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The interaction of external and internal receptors on the feeding behaviour of the blowfly, Phormia regina
J. InsectPhysiol.,1972, Vol. 18, pi. 1673 to 1681. PerpnwnPress.Piintedin GreatBdain
THE INTERACTION OF EXTERNAL AND INTERNAL RECEPTORS ON THE FEEDING BEHAVIOUR OF THE BLOWFLY, PHORiWA REGINA P. A. GETTING=
and R. A. STEINHARDT
Department of Zoology, University of California, Berkeley, California (Received 7 March 1972) Abstract-The interaction of aensory activity from internal gut stretch receptors and from external labellar chemoaenao ryhairshasbeenstudiedboth behaviourally and electrophysiologically in the control of proboscis extension of the blowfly, Phomda regina. Labellar thresholds for proboscis extension, tested behaviourally, do not change significantly up to an hour after feeding in contrast to tarsal thresholds which rise quickly after feeding. Motor activity of the extensor muscle of the haustellum was recorded simultaneously with senaory activity from labellar sensilla. The mean number of muscle spikes per response and the sensory input neceaaary to trigger a reaponse do not vary with starvation, feeding, or sectioning of the recurrent nerve. Activity of internal stretch receptors seem to interact with tarsal sensory input but apparently do not modulate motor responses triggered by labellar sensory input. INTRODUCTION
interaction of information from internal and external receptor systems is commonly used to produce behaviours appropriate to an animal’s needs at the time. Feeding behaviour is a prime example of such a homeostatic mechanism. Information from receptors signalling the animal’s internal state such as nutritional condition or gut capacity can be used to modulate sensory input from external receptor systems signalling the presence of food. The feeding behaviour of the blowfly, Phti regtiu, provides an excellent opportunity for an electrophysiological study of the interaction between internal and external sensory systems in the control of behaviour. The complete feeding behaviour of the blowfly consists of a series of distinct behavioural acts controlled by three sets of external contact chemoreceptor systems. Chemosensory hairs located on the tarsi of all six legs are used to localize and characterize potential food. When stimulated with certain carbohydrates, they initiate extension of the retractable proboscis. Extension of the proboscis can result in the stimulation of the labellar chemosensory hairs which cause further extension of the proboscis, spreading of the oral lobes and initiation of sucking. Proboscis extension and sucking appear to be maintained by the stimulation of the interpseudotracheal papillae located on the inner surfaces of the oral cavity. The THE NEURONAL
* Present address : Department of Zoology, University of Washington, Seattle, Washington 98105. 1673
1674
P. A. GETTING AND R. A. STEINELUXDT
sensory control of proboscis extension by stimulation of the tarsal or labellar chemoreceptors has been extensively studied behaviourally (DETHIER, 1969) and electrophysiologically (GETTING, 1971; D~HIER, 1968). Clearly, feeding, once initiated, does not proceed indefinitely. At the cessation of feeding the fly has consumed a distinct quantity of food depending upon the type and concentration of the sugar (DETHIER et al., 1956). Immediately after feeding both tarsal and labellar thresholds are high due to adaptation, both receptor and central. However, the threshold for tarsal stimulation remains high well after a period suflicient for disadaptation (EVANSand DETHIER, 1957; EVANSand BARTONBROWN, 1960). Tarsal thresholds attain a maximum value between one and six hours after feeding and then decline over a period of days. Similar increases in labellar thresholds with feeding have been reported (ARAB, 1957). However, MINNICH (1931) reports no change in labellar thresholds with starvation in a closely related blowfly, Caliiphora vommitoria. Two sets of internal receptors have been identified which are thought to modulate the excitability of the feeding response to stimulation of the tarsal and labellar chemoreceptors. Denervation of the foregut by section of the recurrent nerve results in increased consumption and in some cases hyperphagia (DBTHIER and BODENSTEIN, 1958; EVANSand BARTONBROWNI,1960; DETHIERand GELPERIN,1967). GELPERIN(1%7) reports the identification in the foregut of stretch receptors which respond to distension with a tonic discharge pattern. These receptors send information to the brain via the recurrent nerve signalling the presence of food in the foregut and are hypothesized to be inhibitory to the chemoreceptor inputs (GELPERIN and DBTHIER, 1967). In addition, section of the ventral nerve cord or median abdominal nerve results in increased feeding suggesting that the activity of identified stretch receptors in the abdominal nerve network is also inhibitory to feeding (DHIIIIER and GELPERIN, 1967; GELPERIN, 1971). This study was undertaken to observe the influence of internal stretch receptors on the excitability of motor neurons involved in proboscis extension to sensory input from iabellar sugar receptors. Proboscis extension to labellar stimulation was investigated behaviourally before and after feeding known quantities of food. In addition the effects of feeding, starvation, and section of the recurrent nerve were investigated electrophysiologically by recording single unit motor output to the extensor muscle of the haustellum while simultaneously recording single unit sensory input from the labellar chemosensory hairs. This electrophysiological approach has several advantages over the behavioural measurement of feeding thresholds. By directly recording all the sensory input involved in initiating a motor act we were able to exclude possible effects of receptor adaptation and insure stable sensory frequencies throughout each experiment. In addition, by direct recording of the motor activity we had a quantitative measure which allowed us to time our test stimulations to avoid the effects of central habituation (GETTING, 1971). We found that the excitability of the feeding response to labellar stimulation does not appear to be modulated by either foregut or abdominal stretch receptor activities.
