Comp. Biochem. Physiol., 1973, Vol. 46A, pp. 767 to 783. Pergamon Press. Printed in Great Britain GASTRIC MILL RHYTHMS IN INTACT CRABS LAWRENCE WADE POWERS* Department of Biology, University of Oregon, Eugene, Oregon 97403, U.S.A. (Received 8 January 1973) Abstract-l. Foregut muscle activity (the gastric mill cycle) was monitored with extracellular electrodes implanted in free-moving crabs. 2. The dominant gastric mill component in Cancer magister and C. productus consisted of tonic cardiopyloric muscle (CPM) contractions of about 5 set duration and a cycle time of about 15 sec. 3. CPM activity reciprocated with anterior gastric muscle activity and CPM burst parameters varied in relation to other foregut muscle activities. INTRODUCTION THE GASTRIC mill of decapod Crustacea serves as a food-grinding organ, analogous to the gizzard of birds. The mill processes coarse food particles in the cardiac portion of the foregut prior to selective filtration by the pyloric foregut and passage to the digestive caeca for absorption. The mechanical aspects of mill function have been described for various decapods by Mocquard (1883), Yonge (1924), Patwardhan (1935a-c), Reddy (1935) and Schaefer (1970). The foregut walls consist of striated skeletal muscles attached to small chitinous ossicles (intrinsic muscles). Other muscles insert on the foregut wall and originate on the internal surface of the carapace (extrinsic muscles). Several large ossicles, bearing denticles and serrated edges that function as triturating surfaces, project into the foregut lumen. The foregut muscles contract in response to activity in the stomatogastric nervous system (SGNS), producing ossicle movements that result in food maceration. Maynard (1962, 1965, 1969, 1972) h as studied pattern generation in the stomato- gastric ganglion, distinguishing two distinct metachronal rhythms of motor output, one driving the pyloric mucles and the other controlling gastric mill movements. The pyloric rhythm has been characterized in some detail, mainly from preparations of the isolated nervous system. Commensurate with the continuous process of filtration, pyloric muscles contract repeatedly in discrete, highly stereo- typed bursts, varying little in cycle time or burst duration. Maynard (1965, 1972) has monitored the pyloric rhythm in the stomatogastric ganglion, correlating intracellular activity in distinct sets of neurons with at least three components of motor output. The pyloric rhythm is probably generated entirely within the stomatogastric ganglion and the rhythm’s qualitative properties are not noticeably * Present address: Department of Zoology, University of Texas, Austin, Texas 78712. 767
Transcript
Comp. Biochem. Physiol., 1973, Vol. 46A, pp. 767 to 783. Pergamon Press. Printed in Great Britain
GASTRIC MILL RHYTHMS IN INTACT CRABS
LAWRENCE WADE POWERS*
Department of Biology, University of Oregon, Eugene, Oregon 97403, U.S.A.
(Received 8 January 1973)
Abstract-l. Foregut muscle activity (the gastric mill cycle) was monitored with extracellular electrodes implanted in free-moving crabs.
2. The dominant gastric mill component in Cancer magister and C. productus consisted of tonic cardiopyloric muscle (CPM) contractions of about 5 set duration and a cycle time of about 15 sec.
3. CPM activity reciprocated with anterior gastric muscle activity and CPM burst parameters varied in relation to other foregut muscle activities.
INTRODUCTION
THE GASTRIC mill of decapod Crustacea serves as a food-grinding organ, analogous to the gizzard of birds. The mill processes coarse food particles in the cardiac portion of the foregut prior to selective filtration by the pyloric foregut and passage to the digestive caeca for absorption. The mechanical aspects of mill function have been described for various decapods by Mocquard (1883), Yonge (1924), Patwardhan (1935a-c), Reddy (1935) and Schaefer (1970). The foregut walls consist of striated skeletal muscles attached to small chitinous ossicles (intrinsic muscles). Other muscles insert on the foregut wall and originate on the internal surface of the carapace (extrinsic muscles). Several large ossicles, bearing denticles and serrated edges that function as triturating surfaces, project into the foregut lumen. The foregut muscles contract in response to activity in the stomatogastric nervous system (SGNS), producing ossicle movements that result in food maceration. Maynard (1962, 1965, 1969, 1972) h as studied pattern generation in the stomato- gastric ganglion, distinguishing two distinct metachronal rhythms of motor output, one driving the pyloric mucles and the other controlling gastric mill movements.
