Motor Innervation of the Pharynx Levator Muscle of the Snail, Helix Pomatia: Physiological and Histological Properties M. PETERS Institut fur Zoologie, R WTH Aachen, 51 Aachen, West Germany Received December 14, 1977; revised April 7,1978
SUMMARY
(1) Motor innervation of the pharynx levator muscle of Helix pomatia was investigated with intracellular recording and axonal iontophoresis of cobalt chloride. (2) Muscle fibers respond to direct electrical stimulation of the muscle with active graded responses or non-overshooting spike potentials. (3) Each fiber is innervated via the external and internal lip nerves by several (mostly 3) excitatory nerve fibers each. Two types of EPSPs can be distinguished according to amplitude, duration, and facilitation. (4) Axonal CoC12-stainingvia an external lip nerve branch revealed many nerve fibers entering the muscle and branching there into a rich network of blebbed fibers of various diameters. INTRODUCTION
Much of what we know about molluscan neuromuscular junctions stems from investigations on lamellibranch preparations, namely the anterior byssus retractor muscle (ABRM) of Mytilus (see e.g., Twarog 1976). On the other hand there is comparatively little information about molluscan muscles other than the ABRM. Only in the last ten years have intracellular recordings been obtained from a few gastropod muscle preparations (e.g., Kater et al., 1971;Heyer et al. 1973; Carew et al. 1974; Banks, 1975; Orkand and Orkand, 1975; Weiss et al. 1975). In order to find properties which characterize molluscan muscles in general it would be desirable to obtain data on various muscles in different species. The present study was undertaken to investigate the motor innervation of the pharynx levator muscle of Helix pomatia. In contrast to the large body of knowledge which exists about individual central neurons in Helix, nothing is known about intracellular potential characteristics in muscles. The muscle which was chosen for this study is involved in feeding behavior and innervation is therefore interesting from a neuroethological point of view. The paired Journal of Neurobiology, Vol. 10, No. 2, pp. 137-152 (1979) 01979, John Wiley 81 Sons, Inc.
0022-3034/79/0010-0137$01.00
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muscles tilt the pharynx (=buccal-mass) forward and upwards. In addition this preparation allowed axonal staining of part of the motor innervation with CoC12, thus allowing comparison of electrophysiological and light-microscopical results. METHODS Adult snails (Helixpornatia) collected locally or obtained from a commercial supplier were used in all experiments. Hibernating snails were awakened a t least 1week before experimentation and maintained in glass vessels in the laboratory. They were fed daily on lettuce. All experiments were carried out a t room temperature. During dissection and throughout the experiments the preparation was bathed in a saline corresponding with the ionic composition of the blood of Helix aspersa as determined by Burton (1968). The saline had the following composition (mM): NaC1,51; CaClp, 10; KCI, 3.7; MgClp, 12.6; Glucose, 5; Tris, 5; pH, 7.4. The pharynx levator muscle (for position of the muscle see Trappmann 1916) was removed from the snail together with its nerves. Pieces of pharynx musculature and body wall, respectively, were left attached to the ends of it to allow easier handling. The muscle was then pinned down with stainless-steel needles in a chamber with a Sylgard (Dow Corning) covered floor for physiological experiments. To allow stable intracellular recordings it was necessary to fix the muscle a t the recording site. This was accomplished by holding the muscle from below with a bent suction electrode. The suction electrode also served for extracellular electrical stimulation of the muscle. Microelectrodes were pulled from filament capillaries of 1.5 mm outer diameter (Hilgenberg, Malsfeld, Germany) by a vertical puller modified for production of extremely fine electrode tips (Chowdhury, 1969). They were filled from the top with 2.5M KC1 solution and had resistances of 30-100 MQ. A WPI M 701 amplifier served as input stage. Recordings were photographed from the screen of a Tektronix 5103 N storage oscilloscope. Sometimes impalement of the cells was facilitated by vibration (Peters, 1977) of the electrode in a longitudinal direction. Stimulation of nerves was by fire-polished glass suction electrodes. For light microscopical demonstration of peripheral nerve endings axonal iontophoresis (Iles and Mulloney, 1971) of CoClp (Pitman et al., 1972) was applied. An oil gap electrode (Peters, 1976) served for this purpose. After cobalt-staining muscles were submitted to slight pressure under a coverslip to obtain a more flat and thin preparation for microscopical examination. For this reason the arrangement of nerve fibers in histological preparations is somewhat distorted. Either of three fix,atives, 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.6), ethanol-acetic acid 75:25, or 70% ethanol was then perfused over the preparation for several minutes. The muscles were then left in the same fixative for 1 h in the cold. They were then treated with a modification of Timm’s sulphide-silver stain (Tyrer and Bell, 1974) for whole mounts (Strausfeld and Obermayer, 1976) to intensify cobalt-filled profiles.
RESULTS
Intracellular Measurements Electrical properties of muscle fibers. Resting potentials in individual muscle fibers of the pharynx levator muscle ranged between 55 and 90 mV. Potentials lower than 60 mV tended to drift towards the zero level, suggesting that the membrane had been damaged by the microelectrode. There seemed to be some correlation between electrode resistance and value of the resting
MOTOR INNERVATION OF HELIX POMATIA
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potential, higher resistances (and supposedly smaller electrode tips) resulting in the registration of higher resting potentials. Most values, however, were in the range of 65 to 70 mV and remained stable for up to one hour in favorable cases. Recordings often showed spontaneously occurring “miniature” depolarizing potentials [Fig. l(a)], suggesting that the recording electrode was situated close to a neuromuscular synapse. On the other hand, upon stimulation of the lowest threshold motor nerve fibers EPSPs with a comparable amplitude were elicited (see below). So the possibility remains that these “miniature” EPSPs are not of the classical type but represent spontaneous firing of severed motor axons or even activity of peripheral motor nerve cells. Upon depolarizing electrical stimulation via the holding suction electrode muscle fibers showed two different types of membrane response: (a) a spike-like depolarization which did not overshoot the zero level [Fig. l(b)] and (b) a graded active membrane response [Fig. l(c)]. The amplitude of the spikes was variable even in one single cell upon repetitive stimulation. The spikes were subject to rapid fatigue and eventually, after some time, only responses of type (b) were elicited. Some fibers never showed spike responses a t all. It could not be decided, however, whether this indicates the existence of two kinds of muscle fibers with different membrane properties. If the duration of the stimulus was increased, repetitive discharges could often be observed [Fig. l(d)].
C
Fig. 1. Intracellular recording from pharynx levator muscle fibers. (a) Spontaneous “miniature” excitatory potentials. (b) Superimposed responses of muscle fiber upon depolarizing stimulation a t different voltages via holding suction electrode: with increasing stimulus amplitude the muscle fiber responds firstly with a graded depolarization and then with spikes. Note that because of the arrangement of stimulus and recording electrodes part of the stimulus current apparently flows through the recording electrode and produces a DC-artifact (arrows). (c) Graded active membrane responses upon depolarizing stimulation of the muscle with stimuli of increasing strength. Stimulus artifact indicated by arrows. (d) Repetitive regenerative membrane responses upon stimulation of the muscle of longer duration. [Calibrations: vertical bar, 10 mV; horizontal bar (a) 2 sec; (h) and (c) 100 msec; (d) 500 msec. Resting potentials: (b) 67 mV; (c) 60 mV; (d) 66 mV.]
140
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Responses to nerve stimulation The pharynx levator muscle can be supposed to be innervated via the internal lip nerve and a branch of the external lip nerve on anatomical grounds. Additional innervation via other nerves, however, has not been tested and thus can not be ruled out. External lip nerve. Excitatory potentials were elicited in muscle fibers of the pharynx levator muscle upon stimulation of the external lip nerve branch and may be classified as two types: (1) Stimulation of nerve fibers with the lowest thresholds evoke EPSPs of low amplitude (up to 4 mV), with a delay of about 50 msec [Fig. 2(a)] which show little facilitation at low frequencies of stimulation [Fig. 2(b)]. Facilitation after a single priming impulse only lasts about 10 sec (Fig. 3). At higher frequencies summation and facilitation become prominent [Fig. 2(b)] and depolarization finally may trigger one or several spike responses [Fig. 2(d)]. In this case also the spike mechanism is readily fatigued. After some cycles of stimulation with trains of impulses the response is first altered to a slow depolarization of low amplitude [Fig. 2(e)] and finally after the end of stimulation there is only a smooth decline of the membrane potential towards the resting level [Fig. 2(f)]. Some fibers apparently are innervated by two nerve fibers eliciting EPSPs of relatively low amplitude [Fig. 2(c)]. Stimulation of the nerve fiber with the lowest threshold results in EPSPs which show no facilitation at all. If stimulus strength is raised to the threshold of the next fiber, EPSPs as described formerly can be observed.
