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Electrical properties of the dendrite in an insect mechanoreceptor: Effects of antidromic or direct electrical stimulation
J. Insect Physiol., Vol. 26, pp. 755 to 162. OPergamon Press Ltd. 1980. Printed in Great Britain.
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ELECTRICAL PROPERTIES OF THE DENDRITE IN AN INSECT MECHANORECEPTOR: EFFECTS OF ANTIDROMIC OR DIRECT ELECTRICAL STIMULATION J. C. GUILLET, J. BERNARD,J. P. COILLOTand J. J. CALLEC Laboratoire de Physiologie Animale. Campus de Beaulieu, 35042 Rennes Cedex, France (Received 3 March 1980; revised 12 June 1980)
Abstract-A study of the negative phase of the spikes recorded extra cellularly from insect mechanoreceptor has been performed in order to characterize some electrical properties of the dendrite which contains the transducing part of the sensory neuron. These properties have been investigated in mechanoreceptors of the metathoracic leg of the locust Schistocerca gregaria by firing antidromic action potentials both at rest and during mechanical or electrical stimulation. The amplitude of the negative phase of the spike appears to be correlated with the polarization of the dendritic membrane, although when bursts of action potentials are applied, the relation is more complex, including a depressive influence of a given spike on the following spike. The receptor potential and the antidromic dendritic spikes both originate in the same region of the dendrite but they involve different ionic processes. Our results indicate that the dendrite is electrically excitable. The spike which originates in the dendrite has an initial negative phase with a small superimposed positive component. A spike of this shape is never observed under natural stimulation. It is proposed that the negative phase of the antidromic impulse provides a suitable means for studying the variations in electrical polarization of the dendrite which cannot be recorded directly. Ke.v Word Index: Dendrite mechanoreceptor. electrophysiology insect
INTRODUCTION IN THEORIESof sensory
transduction
the dendrite
is
assumed to play a very important part (RICE et al., 1973; MORAN and VARELLA,1971; THURM,1968,1970, 1972,1974; ATEMA,1973). However itsexact role is not yet well defined for external insect receptors, because it is impossible with classical external electrode recordings from the base of the sensilla, to record the dendritic potentials selectively. The records represent both the activity of the dendrite and that of the initiation site of the impulses. Therefore, the study of a parameter reflecting dendritic membrane polarization is of considerable interest. With this end in view we have studied the biphasic action potential, recorded from a mechanoreceptor by an external microelectrode because the shape and the amplitude of its two phases, being dependent on both dendritic and axonal polarization, may reflect the electrical activity at these two sites. Our objective is to study dendritic membrane properties using the negative phase of the action potential as a test of the polarization and the excitability of the dendritic membrane. In our work, we have applied electrical stimulation to the sensory axon at a distance from the transducing region so as not to influence it. We have studied the resulting antidromic action potentials arriving at the dendrite placed in different electrical states, particularly in a steady resting state which has never been observed before. We have also applied electrical stimulation directly to the dendrite and proved that it is electrically excitable. Theoretical
considerations
Let us consider
the preparation
classical recording technique, i.e. with a microelectrode inserted at the base of the external process (BOECKH,1962; THURM, 1962; YAMADA,1967, 1971). This electrode is often inserted in the joint membrane, when it exists (for morphological details on external mechanoreceptors, see MORANet al., 197 1; GNATZY, 1976). The microelectrode,
which is always
more or less broken after its passage through the cuticle is large (about l-5 PM) compared with the diameter of the dendrite (about 5 PM). Thus, it cannot penetrate it and it is always an external electrode. The electrical situation is similar when the electrode is not inserted through the cuticle but brought into contact with the exposed end of a chemoreceptor or a tactile hair (WOLBARSHT, 1960; WOLBARSHT and DETHIER, 1958): the recording electrode is external to the dendrite. In these conditions, various electrical models of mechano-or chemoreceptors have been proposed to explain the physiological significance of the two phases of the extracellularly recorded action potential (MORITA, 1959; MORITA and TAKEDA, 1959; WOLBARSHT, 1958; THURM, 1963; WOLBARSHT and HANSON, 1965; DE MELLON, 1968; YAMADA. 1971;
GUILLETand BERNARD,1972; MAES. 1977). It appears that the initial positive component signals the onset of the impulse at the site of electrogenesis and that the subsequent negative phase reflects the antidromic conduction of the impulse towards the dendrite. From these considerations, we have illustrated the experimental situation for the sensory neuron of a mechanoreceptor (Fig. 1) which receives mechanical stimulation in A and electrical stimulation in B. Membrane polarization is represented in the left half of the neuron. The initiation and conduction of the in the case of a sensory spike is represented in the right half. The scope 155
J. C. GULLET et al.
756 A Met
St
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B
,
Fig. 1. Diagram of a sensory neuron showing how the spike is induced and recorded during mechanical stimulation (A) and during electrical stimulation (B). The tip of the recording micropipette is near the dendrite, the reference electrode being in the conductive medium. A. The left part of the neuron shows its electrical state when mechanical stimulation (Met. St.) induces a receptor potential (R.P.) in the transducing zone of the dendrite(D). The R.P., which is a local but sustained depolarization decreasing electrotonically (indicated by the thickness of the dark areas), induces the impulses at the initiation site (1,s.) some distance from the transducing zone. The right part of the neuron shows the spike when it is initiated (time 1) and a moment later (time 2) after orthodromic conduction along the axon and antidromically along the dendrite. The scope record of this spike shows the two phases corresponding to time 1 (+) and to time 2 (-). The second phase, which is a dendritic phase, has the same polarity as the dendritic R.P. B. Same presentation as in A. Electrical stimulation (El. St.) is applied via the positive microelectrode. The current enters the dendrite and exits at the initiation site. In the left part of the neuron, the micraelectrode being positive, we see the current hyperpolarizing the dendrite and depolarizing the initiation site, thus triggering impulses. The hyperpolarized zone is illustrated by dashed areas and the depolarized zone is illustrated by dark areas. Their thickness represents the amplitude of polarization. Comparison of the two spikes shows that the second (negative) phase is small in A because the dendrite is depolarized but large in B because the dendrite is hyperpolarized.
record considers only this spike. The micropipette (the active electrode) is placed on the outside of the sensory neuron, near the dendrite where the receptor potential (R. P.), corresponding to a depolarization of the appears during sensilla stimulation dendrite, (Fig. 1A). This R. P. is therefore recorded as a negative variation of potential, not represented in the scope record. Action potentials, induced by the R. P., appear some distance from the transducing region. They are therefore recorded initially (time 1) as positive variations of potential (upward deflection in the scope record of Fig. 1A). Then (time 2) they are conducted orthodromically towards the central nervous system, and, under certain conditions which are analysed in this paper, antidromically towards the dendritic *We place the initiation site on the axon, near the cell body because it appears to us that the structure of this short (20-30 pm) dendritic bipolar neuron is anatomically close to that of a vertebrate motoneurone (ECCLES, 1957) or to that of certain crustacean proprioreceptors (EDWARDSand OTTOSON, 1958)
where the action potential appears at the axon hillock. It differs from the long dendrite (about 300 pm) of the P.D. organ of crustacean (HARTMANand BOETTIGER,1967; MENDELSON, 1966) or even more from the very long dendrite of sensory neuron of vertebrate where the spikes appear on the dendrite. However. it would have been possible to place the initiation site on the dendrite as illustrated by MORITA (1959) YAMADA(~~~~) and MAF..s( 1977). Thisdoes not affect the most important point to be considered here: the R.P. and the negative phase of the spike are produced in the same part of the dendrite which, when it depolarizes, gives a negative potential in our recording conditions.
