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Motoneuron reinnervation of phasic uropod muscles in crayfish
DEVELOPMENTAL BRAIN RESEARCH
ELSEVIER
Developmental Brain Research 87 (1995) 179-187
Research report
Motoneuron reinnervation of phasic uropod muscles in crayfish How-Jing Lee 1, Charles H. Page * Department of Biological Sciences, and Bureau of Biological Research, Rutgers University, Piscataway, NJ 08855, USA Accepted 11 April 1995
Abstract Motoneuron reinnervation of the lateral abductor and adductor muscles in the exopodite of the crayfish uropod was obtained by cross-tying the cut proximal ends of one set of uropod nerve roots to the cut distal ends of their contralateral homologues. In normal (nonsurgically treated) animals the abductor was innervated by two excitatory motoneurons and an inhibitor while two excitors innervated the adductor. For each muscle one of the excitors produced large excitatory junction potentials (EJPs) while the other evoked small EJPs. When stimulated repetitively only the smaller EJP in the adductor generated a facilitating response. Within less than 10 weeks postsurgery the muscles were each reinnervated by two excitatory motoneurons. While the abductor motoneurons generated synaptic potentials with similar amplitudes and time courses to those of normal animals, they differed from those in normal animals in that they facilitated when stimulated repetitively. In contrast to the large and small EJPs evoked in the normal animal, the two motoneurons that reinnervated the adductor muscle elicited similar amplitude EJPs, neither of which facilitated in response to repetitive stimulation. Keywords: Motoneuron regeneration; Neuromuscular reinnervation; Synaptic plasticity; Crayfish; Uropod
1. Introduction
Surgical interruption of motor axons in a peripheral nerve initiates a regenerative response whereby the sectioned motoneurons reinnervate the muscles that they originally innervated [8]. Typically this process involves rapid degeneration of the distal segments of the severed motor axons and outgrowth from their proximal cut ends to form synaptic contacts on the denervated muscle fibers. In contrast to vertebrates and most other invertebrates, in crustaceans degeneration of the distal axon stump is often very slow, taking up to several months to complete [4,6]. In some circumstances the distal stump can remain viable for many months following the cutting of the motor axon, with or without muscle reinnervation, while in other instances the stump degenerates within several weeks [3,4,16]. As a result, although some investigations of motoneuron regeneration and muscle reinnervation in crustaceans have reported the reestablishment of the original pattern of neuromuscular innervation, there is considerable
* Corresponding author. 1 Present address, Department of Plant Pathology and Entomology, National Taiwan University, Taipei, Taiwan, R.O.C. 0165-3806/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 3 8 0 6 ( 9 5 ) 0 0 0 7 4 - 7
variability in the regenerative capacity of different preparations and their time course for neuromuscular reinnervation [3,7,17,29]. Critical factors include where the axon is sectioned (within the nerve cord or at various points along the peripheral nerve including axon branches on the muscle surface), and the length to which the severed proximal stump is brought into contact with either the distal stump or the denervated muscle fibers [4,5,15,16]. The uropod appendages and caudalmost segments of the crayfish abdomen are innervated by nerves (roots) that project from the terminal (sixth) abdominal ganglion in a fan-like arrangement. Although the distal and proximal ends of these roots retract when cut and therefore cannot be easily brought back into contact by tieing, the projection of the roots in a radial pattern from the ganglion enables the long proximal end of one sectioned root to be cross-tied to the long distal end of its contralateral homologue. This surgical procedure was developed originally to study sensory neuron reinnervation of the terminal ganglion [20]. Each uropod consists of a basal protopodite with attached exopodite and endopodite. Their muscles are innervated by motoneurons that originate in the terminal ganglion, and run in the 2nd and 3rd roots to innervate the uropod [13,14]. Since previous investigations in crayfish
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and insects have concluded that a motoneuron can innervate a denervated muscle that is the contralateral homologue of the normally innervated muscle [9,16,18], crosstying the roots that innervate the uropods should provide a useful preparation for examining motoneuron reinnervation of selected uropod muscles. The physiological experiments described in this report focus on the innervation and reinnervation of the large phasic lateral abductor and adductor muscles which respectively open and close the uropod exopodite.
2. Materials and methods 2.1. The crayfish Procambarus clarkii Girard, of both sexes with carapace lengths of 3 - 5 c m were obtained from Wauban Laboratories (Schriever, LA). Upon receipt their chelae were autotomized to reduce cannibalism. The animals were maintained in large tanks containing 23-25°C dechlorinated water that was continuously aerated and filtered, and fed dried dog food twice a week. 2.2. Analysis of normal muscle innervation Innervation of the lateral abductor and adductor muscles was analyzed by intracellular recording of muscle fiber responses evoked by stimulating motoneurons in the 2nd and 3rd roots which exit the terminal (sixth) abdominal ganglion to innervate the uropods. All experiments were performed on an isolated abdomen, pinned ventral side up on a wax block in an experimental chamber filled with oxygenated (12-15°C) crayfish saline [33]. The nerve cord and uropod roots were exposed for stimulation by removing the overlying cuticle. Abductor and adductor motoneuron impulses were initiated by stimulating the cut distal end of the root running to the muscle with a saline filled suction electrode. A Grass S-48 stimulator generated the stimulus pulses and pulse trains. Two small windows were cut in the ventral cuticle of the uropod exopodite for microelectrode penetration of abductor and adductor muscle fibers. Movement artifacts were minimized by breaking the shank of the 3M KC1 microelectrode (10-20 MY2) near the tip and suspending the tip from a fine silver wire before impaling a muscle fiber. Muscle potentials were fed into a S-7100A WPI electrometer, displayed on a storage oscilloscope and photographed with a Polaroid oscilloscope camera. Positive images were obtained from the negative prints by scanning the prints and then reversing them using commercial software (PhotoShop). In some experiments the GABA antagonist picrotoxin (Sigma) was infused into the bath to test whether peripheral inhibitor activity was responsible for reducing the
amplitude of an excitatory junction potential (EJP). To avoid dislodging the microelectrode from the impaled muscle fiber when changing solutions, a modified chamber with a volume of 25 ml was used in which saline containing 57/~M picrotoxin was introduced on one side and after flowing over the preparation was drained from the other side. The flow rate was about 80 ml/min. 2.3. Muscle denervation procedure The surgical protocol was modified from Krasne and Lee [20]. Healthy (active and alert) animals selected for surgery were immobilized in 5°C water before securing them ventral side up in a chamber filled with 8°C oxygenated saline. The terminal abdominal ganglion and its 2nd and 3rd roots were exposed by: (1) cutting the abdominal sternite overlying the ganglion at its lateral margins; (2) making a U-shaped incision in the soft cuticle, extending posteriorly from the sternite cuts to the base of the uropod protopodites and then medially along the proximal edge of the telson; and (3) folding back the U-shaped flap of soft cuticle freed by the incision. A single strand of dental floss, stained with methylene blue to enhance visibility, was tied around the left pair of 2nd and 3rd roots at a point near the ganglion. After cutting the roots between the tie and the ganglion, the long distal stump was gently pulled over to the still intact right (contralateral) pair of roots and tied close to the contralateral uropod. Finally the intact right roots were cut between the tie and the right uropod, thereby physically cross-connecting the left uropod to the right side of the ganglion. As a result of cross-connecting the left uropod to the contralateral side of the ganglion, the right uropod was separated from the ganglion by a gap of several mm. Throughout surgery care was taken to avoid puncturing the artery that runs around the posterior end of the ganglion. Following completion of surgery the wound was closed by positioning the cuticular flap in place and allowing the wound to clot in air for 20min. The animals were placed in individual containers of 50% saline for 7 days and then returned to a flesh water holding aquarium for 7 - 9 additional weeks, before recording the synaptic responses produced in their reinnervated muscle fibers with intracellular microelectrodes. The survival rate was 75% at 8-10 weeks postsurgery. Microelectrode recordings were obtained from the reinnervated muscle fibers of 21 animals, 8 of which molted successfully during the postsurgical period. Whether or not an animal molted in the period following surgery had no noticeable effect on the synaptic responses of the reinnervated muscle fibers. 2.4. Analysis of muscle reinnervation Electromyograms were recorded periodically from the denervated muscles to monitor their reinnervation. Recordings were obtained by inserting pairs of 100 /xm insulated
H. -J. Lee, C.H. Page/Developmental Brain Research 87 (1995) 179-187
181
A
B
E
F 10 ms
Fig. 1. Intracellular recordings of muscle potentials generated in the lateral abductor and adductor muscles of normal crayfish. A: successive increments in the intensity of 3rd root stimulation (3.0, 3.1 and 3.2 V) activated the three lateral abductor motoneurons. The lower voltages were threshold stimuli for eliciting respectively a small EJP and a large compound EJP. Further increase in stimulus strength (3.2 V) reduced the amplitude and duration of the compound EJP, presumably reflecting activation of the peripheral inhibitor (middle trace). B: in another preparation, the lowest threshold 3rd root stimulus (3.2 V) produced a large abductor EJP. When the stimulus voltage was raised (3.4 V) to activate the second excitor a larger compound EJP was generated. Further increase in stimulus voltage (4.0 V) excited the peripheral inhibitor thereby decreasing the amplitude and duration of the compound EJP (middle trace). C: a spike-like response generated by an abductor excitor. D: stimulation of an adductor motoneuron in the 2nd root produced a large EJP. E: a spike-like response generated by the adductor excitor in the 2nd root. F: a small adductor EJP evoked by 3rd root stimulation. For C and E the slow potential following the spike-like response is a mechanical artifact. Muscle responses were evoked by 3 - 7 V, 0.2 ms pulses applied to the cut distal end of the 2nd or 3rd root of the terminal abdominal ganglion. The stimulus triggered the oscilloscope trace.
