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Summation and gradient characteristics of local electroantennogram response of the European corn borer, Ostrinia nubilalis
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
24, 32-39 (1985)
Summation and Gradient Characteristics of Local Electroantennogram Response of the European Corn Borer, Osirinia nubilaiis TOSHIO Agriculture
NAGAI
Canada, Research Centre. University Sub Post Office, London, Ontario N6A 5B7, Canada Received March 21, 1984: accepted August 20. 1984
Summation and gradient characteristics of the electroantennogram (EAG) responses of male moths of the European corn borer (Ostrinia nubilalis) to their pheromone, Q-11-tetradecenyl acetate, were studied. Direct evidence that the summation of the locally elicited EAGs contribute to the overall response amplitude is presented; if two simultaneously stimulated loci were separated by more than 1 mm, the observed response was the summation of the two independent local EAGs. Configuration of the local EAG response was obtained at various loci of the antenna. It was demonstrated that a gradient in the amplitude of the local EAG exists along the antenna1 axis and that this gradient has its origin in the epithelial layer of the antenna. Several possible morphological and physiological factors affecting the response characteristics were considered. o 198s Academic Press. Inc.
(7). This finding suggested that the EAG is a summed result of local responses generated between the two electrodes; i.e., the greater the antenna1 length between the electrodes, the larger the number of receptor units which might be involved. As the next step of the investigation, local stimulation was applied to study the distribution of potentials along the antenna. It has been found that a gradient exists in the EAG along the antenna1 axis (7, 8); the response amplitude becomes larger when the locally stimulated point is shifted closer to the distal tip. Moreover, the locally generated response spreads from the excited point in an asymmetric manner, predominantly in the proximal direction. An equivalent circuit diagram has been proposed, which includes a mechanism for generating the local EAG characteristics (8). It still remains to be demonstrated, however, whether the local potentials in the antenna are actually additive. Furthermore, there is a question of the effect of different biological factors on the EAG gradient. The following experiments demonstrate
INTRODUCTION
The electroantennogram (EAG) has been used as a tool to study antenna1 receptor function for sex pheromones of moths. The EAG response is assumed to be the summation of bioelectrical potentials generated by many olfactory receptor cells responding to odor stimulants. Although the bioelectrical activities have been discussed extensively at the level of the receptor unit in a sensillum [e.g., (l-6)], very few of these studies have described the summation aspect of EAG responses. The EAG is usually registered by a pair of electrodes placed at the distal region and proximal base of an isolated antenna. The European corn borer antenna is relatively easy to use for EAG studies because of its thread-like simple external form. Recently, several interesting characteristics have been found in the EAG response along the axis of the antenna. When the whole preparation is evenly stimulated, the response amplitude is approximately proportional to the distance between the recording and the indifferent electrodes placed on the antenna 32 0048-3575185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
ELECTROANTENNOGRAM
RESPONSE
in detail the unique pattern of the local response. Further studies on summation phenomena were conducted by simultaneously stimulating two separate loci on the antenna. Several morphological and physiological factors such as local excitability difference, slant angle of sensory hair, electric resistance of hemolymph space, and axonal activity were examined to determine their effect on the response gradient. MATERIALS
AND
METHODS
Antennae from 3-day-old adult male European corn borer moths, Ostrinia nubilalis, were used. The experimental procedures and apparatus used in recording the EAG were basically the same as those previously described (8). The isolated antenna was mounted on a wax plate. Compressed air was passed constantly over the whole preparation perpendicularly to the antenna1 axis at a speed of 30 cmisec. into this air stream, a fine air jet with a speed of 30 cm/set was introduced through a small funnel (inside diameter at the outlet: 0.2 mm), and was aimed at a specific point on the antenna1 surface. Pure air in the jet was switched to stimulant-containing air for a measured time by means of a rotary valve controlled by a solenoid (8). Another identical jet was also used when needed for simultaneous stimulation of a second location. Preparation of the stimulant pheromone, (Z)-IItetradecenyl acetate, has been given elsewhere (9). For measurement of electric resistance through the antenna a modified Wheatstone bridge (10, 11) was used. In some preparations a thin tungsten wire was inserted through the core of the antenna from the proximal cut end. The wire had a diameter of 25 p.m and the tip was slightly tapered by flame to make the insertion easier. Some preparations were perfused internally through the hemolymph space with physiological saline (12) or a modified saline containing tetrodotoxin (l- 10 x lop5 M) at a rate of 0.3-0.8 x 10e3 ml/
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min. Tetrodotoxin was used to block the action potential of the antenna1 axons. A glass micropipet (outside diameter at the outlet: 75-80 km) was inserted into the proximal cut end of the antenna for the perfusion, and the outflowing saline was withdrawn at the distal cut end. All recordings were at room temperature, 22°C. RESULTS
AND
DISCUSSION
Gradient in the Local EAG Response EAG potentials evoked by stimulant chemicals appear as negative changes in the resting potential (9). These are easily detected by an electrode placed at the cut tip together with an indifferent electrode at the cut proximal end. This is the conventional device used by most EAG investigators. When the preparation was locally excited, the recorded EAG response to a given intensity of stimulus was found to depend on the locus of the antenna. Thus, the recorded amplitude became smaller proximally (Figs. 1A and B), approaching zero and frequently reversing its polarity from negative to positive near the proximal end (Fig. 1C). The stimulated location represented by the three points-p, c, and d in Fig. l-was shifted right and left to examine the whole antenna1 length, and the change in the amplitude was found to be gradual from the distal to the proximal end. From these results it can be seen that there is an amplitude gradient in the EAG along the antenna1 axis of the European corn borer. A similar gradient in amplitude was reported in previous work (8). The recorded EAG potential with two electrodes placed at the cut tip and base as described above probably included the sum of the injury potentials at the cut ends in addition to the evoked response potential. Moreover, the resulting potential might be reduced due to a short circuit through the cut ends via the antenna1 hemolymph. Without the injury and short circuit effects, the graded pattern of the EAG response
34
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FIG. 1. EAG responses of European corn borer recorded at the distal end relative to the proximal base to (Z)-11-tetradecenyl acetate (20 ,ug). The distalfive segments were removed. Measurement of three stimulated locations of the antenna-distal(d), central(c), and proximal (p)-are illustrated on the top right. Length calibration for the preparation is on the bottom right; i.e., the distance between d and p was 4 mm. The recording and stimulation arrangements are shown in schematic diagrams to the right of each EAG recording. Vertical arrow indicates recording electrode, and thick horizontal line represents antenna. Same preparation was used for (A) to (E). (A. B. and C) Distal, central, and proximal locations were stimulated, respectively. (D) Two locations, d and c, were stimulated simultaneously. (E) Two locations, d and p, were stimulated simultaneously. Time of stimulation: 1 sec. Response amplitudes measured at the end of stimulation time in (A) to (E) were - 1 S, - 0.6. fO.3, -2.1, and -1.2 mV, respectively. EAG calibration is at the bottom center. Time after excision: 30 min.