terminal. When they invade this area, beneath the they are recorded as negative microelectrode, variations of potential (downward deflection in scope record of Fig. 1A). Figure 1B shows the experimental situation when the receptor is stimulated electrically. The current is applied by means of the recording micropipette. When this micropipette is positive, the imposed maintained current enters, and thereby hyperpolarizes the dendrite (BERNARD and GUILLET 1972). The current exits through the initiation site* of the sensory neuron, which is depolarized, thus triggering a burst of biphasic action potentials. These spikes always exhibit a large negative phase because the dendrite is hyperpolarized. In contrast, spikes which are induced by mechanical stimulation (in A) have a smaller or non-existent negative phase because, in this case, the dendrite is depolarized. The shape and the amplitude of the two phases may vary under different conditions. This is illustrated, for a phasic mechanoreceptor, in Fig. 2. An imposed and maintained electrical current induces a burst of action potentials. At the beginning of the record, they are biphasic. The second phase is negative, large in amplitude, and increases for some time until finally it disappears. In order to explain this phenomenon, we have proposed (GULLET and BERNARD, 1972) that the inward maintained current progressively increases the dendritic membrane resistance (as an anomalous rectification), therefore increasing membrane hyperpolarization. In this scheme, the negative phase enlarges but, after a time, antidromic invasion of the dendrite by the spike is
Electrical
El. St.
properties
of the dendrite
77 El.St.
Met. St.
c
Antidrom.
1 I
in a mcchanoreceptor
,llN+Y
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____-______ I5mV
A
B
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Fig. 2. The different shapes of the action potentials. An electrical stimulation (El. St.) is applied to the receptor (microelectrode +) from A to the end of the record, inducing a long burst of impulses. Mechanical stimulation (Met. St.) is superimposed in B. For explanation. see text.
blocked as the dendrite becomes too hyperpolarized. A mechanical stimulus (Fig. 2B) induces a superimposed depolarization (R.P.) during which the impulses vary again. They are first positive and monophasic. The absence of the negative phase can be explained in this case by the depolarization of the dendrite. This is confirmed by the fact that during the adapting phase of the receptor potential, the dendrite, in progressively recovering its resting potential, recovers also its excitability and during a time corresponding here to three spikes, the conduction of antidromic action potentials, thus giving transitory biphasic potentials. From the above theoretical considerations, the following main points emerge which are important in explaining our results: (1) the antidromic propagation of action potentials in the dendrite is the origin of the negative phase; (2) the amplitude of this negative phase reflects the variations found in the polarization of the dendritic membrane; (3) under both types of stimulation, mechanical and electrical, the action potential is always observed in an activated sensory cell, the dendrite being either depolarized (mechanical stimulation) or hyperpolarized (electrical stimulation); (4) action potentials are grouped into bursts. We will see that this results in modifications in the amplitude of the negative phase. In this paper, we consider as a whole the dendrite or the part of the dendrite which gives the R.P. and the negative spike because it is impossible to distinguish a priori a transducing region from a spiking one. But this point will be partly discussed. METHODS Experiments were carried out on phasic mechanoreceptors of the locust Schistocerca gregaria. The receptors studied on isolated metathoracic legs, are situated dorsally on the distal part of the head of the femur. Their electrical activity has been previously described (GUILLET, 197.5) as well as stimulation and recording techniques (BERNARDand GUILLET, 1972). These are recapitulated briefly in the theoretical considerations above. The entire preparation is shown in Fig. 3. The microelectrode inserted in the joint membrane at the base of the receptor was used for recording and for electrical stimulation. Mechanical stimulation was effected by a microhook connected to the cone of a
Fig. 3. Experimental arrangement. The phasic mechanorcceptors are situated on the distal part of the metathoracic leg. A microelectrode, inserted at the base of one receptor, was used both to record (R.) and to apply electrical stimulation (El. St.). Mechanical stimulation (Met. St.) was applied with a microhook driven by the membrane of a loudspeaker. The reference electrode was introduced into the leg. The sensory nerve containing the axon of the mechanoreceptor was dissected through a small window in the cuticle and gently placed on two silver wires. In these conditions, it is possible to apply an electrical stimulation (Antidrom. El. St.) to the nerve and thus. to record an antidromic action potential at the level of the receptor.
loud-speaker. Electrical stimulation of the sensory axon was applied via two silver electrodes which were placed under a lateral sensitive branch of the nerve V according to the technique described by COILLOT and BOISTEL(1969). The reference electrode consisted of a silver wire placed in the femur.
RESULTS Electrical stimulation of‘the sensory axon at a distance from the dendrite. Stud-v of the antidromic actiorl potential (a) Dendrite at rest. In stimulating the sensory nerve with a short electric pulse, one can record at the receptor, an antidromic action potential (Fig. 4). This spike is typically biphasic with the initially positive component being approximately l/6 the amplitude of the negative component. The duration of the first phase is less than 1 msec whereas the second is more than 15 msec. The positive phase corresponds to the appearance of the action potential at the initiation site; however, in this case, it is not directly elicited at this initiation site. but indirectly after it has been conducted antidromically along the axon. The second phase corresponds to the spike in the dendrite which, in this particular case. was previously in steady state conditions. Its form and amplitude can
I+ I--2,SmV
10 msec
Fig. 4. Antidromic action potential induced by electrical stimulation of the sensory axon far from the receptor and recorded from a receptor at rest.
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J. C. GULLET et al.
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1 . 10
lOiG&
A
.
.
I
.
50
.
.
(Time)
.
-
)
msec
B
Fig. 5. Records (A) and curves (B) showing the decrease in amplitude of the negative phase of antidromic spikes when they are triggered in bursts. A brief stimulus artefact precedes each spike. F, Frequency of the
stimulation (Hz).
therefore serve as a reference for discerning the state of membrane polarization under other experimental conditions. With repetitive stimulation, the amplitude of the second phase decreases from one impulse to the next. For example, in the upper record of Fig. 5A, the negative phase of the second action potential is reduced by 15% in spite of a time interval of 75 msec between the two impulses. This phenomenon is accentuated during bursts, (Fig. 5A, lower records) and becomes even more evident at higher stimulation frequencies as shown by the graphs in Fig. 5B which correspond to three frequencies of sustained stimulation. The decrease, which appears in the first 100 msec, reaches 50% at a frequency of 100 Hz (low for an axon). Therefore, it appears that an action Antidrom. El. St.
+
“I”“.