copper wires into the lateral abductor a n d / o r adductor muscles of animals restrained in a chamber filled with dechlorinated water. Muscle potentials were differentially amplified (P15 Grass preamplifier) and displayed on a storage oscilloscope. An arbitrary scoring system was devised to quantify the strength of the muscle responses evoked by strong tactile stimulation of the abdomen. An absence of muscle responses to the stimulus was scored 0, muscle potential responses smaller than 50 tzV were scored 1, while those larger than 50 /xV were assigned a score of 2.
A
Intracellular microelectrode recordings were conducted as described above to determine the extent of motoneuron reinnervation of the lateral abductor and adductor muscles at 8 - 1 0 weeks postsurgery. A fine-tip ( 1 2 5 - 2 0 0 /zm diameter) glass pipette containing 4 M NaCI connected to a S-48 Grass stimulator was used to apply voltage pulses to the 2nd or 3rd roots, or the interganglionic connectives, to elicit impulses in the motoneurons that reinnervated the muscles. In a few preparations, the cross-connected roots were cut at the base of the ganglion and the distal ends of 2nd or 3rd roots stimulated with a suction electrode.
C
D
3 0.1s
Fig. 2. Intracellular recordings of lateral abductor and adductor responses to pulse train stimulation in normal crayfish. A: responses produced by the lowest threshold (6.5 V) abductor excitor to train stimulation of the 3rd root. B: compound EJPs produced by 7.3 V pulse trains activated both abductor excitors. A and B recorded from the same muscle fiber. C: following the initial response, continued stimulation of the 2nd root generated constant amplitude adductor EJPs. D: facilitation of small adductor EJPs initiated by train stimulation of 3rd root. Stimulus was a 500-ms train of 0.2-ms pulses at 32/s.
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3. Results
A
3.1. Inner~,ation of lateral abductor and adductor muscles
In agreement with Higuchi [13,14], the lateral abductor and adductor muscles are innervated by motoneurons that exit from the terminal abdominal ganglion in the 2nd and 3rd roots. The 3rd root contains the three lateral abductor motoneurons--two excitors and a peripheral inhibitor (Fig. 1A,B), and one of the two adductor excitors (Fig. 1F). The axon of the other adductor excitor is in the 2rid root (Fig. 1D,E). 3.2. Lateral abductor
The three lateral abductor motoneurons were activated in succession by increasing the voltage of the stimulus pulses applied to the 3rd root. Typically the two excitors had lower thresholds than the peripheral inhibitor. Although the threshold voltages for initiating impulses in the two excitors were similar, the excitatory junction potentials (EJPs) that the two excitors evoked in lateral abductor muscle fibers differed in amplitude; one producing only a small EJP while the other initiated a large EJP (lowest traces in Fig. 1A,B respectively). In each instance a small increase in stimulus voltage activated the second excitor. The resulting compound EJPs (upper traces in Fig. 1A,B) reflect the simultaneous stimulation of the two excitors. The initial response of the larger excitor sometimes included a spike-like potential which decayed during repetitive stimulation (Fig. 2. 1C, 2A,B). Innervation of lateral abductor muscle fibers was further characterized by examining their responsiveness to motoneuron stimulation by stimulus pulse trains. In our preparations, the maximum rate of stimulation for which each stimulus pulse would elicit an EJP was about 30 per s. Muscle responses rapidly diminished in size during the first several stimulus pulses (Fig. 2A,B). Following this initial decrease in size, EJP amplitudes remained constant for the remainder of the pulse train. The threshold voltage required to activate the peripheral inhibitor by 3rd root simulation was higher than that for the excitors. Typical thresholds for initiating impulses in the three motoneurons were: 3.3 V for the first excitor, 3.5 V for the second excitor and 4.0 V for the inhibitor. The primary effects of the peripheral inhibitor were to reduce the amplitude and duration of the compound EJP produced by the two excitors (compare upper and middle traces in Fig. 1A,B). To further investigate whether the decrease in EJP size and duration resulted from activity of a GABAergic inhibitor, we examined the effects of exposing the preparation to picrotoxin, a GABA antagonist. As illustrated in Fig. 3, 57 /xM picrotoxin almost entirely eliminated the inhibitory depression of the EJP. In contrast, exposure to picrotoxin never affected the compound EJP generated by stimulating the two excitors if the stimulus
B
5 ms
Fig. 3. Picrotoxin blocked the peripheral inhibitor response in the lateral abductor. A: responses to successive increments in stimulus intensity of 3.3, 3.5 and 4.0 V which activated respectively a first excitor (middle trace), the peripheral inhibitor (lowest trace), and the second excitor (largest trace). Note that the inhibitor reduced the amplitude and duration of the first excitor's EJP. B: responses recorded from the same three abductor motoneurons after 14 min exposure to 57/zM picrotoxin. Note that the inhibitory reduction in EJP size and duration is almost completely blocked (compare lowest trace with that in A). The membrane potential was constant ( - 74 mV) throughout the period of recording A and B.
pulses were subthreshold for activating the inhibitor. Picrotoxin blocking of EJP inhibition could not be reversed, even after 48 min washout with crayfish saline. There was considerable variability in motoneuron innervation of the lateral abductor muscle fibers. Out of a total of 16 muscle fibers, each examined in a different crayfish, 19% were innervated by all three motoneurons, 56% by a single excitor and an inhibitor, 12% by two excitatory motoneurons only, and 13% by a single excitor. 3.3. Adductor
In contrast to previous reports [13,14], the muscle responses generated by separately stimulating the two adductor excitors differed markedly in size, and responsiveness to high frequency stimulation. A large depolarizing potential that usually included a spike-like component was produced by stimulating the 2nd root motoneuron (Fig. 1D,E, 2C), while the excitor running in the 3rd root always evoked a small to medium size EJP (Fig. 1F, 2D). The responses of the 2nd root excitor to pulse train stimulation were similar to those of the abductor excitors (Fig. 2C). After an initial drop in size, EJP amplitudes remained relatively constant for the duration of the pulse train. In contrast, EJPs of the 3rd root excitor always facilitated when the excitor was repetitively stimulated (Fig. 2D). All of the adductor muscle fibers examined were innervated by both motoneurons. None of the recordings ever provided any evidence for peripheral inhibitor innervation of the adductor. The absence of an adductor inhibitor was supported by the lack of any change in adductor EJP amplitude or duration when the preparation was bathed with 57/zM picrotoxin (not shown).
H.-J. Lee, C.H. Page / Developmental Brain Research 87 (1995) 179-187 Table 1 Electromyogram of sensory evoked muscle responses from the reinnervated uropod Weeks
Reconnected
Unconnected
postsurgery
n
ABD
ADD
ABD
ADD
3-4 5-6 7-8 9-10
17 11 13 9
0.59+0.80 0.91+0.94 0.92_+0.64 1.44+0.73
0.88+0.78 0.73_+0.90 1.08_+0.64 1.44+0.73
0.12+0.49 0 0.15_+0.38 0.56+0.73
0.12+0.49 0 0.15_+0.38 0.56+0.73
Responses (mean + S.E.) to tactile stimulation of ventral abdominal cuticle. Scoring system: 0 = no muscle response; 1 = muscle response smaller than 50 /zV; 2 = muscle response larger than 50 /zV. ABD is lateral abductor; ADD is adductor.