along the antenna1 axis could be measured simply by placing the two electrodes in contact with the intact surface of the antenna. Typical results are shown in Fig. 2. The response amplitude recorded from a distal point d reached the maximum if point d was stimulated (Fig. 2A). When the stimulated locus was shifted from d toward proximal point p, the response amplitude became smaller, frequently changing its polarity near point p, and then reached the other extreme amplitude with reversed polarity
NAGAI
FIG. 2. EAG responses of European corn borer recorded with surface electrodes to (Z)-ll-tetradecenyl acetate (20 ug). The distal five segments were removed. Location of the electrodes (d and p) is illustrated on the top right. Same preparation was used for (A) to (G). (A-E) The stimulated location was shifted stepwise from d to p at intervals of 1 mm. (F) The recording and indifferent electrodes of the preparation in (E) were exchanged. (G) Two locations, d and p. were stimulated simultaneously. Time of stimulation: I sec. Response amplitudes measured at the end of stimulation time in (A) to (G) were - I .6, - I .I, -0.5, + 0.5, f 1.2, - 1.2, and -0.4 mV. respectively. Time after excision: 30 min. For further explanation, see Fig. 1.
(Figs. 2A-E). Thus, the characteristic response gradient along the antenna1 axis was also clearly demonstrated with surface electrodes. If the two electrode positions shown in Fig. 2E were interchanged as in Fig. 2F, i.e., the recording and the indifferent electrodes were placed at p and d, respectively, a mirror image response of the one shown in Fig. 2E could be obtained. The negative polarity of the response in Fig. 2F indicates that regardless of the stimulated region, distal (Fig. 2A) or proximal (Fig. 2F), the EAG recorded at the excited point is al-
ELECTROANTENNOGRAM
RESPONSE
ways negative relative to the unstimulated neighboring surface of the antenna (7, 8). When the response was recorded at different distances from the stimulated point as in Figs. 2B, C, or D, a spread effect of the response from the stimulated region in opposite directions along the antenna1 axis was noted and has been shown previously (8). This resulted in the reversed polarity of the response (Figs. IC and 2D). As discussed in the previous work (S), the locally elicited potential is assumed to be a temporal change in the resting receptor potential of the sensory hair unit circuit. The potential generated in response to stimulation in the unit circuit can be recorded by an electrode placed at the excited point, and is found to be negative relative to an indifferent electrode at the distant region. This seems to indicate that a current flows into the excited region from the resting regions on both sides. Although the local EAG responses shown in the present study (Figs. 2A and F) are not generated from a single sensory unit, their negativity may suggest that the excited local region becomes a sink for the current flow. Summated Configuration EAG Response
of the
When many sensory units were excited at the same time, the elicited local potential changes were summed, and could be recorded as a total EAG response. To demonstrate this summation, two different local regions of an antenna were stimulated simultaneously with two identical stimulation jets, and the resulting response pattern was observed. It was found that the two responses were simply added if the distance of the locally stimulated regions were greater than 1 mm. For example, the response in Fig. ID is the sum of the individual responses at d and c (Figs. IA and B) separated by 2 mm. Likewise the response in Fig. 1E is the sum of the responses at d and p (Figs. IA and C), but is smaller than the single response at d (Fig. 1A) because of the reversed polarity of the
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response at p (Fig. IC). A similar result was also obtained from surface recordings; the summed response in Fig. 2G is smaller than the single response in Fig. 2A. Thus, despite a doubling of the stimulus intensity, the resulting response may be smaller than the single component responses depending on the loci of the stimulations with respect to the recording electrode. In any case, the response recorded by two stimulations simply showed up as the sum of the individual responses. If two locally stimulated regions were closer than 1 mm, the amplitude of the resulting EAG was smaller than the sum of the two individual responses. This reduction may be due to the two jet streams colliding and, with the resulting turbulence, failing to deliver two full stimulant dosages. On the other hand, if the region excited by one jet is approximately 1 mm wide, because of overlap, the total area excited by the two jets would be less than 2 mm wide. At the concentration used in this study, 20 kg, stimulation is maximal (9) and could therefore not be greater in the overlap region in spite of the larger amount of stimulation delivered there. There may also be an active interaction between two neighboring regions which could affect the individual response amplitudes (8) although the underlying system has not yet been clearly determined. A study of single sensory hair stimulation and the response of a single sensillum (ESG) (13) might determine whether such an interaction between responses exists. Possible Factors Affecting the Response Gradient The positions of the recording and indifferent electrodes shown in Figs. 2A and F are reversed at d and p on the same antenna. In both cases, however, the response was recorded from the stimulated region. If the antenna were not functionally directional along its axis, the response in Figs. 2A and F would be identical. This was not the case; however, the response re-
36
TOSHIO
corded at distal point d was larger in the amplitude than that at proximal point p, showing that there is in fact a clear directional difference. There does not appear to be an excitability difference between distal and proximal regions as no specially differentiated sensilla have been found (14). Furthermore, the distribution density of the sensilla trichodea is almost uniform along the European corn borer antenna (7). If the total number of the sensilla per unit length of antenna were to determine the response summation, the amplitude would be smaller at the distal point than at the proximal point as the total number of the sensilla decreases distally because of the smaller diameter of the antenna. However, this was not the case as already shown in Figs. 2A and E There is a possibility that excision of the distal and proximal ends would induce a different amount of injury potential at the two ends and as a consequence affect the responses at p and d. However, this effect seemed to be minor as the response shape did not show any significant difference after cutting the distal tip than before (Fig. 3). While the stimulation jet was directed
AL FIG. 3. EAG responses of European corn borer recorded with surface electrodes to (Z)-11-tetradecenyl acetate (20 ug). (A) Response was recorded before the distal tip was cut off (B) Response was recorded immediately after the distalfive segments were removed. Time interval between (A) and (B) was 2 min. Locations of the two electrodes and stimulation were similar to those in Fig. 2A. Parallel lines show the time of stimulation: 1 sec. Response calibration on the bottom right: I mV and 1 sec.
