Conduction Velocity and In Vivo Studies on the Giant Fibres of the Squid, Loligo Opalescens. byThomas S. Otis Send Carrespondence To: Thomas S. Otis Hopkins Marine Station Paci C Abst the squic Data gathered on the escape resper toligo Opalescens, indicates an output through the giant fibre system that arrives to the muscien essentially synchroncusly (within 2 - 5 msec.). Further, the first In vive recordings ever made on the species indicate that the giant fibre system gives a very stereotyped pattern of discharge each time it fires. The giant fibres do not allow the animal to escape any faster; Howeyer. It is also obseryed that giant fibre spikes do not always occur during jetting, that multiple spike discharges are common, and that the number of spikes per jet control the intensity of the jet. Finally, this data suggests a possible course of evolution for the giant fibre pathway. oduc ti ne giant fibre systen of uid is perbaps the most extensively studied neurobiological preparation. Inargueably, it has produced data on ion moyement and nervous signal condustion that is the basis of all neurobiology. These studies were possible que to the large sized axons that are characteristic of the system. They allow ease in both stimulation and recording, and thus their electrophysiological properties have been known now for over half a century. All of this study, however, has focused on a dissected preparation which is essentially "dead". Certainly no behaviours can be studied, nor their neryous control, in the open-mantle, traditional giant fibre preparation. Honetheless, many evolutionany advantages of the fibre system have been suggested, namely that the system has bettem synchronicity and allows the animal to exhibit a fastem escape mesponse. As J. E. Young wrote, "The value of systems of giant fibres to the mimals possessing them lies presumably either in the greatem apeed of conduction in larger axons, or in that such large fibres make it pessible to tall inte action a large number of effecton agents nearly simultaneously" (J. 2. Young. 1938). The work presented in this paper addresses both of these presuppositions directly, by using the traditional, open-mantle preparation to determine the limit of simultaneity, and by examining the first obtained in vive neural recordings from the stellar nerves of Loligo Opalescens. Date collected suggests that the former could be true, while the latter does not aid in allowing the animal to escape faster. Furthermore, this work indicates a probable evolutionary pathway for the development of the giant fibre system. Haterials and Hethods Lolige Opalescens, the animals used in this study, were chtained from Monterey Bay, Monterey, Ca. They were maintained in large circular holding tanks (ca. 2m in diameter and im deep). Ihe experimental animals were typically 7 to 12 cm in mantle length. Because of the nature of the in vive preparation, only healthy, strong swimming animals with all skin intact were chosen. The study was divided into two experiments. Neasurements for both parts of the study were made with en passant suction alectrodes. The slectrodes consisted of two Agola wires, one inside a syringe, the other wrapped arcund the flexible Nalgene tubing tip. Another AgCla wire was inserted in the seawater bath as a ground. The tips of all recording electrodes were flesible Malgene tubing, drawn out over flame so that the diamater of the and provided the best suction onto the stellar Hecordings for both experiments were passed threugh a preamplifier (Tektronix FM I2 to a storage cecillceroge (Micelet Storage Oeeillsacope 2091!. The first experiment measured conduction velscity. Ic da this the animals mantle was cut ventrally and pinned out in a diseection dish, with the head still intact. Water temperature was maintained at 10 -14 O throughout the experiment. Befgre each experiment, the water was aerated for at least 10 min. Iws suction electrodes as described above were placed on one stellar perye, one on the loose segment of nerve as it leaves the stellate danglion (See Fig. 1), the other where the nerve fibre was barely yisible, presumably close to the motor endplate. The latter electrode was placed by removing the collagen tunic and musele layer ue in the the nerv itting the nerve and electrode tip. The nerve was then stimulated presynaptically via the pallial nerve, with pulses of 10 -80 V lasting from 0.1 to 0.5 msec. Time between action potentials was obtained by using the storage oscilloscope cursor movement function (Os explained in Fig. 2). The distance between electrodes was measured using a piece of wire and calipers. For the in vive experiments, the animals were narcotized in a solution of 1.5 -22 ethanol in natural seawater, for 3 to 5 minutes, or