C
Fig. 2. Response of muscle fibers upon stimulation of motor fibers in the external lip nerve branch with lowest thresholds (type 1 EPSPs). (a) Single EPSP (facilitated) at high sweep speed to show delay between stimulus and response. (b) Superimposed traces showing summation of EPSPs evoked by repetitive stimulation a t different frequencies (1, 2.5, 5, 7.5, 10 pulses/sec). (c) Stimulation a t 2 (lower) and 2.5 (upper) volts (5/sec). Superimposed traces to show occurrence of two EPSPs of type 1 with different amplitudes. Fatigue of spike-mechanism: (d) Summed EPSPs upon repetitive stimulation (10/sec) cross the spike-trigger level. (e) After several cycles of stimulation with trains of impulses spike potentials fail leaving only a slight depolarizing wave. (f) Disappearance of this response upon further stimulation. [Calibrations: (vertical bar) 10 mV; (horizontal bar) (a) 50 msec; (h) and (c) 500 msec; (d)-(f) 1 sec. Resting potential: (d)-(f) 58 mV.] Nerve stimulus-duration throughout this and all following experiments: 1 msec.
MOTOR INNERVATION OF H E L I X POMATIA
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EPSP % Control
L
L
(nee) 1
2
3
4
5
6
7
8
Fig. 3. Time course of facilitation of type 1EPSPs. The external lip nerve branch was stimulated with pairs of pulses at different intervals. Amplitude of the test-EPSP in % of that of the conditioning EPSP is plotted against the interval between conditioning and test EPSP in sec. Each point from one measurement. Curve fitted by eye. Inset: typical recordings from the experiment used for construction of the diagram. [Calibrations: (vertical bar) 10 mV; (horizontal bar) 500, 200, 200 msec.]
(2) A second type of synaptic potential can be evoked at higher stimulus strength. These EPSPs show higher amplitudes and longer duration compared with those evoked by the lower threshold fibers [Fig. 4(a)]. Upon stimulation of the external lip nerve branch with pulses of increasing voltage steplike increases in EPSP-amplitude can be recorded [Fig. 4(b)],which suggests innervation by several motor axons. Usually two nerve fibers evoking EPSPs of type 2 can be found, but some muscle fibers show only one, others three responses of different amplitude. Amplitude and facilitation were variable from muscle to muscle and between fibers of one muscle. Upon repetitive stimulation the amplitude of the EPSPs even in one fiber often showed considerable variations [Fig. 4(c)]. This was the case particularly in preparations from animals which were awakened from hibernation in the winter-months of October to February. EPSPs and action potentials in these muscles were found to be fatigued more easily than in other preparations. If the response to repetitive stimulation was observed over some minutes, after an initial phase of facilitation [Figs. 4(d) and 5(b)] a progressive decline of EPSP-amplitude could be seen [Fig. 4(d)l.
PETERS
142
C
Fig. 4. Response of muscle fibers upon stimulation of external lip nerve branch at higher stimulus strengths (type 2 EPSPs). (a) Superimposed responses upon stimulation of lowest-threshold (type 1) nerve fiber (closed arrow, 3 V) and upon additional stimulation of higher threshold (type 2) fiber (open arrow, 8 V) to show different amplitudes and time courses. (both EPSPs facilitated). (b) Superimposed responses upon stimulation with increasing voltages (2, 2.4, 2.5, 2 X 2.6, 2 X 3, 3.5, 4 , s V). Two EPSPs of type 2 can be distinguished. Stimulation with twin pulses (3 secs interval) because facilitated EPSPs allow better discrimination between different amplitudes. (c) Responses upon repetitive stimulation (0.25/sec) to show variability of type 2 EPSPs at constant stimulus strength. (d) Fatigue of EPSPs upon repetitive stimulation (0.2/sec). After an initial phase of facilitation, EPSP-amplitude declines. [Calibrations: (vertical bar) 10 mV; horizontal bar: (a) El00 msec; (b) 1 sec; (c) 2 sec; (d) 15 sec.] EPSP % O f Control
400 500:
[
a
b
c
d
Fig. 5. Facilitation of type 2 EPSPs evoked by stimulation of external lip nerve branch. (a) Time course of facilitation. Stimulation with twin pulses at different intervals. Amplitude of the second EPSP in 96 of the first is plotted against the interval between conditioning and test impulse. Each point represents one measurement. Curve fitted by eye. (b) Cumulation of facilitation upon repetitive stimulation (0.2/sec). (c-e) Representative recordings from the experiment shown in (a). [Calibrations: (vertical bar) 10 mV; (horizontal bar) (b) 5 sec; (c)-(e) 2 sec.]
Facilitation of type 2 EPSPs after a single priming stimulus declines much slower than that of type 1potentials (Fig. 5) and thus facilitation adds up upon successive stimuli [Fig. 5(b)] even at low frequencies of stimulation. Only after 30-40 sec does the EPSP-amplitude reach the resting-level again. Because of this long-lasting facilitation 1 l/2 min a t least had to pass between each trial in a series of measurements. As it is difficult to hold a single muscle fiber for longer times, facilitation curves were constructed from one measurement for each value only.
MOTOR INNERVATION OF HELIX POMATIA
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Usually, because of the high degree of facilitation, only a few type 2 EPSPs suffice to depolarize the muscle fibers above the threshold for triggering one or several action potentials [Fig. 6(a)]. Often the response fatigued rapidly so that only a burst of spikes was evoked even with persistent repetitive stimulation [Fig. 6(b)]. In many fibers the response to nerve stimulation was somewhat atypical in that it resulted only in a very slow and smooth depolarization [Fig. 6(c)]. Active membrane responses of different amplitudes were frequently elicited even with stimulation at constant strength [Fig. 6(d)]. This may be due either to properties of the excitable membrane at the recording site or by invasion of that site by action potentials originating from different distances. Internal lip nerve. Motor innervation via the internal lip nerve closely resembles that described above: stimulation of the nerve fiber with the lowest threshold results in EPSPs of low amplitude [Fig. 7(a)] which show facilitation and summation [Fig. 7(b)] at higher frequencies (type 1EPSPs). Raising the stimulus amplitude results in a steplike increase of the amplitude of the EPSPs [Fig. 7(c)]. These EPSPs have a higher amplitude and a longer time course and therefore are also classified as type 2 EPSPs. Muscle fibers are mostly innervated by one fiber evoking type 1and two fibers eliciting type 2 EPSPs. Often “miniature” potentials were superimposed upon type 2 EPSPs with an initially high frequency which progressively declined in the falling phase [Fig. 7(d)]. Such a correlation of EPSPs and “miniature” potentials has also been found in chromatophore muscle fibers of the squid (Florey and Kriebel, 1969). In some preparations the whole response had an irregular shape and rather re-
c
d
Fig. 6. Active membrane responses evoked by stimulation of external lip-nerve branch with stimulus intensity above threshold for type 2 EPSPs. (a) Spike-potentials elicited by summating EPSPs. (b) Fatigue of synaptic depolarization and spike responses. (c) In this preparation stimulation of the external lip nerve resulted only in a smooth and slow depolarization which finally triggered a spike responses. (d) Active membrane responses of different amplitudes in one fiber. [Calibrations: (vertical bar) 10 mV; horizontal bar: (a) 2 sec; (b) 5 sec; (c) 500 msec; (d) 2 sec;] Resting potentials: (a) 67 mV; (b) 55 mV; (c) 55 mV; (d) 57 mV.
144
b
PETERS
c
Fig. 7. Stimulation of internal lip nerve. (a) Response to stimulation of lowest threshold nerve fiber (type 1) at higher sweep speed to show delay between stimulus (arrow) and response (EPSP facilitated). (b) Superimposed responses to repetitive stimulation a t different frequencies (0.5; 1; 2.5; 5; 7.5; lO/sec) to show summation of type 1-EPSPs. (c) Superimposed responses to stimulation with increasing voltages (7; 8; 8.2; 8.5; 8.75; 2 X 8,9; 10 V) to show innervation by two fibers of type 2. (d) Single type 2-EPSP with superimposed burst of “miniature” potentials. (e) Spike responses evoked by repetitive stimulation of internal lip nerve with stimulus strength above threshold for type 2 EPSPs. [Calibrations: (vertical bar) 10 mV; (horizontal bar) (a) 50 msec; (b) 500 msec; (12) 1 sec; (d) 500 msec; (e) 2 sec.]
sembled a burst of summated small potentials than one single EPSP of higher amplitude. Repetitive stimulation of the higher threshold fibers in the internal lip nerve often led to depolarization which reached the spike-trigger level after a few stimuli, as was the case also for the type 2 fibers in the external lip nerve branch [Fig. 7(e)]. Type 2 EPSPs evoked by stimulation of the internal lip nerve showed the same degree and time course of facilitation as those stemming from external lip nerve stimulation (Fig. 8). After a single conditioning stimulus EPSPs rose t o about 600% of control. EPSP amplitude returned to resting level only after 30-40 sec. Only in four preparations was there an indication of inhibitory synapses at stimulation of the external lip nerve branch: In two preparations stimulation at the lowest threshold evoked hyperpolarizing potentials of low (2-3 mV) amplitude. These disappeared after some cycles of stimulation which could have been due to the use of KC1 in the microelectrodes. In two other muscles upon raising the stimulus voltage above the threshold for type 2 EPSPs a sudden fall in EPSP amplitude could be observed. This process was steplike and threshold dependent and therefore may be taken as a sign that an inhibitory axon was stimulated.
