The effects of high hydrostatic pressure on transmission at the crustacean neuromuscular junction

Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 133 to 140. Pergamon Press. Printed in Great Britain

THE EFFECTS OF HIGH HYDROSTATIC PRESSURE ON TRANSMISSION AT THE CRUSTACEAN NEUROMUSCULAR JUNCTION* ROBERT B. CAMPENOT Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, U.S.A.

(Received 11 October 1974)

Abstract--1. The effects of high hydrostatic pressure on neuromuscular transmission in a shallow-living and a deep-living crustacean were compared. High hydrostatic pressure (50-200 atm) depresses the amplitude of excitatory junctional potentials (ejp's) at the lobster neuromuscular junction. Pressure causes the input resistance of lobster muscle fibers to increase. Preliminary results suggest that the mechanism of the pressure-induced depression of ejp amplitude is a decrease in the amount of transmitter substance released by the nerve endings. 2. Results of work with the moderately deep-living red crab, Geryon quinquedens (300-1600 m), suggest two mechanisms of adaptation to high pressure. In fibers showing ejp's that are small in amplitude at low frequencies of nerve stimulation, the ejp amplitude is depressed by pressure at low frequencies of stimulation, but not at high frequencies. A pressure-induced enhancement in facilitation is probably responsible. Since these fibers contract only when the nerve is stimulated at high frequency, the ejp amplitude is independent of pressure in the physiological frequency range. Fibers showing large amplitude ejp's and contraction at low frequencies, show pressure-induced depression of ejp amplitude within the physiological frequency range. They have long ejp's that sum together at low frequencies, so that a slight increase in the frequency of nerve impulses can counteract the depressive effects of pressure.

INTRODUCTION ELEVATED hydrostatic pressure up to 200 atm gener-

ally causes shallow-living marine animals to become hyperactive. Many exhibit behavior that is uncoordinated and seizure-like. Pressures in excess of 200 atm immobilize them (Macdonald, et al., 1972; Macdonald, 1972). Hyperactivity is often not observed when deep-living marine animals are pressurized, and higher pressures are required to immobilize them (Macdonald et al., 1972). On the hypothesis that these behavioral responses at least in part reflect the effects of high pressure on synaptic transmission, I have undertaken a comparative study of the effects of pressure on transmission at the neuromuscular junction in the shallow-living lobster, Homarus americanus, and the relatively deep-living red crab, Geryon quinquedens. It was hoped that differences between the effects in these two animals would suggest some interesting mechanisms of adaptation to high lbressure. A few studies of the effects of hydrostatic pressure on the action potential in nerve have appeared in the literature (Grundfest, 1936; Spyropoulos, 1957a,b), but, to my knowledge, there have been no studies of its effects upon synaptic transmission. The crustacean neuromuscular junction was chosen for several reasons: it is a technically simple preparation so that the added difficulties involved in

working with pressure would not be too great. Red crabs are relatively easy to obtain. Also, the observation that pressure immobilizes animals suggests a possible involvement of the neuromuscular junction. Pressure was found to have a depressive effect upon transmission at the lobster neuromuscular junction. Preliminary evidence suggests the depression results from interference with the release of transmitter substance. Pressure-induced depression was also observed at the neuromuscular junction of the red crab at low frequencies of nerve stimulation but not at high frequencies. This along with other results suggest some adaptive mechanisms in the red crab that operate in the physiological range of frequencies to counteract the depressive effect of pressure. The results of experiments with the red crab are preliminary. They are presented now because it is thought the comparative aspect of this work would be of particular interest to the readers of this issue.

MATERIALS AND METHODS

Experimental animals

Schroeder (1955) reported the occurrence of great numbers of the red crab, Geryon quinquedens, on the continental shelf and slope between Nova Scotia and Virginia. The crabs were taken at depths from 300 m to ll00 m, the greatest depth that he trawled. These depths correspond to pressures of about 30 atm and 110 atm. Red * Contribution No. 3472 from the Woods Hole Oceano- crabs have been caught in trawls as deep as 1600 m or graphic Institution. This work was supported by the Mas- 160 atm (Richard Haedrich, unpublished data). About 75 sachusetts Institute of Technology--Woods Hole Oceano- red crabs were collected by trawling from the R/V Knorr graphic Institution Joint Program in Oceanography. It will (cruise 35) in the Hudson Canyon area in November, 1973. form part of a dissertation to be submitted in partial fulfill- The crabs were maintained at 1 atm in aquaria supplied ment of the requirements for the Ph.D. degree. with flowing seawater ranging in temperature between 133

134

ROBERT B. CAMPENOT

about 5 ° and 16°C for the entire winter. They were fed frozen herring. Lobsters, Homarus americanus, occur to a depth of about 520 m or roughly 50 atm pressure (Schroeder, 1955). They were purchased from a local fish market.