OF RBCBFTORS ON BLOWFLY
INTERACTION
FEEDING
BBHAVIOUB
1675
MATERIALS AND METHODS Blowflies, Phormia regina Meigen, age 3 to 18 days, 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 water and sucrose. Both males and females were used. Starvation and feeding were carried out as follows. When starvation was necessary, the flies were taken either directly from the cage or given free access to 100 mM sucrose solutions for 24 hr prior to starvation. Flies were starved at room temperature in small plastic boxes with free access to water. For feeding while the animal was mounted in the apparatus, the end of a 10 ~1 capillary, filled with 1-OM sucrose, was placed against the labellar lobes and the fly allowed to drink 10 to 20 ~1 of solution. This quantity is approximately the mean consumption of intact, free-moving flies (GELPERIN,1971). Behavioural studies Flies were starved 24 hr prior to being mounted on a wax sheet by the wings. Behavioural responses, proboscis extension, were tested by stimulation of single labellar hairs with 100 mM sucrose. The flies were divided into two groups: group 1 was fed 10 ~1 of 1-OM sucrose and tested for proboscis extension 15 and 60 min after feeding, group 2 was fed 15 to 20 ~1 of 1.0 M sucrose and tested immediately and 15 min after feeding. All flies were satiated with water before testing and feeding with sucrose solutions. Electmphysiologica~ studies Electrophysiological recordings from the extensor muscle of the haustellum and from single labellar chemosensory hairs were made using a technique previously described in detail (GETTING,1971). Briefly, the proboscis of a legless fly was fixed in a semi-extended position by inserting the constriction between the haustellum and rostrum into a slot in a silver-chlorided 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 label& hairs was recorded by placing a capillary over the tip of the hair. The capillary, connected to the input of a high impedance amplifier, contained various concentrations of sucrose mixed with 50 mM LiCl to provide electrical conductivity. This concentration of LiCl was insufficient to stimulate the salt receptor (GILLARY,1966). Both sensory and motor signals were amplified by conventional electrophysiological techniques and displayed simultaneously on an oscilloscope. Consistent sensory and motor responses could be recorded for several hours. RESULTS
If the activity of the foregut and abdominal stretch receptors is inhibitory to sensory inputs from the tarsal and labellar chemoreceptors as suggested (GELPEFUN and DJZTHIER, 1967) then the responsivenessof motor neurons controlling proboscis
1676
P.
A. GETTINGAND R. A.
%-RINHARDT
extension should vary with starvation and feeding. behaviourally and electrophysiologically.
This hypothesis was tested both
Behaviowal studies Proboscis extension was tested before and after feeding by applying 100 mM sucrose to single labellar hairs. Before feeding all flies gave a full proboscis extension to the sucrose solution but not to water. The flies were divided into two groups according to the volume of. 1-OM sucrose consumed: one group was allowed to consume 10 4, the other 15 to 204 Table 1 summarizes the responses at various times after feeding. All flies gave a full proboscis extension to TABLE 1. BEI-IAVIOURAL RBBPONSES TO AND AFI’RR FEEDING (-hST
SINGLR LABRLLAR HAIR STIMULATION STIMULUS
100 d’f
BEFORE
SUCROSR)
Behavioural responses Post-feeding (min after feeding)
Volume fed (1.0 M sucrose)
No. of ties tested
Pre-feeding
0
15
60
10/d 1 S-20 /.d
35 25
+ +
n.t. +
+ +*
rift.
+--full extension of the proboscis. n.t.-not tested. *-one fly in this group gave only a partial extension to the test stimulus.
the test stimulus at all times after feeding with one exception. One fly gave only a partial response 15 min after consuming 15 to 20 ~1. All other flies of the same group gave full responses. There are two possible interpretations of this data. Either there is no threshold change due to feeding or the test stimulus of 100 mM sucrose was suprathreshold even after feeding. Previous work (GETTING,1971) has shown that the sensory activity resulting from stimulation of a single labellar hair with 100 mM sucrose is just slightly above the minimum sensory input necessary to initiate proboscis extension for starved flies. Therefore it seems unlikely that a threshold increase due to feeding could have occurred. This conclusion agrees with the findings of MINNICH (1931) that labellar thresholds of Calliphora do not change significantly with starvation. Ekctrophysiologakal studies: effects of starvation These behavioural results were directly confirmed by electrophysiological measurements. The excitability of motor neurons controlling proboscis extension was observed by recording motor activity from the extensor of the haustellum and single unit sensory activity from labellar chemosensory hairs. Motor responsiveness was judged by two parameters ; (1) the sensory input necessary to trigger a motor response, and (2) the number of motor spikes per response. Motor output
INTERACTION OF RRCRF’TORSON BLOWFLY FEEDING BEHAVIOUR
1677
to the extensor of the haustellum can be triggered by three general classes of trigger conditions. Class I is motor activity triggered by a single sensory spike (Fig. 1). Class II triggering requires the temporal summation of only two sensory spikes (Grrrrr~c, 1971). Class III is motor activity triggered by the temporal summation of two or more sensory spikes depending upon the iirst sensory interspike interval (GE?TING, 1971). Flies exhibiting all three trigger classes were observed at all starvation times from 0 to 72 hr indicating no consistent trend towards a lower threshold with starvation.
