Delayed firing of giant axons in the American cockroach

y. Insect Physiol., 1971,Vol. 17, pp. 1565to 1577.Pwgamon Press. Printed in Great Britain

DELAYED

FIRING OF GIANT AXONS AMERICAN COCKROACH

C. LEON

HARRIS*

and T. SMYTH,

IN THE

JR.

Departments of Biophysics and Entomology, The Pennsylvania State University, University Park, Pennsylvania 16802

(Received 16 October 1970) Abstract-Giant intemeurons in the abdominal nerve cord of the American cockroach can give two kinds of response to single pre-synaptic shock stimuli: a conventional post-synaptic response with a short delay, and a delayed response consisting of one or a patterned group of impulses beginning after a delay of at least 10 msec. This delayed response originates within the last abdominal ganglion, but is independent of the cereal nerve to giant fibre synapses. Apparently, it is generated by an additional neural element which also is activated by the cereal nerves and which in turn excites the giant fibres. INTRODUCTION GIANT axons in invertebrates are generally assumed to be major elements in rapid response systems because of their relatively high impulse conduction velocities. Thus, the existence of giant fibres in the longitudinal connectives of cockroaches where space is limited implies the importance of rapid impulse conduction there. ROEDER (1948, 1959) assumed that these fibres have a primary ri%e in an escape response, and quite recently DAGAN and PAFWAS (1970) have reinterpreted their role in evasive behaviour. We were surprised, therefore, to discover that single shocks given the cereal nerves could evoke a response delayed by more than 10 msec (DR), alone or in addition to the expected immediate response (IR). This DR, occurring paradoxically in a system specialized for rapid conduction, demanded further study. MATERIALS

AND METHODS

Adult male American cockroaches, Periplaneta americana, were used for all the observations reported here, although females also show a DR. Each cockroach was chilled to facilitate handling and then immobilized ventrai side up on a wax form. The connectives between the fifth and sixth abdominal ganglia (AS-A6) were exposed by removing portions of the appropriate sternites and the tip of the abdomen was then bent dorsally and pinned so as to expose the cereal nerves (nerve 11 of ROEDERet al., 1960; nerve 8 of GUTHRIE and TINDALL, 1968). The accessory gland and part of the fat body were removed to improve visibility and * Present address: Department of Biological Sciences, State University College, Plattsburgh, New York 12901. 1565

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to reduce transmission of rectal movements to the nerve cord. Connectives A5A6 were gently separated and a glass stabilizer was placed between them and the gut. Exposed tissue was kept moist with a physiological saline (YAMASAKI and NARAHASHI, 1959). Chlorided silver electrodes were usually placed as follows: a monopolar recording electrode under one connective adjacent to ganglion A5, an indifferent electrode in the body cavity, and bipolar stimulating electrodes under both cereal nerves. Sturdy microelectrodes for intracellular recording were inserted through the nerve sheath into axons in an A5-A6 connective adjacent to the last abdominal ganglion using essentially the technique of PICHON and BOISTEL (1968). Giant axons were most easily impaled because of their large diameters. The identity of giants was confirmed by measuring conduction velocities between the intra- and extracellular recording sites. Conduction velocities of 4 to 7 msec were taken as indicative of giants. However, smaller axons with conduction velocities as slow as 2.3 msec also gave both IRS and DRs.

RESULTS Appearance of the DR When a cereal nerve was stimulated by an adequate electric shock, the giant fibre response could be an IR, a DR, or both. In about half of the axons showing a DR the threshold was lower for the IR than for the DR; in the others, as shown in Fig. 1, the threshold was lower for the DR. Upper traces in Fig. 1 show extracellular recordings from the proximal end of the left A5-A6 connective; lower traces, intracellular recordings from the distal end of the same connective. The stimulus was a single O-1 msec shock to the homolateral cereal nerve, increasing in voltage from (a) to (c). The DR consisted of three spikes occurring approximately 13, 32, and 34 msec after the stimulus. In (a) there was only a DR. In (b) there was also an IR. With a substantial increase in stimulus strength in (c) additional post-synaptic fibres were activated as indicated by the larger IR on the extracellular record, but the intracellularly recorded DR was unchanged. Over a wide range of stimulus intensities the DR was essentially constant. DR patterns also generally remained stable with respect to time for as long as it was possible to record them. Sometimes individual spikes in the DR failed to appear, but the remaining spikes occurred on schedule, seldom varying more than 5 msec in their timing after the stimulus. Both IR and DR spikes always propagated in an anterior direction, even when a response was evoked by stimulating an anterior point on the nerve cord. Indeed, a DR was sometimes recorded from the stump of an A5-A6 connective after all connexions to A6 were severed except for one cereal nerve. Following such extensive injury the DR soon failed.. The occurrence of a DR in the nearly isolated ganglion indicates that the DR is not a secondary response to a motor reflex evoked by the stimulus. Small motor branches from the cereal nerve (GUTHRIE and TINDALL, 1968) undoubtedly were cut in the isolation procedure. Further evidence against a reflex origin of the DR