Physiological solutions The ionic composition of red crab blood has been determined (William De Paul, unpublished data). There was wide variability in the blood composition, especially in the case of Ca 2+ which ranged 13-30 raM. Red crab saline of the following composition was made up on the basis of De Paul's data: 405 mM Na +, 20 mM K +, 20 mM Ca 2+, 30 mM Mg 2+, and 525 mM C1 . This saline worked well, experiments often lasting 12 hr or more without sign of deterioration of the preparation. It was buffered with 10 mM Tris buffer. The pH of Tris buffered solutions is practically unaffected by pressure (Disteche, 1972). Tris has a large temperature coefficient (dpH/dI" = -0.028, Bates, 1961). Most of the red crab experiments were conducted at ~ 1 0 ° C and the pH was adjusted to 7.4 (7.3-7.5). Lobster saline composed of 460 mM Na +, 15 mM K +, 26 mM Ca 2+, 8 mM Mg 2+, 527 mM C1 , and 8 mM SO ] - was used. Most lobster experiments were conducted at 1020°C, and the saline was buffered to pH 7.4 (7.3-7-5) with Tris.

Recording and stimulating apparatus A conventional microelectrode recording system was used. Glass microelectrodes filled with 3M KCI were connetted to a WPI model M701 solid state electrometer by means of a silver-silver chloride electrode. The microelectrodes typically had resistances of 2-15Mfl. A silversilver chloride reference electrode connected the saline bath to ground via a 3M KC1 agar bridge. Muscle membrane responses were monitored with a Tektronix 502 oscilloscope and photographed with a Polaroid® camera. A suction electrode connected to a Grass $4 stimulator and stimulus isolation unit was used for nerve stimulation. Pulses had a duration of 0'1 msec and were about 3 6 V in amplitude at the output of the isolation unit. In some experiments a second microelectrode was used to inject current into the muscle fiber. Current was supplied by a constant-current source (New, 1972) driven by pulses from the stimulus isolation unit.

Preparation The stretcher muscle of the propodite of the walking legs was used in all the experiments with the red crab. It was found in the red crab, as in most decapods (Wiersma & Ripley, 1952), that this muscle is innervated by only one excitatory axon which also supplies the only excitatory innervation to the opener of the dactylopodite. The excitatory axon was isolated in the meropodite by carefully separating the nerve fibers with fine needles and stimulating with a pair of platinum electrodes to elicit muscle contractions. Opening of the dactyl was always observed when the bundle containing the excitatory axon was stimulated, but only after the axon was nearly completely isolated was stretching of the propodite reliably elicited. This suggests that the inhibitory axon(s) innervating the stretcher lie close to the excitatory axon. The stretcher muscle was exposed by removing a piece of the shell overlying it. The opener muscle of the dactyl was used in the lobster experiments. It is supplied with one excitatory and one inhibitory axon. These are in separate bundles in the meropodite so that it was not necessary to dissect out the excitatory axon (see Kravitz, Kuffter, Potter & van Gelder, 1963; Kravitz, Kuffler & Potter, 1963). The opener muscle was exposed by removing a small piece of the shell from the dorsal surface of the propodite, adjacent to the joint with the dactyl. With the chelate walking legs this could be accomplished without damaging any of the muscle

fibers, so chelate legs were used most of the time. Usually the carpopodite was opened by removing the ventral part of its shell and the bender muscle of the propodite was removed. This was done to expose the section of the axon in the carpopodite to the saline. If the carpopodite was lell closed, the nerve would usually fail after a few hours. An alternative, better method of preparing the opener of the dactyl was used in some of the experiments reported here (see Kravitz, Kuffler and Potter, 1963). The muscle was exposed by removing the ventral surface of the shell and the closer muscle. The excitatory and inhibitory axons were followed proximally by dissecting into the carpopodite. Just inside the carpopodite, they separate and can be stimulated individually. The major advantage of this method is that it allows access to the fibers at the proximal end of the muscle which are small in diameter and give large responses to nerve stimulation.

Pressure vessel and experimental chamber A pressure vessel of 500 arm capacity was specially constructed for this work. It was designed by Barry Walden of the Department of Ocean Engineering ol the Woods Hole Oceanographic Institution, and was built jointly by the Corey Tool Company of Braintree, Massachusetts and the instrument shop at Woods Hole. It is cylindrical with a 6 in. dia bore 18 in. deep. It is closed by a plug that fits into the bore, sealing on a 45 ° angled face with an "O" ring. The plug is held in place by a nut threaded into the bore. The vessel and plug are ARMCO 22-13-5 stainless steel, and the nut is AMPCO 18 centrifugally cast aluminum bronze. The plug is penetrated by ten electrical leads (Mecca 2006 high pressure electrical penetrators). The vessel and hydraulic system were filled with light mineral oil. Pressure was applied with a Black Hawk hand pump which causes an increment of about half an atm/stroke and was measured with a Heise Bourdon tube gauge. Aminco 1/4 in high pressure tubing, fittings and values were used. The pressure vessel was mounted vertically in a thermally insulated 55-gallon drum that serves as a water bath. The bath is supplied with a cooling coil connected to a refrigeration unit through a thermistor actuated pump. The lag in the temperature control system was such that a rapid cooling of several degrees occurred when the pump was actuated. Therefore, the thermistor actuated pump was turned off at the start of an experiment. Any effects of the slow increase in temperature could not be confused with pressure effects, whereas the effects of fast temperature changes could result in confusion (see below). The preparation was contained in an experimental chamber filled with saline that was placed inside the mineral oil filled pressure vessel (see Fig. 1). The experimental chamber is a half cylinder of Plexiglas, 8 in. long and 4 in. in dia. The crustacean leg was held by rubber bands and dental periphery wax to a Plexiglas shelf (not shown). The opening of the chamber has a Plexiglas flange with a rubber gasket. A Plexiglas lid is held in place and sealed against the gasket with stainless steel clamps that are fastened to the flange with wing screws. A rubber diaphragm on the bottom of the chamber insures that no pressure difference can develop across its walls. A single micrometer drive from a Narashige UM-6 ultramanipulator mounted on the lid at an angle of 45 ° is used to drive the microelectrode. A small hole drilled in each end of the micrometer drive allows the mineral oil to flow into the air spaces in the mechanism during compression. A hole at only one end was not sufficient because oil passing by the dovetail caused movement. The microelectrode penetrates the chamber lid through a hole that is lined with rubber tubing to make a seal. A length of Mecca high pressure cable fitted with a shield driven at the same potential as the microelectrode connects the electrode to one of the penetrators in the pressure vessel plug. After many pressure cycles the insulation of the Mecca penetrators breaks down slightly. If this insulation does