The pyloric rhythm has been characterized in some detail, mainly from preparations of the isolated nervous system. Commensurate with the continuous process of filtration, pyloric muscles contract repeatedly in discrete, highly stereo- typed bursts, varying little in cycle time or burst duration. Maynard (1965, 1972) has monitored the pyloric rhythm in the stomatogastric ganglion, correlating intracellular activity in distinct sets of neurons with at least three components of motor output. The pyloric rhythm is probably generated entirely within the stomatogastric ganglion and the rhythm’s qualitative properties are not noticeably
* Present address: Department of Zoology, University of Texas, Austin, Texas 78712.
767
768 LAWRENCE WADE POWERS
affected by the application of natural chemical or tactile stimuli to peripheral structures (antennula, dactyl, mouthparts, etc.).
In contrast, gastric mill activity patterns are highly variable, respond to peripheral stimuli and may contain components generated by elements in parts of the nervous system outside the stomatogastric ganglion. Using chronically implanted electrodes to monitor stomatogastric ganglion motor output, Morris & Maynard (1970) h ave demonstrated transient gastric mill responses to feeding stimuli in unrestrained lobsters. Additional data on gastric mill pattern variability, particularly in intact preparations observed for long time periods (a few days), have not been available. This report describes activities recorded in several foregut muscles of intact and semi-intact crabs, supplementing the information provided by studies of isolated nervous systems. In addition, the techniques employed in this study may be useful in other studies where chronic monitoring of visceral and skeletal muscle function is required.
1. Animals MATERIALS AND METHODS
The Oregon Institute of Marine Biology (OIMB) at Charleston, Oregon, supplied adult Cancer magister (Dana) and C. productus (Randall) measuring 10-18 cm in carapace width. The crabs were maintained without food in large tanks of natural sea water at temperatures between 12 and 15°C. Most animals were used within 1 month after initial collection from Coos Bay and adjacent offshore points. Animals in molt or late pre-molt were excluded from this study. Recordings were made at room temperature (19-22°C) during day and evening hours.
2. Saline
Crab saline, based on serum ion values for C. magister supplied by Charles Hunter at OIMB, contained the following: 854 ml of 0.54 M NaCl, 148 ml of 0.34 M CaC1,.2H,O, 87 ml of 0.90 M MgS0,.7H,O, 27 ml of 0.44 M Na2S04.10H,0 and 20 ml of 0.54 M KCl. The addition of 35 ml of 0.5 M boric acid and 2 ml of 0.5 M NaOH provided buffer. The final volume was increased to 2 1. with distilled water and the final pH adjusted to about 7.5 ( f 0.05). Saline was made fresh every l-2 weeks and refrigerated until use.
3. Procedure
Animals were prepared for electrode placement by attaching the crabs to a wooden block, ventral side down, with legs immobilized. Packing the animals in ice reduced mortality. The dorsal carapace was air-dried and a light coat of Eastman 910 was applied to a few spots over the gastric region. A small amount of dental wax was melted onto each prepared spot, forming bases for the attachment of a wax superstructure. Dissections were originally performed without submerging the animal, but air bubbles appeared in the dorsal arteries, blocking circulation and limiting survival to a few hours. Subsequent dissections under saline or sea water improved survival and the animals remained alert to external stimuli throughout the experimental period. A piece of carapace, sufficient to expose part or all of the foregut, was removed with the aid of a hand-held rotary saw. The hypodermis was carefully split and underlying connective tissue was parted to expose nerves and muscles for recording. Bleeding was controlled with gauze pads; clots were removed by periodic rinsing with saline. A wax bridge was melted onto the wax base spots, spanning the rectangular opening (Fig. 1) and serving as an anchor for lead wire attachment.
GASTRIC MILL RHYTHMS IN INTACT CRABS 769
IF To pre-amp. -
FIG. 1. Posterior view of electrode attachment on crab dorsal carapace. A wax bridge, containing the insulated lead wires, spans a rectangular opening over the foregut. The electrode leads are easily detached from pre-amplifier leads to permit
animal mobility between recording sessions.
