Involvement of Pedal Peptide in Locomotion in Aplysia: Modulation of Foot Muscle Contractions Jon D. Hall and Philip E. Lloyd* Dept. of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637
SUMMARY Pedal peptide (Pep) is a 15-amino-acid neuropeptide that is localized within the Aplysia central nervous system (CNS) predominantly to a broad band of neurons in each pedal ganglion. Pep-neurons were identified by intracellular staining and immunocytology or by radioimmunoassay ( R I A ) of extracts from identified neurons. RIA reveals that 97% of all Pep-like immunoreactivity (IR-Pep) in pedal nerves is found in the three nerves that innervate the foot. Nearly every Pep-neuron sends a n axon out at least one of these three nerves. Application of Pep to foot muscle causes an increase in the
amplitude and relaxation rate of contractions driven by nerve stimulation or intracellular stimulation of pedal motor neurons. The increase in relaxation rate was the predominant effect. Intracellular recording in “splitfoot” preparations reveals that Pep-neurons increase their overall firing rates and fire in bursts with each step during locomotion. Recovery of IR-Pep from foot perfusate following pedal nerve stimulation increases in a frequency-dependent fashion. Thus it appears that one function of Pep-neurons is to modulate foot muscle contractility during locomotion in Aplysia.
INTRODUCTION
dominant source for the Pep present in the foot are these immunoreactive neurons in the pedal ganglia. The foot is the primary effector organ for locomotion in Aplysia calijbrnica. It encompasses the entire ventral surface of the animal extending from the propodium and mouth to the tail. The foot is composed of a dorsal longitudinal muscle layer and a ventral transverse muscle layer (Kandel. 1979) and is primarily innervated by the anterior ( p l ), middle (p8), and posterior (p9) pedal nerves (Jahan-Parwar and Fredman, 1978). p l arises from four roots and innervates the anterior portion of the foot, p8 leaves the ganglion as one root but immediately branches and innervates the middle of the foot, and p9 also arises from a single root and innervates the posterior foot and the tail (Kandel. 1979). Locomotion involves pedal waves, which are peristaltic contractions of the entire width of the foot. The pedal waves in Apljfsia begin anteriorly and progress rearward lo propel the animal forward (Parker, 1917). Although Pep clearly has biological actions on a number of central neurons in Aplysia (Pearson and Lloyd, 1989), its role in the periphery, and
Pedal peptide (Pep) was recently isolated from Aplysia pedal ganglia and sequenced (Lloyd and Connolly, 1989). Immunocytology using antisera directed towards Pep suggested that the peptide was found widely throughout the central nervous system (CNS) and periphery of Aplysia, and it was heavily concentrated in the foot and in the pedal ganglia, which provide the primary central innervation of the foot (Pearson and Lloyd, 1989). Each of the paired pedal ganglia contains roughly 100 large Pep-immunoreactive neurons localized predominantly in a broad band running along the dorsal surface of each ganglion. Pep was transported in very large quantities down pedal nerves that innervate the foot and Pep was not synthesized in significant quantities by the foot itself (Pearson and Lloyd, 1989; 1990). Thus the preReceived February 6 . 1990, accepted April 16, 1990 Journal ofNeurobiology, Vol. 21, No. 6, pp. 858-868 (1990) Q 1990 John Wiley & Sons, Inc. CCC 0022-3034/90/060858-11$04.00 * To whom correspondence should be addressed.
858
Peptido Modulation of'Aplysia Locomotion particularly in the foot, has not been established. In the present study, we provide evidence that Pep is a locally acting transmitter that is involved in modulating motor-neuron-drivcn contractions during locomotion. Specifically. Pep enhances t h e amplitude and rate of relaxation of foot muscle contractions, Pep-neurons significantly increase their firing rate during locomotion, and Pep is recovered from foot tissue perfusate following pedal
nerve stimulation. METHODS Animals Aplysia cal(omicu (60- I50 gm) were obtained from Marinus, Inc.. maintained in circulating artificial sea water (ASW) tanks at 15"C, and fed dried seaweed at 3-day intervals.
Pedal Nerve Radioimrnunoassay Nine peripheral nerves leave each pedal ganglion and a single, unpaired nerve exits from the parapedal connective. The amount of Pep-like immunoreactivity (IRPep) contained in each was assayed. Animals were immobilized by isotonic MgClz injection, pedal nerves were identified, dissected, weighed, and cxtracted for I0 min at 100°C in 0.02 M trifluoroacetic acid (TFA). Aliquots of these samples were used in an RIA (Pearson and Lloyd, 1990).
Morphological Studies Cell bodies having axons in pl. p8, and p9 were visualized by cobalt chloride backfilling. Standard procedures were used (Hening, Walters, Carew, and Kandel, 1979). Lucifer yellow ( 1 % ) was ionophoresed into individual Pep-neurons in pedal ganglia.
Injection of Pep into Freely Behaving A plysia Synthetic Pep (Applied Biosystems) was taken up in 1 ml ASW and injected into the anterior hemocoel. Injections were either 0.1 or 1.0 nmol peptide/g animal weight. Control injections consisted of the vehicle alone. The behavior of the animals was periodically observed for 3 h after the injections. The injections and subsequent behavioral observations were done blind. In the case of the larger Pep injections, animals were also weighed before and periodically after the injections.
Proteolysis of Pep by Blood and Foot Tissue Labelled Pep was prepared by incubating pedal ganglia with "S-methionine and purifying the labelled peptide
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through two modes of high-pressure liquid chromatography (HPLC) as described prcviously (Lloyd and Connolly, 1989). Lyophiliied labelled Pep was dissolved in either ASW or in blood taken from a cold-anesthetized animal (15 min at 4°C) and incubated at 22°C. For proteolysis experiments involving the foot. lyophilized labelled Pep was dissolved in stenlc ASW and incubated for 50 min with pieces of foot tissue (wet weight 20 mg) that had been washed in several changes of sterile ASW to remove blood. Aliquots of the samples were removed periodically and diluted into 500 ~1 0.02 M TFA spiked with 2 nmol synthetic Pep. This sample was heated to 100°C for 10 min, filtered, and run on HPLC as described previously (Lloyd and Connolly, 1989). The retention times of labelled fractions were determined by liquid scintillation counting.