until respiratory movements were minimal. The Leligg was then transferred to a dissecting dish, the dorsal skin above the stellate ganglions removed, and the mantle tissue above the ganglions carefully removed, so as nat to damage the ganglion. The animal was attatched to a plesiglass restraint (See Fig. 3) with superglue. The squid was clearly able to perform normal respiratory, swimming and jetting movements. Here, the animal was transferred to a plexiglass hox (8"xS"x5") filled with seawater maintained at 10 -14 C. One suction electrode was placed on the second, third or fourth stellar nerve (See Fig. i) at the loose segment as they leave the ganglion. Often the nerve was sucked into the electrede tip, and better quality recording resulted. An absolute pressure transducer (sliding resistor coil) whose resistance varied linearly with pressure, was connected to a tube filled with mineral oil, which was inserted through ansther hole (not above the ganglion) in the squid's mantle. The transducer was wired into a Wheatstone bridge with a variable resistance component, so that output current could be adjusted. Using the "balcen pop test" the transducer was determined to have within a two millisecond respense to a step change in pressure. The transducer was not calibrated. ecord de e rou preamplifier inte an analeg/digital processor which output digital signals to a Betamax video recorder (This method is after Dr. Benzanilla, Biephysical Journal, 1785). The pressure transducer recording was run directly to the analog/digital converter. Stop frame analysis of the recordings was made using the Betamax recorded data converted back to analog signals in the O/D converter and displayed on the storage oscilloscope screen. Ihs records could be maintained on the screen by using an outside triggering pulse of the same frequency as the videotape sample (16 Hz.). Further analysis on a faster time scale was performed by cutputting data stored on Betamax tape through the A/D converter and taped on fast speed on an FM tape recorder (Tandberg, Geries 115). Records were played back on slower speeds to a brush recorder to increase time scale. Unless otherwise mentigned all records displayed in this paper were obtained by this method. Escape responses were induced in the animals by frightening them with a finger, perturbing them with a dissecting togl, ory often they responded spontaneously. Experif ion veleit. lable I shows the data collected in determining conduction velocities for an average animal. As mentioned earlier, the time of each action potential was taken from the storage oscilloscope screen. This method was used in calculating data for three animals, each of which produced data analogous to this example, The arrival of signals at the admittedly very roughly estimated endplate in this case was less than I msec. difference. In no other example were the differences greater than 3 msec, betwsen cutgoing signals of the giant fibres. In angther set of observations, two suction electrodes were placed at the estimated endplates of different nerves, and stimulation was given via the pallial nerve. As can be seen in Table I as well, the differences in arrival of signals using this crude method average merely 3 msec. (he average time of impulse transmission to the estimated endplate in all animals in all stallar nerves (End, 3rd, 4th and Sth) was 2.9 msec. and in no case was the transmission time to endplate greater than 5 msec. Experiment 2: In vive regordings As mentioned earlier, most hard copies of recordings stored on videotape were run out on the brush recorder by using an EM tape recorder to increase time resolution. These records proyed to be the most informative and thus the majority of analysis was performed using hard copy records of different speeds, made at different gains to manipulate comparative resolution of specifis events. cale of the recordings increas and hiche frequency events become distinquishable. In Figure 6, we see the 250 asec. time periods surrounding each of the same jets that are in Fig. S. These sequential recordings allow clear identification of the giant spikes preceeding pressure rises in the mantle cavity (As before, bottom traces). In the first jet of the sequence, one giant spike is evident, 25 msec. before the pressure increase. O pattern of much smaller amplitude, yet still large spikes (500 - 800 uV) are still apparent, on a very similar time scale as in the previbus trace. The pressure records are arranged so that the rises in pressure correspond. In the Srd jet, two giant spikes occur, the first with the same 25 msec. latency Until pressure rises. Large spikes are also steregtypically clustered about the giant fibre discharges. Finally, the pressure recordings for