3.4. Reinnervation of lateral abductor and adductor muscles
Beginning 3 to 4 weeks postsurgery, tactile stimulation of the ventral abdominal surface elicited electromyogram responses in the lateral abductor and adductor muscles of many animals (Table 1). There was a marked difference in recovery between the muscles in the surgically cross-connected (left) and unconnected (right) uropods. Even at 10 weeks postsurgery, sensory evoked responses were rarely recorded from muscles on the unconnected side. As a result our intracellular analysis focused on reinnervation of the cross-connected side, i.e. the abductor and adductor muscles that had been cross-tied to the stumps of the contralateral 2nd and 3rd roots. Dissection of regenerated preparations 9 - 1 0 weeks postsurgery revealed varying amounts of scar tissue surrounding the point where the roots innervating the (left) uropod had been cross-tied to the proximal ends of the contralateral (right) 2nd and 3rd roots. Cross-connection of these roots provided the morphological basis for the physiological responses recorded from the muscles in the
A
183
surgically connected (left) uropod in response to stimulation of the proximal segments of the contralateral roots. Outgrowth from the short stumps of the unconnected (left) 2nd and 3rd roots often ran along the 1st a n d / o r 4th (left) roots to reach the scar tissue. Although it was very difficult to follow these outgrowing axons within the scar tissue matrix, the almost complete absence of muscle responses recorded from the surgically unconnected (right) uropod, indicates that at 10 weeks postsurgery these axons rarely reinnervated either the lateral abductor or the adductor muscles. There was a 15 to 20 mV increase in the membrane potential of the reinnervated muscle fibers at 8-10 weeks postsurgery. Membrane potentials of the lateral abductor shifted from - 6 6 . 4 mV ___11.1 (n = 40) in normal (nonsurgically treated) animals to - 86.5 mV ___12.3 (n = 20) ( P < 0.00002, t-test) for reinnervated preparations, with a similar change for the adductor fibers (-69.9mV___ 11.7 in normals (n = 20) to - 8 6 . 2 mV + 13.9 in reinnervated muscles (n = 20) ( P < 0.01, t-test)). The membrane potentials of muscle fibers from the surgically unconnected (right) uropod underwent a similar though smaller increase in amplitude ( - 7 8 . 3 ___12.5 mV for the lateral abductor and - 7 7 . 2 mV___ 13.7 mV for the adductor, n = 20 for both). At 8 to 10 weeks postsurgery, synaptic responses could be recorded with intracellular microelectrodes from the reinnervated lateral abductor and adductor muscles in 84% (21 out of 25) of the surgically cross-connected preparations. EJPs were obtained from the reinnervated muscles in response to tactile stimulation of the abdomen, electrical stimulation of fiber bundles in the interganglionic connectives, focused stimulation by a fine tip stimulating electrode positioned on the surface of the appropriate 2nd or 3rd root (Fig. 4 and Fig. 5) and in a few preparations suction electrode stimulation of the distal end of a cross-
D
IP
.= _
20 ms B
C 0.1s
Fig. 4. Responses of reinnervated lateral abductor muscle fibers. A: stimulation of the 3rd root evoked an abductor EJP. B and C: in another preparation pulse train stimulation of both abductor excitors elicited EJP facilitation. Excitor thresholds were 11 V and 14 V respectively. D: in a third preparation, following rapid decay of the spike-like response the abductor EJPs undergo slow facilitation. Stimulus was a 0.2 ms pulse train at 3 5 / s for 550 ms (B and C) and 3 0 / s for 540 ms (D). B, C and D have the same time calibration. Animals were 56-61 days postsurgery.
H.-J. Lee, C.H. Page / DeL'elopmental Brain Research 87 (1995) 179-187
184
A
c
B
D
20 ms
0.1 s
Fig. 5. Responses of a reinnervated adductor muscle fiber. A: an adductor EJP elicited by 2nd root stimulation. B: third root stimulation initiated another adductor EJP. C and D: the amplitudes of adductor EJPs remained unchanged during pulse train stimulation of the 2nd (C) and 3rd (D) roots. All recordings were from the same muscle fiber. The stimulus train of 0.2ms pulses was 3 0 / s for a period of 540 ms. Time since surgery was 73 days.
tor EJPs was ever observed that could be attributed to peripheral inhibitor activity. Pulse train stimulation always produced a facilitating response in the abductors (Fig. 4B,C). Even for those responses which began with a large spike-like potential, following an initial rapid decline, EJP amplitude slowly increased for the duration of the stimulus (Fig. 4D). Adductor EJPs of similar size could be initiated by stimulating either the 2nd or 3rd cross-connected roots (Fig. 5). While these 8-15 mV EJPs were smaller than the 30-40 mV EJPs generated by 2nd root stimulation in normal animals (Fig. 1D, 2C), they were somewhat larger than those initiated by stimulating the 3rd root ( 3 - 9 mV)
connected root that was cut close to the ganglion. In contrast, 'spontaneously generated' EJPs were not observed in any of these dissected preparations. Stimulation of the cross-connected 3rd root always initiated one or two different EJPs in the reinnervated lateral abductor muscle fibers (Fig. 4A, B,C). Although their amplitudes and time courses were similar to the abductor EJPs recorded from normal animals (Fig. 1A,B), muscle responses in regenerates differed from those of normal crayfish both in the absence of peripheral inhibition and the development of abductor EJP facilitation. With the exception of an unusual preparation described below (Fig. 6A), no change in the amplitude of the abduc-
A
C
B
D
20 ms
<
0.1s
Fig. 6. Unusual patterns of reinnervation. A and B: hyperinnervation of a lateral abductor. Stimulation of the 3rd root (A) activated two excitors (middle and upper traces) and a peripheral inhibitor (lower trace is compound EJP resulting from decrease in first excitor EJP produced by simultaneous activation of the inhibitor) in the lateral abductor. In the same animal, stimulation of the 2nd root (B) excited two additional abductor excitors. C and D: the sizes of the adductor EJPs elicited by stimulating the 2nd and 3rd roots were reversed from normal crayfish. Second root stimulation produced a small EJP (C) while a large EJP (D) was generated in response to 3rd root stimulation. E: one of the two motoneurons that reinnervated the adductor muscle exited from the ipsilateral side of the terminal ganglion. Stimulation of the crossconnected third root elicited a large EJP, while similar stimulation close to the severed stumps of the ipsilateral (unconnected) roots initiated a very small EJP. Time since surgery ranged from 56 to 61 days.
H.-J. Lee, C.H. Page/ Developmental Brain Research 87 (1995) 179-187
in normal crayfish (Fig. IF, 2D). In contrast to the characteristic facilitation of 3rd root adductor EJPs in normal animals (Fig. 2D), neither the 2nd nor the 3rd root adductor EJPs facilitated in the surgically cross-connected animals (Fig. 5C,D). Typically there was minimal change in the amplitude of the adductor EJPs during pulse train stimulation. Unusual reinnervation patterns were observed in three of the surgically reconnected animals. In one crayfish the lateral abductor muscle was innervated by 5 motoneurons. Stimulation of the cross-connected 3rd root activated two excitatory motoneurons and a peripheral inhibitor in the abductor (Fig. 6A), while stimulation of the 2nd root excited two additional excitatory motoneurons (Fig. 6B). In contrast, reinnervation of the adductor was n o r m a l - motoneurons running in both the 2nd and 3rd roots (not shown). In another animal, a large adductor EJP was generated by 3rd root stimulation and a small EJP when the 2nd root was stimulated (Fig. 6C,D). This is opposite to the pattern observed in normal animals where large and small EJPs are produced respectively by 2nd and 3rd root motoneurons (Fig. 1D,F). Finally, in a third preparation an adductor received weak innervation from the unconnected (ipsilateral) side of the ganglion. Stimulation of the crossconnected 3rd root evoked a large EJP in the adductor (Fig. 6E, upper trace), while a small adductor EJP was generated by stimulating the ipsilateral root stump (Fig. 6E, middle trace).