Acute and chronic types of preparation were employed. Acute preparations involved
dissection which resulted in shorter survival times (6 hr or less) than in the chronic prepara- tions. The nerves were lifted onto paired platinum-iridium hook electrodes; the hooks were guided with micromanipulators. The exposed lead wire surfaces and recording hooks were insulated from the surrounding solution by drops of a petroleum jelly-mineral oil mixture. Muscle electrodes consisted of pairs of 36 gauge copper wire, coated with Teflon (except at the recording tip). Chronic preparations were either restrained or free-moving animals, minimally dissected, with electrodes implanted in one or two muscles. Muscle activity was usually monitored over longer periods of time than in acute preparations.
The implanted electrodes consisted of Teflon-coated platinum-iridium wires of about 100 pm dia., soldered to lead wires about 2 in. long. A micro-amphenol female contact (Relia-Tat 220~S02, Amphenol Corp.) was soldered onto the opposite end of the lead wire; the contact was easily connected to, and disconnected from, a male contact (Relia-Tat, 220-P02) on the pre-amplifier lead. Between recording sessions, crabs were not connected to pre-amplifier leads. Tissue healing, with electrodes in place, was evident in chronic preparations surviving 2 days or more, but the signal-to-noise ratio progressively decreased in recordings made 1 week or longer after electrode implantation.
Muscle electrodes were inserted manually and lead wires were attached to the wax bridge spanning the foregut, in both chronic and acute preparations. Animals were transferred to aerated, 3-gal glass aquaria; acute preparations were suspended in the water on a platform and chronic preparations were allowed to move freely. Water levels were kept below the level of the carapace opening and saline was perfused into the thorax periodically to maintain hemolymph volume. Recording was accomplished with differential input to a.c.-coupled pre-amplifiers (Grass P-511) and displayed on a 4-channel oscilloscope (Tektronix 532 with Type M plug-ins). The CR0 display was filmed with a kymograph camera (Grass C-4). Longer sequences of activity were stored on magnetic tape with a 7-channel recorder (Ampex SP-300) for later display and filming.
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4. Data analysis
LAWRENCE WADE POWERS
Patterns of muscle and nerve spike activity were obtained from paper strip films (Grass, Linograph). Muscle spike activity was characterized in terms of burst duration and burst period when spikes were consistently grouped in dense clusters, alternating with intervals of little or no spike activity. Burst duration was measured from the initiation to the termination of the dense spike group;burst period was measured from the initiation points of successive bursts. In some muscles, spikes were more dispersed, so that spike density (number of spikes/set) was a more reliable measure of activity. Means, standard deviations and confidence limits were calculated and histograms were plotted to show the distribution and variability of activity parameters. To avoid assumptions about normal distributions, non-parametric tests were used to compare activity changes in different foregut elements.
RESULTS
1. Gastric mill anatomy and movements
The foregut musculature and ossicles in C. magister and C. productus are similar to that described by Pearson (1908) and by Dando & Laverack (1969) for C. pagurus. The major dorsal nerves and muscles of interest in this study are diagrammed for C. magister (Fig. 2). Following the terminology of Patwardhan (1935a), the mesocardiac and pterocardiac ossicles are collectively referred to as the anterior
FIG. 2. Major nerves and muscles of the dorsal foregut in C. magister. Ossicles are dotted (McO, mesocardiac ossicle; PtO, pterocardiac ossicle; ZcO, zygocardiac ossicle; ExO, exopyloric ossicle; PpO, propyloric ossicle). Muscles are striped (AGM, anterior gastric m.; PGM, posterior gastric m.; LCPM, lateral cardio- pyloric m. ; MCPM, medial cariopyloric m. ; LCM, lateral cardiac m.). LVN, Lateral ventricular n. ; MVN, medial ventricular n. ; and PSN, posterior stomach n.
GASTRIC MILL RHYTHMS IN INTACT CRABS 771
arch. The posterior arch consists of the exopyloric, pyloric and propyloric ossicles. Foregut muscles are classified as extrinsic (extending between foregut and carapace) or intrinsic (extending between foregut ossicles), in accord with Mocquard (1883).
The medial cardiopyloric muscle (MCPM) attaches anteriorly to the meso- cardiac ossicle and posteriorly to the dorsal surface of the propyloric ossicle. The lateral cardiopyloric muscles (LCPM), one on each side, extend from the meso- cardiac ossicle to attach to the exopyloric ossicles. The urocardiac ossicle, partially fused with the mesocardiac ossicle, projects ventrally into the cardiac foregut lumen as the median tooth. Simultaneous contraction of the three cardiopyloric muscles (CPM) shifts the anterior arch posteriorly, a displacement of about 2-3 mm in adult crabs. Anterior arch movement results in movement of the median tooth in an antero-posterior plane on the midline.