Transmission at the crustacean neuromuscular junction

135

Fig. 1. The experimental chamber (internal details are not shown). It is made from a half cylinder of Plexiglas 8 in. long and 4 in. in diameter. The microelectrode is aimed at the muscle by adjusting the position of the Plexiglas lid which is then secured by tightening the clamps around its edge. After a muscle fiber has been impaled, the sealed chamber is picked up and placed on end inside the pressure vessel. Behind the micrometer drive is a clamp for the cable to the microelectrode. It prevents movement of the cable from moving the microelectrode. not provide a large resistance relative to the microelectrode resistance serious errors can result, especially if the resistance changes with pressure. This resistance was checked frequently using the electrode test circuit of the electrometer and if measurable ( < 1000 megohms), the penetrator was replaced. Nerve stimulation was accomplished with a conventional suction electrode into which the cut end of the nerve was drawn. The entire suction electrode arrangement was contained within the experimental chamber. Soldered connections were cast in epoxy. A small hole in the tubing connecting the suction electrode and syringe prevented the expansion of small bubbles upon decompression from blowing the nerve out of the suction electrode.

Experimental procedures Muscle fibers were impaled in the following manner: the microelectrode was inserted backwards through the hole in the lid of the experimental chamber and clamped to the electrode carrier. The chamber was filled to the top with saline, and the lid was placed in position. The muscle was viewed with a dissecting microscope, and the microelectrode was aimed at the muscle by adjusting the position of the lid and advanced to the surface of the muscle. The chamber lid was then clamped in place. No attempt was made to pick out precisely the muscle fiber to be impaled. as the chamber lid would usually move slightly when the clamps were tightened. The micrometer drive was advanced until a resting potential appeared on the oscilloscope, and the nerve was stimulated to elicit excitatory junctional potentials. Most of the time the experiments were done with fibers on the surface of the muscle, but occasionally deeper fibers were used. A crane rigged over the mouth of the pressure vessel is used to lift the nut and plug, both of which hang suspended when the pressure vessel is open. Two 1/4-in. dia stainless steel support bars were threaded into holes in the inside surface of the plug. The experimental chamber was placed on Plexiglas supports attached to these bars. In its position on the support bars, the experimental chamber stands on end. There is a threaded hole in the top end plugged with a wing screw and "O" ring. The wmg screw was removed and saline was syringed into the hole to remove most of any remaining air. There are always some bubbles adhering to surfaces that cannot be removed. If 1 ml of air remains trapped inside the 600-ml experimental chamber (a generous assumption), then the

dissolved oxygen will increase about 5~, nitrogen 12~ and carbon dioxide 0.1~ if all of the air goes into solution during compression. It is assumed that this is inconsequential. This calculation is based on the amount of dissolved gases in seawater in equilibrium with air at 1 atm and 10°C (Sverdrup et al., 1942). Finally, a small amount of mineral oil was added to the pressure vessel to insure that it would be filled to overflowing when the chamber and plug were lowered into place. Then they were lowered into place, and the nut was lowered into place and screwed down tight. This method worked very well. With only reasonable care, the microelectrode was never dislodged from a muscle fiber during all the manipulation entailed. With this method, successful recordings were made aboard a diesel ship under full power. In some experiments two microelectrodes were inserted into the same muscle fiber, one for current injection and one for recording. This required two independently movable electrodes with precise control of position in all three planes of space. Briefly, the experimental chamber used for these measurements consisted of a Plexiglas dish 4-in. dia with a tightly fitting cover made of 1/2-in. Plexiglas. Two Narashi.qe UM-6 ultramicromanipulators were mounted on the cover and the electrodes entered the dish through a l~-in. dia hole in the cover. When this chamber is placed in the pressure vessel, mineral oil floating on top of the saline fills this hole. Because the electrodes are not so rigidly supported, this chamber must be handled delicately, but it still works quite well. The lead to the current electrode was provided with a shield driven from Vou, of the constant-current source. Also, because a greater length of the electrode than is customary was immersed in the saline, more capacity compensation was built into the constantcurrent source. The insulation of the high pressure penetrator through which the current was passed was checked frequently to insure that it provided immeasurably high resistance. A standard paradigm for the application of pressure [somctimes with slight modifications) was used. It had the fOlln ABACA etc., with A = 1 atm, B = 50 atm, C = 100 atm, etc. Thus, the measurements a t a given pressure were always bracketed by control measurements at 1 arm.

