Jet-Propelled Escape in the Squid Loligo Opalescens: Concerted Control by Giant and Non-Giant Motor Axon Pathways

Home , Ink sac, Siphon (mollusc), Squid giant axon

Proc. Natl. Acad. Sci. USA Vol. 87, pp. 2911-2915, April 1990 Neurobiology Jet-propelled escape in the squid Loligo opalescens: Concerted control by giant and non-giant motor axon pathways (cephalopod/neurophysiology/behavior/swimming) THOMAS S. OTIS* AND W. F. GILLYt Hopkins Marine Station of Stanford University, Pacific Grove, CA 93950 Communicated by Donald Kennedy, January 30, 1990

ABSTRACT Recordings of stellar nerve activity were activity in relation to behavior have not been reported, made during escape responses in living squid. Short-latency however, and studies of escape responses have focused on activation of the giant axons is triggered by light-flash stimu- biomechanical aspects (10-14). lation that elicits a stereotyped startle-escape response and Squid with giant axons also possess a parallel small axon powerful jet. Many other types of stimuli produce a highly system (9), and repetitive activity in the numerous small variable, delayed-escape response with strongjetting primarily motor axons generates graded mantle contractions (15), controlled by a small axon motor pathway. In such cases, probably via a separate type ofcircular muscle (16, 17). These activation of the giant axons is not necessary for a vigorous non-giant responses are resistant to fatigue and are presumed escape jet. When they are utilized, the giant axons are not to mediate weak respiratory contractions (9). What role activated until well after the non-giant system initiates the non-giant motor pathways play in high pressure jet propul- escape response, and excitation is critically timed to boost the sion is unknown (18-20). rise in intramantle pressure. Squid thus show at least two This paper describes electrical recordings made in vivo escape modes in which the giant axons can contribute in from giant and non-giant motor axons during escape jetting. different ways to the control of a highly flexible behavior. Some results reinforce the generally accepted ideas about the functional role of squid giant axons. For example, giant axon Analyses of electrical activity in giant axons and cell bodies activity underlies a startle response that shows the minimum have contributed greatly to our understanding of neural behavioral latency. Other experiments, however, reveal un- control of behavior (1). Where giant axons exist, they most expected complexities and a second way in which the giant often startle- and and axons are utilized. Activation of the giant axons is not control rapid-escape responses are necessary for vigorous escape behavior; strong escape jets primarily responsible for the characteristics of short latency can be carried out by the small axon system acting alone. and synchronous muscle activation (2, 3). Escape swimming Moreover, giant axons can be called into play after ajet cycle has been analyzed in detail in crayfish (4) and in teleosts (5), is initiated by the non-giant pathway. In this case the giant where a single action potential in a giant axon elicits a axons provide a potent boost to the escape jet. complex, but stereotyped, motor output at very short latency (<40 ms). These giant axons are not motor fibers themselves but act as interneurons to orchestrate coordinated discharge MATERIALS AND METHODS of many motoneurons. Adult squid were collected in Monterey Bay, CA, and held in Crayfish and teleosts possess parallel, small axon pathways a 2.5 m x 1.5 m circular tank plumbed with flow-through that can effect rapid escape responses in the absence of giant seawater (15TC). Video observations of free-swimming ani- axon activity, but with a longer latency (50-500 ms; ref. 4). mals were carried out in this tank with a Sony Beta-Cam Interplay between giant and non-giant motor pathways during broadcast-quality apparatus. Taped data were analyzed using escape behavior has not been extensively studied, and such an image-analysis system (Megavision, Santa Barbara, CA) interaction may be important in maximizing flexibility of a that can digitize, store, and manipulate 16 real-time frames limited motoneuron pool. For example, in teleosts a small axon (33 ms per image). system can modify the excitability of motoneurons on which Animals were restrained for electrophysiological record- Mauthner axons synapse, and a fixed command signal in a ings by gluing the dorsal mantle surface to a plastic support- giant axon can thereby lead to different motor responses (6). ing platform with cyanoacrylate cement. This apparatus was Another