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Release of Pep from Foot Tissue ..lp/vsiawere immobilized by MgC12 injection and a dorsal incision made along the length of the animal. All internal organs and the central ganglia were removed. The vasculature was perfused with ASW containing 10 mg/l bovine serum albumin (BSA) and 10 mg/I bacitracin as carriers to improve Pep recovery. Other compounds that were tested but failed to significantly inhibit Pep proteolysis or improve Pep recovery were diprotin A (Peninsula Labs), antipain, aprotinin, chymostatin, E-64, leupeptin. N-carboxymethyl-phe-leu, 1,lO-phenanthroline. phenylmethylsulfonyl fluoride, pepstatin A, and thiorphan (all from Sigma) Suction electrodes were bilaterally placed on p8 and p9 nerves and stimulation intensity was adjusted to be supramaximal as judged by the amplitude of contractions. Initially, the bath contained 20 ml ASW plus carriers and an additional 20 ml of ASW plus carriers was perfused through the foot over a 40-min period (20-min stimulation and 20-min poststimulation washout). For high K + experiments, 20 ml ASW containing 100 m M KCI ( 10 X normal) was used as the stimulus. The entire 40-ml sample was removed and passed through a reverse-phase extraction cartridge ( Sep-Pak). Peptides were eluted with 3 ml 75% CH3CN, 0.01 M TFA, and aliquots used for RIA. Foot tissue was heated at 100°C in 30 ml 0.02 Ail TFA for 10 min and aliquots used in the RIA to dcterrnine total foot IR-Pep.
Blood Content of Pep Similar methods were used to measure 1R-Pep in the blood. Blood was drawn from locomoting animals that were subsequently cold-anesthetized ( I5 min at 2°C). The blood was immediately acidified by addition of 0.02 MTFA, heated to 100°C for 10 min, and centrifuged at 10,000 X g for 10 min. The supernatant was extracted and 1R-Pep measured by RIA as described above.
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Effects of Pep on Foot Contractions Two preparations were used. The first consisted of the entire tegument and foot of the animal with the viscera, buccal mass, and CNS removed. An animal was immobilized by MgC12 injection, and an incision was made from head to tail about 1 cm into the tegument from the left edge of the foot. The preparation was pinned flat and the left pedal artery cannulated and perfused with ASW. A suction electrode was used to stimulate nerve p8 with a paradigm consisting of 5 stimuli (I-1.5-ms pulses) in 200 ms at 60 s intervals. Stimulation intensity was supramaximal as judged from contraction amplitudes. Longitudinal or lateral contractions were measured with a displacement transducer (Bionix Isotonic). Synthetic Pep dissolved in ASW was applied through arterial perfusion. The second preparation consisted of a 1 X 3 cm section of the foot innervated by p8. Contractions were produced by intracellular stimulation of either identified (P2; Hening et al., 1979) or previously unidentified motor neurons. A pedal ganglion was pinned in a chamber with all nerves and connectives severed except p8, which was run through a Vaseline barrier to the foot muscle in a second chamber. The pedal ganglion was superfused with ASW containing 1 m M CaCI2 (0.1 X normal) and 110 m M MgC12 ( 2 X normal) to inhibit synaptic transmission while the foot was superfused with normal ASW. Two intracellular electrodes were used: one to inject current pulses (10 ms, 100-200 nA) to drive individual spikes, and the other to monitor membrane potential. Motor neurons were stimulated at 30 Hz for 3 s every 60 s. Experiments were carried out as described above except only longitudinal contractions were measured and the artery was not cannulated. Pep was applied by superfusion only into the sub-chamber containing the foot. Thus, the pedal ganglion was not exposed to Pep.
lntracellular Recording of Neuronal Activity During Locomotion A split foot preparation similar to that described pre-
viously (Hening et al., 1979; Wallers, Byrne, Carew, and Kandel, 1983) was used. Briefly, the animal was immobilized by MgClz injection and pinned dorsal side up in a Sylgard dish. A midline dorsal incision was made from the anterior edge of the parapodia to the head and the buccal mass, buccal ganglia, abdominal ganglia, and the anterior portion of the gut were removed. A midline longitudinal incision was then made in the anterior one-third of the foot. The pedal nerves and the circumesophageal-ring ganglia were pinned to a Sylgard platform and the right pedal ganglion was desheathed for intracellular recording. The abdominal artery was cannulated and perfused with ASW. Pedal waves were measured with a displacement transducer attached to the lateral edge of the foot. Locomotion was defined as waves of contractions starting simultaneously in each
Table 1 Distribution of Pep-Like Immunoreactivity in Pedal Nerves Pep-Like Immunoreactivity'
Pedal Nerve
nmol/g Wet Weight
Pl P2 P3 P4 P5 P(j P7 P8 P9 P10
62.4 f 38.4 1.7 f 0.3 3.5 1.4 7.5 0.2 0.6 2 0.2 3.8 f 0.6 10.2 -+ 1.6 88.9 f 23.5 73.3 f 22.3 0.8 f 0.0
* *
nmol/Nerve 0.71
* 0.21
0.01 k 0.00 0.01 k 0.00
*
0.01 0.00 0.01 IO.00 0.01 f 0.01 0.08 iz 0.02 1.03 t 0.15 1.39 t 0.06 0.00 f 0.00
' Duplicate aliquots from crude extracts were assayed by RIA (Pearson and Lloyd, 1990). Values are mean I S.E.M. half of the head and progressing backward and either occurred spontaneously or was initiated by electrical stimulation through electrodes implanted in the tail (Walters et al., 1983) or by application of NaCl crystals to the parapodia. Intracellular recordings were made using 5 MQ potassium acetate electrodes and modified Getting M5 amplifiers with driven shields. Under these conditions negative capacitance settings did not affect spike amplitude or shape. Pep-neurons were tentatively identified by size, location, and electrophysiological properties (see Results). At the end of select experiments this identification was tested by measuring IR-Pep in extracts of individual neurons by RIA ( Pearson and Lloyd, 1990). Neurons were stained with fast green via an intracellular electrode, dissected in ice-cold 50%)propylene glycol:SO% ASW, and extracted in 0.02 M TFA at 100°C for 10 min.
RESULTS Pep Content of Pedal Nerves The distribution of IR-Pep in pedal nerves as measured by RIA was heavily concentrated in the three major nerves that innervate the foot ( p l , 8, and 9 ) , which contain 97% of all IR-Pep in pedal nerves (Table I ) . Therefore, the predominant destination of Pep synthesized in the pedal ganglia is the foot. HPLC-RIA analyses of extracts from the CNS and foot suggest that nearly all 1R-Pep in these tissues is associated with authentic Pep (Pearson and Lloyd. 1990).