Temperature considerations Temperature is a potential confounding variable in pressure experiments because compression is always accompanied by heating and decompression by cooling. In most

136

ROBERT B. CAMPENOT

experiments the temperature of the saline was monitored by a thermistor mounted in the experimental chamber and recorded with a chart recorder. The adiabatic heating of seawater elevated li'om 1 atm to 200 atm pressure causes an increase in temperature of 0"I°C (von Arx, 1962). The change in the temperature of the saline in these experiments was greater because compression is not carried out in a thermodynamically reversible manner and because mineral oil heats up a great deal more upon compression. After the desired pressure was reached, the saline temperature continued to rise slowly due to heat flow from the mineral oil. After decompression the temperature continued to fall slowly. The maximum rise in temperature incurred during these experiments when 200 atm pressure was applied and held was about I°C. Often the experimental chamber was a few degrees warmer than the mineral oil when it was first placed into the pressure vessel. Equilibration required an hour or more during which time the saline temperature slowly fell. Measurements made at 1 arm during this time can serve as controls for the effects of temperature. Also, part or all of the heat generated by compression goes into reducing the rate of fall of the temperature, so compression can be accomplished with little or no rise in temperature. The results of both these methods indicate that the pressure effects reported in this paper are uncontaminated by temperature effects (see Results).

RESULTS A typical response to nerve stimulation of the membrane potential of a crustacean muscle fiber is shown in Fig. 2(a). The membrane potential undergoes a brief depolarization termed an excitatory junctional potential (ejp). The ejp reflects a local response of the muscle membrane to transmitter substance released by the nerve endings. The entire muscle fiber is involved because nerve endings are distributed widely over its surface (see Fatt & Katz, 1953b). If the ejp is of large enough amplitude, the muscle fiber contracts. The amplitude of the depolarization determines the magnitude of the contraction. The amplitude of the ejp may increase with the frequency of nerve stimulation. In all crustaceans where this facilitation of ejp amplitude has been

2mvl

50 msec

examined, it has been shown to result from an increase in the output of transmitter substance (cf. Dudel & Kuffler, 1961; Frank, 1973). Crustacean muscle fibers vary in the extent to which their ejp's show facilitation. Fibers that contract at low frequencies of nerve stimulation respond with large ejp's that show little facilitation. Others respond with small ejp's at low frequencies and little or no contraction. They show a great deal of facilitation, and the ejp amplitude and the strength of contraction increases with frequency. These are extremes, and fibers of all degrees of facilitation intermediate between them are observed. If the frequency of stimulation is so fast that the interval between nerve impulses is less than the duration of the ejp, successive ejp's will sum with one another further increasing the depolarization and strength of contraction. The properties of facilitation and summation enable the crustacean t o ' o b t a i n a finely graded contraction from a muscle supplied by only one or a few excitatory nerve fibers.

Effect of pressure upon ejp amplitude and duration Experiments with the lobster. Figure 3 shows the effect of pressure upon the amplitude of the ejp recorded from a lobster muscle fiber. The ejp's are small at the start of stimulation and because of facilitation rapidly grow to a plateau. In all experiments the ejp's were recorded after the plateau had been reached. The ordinate in Fig. 3 is an amplitude index with the first measurement at 1 atm for each frequency set at 1 (the amplitude in mV is given in parentheses). This index is used for all of the data reported in this paper. In all of the lobster experiments the measurements were made at. each of the chosen frequencies (usually 1 or 2 and 5 and 10/sec) initially and after each change in pressure. The application of pressure typically required from about 3 6 rain and decompression a minute or two. Pressure was maintained for as long as necessary to record the data, usually about 10 15 min. Pressure causes a striking depression of ejp amplitude. In the experiment of Fig. 3 the magnitude of the depression is somewhat reduced as the frequency

2f

(t2rnv) (36)

(5.6)

IO

O8

06

2mVLom~c 04

02

5m V l _ _ 0.4 s e c Fig. 2. Ejp's recorded from crustacean muscle fibers by means of an intracellular electrode. Records are traced from the original Polaroid ® photographs. The photographs were of several superimposed ejp's, but only one was traced. Upward is the depolarizing (+) direction. (a) Lobster muscle fiber. Nerve stimulation at 10/see. (b) and (e) Red crab muscle fibers. Stimulation at 4'5 and 1/sec respectively.

PRESSURE (ATM}~50 Y ~'Ioo T Yt5020~ TI FREQUENCY OF STI MULATION

I/SEC

50 t00 t5o 200

50 1oo 150200

5/SEC

10/SEC

Fig. 3. Effect of pressure on the amplitude of the ejp recorded from a lobster muscle fiber. Each bar represents the average of about 20 ejp's. The ordinate is an amplitude index with the first average at 1 atm for each frequency set at 1. Its value in mV is given in parentheses. See text for further details.