4. Discussion
The recordings of motoneuron responses from lateral abductor and adductor muscle fibers in normal (nonsurgically treated) crayfish, while confirming the general pattern of motoneuron innervation of these muscles [13,14], provide new information concerning the individual characteristics of their motoneurons. Despite the phasic categorization of these muscles, based on short sarcomere lengths [21] and high levels of myofibrillar ATPase activity [32], only one of the two excitatory motoneurons innervating each muscle has strictly phasic characteristics--large EJPs that undergo an initial depression with repetitive stimulation (Fig. 1B,D, 2B,C). In contrast, the other motoneurons produce smaller EJPs that when repetitively stimulated depress initially in the abductor and facilitate in the adductor (Fig. 1A,F, 2A, D). Our observation that stimulation of the 2nd and 3rd roots elicits respectively large (often with a spike-like component) and small adductor EJPs (Fig. 1D,E,F), differs from previous reports that separate stimulation of these two roots with oil-hook electrodes initiates similar action potentials in adductor muscle fibers [13,14]. Differences in methods of stimulation provide the most likely explanation for these conflicting results. When in preliminary experiments we used wire electrodes to separately stimulate the
185
2nd and 3rd roots, we were unable to initiate a small 3rd root EJP without simultaneously evoking a large 2nd root EJP. Since the 2nd and 3rd roots run in very close contact as they enter the uropod, apparently the stimulus current applied to the 3rd root can spread along the root to the base of the uropod where it activates the 2nd root excitor, producing a large compound F_JP similar to that evoked by direct stimulation of the 2nd root. In contrast, when we stimulated the cut proximal end of the 3rd root with an attached suction electrode, small 3rd root EJPs could be readily elicited without activating the much larger 2nd root EJPs (Fig. 1F, 2D). 4.1. Reinnercation pattern
The muscle responses evoked by tactile stimulation of the abdomen, and electrical stimulation of the nerve cord connectives, prove that the uropod muscles were functionally reinnervated. The pattern of excitatory motoneuron reinnervation of the lateral abductor and adductor muscles was similar to that observed in normal crayfish, except that the reinnervating motoneurons exited from the contralateral half of the terminal ganglion. The similarity in the synaptic responses evoked by focused electrical stimulation of the cross-connected roots, and those initiated by tactile or connective stimulation, indicates that the abductor and adductor responses were evoked by the activation of motor axons that ran in the cross-connected roots to form functional connections between the 6th ganglion and the uropod muscles. The contralateral origin of the regenerated motoneurons presumably resulted from having surgically cross-tied the long distal ends of the uropod's 2nd and 3rd ganglionic roots to the long proximal stumps of the contralateral 2nd and 3rd roots. This conclusion is supported by reports in crayfish and insects that denervated muscles can receive an otherwise normal reinnervation from motoneurons that are contralateral homologues of their normal innervation [9,16,18]. In contrast to the recording of excitor EJPs from almost all regenerates, peripheral inhibitor reinnervation was observed in only one animal at 10 weeks postsurgery. This preparation was atypical, since the lateral abductor was reinnervated by an inhibitor and a supernumery set of excitors (Fig. 6A, B). Therefore either the peripheral inhibitor does not typically reinnervate the lateral abductor, or inhibitory reinnervation of the lateral abductor requires more than 10 weeks. Alternately weak inhibition reinnervation might not have noticeably altered the amplitude of the excitor EJPs and therefore could have been undetectable in our recordings. 4.2. Change in motoneuron phenotype
Although both the lateral abductor and adductor muscles were reinnervated by the same number of excitors as normal animals, the muscle responses generated by these
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regenerated m o t o n e u r o n s differed f r o m those recorded from normal animals, both in the amplitudes o f their E J P s and in their responses to repetitive stimulation. The p h e n o t y p e of each lateral abductor excitor was altered in the regenerates. W h i l e the two abductor excitors still p r o d u c e d small and large EJPs, both of w h i c h w e r e subject to short term depression in normal animals (Fig. 2A,B), f o l l o w i n g reinnervation the EJPs of these two excitors facilitated in response to high f r e q u e n c y stimulus trains (Fig. 4B,C). A l t h o u g h the two adductor m o t o n e u r o n s p r o d u c e d a small facilitating E J P and a large EJP w h i c h initially depressed in normal crayfish (Fig. 2C,D), in regenerates the two excitors e v o k e d intermediate size EJPs that neither depressed nor facilitated in response to short bursts of high f r e q u e n c y stimulation (Fig. 5C,D). That these alterations in lateral abductor and adductor synaptic responses w e r e not simply the result o f a r a n d o m set of excitatory m o t o n e u r o n s reinnervating the lateral abductor and adductor m u s c l e s is indicated by the consistency of our results for all except three of the regenerated preparations. A l t h o u g h EJP amplitudes w e r e u n c h a n g e d f o l l o w i n g reinnervation o f the superficial a b d o m i n a l flexor excitors in crayfish [7,17], investigations focused on the fast closer excitor o f the crayfish c l a w have identified several factors that can alter m o t o n e u r o n phenotype. Phenotypic changes include increasing or decreasing EJP amplitude a n d / o r transforming the extent o f EJP depression or facilitation w h e n stimulated repetitively. C i r c u m s t a n c e s that affect fast closer p h e n o t y p e include: c l a w regeneration [31]; c h a n g i n g the level of m o t o n e u r o n impulse activity [24]; altering sensory input to the m o t o n e u r o n [23,27], and d e v e l o p m e n t a l aging [1,22]. Other preparations w h i c h display similar alterations in n e u r o m u s c u l a r synaptic function include: c h a n g i n g the level o f m o t o r neuron activity for the leg opener [30,34] and phasic a b d o m i n a l extensor motoneurons [26] in crayfish; and the a s y m m e t r i c a l d e v e l o p ment o f lobster c l a w s [10]. M e c h a n i s m s responsible for these changes in n e u r o m u s cular function are presynaptic [1,2,37]. T h e y include structural alterations in synapse size and synaptic ultrastructure [11,12,19,25,36]; changes in Na ÷ a n d / o r Ca 2+ a c c u m u l a tion in the m o t o n e u r o n terminals [25,28,34]; shift o f synapses b e t w e e n ' a c t i v e ' and ' i n a c t i v e ' states [2,35,36]; and neurotransmitter depletion [22,37]. P r e s u m a b l y one or m o r e of these p h e n o m e n a contribute to the alterations in m o t o n e u r o n p h e n o t y p e o b s e r v e d in the reinnervated uropod musculature.
Acknowledgements This w o r k was supported by a Bureau o f B i o l o g i c a l Research, B u s c h M e m o r i a l Research Grant to C . H . P . H . J.L. w a s supported by a grant f r o m the National S c i e n c e Council, R.O.C.
References [1] Atwood, H.L., Age-dependent alterations of synaptic performance and plasticity in crustacean motor systems, Exp. Gerontol., 27 (1992) 51-61. [2] Atwood, H.L. and Wojtowicz, J.M., Short-term and long-term plasticity and physiological differentiation of crustacean motor synapses, Int. Rev. Neurobiol., 28 (1986) 275-262. [3] Atwood, H.L., Dudel, J., Feinstein, N. and Parnas, 1., Long-term survival of decentralized axons and incorporation of satellite cells in motor neurons of rock lobsters, Neurosci. Lett., 101 (1989) 121-126. [4] Bittner, G.D., Degeneration and regeneration in crustacean neuromuscular systems, Am. Zool., 13 (1973) 379-408. [5] Bittner, G.D., Trophic interactions of crustacean neurons. In G. Hoyle (Ed.), Identified Neurons and Behatior, Plenum, New York, 1977, pp. 507-532. [6] Bittner, G.D., Long-term survival of anucleate axons and its implications for nerve regeneration, Trends Neurosci., 14 (1991) 188-193. [7] Ely, P. and Velez, S.J., Regeneration of specific neuromuscular connections in the crayfish. I. Pattern of connections and synaptic strength, J. Neurophysiol., 47 (1982) 656-666. [8] Fawcett, J.W. and Keynes, R.J., Peripheral nerve regeneration, Annu. Reu. Neurosci., 13 (1990) 43-60. [9] Fourtner, C.R., Drewes, C.D. and Holzmann, T.W., Specificity of afferent and efferent regeneration in the cockroach: establishment of a reflex pathway between contralaterally homologous target cells, J. Neurophysiol., 41 (1978) 885-895. [10] Govind, C.K., Development of asymmetry in the neuromuscular system of lobster claws, Biol. Bull., 167 (1984) 94-119. [11] Govind, C.K., Age-related remodeling of lobster neuromuscular terminals, Exp. Gerontol., 27 (1992) 63-74. [12] Govind, C.K., Atwood, H.L. and Lang, F., Synaptic differentiation in