Light microscopy of nerve fibers Axonal cobalt staining via the branch of the external lip nerve revealed that after entering the muscle the nerve split up into a lot of small nerves which
MOTOR INNERVATION OF HELIX POMATIA a
145
b
ESP % of Coatrol
t 600
500 '
400 '
d
300 200
c
loo
Fig. 8. Facilitation of type 2 EPSPs evoked by stimulation of the internal lip nerve. (a) Time course of facilitation. Stimulation with twin-pulses at different intervals. Amplitude of the second EPSP in % of the first is plotted against the interval between conditioning and test impulse. Each point represents one measurement. Curve fitted by eye. (b)-(e) Representative recordings from the experiment shown in (a). [Calibrations: (vertical bar) 10 mV; (horizontal bar) (b) 5 sec; (c)-(e) 2 sec.]
showed further extensive branching [Fig. 9(a)]. These branches were the origin of a rich network of blebbed fibers. Distinct regions seemed to be supplied by separate branches [Fig. 9(b)]. This may be a sign of several motor units. However, as single nerve fibers could not be resolved in the main nerves the possibility remains that there is extensive branching of one single nerve fiber. It may well be that only one fiber or one particular class of nerve fibers together with their motor endings has been stained by axonal iontophoresis. The blebbings had a very irregular shape [Figs. 9(c)-9(e)] and diameter. The kind of blebbed fiber shown in Figs. 9(c)-9(e) is most prominent in cobalt-stained nerves but smaller fibers were also regularly seen [Fig. 9(f)]. However, it could not be decided whether these were terminal ramifications of the fibers shown in Figs. 9(c)-9(e) or whether they represented a second type of neuromuscular junction. Axonal cobalt-staining via the internal lip nerve did not demonstrate nerve endings in the pharynx levator muscle which disagreed with physiological experiments. Only in 3 cases (out of about 20 preparations) could nerve fibers entering the pharynx levator be seen. The reason for this may have been failure of the staining method in this case, the external lip nerve branch offering perhaps a preparation more suitable for cobalt staining of motor nerve endings than other nerves. However a network of blebbed fibers comparable to those in the pharynx levator muscle was stained in the levator labii muscle which is also supplied by the internal lip nerve, indicating that it is possible to fill peripheral nerve endings via the internal lip nerve. Another possibility may be indirect innervation by peripheral motoneurones.
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Fig. 9. Axonal cobalt staining of peripheral nerve endings via the branch of the external lip nerve. (a) Whole mount of a pharynx levator muscle showing the external lip nerve branch (arrow) which enters the muscle and branches there. (b) Typical pattern of innervation by blebbed nerve fibers. The figure was drawn from micrographs taken a t different depths of focus. (The diameter of the blebbed fibers is not in scale.) Fibers leaving the main nerve trunk seem to innervate distinct regions of the muscle. It has to be kept in mind, however, that the arrangement of fibers is somewhat distorted due to the procedure for preparation of whole mounts described in the methods section. (c) and (d) Blebbed fibers as seen most frequently in the pharynx levator muscle. (e) Drawing of same fiber type as shown in (c) and (d) obtained in the same manner as (b). (f) Very thin fiber with smaller blebbings.
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DISCUSSION
The electrical properties of muscle fibers in the pharynx levator muscle are similar to those measured in other molluscan muscles. Properties of muscle fibers and neuromuscular junctions in several mollusc muscles are listed in Table 1 to allow easy comparison of the pharynx levator muscle of Helix with other preparations. From this it can be seen that the resting potential, in most cases 65-70 mV, falls well within the range measured in other muscles. The ability to elicit a spike response seems to be widespread in molluscan muscle fibers also. The pharynx levator muscle has been found to receive excitatory innervation from two nerves by several nerve fibers. By stimulation of the fibers with the lowest thresholds EPSPs of low amplitude are elicited in the muscle fibers. At higher stimulus strengths EPSPs show higher amplitudes and longer time courses. The amplitude of these EPSPs rises in several discrete steps upon raising stimulus intensity, suggesting multiple innervation of the muscle fibers. Multiple innervation seems to be quite common in mollusc muscles as judged from histological and electrophysiological investigations. Double innervation of collumellar muscle fibers in Helix by two different nerve fibers has been claimed after methylene blue staining (Rochling, 1928, Ramsay, 1940). Several nerve profiles making close contact with single muscle fibers have been seen frequently in electron micrographs (e.g., Rogers, 1968;Schlote, 1963;Florey and Kriebel, 1969; McKenna and Rosenbluth, 1973). Partly they contain different populations of vesicles which has been taken as an indication of different transmitter substances. Electrophysiological evidence for excitatory innervation by more than one nerve fiber has been obtained from a variety of muscles (see Table 1). Whereas type 1EPSPs in the pharynx levator resemble excitatory synaptic potentials in other mollusc muscles according to time course and decay time of facilitation, type 2 EPSPs last much longer and decay of facilitation is much slower also. There seems to be a broad range of complexity in the motor innervation of molluscan fibers. Whereas the posterior jugalis muscle of Helisoma is controlled by only one single motoneurone (Kater et al., 1971),in the accessory radula closer muscle of Aplysia each fiber receives excitatory innervation from at least 4 identified motor neurones in the buccal ganglion (Weiss et al., 1975). The functional meaning of the convergence of 6 excitatory nerve fibers on one muscle fiber in the pharynx levator remains to be determined. However, it may be possible that the axons in the internal and external lip nerve are only branches of common motoneurones. Such multiple branching is known from several gastropod neurons as e.g., the serotonergic metacerebral giant cell of Helix (Pentreath, 1976). In Aplysia this cell has a modulatory effect on muscle EPSPs evoked by stimulation of motoneurones (Weiss et al., 1975, 1978). It can not be excluded that a similar process takes place in the pharynx levator muscle. In the experimental procedure used to determine the number of axons innervating a single muscle fiber, stimulus intensity was raised progressively and
(6)g
(5)f
(4)e
(3)d
(2)~
(1)b
Mytilus edulis ABRM Aeyuipecten gibbus Aequipecten irradians adductor muscle Spisula solidissirnu incurrent siphonal valve muscle Rapana thornasiana radula-protractor muscle (sucrose gap) Helisoma triooluis (a) columellar muscle (b) posterior jugalis muscle Aplysia californica gill-muscle
50-75 mV
up to 47 mVa mean 27 mV
45-61 mV
up to 80 mVa
mean 56 mV
55-72 mV
Resting potential
+ graded or
+
overshooting (b) overshooting
(a)
+sometimes overshooting
+non overshooting, often graded +sometimes overshooting
Action potential
3 identified excitatory motoneurones
(a) single excitatory
multiple excitatory
multiple excitatory
multiple excitatory multiple excitatory
Innervation of muscle fibers (units)
mseca LDG2: ca150 mseca
LDGI: ca300
200 msec
ca 100 mseca
ca 50 m s e e
500 msec
EPSP
Half-decay time of
TABLE 1 Electrical Properties of Muscle Fibers and Neuromuscular Junctions in Various Mollusc Muscles
+
+
-
+
Facilitation
Aplysia californica lower extrinsic protractor
Loligo opalescens Chromatophore muscles Helix pornatia pharynx levator muscle
(8)i
(9)J
a
55-90 mV most at 65 mV
up to 50 mVa
60-80 mV
75f8mV
Values taken from figures in the cited papers. Twarog, 1967. De Forrest Mellon, Jr., 1968. Prior, 1976. Kobayashi, 1972.