Morphology of Pep-Neurons of the Pedal Ganglion Dorsal Band The dorsal band of Pep-neurons in the pedal ganglia runs diagonally from the pedal-pedal com-
Peptide Modulation uf Aplysia Locomotion
Pleural Ganglion
Conn.
pa pepneurons p9 Pep neurons
7
,
9
\\ \
Figure 1 Clustering of Pep-neurons that send axons down the same nerve. Diagrammatic representation of the right pedal ganglion showing the dorsal band of Pep-neurons. The Pep-neurons in the medial aspect of the band (cross-hatched circles) send their axons prcdominantly out p l . The lateral aspect of the band contains Pep-neurons (filled circles) that send their axons mainly out p9. Those Pep-neurons intermediate between the other 2 groups (stippled circles) mainly send their axons out p8. Open circles represent Pep-neurons that do not have axons in either p 1, p8, or p9.
missure to a region between the roots of the posterior tegumentary nerve (p5) and p9. This cluster contains more than 60 Pep-immunoreactive neurons. Cobalt backfilling of the pedal nerves that innervate the foot reveals that nearly every Pep-neuron in the dorsal band sends an axon out at least one of these three nerves (Fig. 1 ). In addition, the neurons across the band appear to be segregated into groups that send axons out the same nerve. Neurons in the medial aspect of the band send axons predominantly out p 1. Neurons in the lateral portion of the band send their axons mainly out p9 and the neurons betwecn these groups send axons out p8. Intracellular staining of individual Pep-neurons with Lucifer yellow revealed axons that divide extensively; axonal branching was observed within the ganglion near the Pep-neuron soma or outside the ganglion in pedal nerves. Axons from one neuron often go out every root or branch of one pedal nerve and a few Pep-neurons send axon branches out two nerves. Injection of Pep Produces Postural Changes
Animals injected with small quantities of Pep (0. I nmol/g wet weight, n = 4 ) showed subtle but con-
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sistently observed differences when compared to animals injected with the vehicle alone. Control animals underwent escape locomotion that lasted for several minutes after the injection. whereas animals receiving Pep showed a reduction in the extent of locomotion. Larger injections of Pep ( 1 nmol/g, R = 4) consistently produced dramatic changes in the animal's posture. The foot made poor contact with the substrate, especially in the tail region, and although peristaltic contractions of the foot were sometimes observed, the animals did not locomote effectively. In addition. the parapodia were flared and the entire gill was exposed. The initial effects of larger doses of Pep were very rapid in onset and were observed within 1-2 s after the injections. They were also short-lived as indicated by the return of locomotor behavior within 5 min after the injections. Even with the larger Pep injections it was difficult to distinguish the experimental and control animals by 10-15 min after the injection. Animals were also weighed periodically up to 3 h after the larger injections of Pep. Differences in weight greater than 1% were not observed in either the Pep-injected or control group at any time. If the injectcd Pep was uniformly distributed in the animal, 0.1 nmol/g weight would produce a concentration of 0.1 piM, and I nmol/g weight, a concentration of 1 pM. Presumably, concentrations in the hemocoel immediately after injection would be considerably higher but proteolysis would quickly lower these concentrations (see below). The very rapid effects of injected Pep are likely to be primarily actions on peripheral tissues, presumably, the foot itself. Pep would have to pass through the heart and out the arterial system that supplies the ensheathed ganglia in order to reach central neurons. Apl-ysiu has an open circulatory system with a very large blood volume ( 80% of body weight; Martin, Harrison, Huston, and Stewart, 1958). Of course. later effects of injected Pep might be due to central actions (Cooper, Krontiris-Litowitz. and Walters, 1989).
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Proteolysis of Pep by Blood and Foot Tissue
One possible explanation for the brief nature of the effects of injected Pep is that it might be broken down rapidly. To test for this, purified native Pep labelled in vivo with 35S-methioninewas incubated in ASW, fresh Aplysiu blood, or blood that had previously been boiled. Overall Pep-concentrations were about 1 pM in each case. Periodically, aliquots were taken and run on HPLC to determine the amount of label that remained as native
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Pep. Pep appeared to be relatively stable in ASW, but was broken down in fresh blood with a half-life of about 4 min (Fig. 2 ) . A second experiment using blood from a different animal also gave a half-life of 4 min. Stability of Pep in previously boiled blood was the same as in ASW suggesting that the breakdown in fresh blood was due to enzymatic proteolysis. Proteolysis was also observed when Pep was incubated with foot tissue. With a 10:1 ratio of ASW volume/foot tissue volume, the half-life was about 15 min. In a second similar experiment using foot tissue from a different animal, a half-life of approximately 20 min was obtained. Presumably, physiologically released Pep would be exposed to a much higher ratio of foot tissue and the half-life might be considerably shorter. The HPLC profile of labelled fragments produced by proteolysis was different for blood and foot tissue indicating that different complements of Pep-cleaving proteases must be present in the two tissues. It also indicates that the proteolysis by foot tissue was not due to blood that had been incompletely washed out of the tissue, and suggests that proteolysis by blood and foot would be additive and the half-life of released Pep might be very short indeed. Thus rapid proteolysis of Pep is likely to be one factor underlying the relatively brief effects of injected Pep and makes it unlikely that Pep normally functions as a hormone. Consistent with this conclusion, very low levels of IR-Pep were recovered from blood. RIA measurements from blood taken from 2 animals gave concentrations of 67 and 69 pM. The identity of the immunoreactive species in blood was not investigated further.
125
1
ASW
0
0
200
400
600
Incubation time (s)
Figure 2 Survival of Pedal peptide. Radiolabelled Pep was incubated in either sterile ASW or Aplysiu blood and aliquots were removed after select periods. The amount of label present in Pep was determined by HPLC and liquid scintillation counting. Pep was stable in ASW but had a half-life of about 4 rnin in blood.
""
OHz
6Hz
12Hz
24H z
HighK+
Stimulation
Figure 3 Recovery of IR-Pep following stirnulation of
pedal nerves. The foot was pinned flat and perfused with ASW plus proteolysis inhibitors. Stimulation periods were 20 min and the perfusate was collected and assayed for IR-Pep. The amount of IR-Pep recovcred increased with stimulation frequency and also increased in high K + ASW. Values are mean f S.E.M., n = 4.
Pep Is Released in a StimulationDependent Fashion IR-Pep was recovered from foot tissue perfusate following pedal nerve stimulation and following perfusion with ASW containing 100 m M KCI ( 10X normal). The amount of IR-Pep recovered increased with increasing stimulation frequency to a maximum amount of recovery at 24 Hz or with high K t ASW stimulation (Fig. 3 ) . However, the recovered IR-Pep represents less than 0.1'30 of the total in foot tissue. The recovery may underestimate actual release because, as indicated above. proteolysis of Pep by blood and foot tissue is extremely rapid. Attempts to inhibit this proteolysis by a variety of compounds proved only partially successful. Also, the perfusion of the foot, which transports the carriers into release sites and released Pep out into the bath, was compromised by the dissection procedure in which many major arteries were severed thereby allowing a path of low resistance for the perfusate to follow rather than perfuse evenly through the foot. Pep Modulates Foot Muscle Contractions The response of resting foot muscle to application of Pep was quite variable. High concentrations of Pep ( > l o p 6 M ) usually produced a dramatic decrease in tone followed later by an increase in spontaneous activity. Occasionally, even high concentrations of Pep had no observable effects on resting foot muscle contractile activity, so possible modulatory actions of Pep on evoked contractions were analyzed. Initially, foot muscle contractions were generated by extracellular stimulation of nerve p8. This nerve has the highest concentration
Peptide Modulation of Aplysia Locomotion
M
_I 100s
Peo
Figure 4 Effects of exogenous Pep on contractions evoked by stimulation of p8. The entire foot and tegument was pinned flat and a single pX nerve was stimulated. Longitudinal or lateral contractions from two applications of Pep in the same preparation are shown. 10 -6 M Pep ( 10 ml, solid bar) was perfused through the left pedal artery. These effects were reversed by washout.