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Fig. 6. Antidromic spikes observed in a phasic receptor which was depolarized by mechanical stimulation (Met. St.). In each record, the thin arrow signals the beginning of the maintained mechanical stimulus which induced a receptor potential of phasic type and two superimposed positive monophasic spikes. The thick arrow shows the artefact due to the electrical stimulus (Antidrom. El. St.) which triggers the antidromic spike. From A to D, the antidromic spike was triggered before the receptor potential (A), during the time course of the R.P. (B and C) and after the completion of adaptation (D).
potential which arrives at a dendrite modifies its electrical state for a long time. We do not know the mechanisms which underlie this phenomenon and which are quite different from those observed in an axon. (b) Dendrite depolarized by a mechanical stimulation. It is possible to stimulate the sensory axon at any time during mechanical stimulation. The amplitude of the negative phase can then be studied in a depolarized dendrite. This is illustrated in Fig. 6. In A, B, C, D a weak mechanical stimulus just inducing two monophasic and positive action potentials, was applied to the receptor. An antidromic impulse was triggered before and during this stimulation. In record A, the antidromic potential was observed on a sensory neuron at rest, therefore serving as a reference point. In B it disappears because the antidromic electrical stimulation was applied to the nerve during the refractory period which follows the orthodromic action potentials. In C, it is observed during the adaptation phase of the R.P. It shows a large negative component, which is however lower than that in A. In D, it appears after adaptation is complete. The amplitude of the second phase (2.5 mV) is still lower than that observed in A before stimulation (3.75 mV). This indicates that, even when the dendrite has apparently recovered its resting potential, the influence of the mechanical stimulation is much prolonged and the second pllase provides evidence for this. (c) Dendrite hyperpolarized by electricalstimulation. The applied electric current which hyperpolarizes the dendrite induces a burst of orthodromic potentials during which it is impossible to observe an antidromic dendritic impulse. This is due to the refrattory period as in the case illustrated in Fig. 6B for mechanical stimulation. But after the burst, the antidromic action potential can be observed. The results are illustrated in Fig. 7. As shown in Fig. 7A, current application induced a burst of biphasic action potentials. As shown in B they were monophasic when the current was stronger and hyperpolarized the dendrite membrane sufficiently to render it inexcitable. The action potentials no longer invaded the dendrite. (A
Electrical
properties
Antidrom.
of the dendrite
El. St.
Fig. 7. An electrical stimulus {El. St.) which induced a burst of impulses (microelectrode +) was applied to the receptor from the thin arrow in the record, to the end. The last spike of the burst is marked by an upward pointing arrow. Shortly after this last spike, an antidromic spike was evoked. The electrical stimulus was greater in B than in A. It was just sufficient in B to block the negative phase of all the impulses. The asterisks signal in A some spikes of a second cell which is stimulated by the applied current. It is very difficult in B to distinguish the very small monophasic positive spikes of this second cell.
second cell innervating the sensilla is also sensitive to the applied current and explains the origin of the spikes marked by an asterisk.) After the electrically-evoked activity ceased (marked by an upward pointing arrow), the sensory nerve was stimulated. It is still possible (Fig. 7) to observe an antidromic action potential with the same form and amplitude as that of the last potential in the burst, i.e. biphasic in A but positive and monophasic in B. (d) Influence of a burst ofantidromic actionpotentials on the receptor potential amplitude. The receptor potential and the second phase of the action potential both being electrical phenomena appearing at the dendrite level, show respective amplitudes which are correlated and dependent on the polarization of the dendrite membrane. This was established by applying polarizing currents to the receptor (BERNARDand GULLET. 1972; GULLET and BERNARD,1972) and partly in the above experiments (Fig. 6). Moreover, we have shown the existence of a phenomenon which tends to decrease the amplitude of the second phase in Antidrom. El. St
in a mechanoreceptor
759
a burst of antidromic impulses (Fig. 5). This raises the question as to whether or not the receptor potential and the dendritic action potential affect the same dendritic processes. If this is the case, the phenomenon which decreases the negative phase of action potentials during the burst would be expected to affect the amplitude of the receptor potential similarly. We have never observed a smaller modification in the receptor potential when it is preceded immediately by only one antidromic impulse. However, an antidromic impulse modifies the amplitude of the one that follows (Fig. SA). We applied a series of electrical stimuli to the nerve before mechanical stimulation of the receptor. As shown in Fig. 8A, the receptor potential was preceded by a burst of antidromic impulses with a frequency of 80/set; an attenuation of 8% was experienced. This is much lower than the 52% observed in the last impulse of the burst. In Fig. 8B the burst frequency was 137/set. The attenuation of the receptor potential was 13’& that of the last impulse was 55%. It is clear that there is a discrepency between the two electrical phenomena. Electrical stimulation of the dendrite resulting in an orthodromic dendritic action potential
In all cases studied so far the dendritic action potential was antidromic, invading the transducer zone of the dendrite secondarily. For this reason it was always recorded as a second phase. The question arises as to whether it would possible to trigger the potential at the dendritic level so as to observe an initial negative phase. Isolated negative potentials have been observed but only on deteriorated preparations or in receptors subjected to strong stimulation (MORITA, 1959; WOLBARSHTand HANSON,1965; YAMADA,1971). It is possible that, in the above cases, the positive component was only masked, also it cannot be proved that the dendritic action potential was not antidromic. To remove any such doubt it would be necessary to observe an impulse with a negative first phase followed by a positive phase. (a) Rectangular pulse of long duration. This kind of stimulation gives rise to an action potential only if we adjust precisely the current intensity (negative microelectrode). This is illustrated in Fig. 9A where the pulse induces a negative response (slanting arrow) Met. St.
2Omsec ‘::
Fig. 8. Influence of an antidromic burst of action potentials on the amplitude of the receptor potential (R.P.) The first time (dotted line) a mechanical stimulus alone (Met. St.) was applied to the receptor, inducing a R.P. The second time (full line) the mechanical stimulus was preceded by antidromic electrical stimuli (Antidrom. El. St.) of varying frequencies. A brief stimulus artefact precedes each spike. Frequencies of electrical stimulation: A =80/set; B = 137/set.
760
J. C. GULLET er 01.
A
I
1mV
dmsec J
\
Fig. 9. Comparison of two sorts of spike evoked by direct electrical stimulation of the receptor. The maintained current was applied via the microelectrode with the aid of a Wheatstone bridge. The spikes, at the beginning of the stimulation, appear on a baseline which has not yet stabilized. A, Microelectrode negative. B. Upper truce = microelectrode negative, lower trace = microelectrode positive. Same intensity of the applied current in the two records B.
which appears as an all or nothing spike. The amplitude (1.5 mV) and the duration are identical to those of the second negative phase found in the first situation. We can see, superimposed on this negative potential, a small positive component corresponding with the passage at the initiation site, of the orthodromically conducted impulse. This experiment suggests that the dendrite containing the transducing structure is indeed electrically excitable. Figure 9B compares this orthodromic dendritic action potential with the antidromic one triggered with a pulse of the same intensity but of opposite polarity. In the upper record,
v +
I
1mV
G&
Fig. 10. Spikes triggered in the dendrite
b
by a sinusoMally varying current. Middle record: the positive half of the applied current. A. Corresponding record. The Wheatstone bridge cannot be perfectly balanced because the resistance of the preparation varied with applied current. The dendrite was hyperpolarized initially, then it is returned to its normal resting potential. During the return, two spikes are triggered. They seem monophasic negative. B. On a faster time base, the spikes monophasic negative, but a small appear superimposed positive component can be observed.