NAGAI
perpendicularly to the antenna1 axis, the sensory hairs themselves are slanted about 28” from the perpendicular toward the distal tip. This discrepancy in direction of air flow and hair axis might result in an uneven local stimulation. To examine this possibility, the direction of the jet was altered through an angle of O-40” from the perpendicular toward both the antenna1 tip and the base, while being directed toward the same spot. The configuration of the response was not significantly changed; at an angle of 30” in either direction, however, the amplitude was increased by lo-17% in both cases, but this change was probably due to an increase in the area exposed to the jet. Thus, the response asymmetry along the antenna1 axis appears not to be related to the air flow direction. It was found in the previous work (8) that the local response spreads in both proximal and distal directions from the stimulated point but that the spreading occurs more effectively toward the base. This suggests that more current flows proximally than distally. If so, the increasing cross-sectional area of the antenna in the proximal direction should result in a lower resistance and hence a greater current. To test this possibility the relationship between the antenna1 shape and the resistance was investigated. The gross shape of the male moth antenna of the European corn borer is a slender cone of about 7.8 mm long (range: 7.6 to 8.3 mm) with a circular cross section of 35 pm diameter at the distal end and a rough ellipse with axes of 110 x 130 km at the proximal base, excluding the sensory hairs and scales. The thickness of the epithelial cuticle is 7 km in the distal region and 20 p,rn proximally. The inside hemolymph space has a round form with diameter of 20 pm at the distal end and is a 70 x 90-km oval at the proximal end. These dimensions change in a manner which is proportional to the length from the tip to the base of the antenna. This difference in diameter between distal and proximal regions must increase the electric resistance
ELECTROANTENNOGRAM
RESPONSEOF
of hemolymph space distally in inverse proportion to the cross-sectional area. The resistance along the antenna1 axis between two cut ends was measured. The resistance of the whole antenna with the five distal segments removed was found to be in the range 5 to 7 MLR with considerable variation among preparations. This resistance is probably that of the hemolymph space since the epithelial layer has a much higher resistance (3, 6). Figure 4 shows the resistance-time relations of the separated halves of the same antenna, together with the ratio of their resistances. Although both resistance values decreased with time after excision, the ratio was found to be almost constant: the distal half had 3.4 times the resistance of the proximal half. If the change in taper of the hemolymph space along the antenna1 axis is assumed to be linear, the average cross-sectional area of this slender truncate cone can be calculated for each half of the split antennae. When the major and minor axes of the oval shape were averaged, the cross-sectional diameters of the hemolymph space of the preparations used in Fig. 4 were 80, 50, and 20 km at the proximal end, severed center, and distal end, respectively. The diameter of a cylinder which has the same volume
EUROPEANCORN
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and length as each of the slender truncate cones was calculated. These values were 65.6 pm for the proximal and 36.0 km for the distal half, resulting in average crosssectional areas of 3378 and 1017 pm2, respectively, or a ratio (distal/proximal) of I/ 3.3. Thus the resistance ratio of the two halves of the antenna, 3.4 in Fig. 4, was inversely proportional to that of the crosssectional area. This supports the contention that conductance occurs mainly through the hemolymph space, and that since this conductance increases in the proximal direction, so also will the current. Fifteen preparations gave essentially the same results as that in Fig. 4. To test this conclusion further, an attempt was made to reduce the distal-proximal difference in the hemolymph resistance. To this end, a 25-pm-diameter tungsten wire (having a resistance of approximately 0.1 a/mm) was passed through the core of an antenna from which the distal five to seven segments had been removed. Even after insertion of the wire, the characteristic response difference between distal and proximal regions was still maintained as shown in Fig. 5. Thus the electric resistance difference assumed to be due to the change in the diameter of the hemolymph space of the
12345 05.
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FIG. 4. Resistance-time curves for distal (0) and proximal (0) halves of European corn borer antenna. An excised antenna with the distal five segments remolzed was cut at the center. The length of both halves was 3.9 mm. Calculated ratio of the resistance (distal/ proximal) is shown at the top (a). Time was measured after excision. See text for further explanation.
FIG. 5. EAG responses of European corn borer recorded with surface electrodes to (Z)-11-tetradecenyl acetate (20 ,ug). Locally stimulated locations on the antenna, 1 to 5, are shown in the diagram at the top, with the length calibration for the preparation to the right. Vertical arrow indicates the recording electrode. The distal seven segments were removed. Responses were recorded before (0) and after (0) insertion of a tungsten wire (diam: 25 urn) through the core of the antenna. Recorded potential amplitudes were measured at the end of stimulation time (1 set).