The lateral cardiac muscles (LCM) extend from the zygocardiac ossicles to various ventro-lateral ossicles, externally enclosing the postero-lateral walls of the cardiac foregut. LCM contraction results in depression of the dorso-lateral portion of the foregut (lateral “wings”), associated with movement of the zygo- cardiac ossicles and their lateral teeth toward the midline to interact with the urocardiac median tooth.
The anterior gastric muscles (AGM) are extrinsic, extending from the pro- cephalic shelf (the inward carapace extension behind the eyes) to attach to the dorso-anterior border of the mesocardiac ossicle. AGM contractions pull the anterior arch forward, extending the cardiac foregut and resulting in median tooth movement more extensive than that produced by CPM contractions alone. AGM activity is observed frequently in animals during the first S-10 min after foregut exposure. AGM activity subsequently disappears (no visible signs of contraction and attempts to record ejps are unsuccessful), but in a few preparations vigorous AGM activity returns, alternating with CPM contractions. Various ventral muscles and extrinsic dilator groups produce rapid, complex movements, seen infrequently in this study and not considered further.
2. Cardiopyloric muscle activity
Extracellular ejps were recorded from each of the three cardiopyloric muscles while observing anterior arch movement. CPM activity consists mainly of discrete bursts correlated with a steady posterior movement of the anterior arch. Bursting was usually absent during forward return of the arch, although occasional spikes or very brief bursts during the return phase were seen to result in hesitation or a slight posterior movement of the arch. A burst duration of about 3-4 set was sufficient for the arch to obtain maximum posterior position, but burst durations often exceeded 5 set and occasionally exceeded 10 set in duration. A summary of CPM burst duration and period (cycle time) measurements is presented in Table 1.
A sample of CPM activity (Fig. 3) demonstrates several characteristics typically observed in chronic, intact preparations: (1) most spikes were densely clustered as bursts, associated with active ossicle movement; (2) some spikes appeared between bursts, particularly during long interburst intervals, but were not correlated with
772 LAWRENCE WADE POWERS
TABLE l-CARDIOPYLORIC MUSCLE BURST DURATION AND PERIOD
N is the number of bursts measured, confidence limits are from lower to upper at the 0.95 per cent level. Animal B-14 (a and b) is C. productus, all others are C. mugister. Burst period was measured from onset to onset.
lb --
FIG. 3. Cardiopyloric muscle activity. All traces read from left to right. Trace 1: CPM burst period is short. Trace 2 : the three segments are continuous, cycle time is decreasing with successive bursts. Upper bars indicate burst durations, lower bars indicate sub-bursts. 2a and 2d are not complete bursts. Time mark = 2 sec.
discernible ossicle movements ; (3) occasionally, burst durations were less discrete (as in burst 2b)-bars above the bursts indicate the interval of spikes included, based on an arbitrary but consistently used standard of onset and offset character-
istics; and (4) CPM bursts appeared to consist of underlying spike groups (sub- bursts”), indicated by bars under bursts la and lb, and 2b and 2c.
CPM sub-bursts were present in all preparations that I observed, but they varied in resolution between and within different bursts (Fig. 4a). Sub-bursts were most discrete at the beginning and end of bursts and during bursts of long duration.
GASTRIC MILL RHYTHMS IN INTACT CRABS 773
The bursts in Fig. 4a were taken from the same preparation within an interval of 2 min. Each of the four bursts was fragmented into subgroups of about O-7- 1.0 set in cycle time duration. The distribution of values from a typical preparation (Fig. 4b) shows that sub-burst cycle time duration was stable for many burst cycles.
30
25
(a) 20
No.
I5
IO
5
(b)
. 1 I I I I I
0 0.2 04 0.6 Q6 I.0
Spb- burst cycle time, set
FIG. 4. Cardiopyloric muscle sub-burst periodicity. a. Sub-burst resolution differs between bursts in the same animal. b. Histograms of sub-burst cycle times in one
series of CPM bursts (IV = 58, E = 0.64 set).