a regenerating crab-limb muscle, Proc. Nat. Acad. Sci. USA, 71 (1973) 822-826. [13] Higuchi, T., Topography and distribution of motor neurons supplying uropod muscles of a crayfish, Comp. Biochem. Physiol., 103A (1992) 705-709. [14] Higuchi, T., Dual excitatory innervation through the different ganglionic roots in the uropod adductor muscle of a crayfish, Proeambarus clarkii, Comp. Biochem. Physiol., 109A (1994) 377-383. [15] Hoy, R.R., Degeneration and regeneration in abdominal flexor motor neurons in the crayfish, J. Exp. Zool., 172 (1969) 219-232. [16] Hunt, W.P. and Velez, S.J., Regeneration of specific neuromuscular connections in the crayfish. I. Patterns of reconnection and synaptic strength established in normal and altered target areas, J. Neurobiol., 20 (1982) 666-676. [17] Hunt, W.P. and Velez, S.J., Regeneration of an identifiable motoneuron in the crayfish. I. Patterns of reconnection and synaptic strength established in normal and altered target areas, J. Neurobiol., 20 (1989) 703-717. [18] Hunt, W.P. and Velez, S.J., Regeneration of an identifiable motoneuron in the crayfish. II. Patterns of reconnection and synaptic strength established in the presence of an extra nerve, J. Neurobiol., 20 (1989) 718-730. [19] Katz, P.S., Kirk, M.D. and Govind, C.K., Facilitation and depression at different branches of the same motor axon: evidence for presynaptic differences in release, J. Neurosci., 13 (1993) 3075-3089. [20] Krasne, F.B. and Lee, S.H., Resistance of a crayfish sensory interneurone to hyperinnervation by acceptable afferents, J. Physiol., 331 (1982) 35-49. [21] Larimer, J.L. and Kennedy, D., Innervation patterns of fast and slow muscle in the uropods of crayfish, J. Exp. Biol., 51 (1969) 119-133. [22] Lnenicka, G.A. and Atwood, H.L., Age dependent long-term adaptation of crayfish phasic motor axon synapses to altered activity, J. Neurosci., 5 (1985) 459-467. [23] Lnenicka, G.A. and Atwood, H.L., Long-term changes in neuromus-
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[24]
[25]
[26] [27]
[28]
[29]
[30]
cular synapses with altered sensory input to a crayfish motoneuron, Exp. Neurol., 100 (1988) 437-447. Lnenicka, G.A. and Atwood, H.L., Impulse activity of a crayfish motoneuron regulates its neuromuscular synaptic properties, J. Neurophysiol., 61 (1989) 91-96. Lnenicka, G.A., Atwood, H.L. and Marin, L., Morphological transformation of synaptic terminals of a phasic motoneuron by long-term tonic stimulation, J. Neurosci., 6 (1986) 2252-2258. Mercier, A.J. and Atwood, H.L., Long-term adaptation of a phasic extensor motoneurone in crayfish, J. Exp. Biol., 145 (1989) 9-22. Pahapill, P.A., Lnenicka, G.A. and Atwood, H.L., Asymmetry of motor impulses and neuromuscular synapses produced in crayfish claws by unilateral immobilization, J. Comp. Physiol., 157 (1985) 461-467. Pahapill, P.A., Lnenicka, G.A. and Atwood, H.L., Long-term facilitation and low frequency depression in a crayfish phasic motor axon, J. Comp. Physiol., 161A (1987) 367-375. Parnas, I., Dudel, J. and Atwood, H.L., Synaptic transmission in decentralized axons of rock lobster, J. Neurosci., 11 (1991) 13091315. Sherman, R.G. and Atwood, H.L., Synaptic facilitation: long-term
[31] [32]
[33] [34]
[35]
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[37]
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neuromuscular facilitation in crustaceans, Science, 171 (1971) 1248-1250. Stewart, B.A. and Atwood, H.L., Synaptic plasticity in a regenerated crayfish phasic motoneuron, J. Neurobiol., 23 (1992) 881-889. Takahashi, M. and Hisada, M., The slow muscles in the uropod of crayfish Procambarus clarkii, Comp. Biochem Physiol., 95A (1990) 601-606. Van Harreveld, A.A., A physiological solution for freshwater crustaceans, Proc. Soc. Exp. Biol., 34 (1936) 428-432. Wojtowicz, J.M. and Atwood, H.L., Correlation of presynaptic and postsynaptic events during establishment of long-term facilitation at crayfish neuromuscular junction, J. Neurophysiol., 54 (1985) 220230. Wojtowicz, J.M. and Atwood, H.L., Long-term facilitation alters transmitter releasing properties at the crayfish neuromuscular junction, J. Neurophysiol., 55 (1986) 484-498. Wojtowicz, J.M., Marin, L. and Atwood, H.L., Activity-induced changes in synaptic release sites at the crayfish neuromuscular junction, J. Neurosci., 14 (1994) 3688-3703. Zucker, R.S., Short-term synaptic plasticity, Annu. Rev. Neurosci., 12 (1989) 13-31.
ELSEVIER
Developmental Brain Research 87 (1995) 179-187
Research report
Motoneuron reinnervation of phasic uropod muscles in crayfish How-Jing Lee 1, Charles H. Page * Department of Biological Sciences, and Bureau of Biological Research, Rutgers University, Piscataway, NJ 08855, USA Accepted 11 April 1995
Abstract Motoneuron reinnervation of the lateral abductor and adductor muscles in the exopodite of the crayfish uropod was obtained by cross-tying the cut proximal ends of one set of uropod nerve roots to the cut distal ends of their contralateral homologues. In normal (nonsurgically treated) animals the abductor was innervated by two excitatory motoneurons and an inhibitor while two excitors innervated the adductor. For each muscle one of the excitors produced large excitatory junction potentials (EJPs) while the other evoked small EJPs. When stimulated repetitively only the smaller EJP in the adductor generated a facilitating response. Within less than 10 weeks postsurgery the muscles were each reinnervated by two excitatory motoneurons. While the abductor motoneurons generated synaptic potentials with similar amplitudes and time courses to those of normal animals, they differed from those in normal animals in that they facilitated when stimulated repetitively. In contrast to the large and small EJPs evoked in the normal animal, the two motoneurons that reinnervated the adductor muscle elicited similar amplitude EJPs, neither of which facilitated in response to repetitive stimulation. Keywords: Motoneuron regeneration; Neuromuscular reinnervation; Synaptic plasticity; Crayfish; Uropod
1. Introduction
Surgical interruption of motor axons in a peripheral nerve initiates a regenerative response whereby the sectioned motoneurons reinnervate the muscles that they originally innervated [8]. Typically this process involves rapid degeneration of the distal segments of the severed motor axons and outgrowth from their proximal cut ends to form synaptic contacts on the denervated muscle fibers. In contrast to vertebrates and most other invertebrates, in crustaceans degeneration of the distal axon stump is often very slow, taking up to several months to complete [4,6]. In some circumstances the distal stump can remain viable for many months following the cutting of the motor axon, with or without muscle reinnervation, while in other instances the stump degenerates within several weeks [3,4,16]. As a result, although some investigations of motoneuron regeneration and muscle reinnervation in crustaceans have reported the reestablishment of the original pattern of neuromuscular innervation, there is considerable
* Corresponding author. 1 Present address, Department of Plant Pathology and Entomology, National Taiwan University, Taipei, Taiwan, R.O.C. 0165-3806/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 3 8 0 6 ( 9 5 ) 0 0 0 7 4 - 7
variability in the regenerative capacity of different preparations and their time course for neuromuscular reinnervation [3,7,17,29]. Critical factors include where the axon is sectioned (within the nerve cord or at various points along the peripheral nerve including axon branches on the muscle surface), and the length to which the severed proximal stump is brought into contact with either the distal stump or the denervated muscle fibers [4,5,15,16]. The uropod appendages and caudalmost segments of the crayfish abdomen are innervated by nerves (roots) that project from the terminal (sixth) abdominal ganglion in a fan-like arrangement. Although the distal and proximal ends of these roots retract when cut and therefore cannot be easily brought back into contact by tieing, the projection of the roots in a radial pattern from the ganglion enables the long proximal end of one sectioned root to be cross-tied to the long distal end of its contralateral homologue. This surgical procedure was developed originally to study sensory neuron reinnervation of the terminal ganglion [20]. Each uropod consists of a basal protopodite with attached exopodite and endopodite. Their muscles are innervated by motoneurons that originate in the terminal ganglion, and run in the 2nd and 3rd roots to innervate the uropod [13,14]. Since previous investigations in crayfish
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and insects have concluded that a motoneuron can innervate a denervated muscle that is the contralateral homologue of the normally innervated muscle [9,16,18], crosstying the roots that innervate the uropods should provide a useful preparation for examining motoneuron reinnervation of selected uropod muscles. The physiological experiments described in this report focus on the innervation and reinnervation of the large phasic lateral abductor and adductor muscles which respectively open and close the uropod exopodite.