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antenna does not appear to account for the observed response gradient. On the other hand, the tungsten wire insertion might have damaged structures including axons in the antenna1 core. Therefore, the directional nature of the nervous conduction and the regional difference in the number of passing axons, i.e., more axons proximally than distally, are unlikely to contribute to the asymmetrical EAG response gradient. The possible effect of axonal activity was tested directly by a nerve-blocking chemical. Tetrodotoxin (TTX) has a remarkably specific blocking effect on the action potential in most excitable cells [e.g., (15, 16)]. It was found that TTX-saline (l- 10 x 10-j M> perfused through the hemolymph space had little effect on the EAG generation and on its gradient characteristics; the response amplitude showed a smooth decrease without any abrupt change when TTX was introduced (Fig. 6), and the relationship between the amplitudes of the responses to various stimulated loci (1 to 14 in Fig. 6) was not disturbed by the toxin. It has already been shown that the amplitude
NAGAI
of EAG response gradually decreases throughout the life span of the preparation (9). This present result directly suggests that the underlying mechanism causing the response gradient is not dependent on the axonal activity in the antenna1 hemolymph space. Thus, the main source for the characteristic response seems to be in the epithelial layer. The bioelectrical response in the sensory unit of the antenna may occur in a circuit passing through the receptor cell and the supporting auxillary cells in the sensory hair (3, 6). The local current source in the unit circuit could act like a battery and discharge in response to stimulation. The numerous batteries of the units could be interconnected in parallel or series along the antenna1 axis (17). If these were merely connected in parallel, neither summation nor directional gradient in the response amplitude would be expected. Therefore, there must be a series connection component between adjacent unit circuits (8). The present study demonstrates that the interaction network responsible for the EAG characteristics are mainly in the epithelial layer where all the receptor units are inlaid. However, direct evidence at the cellular level of the receptor remains to be demonstrated. ACKNOWLEDGMENTS
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FIG. 6. EAG
I thank Dr. D. G. R. McLeod for his encouragement and criticism. I am also indebted to Dr. D. M. Miller for reading the manuscript, to Dr. A. N. Starratt for a supply of the chemical, and to Mr. G. R. Driscoll and Mrs. M. E. Stevens for their assistance.
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amplitude-time curves for European corn borer. The preparation was perfused internally from the proximal end: Response during perfusion with normal saline (0) and TTX-saline. 5 X 10m5 M. (@). The distal 12 segments were removed. Time was measured after excision. Locally stimulated locations, I to 4, are illustrated in the diagram at the top, with the length calibration for the preparation to the right. Responses to (Z)-ll-tetradecenyl acetate (20 ,ug were recorded with a surface electrode at position 4 relative to an indifferent electrode at the proximal end.
REFERENCES 1. D. Schneider, Electrophysiological investigation on the olfactory specificity of sexual attracting substances in different species of moths, J. Insect Physiol. 8, 15 (1962). 2. D. Schneider, Insect olfaction: Deciphering system for chemical messages, Science (Wushington, D.C.) 163, 1013 (1969). 3. U. Thurm, Basics of the generation of receptor potentials in epidermal mechanoreceptors of insects, in “Mechanoreception” (.I. Schwartz-
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4.
5.
6.
7.
8.
9.
0.
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kopff, Ed.), p. 355, Abh. Rhein, Westf. Akad. Wiss., Opladen, 1974. K. E. Kaissling, Sensory transduction in insect olfactory receptors, in “Biochemistry of Sensory Functions” (L. Jenicke, Ed.), p. 243, SpringerVerlag, Berlin/New York, 1974. K. E. Kaissling, Recognition of pheromones by moths, especially in Saturniids and Bombyx mori, in “Chemical Ecology: Odour Communication in Animals” (F. J. Ritter, Ed.), p. 43, ElsevieriNorth-Holland, Amsterdam, 1979. K. E. Kaissling and J. Thorson, Insect olfactory sensilla: Structural, chemical and electrical aspects of the functional organization, in “Receptors for Neurotransmitters, Hormones and Pheromones in Insects” (D. B. Satelle et ul., Ed.), p. 261, ElsevierlNorth-Holland, Amsterdam, 1980. T. Nagai, Electroantennogram response gradient on the antenna of the European corn borer, OStrinia nubilalis, 1. Insect Physiol. 27, 889 (1981). T. Nagai, Spread of local electroantennogram response of the European corn borer, Ostrinia nubilalis, Pestic. Biochem. Physiol. 19, 291 (1983). T. Nagai, A. N. Starratt, D. G. R. McLeod. and G. R. Driscoll, Electroantennogram responses of the European corn borer, Ostrinia nubilalis, to (Z)- and (E)-1 I-tetradecenyl acetates, .I. Insect Physiol. 23, 591 (1977). J. Bernard and J. C. Guillet. Changes in the re-
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ceptor potential under polarizing currents in two insect receptors, J. Insect Physiol. 18, 2173 (1972). 11.
12.
13.
14.
15.
16.
17.
M. Kobayashi, C. L. Prosser, and T. Nagai. Electrical properties of intestinal muscle as measured intracellularly and extracellularly. Amer. J. Physiol. 213, 275 (1967). W. L. Roelofs and A. Comeau, Sex pheromone perception: Electroantennogram responses of the red-banded leaf roller moth, J. Insect PhyCo/. 17, 1969 (1971). T. Nagai. On the relationship between the electroantennogram and simultaneously recorded single sensillum response of the European corn borer, Ostrinia nubilalis, Arch. Insect Biochem. Physiol. 1, 85 (1983). M. E. Cornford, W. A. Rowley, and J. A. Klun, Scanning electron microscopy of antenna1 sensilla of the European corn borer, Ostrinia nubilalis, Ann. Entomol. Sot. Amer. 66, 1079 (1973). T. Narahashi, J. W. Moore, and W. R. Scott, Tetrodotoxin blockage of sodium conductance increase in lobster giant axons, f. Gen. Physiol. 47, 965 (1964). Y. Nakamura, S. Nakajima and H. Grundfest, The action of tetrodotoxin on electrogenic components of squid giant axons, J. Gen. Physiol. 48, 985 (1965). K. Kramer, Insect pheromones. in “Taxis and Behavior” (G. L. Hazelbauer, Ed.). p. 205. Chapman & Hall, London, 1978.