FIG. 1. Electrophysiological recordings from a single labellar sensilla stimulated with 25 mM sucrose (S) and the reaultant motor response (M) recorded from the extensor of the hauatellum. The large arrow at the beginning of the sensory record marks an artifact at the moment the stimuhrs solution contacts the hair. Each of the two sugar receptor spikes (small arrows) is followed by a motor response ilhrstrating Class I triggering. Water receptor activity (small spikes of sensory trace) is also present, however, this activity has been shown not to be involved in triggering motor activity of a water-satiated fly (GETTING, 1971). Tune mark, 100 msec.
The mean number of motor spikes per response for 20 flies starved between 0 and 72 hr is summarized in Table 2. No effect of starvationon motor responsiveness is concluded since none of the mean response levels differ significantly. The large standard deviation for the unstarved flies is due to the unusual responses of two flies; one which gave no motor response to the sensory test frequency used, and another which gave an abnormally larger response. TABLE 2. MEAN MOTOR
RESPONSE VB. HOURS OF STARVATION Houra
0 Mean motor response (S.D.) N
22.2 (21.2) 5
starved
24
62
72
16.0 (7.2) 4
18.0
141 (4.0) 8
Y7)
The motor response level (motor spikes/response) was determined at a standardized sensory input of 30 impulses/set. There is no significant difference (P = O-01) between any of the mean responses by a ‘t-test’.
Eflects of feeding on motor output To avoid individual variations in responsiveness and the necessity for interanimal comparisons, we investigatedthe effects of feeding on motor responsiveness.
P. A. GE~TINC AND R. A. STIXNHARDT
1678
The motor response level of a starved fly was determined by several stimulations of a single labellar hair with 100 mM sucrose. The fiy was then fed 20 ~1 of 1-OM sucrose from a pipette. The post-feeding motor response level was again determined by several 100 mM sucrose stimulations at 10 min intervals for up to 30 min after feeding, Figure 2 shows the motor response level as a function of time before and after feeding for one of three experiments. There is no sign&ant change in the motor response level in the tests made for up to 30 min after feeding. The sensory spike frequency was the same for all stimulations.
z
B
r . f .z ,o
.
18 16 1412IO-
l
/\/i-
. t
Fed 20~1 1.0 M sucrose
0’
I
0
I
IO
20 Time
I
30 40 (minutes1
50
60
FIG. 2. Motor response level as a function of time before and after feeding for one of three experiments. At the arrow the fly was fed 20 ~1 of 1-O M sucrose. The sensory spike frequency was the same for all stimulations.
EVANSand BARTON BROWNE(1960) report that ingestion of only 3 4 of 2 M glucose causes observable increases in tarsal thresholds 15 min after feeding presumably by stimulating the foregut stretch receptors. In addition, ingestion of only 6 ~1 of 1-OM sucrose causes a threefold increase in abdominal stretch receptor activity (GELPERIN, 1971). Therefore, the quantity of 1.0 M sucrose ingested by the flies in this study (20 ~1) seems sufBcient to cause significant increases in the activity of both stretch receptor types. However, there is no observed change in motor responsiveness to label& stimulation after feeding. Section of the recurrent nerve As a final check on the possible inhibitory role of the foregut stretch receptors, we investigated the effects of sectioning the recurrent nerve which contains the afIerent axons of the stretch receptors. If the foregut receptor activity is inhibitory to labellar sugar receptor input, then sectioning of the recurrent nerve should result in an increased motor responsiveness. The recurrent nerve was sectioned anterior to the corpora cardiaca by the technique of DETHIERand BODENSTEIN (1958). The mean motor response of flies with severed recurrent nerves is not significantly different (P = O-01) than that of the controls (Table 3).
INTERACTION
TABLE 3. MEAN
OF RECEPTORS ON BLOWFLY
MOTOR
RESPONSE
1679
FEEDING BEHAVIOUR
OF RECURRENT NZRVE sEcTIoNED
FLIm
vs.
CONTROLS
Recurrentnerve Mean motor response (S.D.) N
Control
sectioned
19.4 (159) 9
17.2 (i.7)
The motor response level (motor spikes/response) was determined at a stanThere is no significant difference dardized sensory input of 30 impulses/set. (P = O-01) between the mean responses by a ‘t-test’. The large large standard deviation of the control group is due to the unusual responses of two flies; one which did not respond at the test frequency and another which gave an abnormally large response.
The findings that motor responsiveness to labellar sugar stimulation is not altered by starvation,feeding, or section of the recurrent nerve leads us to conclude that there is no inhibitory feed-back from either the foregut or abdominal stretch receptors to sensory input from the labellar sugar receptors. This does not, however, rule out the possibility of inhibition of feeding activated by sensory input from the tarsalsugar receptors as demonstratedbehaviourally (EVANSand DRIER, 1957; EVANSand BARTONBROWNE,1960). DISCUSSION
Behavioural studies suggest that the feeding behaviour of the fly is modulated in part by activity of at least two sets of internal stretch receptors. The activity of these internal stretch receptors is thought to be inhibitory to the sensory input from both the tarsaland labellar chemoreceptors thus exerting a modulating &ect on at least the first two stages of feeding behaviour, proboscis extension to tarsal and labellar stimulation (GELPEFUN, 1971). The results of this study indicate that proboscis extension to labellar stimulation is not modulated by activity of either of the internal stretch receptor types. Normally, the labellar sensilla will be stimulated only after the proboscis has been extended due to tarsal stimulation. Thus modulation of the tarsal sensory input by the internal stretch receptors is sufficient to prevent inappropriate proboscis extension when the fly is food satiated. Starvation and feeding cause dramatic changes in tarsal thresholds (EVANSand DETHIER,1957), but labellar thresholds appear to be unaffected. The modulation of feeding by the internal receptors is apparently accomplished by the inhibition of the sensory input driving the first step of the behaviour, proboscis extension to tarsal stimulation, but apparently not on subsequent steps of the behaviour, proboscis extension and sucking driven by labellar stimulation (Fig. 3). Such a control system is similar to ‘end-product’ inhibition common to many biosynthetic pathways (CONNand STUMPF,1967). The function of the labellar sensillain feeding behaviour appearsto be twofold ; first the localization and characterizationof potential food once the proboscis has