The interval between successive CPM bursts did not remain constant in most preparations. Cycle times were short in bursts la-lc of Fig. 3, but they were considerably longer in bursts 2a-2d, sampled a few minutes later, showing a gradual shift from long to short burst period or cycle time. Figure 5(a) shows the ranges and median values of burst cycle times (periods) in samples taken 10 min apart in a chronic, intact preparation. Each sample represents several successive burst periods (N = number of cycles per sample) and the entire sequence of samples represents a 2-hr monitoring period. The amount of variation in range (length of vertical line) was not correlated with sample size (N = 13 for sample 7 and iV = 5 for sample 9). Figure (Sb) compares the distribution of burst duration value with burst period values in a preparation that exhibited shifts from one modal level of cycle time to another. This shift was observed in several preparations, but was easily obscured by variations in conditions external to the preparation (changes in temperature, aquarium water aeration and light intensity had affects on CPM cycle time). In addition, the activities of other stomatogastric units were able to produce modulating effects on CPM burst parameters.
774 LAWRENCE WADE POWERS
0 i 2 3 4 5 6 7 8 9 to II 12 13
Sample sequence Ievery 1Omin)
No.
0 2 4 6 8 6 16 24 32 40
tnterwf time. set
FIG. 5. CardiopyIoric muscfe activity. (a). Sequential series of CPM burst samples, taken ICI min apart, from left to right, Vertical bars are the range of burst periods and horizontal bars are the median value of period observed for each sample. iV indicates the number of bursts/sample. (b). CPM burst duration and cycle time histograms in a preparation with discrete modal shifts in cycle time. .N = 109 and
96 for duration and cycle time, respectiveIy.
Although ACM activity was often observed immediately after exposing the foregut, visible contractions and recordable ejps seldom persisted for more than 10 min. Xn a few chronic, minimaIly dissected preparations, AGM activity returned after an absence of 1 hr or Ionger. The reappearance of AGM activity was gradual, beginning with barely discernible muscle twitching, increasing in strength to a
GASTRIC MILL RHYTHMS IN INTACT CRABS 775
plateau of vigorous contractions alternating with CPM contractions. After 10 min or less of activity, AGM contractions gradually weakened and disappeared. Measurements of CPM burst duration and burst cycle time during alternation with AGM bursts were compared with measurements of the same parameters when AGM activity was absent. Table 2 summarizes the differences in CPM activity analyzed by the Mann-Whitney U-test: CPM burst duration with or without AGM interaction was not significantly different, but differences in CPM burst cycle time and the number of sub-bursts per burst during AGM activity were significantly different from the same parameters during AGM inactivity.
TABLE ~-EFFECTS OF AGM ACTIVITY ON CPM ACTIVITY
AGM active AGM inactive U-test results CPM parameter
N = 48 for AGM active and N = 23 for AGM inactive; probabilities are two- tailed.
A typical sequence of CPM-AGM alternation is depicted in Fig. 6; numbered brackets refer to sample records in Fig. 7. The sequence illustrates the transition of CPM cycle time from short durations of relative uniformity to long durations of erratic fluctuation, also evident in AGM activity. The strict one-to-one alternation of AGM and CPM bursts was disrupted (sequence Nos. 55-56,73-74 and 80-81) as both muscle activities increased in cycle time duration. CPM cycle time increased when AGM activity temporarily disappeared (Nos. llO-115), but when AGM bursting returned, CPM cycle time decreased. The gradual decline and subsequent return of AGM bursts is shown by vertical bars without color to indicate the diffuse distribution of spikes during the transition interval (as in Fig. 7~). Spike density (number of spikeslsec), measured in 1 set increments, was found to be a less subjective measure of AGM activity than duration or period during these transitions. The mean number of spikes/set during AGM pre-transition bursts (Nos. 85-103) was 16.47, during transition bursts (Nos. 104-134) was 4.66 and during post- transition bursts (Nos. 135-148) was 12.41, demonstrating variation in the intensity of bursts as well as the duration and cycle time of bursts.