each of the 3 jets are graded in amplitude, with the no giant fibre spike record giving the lowest change in pressure, and the slowest rate of pressure rise in the mantle cavity. As expected, the pressure change for the one diant fibre spike record is higher in amplitude, but still smaller than the record with twe giant fibre spikes. Both pressure tracings show a similar rate of pressure rise. In Figure 7, there are three recordings of jet sequences from the same animal. All three records have 2 parts, each of different gain. The first record shows a weak jet, with no giant fibre spike, followed by a stronger, two giant fibre spike jet, Jet sequence number two shows 3 jets, the first two without giant spikes, the last with only one giant spike. Lastly, the third jet sequence displays one weak, non-giant jet and a jet with 5 giant fibre discharges. The water artifact occuring immediately after this final jet appears considerably larger than in the previous ure displeys s harde e ee re. quence. This record was not run threugh the Fi recorder, but rather was obtained directly from the videotape. O total of six "escape" jets occur during the course of the continuous recording, Perhaps the most salient events in this figure are the artifacts from the two jet sequences. These are the high amplitude, very low frequency events occuring after each unit of three jets. The two most notable pieces of information gained from this record are the regular pattern of activity occuring at 1 to 2 second interyals and the higher amplitude "breaths" seen immediately preceeding each unit of 3 jets. One can also notice small amplitude, low frequency events occuring after each "breath", indicitive of artifactual seawater moyement. Ihe Sth Figure is data obtained from FM re-recordings of a previously videotaped experiment. It is a 3 jet sequence, and the much faster time scale clearly affords better resolution of nigh frequency events. The lower tracing is a record of pressure change in the mantle cavity, and it is evident that a large preszure increase occurs approximately 2E zec, after eome extremely large amplitude electrical action recorded in the usser tracing. Ihis suggests that mantle muscles have contracted, powering the escape jet, and that these muscles have been commanded by the large amplitude cutput occuring just previous to the sudden pressure change. Also apparent is the biphasic nature of the high amplitude, high frequency electrical record preceeding the pressure change. There is a 400 - 700 msec. pericd of high activity, followed by a relitively inactive period of 50 - 60 msec., and then an extremely large amplitude period of 70 - 80 msec. just before pressure change. These consistent patterns become even clearer as the time ne sequence have the Discussion Clearly there are many important conclusions drawn from this data. The conduction velocity data suggests that neurel output to the endplate errives yery nearly simultanegusly. My data indicating a 2 - 3 msec. difference for Loliga of mantle length 10 - 20 cm. is rather consitent with data from others, particularly Sosline, et al (J. Exp. Bio. 1983), who states that his figures indicate better than 10 msec. synchronicity. His animals were larger, thus his differences would be expected to be greater. Certainly my measurements are too few to make definite estimates of latencies, but they are a fine indication that the system is essentially synchronous. Considering the 300 - 800 msec. (Gosline, et al, J. Exp. Bio., 1783) period of muscle activity, this asynchranicity seems rather trivial. The in vive experiments provide much excitng detail about this well studied giant fibre system. The biphasic nature of both respirations and giant fibre induced jetting suggest that Gosline's data an hyperinflation and subsequent jetting (J. ERp. Eio., 1783) is in fact true, given that the 400 - S0o msec. period of large amplitude activity is being carried by fibres innervating the radial muscles. This is consistent with my records because the squid was tethered, and thus wasn't able to move through the water, which normally would allow flow-induced pressures to aid in mantle reinflation during respiration. It would be reasonable to expect the animal to have to work harder in this static situation during respiration and jetting alike. Therefore nervous discharge to radial muscle fibres could be exaggerated. The large, very high frequency spikes clustered in the most 1i activatine circular muscie in the mantle, probably of thes type (Bone and Pulsford, 1948). The giant fibre spikes would then activate faster twitch muscle tissue in the mantle, which isthe most abundant muscle tisse (Sosline. Ei. Am.. 185). The numerous large spikes surrounding every giant spike