P. A. GETTINGAND
1680
R. A.
STEINHARDT
been extended by tarsal stimulation, and second, the initiation of oral lobe spreading and sucking (DETHIER, 1955). Stimulation of the tarsal receptors indicates the presence of food but does not localize the food under the labellum. The localixation is probably accomplished by the labellar sensilla. Observation of feeding flies LAIELLAR SUGAR RECEPTORS
INlERNtUROH +
L
+
. .
f TARSAL
MOTOR WEURON
+
SUSAR
:%
SECEPTORS
IYTEnIEuaow
_-o_____-_--
q*
_*-______
qJ_j
,-. yl' ‘7;"
+r , /
-_=
.
k--3z!z
--G-
_______qoJ
FOlEEUT1 AIDOYIIAL
EXTENSOR
MUSCLE
StRETCH IECLPTORS
FIG. 3. A neuronal model to account for motor activity in the extensor of the hauatellum in response to sucrose stimulation of labellar and tarsal chemosensory hairs. The labehar sugar recepton converge on intemeuron L permitting summation between receptor inputs. Activation of interneuron L causes proboscis extension via the motor neuron, thus interneuron L is the ‘decision making’ element of the network. A similar but independent pathway is shown for tarsal sugar receptor input. Tarsal threshold regulation is probably achieved by inhibitory input from the foregut and abdominal stretch receptors onto the ‘decision making’ intemeuron T. Excitatory synapsea are ahown with + sign; inhibitory with - sign.
supports this view. Stimulation of labellar sensilla on the anterior portion of the 1abelIum causes anteriorly directed proboscis extension. Similarly, stimulation of sensiha on the posterior portion results in posteriorly directed extension. In addition, stimulation of sensilla on one lobe of the labellum can cause spreading of the ipsilateral lobe but not the contralateral. Direct observation of position labelling of the labellar se&la could be demonstrated by recording motor activity from both extensor muscles simultaneously. Differential activation of these two muscles would indicate an asymetrical extension of the proboscis. Acknoudedgements-Th research was funded in part by the following grants: a predoctoral training grant to P. A. G., USPHS grant No. S-TOl-GMO0829, and a Biomedical Support Grant and USPHS grant No. GM1021-08 to R. A. S. We wish to thank SUZANNB STANFORDfor her assistance with the behavioural experiments and EMILY REID for the preparation of the figures and drawings.
INTERACZIONOF REUE’TORSON BLOWFLY PeEDINGBEI-IAVIOUR
1681
REFERENCES ARAB Y. M. (1957) A study of some aspects of contact chemoreception in the blowfly. Ph.D. thesis, Johns Hopkins University, Baltimore, Maryland. CONN E. E. and .%JMPF P. F.‘(1967) In Outlines of Biochemistry. John Wiley, New York. DETHIER V. G. (1955) The physiology and histology of contact chemoreceptors of the blowfly. Quart. Rev. Biol. 30, 348-371. DBI’HIERV. G. (1968) Chemosensory input and taste discrimination in the blowfly. Scimce, Wash. 161, 389-391. DOUGHIER V. G. (1969) Feeding behavior of the blowfly. Ado. tim. Behau. 2,111-266. DETHIER V. G. and BODENSTEIND. (1958) Hunger in the blowfly. 2. TierpgdaoE. 15, 129-140. DETHIER V. G., EVANB D. R and RHOADESM. V. (1956) Some factors controlling the ingestion of carbohydrates by the blowfly. Biol. Bull., Woods Hole, 111, 204-222. DETHIER V. G. and GELPERIN A. (1967) Hyperphagia in the blowfly. J. exp. Biol. 47, 191-200. EVANS D. R. and BARTON BROWNE L. (1960) Physiology of hunger in the blowfly. Am.
Mid. Nat. 64,282-300. EVANSD. R. and DETHIERV. G. (1957) The regulation of taste thresholds for sugars in the blowfly. J. Insect Physiol. 1, 3-17. GELPBRIN A. (1967) Stretch receptors in the foregut of the blowfly. Science, Wash. 157,
208-210. GELPERINA. (1971) Abdominal sensory neurons providing negative feedback to the feeding behaviour of the blowfly. Z. vergl. Physiol. 72, 17-31. GELPFXINA. and DETHIER V. G. (1967) Long-term regulation of sugar intake by the blowfly. Physiol. Z&l. 40, 218-228. GOING P. A. (1971) The sensory control of motor output in fly proboscis extension. , Z. vergl. Physiol. 74, 103-120. GILLARY H. L. (1966) Stimulation of the salt receptor of the blowfIy-111. The alkali halides. J. gen. Physiol. 50, 359-368. MINNICH D. E. (1931) The sensitivity of the oral lobes of the proboscis of the blowfly, Calliphora uomitoria Linn., to various sugars. g. exp. 2001. 60,121-139.