Transmission at the crustacean neuromuscular junction

137

Table 1. Effect of pressure on the amplitude of ejp's recorded from eight lobster muscle fibers Frequency of stimulation 5/see

1 or 2/see Pressure (atm) 1

50 l 100 1 150 1 200 1

X* 1.00

0.78 1"05 0.56 0"98 0.51 0"98 0.22 0"92

S.E. --

0"06 0-06 0"06 0"04 0"11 0"07 ---

N

X

8

1.00

6 5 8 7 3 4 1 1

0.79 1"03 0.61 0"97 0.41 1"06 0.35 1"07

S.E. --

0"03 0.04 0"05 0"04 0-09 0-09 0"0l 0"01

10/see N

,X

7

1 "00

5 5 7 7 3 3 2 2

0.83 1"05 0.66 1'01 0"48 1"03 0.40 1"08

S.E.

N 6

0"03 0"04 0"06 0"04 0"08 0"05 0"01 0-01

4 4 6 6 3 3 2 2

* X is the mean ejp amplitude expressed relative to the first measurement at 1 atm at each frequency. The data from each fiber were converted to this form before computation of X and S.E. (the standard error). is increased. In o n e experiment the effect of pressure on ejp amplitude showed substantially more frequency dependence, with 100 atm reducing the amplitude to 0.58 at 1/see and only 0-9 at 10/see, but in most experiments the frequency dependence was slight or not observed at all. Table 1 summarizes the data from these two muscle fibers and six others. The ejp amplitudes at 1 atm ranged from 0.23-4.06 mV at 1 and 2/see and 0"36-8"62 mV at 5 and 10/see. The resting potentials ranged 55-80 mV and averaged 65 mV. All the fibers showed pressure-induced depression which, on the average, was independent of stimulation frequency. The depression was reversible in all these experiments. In some other experiments it was not reversed. This was attributable in most instances to movements of the microelectrode. Some of the records from the experiment of Fig. 3 are shown in Fig. 4. Figures 4a and b are ejp's recorded at 1 atm pressure and stimulation frequencies of 1/sec and 5/sec respectively. The amplitude at 5/sec is greater than the amplitude at 1/sec (note change in scale), and the variability is less. The average amplitude was 1-00 mV at 1/sec and 3"92 mV at 5/sec (N = 200). The coefficient of variation (CV = s/X, the standard deviation divided by the mean amplitude) was 0.16 at 1/sec and 0-08 at 5/sec. As long as the amplitude is a small fraction of the resting potential (assuming the ejp reversal potential is close to zero), this difference in the CV can be interpreted as reflecting a difference in the quantal content of the ejp; i.e. a difference in the amount of transmitter released by the nerve endings (del Castello & Katz, 1954). The resting potential was 71 mV at the end of this experiment so the mean amplitude at 5/sec was 5.5% of the resting potential which is probably marginal (see del Castello & Katz, 1954). The difference in the CV may reflect the mixed effects of a difference in the quantal content and of non-linear summation of the quantal units that make up the larger amplitude ejp (see Martin, 1955). If the pressure-induced depression results from interference with the secretion of transmitter substance, then an identical change in the coefficient of variation should result if the 5/sec response is depressed to the amplitude of the 1/sec response by applying pressure rather than by reducing the frequency. Figure 4c shows the result of that experiment. The mean amplitude was 1-04 mV and the CV was

0-15, in excellent agreement with this hypothesis. Because of the assumption that a change in quantal content is responsible for most of the change in the CV, this evidence must be regarded as suggestive. The experiment of Fig. 3 serves to illustrate that temperature changes associated with compression and decompression cannot be responsible for the observed effects. The pressure vessel was at a temperature of 18°C during this experiment, and the dissection and set-up procedures were done at room temperature. The data were recorded over a period of about 3 hr during which time the saline temperature fell about 5°C. Note that the average ejp amplitude at 1 atm was unaffected by the change in temperature. No increase in temperature occurred upon compression to 50 or 100 atm. The heating caused by compression served only to reduce the rate of fall of temperature. Upon compression to 150 and 200 atm an increase in temperature of about 0.5 ° and 0-8°C respectively was observed. The ejp duration ranged 50-100 msec in these lobster muscle fibers, with 12-42 msec for decay from peak to half amplitude. Pressure reversibly increased the duration of the ejp. The magnitude of the increase Q

1/SEC 1ATM

m

1mY20

sec

b

5/SEC1ATM ~ 1

4myI 5 sec

C

200ATM

~

1mV 5

sec

Fig. 4. Records from the muscle fiber of Fig. 3 traced from the original Polaroid® photographs. The pressure and frequency of nerve stimulation are given at the left. Note changes in scale.