2. Materials and methods 2.1. The crayfish Procambarus clarkii Girard, of both sexes with carapace lengths of 3 - 5 c m were obtained from Wauban Laboratories (Schriever, LA). Upon receipt their chelae were autotomized to reduce cannibalism. The animals were maintained in large tanks containing 23-25°C dechlorinated water that was continuously aerated and filtered, and fed dried dog food twice a week. 2.2. Analysis of normal muscle innervation Innervation of the lateral abductor and adductor muscles was analyzed by intracellular recording of muscle fiber responses evoked by stimulating motoneurons in the 2nd and 3rd roots which exit the terminal (sixth) abdominal ganglion to innervate the uropods. All experiments were performed on an isolated abdomen, pinned ventral side up on a wax block in an experimental chamber filled with oxygenated (12-15°C) crayfish saline [33]. The nerve cord and uropod roots were exposed for stimulation by removing the overlying cuticle. Abductor and adductor motoneuron impulses were initiated by stimulating the cut distal end of the root running to the muscle with a saline filled suction electrode. A Grass S-48 stimulator generated the stimulus pulses and pulse trains. Two small windows were cut in the ventral cuticle of the uropod exopodite for microelectrode penetration of abductor and adductor muscle fibers. Movement artifacts were minimized by breaking the shank of the 3M KC1 microelectrode (10-20 MY2) near the tip and suspending the tip from a fine silver wire before impaling a muscle fiber. Muscle potentials were fed into a S-7100A WPI electrometer, displayed on a storage oscilloscope and photographed with a Polaroid oscilloscope camera. Positive images were obtained from the negative prints by scanning the prints and then reversing them using commercial software (PhotoShop). In some experiments the GABA antagonist picrotoxin (Sigma) was infused into the bath to test whether peripheral inhibitor activity was responsible for reducing the
amplitude of an excitatory junction potential (EJP). To avoid dislodging the microelectrode from the impaled muscle fiber when changing solutions, a modified chamber with a volume of 25 ml was used in which saline containing 57/~M picrotoxin was introduced on one side and after flowing over the preparation was drained from the other side. The flow rate was about 80 ml/min. 2.3. Muscle denervation procedure The surgical protocol was modified from Krasne and Lee [20]. Healthy (active and alert) animals selected for surgery were immobilized in 5°C water before securing them ventral side up in a chamber filled with 8°C oxygenated saline. The terminal abdominal ganglion and its 2nd and 3rd roots were exposed by: (1) cutting the abdominal sternite overlying the ganglion at its lateral margins; (2) making a U-shaped incision in the soft cuticle, extending posteriorly from the sternite cuts to the base of the uropod protopodites and then medially along the proximal edge of the telson; and (3) folding back the U-shaped flap of soft cuticle freed by the incision. A single strand of dental floss, stained with methylene blue to enhance visibility, was tied around the left pair of 2nd and 3rd roots at a point near the ganglion. After cutting the roots between the tie and the ganglion, the long distal stump was gently pulled over to the still intact right (contralateral) pair of roots and tied close to the contralateral uropod. Finally the intact right roots were cut between the tie and the right uropod, thereby physically cross-connecting the left uropod to the right side of the ganglion. As a result of cross-connecting the left uropod to the contralateral side of the ganglion, the right uropod was separated from the ganglion by a gap of several mm. Throughout surgery care was taken to avoid puncturing the artery that runs around the posterior end of the ganglion. Following completion of surgery the wound was closed by positioning the cuticular flap in place and allowing the wound to clot in air for 20min. The animals were placed in individual containers of 50% saline for 7 days and then returned to a flesh water holding aquarium for 7 - 9 additional weeks, before recording the synaptic responses produced in their reinnervated muscle fibers with intracellular microelectrodes. The survival rate was 75% at 8-10 weeks postsurgery. Microelectrode recordings were obtained from the reinnervated muscle fibers of 21 animals, 8 of which molted successfully during the postsurgical period. Whether or not an animal molted in the period following surgery had no noticeable effect on the synaptic responses of the reinnervated muscle fibers. 2.4. Analysis of muscle reinnervation Electromyograms were recorded periodically from the denervated muscles to monitor their reinnervation. Recordings were obtained by inserting pairs of 100 /xm insulated
H. -J. Lee, C.H. Page/Developmental Brain Research 87 (1995) 179-187
181
A
B
E
F 10 ms
Fig. 1. Intracellular recordings of muscle potentials generated in the lateral abductor and adductor muscles of normal crayfish. A: successive increments in the intensity of 3rd root stimulation (3.0, 3.1 and 3.2 V) activated the three lateral abductor motoneurons. The lower voltages were threshold stimuli for eliciting respectively a small EJP and a large compound EJP. Further increase in stimulus strength (3.2 V) reduced the amplitude and duration of the compound EJP, presumably reflecting activation of the peripheral inhibitor (middle trace). B: in another preparation, the lowest threshold 3rd root stimulus (3.2 V) produced a large abductor EJP. When the stimulus voltage was raised (3.4 V) to activate the second excitor a larger compound EJP was generated. Further increase in stimulus voltage (4.0 V) excited the peripheral inhibitor thereby decreasing the amplitude and duration of the compound EJP (middle trace). C: a spike-like response generated by an abductor excitor. D: stimulation of an adductor motoneuron in the 2nd root produced a large EJP. E: a spike-like response generated by the adductor excitor in the 2nd root. F: a small adductor EJP evoked by 3rd root stimulation. For C and E the slow potential following the spike-like response is a mechanical artifact. Muscle responses were evoked by 3 - 7 V, 0.2 ms pulses applied to the cut distal end of the 2nd or 3rd root of the terminal abdominal ganglion. The stimulus triggered the oscilloscope trace.
copper wires into the lateral abductor a n d / o r adductor muscles of animals restrained in a chamber filled with dechlorinated water. Muscle potentials were differentially amplified (P15 Grass preamplifier) and displayed on a storage oscilloscope. An arbitrary scoring system was devised to quantify the strength of the muscle responses evoked by strong tactile stimulation of the abdomen. An absence of muscle responses to the stimulus was scored 0, muscle potential responses smaller than 50 tzV were scored 1, while those larger than 50 /xV were assigned a score of 2.
A
Intracellular microelectrode recordings were conducted as described above to determine the extent of motoneuron reinnervation of the lateral abductor and adductor muscles at 8 - 1 0 weeks postsurgery. A fine-tip ( 1 2 5 - 2 0 0 /zm diameter) glass pipette containing 4 M NaCI connected to a S-48 Grass stimulator was used to apply voltage pulses to the 2nd or 3rd roots, or the interganglionic connectives, to elicit impulses in the motoneurons that reinnervated the muscles. In a few preparations, the cross-connected roots were cut at the base of the ganglion and the distal ends of 2nd or 3rd roots stimulated with a suction electrode.
C
D
3 0.1s
Fig. 2. Intracellular recordings of lateral abductor and adductor responses to pulse train stimulation in normal crayfish. A: responses produced by the lowest threshold (6.5 V) abductor excitor to train stimulation of the 3rd root. B: compound EJPs produced by 7.3 V pulse trains activated both abductor excitors. A and B recorded from the same muscle fiber. C: following the initial response, continued stimulation of the 2nd root generated constant amplitude adductor EJPs. D: facilitation of small adductor EJPs initiated by train stimulation of 3rd root. Stimulus was a 500-ms train of 0.2-ms pulses at 32/s.
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3. Results
A
3.1. Inner~,ation of lateral abductor and adductor muscles
In agreement with Higuchi [13,14], the lateral abductor and adductor muscles are innervated by motoneurons that exit from the terminal abdominal ganglion in the 2nd and 3rd roots. The 3rd root contains the three lateral abductor motoneurons--two excitors and a peripheral inhibitor (Fig. 1A,B), and one of the two adductor excitors (Fig. 1F). The axon of the other adductor excitor is in the 2rid root (Fig. 1D,E). 3.2. Lateral abductor
The three lateral abductor motoneurons were activated in succession by increasing the voltage of the stimulus pulses applied to the 3rd root. Typically the two excitors had lower thresholds than the peripheral inhibitor. Although the threshold voltages for initiating impulses in the two excitors were similar, the excitatory junction potentials (EJPs) that the two excitors evoked in lateral abductor muscle fibers differed in amplitude; one producing only a small EJP while the other initiated a large EJP (lowest traces in Fig. 1A,B respectively). In each instance a small increase in stimulus voltage activated the second excitor. The resulting compound EJPs (upper traces in Fig. 1A,B) reflect the simultaneous stimulation of the two excitors. The initial response of the larger excitor sometimes included a spike-like potential which decayed during repetitive stimulation (Fig. 2. 1C, 2A,B). Innervation of lateral abductor muscle fibers was further characterized by examining their responsiveness to motoneuron stimulation by stimulus pulse trains. In our preparations, the maximum rate of stimulation for which each stimulus pulse would elicit an EJP was about 30 per s. Muscle responses rapidly diminished in size during the first several stimulus pulses (Fig. 2A,B). Following this initial decrease in size, EJP amplitudes remained constant for the remainder of the pulse train. The threshold voltage required to activate the peripheral inhibitor by 3rd root simulation was higher than that for the excitors. Typical thresholds for initiating impulses in the three motoneurons were: 3.3 V for the first excitor, 3.5 V for the second excitor and 4.0 V for the inhibitor. The primary effects of the peripheral inhibitor were to reduce the amplitude and duration of the compound EJP produced by the two excitors (compare upper and middle traces in Fig. 1A,B). To further investigate whether the decrease in EJP size and duration resulted from activity of a GABAergic inhibitor, we examined the effects of exposing the preparation to picrotoxin, a GABA antagonist. As illustrated in Fig. 3, 57 /xM picrotoxin almost entirely eliminated the inhibitory depression of the EJP. In contrast, exposure to picrotoxin never affected the compound EJP generated by stimulating the two excitors if the stimulus
B
5 ms
Fig. 3. Picrotoxin blocked the peripheral inhibitor response in the lateral abductor. A: responses to successive increments in stimulus intensity of 3.3, 3.5 and 4.0 V which activated respectively a first excitor (middle trace), the peripheral inhibitor (lowest trace), and the second excitor (largest trace). Note that the inhibitor reduced the amplitude and duration of the first excitor's EJP. B: responses recorded from the same three abductor motoneurons after 14 min exposure to 57/zM picrotoxin. Note that the inhibitory reduction in EJP size and duration is almost completely blocked (compare lowest trace with that in A). The membrane potential was constant ( - 74 mV) throughout the period of recording A and B.