the microelectrode was negative resulting in an action potential triggered at the dendritic level. The first phase is negative. In the lower record, the microelectrode is positive resulting in an action potential which appears at the initiation site. The latter is typically biphasic (positive then negative). The negative phases of the two spikes are different in amplitude, because the dendrite was depolarized in the upper record and hyperpolarized in the lower. (b) Shu.dation by a sinusoidal current. It was possible to increase the amplitude of the orthodromic negative potential appearing at the dendrite by previous hyperpolarization. For this purpose, we utilized the positive half of a sinusoi’dal wave, rendering the microelectrode positive (Fig. IOA). During the recovery which follows the maximum level ofhyperpolarization, twonegativedeviationscan be observed (Fig. 10A). They represent two dendritic action potentials as illustrated by the record 10B on a faster time base. The amplitude of the negative component reaches 3 mV which is about twice the amplitude of the action potential induced by cathodal stimulation (Fig. 9A). A small positive inflection which corresponds with the passage of the orthodromic action potential at the initiation site can be seen in the lower part of the curve. DISCUSSION The action potential triggered at the initiation site following mechanical or electrical stimulation of the dendrite appears always on an excited neuron and the moment of its origin cannot be controlled. However, by contrast, an antidromic impulse electrically evoked from the axon, far from the receptor terminal can be observed on neurons at any time, particularly when the initiation site does not induce spikes, i.e. when it is at rest (Fig. 4) or fully adapted following mechanical stimulation (Figs. 6D and 7). This should permit the amplitude of a second phase which closely reflects the dendritic membrane polarization to be followed at any moment, apart from the period during which a burst of sensory impulses is evoked. At this time collisions could be produced between the ortho and antidromic potentials (Fig. 6B). Our experiments have shown that, in a burst of action potentials, there is a large depressive influence of a spike on the one that follows (Fig. 5). At present we cannot explain this result. It is probably due to specific properties of the dendritic membrane because we do not detect such changes at the axonal level. Even though there is a clear relation (GUILLET and BERNARD, 1972) between the amplitude of the receptor potential and the amplitude of the negative phase which indicates that both affect the dendritic region, it appears in our experiments that they are not due to the same ionic processes. In fact, the phenomena that cause the marked decrease in impulses amplitude during a burst scarcely affect the receptor potential amplitude (Fig. 8). This supports the notion of WOLBARSHTand HANSON(1965) who consider that the invasion of the dendrite by impulses does not necessarily imply an invasion of the transducing surface. Moreover, studying the antidromic potential arriving at a receptor adapted to maintained
Electrical properties of the dendrite in a mechanoreceptor mechanical stimulation (Fig. 6) shows that even though its repolarization seems complete, the dendrite has not yet recovered its normal action potential. Indeed, its second phase reaches only a part of the amplitude obtained before stimulation. Concerning the question on the possible existence of two distinct parts in the dendrite, a transducing one giving the R.P. and a spiking one giving the negative phase of the action potential, we can only give the above arguments. But if they exist, we may expect, considering the small size of the dendrite, that their membrane voltage is the same and, up to a point varies in similar way (see below). Electrical stimulation of the receptor (Fig. 7) allows us to have a hyperpolarized dendrite and a depolarized initiation site. Even when the latter is totally adapted to the depolarizing current and no longer generates any more impulses, it is still capable of activity because it conducts the antidromic potential. With reference to the hyperpolarized dendrite, we have noticed as in previous work (GULLET and BERNARD, 1972) that the negative phase is enlarged by a small hyperpolarization but blocked by a larger hyperpolarization (Fig. 7). Our experiments clearly demonstrate that the dendrite and the initiation site are separately excitable since the two action potential phases can be completely dissociated from each other. Indeed the negative phase which has always been described until now as a second phase (see Fig. 4) can become a first phase (Fig. 9). This proves that the positive component cannot correspond to the recording of positive action currents induced into the conduction medium by the action potential propagation as YAMADA (1971) and MAES (1977) supposed. Both phases correspond to a true activation of the structures concerned (initiation site and dendrite). both being situated in two independent compartments. In natural stimulative conditions, a positive spike with a superimposed negative component has never been observed, which means that natural action potentials are never elicited in the dendritic part of the sensory neuron. This part has certainly a higher threshold than the natural initiation site. The antidromic propagation of the action potential towards the transducer region of the dendrite is probably not continuous as in the case of an unmyelinated axon, but more likely of a saltatory type. It seems that there is a non conducting zone between the compartment of the initiation site and that of the dendrite. This is indicated first of all by the net dissociation of the positive and negative components that also have different forms. This is seen also with electrical polarization (Fig. 2) which, in progressively hyperpolarizing the dendrite, should also progressively arrest the antidromic propagation of the impulse. thus resulting in a progressive decrease of the second phase. On the contrary, most often, the second phase suddenly disappears as if the dendritic compartment suddenly became as a whole electrically inexcitable resulting from the increase in its threshold. (However, in some cases, a small second negative phase may persist in one or two spikes after the disappearance of the large negative phase, as illustrated in part A of Fig. 2. Its seems that this small negative phase may be attributed to a second sensitive
761
cell which is stimulated by the electrical current and less synchronously with the fires more or mechanosensitive one.) The question of the presence or absence of a non-conducting zone remains as a working hypothesis. Finally. we can conclude that there is in the dendrite a region containing both a transducing structure and a structure capable of regenerative activity. In natural stimulating conditions. the spikes are not induced in this region but far from it, at the initiation site. It is impossible, morphologically, to distinguish between the two dendritic structures. But the ionic processes which underlie the R.P. are not the same as those generating the negative phase. Nevertheless, it appears that the amplitude of the negative phase reflects the polarization of the dendritic region which contains the two structures and thus, that the antidromic action potential can serve as a test to follow its variations. The reference action potential must be the one that is initiated on a sensory neuron at rest. There are limits to this test: the action potential must be triggered alone and outside of a burst of ortho or antidromic impulses. It could serve for example for following the variations in the membrane potential of the dendrite in response to changes in the ionic environment or to the addition of pharmacological substances. This would enable further analysis of specific properties of the transducer membrane if, as we expect for this short dendrite. the voltage is the same for the membrane performing mechano-electric transduction and the membrane giving rise to the negative phase of the spike. Acknowledgrmenf.s-
We wish to thank Dr D. B. SAI'TLLI.~. and
and Dr I. D. HARROW for their useful corrections criticisms.
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MAES F. W. (1977) Simultaneous chemical and electrical stimulation of labellar taste hairs of the blowfly Calliphora vicina. J. Insect Physiol. 23, 453-460.
MELLONDE F. (1968) The Physiology of Sense Organs. Univ. Reviews in Biol. Oliver & Boyd, London. MENDEL~ONM. (1966) The site of impulse initiation in bipolar receptor neurons of Callinectes sapidus L. J. exp. Biol. 45,411420. MORAND. T. and VARELLAF. G. (1971) Microtubules and sensory transduction. Proc. Nat. Acad. Sci., U.S.A. 68, 757. MORAND. T. and ROWLEYJ. C. (1975) Hight voltage and scanning electron microscopy of the site of stimulus reception of an insect mechanoreceptor. J. Ultrastruct. Res. 50, 38, 46.
MORAND. T.. CHAPMANK. M. and ELLISR. A. (1971) The fine structure of cockroach campaniform sensfila. J: Cell Biol. 48, 155-173. MORITAH. (1959) Initiation of spike potentials in contact chemosensory hairs of insects. III. D.C. stimulation and generator potential of labellar chemoreceptor of Calliphora. J. Cell. camp. Physiol. 54, 189. MORITA H. and TAKEDA K. (1959) Initiation of snike potentials in contact chemosensory iairs of insects. II.‘The effect of electric current on tarsal chemosensory hairs of Vanessa. J. Cell. romp. Physiol. 54, 177. RICE M. J. (1975) Insect mechanoreceptor mechanisms. In Sensory Physiology and Behavior (Ed. by GALUN R., HILLMANP., PARNASI. and WERMANR.) Plenum Press, New York. RICE M. J., GALUN R. and FINLAYSONL. H. (1973) Mechanotransduction in insect neurones. Nature, New Biol. 241. 286-288. THURM U. ’ (1962) Ableitung der Rezeptorpotentiale und Nervimpulse einzelner Cuticula-sensillen bei Insekten. Z. Natbforsch.