138

ROBERT B. CAMPENOT

was quite variable. A pressure of 150 atm increased the time for decay to half amplitude to 1.39 of the time at 1 atm (S.E. = 0.15, N = 3).

in progress. Because of the rebound and potentiation each frequency had to be tested during a separate pressure cycle in the red crab experiments. Table 2 summarizes the amplitude data from this experiment and four others. In all of these fibers the ejp amplitude was small at low frequencies of nerve stimulation and showed considerable facilitation. Ejp amplitudes ranged 0'28-1'07 mV at 1 or 2/sec and 4.89-5.69 mV at 10/see. Resting potentials ranged 55-80 mV, averaging 66 inV. In all of the fibers tested the pressureinduced depression of ejp amplitude was much less at stimulation frequencies of 7 or 10/see than at 1 or 2/see, and the rebound upon decompression was observed. In one experiment besides the experiment illustrated in Fig. 5 low frequency measurements were made both before and after high frequency measurements, and potentiation was observed. The amplitude at l/see and I atm was increased to 1-17 after stimulation at 7/see under pressure. The duration of the ejp in these fibers ranged 90-200 msec with 30-70 msec for decay to half amplitude. The effect of pressure upon ejp duration was not tested.

Experiments with the red crab The effect of pressure upon the ejp amplitude in a red crab muscle fiber is shown in Fig. 5. Except in a few respects the procedure was the same as in the lobster experiments. Data were usually recorded twice at each pressure with about 15 rain intervening. Special care was taken to record the data at a precise time after starting the stimulation. At high frequencies the ejp's would rapidly grow to an apparent plateau, but if responses were recorded about 10- 20 see after the plateau had been reached, it was found they had grown 10-20% more. For reasons given below, each frequency was always tested during a separate pressure cycle. The sequence in which the chosen frequencies were tested turned out to be very important. The experiment illustrated in Fig. 5 was conducted in the following sequence: frequency at 1/see and pressures of 1, 100, and 1 atm; frequency at 5/see and pressures of 1, 100, and 1 atm; frequency at 10/sec and pressures of 1, 100, 1, 150 and 1 atm; frequency at 1/see and pressures of 1, 150, and 1 atm; and finally, frequency at 5/see and pressures of 1, 150, and 1 atm. At stimulation frequencies of 1 and 5/see pressure depresses the ejp amplitude, but little or no pressure-induced depression is observed at a frequency of 10/see. Also, at a frequency of 10/sec the responses at 1 atm recorded after decompression are larger than the responses at 1 atm recorded before compression; i.e. the resistance to pressure-induced depression is accompanied by a rebound of ejp amplitude upon decompression to above its initial value. This suggests a pressure-induced enhancement in facilitation that persists for awhile after decompression. The responses at 1/sec and 5/see at 1 atm recorded after the measurements at 10/see are larger than before the 10/ sec measurements (compare the amplitudes given in parentheses in Fig. 5). Experiments to determine whether this potentiation results from stimulation at 10/see alone or from stimulation under pressure are

( 0 . 6 3 my)

(089)

r......lo PRESSURE(ATM) FREQUENCY OF STIMULATION

I I00 ~

I ~50 I

I tO0 I

I/SEC

I 150 I

I I00 I 150 1

5/SEC

IOtSEC

Fig. 5. Effect of pressure on the amplitude of the ejp recorded from a red crab muscle fiber. Each bar represents the average of about 40 ejp's. Measurements were made twice at each pressure with about 15 min intervening. The ordinate is the same as in Fig. 3. See text for further details.

Table 2. Effect of pressure on the amplitude of ejp's recorded from five red crab muscle fibers Frequency of stimulation 5/see

I or 2/scc Pressure (atm) 1

X* 1.00

1-03 100

0.62 0.62

1 150 1 200 1

0.87 0"88 0.42 0.49 0-88 0-89

S.E.

N

--

5

1.00

5 5 5 5 4 4 4 4 4

0.99 0.81 0.78 0-99 1'02 0.24 0.20 0"99 1'01

0.03 0"05 0-06 0.04 0.04 0-09 0-18 0"03 0"04

X

7 or 10/see

S.E.

N

--

2

1-00

2 2 2 2 2 t 1 1 1

1.02 0.92 0.95 1"12 1"06 0.80 0.87 1-14 1.07

0"05 0' 10 0'01 0"04 0"07 ---

X

S.E.

N

0.02 0"04 0-02 0'02 0"06 0"03 0"02 0"03 0.06

3 3 3 3 3 3 3 3 3

3

0-34 0.39

---

1 1

0-70 0-78

---

1 l

0"90 0"86

---

1 1

1"16 1.06

--

l 1

* X is the mean ejp amplitude expressed relative to the first measurement at 1 atm at each frequency. The data from each fiber were converted to this form before computation of X and S.E. (the standard error). The data were recorded twice at each frequency with about 15 min intervening.

Transmission at the crustacean neuromuscular junction Many fibers with large amplitude ejp's at low frequencies of stimulation were observed. All had ejp's of very long duration (see Fig. 2c). Movements of the propodite were observed at frequencies of 2 or 3/sec, and contraction of these fibers must have been responsible. Pressure experiments were performed with three such fibers. The duration of the ejp's ranged 0-5-0.7 sec with 0-17-0.22 sec for decay from peak to half amplitude. The ejp amplitudes could only be measured at frequencies of about 2/see or less because these long ejp's sum at higher frequencies. The amplitudes at 1 atm ranged 3'92-4"21 mV at 0.2/see and 5"56-7'87 mV at 1 or 2/see. The resting potential of these fibers averaged 79 mV (70-95 mV). All of the fibers showed pressure-induced depression of ejp amplitude. A pressure of 100 atm depressed the ejp amplitude to about 0.69 (S.E. = 0.04, N = 3) at a stimulation frequency of 0.2/see, and to 0.75 in two fibers tested at 1 or 2/see. These fibers showed facilitation within the frequency range tested, but the data are not sufficient to decide if the magnitude of the depression decreases with increasing frequency. The effect of pressure upon the duration of the ejp in these fibers was not tested.