FIG:. 1. from

DR

following stimulus to homolateral cereal nerve. Stimulus voltage increases In (b) and (c) an IR also appears. Voltage calibration applies only to the intracellular recordings, lower traces. Stimulus trigjyzrs oscilloscope sweep.

(a) to (c).

156X

Se 75 mV

L 15 msec

Frti. 2. Responses of a single giant to stimuli applied at different locations : (a) homolateral cercus-IR and DR; (b) heterolateral cercus-DR only; (c) and (d) T3-Al connectives and a cervical connective, respectively-descending spike indicated by pointer and late Voltage calibration applies only to ascending spike; (e) air puff directed to left cercus. Stimulus triggers oscilloscope sweep. the intracellular recordings, lower traces.

156’)

hi. 3. Synaptic potentials monitored in a giant axon near ganglion A6. With a weak stimulus (a) the IR is an EPSP. Stronger stimuli (b, c, d) evoke an IR spike. The DR is associated with a slow, prolonged depolarization. When DR spike components fail to appear there may be instead a brief additional depolarization (c, d). Voltage calibration applies only to the intracellular recordings, lower traces. Stimulus triggers oscilloscope sweep.

7.5 mV

L 15 msec

FIG. 4. Effects of picrotoxin and eserine. (a) Before and (b) 30 min after application of 2 x 10. d M picrotoxin. (c) Before and (d) 15 min after application of 3 x IO-” M eserine sulphate. Voltage calibration applies only to the intracellular recordings, lower traces. Stimulus triggers oscilloscope sweep.

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75

)

mV

15 msec

l-lower

trace

FIG. 5. Resetting the DR by a second stimulus (dot). First stimulus starts oscilloscope (a) DR following first simulus; (b, c) DR to first stimulus cancelled by second sweep. stimulus which restarts the DR, seen delayed in (b), off screen in (c). In (d) the second stimulus and an IR follow the DR to the first stimulus. In this experiment the second stimulus was slightly stronger than the first. Voltage calibration applies only to the intracellular recordings, lower traces.

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was obtained from cockroaches in which the muscles were paralysed with ryanodine (supplied by courtesy of S. B. Penick and Company), and from cockroaches it immobilized in paraflin. Neither procedure affected the DR. Therefore, appears that the long latency and the pattern of the DR arise entirely within the last abdominal ganglion. Sites for evoking the DR

The DR could be evoked by stimulating either cereal nerve. An ascending action potential originating in the terminal ganglion could also be evoked by stimulating an anterior point on the nerve cord such as a connective between the first and second abdominal ganglia (Al-A2) or a cervical connective. Fig. 2 shows responses of a preparation in which the DR consisted of a single spike. Stimulating the homolateral cereal nerve evoked an IR and also a DR with 18 msec latency (Fig. 2a). Stimulating the heterolateral cereal nerve evoked only a DR, latency 17 msec (Fig. 2b). When an Al-A2 connective or cervical connective was stimulated (Fig. 2c, 2d), there was a descending spike in the giant fibre followed after about 40 msec by an ascending spike which was probably a DR evoked by descending small fibre activity. In another preparation where Al-A2 was stimulated the DR followed the stimulus by only 24 msec. Fig. 2(e) illustrates a typical response to a puff of air directed at the cerci. In this case the IR consisted of two impulses in quick succession. A DR was never found when the stimulus was an air puff, sound, or other mechanical disturbance of the cerci. Of 13 axons in which the DR was evoked by stimulating the homolateral cereal nerve, the threshold for the DR was lower than for the IR in six, higher in five, equal in one and undetermined in one. All axons giving a DR could also be made to give an IR. In about half of the axons the DR consisted of one spike, the others giving a pattern of two or three. Of five axons giving a DR when the heterolateral cereal nerve was stimulated, three showed only a DR, the others both IR and DR with a lower threshold for the IR. There were one or two spikes in each DR. When a DR was evoked by stimulating Al-A2 or a cervical connective, its threshold was higher than the threshold for initiating a descending impulse in the giant. This suggests that the DR is not generated by some persisting excitability or rebound in the giant itself but depends on activation of other (descending) axons which, being smaller than the giants, should have higher thresholds for external electrical stimulation. Synaptic potentials