BIOCHEMISTRY
AND
PHYSIOLOGY
24, 32-39 (1985)
Summation and Gradient Characteristics of Local Electroantennogram Response of the European Corn Borer, Osirinia nubilaiis TOSHIO Agriculture
NAGAI
Canada, Research Centre. University Sub Post Office, London, Ontario N6A 5B7, Canada Received March 21, 1984: accepted August 20. 1984
Summation and gradient characteristics of the electroantennogram (EAG) responses of male moths of the European corn borer (Ostrinia nubilalis) to their pheromone, Q-11-tetradecenyl acetate, were studied. Direct evidence that the summation of the locally elicited EAGs contribute to the overall response amplitude is presented; if two simultaneously stimulated loci were separated by more than 1 mm, the observed response was the summation of the two independent local EAGs. Configuration of the local EAG response was obtained at various loci of the antenna. It was demonstrated that a gradient in the amplitude of the local EAG exists along the antenna1 axis and that this gradient has its origin in the epithelial layer of the antenna. Several possible morphological and physiological factors affecting the response characteristics were considered. o 198s Academic Press. Inc.
(7). This finding suggested that the EAG is a summed result of local responses generated between the two electrodes; i.e., the greater the antenna1 length between the electrodes, the larger the number of receptor units which might be involved. As the next step of the investigation, local stimulation was applied to study the distribution of potentials along the antenna. It has been found that a gradient exists in the EAG along the antenna1 axis (7, 8); the response amplitude becomes larger when the locally stimulated point is shifted closer to the distal tip. Moreover, the locally generated response spreads from the excited point in an asymmetric manner, predominantly in the proximal direction. An equivalent circuit diagram has been proposed, which includes a mechanism for generating the local EAG characteristics (8). It still remains to be demonstrated, however, whether the local potentials in the antenna are actually additive. Furthermore, there is a question of the effect of different biological factors on the EAG gradient. The following experiments demonstrate
INTRODUCTION
The electroantennogram (EAG) has been used as a tool to study antenna1 receptor function for sex pheromones of moths. The EAG response is assumed to be the summation of bioelectrical potentials generated by many olfactory receptor cells responding to odor stimulants. Although the bioelectrical activities have been discussed extensively at the level of the receptor unit in a sensillum [e.g., (l-6)], very few of these studies have described the summation aspect of EAG responses. The EAG is usually registered by a pair of electrodes placed at the distal region and proximal base of an isolated antenna. The European corn borer antenna is relatively easy to use for EAG studies because of its thread-like simple external form. Recently, several interesting characteristics have been found in the EAG response along the axis of the antenna. When the whole preparation is evenly stimulated, the response amplitude is approximately proportional to the distance between the recording and the indifferent electrodes placed on the antenna 32 0048-3575185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
ELECTROANTENNOGRAM
RESPONSE
in detail the unique pattern of the local response. Further studies on summation phenomena were conducted by simultaneously stimulating two separate loci on the antenna. Several morphological and physiological factors such as local excitability difference, slant angle of sensory hair, electric resistance of hemolymph space, and axonal activity were examined to determine their effect on the response gradient. MATERIALS
AND
METHODS
Antennae from 3-day-old adult male European corn borer moths, Ostrinia nubilalis, were used. The experimental procedures and apparatus used in recording the EAG were basically the same as those previously described (8). The isolated antenna was mounted on a wax plate. Compressed air was passed constantly over the whole preparation perpendicularly to the antenna1 axis at a speed of 30 cmisec. into this air stream, a fine air jet with a speed of 30 cm/set was introduced through a small funnel (inside diameter at the outlet: 0.2 mm), and was aimed at a specific point on the antenna1 surface. Pure air in the jet was switched to stimulant-containing air for a measured time by means of a rotary valve controlled by a solenoid (8). Another identical jet was also used when needed for simultaneous stimulation of a second location. Preparation of the stimulant pheromone, (Z)-IItetradecenyl acetate, has been given elsewhere (9). For measurement of electric resistance through the antenna a modified Wheatstone bridge (10, 11) was used. In some preparations a thin tungsten wire was inserted through the core of the antenna from the proximal cut end. The wire had a diameter of 25 p.m and the tip was slightly tapered by flame to make the insertion easier. Some preparations were perfused internally through the hemolymph space with physiological saline (12) or a modified saline containing tetrodotoxin (l- 10 x lop5 M) at a rate of 0.3-0.8 x 10e3 ml/
OF
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min. Tetrodotoxin was used to block the action potential of the antenna1 axons. A glass micropipet (outside diameter at the outlet: 75-80 km) was inserted into the proximal cut end of the antenna for the perfusion, and the outflowing saline was withdrawn at the distal cut end. All recordings were at room temperature, 22°C. RESULTS
AND
DISCUSSION
Gradient in the Local EAG Response EAG potentials evoked by stimulant chemicals appear as negative changes in the resting potential (9). These are easily detected by an electrode placed at the cut tip together with an indifferent electrode at the cut proximal end. This is the conventional device used by most EAG investigators. When the preparation was locally excited, the recorded EAG response to a given intensity of stimulus was found to depend on the locus of the antenna. Thus, the recorded amplitude became smaller proximally (Figs. 1A and B), approaching zero and frequently reversing its polarity from negative to positive near the proximal end (Fig. 1C). The stimulated location represented by the three points-p, c, and d in Fig. l-was shifted right and left to examine the whole antenna1 length, and the change in the amplitude was found to be gradual from the distal to the proximal end. From these results it can be seen that there is an amplitude gradient in the EAG along the antenna1 axis of the European corn borer. A similar gradient in amplitude was reported in previous work (8). The recorded EAG potential with two electrodes placed at the cut tip and base as described above probably included the sum of the injury potentials at the cut ends in addition to the evoked response potential. Moreover, the resulting potential might be reduced due to a short circuit through the cut ends via the antenna1 hemolymph. Without the injury and short circuit effects, the graded pattern of the EAG response
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FIG. 1. EAG responses of European corn borer recorded at the distal end relative to the proximal base to (Z)-11-tetradecenyl acetate (20 ,ug). The distalfive segments were removed. Measurement of three stimulated locations of the antenna-distal(d), central(c), and proximal (p)-are illustrated on the top right. Length calibration for the preparation is on the bottom right; i.e., the distance between d and p was 4 mm. The recording and stimulation arrangements are shown in schematic diagrams to the right of each EAG recording. Vertical arrow indicates recording electrode, and thick horizontal line represents antenna. Same preparation was used for (A) to (E). (A. B. and C) Distal, central, and proximal locations were stimulated, respectively. (D) Two locations, d and c, were stimulated simultaneously. (E) Two locations, d and p, were stimulated simultaneously. Time of stimulation: 1 sec. Response amplitudes measured at the end of stimulation time in (A) to (E) were - 1 S, - 0.6. fO.3, -2.1, and -1.2 mV, respectively. EAG calibration is at the bottom center. Time after excision: 30 min.