of Pep of the pedal nerves (Table 1 ) and contains large Pep-immunoreactive axons ( Pearson and Lloyd, 1989). To minimize release of endogenous Pep, contractions were induced by a limited stimulation paradigm ( 5 stimuli in 200 ms at 60 s intervals). This paradigm produced contractions that were rapid in onset and relaxed slowly. Perfusion of Pep through the foot's arterial system had reliable effects on these contractions. There was a moderate increase in the amplitude of contrsctions and occasional double contractions were observed indicating an increase in excitability of the muscle. The most pronounced effect, however, was an increase in the rate of relaxation of each contraction. The effects of Pep on both longitudinal and lateral contractions were similar (Fig. 4 ) . Although threshold effects were sometimes observed at lo-' M , higher concentrations (> lo-' M ) of Pep were often required lo produce clearly observable modulation of foot contractions. Protease inhibitors were not used in these experiments, however, so thc concentrations of Pcp at receptors in the foot may be significantly lower than those applied. Because extracellular stimulation of p8 may release Pep, an analysis of the effects of exogenous Pep on foot muscle contractions produced by intracellular stimulation of pedal motor neurons was performed. Even sustained, high frequency stimulation of individual motor neurons produced smaller contractions than nerve stimulation, so spontaneous contractions were often of similar amplitude to driven contractions. To minimize spontaneous contractions, a reduced preparation
863
consisting of the small piece of foot innervated by p8 was used. In these experiments, the pedal ganglia were placed in a separate chamber superfused with low CaL+,high Mg2' ASW to inhibit synaptic activity. This largely prevented spontaneous firing in Pep-neurons. In these preparations, superfusion of Pep also produced small increases in contraction amplitude and a pronounced increase in the rate of relaxation of driven contractions (Fig. 5 ). The effects of Pep on two other neuromuscular preparations were also analyzed. The first preparation consisted of the columellar muscles, a pair of body wall muscles that run from head to tail and which are innervated primarily by the pedal ganglia via nerve p5. Analysis by RIA indicated that p5 had the lowest concentration of Pep of any of the pedal nerves. Furthermore, the columellar muscles themselves contained nearly 100-fold lowcr concentrations of Pep than did foot muscle (Pearson and Lloyd, 1990). Stimulation of p5 with the same parameters described above produce columellar muscle contractions that are rapid in onset and relax slowly. However, application of Pep up to A4 had no observable effects on these contractions ( N = 3 preparations), whereas serotonin application had marked modulatory effects on the contractions consisting of enhanced contraction amplitude and relaxation rate (data not shown). The second preparation consisted of identified buccal motor neurons B 15 and B 16 and the muscle (15 ) they inncrvate (Cohen, Weiss, and Kupfermann, 1978). Again, there is no evidence for significant innervation from Pep-neurons nor were there significant levels of Pep in the musclc (Lloyd, 1988; Pearson and Lloyd, 1990). Pep applied at levels up to A4 had no effects on this neuromuscular preparation even though the same muscles were sensitive to other modulatory peptides at concentrations below lo-' M [e.g., the small cardioactive peptides ( SCPB)]. These results suggest that the effects of Pep may be confined to muscles in which it is found. A similar segregation of effect4 was observed in the CNS, where neurons that received Pep innervation as evidenced by immunoreactive varicosities on their cell bodies, were much more sensitive to Pep than neurons lacking these varicosities ( Lloyd and Connolly, 1989). Properties of Pep-Neurons in the Pedal Ganglia Identification of Pep-neurons in the pedal ganglia was aided by the observation that each of these
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3
0.4
rnrn
MN
5 x 10-’M Pep
B
100s
0.4 mrn
4s
Figure 5 Effects of exogenous Pep on contractions driven by intracellular stimulation of a motor neuron ( M N ) . ( A ) Superfusion of 20 ml of 5 X M P e p (solid bar) over the surface of the foot caused an increase in the rate of relaxation of the driven contractions (Foot). ( B ) Superimposition of higher speed recording of the indicated contractions from A. Note the increase in relaxation rate caused by Pep. Records were triggered from the first spike in the motor neuron burst. The motor neuron in this experiment has not been identified. The persistant basal relaxation seen following Pep superfusion was consistently observed and is thought to be due to the enhanced relaxation rate of the contractions. The decline of the depolarizing afterpotential of the motor neuron was not a consistent observation.
neurons had similar physical and electrophysiological characteristics. In a medium-sized Aplysiu ( 100 g), the dorsal band contains more than 60 Pep-neurons with diameters between 120 and 160 pm. Pep-neurons could be identified in live ganglia by their location, large size, and pale appearance compared to other pedal neurons. In pedal ganglia from 2 animals, the positions of all the neurons on the dorsal surface that appeared, from visual criteria, to be Pep-neurons were plotted. Subsequent immunocytology revealed an absolute correlation between these putative Pep-neurons and the immunoreactive neurons. These results, and those obtained from RIA analysis (see below). confirm that Pep-neurons could be reproducibly identified in live ganglia. Pep-neurons typically had rest potentials near -45 mV and broad action potentials with pronounced shoulders (Fig. 6 ) . In one ganglion, stable recordings were obtained from about onethird of all the dorsal Pep-neurons and the mean spike duration (at half-maximal amplitude) was 6.7 f 1.7 ms (S.D.,n = 20) during low frequency firing ( 1 Hz). Spikes of other neurons on the dorsal surface of the pedal ganglia were of shorter
-
duration and smaller amplitude. For example. i n the same preparation the large identified motor neurons, P2 and P3, had spike durations from 3.5-5.0 ms without any sign of a shoulder. In these studies, and those described below, the
5 ins
Figure 6 Comparison of the large, broad spike of a Pep-neuron ( P N ) and the smaller, faster spike of a motor neuron ( M N ) from the dorsal surface of a pedal ganglion. These spikes arc typical of those observed repeatedly in the two populations of neurons.