(10)
Aplysia californica accessory radula closer muscle
(7)h
typically 6 excitatory axons via 2 nerves, two types of EPSPs (1and 2)
ca 200 m s e e
type 1 ca 300 msec type 2 ca 2 sec
ca 50 mseca
100-200 mseca
B16
B15
Kater et al., 1971; Heyer et al., 1973. Carew et al., 1974; Jacklet and Rine, 1977. Weiss et al., 1975; Cohen et al., 1978, Weiss et al., 1978. Orkand and Orkand, 1975; Banks, 1975. J Florey and Kriebel, 1969.
overshooting
+ non
+sometimes overshooting
4 identified excitatory motoneurones 1 modulatory typically 4 excitatory axons via 2 nerves 1 inhibitory multiple excitatory
+type 2: long lasting (30-50 sec)
+type 1: short lasting (ca 10 sec)
rarely
+
+Bls: large +BIG: small
150
PETERS
the resulting alterations in EPSP amplitudes were observed. Upon raising stimulus strength more fibers are recruited according to their threshold and are stimulated in addition to lower threshold fibers. Thus the highest EPSP in a muscle fiber generally results from the synchroneous activation of 3-4 nerve fibers. It can be speculated then that the properties of the type 2 EPSPs result from a modulatory pre- or postsynaptic effect of higher threshold axons. Some of the properties of the synaptic potentials may be explained by the assumption, that the muscle contains several motor units which are coupled electrically via the muscle fibers. Multiple innervation by axons then might hold true for a bundle of coupled muscle fibers rather than a single fiber, each single fiber receiving perhaps only double or triple innervation. There is some evidence for electrical coupling between smooth muscle fibers in molluscs. Nexal junctions between muscle fibers can be seen with the electron miscroscope in the ABRM (Twarog et al., 1973; Gilloteaux, 1976) as well as in the lower extrinsic protractor muscle in Aplysia (Orkand and Orkand, 1975). In both cases electrical coupling between muscle cells has also been shown by electrophysiological methods. Similar results have been obtained from chromatophore muscle fibers of Loligo (Florey and Kriebel, 1969) and the accessory radula closer muscle of Aplysia (Cohen et al., 1978). Electrical coupling between muscle cells would e.g., explain the very slow rise of the EPSPs which occurred frequently. An alternative explanation could he the release of a transmitter from nerve terminals some distance away from the receptive membrane, thus introducing a diffusional delay. Since I did not succeed in impaling two cells simultaneously the question of electrotonic coupling is unresolved as yet. The electrophysiological results suggest multiple innervation of the muscle fibers of the pharynx levator muscle. Although the results of cobalt-staining support this idea for the muscle as a whole, there was no definite histological proof of innervation by two types of nerve fibers as has been described by Gilloteaux (1972) in the ABRM of Mytilus with osmium-zinc-iodide staining. Cobailtstaining via the external lip nerve revealed a lot of nerve branches entering the muscle. These were the origin of a rich network of fine blebbed fibers. Fibers of this kind have also been seen after methylene blue staining in the penis retractor muscle (Foh and Bogusch, 1969) and the columellar muscle (Rochling, 1928) as well as in the heart of Helix (Cottrell and Osborne, 1969). After formaldehyde treatment for histochemical demonstration of catecholamines varicose fibers were seen in the penis retractor muscle of Helix (Foh and Bogusch, 1969) and in the ABRM of Mytilus (McKenna and Rosenbluth, 1973). Nerve endings stained with osmium-zinc-iodide in the ABRM (Gilloteaux, 1972) also had a similar appearance. So these varicosities are probably not an artifact introduced by the procedure of cobalt staining. In all cases mentioned above the varicose fibers have been supposed to be motor. The blebbings, then, may represent sites of transmitter release as suggested for vertebrate autonomic nerve fibers. However, it can not be ruled out that some of the stained fibers in the pharynx levator muscle were branches of sensory cells. In electron microscopical pictures of various mollusc muscles presumed neu-
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romuscular junctions do not exceed a diameter of approximately 1.5 ym (e.g., Rogers, 1968;Florey and Kriebel, 1969;McKenna and Rosenbluth, 1973;Wabnitz, 1976). This corresponds well with the fine blebbing in the pharynx levator muscle which are seen most frequently. However, there were also thickenings of the nerve fibers, with diameters reaching up to 5 ym. There may be several sources of error in the interpretation of the results of Co-staining which could be the reason for this discrepancy: (1)Co may induce swelling of the axons and thus dimensions may be distorted. (2) Fine nerve branches may be misinterpreted as single nerve fibers. (3) Glial cells may be stained together with the axon due to leakage of Co, the large blebbings thus representing glial cell bodies rather than expansions of an axon. I hope that electron-microscopicaland pharmacological investigations, which are under way now, will answer some of the questions posed. I wish t o thank I. Holtkamp for technical assistance. REFERENCES BANKS,F. W. (1975). Inhibitory transmission a t a molluscan neuromuscular junction. J. Neurobiol. 6: 429-433. BURTON,R. F. (1968). Ionic balance in the blood of Pulmonata. Comp. Biochern. Physiol. 25: 509-516. H., RUBINSON,K., and KANDEL,E. R. (1974). Physiological and bioCAREW,J. I., PINSKER, chemical properties of neuromuscular transmission between identified motoneurons and gill muscle in Aplysia. J. Neurophysiol. 27: 1020-1040. T. K. (1969). Techniques of intracellular microinjection. In Glass Microelectrodes, CHOWDHURY, M. Lavalee, 0. F. Schanne, and N. C. HBbert, Eds, Wiley and Sons, New York, pp. 404-423. I. (1978). Motor control of buccal muscles in A p COHEN,I. L., WEISS,K. R., and KUPFERMANN, lysia. J. Neurophysiol. 41: 157-180. COTTRELL,G. A. and OSBORNE,N. (1969). A neurosecretory system terminating in the Helix heart. Comp. Biochem. Physiol. 2 8 1455-1459. MELLON,JR. (1968). Junctional physiology and motor nerve distribution in the fast DE FOREST adductor muscle of the scallop. Science 160: 1018-1020. FLOREY,E. and KRIEBEL,M. E. (1969). Electrical and mechanical responses of chromatophore muscle fibers of the squid, Loligo opalescens, to nerve stimulation and drugs. Z. Physiol. 65: 98-130. FOH, E. and BOGUSCH,G. (1969). Die Nervennetze im Penisretraktor von Helix pomatia. Z. Zellforsch. 93: 439-446. GILLOTEAUX,J. (1972). Innervation of the anterior byssal retractor muscle in Mytilas edulis L. Z. Zellforsch. 124: 204-216. GILLOTEAUX,J. (1976). Intercellular connections of a smooth muscle: Ultrastructure of the anterior byssal retractor muscle (ABRM) of Mytilus edulis L (Mollusca Pelecypoda). Cytobiol. 12: 457-472. HEYER,C. B., KATER,S. B., and KARLSSON,K. L. (1973). Neuromuscular systems in molluscs. Am. Zool. 13: 247-270. ILES, J. F. and MULLONEY,B. M. (1971). Procion yellow staining of cockroach motor neurones without the use of microelectrodes. Brain Res. 3 0 397-400. JACKLET,J . W. and RINE, J . (1977). Facilitation a t neuromuscular junctions: Contribution to habituation and dishabituation of the Aplysia gill withdrawal reflex. Proc. Natl. Acad. Sci. 74: 1267-1271. J. P. (1971). Neuromuscular transmission in the gastropod KATER,S. B., HEYER,C. and HEGMANN, mollusc Helisoma triuoluis: Identification of motoneurons. 2. Physiol. 74: 127-139.