With intracellular electrodes placed very close to the last abdominal ganglion it was possible to observe non-propagated synaptic potentials. Fig. 3(a) shows a DR evoked by weak stimulation of the homolateral cereal nerve. There is no IR, but in place of it there is a small EPSP. As the stimulus voltage was increased this became stiil larger until a spike was generated (Fig. 3b). When individual spikes in the DR failed to occur, as in Fig. 3(c) and 3(d), there was also usually a small sharp depolarization (an EPSP or possibly an abortive spike) superimposed

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on a slower depolarization. This slow depolarization did not depend on the IR or its EPSP, but arose later and was associated only with the DR. Pharmacological observations

There have been several studies of the pharmacology and synaptic mechanisms of ganglion A6, and they point to the conclusion that acetylcholine and GABA serve as synaptic transmitters (KERKUTet al., 1969). Unfortunately, in most cases it is not clear just which synapses were studied. The excitatory synapses from cereal afferent nerves to the giant interneurons appear to be cholinergic (TWAROG and ROEDER,1957) and the response of the giants can be inhibited by high concentrations of GABA (GAHERRYand BOISTEL,1965). Since it seemed possible that the IR-DR response pattern of a neuron might be the result of a unique balance of excitatory and inhibitory synaptic inputs, we partially desheathed the terminal ganglion and applied chemical agents which should be expected to alter the function of the cholinergic and GABA-mediated synapses. Picrotoxin blocks GABA-mediated synapses in the cockroach (USHERWOOD and GRUNDFEST,1965). In four preparations picrotoxin at 2 x 1O-4 M greatly increased the number of fibres showing a DR without affecting the typical appearance or timing of the DR in individual giants. Fig. 4(a) shows an extracellular recording of an IR and DR before picrotoxin. The upper trace of Fig. 4(b) illustrates the increase in number of fibres responding 30 min after application of picrotoxin, and the lower trace shows a typical DR recorded intracellularly at that time. The fact that typical DRs occur in individual giants following application of sufficient picrotoxin to increase greatly the total activity of the ganglion indicates that GABA-mediated synapses are probably not involved in the generation of the DR. The anticholinesterase eserine first potentiates and then firmly blocks the cereal to giant synapses (TWAROGand ROEDER,1957). In four preparations eserine first potentiated the IR without altering the DR. Later, both IR and DR were blocked. Fig. 4(c) is an extracellular recording before eserine. Fig. 4(d), 15 min after application of 3 x 1O-6 M eserine sulphate, shows a similar DR. The simultaneous intracellular recording in Fig. 4(d) illustrates the enhancement of the IR (two spikes in this example). Thus, generation of the IR and activation of the DR-generating mechanism appear to be cholinergic processes, but the DR itself apparently is not. These observations indicate that the DR is not a direct result of synaptic interaction between the cereal afferents and the giants. Search for a reverberant circuit

Although reverberant circuits have frequently been invoked in theoretical discussions of nervous function, their actual existence in real animals is disputed. Nevertheless, it seemed possible that the DR might be generated by such a circuit. We tested for this possibility by a method described by WALL (1959) and PRESTON and KENNEDY(1962). The rationale is that once reverberant activity is established