Peptide Modulation of Aplysia Locomotion
amount of Pep in neuronal cell bodies was measured at the end of select experiments. Putative Pep-neurons, and in some instances motor neurons, were stained with fast green via an intracellular electrode, dissected manually from the ganglia, extracted, and Pep-measured by RIA. As expected, the amount of Pep per neuron varied with the diameter of the neuron. Values for IR-Pep per neuron were 0.6 1 f 0.1 1 pmol (S.E.M.; n = 9) for 100-120 pm cell bodies; 0.84 k 0.15 pmol ( n = 17) for 121-160 pm cells: and 1.86 k 0.87 pmol ( n = 4 ) for 161-1 80 pm cells. In addition, 13 motor neurons (including identified P2 and P3 motor neurons) were also tested and all were found to be negative in the RIA (
SUMMARY Pedal peptide (Pep) is a 15-amino-acid neuropeptide that is localized within the Aplysia central nervous system (CNS) predominantly to a broad band of neurons in each pedal ganglion. Pep-neurons were identified by intracellular staining and immunocytology or by radioimmunoassay ( R I A ) of extracts from identified neurons. RIA reveals that 97% of all Pep-like immunoreactivity (IR-Pep) in pedal nerves is found in the three nerves that innervate the foot. Nearly every Pep-neuron sends a n axon out at least one of these three nerves. Application of Pep to foot muscle causes an increase in the
amplitude and relaxation rate of contractions driven by nerve stimulation or intracellular stimulation of pedal motor neurons. The increase in relaxation rate was the predominant effect. Intracellular recording in “splitfoot” preparations reveals that Pep-neurons increase their overall firing rates and fire in bursts with each step during locomotion. Recovery of IR-Pep from foot perfusate following pedal nerve stimulation increases in a frequency-dependent fashion. Thus it appears that one function of Pep-neurons is to modulate foot muscle contractility during locomotion in Aplysia.
INTRODUCTION
dominant source for the Pep present in the foot are these immunoreactive neurons in the pedal ganglia. The foot is the primary effector organ for locomotion in Aplysia calijbrnica. It encompasses the entire ventral surface of the animal extending from the propodium and mouth to the tail. The foot is composed of a dorsal longitudinal muscle layer and a ventral transverse muscle layer (Kandel. 1979) and is primarily innervated by the anterior ( p l ), middle (p8), and posterior (p9) pedal nerves (Jahan-Parwar and Fredman, 1978). p l arises from four roots and innervates the anterior portion of the foot, p8 leaves the ganglion as one root but immediately branches and innervates the middle of the foot, and p9 also arises from a single root and innervates the posterior foot and the tail (Kandel. 1979). Locomotion involves pedal waves, which are peristaltic contractions of the entire width of the foot. The pedal waves in Apljfsia begin anteriorly and progress rearward lo propel the animal forward (Parker, 1917). Although Pep clearly has biological actions on a number of central neurons in Aplysia (Pearson and Lloyd, 1989), its role in the periphery, and
Pedal peptide (Pep) was recently isolated from Aplysia pedal ganglia and sequenced (Lloyd and Connolly, 1989). Immunocytology using antisera directed towards Pep suggested that the peptide was found widely throughout the central nervous system (CNS) and periphery of Aplysia, and it was heavily concentrated in the foot and in the pedal ganglia, which provide the primary central innervation of the foot (Pearson and Lloyd, 1989). Each of the paired pedal ganglia contains roughly 100 large Pep-immunoreactive neurons localized predominantly in a broad band running along the dorsal surface of each ganglion. Pep was transported in very large quantities down pedal nerves that innervate the foot and Pep was not synthesized in significant quantities by the foot itself (Pearson and Lloyd, 1989; 1990). Thus the preReceived February 6 . 1990, accepted April 16, 1990 Journal ofNeurobiology, Vol. 21, No. 6, pp. 858-868 (1990) Q 1990 John Wiley & Sons, Inc. CCC 0022-3034/90/060858-11$04.00 * To whom correspondence should be addressed.
858
Peptido Modulation of'Aplysia Locomotion particularly in the foot, has not been established. In the present study, we provide evidence that Pep is a locally acting transmitter that is involved in modulating motor-neuron-drivcn contractions during locomotion. Specifically. Pep enhances t h e amplitude and rate of relaxation of foot muscle contractions, Pep-neurons significantly increase their firing rate during locomotion, and Pep is recovered from foot tissue perfusate following pedal
nerve stimulation. METHODS Animals Aplysia cal(omicu (60- I50 gm) were obtained from Marinus, Inc.. maintained in circulating artificial sea water (ASW) tanks at 15"C, and fed dried seaweed at 3-day intervals.
Pedal Nerve Radioimrnunoassay Nine peripheral nerves leave each pedal ganglion and a single, unpaired nerve exits from the parapedal connective. The amount of Pep-like immunoreactivity (IRPep) contained in each was assayed. Animals were immobilized by isotonic MgClz injection, pedal nerves were identified, dissected, weighed, and cxtracted for I0 min at 100°C in 0.02 M trifluoroacetic acid (TFA). Aliquots of these samples were used in an RIA (Pearson and Lloyd, 1990).
Morphological Studies Cell bodies having axons in pl. p8, and p9 were visualized by cobalt chloride backfilling. Standard procedures were used (Hening, Walters, Carew, and Kandel, 1979). Lucifer yellow ( 1 % ) was ionophoresed into individual Pep-neurons in pedal ganglia.
Injection of Pep into Freely Behaving A plysia Synthetic Pep (Applied Biosystems) was taken up in 1 ml ASW and injected into the anterior hemocoel. Injections were either 0.1 or 1.0 nmol peptide/g animal weight. Control injections consisted of the vehicle alone. The behavior of the animals was periodically observed for 3 h after the injections. The injections and subsequent behavioral observations were done blind. In the case of the larger Pep injections, animals were also weighed before and periodically after the injections.
Proteolysis of Pep by Blood and Foot Tissue Labelled Pep was prepared by incubating pedal ganglia with "S-methionine and purifying the labelled peptide
859
through two modes of high-pressure liquid chromatography (HPLC) as described prcviously (Lloyd and Connolly, 1989). Lyophiliied labelled Pep was dissolved in either ASW or in blood taken from a cold-anesthetized animal (15 min at 4°C) and incubated at 22°C. For proteolysis experiments involving the foot. lyophilized labelled Pep was dissolved in stenlc ASW and incubated for 50 min with pieces of foot tissue (wet weight 20 mg) that had been washed in several changes of sterile ASW to remove blood. Aliquots of the samples were removed periodically and diluted into 500 ~1 0.02 M TFA spiked with 2 nmol synthetic Pep. This sample was heated to 100°C for 10 min, filtered, and run on HPLC as described previously (Lloyd and Connolly, 1989). The retention times of labelled fractions were determined by liquid scintillation counting.
-
Release of Pep from Foot Tissue ..lp/vsiawere immobilized by MgC12 injection and a dorsal incision made along the length of the animal. All internal organs and the central ganglia were removed. The vasculature was perfused with ASW containing 10 mg/l bovine serum albumin (BSA) and 10 mg/I bacitracin as carriers to improve Pep recovery. Other compounds that were tested but failed to significantly inhibit Pep proteolysis or improve Pep recovery were diprotin A (Peninsula Labs), antipain, aprotinin, chymostatin, E-64, leupeptin. N-carboxymethyl-phe-leu, 1,lO-phenanthroline. phenylmethylsulfonyl fluoride, pepstatin A, and thiorphan (all from Sigma) Suction electrodes were bilaterally placed on p8 and p9 nerves and stimulation intensity was adjusted to be supramaximal as judged by the amplitude of contractions. Initially, the bath contained 20 ml ASW plus carriers and an additional 20 ml of ASW plus carriers was perfused through the foot over a 40-min period (20-min stimulation and 20-min poststimulation washout). For high K + experiments, 20 ml ASW containing 100 m M KCI ( 10 X normal) was used as the stimulus. The entire 40-ml sample was removed and passed through a reverse-phase extraction cartridge ( Sep-Pak). Peptides were eluted with 3 ml 75% CH3CN, 0.01 M TFA, and aliquots used for RIA. Foot tissue was heated at 100°C in 30 ml 0.02 Ail TFA for 10 min and aliquots used in the RIA to dcterrnine total foot IR-Pep.