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MCKENNA,0. C. and ROSENBLUTH,I. (1973). Myoneural and intermuscular junctions in a molluscan smooth muscle. J. Ultrastruct. Res. 4 2 434-450. P. M. and ORKAND,R. K. (1975). Neuromuscular junctions in the buccal mass of Aplysia: ORKAND, Fine structure and electrophysiology of excitatory transmission. J . Neurobiol. 6: 531-548. PENTREATH,V. W. (1976). Ultrastructure of the terminals of an identified 5-hydroxytryptamine-containing neurone marked by intracellular injection of radioactive 5-hydroxytryptamine. J. Neurocytol. 5: 43-51. PETERS, M. (1976). Cobalt staining of motor-nerve endings in the locust (Locusta rnigratoria). Experientia 32: 264-265. PETERS, M. (1977). Innervation of the ventral diaphragm of the locust (Locusta migratoria). J. Exp. Biol. 69: 23-32. PITMAN, R. M., TWEEDLE, C. A., and COHEN,M. J. (1972). Branching of central neurons: Intracellular cobalt injection for light and electron microscopy. Science, 176: 412-414. PRIOR,D. J. (1975). A study of the electrophysiological properties of the incurrent siphonal valve muscle of the surf clam, Spisula solidissirno. Cornp. Biochem, Physiol. 52A, 607-610. ROCHLING,E. (1928). Der Kolumellarmuskel von Helix porn. und seine Beziehung zur Schale. 2. Wiss. Zool. 119: 485-525. ROGERS,D. C. (1968). Fine structure of smooth muscle and neuromuscular junctions in the optic tentacles of Helix aspersa and Lirnax flauus. Z. Zellforsch. 89: 80-94. RAMSAY,J. A. (1940). A nerve-muscle preparation from the snail. J . Exp. Biol. 17: 96-115. SCHLOTE, F. W. (1963). Neurosekretartige Grana in den peripheren Nerven und in den NervMuskel Verbindungen von Helix pornatia. Z. Zellforsch. 60: 325-347. M. (1976). Resolution of intraneuronal and transsynaptic STRAUSFELD, N. J. and OBERMAYER, migration of cobalt in the insect visual and central nervous systems. J . Cornp. Physiol. 110: 1-12. W. (1916). Die Muskulatur von Helix pornatia L. Z. Wiss. 2001.115: 489-585. TRAPPMANN, TWAROG,B. M. (1967). Excitation in mytilus smooth muscle. J. Physiol. 192: 857-868. TWAROG,B. M., DEWEY,M. M., and HIDAKA,T. (1973). The structure of mytilus smooth muscle and the electrical constants of the resting muscle. J . Cen. Physiol. 61: 207-221. TWAROG,B. M. (1976). Aspects of smooth muscle function in molluscan catch muscle. Physiol. Reu. 56: 829-838. TYRER, N. M. and BELL, E. M. (1974). The intensification of cobalt-filled neurone profiles using a modification of Timm’s sulphide-silver method. Brain Res. 73: 151-155. WABNITZ,R. (1976). Mechanical and electromyographic study of the penis retractor muscle (PRM) of Helix pornatia. Cornp. Biochern. Physiol. 5 5 A 253-259. I. (1975). Potentiation of muscle contraction: a WEISS, K. R., COHEN,J., and KUPFERMANN, possible modulatory function of an identified serotonergic cell in Aplysia. Brain Res. 9 9 381386. I. (1978). Modulatory control of buccal musculature WEISS,K. R., COHEN,I. L., and KUPFERMANN, by a serotonergic neuron (metacerebral cell) in Aplysia. J. Neurophysiol. 41: 181-203.
SUMMARY
(1) Motor innervation of the pharynx levator muscle of Helix pomatia was investigated with intracellular recording and axonal iontophoresis of cobalt chloride. (2) Muscle fibers respond to direct electrical stimulation of the muscle with active graded responses or non-overshooting spike potentials. (3) Each fiber is innervated via the external and internal lip nerves by several (mostly 3) excitatory nerve fibers each. Two types of EPSPs can be distinguished according to amplitude, duration, and facilitation. (4) Axonal CoC12-stainingvia an external lip nerve branch revealed many nerve fibers entering the muscle and branching there into a rich network of blebbed fibers of various diameters. INTRODUCTION
Much of what we know about molluscan neuromuscular junctions stems from investigations on lamellibranch preparations, namely the anterior byssus retractor muscle (ABRM) of Mytilus (see e.g., Twarog 1976). On the other hand there is comparatively little information about molluscan muscles other than the ABRM. Only in the last ten years have intracellular recordings been obtained from a few gastropod muscle preparations (e.g., Kater et al., 1971;Heyer et al. 1973; Carew et al. 1974; Banks, 1975; Orkand and Orkand, 1975; Weiss et al. 1975). In order to find properties which characterize molluscan muscles in general it would be desirable to obtain data on various muscles in different species. The present study was undertaken to investigate the motor innervation of the pharynx levator muscle of Helix pomatia. In contrast to the large body of knowledge which exists about individual central neurons in Helix, nothing is known about intracellular potential characteristics in muscles. The muscle which was chosen for this study is involved in feeding behavior and innervation is therefore interesting from a neuroethological point of view. The paired Journal of Neurobiology, Vol. 10, No. 2, pp. 137-152 (1979) 01979, John Wiley 81 Sons, Inc.
0022-3034/79/0010-0137$01.00
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muscles tilt the pharynx (=buccal-mass) forward and upwards. In addition this preparation allowed axonal staining of part of the motor innervation with CoC12, thus allowing comparison of electrophysiological and light-microscopical results. METHODS Adult snails (Helixpornatia) collected locally or obtained from a commercial supplier were used in all experiments. Hibernating snails were awakened a t least 1week before experimentation and maintained in glass vessels in the laboratory. They were fed daily on lettuce. All experiments were carried out a t room temperature. During dissection and throughout the experiments the preparation was bathed in a saline corresponding with the ionic composition of the blood of Helix aspersa as determined by Burton (1968). The saline had the following composition (mM): NaC1,51; CaClp, 10; KCI, 3.7; MgClp, 12.6; Glucose, 5; Tris, 5; pH, 7.4. The pharynx levator muscle (for position of the muscle see Trappmann 1916) was removed from the snail together with its nerves. Pieces of pharynx musculature and body wall, respectively, were left attached to the ends of it to allow easier handling. The muscle was then pinned down with stainless-steel needles in a chamber with a Sylgard (Dow Corning) covered floor for physiological experiments. To allow stable intracellular recordings it was necessary to fix the muscle a t the recording site. This was accomplished by holding the muscle from below with a bent suction electrode. The suction electrode also served for extracellular electrical stimulation of the muscle. Microelectrodes were pulled from filament capillaries of 1.5 mm outer diameter (Hilgenberg, Malsfeld, Germany) by a vertical puller modified for production of extremely fine electrode tips (Chowdhury, 1969). They were filled from the top with 2.5M KC1 solution and had resistances of 30-100 MQ. A WPI M 701 amplifier served as input stage. Recordings were photographed from the screen of a Tektronix 5103 N storage oscilloscope. Sometimes impalement of the cells was facilitated by vibration (Peters, 1977) of the electrode in a longitudinal direction. Stimulation of nerves was by fire-polished glass suction electrodes. For light microscopical demonstration of peripheral nerve endings axonal iontophoresis (Iles and Mulloney, 1971) of CoClp (Pitman et al., 1972) was applied. An oil gap electrode (Peters, 1976) served for this purpose. After cobalt-staining muscles were submitted to slight pressure under a coverslip to obtain a more flat and thin preparation for microscopical examination. For this reason the arrangement of nerve fibers in histological preparations is somewhat distorted. Either of three fix,atives, 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.6), ethanol-acetic acid 75:25, or 70% ethanol was then perfused over the preparation for several minutes. The muscles were then left in the same fixative for 1 h in the cold. They were then treated with a modification of Timm’s sulphide-silver stain (Tyrer and Bell, 1974) for whole mounts (Strausfeld and Obermayer, 1976) to intensify cobalt-filled profiles.
RESULTS
Intracellular Measurements Electrical properties of muscle fibers. Resting potentials in individual muscle fibers of the pharynx levator muscle ranged between 55 and 90 mV. Potentials lower than 60 mV tended to drift towards the zero level, suggesting that the membrane had been damaged by the microelectrode. There seemed to be some correlation between electrode resistance and value of the resting
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potential, higher resistances (and supposedly smaller electrode tips) resulting in the registration of higher resting potentials. Most values, however, were in the range of 65 to 70 mV and remained stable for up to one hour in favorable cases. Recordings often showed spontaneously occurring “miniature” depolarizing potentials [Fig. l(a)], suggesting that the recording electrode was situated close to a neuromuscular synapse. On the other hand, upon stimulation of the lowest threshold motor nerve fibers EPSPs with a comparable amplitude were elicited (see below). So the possibility remains that these “miniature” EPSPs are not of the classical type but represent spontaneous firing of severed motor axons or even activity of peripheral motor nerve cells. Upon depolarizing electrical stimulation via the holding suction electrode muscle fibers showed two different types of membrane response: (a) a spike-like depolarization which did not overshoot the zero level [Fig. l(b)] and (b) a graded active membrane response [Fig. l(c)]. The amplitude of the spikes was variable even in one single cell upon repetitive stimulation. The spikes were subject to rapid fatigue and eventually, after some time, only responses of type (b) were elicited. Some fibers never showed spike responses a t all. It could not be decided, however, whether this indicates the existence of two kinds of muscle fibers with different membrane properties. If the duration of the stimulus was increased, repetitive discharges could often be observed [Fig. l(d)].
C
Fig. 1. Intracellular recording from pharynx levator muscle fibers. (a) Spontaneous “miniature” excitatory potentials. (b) Superimposed responses of muscle fiber upon depolarizing stimulation a t different voltages via holding suction electrode: with increasing stimulus amplitude the muscle fiber responds firstly with a graded depolarization and then with spikes. Note that because of the arrangement of stimulus and recording electrodes part of the stimulus current apparently flows through the recording electrode and produces a DC-artifact (arrows). (c) Graded active membrane responses upon depolarizing stimulation of the muscle with stimuli of increasing strength. Stimulus artifact indicated by arrows. (d) Repetitive regenerative membrane responses upon stimulation of the muscle of longer duration. [Calibrations: vertical bar, 10 mV; horizontal bar (a) 2 sec; (h) and (c) 100 msec; (d) 500 msec. Resting potentials: (b) 67 mV; (c) 60 mV; (d) 66 mV.]