pulses were subthreshold for activating the inhibitor. Picrotoxin blocking of EJP inhibition could not be reversed, even after 48 min washout with crayfish saline. There was considerable variability in motoneuron innervation of the lateral abductor muscle fibers. Out of a total of 16 muscle fibers, each examined in a different crayfish, 19% were innervated by all three motoneurons, 56% by a single excitor and an inhibitor, 12% by two excitatory motoneurons only, and 13% by a single excitor. 3.3. Adductor
In contrast to previous reports [13,14], the muscle responses generated by separately stimulating the two adductor excitors differed markedly in size, and responsiveness to high frequency stimulation. A large depolarizing potential that usually included a spike-like component was produced by stimulating the 2nd root motoneuron (Fig. 1D,E, 2C), while the excitor running in the 3rd root always evoked a small to medium size EJP (Fig. 1F, 2D). The responses of the 2nd root excitor to pulse train stimulation were similar to those of the abductor excitors (Fig. 2C). After an initial drop in size, EJP amplitudes remained relatively constant for the duration of the pulse train. In contrast, EJPs of the 3rd root excitor always facilitated when the excitor was repetitively stimulated (Fig. 2D). All of the adductor muscle fibers examined were innervated by both motoneurons. None of the recordings ever provided any evidence for peripheral inhibitor innervation of the adductor. The absence of an adductor inhibitor was supported by the lack of any change in adductor EJP amplitude or duration when the preparation was bathed with 57/zM picrotoxin (not shown).
H.-J. Lee, C.H. Page / Developmental Brain Research 87 (1995) 179-187 Table 1 Electromyogram of sensory evoked muscle responses from the reinnervated uropod Weeks
Reconnected
Unconnected
postsurgery
n
ABD
ADD
ABD
ADD
3-4 5-6 7-8 9-10
17 11 13 9
0.59+0.80 0.91+0.94 0.92_+0.64 1.44+0.73
0.88+0.78 0.73_+0.90 1.08_+0.64 1.44+0.73
0.12+0.49 0 0.15_+0.38 0.56+0.73
0.12+0.49 0 0.15_+0.38 0.56+0.73
Responses (mean + S.E.) to tactile stimulation of ventral abdominal cuticle. Scoring system: 0 = no muscle response; 1 = muscle response smaller than 50 /zV; 2 = muscle response larger than 50 /zV. ABD is lateral abductor; ADD is adductor.
3.4. Reinnervation of lateral abductor and adductor muscles
Beginning 3 to 4 weeks postsurgery, tactile stimulation of the ventral abdominal surface elicited electromyogram responses in the lateral abductor and adductor muscles of many animals (Table 1). There was a marked difference in recovery between the muscles in the surgically cross-connected (left) and unconnected (right) uropods. Even at 10 weeks postsurgery, sensory evoked responses were rarely recorded from muscles on the unconnected side. As a result our intracellular analysis focused on reinnervation of the cross-connected side, i.e. the abductor and adductor muscles that had been cross-tied to the stumps of the contralateral 2nd and 3rd roots. Dissection of regenerated preparations 9 - 1 0 weeks postsurgery revealed varying amounts of scar tissue surrounding the point where the roots innervating the (left) uropod had been cross-tied to the proximal ends of the contralateral (right) 2nd and 3rd roots. Cross-connection of these roots provided the morphological basis for the physiological responses recorded from the muscles in the
A
183
surgically connected (left) uropod in response to stimulation of the proximal segments of the contralateral roots. Outgrowth from the short stumps of the unconnected (left) 2nd and 3rd roots often ran along the 1st a n d / o r 4th (left) roots to reach the scar tissue. Although it was very difficult to follow these outgrowing axons within the scar tissue matrix, the almost complete absence of muscle responses recorded from the surgically unconnected (right) uropod, indicates that at 10 weeks postsurgery these axons rarely reinnervated either the lateral abductor or the adductor muscles. There was a 15 to 20 mV increase in the membrane potential of the reinnervated muscle fibers at 8-10 weeks postsurgery. Membrane potentials of the lateral abductor shifted from - 6 6 . 4 mV ___11.1 (n = 40) in normal (nonsurgically treated) animals to - 86.5 mV ___12.3 (n = 20) ( P < 0.00002, t-test) for reinnervated preparations, with a similar change for the adductor fibers (-69.9mV___ 11.7 in normals (n = 20) to - 8 6 . 2 mV + 13.9 in reinnervated muscles (n = 20) ( P < 0.01, t-test)). The membrane potentials of muscle fibers from the surgically unconnected (right) uropod underwent a similar though smaller increase in amplitude ( - 7 8 . 3 ___12.5 mV for the lateral abductor and - 7 7 . 2 mV___ 13.7 mV for the adductor, n = 20 for both). At 8 to 10 weeks postsurgery, synaptic responses could be recorded with intracellular microelectrodes from the reinnervated lateral abductor and adductor muscles in 84% (21 out of 25) of the surgically cross-connected preparations. EJPs were obtained from the reinnervated muscles in response to tactile stimulation of the abdomen, electrical stimulation of fiber bundles in the interganglionic connectives, focused stimulation by a fine tip stimulating electrode positioned on the surface of the appropriate 2nd or 3rd root (Fig. 4 and Fig. 5) and in a few preparations suction electrode stimulation of the distal end of a cross-
D
IP
.= _
20 ms B
C 0.1s
Fig. 4. Responses of reinnervated lateral abductor muscle fibers. A: stimulation of the 3rd root evoked an abductor EJP. B and C: in another preparation pulse train stimulation of both abductor excitors elicited EJP facilitation. Excitor thresholds were 11 V and 14 V respectively. D: in a third preparation, following rapid decay of the spike-like response the abductor EJPs undergo slow facilitation. Stimulus was a 0.2 ms pulse train at 3 5 / s for 550 ms (B and C) and 3 0 / s for 540 ms (D). B, C and D have the same time calibration. Animals were 56-61 days postsurgery.
H.-J. Lee, C.H. Page / DeL'elopmental Brain Research 87 (1995) 179-187
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A
c
B
D
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Fig. 5. Responses of a reinnervated adductor muscle fiber. A: an adductor EJP elicited by 2nd root stimulation. B: third root stimulation initiated another adductor EJP. C and D: the amplitudes of adductor EJPs remained unchanged during pulse train stimulation of the 2nd (C) and 3rd (D) roots. All recordings were from the same muscle fiber. The stimulus train of 0.2ms pulses was 3 0 / s for a period of 540 ms. Time since surgery was 73 days.
tor EJPs was ever observed that could be attributed to peripheral inhibitor activity. Pulse train stimulation always produced a facilitating response in the abductors (Fig. 4B,C). Even for those responses which began with a large spike-like potential, following an initial rapid decline, EJP amplitude slowly increased for the duration of the stimulus (Fig. 4D). Adductor EJPs of similar size could be initiated by stimulating either the 2nd or 3rd cross-connected roots (Fig. 5). While these 8-15 mV EJPs were smaller than the 30-40 mV EJPs generated by 2nd root stimulation in normal animals (Fig. 1D, 2C), they were somewhat larger than those initiated by stimulating the 3rd root ( 3 - 9 mV)
connected root that was cut close to the ganglion. In contrast, 'spontaneously generated' EJPs were not observed in any of these dissected preparations. Stimulation of the cross-connected 3rd root always initiated one or two different EJPs in the reinnervated lateral abductor muscle fibers (Fig. 4A, B,C). Although their amplitudes and time courses were similar to the abductor EJPs recorded from normal animals (Fig. 1A,B), muscle responses in regenerates differed from those of normal crayfish both in the absence of peripheral inhibition and the development of abductor EJP facilitation. With the exception of an unusual preparation described below (Fig. 6A), no change in the amplitude of the abduc-
A
C
B
D
20 ms
<
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Fig. 6. Unusual patterns of reinnervation. A and B: hyperinnervation of a lateral abductor. Stimulation of the 3rd root (A) activated two excitors (middle and upper traces) and a peripheral inhibitor (lower trace is compound EJP resulting from decrease in first excitor EJP produced by simultaneous activation of the inhibitor) in the lateral abductor. In the same animal, stimulation of the 2nd root (B) excited two additional abductor excitors. C and D: the sizes of the adductor EJPs elicited by stimulating the 2nd and 3rd roots were reversed from normal crayfish. Second root stimulation produced a small EJP (C) while a large EJP (D) was generated in response to 3rd root stimulation. E: one of the two motoneurons that reinnervated the adductor muscle exited from the ipsilateral side of the terminal ganglion. Stimulation of the crossconnected third root elicited a large EJP, while similar stimulation close to the severed stumps of the ipsilateral (unconnected) roots initiated a very small EJP. Time since surgery ranged from 56 to 61 days.