THE INTERACTION OF EXTERNAL AND INTERNAL RECEPTORS ON THE FEEDING BEHAVIOUR OF THE BLOWFLY, PHORiWA REGINA P. A. GETTING=
and R. A. STEINHARDT
Department of Zoology, University of California, Berkeley, California (Received 7 March 1972) Abstract-The interaction of aensory activity from internal gut stretch receptors and from external labellar chemoaenao ryhairshasbeenstudiedboth behaviourally and electrophysiologically in the control of proboscis extension of the blowfly, Phomda regina. Labellar thresholds for proboscis extension, tested behaviourally, do not change significantly up to an hour after feeding in contrast to tarsal thresholds which rise quickly after feeding. Motor activity of the extensor muscle of the haustellum was recorded simultaneously with senaory activity from labellar sensilla. The mean number of muscle spikes per response and the sensory input neceaaary to trigger a reaponse do not vary with starvation, feeding, or sectioning of the recurrent nerve. Activity of internal stretch receptors seem to interact with tarsal sensory input but apparently do not modulate motor responses triggered by labellar sensory input. INTRODUCTION
interaction of information from internal and external receptor systems is commonly used to produce behaviours appropriate to an animal’s needs at the time. Feeding behaviour is a prime example of such a homeostatic mechanism. Information from receptors signalling the animal’s internal state such as nutritional condition or gut capacity can be used to modulate sensory input from external receptor systems signalling the presence of food. The feeding behaviour of the blowfly, Phti regtiu, provides an excellent opportunity for an electrophysiological study of the interaction between internal and external sensory systems in the control of behaviour. The complete feeding behaviour of the blowfly consists of a series of distinct behavioural acts controlled by three sets of external contact chemoreceptor systems. Chemosensory hairs located on the tarsi of all six legs are used to localize and characterize potential food. When stimulated with certain carbohydrates, they initiate extension of the retractable proboscis. Extension of the proboscis can result in the stimulation of the labellar chemosensory hairs which cause further extension of the proboscis, spreading of the oral lobes and initiation of sucking. Proboscis extension and sucking appear to be maintained by the stimulation of the interpseudotracheal papillae located on the inner surfaces of the oral cavity. The THE NEURONAL
* Present address : Department of Zoology, University of Washington, Seattle, Washington 98105. 1673
1674
P. A. GETTING AND R. A. STEINELUXDT
sensory control of proboscis extension by stimulation of the tarsal or labellar chemoreceptors has been extensively studied behaviourally (DETHIER, 1969) and electrophysiologically (GETTING, 1971; D~HIER, 1968). Clearly, feeding, once initiated, does not proceed indefinitely. At the cessation of feeding the fly has consumed a distinct quantity of food depending upon the type and concentration of the sugar (DETHIER et al., 1956). Immediately after feeding both tarsal and labellar thresholds are high due to adaptation, both receptor and central. However, the threshold for tarsal stimulation remains high well after a period suflicient for disadaptation (EVANSand DETHIER, 1957; EVANSand BARTONBROWN, 1960). Tarsal thresholds attain a maximum value between one and six hours after feeding and then decline over a period of days. Similar increases in labellar thresholds with feeding have been reported (ARAB, 1957). However, MINNICH (1931) reports no change in labellar thresholds with starvation in a closely related blowfly, Caliiphora vommitoria. Two sets of internal receptors have been identified which are thought to modulate the excitability of the feeding response to stimulation of the tarsal and labellar chemoreceptors. Denervation of the foregut by section of the recurrent nerve results in increased consumption and in some cases hyperphagia (DBTHIER and BODENSTEIN, 1958; EVANSand BARTONBROWNI,1960; DETHIERand GELPERIN,1967). GELPERIN(1%7) reports the identification in the foregut of stretch receptors which respond to distension with a tonic discharge pattern. These receptors send information to the brain via the recurrent nerve signalling the presence of food in the foregut and are hypothesized to be inhibitory to the chemoreceptor inputs (GELPERIN and DBTHIER, 1967). In addition, section of the ventral nerve cord or median abdominal nerve results in increased feeding suggesting that the activity of identified stretch receptors in the abdominal nerve network is also inhibitory to feeding (DHIIIIER and GELPERIN, 1967; GELPERIN, 1971). This study was undertaken to observe the influence of internal stretch receptors on the excitability of motor neurons involved in proboscis extension to sensory input from iabellar sugar receptors. Proboscis extension to labellar stimulation was investigated behaviourally before and after feeding known quantities of food. In addition the effects of feeding, starvation, and section of the recurrent nerve were investigated electrophysiologically by recording single unit motor output to the extensor muscle of the haustellum while simultaneously recording single unit sensory input from the labellar chemosensory hairs. This electrophysiological approach has several advantages over the behavioural measurement of feeding thresholds. By directly recording all the sensory input involved in initiating a motor act we were able to exclude possible effects of receptor adaptation and insure stable sensory frequencies throughout each experiment. In addition, by direct recording of the motor activity we had a quantitative measure which allowed us to time our test stimulations to avoid the effects of central habituation (GETTING, 1971). We found that the excitability of the feeding response to labellar stimulation does not appear to be modulated by either foregut or abdominal stretch receptor activities.