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Responses to nerve stimulation The pharynx levator muscle can be supposed to be innervated via the internal lip nerve and a branch of the external lip nerve on anatomical grounds. Additional innervation via other nerves, however, has not been tested and thus can not be ruled out. External lip nerve. Excitatory potentials were elicited in muscle fibers of the pharynx levator muscle upon stimulation of the external lip nerve branch and may be classified as two types: (1) Stimulation of nerve fibers with the lowest thresholds evoke EPSPs of low amplitude (up to 4 mV), with a delay of about 50 msec [Fig. 2(a)] which show little facilitation at low frequencies of stimulation [Fig. 2(b)]. Facilitation after a single priming impulse only lasts about 10 sec (Fig. 3). At higher frequencies summation and facilitation become prominent [Fig. 2(b)] and depolarization finally may trigger one or several spike responses [Fig. 2(d)]. In this case also the spike mechanism is readily fatigued. After some cycles of stimulation with trains of impulses the response is first altered to a slow depolarization of low amplitude [Fig. 2(e)] and finally after the end of stimulation there is only a smooth decline of the membrane potential towards the resting level [Fig. 2(f)]. Some fibers apparently are innervated by two nerve fibers eliciting EPSPs of relatively low amplitude [Fig. 2(c)]. Stimulation of the nerve fiber with the lowest threshold results in EPSPs which show no facilitation at all. If stimulus strength is raised to the threshold of the next fiber, EPSPs as described formerly can be observed.
C
Fig. 2. Response of muscle fibers upon stimulation of motor fibers in the external lip nerve branch with lowest thresholds (type 1 EPSPs). (a) Single EPSP (facilitated) at high sweep speed to show delay between stimulus and response. (b) Superimposed traces showing summation of EPSPs evoked by repetitive stimulation a t different frequencies (1, 2.5, 5, 7.5, 10 pulses/sec). (c) Stimulation a t 2 (lower) and 2.5 (upper) volts (5/sec). Superimposed traces to show occurrence of two EPSPs of type 1 with different amplitudes. Fatigue of spike-mechanism: (d) Summed EPSPs upon repetitive stimulation (10/sec) cross the spike-trigger level. (e) After several cycles of stimulation with trains of impulses spike potentials fail leaving only a slight depolarizing wave. (f) Disappearance of this response upon further stimulation. [Calibrations: (vertical bar) 10 mV; (horizontal bar) (a) 50 msec; (h) and (c) 500 msec; (d)-(f) 1 sec. Resting potential: (d)-(f) 58 mV.] Nerve stimulus-duration throughout this and all following experiments: 1 msec.
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EPSP % Control
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Fig. 3. Time course of facilitation of type 1EPSPs. The external lip nerve branch was stimulated with pairs of pulses at different intervals. Amplitude of the test-EPSP in % of that of the conditioning EPSP is plotted against the interval between conditioning and test EPSP in sec. Each point from one measurement. Curve fitted by eye. Inset: typical recordings from the experiment used for construction of the diagram. [Calibrations: (vertical bar) 10 mV; (horizontal bar) 500, 200, 200 msec.]
(2) A second type of synaptic potential can be evoked at higher stimulus strength. These EPSPs show higher amplitudes and longer duration compared with those evoked by the lower threshold fibers [Fig. 4(a)]. Upon stimulation of the external lip nerve branch with pulses of increasing voltage steplike increases in EPSP-amplitude can be recorded [Fig. 4(b)],which suggests innervation by several motor axons. Usually two nerve fibers evoking EPSPs of type 2 can be found, but some muscle fibers show only one, others three responses of different amplitude. Amplitude and facilitation were variable from muscle to muscle and between fibers of one muscle. Upon repetitive stimulation the amplitude of the EPSPs even in one fiber often showed considerable variations [Fig. 4(c)]. This was the case particularly in preparations from animals which were awakened from hibernation in the winter-months of October to February. EPSPs and action potentials in these muscles were found to be fatigued more easily than in other preparations. If the response to repetitive stimulation was observed over some minutes, after an initial phase of facilitation [Figs. 4(d) and 5(b)] a progressive decline of EPSP-amplitude could be seen [Fig. 4(d)l.
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Fig. 4. Response of muscle fibers upon stimulation of external lip nerve branch at higher stimulus strengths (type 2 EPSPs). (a) Superimposed responses upon stimulation of lowest-threshold (type 1) nerve fiber (closed arrow, 3 V) and upon additional stimulation of higher threshold (type 2) fiber (open arrow, 8 V) to show different amplitudes and time courses. (both EPSPs facilitated). (b) Superimposed responses upon stimulation with increasing voltages (2, 2.4, 2.5, 2 X 2.6, 2 X 3, 3.5, 4 , s V). Two EPSPs of type 2 can be distinguished. Stimulation with twin pulses (3 secs interval) because facilitated EPSPs allow better discrimination between different amplitudes. (c) Responses upon repetitive stimulation (0.25/sec) to show variability of type 2 EPSPs at constant stimulus strength. (d) Fatigue of EPSPs upon repetitive stimulation (0.2/sec). After an initial phase of facilitation, EPSP-amplitude declines. [Calibrations: (vertical bar) 10 mV; horizontal bar: (a) El00 msec; (b) 1 sec; (c) 2 sec; (d) 15 sec.] EPSP % O f Control
400 500:
[
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Fig. 5. Facilitation of type 2 EPSPs evoked by stimulation of external lip nerve branch. (a) Time course of facilitation. Stimulation with twin pulses at different intervals. Amplitude of the second EPSP in 96 of the first is plotted against the interval between conditioning and test impulse. Each point represents one measurement. Curve fitted by eye. (b) Cumulation of facilitation upon repetitive stimulation (0.2/sec). (c-e) Representative recordings from the experiment shown in (a). [Calibrations: (vertical bar) 10 mV; (horizontal bar) (b) 5 sec; (c)-(e) 2 sec.]
Facilitation of type 2 EPSPs after a single priming stimulus declines much slower than that of type 1potentials (Fig. 5) and thus facilitation adds up upon successive stimuli [Fig. 5(b)] even at low frequencies of stimulation. Only after 30-40 sec does the EPSP-amplitude reach the resting-level again. Because of this long-lasting facilitation 1 l/2 min a t least had to pass between each trial in a series of measurements. As it is difficult to hold a single muscle fiber for longer times, facilitation curves were constructed from one measurement for each value only.
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Usually, because of the high degree of facilitation, only a few type 2 EPSPs suffice to depolarize the muscle fibers above the threshold for triggering one or several action potentials [Fig. 6(a)]. Often the response fatigued rapidly so that only a burst of spikes was evoked even with persistent repetitive stimulation [Fig. 6(b)]. In many fibers the response to nerve stimulation was somewhat atypical in that it resulted only in a very slow and smooth depolarization [Fig. 6(c)]. Active membrane responses of different amplitudes were frequently elicited even with stimulation at constant strength [Fig. 6(d)]. This may be due either to properties of the excitable membrane at the recording site or by invasion of that site by action potentials originating from different distances. Internal lip nerve. Motor innervation via the internal lip nerve closely resembles that described above: stimulation of the nerve fiber with the lowest threshold results in EPSPs of low amplitude [Fig. 7(a)] which show facilitation and summation [Fig. 7(b)] at higher frequencies (type 1EPSPs). Raising the stimulus amplitude results in a steplike increase of the amplitude of the EPSPs [Fig. 7(c)]. These EPSPs have a higher amplitude and a longer time course and therefore are also classified as type 2 EPSPs. Muscle fibers are mostly innervated by one fiber evoking type 1and two fibers eliciting type 2 EPSPs. Often “miniature” potentials were superimposed upon type 2 EPSPs with an initially high frequency which progressively declined in the falling phase [Fig. 7(d)]. Such a correlation of EPSPs and “miniature” potentials has also been found in chromatophore muscle fibers of the squid (Florey and Kriebel, 1969). In some preparations the whole response had an irregular shape and rather re-
c
d
Fig. 6. Active membrane responses evoked by stimulation of external lip-nerve branch with stimulus intensity above threshold for type 2 EPSPs. (a) Spike-potentials elicited by summating EPSPs. (b) Fatigue of synaptic depolarization and spike responses. (c) In this preparation stimulation of the external lip nerve resulted only in a smooth and slow depolarization which finally triggered a spike responses. (d) Active membrane responses of different amplitudes in one fiber. [Calibrations: (vertical bar) 10 mV; horizontal bar: (a) 2 sec; (b) 5 sec; (c) 500 msec; (d) 2 sec;] Resting potentials: (a) 67 mV; (b) 55 mV; (c) 55 mV; (d) 57 mV.