17b, 285-286.
THURMU. (1963) Die Beziehungen Zwischen Mechanischen Reisgrijssen und Stationlren erregung>zustlnden bei Borstenfeld-sensillen von Bienen. Z. Vergl. Physiol. 46, 35 l-382.
THURM U. (1968) Steps in the transducer process of mechanoreceptors. Symp. Zoo/. Sot., Lond. 23, 199. THURM U. (1970) Untersuchungen zur funktionellen Organisation sensorischer ZellverbHnde. Verhandl. Drsch. Zool. Ges. 64, 79-88. THURMU. (1972) The generation of receptor potentials in epithelial receptors. In O/jaction and Taste Vol. IV. Pergamon Press, Oxford. THURM U. (1974) Basics of the generation of receptor potentials in epidermal mechanoreceptors of Insects. In ‘Mechanoreception’, Symposium kochurn. Abhdlg. Rhein. Westf. Akad. Wissensch.. Westd. Verl. Ooladen. WOLBARSHTM. L. (1958) Electrical activity *in the chemoreceptors of the blowfly. II. Responses to electrical stimulation. J. gen. Physiol. 42, 413-428. WOLBARSHT M. L. (1960) Electrical characteristics of insect mechanoreceptors. J. gen. Physiol. 44, 105-l 22. WOLBARSHTM. L. and DETHIERV. G. (1958) Electrical activity in the chemoreceptors of the blowfly. 1. J. ~cn. Physiol. 42, 393412.
WOLBARSHTM. L. and HANSON F. E. (1965) Electrical activity in thechemoreceptorsofthe blowfly. III. Dendritic action potentials. J. gen. Physiol. 48, 673-683. YAMADA M. (1967) The electrical activity recorded from sensilla basiconica on antenna ofthe rice stem borer larvae. Chilo suppress&s (Lepidoptera: Pyralidae). .4pf/. ,%t. Zool. 2, 22-30. YAMADA M. (1971) The dendritic action potentials in an olfactory hair of the fruit-piercing moth Orae.\iae~cavafu. J. Insect Phvsiol. 17, 169-179
0022-1910/80/l
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ELECTRICAL PROPERTIES OF THE DENDRITE IN AN INSECT MECHANORECEPTOR: EFFECTS OF ANTIDROMIC OR DIRECT ELECTRICAL STIMULATION J. C. GUILLET, J. BERNARD,J. P. COILLOTand J. J. CALLEC Laboratoire de Physiologie Animale. Campus de Beaulieu, 35042 Rennes Cedex, France (Received 3 March 1980; revised 12 June 1980)
Abstract-A study of the negative phase of the spikes recorded extra cellularly from insect mechanoreceptor has been performed in order to characterize some electrical properties of the dendrite which contains the transducing part of the sensory neuron. These properties have been investigated in mechanoreceptors of the metathoracic leg of the locust Schistocerca gregaria by firing antidromic action potentials both at rest and during mechanical or electrical stimulation. The amplitude of the negative phase of the spike appears to be correlated with the polarization of the dendritic membrane, although when bursts of action potentials are applied, the relation is more complex, including a depressive influence of a given spike on the following spike. The receptor potential and the antidromic dendritic spikes both originate in the same region of the dendrite but they involve different ionic processes. Our results indicate that the dendrite is electrically excitable. The spike which originates in the dendrite has an initial negative phase with a small superimposed positive component. A spike of this shape is never observed under natural stimulation. It is proposed that the negative phase of the antidromic impulse provides a suitable means for studying the variations in electrical polarization of the dendrite which cannot be recorded directly. Ke.v Word Index: Dendrite mechanoreceptor. electrophysiology insect
INTRODUCTION IN THEORIESof sensory
transduction
the dendrite
is
assumed to play a very important part (RICE et al., 1973; MORAN and VARELLA,1971; THURM,1968,1970, 1972,1974; ATEMA,1973). However itsexact role is not yet well defined for external insect receptors, because it is impossible with classical external electrode recordings from the base of the sensilla, to record the dendritic potentials selectively. The records represent both the activity of the dendrite and that of the initiation site of the impulses. Therefore, the study of a parameter reflecting dendritic membrane polarization is of considerable interest. With this end in view we have studied the biphasic action potential, recorded from a mechanoreceptor by an external microelectrode because the shape and the amplitude of its two phases, being dependent on both dendritic and axonal polarization, may reflect the electrical activity at these two sites. Our objective is to study dendritic membrane properties using the negative phase of the action potential as a test of the polarization and the excitability of the dendritic membrane. In our work, we have applied electrical stimulation to the sensory axon at a distance from the transducing region so as not to influence it. We have studied the resulting antidromic action potentials arriving at the dendrite placed in different electrical states, particularly in a steady resting state which has never been observed before. We have also applied electrical stimulation directly to the dendrite and proved that it is electrically excitable. Theoretical
considerations
Let us consider
the preparation
classical recording technique, i.e. with a microelectrode inserted at the base of the external process (BOECKH,1962; THURM, 1962; YAMADA,1967, 1971). This electrode is often inserted in the joint membrane, when it exists (for morphological details on external mechanoreceptors, see MORANet al., 197 1; GNATZY, 1976). The microelectrode,
which is always
more or less broken after its passage through the cuticle is large (about l-5 PM) compared with the diameter of the dendrite (about 5 PM). Thus, it cannot penetrate it and it is always an external electrode. The electrical situation is similar when the electrode is not inserted through the cuticle but brought into contact with the exposed end of a chemoreceptor or a tactile hair (WOLBARSHT, 1960; WOLBARSHT and DETHIER, 1958): the recording electrode is external to the dendrite. In these conditions, various electrical models of mechano-or chemoreceptors have been proposed to explain the physiological significance of the two phases of the extracellularly recorded action potential (MORITA, 1959; MORITA and TAKEDA, 1959; WOLBARSHT, 1958; THURM, 1963; WOLBARSHT and HANSON, 1965; DE MELLON, 1968; YAMADA. 1971;
GUILLETand BERNARD,1972; MAES. 1977). It appears that the initial positive component signals the onset of the impulse at the site of electrogenesis and that the subsequent negative phase reflects the antidromic conduction of the impulse towards the dendrite. From these considerations, we have illustrated the experimental situation for the sensory neuron of a mechanoreceptor (Fig. 1) which receives mechanical stimulation in A and electrical stimulation in B. Membrane polarization is represented in the left half of the neuron. The initiation and conduction of the in the case of a sensory spike is represented in the right half. The scope 155
J. C. GULLET et al.
756 A Met
St
\
ElSt+\
I
B
,
Fig. 1. Diagram of a sensory neuron showing how the spike is induced and recorded during mechanical stimulation (A) and during electrical stimulation (B). The tip of the recording micropipette is near the dendrite, the reference electrode being in the conductive medium. A. The left part of the neuron shows its electrical state when mechanical stimulation (Met. St.) induces a receptor potential (R.P.) in the transducing zone of the dendrite(D). The R.P., which is a local but sustained depolarization decreasing electrotonically (indicated by the thickness of the dark areas), induces the impulses at the initiation site (1,s.) some distance from the transducing zone. The right part of the neuron shows the spike when it is initiated (time 1) and a moment later (time 2) after orthodromic conduction along the axon and antidromically along the dendrite. The scope record of this spike shows the two phases corresponding to time 1 (+) and to time 2 (-). The second phase, which is a dendritic phase, has the same polarity as the dendritic R.P. B. Same presentation as in A. Electrical stimulation (El. St.) is applied via the positive microelectrode. The current enters the dendrite and exits at the initiation site. In the left part of the neuron, the micraelectrode being positive, we see the current hyperpolarizing the dendrite and depolarizing the initiation site, thus triggering impulses. The hyperpolarized zone is illustrated by dashed areas and the depolarized zone is illustrated by dark areas. Their thickness represents the amplitude of polarization. Comparison of the two spikes shows that the second (negative) phase is small in A because the dendrite is depolarized but large in B because the dendrite is hyperpolarized.