OF RBCBFTORS ON BLOWFLY
INTERACTION
FEEDING
BBHAVIOUB
1675
MATERIALS AND METHODS Blowflies, Phormia regina Meigen, age 3 to 18 days, 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 water and sucrose. Both males and females were used. Starvation and feeding were carried out as follows. When starvation was necessary, the flies were taken either directly from the cage or given free access to 100 mM sucrose solutions for 24 hr prior to starvation. Flies were starved at room temperature in small plastic boxes with free access to water. For feeding while the animal was mounted in the apparatus, the end of a 10 ~1 capillary, filled with 1-OM sucrose, was placed against the labellar lobes and the fly allowed to drink 10 to 20 ~1 of solution. This quantity is approximately the mean consumption of intact, free-moving flies (GELPERIN,1971). Behavioural studies Flies were starved 24 hr prior to being mounted on a wax sheet by the wings. Behavioural responses, proboscis extension, were tested by stimulation of single labellar hairs with 100 mM sucrose. The flies were divided into two groups: group 1 was fed 10 ~1 of 1-OM sucrose and tested for proboscis extension 15 and 60 min after feeding, group 2 was fed 15 to 20 ~1 of 1.0 M sucrose and tested immediately and 15 min after feeding. All flies were satiated with water before testing and feeding with sucrose solutions. Electmphysiologica~ studies Electrophysiological recordings from the extensor muscle of the haustellum and from single labellar chemosensory hairs were made using a technique previously described in detail (GETTING,1971). Briefly, the proboscis of a legless fly was fixed in a semi-extended position by inserting the constriction between the haustellum and rostrum into a slot in a silver-chlorided 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 label& hairs was recorded by placing a capillary over the tip of the hair. The capillary, connected to the input of a high impedance amplifier, contained various concentrations of sucrose mixed with 50 mM LiCl to provide electrical conductivity. This concentration of LiCl was insufficient to stimulate the salt receptor (GILLARY,1966). Both sensory and motor signals were amplified by conventional electrophysiological techniques and displayed simultaneously on an oscilloscope. Consistent sensory and motor responses could be recorded for several hours. RESULTS
If the activity of the foregut and abdominal stretch receptors is inhibitory to sensory inputs from the tarsal and labellar chemoreceptors as suggested (GELPEFUN and DJZTHIER, 1967) then the responsivenessof motor neurons controlling proboscis
1676
P.
A. GETTINGAND R. A.
%-RINHARDT
extension should vary with starvation and feeding. behaviourally and electrophysiologically.
This hypothesis was tested both
Behaviowal studies Proboscis extension was tested before and after feeding by applying 100 mM sucrose to single labellar hairs. Before feeding all flies gave a full proboscis extension to the sucrose solution but not to water. The flies were divided into two groups according to the volume of. 1-OM sucrose consumed: one group was allowed to consume 10 4, the other 15 to 204 Table 1 summarizes the responses at various times after feeding. All flies gave a full proboscis extension to TABLE 1. BEI-IAVIOURAL RBBPONSES TO AND AFI’RR FEEDING (-hST
SINGLR LABRLLAR HAIR STIMULATION STIMULUS
100 d’f
BEFORE
SUCROSR)
Behavioural responses Post-feeding (min after feeding)
Volume fed (1.0 M sucrose)
No. of ties tested
Pre-feeding
0
15
60
10/d 1 S-20 /.d
35 25
+ +
n.t. +
+ +*
rift.
+--full extension of the proboscis. n.t.-not tested. *-one fly in this group gave only a partial extension to the test stimulus.
the test stimulus at all times after feeding with one exception. One fly gave only a partial response 15 min after consuming 15 to 20 ~1. All other flies of the same group gave full responses. There are two possible interpretations of this data. Either there is no threshold change due to feeding or the test stimulus of 100 mM sucrose was suprathreshold even after feeding. Previous work (GETTING,1971) has shown that the sensory activity resulting from stimulation of a single labellar hair with 100 mM sucrose is just slightly above the minimum sensory input necessary to initiate proboscis extension for starved flies. Therefore it seems unlikely that a threshold increase due to feeding could have occurred. This conclusion agrees with the findings of MINNICH (1931) that labellar thresholds of Calliphora do not change significantly with starvation. Ekctrophysiologakal studies: effects of starvation These behavioural results were directly confirmed by electrophysiological measurements. The excitability of motor neurons controlling proboscis extension was observed by recording motor activity from the extensor of the haustellum and single unit sensory activity from labellar chemosensory hairs. Motor responsiveness was judged by two parameters ; (1) the sensory input necessary to trigger a motor response, and (2) the number of motor spikes per response. Motor output
INTERACTION OF RRCRF’TORSON BLOWFLY FEEDING BEHAVIOUR
1677
to the extensor of the haustellum can be triggered by three general classes of trigger conditions. Class I is motor activity triggered by a single sensory spike (Fig. 1). Class II triggering requires the temporal summation of only two sensory spikes (Grrrrr~c, 1971). Class III is motor activity triggered by the temporal summation of two or more sensory spikes depending upon the iirst sensory interspike interval (GE?TING, 1971). Flies exhibiting all three trigger classes were observed at all starvation times from 0 to 72 hr indicating no consistent trend towards a lower threshold with starvation.