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Fig. 7. Stimulation of internal lip nerve. (a) Response to stimulation of lowest threshold nerve fiber (type 1) at higher sweep speed to show delay between stimulus (arrow) and response (EPSP facilitated). (b) Superimposed responses to repetitive stimulation a t different frequencies (0.5; 1; 2.5; 5; 7.5; lO/sec) to show summation of type 1-EPSPs. (c) Superimposed responses to stimulation with increasing voltages (7; 8; 8.2; 8.5; 8.75; 2 X 8,9; 10 V) to show innervation by two fibers of type 2. (d) Single type 2-EPSP with superimposed burst of “miniature” potentials. (e) Spike responses evoked by repetitive stimulation of internal lip nerve with stimulus strength above threshold for type 2 EPSPs. [Calibrations: (vertical bar) 10 mV; (horizontal bar) (a) 50 msec; (b) 500 msec; (12) 1 sec; (d) 500 msec; (e) 2 sec.]
sembled a burst of summated small potentials than one single EPSP of higher amplitude. Repetitive stimulation of the higher threshold fibers in the internal lip nerve often led to depolarization which reached the spike-trigger level after a few stimuli, as was the case also for the type 2 fibers in the external lip nerve branch [Fig. 7(e)]. Type 2 EPSPs evoked by stimulation of the internal lip nerve showed the same degree and time course of facilitation as those stemming from external lip nerve stimulation (Fig. 8). After a single conditioning stimulus EPSPs rose t o about 600% of control. EPSP amplitude returned to resting level only after 30-40 sec. Only in four preparations was there an indication of inhibitory synapses at stimulation of the external lip nerve branch: In two preparations stimulation at the lowest threshold evoked hyperpolarizing potentials of low (2-3 mV) amplitude. These disappeared after some cycles of stimulation which could have been due to the use of KC1 in the microelectrodes. In two other muscles upon raising the stimulus voltage above the threshold for type 2 EPSPs a sudden fall in EPSP amplitude could be observed. This process was steplike and threshold dependent and therefore may be taken as a sign that an inhibitory axon was stimulated.
Light microscopy of nerve fibers Axonal cobalt staining via the branch of the external lip nerve revealed that after entering the muscle the nerve split up into a lot of small nerves which
MOTOR INNERVATION OF HELIX POMATIA a
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b
ESP % of Coatrol
t 600
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Fig. 8. Facilitation of type 2 EPSPs evoked by stimulation of the internal lip nerve. (a) Time course of facilitation. Stimulation with twin-pulses at different intervals. Amplitude of the second EPSP in % of the first is plotted against the interval between conditioning and test impulse. Each point represents one measurement. Curve fitted by eye. (b)-(e) Representative recordings from the experiment shown in (a). [Calibrations: (vertical bar) 10 mV; (horizontal bar) (b) 5 sec; (c)-(e) 2 sec.]
showed further extensive branching [Fig. 9(a)]. These branches were the origin of a rich network of blebbed fibers. Distinct regions seemed to be supplied by separate branches [Fig. 9(b)]. This may be a sign of several motor units. However, as single nerve fibers could not be resolved in the main nerves the possibility remains that there is extensive branching of one single nerve fiber. It may well be that only one fiber or one particular class of nerve fibers together with their motor endings has been stained by axonal iontophoresis. The blebbings had a very irregular shape [Figs. 9(c)-9(e)] and diameter. The kind of blebbed fiber shown in Figs. 9(c)-9(e) is most prominent in cobalt-stained nerves but smaller fibers were also regularly seen [Fig. 9(f)]. However, it could not be decided whether these were terminal ramifications of the fibers shown in Figs. 9(c)-9(e) or whether they represented a second type of neuromuscular junction. Axonal cobalt-staining via the internal lip nerve did not demonstrate nerve endings in the pharynx levator muscle which disagreed with physiological experiments. Only in 3 cases (out of about 20 preparations) could nerve fibers entering the pharynx levator be seen. The reason for this may have been failure of the staining method in this case, the external lip nerve branch offering perhaps a preparation more suitable for cobalt staining of motor nerve endings than other nerves. However a network of blebbed fibers comparable to those in the pharynx levator muscle was stained in the levator labii muscle which is also supplied by the internal lip nerve, indicating that it is possible to fill peripheral nerve endings via the internal lip nerve. Another possibility may be indirect innervation by peripheral motoneurones.
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Fig. 9. Axonal cobalt staining of peripheral nerve endings via the branch of the external lip nerve. (a) Whole mount of a pharynx levator muscle showing the external lip nerve branch (arrow) which enters the muscle and branches there. (b) Typical pattern of innervation by blebbed nerve fibers. The figure was drawn from micrographs taken a t different depths of focus. (The diameter of the blebbed fibers is not in scale.) Fibers leaving the main nerve trunk seem to innervate distinct regions of the muscle. It has to be kept in mind, however, that the arrangement of fibers is somewhat distorted due to the procedure for preparation of whole mounts described in the methods section. (c) and (d) Blebbed fibers as seen most frequently in the pharynx levator muscle. (e) Drawing of same fiber type as shown in (c) and (d) obtained in the same manner as (b). (f) Very thin fiber with smaller blebbings.
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DISCUSSION
The electrical properties of muscle fibers in the pharynx levator muscle are similar to those measured in other molluscan muscles. Properties of muscle fibers and neuromuscular junctions in several mollusc muscles are listed in Table 1 to allow easy comparison of the pharynx levator muscle of Helix with other preparations. From this it can be seen that the resting potential, in most cases 65-70 mV, falls well within the range measured in other muscles. The ability to elicit a spike response seems to be widespread in molluscan muscle fibers also. The pharynx levator muscle has been found to receive excitatory innervation from two nerves by several nerve fibers. By stimulation of the fibers with the lowest thresholds EPSPs of low amplitude are elicited in the muscle fibers. At higher stimulus strengths EPSPs show higher amplitudes and longer time courses. The amplitude of these EPSPs rises in several discrete steps upon raising stimulus intensity, suggesting multiple innervation of the muscle fibers. Multiple innervation seems to be quite common in mollusc muscles as judged from histological and electrophysiological investigations. Double innervation of collumellar muscle fibers in Helix by two different nerve fibers has been claimed after methylene blue staining (Rochling, 1928, Ramsay, 1940). Several nerve profiles making close contact with single muscle fibers have been seen frequently in electron micrographs (e.g., Rogers, 1968;Schlote, 1963;Florey and Kriebel, 1969; McKenna and Rosenbluth, 1973). Partly they contain different populations of vesicles which has been taken as an indication of different transmitter substances. Electrophysiological evidence for excitatory innervation by more than one nerve fiber has been obtained from a variety of muscles (see Table 1). Whereas type 1EPSPs in the pharynx levator resemble excitatory synaptic potentials in other mollusc muscles according to time course and decay time of facilitation, type 2 EPSPs last much longer and decay of facilitation is much slower also. There seems to be a broad range of complexity in the motor innervation of molluscan fibers. Whereas the posterior jugalis muscle of Helisoma is controlled by only one single motoneurone (Kater et al., 1971),in the accessory radula closer muscle of Aplysia each fiber receives excitatory innervation from at least 4 identified motor neurones in the buccal ganglion (Weiss et al., 1975). The functional meaning of the convergence of 6 excitatory nerve fibers on one muscle fiber in the pharynx levator remains to be determined. However, it may be possible that the axons in the internal and external lip nerve are only branches of common motoneurones. Such multiple branching is known from several gastropod neurons as e.g., the serotonergic metacerebral giant cell of Helix (Pentreath, 1976). In Aplysia this cell has a modulatory effect on muscle EPSPs evoked by stimulation of motoneurones (Weiss et al., 1975, 1978). It can not be excluded that a similar process takes place in the pharynx levator muscle. In the experimental procedure used to determine the number of axons innervating a single muscle fiber, stimulus intensity was raised progressively and
(6)g
(5)f
(4)e
(3)d
(2)~
(1)b
Mytilus edulis ABRM Aeyuipecten gibbus Aequipecten irradians adductor muscle Spisula solidissirnu incurrent siphonal valve muscle Rapana thornasiana radula-protractor muscle (sucrose gap) Helisoma triooluis (a) columellar muscle (b) posterior jugalis muscle Aplysia californica gill-muscle
50-75 mV
up to 47 mVa mean 27 mV
45-61 mV
up to 80 mVa
mean 56 mV
55-72 mV
Resting potential
+ graded or
+
overshooting (b) overshooting
(a)
+sometimes overshooting
+non overshooting, often graded +sometimes overshooting
Action potential
3 identified excitatory motoneurones
(a) single excitatory
multiple excitatory
multiple excitatory
multiple excitatory multiple excitatory
Innervation of muscle fibers (units)
mseca LDG2: ca150 mseca
LDGI: ca300
200 msec
ca 100 mseca
ca 50 m s e e
500 msec
EPSP
Half-decay time of
TABLE 1 Electrical Properties of Muscle Fibers and Neuromuscular Junctions in Various Mollusc Muscles
+
+
-
+
Facilitation
Aplysia californica lower extrinsic protractor
Loligo opalescens Chromatophore muscles Helix pornatia pharynx levator muscle
(8)i
(9)J
a
55-90 mV most at 65 mV
up to 50 mVa
60-80 mV
75f8mV
Values taken from figures in the cited papers. Twarog, 1967. De Forrest Mellon, Jr., 1968. Prior, 1976. Kobayashi, 1972.