type of interplay may involve concerted control suspended in an aquarium filled with seawater (15-18'C) over the simultaneous usage ofgiant and non-giant pathways. bubbled continuously with pure 02 Squid thus studied could If a giant axon could be called into play in a "thoughtful," be removed and returned to the holding tank where they nonobligatory way during activity independently mediated displayed normal swimming behavior and survived for sev- by non-giant processes, its utility would be greatly enhanced. eral days. Efforts to demonstrate such a dual usage ofgiant axons have Pressure was recorded from within the mantle cavity with been equivocal, however (4, 7). an analog transducer (0- to 140-kPa range) attached, via an We describe here experiments that shed new light on this oil-filled rigid tube, to a hypodermic needle penetrating the idea. Although the squid giant axon has been extensively mantle (frequency response of 50 Hz). The staircase-like studied from numerous points of view, little is known about rise and fall of records in Figs. 3 and 4 is inherent to the the axon's in vivo role in escape behavior. All-or-none reflex transducer. activation of escape jetting was inferred 50 years ago from Conventional extracellular recordings of nerve activity studies of stellar nerve-mantle preparations (8, 9), and this were made with glass suction electrodes (21) through holes (5 idea remains widely accepted. Recordings of giant axon mm in diameter) in the mantle over the stellate ganglia. Each

The publication costs of this article were defrayed in part by page charge *Present address: Department of Neurology, Stanford University payment. This article must therefore be hereby marked "advertisement" Medical School, Stanford, CA 94305. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2911 Downloaded by guest on October 2, 2021 2912 Neurobiology: Otis and Gilly Proc. Natl. Acad Sci. USA 87 (1990) stellar nerve contains one giant axon, and recordings were In our experiments only light flashes elicited escape re- made en passant from the hindmost or second-hindmost sponses showing the short-latency component. All other nerve near its emergence from the ganglion. Data were stimuli investigated produced responses that lacked the continuously digitized at 40 kHz with a modified audio short-latency component and instead showed delays of at processor (Unitrade, Philadelphia) and stored on videotape least 200 ms and variablejets such as those during the second for subsequent sampling (10 kHz) and analysis using a phase in Fig. 1B. An example of such a "delayed-escape" laboratory computer. evoked by an electrical shock is illustrated in Fig. 1C. Chemical stimulation of the olfactory organ (22) was car- Vibrational stimuli generated by tapping the aquarium with a ried out by pressure ejection of selectable substances in the solenoid-driven metal rod produced similar responses. immediate vicinity of the organ, which lies just posterior to Chemical stimulation of the olfactory organ leads to de- the orbit. Seventy-five- to 300-ms-duration pulses [20 psi (1 layed-escapes with even longer latencies. Fig. iD shows the psi = 6.89 kPa)] delivered fluid at a rate of 1 LI/ms from a port reaction to a puff of 20 ,uM propyl paraben delivered to the 0.65 mm in diameter. The olfactory organ. A separate series of experiments has dem- stimulating probe assembly also onstrated that this substance, along with others that evoke contained a pair of small Pt wires and an optic fiber to escape responses in vivo, can block voltage-controlled K transmit electrical and visual stimuli. Brief shocks (several channels in receptor cells isolated from the sensory epithe- milliseconds in duration) were delivered near the base of the lium and studied with patch clamp techniques. A physiolog- arms. ical role for this organ has not been previously identified (18), and this work will be described in detail elsewhere. RESULTS Squid escape behavior thus has two major components that can be executed independently. Depending on the exact Squid react to many kinds of stimuli with a powerful mantle nature of an unexpected stimulus, a short-latency pressure contraction that leads to jet-propelled escape as seawater is pulse ofrelatively fixed amplitude may or may not occur, and ejected out the siphon. The most simple kind of escape this initialjet may or may not be followed by a series ofstrong response is evoked by a sudden visual stimulus. Fig. lA jets of variable timing and amplitude. This delay component shows intramantle pressure in a restrained animal following can also occur by itself. a strobe flash. The large, rapid