139

104-4.4 x l0 s f~. Table 3 shows the effects of pressure on input resistance and time constant. The input resistance increased with pressure, 150atm causing an increase to about 1.42. In one experiment, 500 atm was applied causing a reversible increase to 3.64. This result supports the hypothesis that the mechanism of the pressure-induced depression of ejp amplitude is a decrease in the release of transmitter substance. It eliminates another possible mechanism, that of a pressure-induced decrease in muscle membrane resistance. Time constants were measured as the time of rise to 0"85 of the maximal amplitude. The effect of pressure on the time constant was much more variable than the effect upon input resistance, and generally of larger magnitude (see Table 3). DISCUSSION

Two mechanisms of adaptation to high pressure are suggested by the experiments with the red crab. In fibers that have small ejp's at low frequencies of stimulation, the ejp amplitude at low frequencies of stimulation is depressed by pressure, but shows little or no depression at high frequencies. Since high frequency nerve stimulation is required to elicit contracEffect of pressure upon resting potential, input resis- tion of these fibers, the ejp amplitude is independent tance, and time constant of pressure in the physiological frequency range. A In some of the lobster experiments and all of the pressure-induced enhancement in facilitation is probred crab experiments, pressure often caused large and ably the underlying mechanism of this adaptation. erratic d.c. changes. Certain features of the apparatus Fibers that respond with large ejp's and contraction proved to be the source of these artifacts. Some lobs- at low frequencies of nerve stimulation probably show ter experiments were performed after these sources pressure-induced depression of ejp amplitude at freof artifact had been eliminated. In some of these ex- quencies within the physiological range. These fibers periments pressure caused a small reversible depolari- have long ejp's that sum with one another at low zation of the membrane potential. The magnitude of frequencies, so the crab can make up for the pressurethe depolarization was about 1 mV at 100 atm and induced depression by slightly increasing the fre3 mV at 200 arm. When observed, the depolarization quency of nerve impulses. was highly variable. It was not observed in every exIf, as the data suggest, the mechanism of the presperiment, and may be artifact. sure-induced depression of ejp amplitude is interferIn four lobster experiments two electrodes were in- ence with the secretion of transmitter substance, then serted into the same muscle fiber, one to pass current the nerve impulse or some step in the processes linkand one to monitor the change in membrane poten- ing the nerve impulse and transmitter release must tial (see Fig. 6). In this way the input resistance and be depressed by pressure. Spyropoulos (1957a,b) time constant can be measured (see Fatt & Katz, found that pressure increased the duration of the 1953a). Both measurements assume zero separation action potential in squid axon and toad nerve and between the electrodes, and although the separation had no effect upon the amplitude. If pressure similarly was not measured accurately, the electrodes were in- effects the action potential in crustacean nerve terserted into the muscle fibers as close together as minals, this would tend to increase the release of possible. In any event, it is the values of these quanti- transmitter, not depress it, and the mechanism of the ties under pressure relative to their values at 1 atm pressure-induced depression would have to involve that are of interest, not their absolute values. Elec- some step in the link between nerve impulse and sectrodes usually had resistances of 10 Mfl or higher. retion. If the depressed step is common to all synapses Sometimes a small depolarization ( < l m V ) accom- (e.g., if the process of exocytosis were depressed), then panied insertion of the current electrode. The current a similar effect of pressure would be expected at all injected ranged 5 x 10-s-2 x 10-TA and at l a t m synapses, and the adaptive mechanisms suggested by caused hyperpolarization of the membrane potential the red crab experiments might be the principal ranging 2"8-22 mV. The input resistance ranged 3"5 x mechanisms by which the nervous systems of all moderately deep-living animals are adapted to pressure. The same processes of facilitation and summation of local membrane responses that control the tension developed by the crustacean muscle are important msec components for integration of synaptic influences in Fig. 6. Response of the membrane potential of a lobster the nervous systems of all animals. Since these adaptmuscle fiber to hyperpolarizing current. The record was ive mechanisms confer an insensitivity to pressures traced from the original Polaroid® photograph. The bot- in the range 1-200 atm they would be suitable tom trace monitors the current which was 5 x 10 8 A. mechanisms for the vertically migrating midwater aniThe top trace is the response of the membrane potential. mals.