(10)
Aplysia californica accessory radula closer muscle
(7)h
typically 6 excitatory axons via 2 nerves, two types of EPSPs (1and 2)
ca 200 m s e e
type 1 ca 300 msec type 2 ca 2 sec
ca 50 mseca
100-200 mseca
B16
B15
Kater et al., 1971; Heyer et al., 1973. Carew et al., 1974; Jacklet and Rine, 1977. Weiss et al., 1975; Cohen et al., 1978, Weiss et al., 1978. Orkand and Orkand, 1975; Banks, 1975. J Florey and Kriebel, 1969.
overshooting
+ non
+sometimes overshooting
4 identified excitatory motoneurones 1 modulatory typically 4 excitatory axons via 2 nerves 1 inhibitory multiple excitatory
+type 2: long lasting (30-50 sec)
+type 1: short lasting (ca 10 sec)
rarely
+
+Bls: large +BIG: small
150
PETERS
the resulting alterations in EPSP amplitudes were observed. Upon raising stimulus strength more fibers are recruited according to their threshold and are stimulated in addition to lower threshold fibers. Thus the highest EPSP in a muscle fiber generally results from the synchroneous activation of 3-4 nerve fibers. It can be speculated then that the properties of the type 2 EPSPs result from a modulatory pre- or postsynaptic effect of higher threshold axons. Some of the properties of the synaptic potentials may be explained by the assumption, that the muscle contains several motor units which are coupled electrically via the muscle fibers. Multiple innervation by axons then might hold true for a bundle of coupled muscle fibers rather than a single fiber, each single fiber receiving perhaps only double or triple innervation. There is some evidence for electrical coupling between smooth muscle fibers in molluscs. Nexal junctions between muscle fibers can be seen with the electron miscroscope in the ABRM (Twarog et al., 1973; Gilloteaux, 1976) as well as in the lower extrinsic protractor muscle in Aplysia (Orkand and Orkand, 1975). In both cases electrical coupling between muscle cells has also been shown by electrophysiological methods. Similar results have been obtained from chromatophore muscle fibers of Loligo (Florey and Kriebel, 1969) and the accessory radula closer muscle of Aplysia (Cohen et al., 1978). Electrical coupling between muscle cells would e.g., explain the very slow rise of the EPSPs which occurred frequently. An alternative explanation could he the release of a transmitter from nerve terminals some distance away from the receptive membrane, thus introducing a diffusional delay. Since I did not succeed in impaling two cells simultaneously the question of electrotonic coupling is unresolved as yet. The electrophysiological results suggest multiple innervation of the muscle fibers of the pharynx levator muscle. Although the results of cobalt-staining support this idea for the muscle as a whole, there was no definite histological proof of innervation by two types of nerve fibers as has been described by Gilloteaux (1972) in the ABRM of Mytilus with osmium-zinc-iodide staining. Cobailtstaining via the external lip nerve revealed a lot of nerve branches entering the muscle. These were the origin of a rich network of fine blebbed fibers. Fibers of this kind have also been seen after methylene blue staining in the penis retractor muscle (Foh and Bogusch, 1969) and the columellar muscle (Rochling, 1928) as well as in the heart of Helix (Cottrell and Osborne, 1969). After formaldehyde treatment for histochemical demonstration of catecholamines varicose fibers were seen in the penis retractor muscle of Helix (Foh and Bogusch, 1969) and in the ABRM of Mytilus (McKenna and Rosenbluth, 1973). Nerve endings stained with osmium-zinc-iodide in the ABRM (Gilloteaux, 1972) also had a similar appearance. So these varicosities are probably not an artifact introduced by the procedure of cobalt staining. In all cases mentioned above the varicose fibers have been supposed to be motor. The blebbings, then, may represent sites of transmitter release as suggested for vertebrate autonomic nerve fibers. However, it can not be ruled out that some of the stained fibers in the pharynx levator muscle were branches of sensory cells. In electron microscopical pictures of various mollusc muscles presumed neu-
MOTOR INNERVATION OF HELIX POMATIA
151
romuscular junctions do not exceed a diameter of approximately 1.5 ym (e.g., Rogers, 1968;Florey and Kriebel, 1969;McKenna and Rosenbluth, 1973;Wabnitz, 1976). This corresponds well with the fine blebbing in the pharynx levator muscle which are seen most frequently. However, there were also thickenings of the nerve fibers, with diameters reaching up to 5 ym. There may be several sources of error in the interpretation of the results of Co-staining which could be the reason for this discrepancy: (1)Co may induce swelling of the axons and thus dimensions may be distorted. (2) Fine nerve branches may be misinterpreted as single nerve fibers. (3) Glial cells may be stained together with the axon due to leakage of Co, the large blebbings thus representing glial cell bodies rather than expansions of an axon. I hope that electron-microscopicaland pharmacological investigations, which are under way now, will answer some of the questions posed. I wish t o thank I. Holtkamp for technical assistance. REFERENCES BANKS,F. W. (1975). Inhibitory transmission a t a molluscan neuromuscular junction. J. Neurobiol. 6: 429-433. BURTON,R. F. (1968). Ionic balance in the blood of Pulmonata. Comp. Biochern. Physiol. 25: 509-516. H., RUBINSON,K., and KANDEL,E. R. (1974). Physiological and bioCAREW,J. I., PINSKER, chemical properties of neuromuscular transmission between identified motoneurons and gill muscle in Aplysia. J. Neurophysiol. 27: 1020-1040. T. K. (1969). Techniques of intracellular microinjection. In Glass Microelectrodes, CHOWDHURY, M. Lavalee, 0. F. Schanne, and N. C. HBbert, Eds, Wiley and Sons, New York, pp. 404-423. I. (1978). Motor control of buccal muscles in A p COHEN,I. L., WEISS,K. R., and KUPFERMANN, lysia. J. Neurophysiol. 41: 157-180. COTTRELL,G. A. and OSBORNE,N. (1969). A neurosecretory system terminating in the Helix heart. Comp. Biochem. Physiol. 2 8 1455-1459. MELLON,JR. (1968). Junctional physiology and motor nerve distribution in the fast DE FOREST adductor muscle of the scallop. Science 160: 1018-1020. FLOREY,E. and KRIEBEL,M. E. (1969). Electrical and mechanical responses of chromatophore muscle fibers of the squid, Loligo opalescens, to nerve stimulation and drugs. Z. Physiol. 65: 98-130. FOH, E. and BOGUSCH,G. (1969). Die Nervennetze im Penisretraktor von Helix pomatia. Z. Zellforsch. 93: 439-446. GILLOTEAUX,J. (1972). Innervation of the anterior byssal retractor muscle in Mytilas edulis L. Z. Zellforsch. 124: 204-216. GILLOTEAUX,J. (1976). Intercellular connections of a smooth muscle: Ultrastructure of the anterior byssal retractor muscle (ABRM) of Mytilus edulis L (Mollusca Pelecypoda). Cytobiol. 12: 457-472. HEYER,C. B., KATER,S. B., and KARLSSON,K. L. (1973). Neuromuscular systems in molluscs. Am. Zool. 13: 247-270. ILES, J. F. and MULLONEY,B. M. (1971). Procion yellow staining of cockroach motor neurones without the use of microelectrodes. Brain Res. 3 0 397-400. JACKLET,J . W. and RINE, J . (1977). Facilitation a t neuromuscular junctions: Contribution to habituation and dishabituation of the Aplysia gill withdrawal reflex. Proc. Natl. Acad. Sci. 74: 1267-1271. J. P. (1971). Neuromuscular transmission in the gastropod KATER,S. B., HEYER,C. and HEGMANN, mollusc Helisoma triuoluis: Identification of motoneurons. 2. Physiol. 74: 127-139.
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