pressure pulse at a latency of Complex escape behavior and a latency of50-75 ms for the 50-75 ms characterizes this stereotyped "startle-escape." A startle response also occur in free-swimming squid. Video- light flash can also trigger a more complex escape response taping experiments revealed a minimum latency of two in which the initial short-latency component is followed by frames, or 66 ms (i.e., motion due to mantle contraction was multiple jet cycles of highly variable number, timing, and first detectable in the third frame following a strobe stimulus). amplitude (Fig. 1B). Because the flash was not synchronized with the video framing, and because the actual onset of movement during the third frame is not definable, 66 ms represents an approx- imate value. A lower limit can be set at 33 ms, however, because movement in the first frame following the flash was never detected. Such startle-escapes could be repeatedly produced at 10-sec intervals with no apparent sign of habit- uation. B As with restrained animals, a strobe flash was the only stimulus found capable ofevoking short-latency responses in free-swimming squid. A splash on the surface created by a falling marble produced a longer and more variable delay, as did banging the side of the tank. Responses to these latter stimuli readily habituate, and the animals appear to ignore the stimulus after a few trials. Stimuli with obvious biological relevance are difficult to present in a sudden and controlled way to a school of free-swimming squid, but despite this limitation these experiments show that nonrestrained animals also display different types of escape behavior in reaction to particular stimuli. Most important, squid do not always exhibit the minimum latency of which they are capable. Electrical recordings ofstellar nerve activity during escape D responses reveal the roles played by giant axons in both types of escape. Giant axon activity in the stereotyped startle- escape is relatively straightforward, and Fig. 2A shows the discharge in right and left stellar nerves ofa restrained animal following a flash. In either nerve, the first event is a giant --F- axon spike at a delay of 50 ms, and this is immediately followed by a large movement artifact as seawater inside the I I I mantle cavity is pushed up through the holes that accommo- oIs 0 5 l1 date the recording electrodes. If successful at all, strobe stimulation guarantees short-latency firing of the giant axons FIG. 1. Pressure transients during escape responses in a living, and a stereotyped initial cycle of escape jetting. Delayed- restrained squid. (A) A strobe flash elicits a stereotyped startle- escapes are never produced. escape of short latency. (B) A multiple cycle response can also be produced by flash stimulation. (C) A delayed-escape follows a brief The 50-ms delay for giant axon excitation in the startle- electrical shock and is characterized by a long latency and a complex escape is similar to that for mantle contraction, indicating series of pressure transients. (D) A delayed-escape can also be that the major source of behavioral delay lies in the central elicited by chemical stimulation of the olfactory organ. Downward nervous system and not in conduction time along the giant deflections in each trace indicate stimulus timing. axon (<10 ms) or muscle activation. Some neurons in the Downloaded by guest on October 2, 2021 Neurobiology: Otis and Gilly Proc. Natl. Acad. Sci. USA 87 (1990) 2913 A reflect information processing by giant interneurons that drive the giant motor axons in the stellar nerves. Short, large axons from the optic lobe impinge on a single (bilaterally MV ow ~~~~I fused) giant interneuron in the magnocellular lobe (24). The first-order giant cell projects one short axon on each side onto a second-order giant cell in the palliovisceral lobe (25). A FLASH giant axon leading from each second-order cell contacts all (7 or 8) giant motor axons in the ipsilateral stellate ganglion. In 2 Ms addition, the first-order giant neuron also drives other (bilaterally symmetrical) second-order giant cells in the palliovisceral lobe that are motoneurons and directly innervate head retractor, siphon, and ink sac muscles. Important decision-making may occur at the level of the first- and second-order giants, and selective activation of different second-order neurons may be an important feature 0 100 200 ms of escape behavior. For example, squid can produce power- B ful escapejets without inking, and the siphon may or may not change orientation. On the other hand, head retraction ap- - 1.0 0 pears to always accompany jetting and actually precedes 000o 00 0 .0 mantle contraction as the first detectable motor output even