could be signals sent to slower acting circular muscle. It is clearly evident that the giant spike, if one occurs during a jet, is "held back", perhaps so that large spike output to the circular muscles can arrive synchronously with the much faster conducting giant fibre cutput. Mast exciting is the observation that the giant fibre system does not make the escape responses any faster as J. E. Young suppossed. Rather it seems that the system can increase the strength of the jet and probably developed to increase synchronicity of a strong muscle cantraction. The records showing multiple giant spikes are equally remarkable, because they indicate that strength of contraction can he graded by the numbem of spikes fired as mell. Undoubtedly, more careful experimentation will be done with pressure traneducens to elucidate the specific montribution of a system capable of multiple spikes. Lastly, the fact that the giant fibre system does not aid the animal in quikening the response time to a threatening stimulus indicates that it probably developed in animals which already had an escape response (most likely mediated by the same large amplitude fibres seen in my recordings). Thus the newly evolved giant fibre network would have to "hold back" to be synchronous with the slower, previously developed system. Acknoul edgenents Dr. William Gilly deserves more thanks than I am able to give, here in my utterly inadequate "Ack" section. He simply was always there, to such an extent, that halfway through the quarter, another undergraduate and I began to speculate on whether he had another job here or not. I am also very thankful that there is a Dr. Stuart Thompson at H.M.S. Though I have trouble spelling his name, his timely donations of equiptment and expertise allowed me to complete the project. Finally, Bruce Hopkins, and Carol Marzuola are no less wonderful for dealing with hordes of crucial things (like supplying the animals). Their time and attention was much appreciated. References Penzenilla, J. (1985) Biophysical Journaly March. 1EE. Bone, 0. et al. (1980) The Role of L-glutamate in Neuromuscular Transmission in some Mollusks. J. Mar. Bic. Ass. U.K. 60, 619 - 626. Bryant. S. H. (1958) Transmission in Squid Giant Synapses: The Importance of Oxygen Supply and the Effects of Drugs. J. Gen. Ehys. 41, No.3, 473-484, Bryant. S. H. (1759) The Function of the Proximal Synapses of the Squid Stellate Ganglion. J. Gen. Ehys. 42, No.3, 609-616. Dorsett, D.A. (1984) Design and Function of Giant Fibre Systems. TINE Perguson, G.P. et al. (in press) Motor Fields and in yiys Hator Programs of Fin and Stellar Nerves in the Squid, L. Breviz. Gosline. J.M. et al. (1983) Patterns of Circular and Radial Muscle Activity in Respiration and Jetting of the Squid. L. Upalescens. J. Exp. Bic. 104, 97-109. Wilson, Donald M. (1960) Nervous Control of Movement in Cephalopods. J. Exp. Bio. 37, 57-71. Yeung, J.Z. (1938) The Functioning of the Giant Nerye Fibres of the Squid. J. Exp. Bio. 15, 171-185. Fig¬ Palli Giant Hber Loc Nerve Stellate Ganghon . - 15 1.. t 4 5 44 2ro 2rd e qa Condudn was masvred b takim he differene of Cursor valves vhen he cusor is at the hest sage peak of each ocrun potenthal, in mnis exovapse — nere and Fig:2 Fyoæ 3. Restroint Suchon Bechodes a0d Pressute Horsducet eted here 1 Box OSt- —---- -8" Gad Elechode To Cg B 25 oohng Elment - : 6 Fig. . - - — — — .. - - —.— ——- ————— ——- — + + -- —--— —.—. + - -- + —--- — - -+ — - —-- - ----- - --- — — -— — 6 a- e e e ee e aatataa couots Coonanoe 0 vo . . . S e me taaataa- 1S4111-2923-32 — — — SOOMS a + katakkvva- 590145 Fig 7. ) & a a 82 255 ä a a . 625 + ZuN 1 00 — A X co To 00 00 0 00 O Figure Legends Figure 1: Pallial nerve and stellate ganglion, with the "loose" segments of each nerve showing. Figure 2: Oscilloscope picture, illustrating the method used to determine conduction velocities. Eigure : Restraint and holding bo used for in vivo experiments. Figure 4: Data recorded using suction electrodes, taken directly from the Betamax recording. "Q" - water movement artifact, "B" - breath, "J" - jet. Eigure 5: Sequential three jet record, with electrical record on top, and pressure changee in mantle on the bottom. "G" - giant spike, "K" - proposed output to radial muscles, "C" proposed output to circular muscles. Time bar is 100 msec. Eigure 6: Same three jets as in Fig. 5, on an expanded time scale. The records are lined up to highlight the stereotyped electrical activity, and corresponding pressure rise. Time bar is 10 msec. Figure 7: Ihree different jet sequences from other animals. Both records (top and bottom) are the same, but played out on different gain. Ihe key is as in Fig. 5. Lable 1: Conduction velocity measurements tro C one animal. a - action potential.