along the antenna1 axis could be measured simply by placing the two electrodes in contact with the intact surface of the antenna. Typical results are shown in Fig. 2. The response amplitude recorded from a distal point d reached the maximum if point d was stimulated (Fig. 2A). When the stimulated locus was shifted from d toward proximal point p, the response amplitude became smaller, frequently changing its polarity near point p, and then reached the other extreme amplitude with reversed polarity
NAGAI
FIG. 2. EAG responses of European corn borer recorded with surface electrodes to (Z)-ll-tetradecenyl acetate (20 ug). The distal five segments were removed. Location of the electrodes (d and p) is illustrated on the top right. Same preparation was used for (A) to (G). (A-E) The stimulated location was shifted stepwise from d to p at intervals of 1 mm. (F) The recording and indifferent electrodes of the preparation in (E) were exchanged. (G) Two locations, d and p. were stimulated simultaneously. Time of stimulation: I sec. Response amplitudes measured at the end of stimulation time in (A) to (G) were - I .6, - I .I, -0.5, + 0.5, f 1.2, - 1.2, and -0.4 mV. respectively. Time after excision: 30 min. For further explanation, see Fig. 1.
(Figs. 2A-E). Thus, the characteristic response gradient along the antenna1 axis was also clearly demonstrated with surface electrodes. If the two electrode positions shown in Fig. 2E were interchanged as in Fig. 2F, i.e., the recording and the indifferent electrodes were placed at p and d, respectively, a mirror image response of the one shown in Fig. 2E could be obtained. The negative polarity of the response in Fig. 2F indicates that regardless of the stimulated region, distal (Fig. 2A) or proximal (Fig. 2F), the EAG recorded at the excited point is al-
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ways negative relative to the unstimulated neighboring surface of the antenna (7, 8). When the response was recorded at different distances from the stimulated point as in Figs. 2B, C, or D, a spread effect of the response from the stimulated region in opposite directions along the antenna1 axis was noted and has been shown previously (8). This resulted in the reversed polarity of the response (Figs. IC and 2D). As discussed in the previous work (S), the locally elicited potential is assumed to be a temporal change in the resting receptor potential of the sensory hair unit circuit. The potential generated in response to stimulation in the unit circuit can be recorded by an electrode placed at the excited point, and is found to be negative relative to an indifferent electrode at the distant region. This seems to indicate that a current flows into the excited region from the resting regions on both sides. Although the local EAG responses shown in the present study (Figs. 2A and F) are not generated from a single sensory unit, their negativity may suggest that the excited local region becomes a sink for the current flow. Summated Configuration EAG Response
of the
When many sensory units were excited at the same time, the elicited local potential changes were summed, and could be recorded as a total EAG response. To demonstrate this summation, two different local regions of an antenna were stimulated simultaneously with two identical stimulation jets, and the resulting response pattern was observed. It was found that the two responses were simply added if the distance of the locally stimulated regions were greater than 1 mm. For example, the response in Fig. ID is the sum of the individual responses at d and c (Figs. IA and B) separated by 2 mm. Likewise the response in Fig. 1E is the sum of the responses at d and p (Figs. IA and C), but is smaller than the single response at d (Fig. 1A) because of the reversed polarity of the
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response at p (Fig. IC). A similar result was also obtained from surface recordings; the summed response in Fig. 2G is smaller than the single response in Fig. 2A. Thus, despite a doubling of the stimulus intensity, the resulting response may be smaller than the single component responses depending on the loci of the stimulations with respect to the recording electrode. In any case, the response recorded by two stimulations simply showed up as the sum of the individual responses. If two locally stimulated regions were closer than 1 mm, the amplitude of the resulting EAG was smaller than the sum of the two individual responses. This reduction may be due to the two jet streams colliding and, with the resulting turbulence, failing to deliver two full stimulant dosages. On the other hand, if the region excited by one jet is approximately 1 mm wide, because of overlap, the total area excited by the two jets would be less than 2 mm wide. At the concentration used in this study, 20 kg, stimulation is maximal (9) and could therefore not be greater in the overlap region in spite of the larger amount of stimulation delivered there. There may also be an active interaction between two neighboring regions which could affect the individual response amplitudes (8) although the underlying system has not yet been clearly determined. A study of single sensory hair stimulation and the response of a single sensillum (ESG) (13) might determine whether such an interaction between responses exists. Possible Factors Affecting the Response Gradient The positions of the recording and indifferent electrodes shown in Figs. 2A and F are reversed at d and p on the same antenna. In both cases, however, the response was recorded from the stimulated region. If the antenna were not functionally directional along its axis, the response in Figs. 2A and F would be identical. This was not the case; however, the response re-
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corded at distal point d was larger in the amplitude than that at proximal point p, showing that there is in fact a clear directional difference. There does not appear to be an excitability difference between distal and proximal regions as no specially differentiated sensilla have been found (14). Furthermore, the distribution density of the sensilla trichodea is almost uniform along the European corn borer antenna (7). If the total number of the sensilla per unit length of antenna were to determine the response summation, the amplitude would be smaller at the distal point than at the proximal point as the total number of the sensilla decreases distally because of the smaller diameter of the antenna. However, this was not the case as already shown in Figs. 2A and E There is a possibility that excision of the distal and proximal ends would induce a different amount of injury potential at the two ends and as a consequence affect the responses at p and d. However, this effect seemed to be minor as the response shape did not show any significant difference after cutting the distal tip than before (Fig. 3). While the stimulation jet was directed
AL FIG. 3. EAG responses of European corn borer recorded with surface electrodes to (Z)-11-tetradecenyl acetate (20 ug). (A) Response was recorded before the distal tip was cut off (B) Response was recorded immediately after the distalfive segments were removed. Time interval between (A) and (B) was 2 min. Locations of the two electrodes and stimulation were similar to those in Fig. 2A. Parallel lines show the time of stimulation: 1 sec. Response calibration on the bottom right: I mV and 1 sec.