0 - 0 00 0 in visually evoked startle responses. Fig. 2B indicates that c CL 0 head retraction precedes mantle contraction by approxi- 0 0 0 0 0 mately one video frame or 33 ms, more than twice the time cr expected for down - 0.5- 0 0 conduction the giant axon and across the 0 00 one extra synapse. Although the discrepancy is small, it 0 - Cd0 0 ~~~0 reinforces the idea that activation of all second-order giant Li. - neurons, including that one driving the giant motor axons, is Flash 0 under a high degree of active control. Stringent control over the giant motor axons is readily lilQ*~ apparent during delayed-escapes. Stellar nerve activity and . ,.1.VIIII., ...I I ., .,I .-[-T 0 5 10 15 mantle pressure following an electrical stimulus are illus- Video Frame Number trated in Fig. 3A. During the long delay to the first jet the squid processes sensory inputs, makes the decision to es- FIG. 2. Stellar nerve activity and behavioral outputs accompa- cape, and then initiates escape behavior with an abnormally nying a visually evoked startle-escape. (A) A flash elicits a single strong cycle of with giant axon spike at short latency in right and left stellar nerves. (Inset) mantle-filling (visually correlated the Time course of giant axon spike. (B) Time course of head retraction "inflation" period of heightened neural activity; cf. refs. 13 (o) and mantle contraction (0) following a flash in another animal. and 14). Nerve activity then ceases for 150 ms after which a Head retraction was measured from videotaped data as the distance short-duration "jet" burst of high-frequency activity in small from the posterior orbit to the anterior mantle edge. Mantle diameter axons occurs and apparently serves to drive the pressurejet. was measured at its widest point. This cycle is then repeated, and the pattern of neuronal activity appears unchanged-except for the occurrence of optic lobe of the brain respond within 25 ms following a flash two giant axon spikesjust before the second pressurejet. Fig. stimulus (23), and much of the remaining delay time may 3 B and C display neural activity on an expanded time scale

Shock Inflation

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FIG. 3. Stellar nerve activity during a delayed-escape. (A) The upper B trace shows activity in one nerve dur- v----OP N AL. C Wq ing two cycles of escape jetting Right triggered by electrical stimulation. The 100 lower trace is pressure. (B) Small unit ms activity in the right and left stellar nerves during the 300 ms prior to the -NW wpwtob - - 1"T N-r WE rise of pressure in cycle 1 of A is Left displayed on an expanded time scale. l Giant axon spikes are absent. (C) Re- cordings analogous to those in B are shown for cycle 2. Two giant axon spikes are present. Downloaded by guest on October 2, 2021 2914 Neurobiology: Otis and Gilly Proc. Nati. Acad. Sci. USA 87 (1990) recordings are aligned so that the onsets of all pressure 2 mV transients coincide. The small axon burst beginning 50-60 ms cycle before the rise in pressure is common to all three cycles, but the number ofgiant axon spikes varies from 0 to 2. Thus, the delayed-escape is primarily defined by repetitive activity in 3 1~~~* non-giant motor axons. Giant axons are important but op- 1 ~~~~~2 tional boosting elements. When utilized they can additively I kPa contribute force equal to or greater than that provided by the pressure small axon system. 50ms Timing of giant axon activity during delayed-escapes al- Im I ways occurs in phase with the small unit burst as shown in cycle 2 Fig. 4, but the number of giant axon spikes in each cycle can vary. Fig. SA plots peak pressure vs. the number of giant axon spikes during individual cycles of delayed-escapes in two different animals. All points are normalized to the mean value ofthe startle-escapejet, driven by one giant axon spike, cycle 3 determined for each animal. Pooled mean values (±1 SEM) are also given. Although variability exists in these data, two points are evident. (i) Substantial pressure can be generated by the non-giant system acting alone, but the mean value is less than that for the average startle response (P < 0.01 by t test). (it) A single giant axon spike acting in conjunction with FIG. 4. Timing ofthe small axon discharge in relation to the onset the non-giant system can produce a stronger jet than it does ofpressure rise and firing ofthe giant axons. Threejet cycles (single during a startle-escape when it (presumably) acts alone (P < shock stimulus, not illustrated) were generated in the order indicated, 0.05 by t test). Increasing