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in a closed loop of neurons, the interpolation of an additional spike will not affect the timing of the output from the cycle already started. Our method was to apply pairs of shocks to the cereal nerves and observe whether the second shock affected the DR evoked by the first. Fig. 5(a) shows the DR evoked by a single shock to the homolateral cereal nerve. In this case there was no IR, although the results were no different when a stronger stimulus was used and an IR appeared. In Fig. 5(b) a second shock of slightly higher voltage was applied to the same cereal nerve 10 msec after the first. It evoked an IR and then a DR, but the DR to the first shock failed to appear. In Fig. S(c) the second stimulus was given 23 msec after the first, and the DR to the first shock again failed to appear. The DR to the second occurred beyond the end of the record. In Fig. 5(d) the second shock was given just after the DR to the first shock and again its DR was off screen. Similar results were obtained when one shock was given to one cereal nerve and the second to the other. In summary, a second stimulus given during the IR-DR interval abolished the DR to the first stimulus and generated a DR anew. These results are contrary to what should be expected of a reverberant circuit, but imply, instead, a single re-entry unit. Observations on freely moving cockroaches

Several cockroaches were prepared with fine silver wire stimulating electrodes implanted across a cereal nerve and recording electrodes on abdominal connectives. These individuals were relatively free to move about, tethered by the light wire leads. They never gave a DR when stimulated by an air puff, sound or touch, although electrical shock stimuli to the cereal nerves effectively evoked a DR. No behaviour specifically related to the DR was observed. It might be noted that the giant fibres were quite busy whenever the cockroach was active, and especially so during mating. DISCUSSION

The delayed response described in this paper has a fixed latency and pattern and occurs in individual giant and intermediate size axons which can also respond immediately to appropriate stimuli. A delayed response seen by TWAROG and ROEDER(1957) un d er similar conditions of stimulation and extracellular recording was described by them as consisting of small fibre activity, but not giants. Actually, in our own extracellular records, some of the giant fibre activity might not have been recognized if there had not been concurrent intracellular recordings and conduction velocity measurements. The search for the DR generating mechanism has proceeded largely by a process of elimination. In the previous section evidence has been presented that excludes: (a) a reflex or reverberant loop, either motor, neural involving other ganglia, or neural within ganglion A6 (which develops by fusion of ganglionic elements from several embryonic segments) ; (b) prolonged synaptic action by the afferent fibres; (c) a complicated pattern of current flow within the giant-like

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that described by TAUC (1962) in Aplysia where current from active membrane could invade the inexcitable soma of a neuron and then electrotonically return to generate a second spike. Therefore, we must conclude that the DR generator lies within the last abdominal ganglion and that it involves a neural element in addition to the cereal nerves and ascending interneurons, but that it is not a chain of neurons. It can be activated by more than one neural input, but its output is independent of the path of activation. Since eserine affects the IR and the activation of the DR generator but not the nature of the DR, it seems possible that the DR generator excites ascending interneurons by a non-cholinergic mechanism. This is further suggested by the slow depolarization associated with the DR in contrast with the brief EPSP related to the IR. CALLEC and BOISTEL (1968), recording in the same ganglion, have also reported two kinds of synaptic potentials, a brief EPSP peaking within 2 msec and related to giant fibre activation (our IR), and a slower EPSP lasting up to 120 msec and apparently associated with smaller neurons. The nature of the transmitter is unknown. Adrenergic agents (TWAROG and ROEDER, 1957) and 3-hydroxytyramine (GAHERY and BOISTEL, 1965) reportedly stimulate small Glutamic acid and its pharmacological relatives seem fibres but not the giants. not to have been tested in this ganglion. Electrical synapses have not yet been reported in insects. The presence of a neural mechanism for the generation of DRs by giant fibres implies a function for the mechanism in the normal behaviour of the cockroach, but we have not yet found it. Nor have we discovered how the DR generator may However, it is obviously too early to expect to be activated by natural stimuli. understand the role of the DR while the function of the giants is still uncertain. The commonly held notion that the giants serve as command interneurons for escape movements is contrary to the recent report by DAGAN and PARNAS (1970) that smaller interneurons, but not the giants, can activate the motor mechanism of the legs. They have suggested that the role of the giants might be to inhibit ongoing activity and possibly also to alert other parts of the neuromotor and sensory systems to function appropriately during evasive behaviour. The output of the DR generators could serve to prolong such effects. Another possible role for the DR could be to provide critical timing for sensitivity to other sensory inputs or for the co-ordination of sequential motor outputs. The timing of the DRs is different among the several pairs of giants, and it is not certain that all giants are activated by DR generators. It seems probable that the various giant fibres have different functions, and therefore it is possible that the DRs may have different functional significance to the several rapidly conducting neural pathways.

Acknowuledgement-C. Public Health Service.

L. H. was an NIH

pre- and post-doctoral

trainee of the U.S.

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