record considers only this spike. The micropipette (the active electrode) is placed on the outside of the sensory neuron, near the dendrite where the receptor potential (R. P.), corresponding to a depolarization of the appears during sensilla stimulation dendrite, (Fig. 1A). This R. P. is therefore recorded as a negative variation of potential, not represented in the scope record. Action potentials, induced by the R. P., appear some distance from the transducing region. They are therefore recorded initially (time 1) as positive variations of potential (upward deflection in the scope record of Fig. 1A). Then (time 2) they are conducted orthodromically towards the central nervous system, and, under certain conditions which are analysed in this paper, antidromically towards the dendritic *We place the initiation site on the axon, near the cell body because it appears to us that the structure of this short (20-30 pm) dendritic bipolar neuron is anatomically close to that of a vertebrate motoneurone (ECCLES, 1957) or to that of certain crustacean proprioreceptors (EDWARDSand OTTOSON, 1958)
where the action potential appears at the axon hillock. It differs from the long dendrite (about 300 pm) of the P.D. organ of crustacean (HARTMANand BOETTIGER,1967; MENDELSON, 1966) or even more from the very long dendrite of sensory neuron of vertebrate where the spikes appear on the dendrite. However. it would have been possible to place the initiation site on the dendrite as illustrated by MORITA (1959) YAMADA(~~~~) and MAF..s( 1977). Thisdoes not affect the most important point to be considered here: the R.P. and the negative phase of the spike are produced in the same part of the dendrite which, when it depolarizes, gives a negative potential in our recording conditions.
terminal. When they invade this area, beneath the they are recorded as negative microelectrode, variations of potential (downward deflection in scope record of Fig. 1A). Figure 1B shows the experimental situation when the receptor is stimulated electrically. The current is applied by means of the recording micropipette. When this micropipette is positive, the imposed maintained current enters, and thereby hyperpolarizes the dendrite (BERNARD and GUILLET 1972). The current exits through the initiation site* of the sensory neuron, which is depolarized, thus triggering a burst of biphasic action potentials. These spikes always exhibit a large negative phase because the dendrite is hyperpolarized. In contrast, spikes which are induced by mechanical stimulation (in A) have a smaller or non-existent negative phase because, in this case, the dendrite is depolarized. The shape and the amplitude of the two phases may vary under different conditions. This is illustrated, for a phasic mechanoreceptor, in Fig. 2. An imposed and maintained electrical current induces a burst of action potentials. At the beginning of the record, they are biphasic. The second phase is negative, large in amplitude, and increases for some time until finally it disappears. In order to explain this phenomenon, we have proposed (GULLET and BERNARD, 1972) that the inward maintained current progressively increases the dendritic membrane resistance (as an anomalous rectification), therefore increasing membrane hyperpolarization. In this scheme, the negative phase enlarges but, after a time, antidromic invasion of the dendrite by the spike is
Electrical
El. St.
properties
of the dendrite
77 El.St.
Met. St.
c
Antidrom.
1 I
in a mcchanoreceptor
,llN+Y
c------
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____-______ I5mV
A
B
5Xc
Fig. 2. The different shapes of the action potentials. An electrical stimulation (El. St.) is applied to the receptor (microelectrode +) from A to the end of the record, inducing a long burst of impulses. Mechanical stimulation (Met. St.) is superimposed in B. For explanation. see text.
blocked as the dendrite becomes too hyperpolarized. A mechanical stimulus (Fig. 2B) induces a superimposed depolarization (R.P.) during which the impulses vary again. They are first positive and monophasic. The absence of the negative phase can be explained in this case by the depolarization of the dendrite. This is confirmed by the fact that during the adapting phase of the receptor potential, the dendrite, in progressively recovering its resting potential, recovers also its excitability and during a time corresponding here to three spikes, the conduction of antidromic action potentials, thus giving transitory biphasic potentials. From the above theoretical considerations, the following main points emerge which are important in explaining our results: (1) the antidromic propagation of action potentials in the dendrite is the origin of the negative phase; (2) the amplitude of this negative phase reflects the variations found in the polarization of the dendritic membrane; (3) under both types of stimulation, mechanical and electrical, the action potential is always observed in an activated sensory cell, the dendrite being either depolarized (mechanical stimulation) or hyperpolarized (electrical stimulation); (4) action potentials are grouped into bursts. We will see that this results in modifications in the amplitude of the negative phase. In this paper, we consider as a whole the dendrite or the part of the dendrite which gives the R.P. and the negative spike because it is impossible to distinguish a priori a transducing region from a spiking one. But this point will be partly discussed. METHODS Experiments were carried out on phasic mechanoreceptors of the locust Schistocerca gregaria. The receptors studied on isolated metathoracic legs, are situated dorsally on the distal part of the head of the femur. Their electrical activity has been previously described (GUILLET, 197.5) as well as stimulation and recording techniques (BERNARDand GUILLET, 1972). These are recapitulated briefly in the theoretical considerations above. The entire preparation is shown in Fig. 3. The microelectrode inserted in the joint membrane at the base of the receptor was used for recording and for electrical stimulation. Mechanical stimulation was effected by a microhook connected to the cone of a
Fig. 3. Experimental arrangement. The phasic mechanorcceptors are situated on the distal part of the metathoracic leg. A microelectrode, inserted at the base of one receptor, was used both to record (R.) and to apply electrical stimulation (El. St.). Mechanical stimulation (Met. St.) was applied with a microhook driven by the membrane of a loudspeaker. The reference electrode was introduced into the leg. The sensory nerve containing the axon of the mechanoreceptor was dissected through a small window in the cuticle and gently placed on two silver wires. In these conditions, it is possible to apply an electrical stimulation (Antidrom. El. St.) to the nerve and thus. to record an antidromic action potential at the level of the receptor.
loud-speaker. Electrical stimulation of the sensory axon was applied via two silver electrodes which were placed under a lateral sensitive branch of the nerve V according to the technique described by COILLOT and BOISTEL(1969). The reference electrode consisted of a silver wire placed in the femur.
RESULTS Electrical stimulation of‘the sensory axon at a distance from the dendrite. Stud-v of the antidromic actiorl potential (a) Dendrite at rest. In stimulating the sensory nerve with a short electric pulse, one can record at the receptor, an antidromic action potential (Fig. 4). This spike is typically biphasic with the initially positive component being approximately l/6 the amplitude of the negative component. The duration of the first phase is less than 1 msec whereas the second is more than 15 msec. The positive phase corresponds to the appearance of the action potential at the initiation site; however, in this case, it is not directly elicited at this initiation site. but indirectly after it has been conducted antidromically along the axon. The second phase corresponds to the spike in the dendrite which, in this particular case. was previously in steady state conditions. Its form and amplitude can
I+ I--2,SmV
10 msec
Fig. 4. Antidromic action potential induced by electrical stimulation of the sensory axon far from the receptor and recorded from a receptor at rest.
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0
1 . 10
lOiG&
A
.