the number ofgiant axon spikes to and neural recordings are aligned so the onsets of the pressure two or three may provide an additional increase in peak transients coincide. The neural activity common to all cycles is the pressure, but this must be regarded as tentative. burst of small unit activity commencing 50 ms before the pressure Velocity (V) of the seawater jet (with respect to the body) rise. Giant axon spikes augment the rate of pressure rise. Same ejected through a fixed siphon aperture is proportional to the animal as illustrated in Fig. 2A. square root ofpressure (P; ref. 20), and thisjet accelerates the the indicated interval before each animal. Rate of change of the jet velocity (dV/dt) is propor- during pressure transient. tional to p-1/2 (dP/dt), and an increase in dP/dt would thus Giant axon spikes are absent in Fig. 3B and present in Fig. 3C, also aid escape performance. Fig. SB shows that a single giant but the small unit jet burst occurs in both cases. axon spike markedly boosts the acceleration provided by the Strong delayed-escape jetting can thus be driven by a small axon system acting independently. Repetitive firing of non-giant motor system acting alone, but concerted giant the giant axons produces only a marginal additional affect. axon activation can dramatically boost the pressure transient without greatly altering its time course or its time of occurrence as defined by the underlying cycle ofnon-giant activity. DISCUSSION These points are illustrated in Fig. 4, which compares the For many years it has been assumed that the role ofthe squid timing of the small axon jet burst, giant axon spikes, and giant axon in escape behavior is a straightforward one of pressure rise during each of the three cycles that constituted maximally speeding the motor nerve impulse to the mantle a delayed-escape in response to an electrical stimulus. Neural muscle in order to guarantee the quickest response to a o A B 2.0- -nl_ 0

0 ._ 0 0 0 0 a 0. - Q50 a 0 a _ _ + en a- 0 0 * 1.0- At~A 0_ao10

I i I I 0 1 2 3 0 1 2 3 Giant Axon Spikes Giant Axon Spikes

FIG. 5. Repetitive firing of giant axons and its relationship tojet thrust. (A) Peak pressure during individual cycles of delayed-escape jetting (single shock stimuli) in two different squid (a, o) is plotted against the number of giant axon spikes. All values have been normalized to the mean pressure of visually evoked startle-escapes [1 SEM = 0.064 (0), n = 2, and 0.043 (0), n = 12]. Pooled means (±1 SEM) are also plotted (x). (B) Analysis of the rate of pressure rise vs. giant axon spikes is presented for the same delayed-escapes described in conjunction with A. Downloaded by guest on October 2, 2021 Neurobiology: Otis and Gilly Proc. Nati. Acad. Sci. USA 87 (1990) 2915 threatening situation. Results in this paper show that this idea such behavior, free-swimming squid can jet "forward" dur- is oversimplified. Squid show two distinct modes of escape ing prey capture as rapidly as they can during "backward" when presented with an alarming stimulus. The two modes escape responses (10). Giant axons are thus likely to be can be combined in a variety of ways, and giant axons are involved in feeding, and, if they are, stringent control over employed in totally different ways in the two cases. their recruitment is an obvious necessity. In a flash-evoked startle-escape, giant axons function Is the picture presented here for squid unique? In many much as we would expect them to and are primarily respon- other species, latencies are briefer, non-giant and giant motor sible for a powerful, short-latency jet. Minimum latency for pathways are thought to be more independently utilized, and giant fiber activation is 50 ms, roughly 10 times longer than giant axons appear to be employed only in stereotyped that produced by tactile stimulation in crayfish (4). Latencies escape behavior. Few studies have specifically addressed the for muscular response (jet vs. tail flip) in the two species last two points, however (6). Given the advantages that the differ by a much smaller factor. Thus, the "slow" steps in "smart" type of giant axon system appears to confer to crayfish startle-escape must occur after the giant axon spike, squid, it would be surprising if concerted interplay of giant whereas in squid the analogous events take place in the and non-giant motor pathways is not more widespread than central nervous system prior to giant axon activation. generally thought. Squid giant axons play a remarkable role in delayed-escape responses that are evoked by many stimuli and that always We thank Mark Shelly (Sea Studios, Monterey, CA) for video show a latency ofat least 200 ms. In this case non-giant motor recordings of free-swimming squid and Charles Baxter, Michael axons are the primary motor effectors, and their activity Bennett, George Mackie, and Jeffrey Wine for comments on the defines a cycle of escape jetting. During each cycle the giant manuscript. This work was supported by the Whitehall Foundation axons can be (but need not be) called into play precisely at the (J86-110). right moment with respect to non-giant activity to boost the rate ofrise in mantle pressure and the peak pressure attained. 