At least two units of different amplitude are apparent in the AGM traces of Fig. 7 : the smaller units appear to be contained within the burst limits of the larger units, but resolution is difficult due to amplitude shifts associated with muscle movements. During long AGM bursts, the smaller units display periodicities similar in duration to those of CPM sub-bursts, but the larger AGM units appear more evently spaced (Fig. 7e). The posterior stomach nerve (PSN) trace on the top
776 LAWRENCE WADE POWERS
I
30 I / I I i I I ’
AGM
-J40
Sequence No. J-5o 100
FIG. 6. AGM and CPM interaction: sequence from left to right. Total vertical displacement from line of origin represents burst cycle time; solid-colored vertical bars represent burst duration within that cycle time. Vertical bars without solid segment represent scattered spike activity. CPM burst cycle time Nos. 112 and 115 are 56.2 and 79.0 set, respectively. Numbered bars indicate sample myograms
FIG. 7. Myograms of AGM and CPM interaction. Traces correspond to bracketed intervals depicted in Fig. 6, except for (e), which was recorded after AGM activity had fully returned to regular bursting. CPM activity is not apparent due to
electrode dislocation. Time mark = 2 sec.
GASTRIC MILL RHYTHMS IN INTACT CRABS 777
of each sample in Fig. 7 shows a slight increase in activity during AGM bursts (see below).
Time intervals between AGM and CPM bursts were measured for forty-eight pairs of interactions (one pair = AGM to CPM time, and CPM to AGM time). The average time from the end of a CPM burst to the start of an AGM burst was 0.31 set and the average time from the end of an AGM burst to the start of a CPM burst was only O-025 sec. The AGM to CPM time average included six values (of forty-eight) in which the CPM burst began before the AGM burst terminated. Overlap from CPM burst to AGM burst was not observed. Thus, CPM bursts could begin during the end of, and at variable times after, AGM bursts. In contrast, AGM bursts did not start until CPM bursts terminated. In a few cases, AGM spikes were correlated with CPM burst interruptions (Fig. 7d), but in no case observed did the AGM activity continue as a burst of more than a few spikes, indicating a likely inhibitory interaction of CPM neural units on the AGM neural units. An observed overlap of as much as O-7 set from AGM bursts to CPM bursts may indicate that the interaction is indirect and complex, supporting the intracellular data presented by Maynard (1972) on neuron interactions in the stomatogastric ganglion.
4. Activity in other muscles
(a) Lateralcardiac muscle. LCM activity appeared sporadically and infrequently during an experiment, and like AGM activity, began as scattered spikes, became more organized into discrete bursts of increasing spike density, then gradually declined. The bursts were not as discrete as AGM and CPM bursts and LCM burst duration and cycle time were extremely variable (Fig. 8). The interaction of LCM activity and CPM activity was measured and compared by the Mann- Whitney U-test, summarized in Table 3. As with AGM-CPM comparisons, but to
TABLE ~-COMPARISON OF LCM ACTIVITY AND CPM ACTIVITY
LCM active LCM inactive U test results CPM parameter
N = 50 for LCM active and N = 56 for LCM inactive; probabilities are two- tailed.
a lesser extent, CPM cycle time varied more than CPM burst duration. When CPM cycle times were long (greater than 20 set), CPM and LCM bursts alternated in discrete fashion; during short CPM cycle times (less than 20 set) and increased LCM activity (Fig. g), LCM bursts often occurred during long CPM bursts and reciprocal bursting was not apparent. The lack of discrete reciprocal LCM-CPM activity during short CPM cycle times is the reverse of the reciprocity seen in
778 LAWRENCE WADE POWERS
FIG. 8. LCM and CPM activity. The two samples are not continuous. Time mark = 2 sec.
AGM-CPM activity comparisons. No definitive statements about the causal
relations of these activities can be made from the present data. (b) Lateralpyloric muscle. In contrast to gastric mill muscles, pyloric muscles
contracted rhythmically at stable frequencies and displayed little indication of modulation by sensory input or by interaction with other stomatogastric elements. LPM activity presented little variation in burst duration, cycle time or in the number of spikes per burst, regardless of the activity states of gastric mill muscles
simultaneously observed (CPM, AGM and LCM). Table 4 contains data on LPM activity and Fig. 9(a) is a sample trace showing discrete, regular LPM bursts re- corded from intact crabs (compare with AGM and LCM activity). Despite absolute
differences between the two preparations in Table 4, the standard deviations of
TABLE ~-LATERAL PYLORIC MUSCLE ACTIVITY
Animal N
B-15 26 B-5 90
Burst duration (set) Burst cycle time (set)
Confidence Confidence Mean S.D. limits Mean SD. limits
burst parameters are small, indicating a high degree of stability in LPM activity for a given animal. Figure 9(b) is a histogram of LPM burst duration and cycle time, and indicates that burst cycle time varied less than duration (compare standard deviations in both preparations of Table 4), the reverse of measures of CPM and AGM activities. In addition, the number of spikes/LPM burst is relatively constant, ranging from 7 to 9 (average = 8.15) for the bursts shown in Fig. 9(b) (animal B-15, N = 26). The highly stereotyped quality of LPM activity recorded in intact, chronically maintained crabs correlates well with the regularity seen by Maynard (1972) for pyloric elements in the isolated stomatogastric nervous system.