Blood Content of Pep Similar methods were used to measure 1R-Pep in the blood. Blood was drawn from locomoting animals that were subsequently cold-anesthetized ( I5 min at 2°C). The blood was immediately acidified by addition of 0.02 MTFA, heated to 100°C for 10 min, and centrifuged at 10,000 X g for 10 min. The supernatant was extracted and 1R-Pep measured by RIA as described above.
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Effects of Pep on Foot Contractions Two preparations were used. The first consisted of the entire tegument and foot of the animal with the viscera, buccal mass, and CNS removed. An animal was immobilized by MgC12 injection, and an incision was made from head to tail about 1 cm into the tegument from the left edge of the foot. The preparation was pinned flat and the left pedal artery cannulated and perfused with ASW. A suction electrode was used to stimulate nerve p8 with a paradigm consisting of 5 stimuli (I-1.5-ms pulses) in 200 ms at 60 s intervals. Stimulation intensity was supramaximal as judged from contraction amplitudes. Longitudinal or lateral contractions were measured with a displacement transducer (Bionix Isotonic). Synthetic Pep dissolved in ASW was applied through arterial perfusion. The second preparation consisted of a 1 X 3 cm section of the foot innervated by p8. Contractions were produced by intracellular stimulation of either identified (P2; Hening et al., 1979) or previously unidentified motor neurons. A pedal ganglion was pinned in a chamber with all nerves and connectives severed except p8, which was run through a Vaseline barrier to the foot muscle in a second chamber. The pedal ganglion was superfused with ASW containing 1 m M CaCI2 (0.1 X normal) and 110 m M MgC12 ( 2 X normal) to inhibit synaptic transmission while the foot was superfused with normal ASW. Two intracellular electrodes were used: one to inject current pulses (10 ms, 100-200 nA) to drive individual spikes, and the other to monitor membrane potential. Motor neurons were stimulated at 30 Hz for 3 s every 60 s. Experiments were carried out as described above except only longitudinal contractions were measured and the artery was not cannulated. Pep was applied by superfusion only into the sub-chamber containing the foot. Thus, the pedal ganglion was not exposed to Pep.
lntracellular Recording of Neuronal Activity During Locomotion A split foot preparation similar to that described pre-
viously (Hening et al., 1979; Wallers, Byrne, Carew, and Kandel, 1983) was used. Briefly, the animal was immobilized by MgClz injection and pinned dorsal side up in a Sylgard dish. A midline dorsal incision was made from the anterior edge of the parapodia to the head and the buccal mass, buccal ganglia, abdominal ganglia, and the anterior portion of the gut were removed. A midline longitudinal incision was then made in the anterior one-third of the foot. The pedal nerves and the circumesophageal-ring ganglia were pinned to a Sylgard platform and the right pedal ganglion was desheathed for intracellular recording. The abdominal artery was cannulated and perfused with ASW. Pedal waves were measured with a displacement transducer attached to the lateral edge of the foot. Locomotion was defined as waves of contractions starting simultaneously in each
Table 1 Distribution of Pep-Like Immunoreactivity in Pedal Nerves Pep-Like Immunoreactivity'
Pedal Nerve
nmol/g Wet Weight
Pl P2 P3 P4 P5 P(j P7 P8 P9 P10
62.4 f 38.4 1.7 f 0.3 3.5 1.4 7.5 0.2 0.6 2 0.2 3.8 f 0.6 10.2 -+ 1.6 88.9 f 23.5 73.3 f 22.3 0.8 f 0.0
* *
nmol/Nerve 0.71
* 0.21
0.01 k 0.00 0.01 k 0.00
*
0.01 0.00 0.01 IO.00 0.01 f 0.01 0.08 iz 0.02 1.03 t 0.15 1.39 t 0.06 0.00 f 0.00
' Duplicate aliquots from crude extracts were assayed by RIA (Pearson and Lloyd, 1990). Values are mean I S.E.M. half of the head and progressing backward and either occurred spontaneously or was initiated by electrical stimulation through electrodes implanted in the tail (Walters et al., 1983) or by application of NaCl crystals to the parapodia. Intracellular recordings were made using 5 MQ potassium acetate electrodes and modified Getting M5 amplifiers with driven shields. Under these conditions negative capacitance settings did not affect spike amplitude or shape. Pep-neurons were tentatively identified by size, location, and electrophysiological properties (see Results). At the end of select experiments this identification was tested by measuring IR-Pep in extracts of individual neurons by RIA ( Pearson and Lloyd, 1990). Neurons were stained with fast green via an intracellular electrode, dissected in ice-cold 50%)propylene glycol:SO% ASW, and extracted in 0.02 M TFA at 100°C for 10 min.
RESULTS Pep Content of Pedal Nerves The distribution of IR-Pep in pedal nerves as measured by RIA was heavily concentrated in the three major nerves that innervate the foot ( p l , 8, and 9 ) , which contain 97% of all IR-Pep in pedal nerves (Table I ) . Therefore, the predominant destination of Pep synthesized in the pedal ganglia is the foot. HPLC-RIA analyses of extracts from the CNS and foot suggest that nearly all 1R-Pep in these tissues is associated with authentic Pep (Pearson and Lloyd. 1990).