1. Eaton, R. C., ed. (1984) Neural Mechanisms ofStartle Behav- The role of repetitive firing of the giant axons during an ior (Plenum, New York). individual escape jet cycle remains to be elucidated, but one 2. Dorset, D. A. (1980) Trends Neurosci. 3, 205-208. possibility would be to compensate for fatigue in neuromus- 3. Bullock, T. H. (1984) in Neural Mechanisms ofStartle Behav- ior, ed. Eaton, R. C. (Plenum, New York), pp. 1-13. cular transmision (15). 4. Krasne, F. B. & Wine, J. J. (1984) in Neural Mechanisms of Interplay of giant and non-giant motor systems permits a Startle Behavior, ed. Eaton, R. C. (Plenum, New York), pp. highly flexible escape behavior that is adjustable by alter- 179-212. ations in the firing pattern of the giant axons (and possibly of 5. Eaton, R. C. & Hackett, J. T. (1984) in Neural Mechanisms of the small axons as well) while it is being executed, presum- Startle Behavior, ed. Eaton, R. C. (Plenum, New York), pp. ably as the necessity of a situation dictates. This capability 213-266. appears to require tight control over recruitment of giant 6. Bennett, M. V. L. (1984) in Neural Mechanisms of Startle axons, which is best described as critically timed excitation. Behavior, ed. Eaton, R. C. (Plenum, New York), pp. 353-363. An idea of the processing speed in the central nervous 7. Schrameck, J. E. (1970) Science 169, 698-700. can 8. Prosser, C. L. & Young, J. Z. (1937) Biol. Bull. 73, 237-241. system effecting this control be obtained by considering 9. Young, J. Z. (1938) J. Exp. Biol. 15, 170-185. the timing ofneural events in the escape response. During the 10. Packard, A. (1969) Nature (London) 221, 875-877. latent period of a delayed-escape, central integration con- 11. Ward, D. V. (1972) J. Zool. London 167, 487-499. cerning stimulus quality, intensity, and localization must 12. Packard, A. & Trueman, E. R. (1974) J. Exp. Biol. 61, 411-419. transpire prior to initiation ofthe small axonjet burst (50-100 13. Gosline, J. M., Steeves, J. D., Harman, A. D. & Demon, ms in duration). Because impulse conduction along the giant M. E. (1983) J. Exp. Biol. 104, 97-109. axon is so fast (<10 ms), at least 40 ms of additional time 14. Gosline, J. M. & Shadwick, R. E. (1983) Can. J. Zool. 61, would exist-after the primary decision to escape had already 1421-1431. been made-to make a secondary choice ofwhether or not to 15. Wilson, D. M. (1960) J. Exp. Biol. 37, 57-72. via the axons. 16. Bone, Q., Pulsford, A. & Chubb, A. D. (1981) J. Mar. Biol. boostjetting giant This amount ofhypothetical Assoc. U.K. 61, 327-342. processing time is similar to that discussed above in con- 17. Mommsen, T. P., Ballantyne, J., MacDonald, D., Gosline, J. & junction with Fig. 2B and the startle response. In both cases Hochachka, P. W. (1981) Proc. Natl. Acad. Sci. USA 78, the first- and second-order giant cells are probably key sites 3274-3278. ofintegration, but nothing is presently known about electrical 18. Boyle, P. R. (1986) in The Mollusca, ed. Willows, A. 0. D. activity in these elements, or about the possible sources of (Academic, New York), Vol. 9, Pt. 2, pp. 1-99. inhibition that may underlie the apparently controlled repres- 19. Mackie, G. 0. (1990) Can. J. Zool. 68, 799-805. sion of giant axon activation during delayed-escape re- 20. O'Dor, R. K. (1988) J. Exp. Biol. 137, 421-442. sponses. 21. Yee, A., Burkhard, J. & Gilly, W. F. (1987) J. Exp. Biol. 128, Escape behavior in squid is not simple. Concerted control 287-305. 22. Emery, D. G. (1975) Tissue Cell 7, 357-367. over giant and non-giant motor pathways leads to obvious 23. MacNichol, E. F. & Love, W. E. (1960) Science 132, 737-738. advantages in fine tuning escape responses. It probably also 24. Young, J. Z. (1974) Philos. Trans. R. Soc. London Ser. B 267, provides the means to effectively exploit this pathway during 263-302. other high-speedjet-propelled activities such as prey capture. 25. Young, J. Z. (1939) Philos. Trans. R. Soc. London Ser. B 229, Although we have not recorded giant axon activity during 465-503. Downloaded by guest on October 2, 2021