5. Changes in patterns of nerve activity
Gastric mill rhythms can be monitored extracellularly in several prominent dorsal nerves (refer to Fig. 2). Morris & Maynard (1970) recorded from the
GASTRIC MILL RHYTHMS IN INTACT CRABS 779
IO -
Duration Cycle J- time
No. 5 -
0
I- i I I I I
0. I 0.2 0.3 0.4 0.5 0.6
Interval time, set
(b>
FIG. 9. LPM activity. (a). Myogram of LPM bursts, showing regularity of cycle time and spikes/burst. (b). Histogram of LPM burst duration and frequency:
cycle time is less variable than duration. Time mark = 2 sec.
lateral ventricular nerve (LVN) in intact lobsters and Dando & Laverack (1969) recorded from the posterior stomach nerve (PSN) in C. pagurus. The PSN is believed to be purely sensory (Dando, personal communication), innervating the posterior arch region in general and the posterior gastric muscles (PGM) in particular.
The median ventricular nerve (MVN), or outer lateral nerve of Larimer & Kennedy (1966), contains motor fibers to several anterior foregut mucles as well as to elements in the lateral pyloric region. Patterned activity in the MVN comprises part of the pyloric cycle but differs with changes in CPM activity (Fig. 10). Cycle times were short, MVN activity was relatively regular and consisted of one to three large units alternating with two to four smaller units (Fig. 1Oa). When CPM cycle times were longer, CPM burst onset was correlated with an absence of MVN
FIG. 10. MVN activity patterns. (a). CPM cycle time is short and MVN activity is regular. (b). CPM cycle time is long and MVN activity is irregular with long periods of inactivity and shorter periods of inactivity correlated with the first few
seconds of CPM burst. Time mark = 2 sec.
780 LAWRENCE WADE POWERS
spikes lasting 2-5 set, followed by a transient period of regular, alternating MVN spikes, and finally decreased to a few scattered spikes (Fig. lob). Stimulation of the LVN (6 V at 10 Hz) increased the CPM burst pattern from a long cycle time to a
short cycle time, associated with changes from irregular to regular spike patterns in the MVN, respectively. The same results were obtained by stimulating the cut
central ends of the PSN, possibly indicating that pattern changes in the MVN reflect neural interactions between stomatogastric units involving the cardiopyloric muscles (gastric mill) and motor output to pyloric elements (via the MVN). Table 5 is a quantitative summary of MVN spike activity in relation to CPM burst cycle
time.
TABLE S-CPM CYCLE TIME AND CHANGES IN MVN SPIKE ACTIVITY
Mean number of MVN spikes
Short CPM Long CPM iv n cycle time cycle time
During CPM bursts 15 1365 58.4 32.6 Between CPM bursts 15 1414 61.4 32.9
Total 30 2779 59.9 32.75
N = number of CPM bursts samples, n = number of MVN spikes counted. Short CPM cycle times are less than 20 set, long CPM cycle times are greater than 20 sec.
DISCUSSION
Because the gastric mill cycle is sensitive to SGNS isolation, more information from intact and semi-intact preparations is needed in order to describe the full
range of SGG integrative activities, CNS-SGG interactions and the relationships of foregut rhythms to other digestive activities and to the behavior of the animal in general. The results described in the present study provide some information on gastric mill activities in intact preparations, emphasizing a component given little attention in previous reports-the cardiopyloric rhythm. CPM junction potentials occur regularly and remain stable after foregut exposure ; in C. magister and C. productus, the CPM rhythm is the most dominant indication of foregut activity after AGM contractions cease.