FIG. 1. Electrophysiological recordings from a single labellar sensilla stimulated with 25 mM sucrose (S) and the reaultant motor response (M) recorded from the extensor of the hauatellum. The large arrow at the beginning of the sensory record marks an artifact at the moment the stimuhrs solution contacts the hair. Each of the two sugar receptor spikes (small arrows) is followed by a motor response ilhrstrating Class I triggering. Water receptor activity (small spikes of sensory trace) is also present, however, this activity has been shown not to be involved in triggering motor activity of a water-satiated fly (GETTING, 1971). Tune mark, 100 msec.
The mean number of motor spikes per response for 20 flies starved between 0 and 72 hr is summarized in Table 2. No effect of starvationon motor responsiveness is concluded since none of the mean response levels differ significantly. The large standard deviation for the unstarved flies is due to the unusual responses of two flies; one which gave no motor response to the sensory test frequency used, and another which gave an abnormally larger response. TABLE 2. MEAN MOTOR
RESPONSE VB. HOURS OF STARVATION Houra
0 Mean motor response (S.D.) N
22.2 (21.2) 5
starved
24
62
72
16.0 (7.2) 4
18.0
141 (4.0) 8
Y7)
The motor response level (motor spikes/response) was determined at a standardized sensory input of 30 impulses/set. There is no significant difference (P = O-01) between any of the mean responses by a ‘t-test’.
Eflects of feeding on motor output To avoid individual variations in responsiveness and the necessity for interanimal comparisons, we investigatedthe effects of feeding on motor responsiveness.
P. A. GE~TINC AND R. A. STIXNHARDT
1678
The motor response level of a starved fly was determined by several stimulations of a single labellar hair with 100 mM sucrose. The fiy was then fed 20 ~1 of 1-OM sucrose from a pipette. The post-feeding motor response level was again determined by several 100 mM sucrose stimulations at 10 min intervals for up to 30 min after feeding, Figure 2 shows the motor response level as a function of time before and after feeding for one of three experiments. There is no sign&ant change in the motor response level in the tests made for up to 30 min after feeding. The sensory spike frequency was the same for all stimulations.
z
B
r . f .z ,o
.
18 16 1412IO-
l
/\/i-
. t
Fed 20~1 1.0 M sucrose
0’
I
0
I
IO
20 Time
I
30 40 (minutes1
50
60
FIG. 2. Motor response level as a function of time before and after feeding for one of three experiments. At the arrow the fly was fed 20 ~1 of 1-O M sucrose. The sensory spike frequency was the same for all stimulations.
EVANSand BARTON BROWNE(1960) report that ingestion of only 3 4 of 2 M glucose causes observable increases in tarsal thresholds 15 min after feeding presumably by stimulating the foregut stretch receptors. In addition, ingestion of only 6 ~1 of 1-OM sucrose causes a threefold increase in abdominal stretch receptor activity (GELPERIN, 1971). Therefore, the quantity of 1.0 M sucrose ingested by the flies in this study (20 ~1) seems sufBcient to cause significant increases in the activity of both stretch receptor types. However, there is no observed change in motor responsiveness to label& stimulation after feeding. Section of the recurrent nerve As a final check on the possible inhibitory role of the foregut stretch receptors, we investigated the effects of sectioning the recurrent nerve which contains the afIerent axons of the stretch receptors. If the foregut receptor activity is inhibitory to labellar sugar receptor input, then sectioning of the recurrent nerve should result in an increased motor responsiveness. The recurrent nerve was sectioned anterior to the corpora cardiaca by the technique of DETHIERand BODENSTEIN (1958). The mean motor response of flies with severed recurrent nerves is not significantly different (P = O-01) than that of the controls (Table 3).
INTERACTION
TABLE 3. MEAN
OF RECEPTORS ON BLOWFLY
MOTOR
RESPONSE
1679
FEEDING BEHAVIOUR
OF RECURRENT NZRVE sEcTIoNED
FLIm
vs.
CONTROLS
Recurrentnerve Mean motor response (S.D.) N
Control
sectioned
19.4 (159) 9
17.2 (i.7)
The motor response level (motor spikes/response) was determined at a stanThere is no significant difference dardized sensory input of 30 impulses/set. (P = O-01) between the mean responses by a ‘t-test’. The large large standard deviation of the control group is due to the unusual responses of two flies; one which did not respond at the test frequency and another which gave an abnormally large response.
The findings that motor responsiveness to labellar sugar stimulation is not altered by starvation,feeding, or section of the recurrent nerve leads us to conclude that there is no inhibitory feed-back from either the foregut or abdominal stretch receptors to sensory input from the labellar sugar receptors. This does not, however, rule out the possibility of inhibition of feeding activated by sensory input from the tarsalsugar receptors as demonstratedbehaviourally (EVANSand DRIER, 1957; EVANSand BARTONBROWNE,1960). DISCUSSION
Behavioural studies suggest that the feeding behaviour of the fly is modulated in part by activity of at least two sets of internal stretch receptors. The activity of these internal stretch receptors is thought to be inhibitory to the sensory input from both the tarsaland labellar chemoreceptors thus exerting a modulating &ect on at least the first two stages of feeding behaviour, proboscis extension to tarsal and labellar stimulation (GELPEFUN, 1971). The results of this study indicate that proboscis extension to labellar stimulation is not modulated by activity of either of the internal stretch receptor types. Normally, the labellar sensilla will be stimulated only after the proboscis has been extended due to tarsal stimulation. Thus modulation of the tarsal sensory input by the internal stretch receptors is sufficient to prevent inappropriate proboscis extension when the fly is food satiated. Starvation and feeding cause dramatic changes in tarsal thresholds (EVANSand DETHIER,1957), but labellar thresholds appear to be unaffected. The modulation of feeding by the internal receptors is apparently accomplished by the inhibition of the sensory input driving the first step of the behaviour, proboscis extension to tarsal stimulation, but apparently not on subsequent steps of the behaviour, proboscis extension and sucking driven by labellar stimulation (Fig. 3). Such a control system is similar to ‘end-product’ inhibition common to many biosynthetic pathways (CONNand STUMPF,1967). The function of the labellar sensillain feeding behaviour appearsto be twofold ; first the localization and characterizationof potential food once the proboscis has