-~

F 20mV[ 200

140

ROBERT B. CAMPENOT

Table 3. Effect of pressure on the input resistance and time constant of four lobster muscle fibers Pressure

,X*

Input resistance S.E. N

X

1

1 '00

--

4

1.00

50 1 100 1 150 1 200 1

1-13 0'98 1.26 1.03 1.39 1"10 1.58 1"07

0"02 0.02 0.02 0"03 0"06 0"01 O"13 0"02

4 3 4 3 3 2 2 2

| .07 1.00 1.63 1.04 1.88 1'20 2-37 1"00

Time constant S.E. N 4

0-10 0.08 0.13 0.12 0"28 0'20 O'54 0"00

4 3 4 3 3 2 2 2

* X is the mean relative to first measurement at 1 atm. The data from each fiber were converted to this form before computation of X and S.E. (the standard error).

Hyperactivity is observed in shallow-living animals at pressures m u c h lower than are required to immobilize them. Pressure has been reported to reduce the firing threshold in squid axon (Spyropoulos, 1957b) a n d frog nerve (Grundfest, 1936). A slight increase in the threshold was found in the case of toad nerve (Spyropoulos, 1957a). Effects o n threshold may be responsible for the hyperactivity, but a n o t h e r possibility is depression of transmission at inhibitory synapses. A greater sensitivity to pressure of inhibitory synapses or differences in the way inhibition a n d excitation relate to behavior could account for the lower pressures at which the effect u p o n inhibitory synapses is expressed in behavior. W o r k is now in progress to further detail the effects of pressure u p o n neuromuscular transmission a n d to get additional clues a b o u t the mechanisms underlying the effects. W o r k with other synaptic preparations is planned for the future. The purpose is b o t h to understand how nervous systems function at high pressure in the deep-sea a n d to determine if pressure studies can contribute to the understanding of the basic physiology of synapses. Acknowledgements--The author thanks John M. Teal, Francis G. Carey, Edwin J. Furshpan, Jerome Lettvin, and Fred Lang for their advice and encouragement and Richard L. Haedrich for providing the red crabs.

REFERENCES

BATES R. G. (1961) Amine buffers for pH control. Ann. N.Y. Acad. Sci., U.S. 92, 341 356. DEL CASTELLO J. & KA'TZ B. (1954) Quantal components of the end-plate potential. J. Physiol., Lond. 124, 560 573. DlSTECHE A. (1972) Effects of pressure on the dissociation of weak acids. Syrup. Soc. exp. Biol. XXVI, 27-60. DUDEL J. & KUFFLER S. W. (1961) Mechanism of facilitation at the crayfish neuromuscular junction. J. Physiol., Lond. 155, 530-542. FATT P. & KATZ B. (1953a) The electrical properties of crustacean muscle fibers. J. Physiol., Lond. 120. 171-204.

FATT P. & KATZ B. (1953b) Distributed 'end-plate potentials' of crustacean muscle fibers. J. exp. Biol. 30, 4 3 9 439. FRANK E. (1973) Matching of facilitation at the neuromuscular junction of the lobster: a possible case for influence of muscle on nerve. J. Physiol., Lond. 233, 635658. GRUNDFEST H. (1936) Effects of hydrostatic pressures upon the excitability, the recovery, and the potential sequence of frog nerve. Cold Spring Harb. Symp. quant. Biol. 5, 17~187. KRAVlTZ E. A., KUEELZR S. W., POTTER D. D. & VAN GELDER N. M. (1963) Gamma-aminobutyric acid and other blocking compounds in crustacea--II. Peripheral nervous system. J. Neurophysiol. 26, 729-738. KRAV1TZ E. A,, KUFFLER S. W. & POTTER D. D. (1963) Gamma-aminobutyric acid and other blocking compounds in crustacea--III. Their relative concentrations m separated motor and inhibitory axons. J. Neurophysiol. 26, 739-751. MACDONALD A. G. (1972) The role of high hydrostatic pressure in the physiology of marine animals. Symp. Soc. exp. Biol. 26, 209-232. MACDONALD A. G., GILCHRIST I. & TtiAL J. M. (1972) Some observations on the tolerance of oceanic plankton to high hydrostatic pressure. J. mar. biol. Ass. U.K. 52, 213223. MARTIN A. R. (1955) A further study of the statistical composition of the end-plate potential. J. Physiol., Lond. 130, 114-122. NEW JR. W. (1972) Constant-current source for microelectrodes. J. appl. Physiol. 32, 885 887. SCHROEDERW. C. (1955) Report on the results of exploratory otter-trawling along the continental shelf and slope between Nova Scotia and Virginia during the summers of 1952 and 1953. Suppl. to Deep-Sea Res. 3, 358-372. SPYROPOULOS C. S. (1957a) Response of single nerve fibers at different hydrostatic pressures. Am. J. Physiol. 189, 214-218. SP~q~OPOULOSC. S. (1957b) The effects of hydrostatic pressure upon the normal and narcotized nerve fiber. J. gen. Physiol. 40, 849-857. SVERDRUP H. U., JOHNSON M. W. & FLEMING R. H. (1942) The Oceans, Their Physics, Chemistry, and General Biology, pp. 188 191. Prentice-Hall, Englewood Cliffs, N.J. YON ARX W. S. (1962) An Introduction to Physical Oceanography, p. 399. Addison Wesley, Reading, Mass. WIERSMA C. A. G. & RmLE¥ S. H. (1952) Innervation patterns of crustacean limbs. Physiologia comp Oecol 2, 391 405.