.
I
.
50
.
.
(Time)
.
-
)
msec
B
Fig. 5. Records (A) and curves (B) showing the decrease in amplitude of the negative phase of antidromic spikes when they are triggered in bursts. A brief stimulus artefact precedes each spike. F, Frequency of the
stimulation (Hz).
therefore serve as a reference for discerning the state of membrane polarization under other experimental conditions. With repetitive stimulation, the amplitude of the second phase decreases from one impulse to the next. For example, in the upper record of Fig. 5A, the negative phase of the second action potential is reduced by 15% in spite of a time interval of 75 msec between the two impulses. This phenomenon is accentuated during bursts, (Fig. 5A, lower records) and becomes even more evident at higher stimulation frequencies as shown by the graphs in Fig. 5B which correspond to three frequencies of sustained stimulation. The decrease, which appears in the first 100 msec, reaches 50% at a frequency of 100 Hz (low for an axon). Therefore, it appears that an action Antidrom. El. St.
+
“I”“.
;y--$p-
Fig. 6. Antidromic spikes observed in a phasic receptor which was depolarized by mechanical stimulation (Met. St.). In each record, the thin arrow signals the beginning of the maintained mechanical stimulus which induced a receptor potential of phasic type and two superimposed positive monophasic spikes. The thick arrow shows the artefact due to the electrical stimulus (Antidrom. El. St.) which triggers the antidromic spike. From A to D, the antidromic spike was triggered before the receptor potential (A), during the time course of the R.P. (B and C) and after the completion of adaptation (D).
potential which arrives at a dendrite modifies its electrical state for a long time. We do not know the mechanisms which underlie this phenomenon and which are quite different from those observed in an axon. (b) Dendrite depolarized by a mechanical stimulation. It is possible to stimulate the sensory axon at any time during mechanical stimulation. The amplitude of the negative phase can then be studied in a depolarized dendrite. This is illustrated in Fig. 6. In A, B, C, D a weak mechanical stimulus just inducing two monophasic and positive action potentials, was applied to the receptor. An antidromic impulse was triggered before and during this stimulation. In record A, the antidromic potential was observed on a sensory neuron at rest, therefore serving as a reference point. In B it disappears because the antidromic electrical stimulation was applied to the nerve during the refractory period which follows the orthodromic action potentials. In C, it is observed during the adaptation phase of the R.P. It shows a large negative component, which is however lower than that in A. In D, it appears after adaptation is complete. The amplitude of the second phase (2.5 mV) is still lower than that observed in A before stimulation (3.75 mV). This indicates that, even when the dendrite has apparently recovered its resting potential, the influence of the mechanical stimulation is much prolonged and the second pllase provides evidence for this. (c) Dendrite hyperpolarized by electricalstimulation. The applied electric current which hyperpolarizes the dendrite induces a burst of orthodromic potentials during which it is impossible to observe an antidromic dendritic impulse. This is due to the refrattory period as in the case illustrated in Fig. 6B for mechanical stimulation. But after the burst, the antidromic action potential can be observed. The results are illustrated in Fig. 7. As shown in Fig. 7A, current application induced a burst of biphasic action potentials. As shown in B they were monophasic when the current was stronger and hyperpolarized the dendrite membrane sufficiently to render it inexcitable. The action potentials no longer invaded the dendrite. (A
Electrical
properties
Antidrom.
of the dendrite
El. St.
Fig. 7. An electrical stimulus {El. St.) which induced a burst of impulses (microelectrode +) was applied to the receptor from the thin arrow in the record, to the end. The last spike of the burst is marked by an upward pointing arrow. Shortly after this last spike, an antidromic spike was evoked. The electrical stimulus was greater in B than in A. It was just sufficient in B to block the negative phase of all the impulses. The asterisks signal in A some spikes of a second cell which is stimulated by the applied current. It is very difficult in B to distinguish the very small monophasic positive spikes of this second cell.
second cell innervating the sensilla is also sensitive to the applied current and explains the origin of the spikes marked by an asterisk.) After the electrically-evoked activity ceased (marked by an upward pointing arrow), the sensory nerve was stimulated. It is still possible (Fig. 7) to observe an antidromic action potential with the same form and amplitude as that of the last potential in the burst, i.e. biphasic in A but positive and monophasic in B. (d) Influence of a burst ofantidromic actionpotentials on the receptor potential amplitude. The receptor potential and the second phase of the action potential both being electrical phenomena appearing at the dendrite level, show respective amplitudes which are correlated and dependent on the polarization of the dendrite membrane. This was established by applying polarizing currents to the receptor (BERNARDand GULLET. 1972; GULLET and BERNARD,1972) and partly in the above experiments (Fig. 6). Moreover, we have shown the existence of a phenomenon which tends to decrease the amplitude of the second phase in Antidrom. El. St
in a mechanoreceptor
759
a burst of antidromic impulses (Fig. 5). This raises the question as to whether or not the receptor potential and the dendritic action potential affect the same dendritic processes. If this is the case, the phenomenon which decreases the negative phase of action potentials during the burst would be expected to affect the amplitude of the receptor potential similarly. We have never observed a smaller modification in the receptor potential when it is preceded immediately by only one antidromic impulse. However, an antidromic impulse modifies the amplitude of the one that follows (Fig. SA). We applied a series of electrical stimuli to the nerve before mechanical stimulation of the receptor. As shown in Fig. 8A, the receptor potential was preceded by a burst of antidromic impulses with a frequency of 80/set; an attenuation of 8% was experienced. This is much lower than the 52% observed in the last impulse of the burst. In Fig. 8B the burst frequency was 137/set. The attenuation of the receptor potential was 13’& that of the last impulse was 55%. It is clear that there is a discrepency between the two electrical phenomena. Electrical stimulation of the dendrite resulting in an orthodromic dendritic action potential
In all cases studied so far the dendritic action potential was antidromic, invading the transducer zone of the dendrite secondarily. For this reason it was always recorded as a second phase. The question arises as to whether it would possible to trigger the potential at the dendritic level so as to observe an initial negative phase. Isolated negative potentials have been observed but only on deteriorated preparations or in receptors subjected to strong stimulation (MORITA, 1959; WOLBARSHTand HANSON,1965; YAMADA,1971). It is possible that, in the above cases, the positive component was only masked, also it cannot be proved that the dendritic action potential was not antidromic. To remove any such doubt it would be necessary to observe an impulse with a negative first phase followed by a positive phase. (a) Rectangular pulse of long duration. This kind of stimulation gives rise to an action potential only if we adjust precisely the current intensity (negative microelectrode). This is illustrated in Fig. 9A where the pulse induces a negative response (slanting arrow) Met. St.
2Omsec ‘::
Fig. 8. Influence of an antidromic burst of action potentials on the amplitude of the receptor potential (R.P.) The first time (dotted line) a mechanical stimulus alone (Met. St.) was applied to the receptor, inducing a R.P. The second time (full line) the mechanical stimulus was preceded by antidromic electrical stimuli (Antidrom. El. St.) of varying frequencies. A brief stimulus artefact precedes each spike. Frequencies of electrical stimulation: A =80/set; B = 137/set.