P. A. GETTINGAND
1680
R. A.
STEINHARDT
been extended by tarsal stimulation, and second, the initiation of oral lobe spreading and sucking (DETHIER, 1955). Stimulation of the tarsal receptors indicates the presence of food but does not localize the food under the labellum. The localixation is probably accomplished by the labellar sensilla. Observation of feeding flies LAIELLAR SUGAR RECEPTORS
INlERNtUROH +
L
+
. .
f TARSAL
MOTOR WEURON
+
SUSAR
:%
SECEPTORS
IYTEnIEuaow
_-o_____-_--
q*
_*-______
qJ_j
,-. yl' ‘7;"
+r , /
-_=
.
k--3z!z
--G-
_______qoJ
FOlEEUT1 AIDOYIIAL
EXTENSOR
MUSCLE
StRETCH IECLPTORS
FIG. 3. A neuronal model to account for motor activity in the extensor of the hauatellum in response to sucrose stimulation of labellar and tarsal chemosensory hairs. The labehar sugar recepton converge on intemeuron L permitting summation between receptor inputs. Activation of interneuron L causes proboscis extension via the motor neuron, thus interneuron L is the ‘decision making’ element of the network. A similar but independent pathway is shown for tarsal sugar receptor input. Tarsal threshold regulation is probably achieved by inhibitory input from the foregut and abdominal stretch receptors onto the ‘decision making’ intemeuron T. Excitatory synapsea are ahown with + sign; inhibitory with - sign.
supports this view. Stimulation of labellar sensilla on the anterior portion of the 1abelIum causes anteriorly directed proboscis extension. Similarly, stimulation of sensiha on the posterior portion results in posteriorly directed extension. In addition, stimulation of sensilla on one lobe of the labellum can cause spreading of the ipsilateral lobe but not the contralateral. Direct observation of position labelling of the labellar se&la could be demonstrated by recording motor activity from both extensor muscles simultaneously. Differential activation of these two muscles would indicate an asymetrical extension of the proboscis. Acknoudedgements-Th research was funded in part by the following grants: a predoctoral training grant to P. A. G., USPHS grant No. S-TOl-GMO0829, and a Biomedical Support Grant and USPHS grant No. GM1021-08 to R. A. S. We wish to thank SUZANNB STANFORDfor her assistance with the behavioural experiments and EMILY REID for the preparation of the figures and drawings.
INTERACZIONOF REUE’TORSON BLOWFLY PeEDINGBEI-IAVIOUR
1681
REFERENCES ARAB Y. M. (1957) A study of some aspects of contact chemoreception in the blowfly. Ph.D. thesis, Johns Hopkins University, Baltimore, Maryland. CONN E. E. and .%JMPF P. F.‘(1967) In Outlines of Biochemistry. John Wiley, New York. DETHIER V. G. (1955) The physiology and histology of contact chemoreceptors of the blowfly. Quart. Rev. Biol. 30, 348-371. DBI’HIERV. G. (1968) Chemosensory input and taste discrimination in the blowfly. Scimce, Wash. 161, 389-391. DOUGHIER V. G. (1969) Feeding behavior of the blowfly. Ado. tim. Behau. 2,111-266. DETHIER V. G. and BODENSTEIND. (1958) Hunger in the blowfly. 2. TierpgdaoE. 15, 129-140. DETHIER V. G., EVANB D. R and RHOADESM. V. (1956) Some factors controlling the ingestion of carbohydrates by the blowfly. Biol. Bull., Woods Hole, 111, 204-222. DETHIER V. G. and GELPERIN A. (1967) Hyperphagia in the blowfly. J. exp. Biol. 47, 191-200. EVANS D. R. and BARTON BROWNE L. (1960) Physiology of hunger in the blowfly. Am.
Mid. Nat. 64,282-300. EVANSD. R. and DETHIERV. G. (1957) The regulation of taste thresholds for sugars in the blowfly. J. Insect Physiol. 1, 3-17. GELPBRIN A. (1967) Stretch receptors in the foregut of the blowfly. Science, Wash. 157,
208-210. GELPERINA. (1971) Abdominal sensory neurons providing negative feedback to the feeding behaviour of the blowfly. Z. vergl. Physiol. 72, 17-31. GELPFXINA. and DETHIER V. G. (1967) Long-term regulation of sugar intake by the blowfly. Physiol. Z&l. 40, 218-228. GOING P. A. (1971) The sensory control of motor output in fly proboscis extension. , Z. vergl. Physiol. 74, 103-120. GILLARY H. L. (1966) Stimulation of the salt receptor of the blowfIy-111. The alkali halides. J. gen. Physiol. 50, 359-368. MINNICH D. E. (1931) The sensitivity of the oral lobes of the proboscis of the blowfly, Calliphora uomitoria Linn., to various sugars. g. exp. 2001. 60,121-139.