Morphology of Pep-Neurons of the Pedal Ganglion Dorsal Band The dorsal band of Pep-neurons in the pedal ganglia runs diagonally from the pedal-pedal com-
Peptide Modulation uf Aplysia Locomotion
Pleural Ganglion
Conn.
pa pepneurons p9 Pep neurons
7
,
9
\\ \
Figure 1 Clustering of Pep-neurons that send axons down the same nerve. Diagrammatic representation of the right pedal ganglion showing the dorsal band of Pep-neurons. The Pep-neurons in the medial aspect of the band (cross-hatched circles) send their axons prcdominantly out p l . The lateral aspect of the band contains Pep-neurons (filled circles) that send their axons mainly out p9. Those Pep-neurons intermediate between the other 2 groups (stippled circles) mainly send their axons out p8. Open circles represent Pep-neurons that do not have axons in either p 1, p8, or p9.
missure to a region between the roots of the posterior tegumentary nerve (p5) and p9. This cluster contains more than 60 Pep-immunoreactive neurons. Cobalt backfilling of the pedal nerves that innervate the foot reveals that nearly every Pep-neuron in the dorsal band sends an axon out at least one of these three nerves (Fig. 1 ). In addition, the neurons across the band appear to be segregated into groups that send axons out the same nerve. Neurons in the medial aspect of the band send axons predominantly out p 1. Neurons in the lateral portion of the band send their axons mainly out p9 and the neurons betwecn these groups send axons out p8. Intracellular staining of individual Pep-neurons with Lucifer yellow revealed axons that divide extensively; axonal branching was observed within the ganglion near the Pep-neuron soma or outside the ganglion in pedal nerves. Axons from one neuron often go out every root or branch of one pedal nerve and a few Pep-neurons send axon branches out two nerves. Injection of Pep Produces Postural Changes
Animals injected with small quantities of Pep (0. I nmol/g wet weight, n = 4 ) showed subtle but con-
861
sistently observed differences when compared to animals injected with the vehicle alone. Control animals underwent escape locomotion that lasted for several minutes after the injection. whereas animals receiving Pep showed a reduction in the extent of locomotion. Larger injections of Pep ( 1 nmol/g, R = 4) consistently produced dramatic changes in the animal's posture. The foot made poor contact with the substrate, especially in the tail region, and although peristaltic contractions of the foot were sometimes observed, the animals did not locomote effectively. In addition. the parapodia were flared and the entire gill was exposed. The initial effects of larger doses of Pep were very rapid in onset and were observed within 1-2 s after the injections. They were also short-lived as indicated by the return of locomotor behavior within 5 min after the injections. Even with the larger Pep injections it was difficult to distinguish the experimental and control animals by 10-15 min after the injection. Animals were also weighed periodically up to 3 h after the larger injections of Pep. Differences in weight greater than 1% were not observed in either the Pep-injected or control group at any time. If the injectcd Pep was uniformly distributed in the animal, 0.1 nmol/g weight would produce a concentration of 0.1 piM, and I nmol/g weight, a concentration of 1 pM. Presumably, concentrations in the hemocoel immediately after injection would be considerably higher but proteolysis would quickly lower these concentrations (see below). The very rapid effects of injected Pep are likely to be primarily actions on peripheral tissues, presumably, the foot itself. Pep would have to pass through the heart and out the arterial system that supplies the ensheathed ganglia in order to reach central neurons. Apl-ysiu has an open circulatory system with a very large blood volume ( 80% of body weight; Martin, Harrison, Huston, and Stewart, 1958). Of course. later effects of injected Pep might be due to central actions (Cooper, Krontiris-Litowitz. and Walters, 1989).
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Proteolysis of Pep by Blood and Foot Tissue
One possible explanation for the brief nature of the effects of injected Pep is that it might be broken down rapidly. To test for this, purified native Pep labelled in vivo with 35S-methioninewas incubated in ASW, fresh Aplysiu blood, or blood that had previously been boiled. Overall Pep-concentrations were about 1 pM in each case. Periodically, aliquots were taken and run on HPLC to determine the amount of label that remained as native
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Pep. Pep appeared to be relatively stable in ASW, but was broken down in fresh blood with a half-life of about 4 min (Fig. 2 ) . A second experiment using blood from a different animal also gave a half-life of 4 min. Stability of Pep in previously boiled blood was the same as in ASW suggesting that the breakdown in fresh blood was due to enzymatic proteolysis. Proteolysis was also observed when Pep was incubated with foot tissue. With a 10:1 ratio of ASW volume/foot tissue volume, the half-life was about 15 min. In a second similar experiment using foot tissue from a different animal, a half-life of approximately 20 min was obtained. Presumably, physiologically released Pep would be exposed to a much higher ratio of foot tissue and the half-life might be considerably shorter. The HPLC profile of labelled fragments produced by proteolysis was different for blood and foot tissue indicating that different complements of Pep-cleaving proteases must be present in the two tissues. It also indicates that the proteolysis by foot tissue was not due to blood that had been incompletely washed out of the tissue, and suggests that proteolysis by blood and foot would be additive and the half-life of released Pep might be very short indeed. Thus rapid proteolysis of Pep is likely to be one factor underlying the relatively brief effects of injected Pep and makes it unlikely that Pep normally functions as a hormone. Consistent with this conclusion, very low levels of IR-Pep were recovered from blood. RIA measurements from blood taken from 2 animals gave concentrations of 67 and 69 pM. The identity of the immunoreactive species in blood was not investigated further.
125
1
ASW
0
0
200
400
600
Incubation time (s)
Figure 2 Survival of Pedal peptide. Radiolabelled Pep was incubated in either sterile ASW or Aplysiu blood and aliquots were removed after select periods. The amount of label present in Pep was determined by HPLC and liquid scintillation counting. Pep was stable in ASW but had a half-life of about 4 rnin in blood.
""
OHz
6Hz
12Hz
24H z
HighK+
Stimulation
Figure 3 Recovery of IR-Pep following stirnulation of
pedal nerves. The foot was pinned flat and perfused with ASW plus proteolysis inhibitors. Stimulation periods were 20 min and the perfusate was collected and assayed for IR-Pep. The amount of IR-Pep recovcred increased with stimulation frequency and also increased in high K + ASW. Values are mean f S.E.M., n = 4.
Pep Is Released in a StimulationDependent Fashion IR-Pep was recovered from foot tissue perfusate following pedal nerve stimulation and following perfusion with ASW containing 100 m M KCI ( 10X normal). The amount of IR-Pep recovered increased with increasing stimulation frequency to a maximum amount of recovery at 24 Hz or with high K t ASW stimulation (Fig. 3 ) . However, the recovered IR-Pep represents less than 0.1'30 of the total in foot tissue. The recovery may underestimate actual release because, as indicated above. proteolysis of Pep by blood and foot tissue is extremely rapid. Attempts to inhibit this proteolysis by a variety of compounds proved only partially successful. Also, the perfusion of the foot, which transports the carriers into release sites and released Pep out into the bath, was compromised by the dissection procedure in which many major arteries were severed thereby allowing a path of low resistance for the perfusate to follow rather than perfuse evenly through the foot. Pep Modulates Foot Muscle Contractions The response of resting foot muscle to application of Pep was quite variable. High concentrations of Pep ( > l o p 6 M ) usually produced a dramatic decrease in tone followed later by an increase in spontaneous activity. Occasionally, even high concentrations of Pep had no observable effects on resting foot muscle contractile activity, so possible modulatory actions of Pep on evoked contractions were analyzed. Initially, foot muscle contractions were generated by extracellular stimulation of nerve p8. This nerve has the highest concentration
Peptide Modulation of Aplysia Locomotion
M
_I 100s
Peo
Figure 4 Effects of exogenous Pep on contractions evoked by stimulation of p8. The entire foot and tegument was pinned flat and a single pX nerve was stimulated. Longitudinal or lateral contractions from two applications of Pep in the same preparation are shown. 10 -6 M Pep ( 10 ml, solid bar) was perfused through the left pedal artery. These effects were reversed by washout.