A single neuron (CP cell) in the SGG sends axon branches via the superior and inferior bundles of the LVN to the three CPM muscles (see Fig. 2). Maynard (1972) has not indicated a specific SGG neuronal input to the CP cell, but he does indicate an inhibitory action of the CP cell on the AM cell, which in turn inhibits at least two other groups of gastric mill cells. One of these latter groups, the four GM cells, innervate the anterior gastric muscles (AGM). Indirect interaction between
GASTRIC MILL RHYTHMS IN INTACT CRABS 781
the CP neuron and the GM cells might produce the type of reciprocal muscle activity seen in Figs. 6 and 7, but other elements than those indicated above must be involved. Concurrent intracellular and extracellular studies of the cardiopyloric rhythm have not been attempted in intact animals.
Spontaneous shifts in CPM cycle time are common (Figs. 3 and Sa) and some preparations display modal steps in the distribution of burst cycle time measure- ments (Fig. 5b). The causative factors and specific neuronal circuitry involved in CPM burst frequency modulation are not known, but my observations on another crab suggests that peripheral sensory input is one source of modulation. I have been able to initiate CPM bursting in chronic, free-moving CaZZinectes sapidus (the edible blue crab) by the application of fish meat or fish juice to the walking legs or into the inhalent respiratory current. In Callinectes, the cardiopyloric muscles. do not discharge in regular bursts ; ongoing CPM activity appears as scattered individual spikes. Chemical (and occasionally, tactical) stimulation of mouthparts, antennulae and dactyls in CaZZinectes results in the sudden onset of dense bursts of spikes of 2-4 set duration and a frequency of 80-100 c/min. The response fades after ten to fifteen burst cycles and CPM activity returns to scattered single spiking. Repeated stimulation of the same peripheral structures leads to a rapid decline in the burst response (probably habituation), whereas stimulation via a different peripheral structure evokes CPM bursting. Other foregut muscle activities in Callinectes have not been monitored and attempts to modulate ongoing CPM activity in Cancer have produced equivocal results. A correlation between CPM burst rate and MVN spike pattern is shown in Fig. 10 and Table 5 (see Dando & Laverack, 1969, for observa- tions of PSN stimulation and MVN spike pattern). Increased CPM burst frequency (decreased cycle time) shows a significant correlation with increased MVN spike frequency. The MVN sends axons from the VD and IC cells to ventral cardiac muscles CVl and CV2, components of the pyloric cycle (Maynard & Dando, 1973), thus CPM activity and the motor pattern to ventral cardiac foregut dilators show a possible relationship. The origin of CPM sub-bursts is unknown, but their measured regularity (Fig. 4b) suggests that they may be additional indica- tions of interaction between the CP cell and other SGG neurons. The lateral pyloric muscle bursts with similar regularity and frequency as the CPM sub-burst cycle time; simultaneous records of LPM and CPM activity would be of interest and might provide further clues as to the relationship between the CPM rhythm and pyloric activities.
The results are a partial description of some foregut activities, recorded extracellularly in intact animals. Some of the preparations utilized chronic implanted electrodes and the animal was free to move and feed while foregut muscle contractions were monitored. The use of muscle electrodes overcomes many of the technical problems inherent in the method of Morris & Maynard (1970). Maintain- ing good signal-to-noise ratios in chronic nerve implants is not easy and the bulky polyethylene sleeve they used to provide nerve-electrode contact limits the number of nerves readily accessible by minimal dissection. Muscle electrodes can be implanted quickly, with little trauma, and into a number of different units. The use
782 LAWRENCE WADE POWERS
of intact, behaving animals overcomes some of the problems with isolated prepara- tions. Depending on one’s point of view, the simplified, stereotyped pattern of activity seen in the isolated SGNS is either ideal for analysis because of the
constancy and stability of neural function, or it is less than representative of the actual relationships and cellular interactions characteristic of the system in situ.
Both approaches are necessary in order to understand the control and regulation of foregut activities. Further study of the gastric mill cycle in isolated and intact
preparations will be necessary to define the role of the cardiopyloric rhythm, the extent of regulation of SGG pattern by CNS command elements and the pathways of reflex feedback in co-ordinating foregut activities with the rest of the digestive
system.
Acknowledgements-The advice and constructive criticism of Don Maynard at the University of Oregon, Eugene, Oregon and of Malcolm Dando at the Gatty Marine Laboratory, University of St. Andrews, Scotland, were greatly appreciated during the research and during preparation of the manuscript. My special thanks to numerous colleagues and friends at the University of Oregon and the University of Texas at Austin for their helpful comments. This work was supported in part by an NIH grant to Don Maynard (No. NS-09474-02) and a PHS training grant (No. 2T016M00336) to the author.
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