NAGAI
perpendicularly to the antenna1 axis, the sensory hairs themselves are slanted about 28” from the perpendicular toward the distal tip. This discrepancy in direction of air flow and hair axis might result in an uneven local stimulation. To examine this possibility, the direction of the jet was altered through an angle of O-40” from the perpendicular toward both the antenna1 tip and the base, while being directed toward the same spot. The configuration of the response was not significantly changed; at an angle of 30” in either direction, however, the amplitude was increased by lo-17% in both cases, but this change was probably due to an increase in the area exposed to the jet. Thus, the response asymmetry along the antenna1 axis appears not to be related to the air flow direction. It was found in the previous work (8) that the local response spreads in both proximal and distal directions from the stimulated point but that the spreading occurs more effectively toward the base. This suggests that more current flows proximally than distally. If so, the increasing cross-sectional area of the antenna in the proximal direction should result in a lower resistance and hence a greater current. To test this possibility the relationship between the antenna1 shape and the resistance was investigated. The gross shape of the male moth antenna of the European corn borer is a slender cone of about 7.8 mm long (range: 7.6 to 8.3 mm) with a circular cross section of 35 pm diameter at the distal end and a rough ellipse with axes of 110 x 130 km at the proximal base, excluding the sensory hairs and scales. The thickness of the epithelial cuticle is 7 km in the distal region and 20 p,rn proximally. The inside hemolymph space has a round form with diameter of 20 pm at the distal end and is a 70 x 90-km oval at the proximal end. These dimensions change in a manner which is proportional to the length from the tip to the base of the antenna. This difference in diameter between distal and proximal regions must increase the electric resistance
ELECTROANTENNOGRAM
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of hemolymph space distally in inverse proportion to the cross-sectional area. The resistance along the antenna1 axis between two cut ends was measured. The resistance of the whole antenna with the five distal segments removed was found to be in the range 5 to 7 MLR with considerable variation among preparations. This resistance is probably that of the hemolymph space since the epithelial layer has a much higher resistance (3, 6). Figure 4 shows the resistance-time relations of the separated halves of the same antenna, together with the ratio of their resistances. Although both resistance values decreased with time after excision, the ratio was found to be almost constant: the distal half had 3.4 times the resistance of the proximal half. If the change in taper of the hemolymph space along the antenna1 axis is assumed to be linear, the average cross-sectional area of this slender truncate cone can be calculated for each half of the split antennae. When the major and minor axes of the oval shape were averaged, the cross-sectional diameters of the hemolymph space of the preparations used in Fig. 4 were 80, 50, and 20 km at the proximal end, severed center, and distal end, respectively. The diameter of a cylinder which has the same volume
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and length as each of the slender truncate cones was calculated. These values were 65.6 pm for the proximal and 36.0 km for the distal half, resulting in average crosssectional areas of 3378 and 1017 pm2, respectively, or a ratio (distal/proximal) of I/ 3.3. Thus the resistance ratio of the two halves of the antenna, 3.4 in Fig. 4, was inversely proportional to that of the crosssectional area. This supports the contention that conductance occurs mainly through the hemolymph space, and that since this conductance increases in the proximal direction, so also will the current. Fifteen preparations gave essentially the same results as that in Fig. 4. To test this conclusion further, an attempt was made to reduce the distal-proximal difference in the hemolymph resistance. To this end, a 25-pm-diameter tungsten wire (having a resistance of approximately 0.1 a/mm) was passed through the core of an antenna from which the distal five to seven segments had been removed. Even after insertion of the wire, the characteristic response difference between distal and proximal regions was still maintained as shown in Fig. 5. Thus the electric resistance difference assumed to be due to the change in the diameter of the hemolymph space of the
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FIG. 4. Resistance-time curves for distal (0) and proximal (0) halves of European corn borer antenna. An excised antenna with the distal five segments remolzed was cut at the center. The length of both halves was 3.9 mm. Calculated ratio of the resistance (distal/ proximal) is shown at the top (a). Time was measured after excision. See text for further explanation.