760
J. C. GULLET er 01.
A
I
1mV
dmsec J
\
Fig. 9. Comparison of two sorts of spike evoked by direct electrical stimulation of the receptor. The maintained current was applied via the microelectrode with the aid of a Wheatstone bridge. The spikes, at the beginning of the stimulation, appear on a baseline which has not yet stabilized. A, Microelectrode negative. B. Upper truce = microelectrode negative, lower trace = microelectrode positive. Same intensity of the applied current in the two records B.
which appears as an all or nothing spike. The amplitude (1.5 mV) and the duration are identical to those of the second negative phase found in the first situation. We can see, superimposed on this negative potential, a small positive component corresponding with the passage at the initiation site, of the orthodromically conducted impulse. This experiment suggests that the dendrite containing the transducing structure is indeed electrically excitable. Figure 9B compares this orthodromic dendritic action potential with the antidromic one triggered with a pulse of the same intensity but of opposite polarity. In the upper record,
v +
I
1mV
G&
Fig. 10. Spikes triggered in the dendrite
b
by a sinusoMally varying current. Middle record: the positive half of the applied current. A. Corresponding record. The Wheatstone bridge cannot be perfectly balanced because the resistance of the preparation varied with applied current. The dendrite was hyperpolarized initially, then it is returned to its normal resting potential. During the return, two spikes are triggered. They seem monophasic negative. B. On a faster time base, the spikes monophasic negative, but a small appear superimposed positive component can be observed.
the microelectrode was negative resulting in an action potential triggered at the dendritic level. The first phase is negative. In the lower record, the microelectrode is positive resulting in an action potential which appears at the initiation site. The latter is typically biphasic (positive then negative). The negative phases of the two spikes are different in amplitude, because the dendrite was depolarized in the upper record and hyperpolarized in the lower. (b) Shu.dation by a sinusoidal current. It was possible to increase the amplitude of the orthodromic negative potential appearing at the dendrite by previous hyperpolarization. For this purpose, we utilized the positive half of a sinusoi’dal wave, rendering the microelectrode positive (Fig. IOA). During the recovery which follows the maximum level ofhyperpolarization, twonegativedeviationscan be observed (Fig. 10A). They represent two dendritic action potentials as illustrated by the record 10B on a faster time base. The amplitude of the negative component reaches 3 mV which is about twice the amplitude of the action potential induced by cathodal stimulation (Fig. 9A). A small positive inflection which corresponds with the passage of the orthodromic action potential at the initiation site can be seen in the lower part of the curve. DISCUSSION The action potential triggered at the initiation site following mechanical or electrical stimulation of the dendrite appears always on an excited neuron and the moment of its origin cannot be controlled. However, by contrast, an antidromic impulse electrically evoked from the axon, far from the receptor terminal can be observed on neurons at any time, particularly when the initiation site does not induce spikes, i.e. when it is at rest (Fig. 4) or fully adapted following mechanical stimulation (Figs. 6D and 7). This should permit the amplitude of a second phase which closely reflects the dendritic membrane polarization to be followed at any moment, apart from the period during which a burst of sensory impulses is evoked. At this time collisions could be produced between the ortho and antidromic potentials (Fig. 6B). Our experiments have shown that, in a burst of action potentials, there is a large depressive influence of a spike on the one that follows (Fig. 5). At present we cannot explain this result. It is probably due to specific properties of the dendritic membrane because we do not detect such changes at the axonal level. Even though there is a clear relation (GUILLET and BERNARD, 1972) between the amplitude of the receptor potential and the amplitude of the negative phase which indicates that both affect the dendritic region, it appears in our experiments that they are not due to the same ionic processes. In fact, the phenomena that cause the marked decrease in impulses amplitude during a burst scarcely affect the receptor potential amplitude (Fig. 8). This supports the notion of WOLBARSHTand HANSON(1965) who consider that the invasion of the dendrite by impulses does not necessarily imply an invasion of the transducing surface. Moreover, studying the antidromic potential arriving at a receptor adapted to maintained
Electrical properties of the dendrite in a mechanoreceptor mechanical stimulation (Fig. 6) shows that even though its repolarization seems complete, the dendrite has not yet recovered its normal action potential. Indeed, its second phase reaches only a part of the amplitude obtained before stimulation. Concerning the question on the possible existence of two distinct parts in the dendrite, a transducing one giving the R.P. and a spiking one giving the negative phase of the action potential, we can only give the above arguments. But if they exist, we may expect, considering the small size of the dendrite, that their membrane voltage is the same and, up to a point varies in similar way (see below). Electrical stimulation of the receptor (Fig. 7) allows us to have a hyperpolarized dendrite and a depolarized initiation site. Even when the latter is totally adapted to the depolarizing current and no longer generates any more impulses, it is still capable of activity because it conducts the antidromic potential. With reference to the hyperpolarized dendrite, we have noticed as in previous work (GULLET and BERNARD, 1972) that the negative phase is enlarged by a small hyperpolarization but blocked by a larger hyperpolarization (Fig. 7). Our experiments clearly demonstrate that the dendrite and the initiation site are separately excitable since the two action potential phases can be completely dissociated from each other. Indeed the negative phase which has always been described until now as a second phase (see Fig. 4) can become a first phase (Fig. 9). This proves that the positive component cannot correspond to the recording of positive action currents induced into the conduction medium by the action potential propagation as YAMADA (1971) and MAES (1977) supposed. Both phases correspond to a true activation of the structures concerned (initiation site and dendrite). both being situated in two independent compartments. In natural stimulative conditions, a positive spike with a superimposed negative component has never been observed, which means that natural action potentials are never elicited in the dendritic part of the sensory neuron. This part has certainly a higher threshold than the natural initiation site. The antidromic propagation of the action potential towards the transducer region of the dendrite is probably not continuous as in the case of an unmyelinated axon, but more likely of a saltatory type. It seems that there is a non conducting zone between the compartment of the initiation site and that of the dendrite. This is indicated first of all by the net dissociation of the positive and negative components that also have different forms. This is seen also with electrical polarization (Fig. 2) which, in progressively hyperpolarizing the dendrite, should also progressively arrest the antidromic propagation of the impulse. thus resulting in a progressive decrease of the second phase. On the contrary, most often, the second phase suddenly disappears as if the dendritic compartment suddenly became as a whole electrically inexcitable resulting from the increase in its threshold. (However, in some cases, a small second negative phase may persist in one or two spikes after the disappearance of the large negative phase, as illustrated in part A of Fig. 2. Its seems that this small negative phase may be attributed to a second sensitive
761
cell which is stimulated by the electrical current and less synchronously with the fires more or mechanosensitive one.) The question of the presence or absence of a non-conducting zone remains as a working hypothesis. Finally. we can conclude that there is in the dendrite a region containing both a transducing structure and a structure capable of regenerative activity. In natural stimulating conditions. the spikes are not induced in this region but far from it, at the initiation site. It is impossible, morphologically, to distinguish between the two dendritic structures. But the ionic processes which underlie the R.P. are not the same as those generating the negative phase. Nevertheless, it appears that the amplitude of the negative phase reflects the polarization of the dendritic region which contains the two structures and thus, that the antidromic action potential can serve as a test to follow its variations. The reference action potential must be the one that is initiated on a sensory neuron at rest. There are limits to this test: the action potential must be triggered alone and outside of a burst of ortho or antidromic impulses. It could serve for example for following the variations in the membrane potential of the dendrite in response to changes in the ionic environment or to the addition of pharmacological substances. This would enable further analysis of specific properties of the transducer membrane if, as we expect for this short dendrite. the voltage is the same for the membrane performing mechano-electric transduction and the membrane giving rise to the negative phase of the spike. Acknowledgrmenf.s-
We wish to thank Dr D. B. SAI'TLLI.~. and
and Dr I. D. HARROW for their useful corrections criticisms.
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