to interneuron B from other motor centres could explain changes in responsiveness following periods when the motor units were spontaneously active; inhibitory input, presumably from neurons controlling the retractor muscle, would prevent extension during stimulation with concentrated salt alone. Another source of excitatory and inhibitory input to interneuron B might be from neurons relaying information from the chemoreceptors on the tarsi. Acknowleclgements-This work was funded in part by a USPHS predoctoral fellowship, and a USPHS grant No. GM 1021-11 to RICHARDA. STEINI-IARDT.I wish to thank Dr. C. H. F. ROWELL, Dr. M. R. O’SHEA, Dr. D. A. EWALD, and especially Dr. STEINHARDT for their helpful discussions; Dr D. R. BENTLEY and Dr. E. LEWIS for their criticism of the manuscript; Ms. E. REID for preparing the figures, and my wife Marsha for her constant encouragement. REFERENCES DETHIERV. G. (1952) Adaptation to chemical stimuli of the tarsal receptors of the blowfly. Biol. Bull., Woods Hole 103, 178-189. DETHIERV. G. (1955) The physiology and histology of the contact chemoreceptors of the blowfly. Quart. Rev. Biol. 30, 348-371. DETHIERV. G. (1968) Chemosensory input and taste discrimination in the blowfly. Science, Wash. 161. 389-391. DETHIER V. G. and EVANSD. (1961) The physiological control of water ingestion in the blowfly. Biol. Bull., Woods Hole 121, 108-l 16. DETHIERV. G., SOLOMONR., and TURNER L. H. (1965) Sensory input and central excitation and inhibition in the blowfly. J. camp. physiol. Psychol. 60, 303-313. DETHIERV. G., SOLOMON R., and TURNER L. H. (1968) Central inhibition in the blowfly. J. camp. physiol. Psychol. 66, 144-150. EVANSD. (1961) Control of the responsiveness of the blowfly to water. Nature, Lond. 190, 1132-1133. EVANSD. and MELLON DeF (1962) Electrophysiological studies of a water receptor associated with the taste sensilla of the blowfly. J. gen. Physiol. 45, 487-500. FREDMANS. M. and STEINHARDTR. A. (1973) Mechanism of the inhibitory action by salts in the feeding behaviour of the blowfly, Phormia regina. J. Insect Physiol. 19, 781-790.

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GBTTINGP. A. (1971) The sensory control of motor output in fly proboscis extension. 2. vergi. Physic% 74, 103-120. GETTING P. A. and STEINHARDT R. A. (1972) The interaction of the external and internal receptors in the feeding behaviour of the blowfly, Phormia regina. J. Insect Physiol. 18, 1673-1681. GILLARY H. L. (1966a) Stimulation of the salt receptor of the blowfly-I. NaCl. J. gen. Physiol. 50, 337-350. GILLARY H. L. (1966b) Stimulation of the salt receptor of the blowfly-III. The alkali halides. J. gen. Physiol. 50, 359-368. HODIXON E. S. (1957) Electrophysiological studies of arthropod chemoreception-II. Responses of labellar chemoreceptors of the bIowAy to stimulation by carbohydrates. J. Insect Physiol. 1, 240-247. HODGSON E. S., LETTVIN J. Y., and ROEDERK. D. (1955) The physiology of a primary chemoreceptor unit. Science, Wash. 122, 417-418. MINNICH D. E. (1929) The chemical sensitivity of the legs of the blowfly, Calliphora vomitoria Linn. to various sugars. 2. vergl. Physiol. 11, 1-55. MINNICH D. E. (1931) The sensitivity of the oral lobes of the proboscis of the blowfly, Calliphora vomitoria Linn. to various sugars. J. exp. 2001. 60, 121-139. OMANDE. (1971) A peripheral sensory basis for behavioral regulation. Camp. biochem. Physiol. 38A, 265-278. OMAND E. and DETHIER V. G. (1969) An electrophysiological analysis of the action of carbohydrates on the sugar receptor of the blowfly. Proc. nat. Acad. Sci. U.S.A. 62, 136-143. F&ES C. J. C. (1970) The primary process of reception in the type 3 (‘H,O’) receptor cell of the By Phormia terranovae. Proc. R. Sot. Lond. (B) 174,496490. SHIRAISHIA. and MORITAH. (1969) The effects of pH on the labellar sugar receptor of the fleshily. J. gen. Physiol. 53, 450-470. STEINHARDTR. A. (1965) Cation and anion stimulation of electrolyte receptors of the blowfly, Phormia regina. Am. Zoologist 5, 651-652. STEINHARDT R. A., MORITAH., and HODGSONE. S. (1966) Mode of action of straight chain hydrocarbons on primary chemoreceptors of the blowfly, Phormia regina. J. cell. Physiol. 67, 53-62. WACHTEL H. and KANDEL E. R. (1967) A direct synaptic connection mediating both excitation and inhibition. Science, Wash. 158, 1206-1208. WACHTELH. and KANDELE. R. (1971) Coversion of synaptic excitation to inhibition at a dual chemical synapse. r. Neurophysiol. 34, 56-68.