FIG. 5. EAG responses of European corn borer recorded with surface electrodes to (Z)-11-tetradecenyl acetate (20 ,ug). Locally stimulated locations on the antenna, 1 to 5, are shown in the diagram at the top, with the length calibration for the preparation to the right. Vertical arrow indicates the recording electrode. The distal seven segments were removed. Responses were recorded before (0) and after (0) insertion of a tungsten wire (diam: 25 urn) through the core of the antenna. Recorded potential amplitudes were measured at the end of stimulation time (1 set).
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antenna does not appear to account for the observed response gradient. On the other hand, the tungsten wire insertion might have damaged structures including axons in the antenna1 core. Therefore, the directional nature of the nervous conduction and the regional difference in the number of passing axons, i.e., more axons proximally than distally, are unlikely to contribute to the asymmetrical EAG response gradient. The possible effect of axonal activity was tested directly by a nerve-blocking chemical. Tetrodotoxin (TTX) has a remarkably specific blocking effect on the action potential in most excitable cells [e.g., (15, 16)]. It was found that TTX-saline (l- 10 x 10-j M> perfused through the hemolymph space had little effect on the EAG generation and on its gradient characteristics; the response amplitude showed a smooth decrease without any abrupt change when TTX was introduced (Fig. 6), and the relationship between the amplitudes of the responses to various stimulated loci (1 to 14 in Fig. 6) was not disturbed by the toxin. It has already been shown that the amplitude
NAGAI
of EAG response gradually decreases throughout the life span of the preparation (9). This present result directly suggests that the underlying mechanism causing the response gradient is not dependent on the axonal activity in the antenna1 hemolymph space. Thus, the main source for the characteristic response seems to be in the epithelial layer. The bioelectrical response in the sensory unit of the antenna may occur in a circuit passing through the receptor cell and the supporting auxillary cells in the sensory hair (3, 6). The local current source in the unit circuit could act like a battery and discharge in response to stimulation. The numerous batteries of the units could be interconnected in parallel or series along the antenna1 axis (17). If these were merely connected in parallel, neither summation nor directional gradient in the response amplitude would be expected. Therefore, there must be a series connection component between adjacent unit circuits (8). The present study demonstrates that the interaction network responsible for the EAG characteristics are mainly in the epithelial layer where all the receptor units are inlaid. However, direct evidence at the cellular level of the receptor remains to be demonstrated. ACKNOWLEDGMENTS
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I thank Dr. D. G. R. McLeod for his encouragement and criticism. I am also indebted to Dr. D. M. Miller for reading the manuscript, to Dr. A. N. Starratt for a supply of the chemical, and to Mr. G. R. Driscoll and Mrs. M. E. Stevens for their assistance.
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amplitude-time curves for European corn borer. The preparation was perfused internally from the proximal end: Response during perfusion with normal saline (0) and TTX-saline. 5 X 10m5 M. (@). The distal 12 segments were removed. Time was measured after excision. Locally stimulated locations, I to 4, are illustrated in the diagram at the top, with the length calibration for the preparation to the right. Responses to (Z)-ll-tetradecenyl acetate (20 ,ug were recorded with a surface electrode at position 4 relative to an indifferent electrode at the proximal end.
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kopff, Ed.), p. 355, Abh. Rhein, Westf. Akad. Wiss., Opladen, 1974. K. E. Kaissling, Sensory transduction in insect olfactory receptors, in “Biochemistry of Sensory Functions” (L. Jenicke, Ed.), p. 243, SpringerVerlag, Berlin/New York, 1974. K. E. Kaissling, Recognition of pheromones by moths, especially in Saturniids and Bombyx mori, in “Chemical Ecology: Odour Communication in Animals” (F. J. Ritter, Ed.), p. 43, ElsevieriNorth-Holland, Amsterdam, 1979. K. E. Kaissling and J. Thorson, Insect olfactory sensilla: Structural, chemical and electrical aspects of the functional organization, in “Receptors for Neurotransmitters, Hormones and Pheromones in Insects” (D. B. Satelle et ul., Ed.), p. 261, ElsevierlNorth-Holland, Amsterdam, 1980. T. Nagai, Electroantennogram response gradient on the antenna of the European corn borer, OStrinia nubilalis, 1. Insect Physiol. 27, 889 (1981). T. Nagai, Spread of local electroantennogram response of the European corn borer, Ostrinia nubilalis, Pestic. Biochem. Physiol. 19, 291 (1983). T. Nagai, A. N. Starratt, D. G. R. McLeod. and G. R. Driscoll, Electroantennogram responses of the European corn borer, Ostrinia nubilalis, to (Z)- and (E)-1 I-tetradecenyl acetates, .I. Insect Physiol. 23, 591 (1977). J. Bernard and J. C. Guillet. Changes in the re-
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M. Kobayashi, C. L. Prosser, and T. Nagai. Electrical properties of intestinal muscle as measured intracellularly and extracellularly. Amer. J. Physiol. 213, 275 (1967). W. L. Roelofs and A. Comeau, Sex pheromone perception: Electroantennogram responses of the red-banded leaf roller moth, J. Insect PhyCo/. 17, 1969 (1971). T. Nagai. On the relationship between the electroantennogram and simultaneously recorded single sensillum response of the European corn borer, Ostrinia nubilalis, Arch. Insect Biochem. Physiol. 1, 85 (1983). M. E. Cornford, W. A. Rowley, and J. A. Klun, Scanning electron microscopy of antenna1 sensilla of the European corn borer, Ostrinia nubilalis, Ann. Entomol. Sot. Amer. 66, 1079 (1973). T. Narahashi, J. W. Moore, and W. R. Scott, Tetrodotoxin blockage of sodium conductance increase in lobster giant axons, f. Gen. Physiol. 47, 965 (1964). Y. Nakamura, S. Nakajima and H. Grundfest, The action of tetrodotoxin on electrogenic components of squid giant axons, J. Gen. Physiol. 48, 985 (1965). K. Kramer, Insect pheromones. in “Taxis and Behavior” (G. L. Hazelbauer, Ed.). p. 205. Chapman & Hall, London, 1978.