of Pep of the pedal nerves (Table 1 ) and contains large Pep-immunoreactive axons ( Pearson and Lloyd, 1989). To minimize release of endogenous Pep, contractions were induced by a limited stimulation paradigm ( 5 stimuli in 200 ms at 60 s intervals). This paradigm produced contractions that were rapid in onset and relaxed slowly. Perfusion of Pep through the foot's arterial system had reliable effects on these contractions. There was a moderate increase in the amplitude of contrsctions and occasional double contractions were observed indicating an increase in excitability of the muscle. The most pronounced effect, however, was an increase in the rate of relaxation of each contraction. The effects of Pep on both longitudinal and lateral contractions were similar (Fig. 4 ) . Although threshold effects were sometimes observed at lo-' M , higher concentrations (> lo-' M ) of Pep were often required lo produce clearly observable modulation of foot contractions. Protease inhibitors were not used in these experiments, however, so thc concentrations of Pcp at receptors in the foot may be significantly lower than those applied. Because extracellular stimulation of p8 may release Pep, an analysis of the effects of exogenous Pep on foot muscle contractions produced by intracellular stimulation of pedal motor neurons was performed. Even sustained, high frequency stimulation of individual motor neurons produced smaller contractions than nerve stimulation, so spontaneous contractions were often of similar amplitude to driven contractions. To minimize spontaneous contractions, a reduced preparation
863
consisting of the small piece of foot innervated by p8 was used. In these experiments, the pedal ganglia were placed in a separate chamber superfused with low CaL+,high Mg2' ASW to inhibit synaptic activity. This largely prevented spontaneous firing in Pep-neurons. In these preparations, superfusion of Pep also produced small increases in contraction amplitude and a pronounced increase in the rate of relaxation of driven contractions (Fig. 5 ). The effects of Pep on two other neuromuscular preparations were also analyzed. The first preparation consisted of the columellar muscles, a pair of body wall muscles that run from head to tail and which are innervated primarily by the pedal ganglia via nerve p5. Analysis by RIA indicated that p5 had the lowest concentration of Pep of any of the pedal nerves. Furthermore, the columellar muscles themselves contained nearly 100-fold lowcr concentrations of Pep than did foot muscle (Pearson and Lloyd, 1990). Stimulation of p5 with the same parameters described above produce columellar muscle contractions that are rapid in onset and relax slowly. However, application of Pep up to A4 had no observable effects on these contractions ( N = 3 preparations), whereas serotonin application had marked modulatory effects on the contractions consisting of enhanced contraction amplitude and relaxation rate (data not shown). The second preparation consisted of identified buccal motor neurons B 15 and B 16 and the muscle (15 ) they inncrvate (Cohen, Weiss, and Kupfermann, 1978). Again, there is no evidence for significant innervation from Pep-neurons nor were there significant levels of Pep in the musclc (Lloyd, 1988; Pearson and Lloyd, 1990). Pep applied at levels up to A4 had no effects on this neuromuscular preparation even though the same muscles were sensitive to other modulatory peptides at concentrations below lo-' M [e.g., the small cardioactive peptides ( SCPB)]. These results suggest that the effects of Pep may be confined to muscles in which it is found. A similar segregation of effect4 was observed in the CNS, where neurons that received Pep innervation as evidenced by immunoreactive varicosities on their cell bodies, were much more sensitive to Pep than neurons lacking these varicosities ( Lloyd and Connolly, 1989). Properties of Pep-Neurons in the Pedal Ganglia Identification of Pep-neurons in the pedal ganglia was aided by the observation that each of these
864
Hull und Lloyd
3
0.4
rnrn
MN
5 x 10-’M Pep
B
100s
0.4 mrn
4s
Figure 5 Effects of exogenous Pep on contractions driven by intracellular stimulation of a motor neuron ( M N ) . ( A ) Superfusion of 20 ml of 5 X M P e p (solid bar) over the surface of the foot caused an increase in the rate of relaxation of the driven contractions (Foot). ( B ) Superimposition of higher speed recording of the indicated contractions from A. Note the increase in relaxation rate caused by Pep. Records were triggered from the first spike in the motor neuron burst. The motor neuron in this experiment has not been identified. The persistant basal relaxation seen following Pep superfusion was consistently observed and is thought to be due to the enhanced relaxation rate of the contractions. The decline of the depolarizing afterpotential of the motor neuron was not a consistent observation.
neurons had similar physical and electrophysiological characteristics. In a medium-sized Aplysiu ( 100 g), the dorsal band contains more than 60 Pep-neurons with diameters between 120 and 160 pm. Pep-neurons could be identified in live ganglia by their location, large size, and pale appearance compared to other pedal neurons. In pedal ganglia from 2 animals, the positions of all the neurons on the dorsal surface that appeared, from visual criteria, to be Pep-neurons were plotted. Subsequent immunocytology revealed an absolute correlation between these putative Pep-neurons and the immunoreactive neurons. These results, and those obtained from RIA analysis (see below). confirm that Pep-neurons could be reproducibly identified in live ganglia. Pep-neurons typically had rest potentials near -45 mV and broad action potentials with pronounced shoulders (Fig. 6 ) . In one ganglion, stable recordings were obtained from about onethird of all the dorsal Pep-neurons and the mean spike duration (at half-maximal amplitude) was 6.7 f 1.7 ms (S.D.,n = 20) during low frequency firing ( 1 Hz). Spikes of other neurons on the dorsal surface of the pedal ganglia were of shorter
-
duration and smaller amplitude. For example. i n the same preparation the large identified motor neurons, P2 and P3, had spike durations from 3.5-5.0 ms without any sign of a shoulder. In these studies, and those described below, the
5 ins
Figure 6 Comparison of the large, broad spike of a Pep-neuron ( P N ) and the smaller, faster spike of a motor neuron ( M N ) from the dorsal surface of a pedal ganglion. These spikes arc typical of those observed repeatedly in the two populations of neurons.
Peptide Modulation of Aplysia Locomotion
amount of Pep in neuronal cell bodies was measured at the end of select experiments. Putative Pep-neurons, and in some instances motor neurons, were stained with fast green via an intracellular electrode, dissected manually from the ganglia, extracted, and Pep-measured by RIA. As expected, the amount of Pep per neuron varied with the diameter of the neuron. Values for IR-Pep per neuron were 0.6 1 f 0.1 1 pmol (S.E.M.; n = 9) for 100-120 pm cell bodies; 0.84 k 0.15 pmol ( n = 17) for 121-160 pm cells: and 1.86 k 0.87 pmol ( n = 4 ) for 161-1 80 pm cells. In addition, 13 motor neurons (including identified P2 and P3 motor neurons) were also tested and all were found to be negative in the RIA (