H.-J. Lee, C.H. Page/ Developmental Brain Research 87 (1995) 179-187
in normal crayfish (Fig. IF, 2D). In contrast to the characteristic facilitation of 3rd root adductor EJPs in normal animals (Fig. 2D), neither the 2nd nor the 3rd root adductor EJPs facilitated in the surgically cross-connected animals (Fig. 5C,D). Typically there was minimal change in the amplitude of the adductor EJPs during pulse train stimulation. Unusual reinnervation patterns were observed in three of the surgically reconnected animals. In one crayfish the lateral abductor muscle was innervated by 5 motoneurons. Stimulation of the cross-connected 3rd root activated two excitatory motoneurons and a peripheral inhibitor in the abductor (Fig. 6A), while stimulation of the 2nd root excited two additional excitatory motoneurons (Fig. 6B). In contrast, reinnervation of the adductor was n o r m a l - motoneurons running in both the 2nd and 3rd roots (not shown). In another animal, a large adductor EJP was generated by 3rd root stimulation and a small EJP when the 2nd root was stimulated (Fig. 6C,D). This is opposite to the pattern observed in normal animals where large and small EJPs are produced respectively by 2nd and 3rd root motoneurons (Fig. 1D,F). Finally, in a third preparation an adductor received weak innervation from the unconnected (ipsilateral) side of the ganglion. Stimulation of the crossconnected 3rd root evoked a large EJP in the adductor (Fig. 6E, upper trace), while a small adductor EJP was generated by stimulating the ipsilateral root stump (Fig. 6E, middle trace).
4. Discussion
The recordings of motoneuron responses from lateral abductor and adductor muscle fibers in normal (nonsurgically treated) crayfish, while confirming the general pattern of motoneuron innervation of these muscles [13,14], provide new information concerning the individual characteristics of their motoneurons. Despite the phasic categorization of these muscles, based on short sarcomere lengths [21] and high levels of myofibrillar ATPase activity [32], only one of the two excitatory motoneurons innervating each muscle has strictly phasic characteristics--large EJPs that undergo an initial depression with repetitive stimulation (Fig. 1B,D, 2B,C). In contrast, the other motoneurons produce smaller EJPs that when repetitively stimulated depress initially in the abductor and facilitate in the adductor (Fig. 1A,F, 2A, D). Our observation that stimulation of the 2nd and 3rd roots elicits respectively large (often with a spike-like component) and small adductor EJPs (Fig. 1D,E,F), differs from previous reports that separate stimulation of these two roots with oil-hook electrodes initiates similar action potentials in adductor muscle fibers [13,14]. Differences in methods of stimulation provide the most likely explanation for these conflicting results. When in preliminary experiments we used wire electrodes to separately stimulate the
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2nd and 3rd roots, we were unable to initiate a small 3rd root EJP without simultaneously evoking a large 2nd root EJP. Since the 2nd and 3rd roots run in very close contact as they enter the uropod, apparently the stimulus current applied to the 3rd root can spread along the root to the base of the uropod where it activates the 2nd root excitor, producing a large compound F_JP similar to that evoked by direct stimulation of the 2nd root. In contrast, when we stimulated the cut proximal end of the 3rd root with an attached suction electrode, small 3rd root EJPs could be readily elicited without activating the much larger 2nd root EJPs (Fig. 1F, 2D). 4.1. Reinnercation pattern
The muscle responses evoked by tactile stimulation of the abdomen, and electrical stimulation of the nerve cord connectives, prove that the uropod muscles were functionally reinnervated. The pattern of excitatory motoneuron reinnervation of the lateral abductor and adductor muscles was similar to that observed in normal crayfish, except that the reinnervating motoneurons exited from the contralateral half of the terminal ganglion. The similarity in the synaptic responses evoked by focused electrical stimulation of the cross-connected roots, and those initiated by tactile or connective stimulation, indicates that the abductor and adductor responses were evoked by the activation of motor axons that ran in the cross-connected roots to form functional connections between the 6th ganglion and the uropod muscles. The contralateral origin of the regenerated motoneurons presumably resulted from having surgically cross-tied the long distal ends of the uropod's 2nd and 3rd ganglionic roots to the long proximal stumps of the contralateral 2nd and 3rd roots. This conclusion is supported by reports in crayfish and insects that denervated muscles can receive an otherwise normal reinnervation from motoneurons that are contralateral homologues of their normal innervation [9,16,18]. In contrast to the recording of excitor EJPs from almost all regenerates, peripheral inhibitor reinnervation was observed in only one animal at 10 weeks postsurgery. This preparation was atypical, since the lateral abductor was reinnervated by an inhibitor and a supernumery set of excitors (Fig. 6A, B). Therefore either the peripheral inhibitor does not typically reinnervate the lateral abductor, or inhibitory reinnervation of the lateral abductor requires more than 10 weeks. Alternately weak inhibition reinnervation might not have noticeably altered the amplitude of the excitor EJPs and therefore could have been undetectable in our recordings. 4.2. Change in motoneuron phenotype
Although both the lateral abductor and adductor muscles were reinnervated by the same number of excitors as normal animals, the muscle responses generated by these
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regenerated m o t o n e u r o n s differed f r o m those recorded from normal animals, both in the amplitudes o f their E J P s and in their responses to repetitive stimulation. The p h e n o t y p e of each lateral abductor excitor was altered in the regenerates. W h i l e the two abductor excitors still p r o d u c e d small and large EJPs, both of w h i c h w e r e subject to short term depression in normal animals (Fig. 2A,B), f o l l o w i n g reinnervation the EJPs of these two excitors facilitated in response to high f r e q u e n c y stimulus trains (Fig. 4B,C). A l t h o u g h the two adductor m o t o n e u r o n s p r o d u c e d a small facilitating E J P and a large EJP w h i c h initially depressed in normal crayfish (Fig. 2C,D), in regenerates the two excitors e v o k e d intermediate size EJPs that neither depressed nor facilitated in response to short bursts of high f r e q u e n c y stimulation (Fig. 5C,D). That these alterations in lateral abductor and adductor synaptic responses w e r e not simply the result o f a r a n d o m set of excitatory m o t o n e u r o n s reinnervating the lateral abductor and adductor m u s c l e s is indicated by the consistency of our results for all except three of the regenerated preparations. A l t h o u g h EJP amplitudes w e r e u n c h a n g e d f o l l o w i n g reinnervation o f the superficial a b d o m i n a l flexor excitors in crayfish [7,17], investigations focused on the fast closer excitor o f the crayfish c l a w have identified several factors that can alter m o t o n e u r o n phenotype. Phenotypic changes include increasing or decreasing EJP amplitude a n d / o r transforming the extent o f EJP depression or facilitation w h e n stimulated repetitively. C i r c u m s t a n c e s that affect fast closer p h e n o t y p e include: c l a w regeneration [31]; c h a n g i n g the level of m o t o n e u r o n impulse activity [24]; altering sensory input to the m o t o n e u r o n [23,27], and d e v e l o p m e n t a l aging [1,22]. Other preparations w h i c h display similar alterations in n e u r o m u s c u l a r synaptic function include: c h a n g i n g the level o f m o t o r neuron activity for the leg opener [30,34] and phasic a b d o m i n a l extensor motoneurons [26] in crayfish; and the a s y m m e t r i c a l d e v e l o p ment o f lobster c l a w s [10]. M e c h a n i s m s responsible for these changes in n e u r o m u s cular function are presynaptic [1,2,37]. T h e y include structural alterations in synapse size and synaptic ultrastructure [11,12,19,25,36]; changes in Na ÷ a n d / o r Ca 2+ a c c u m u l a tion in the m o t o n e u r o n terminals [25,28,34]; shift o f synapses b e t w e e n ' a c t i v e ' and ' i n a c t i v e ' states [2,35,36]; and neurotransmitter depletion [22,37]. P r e s u m a b l y one or m o r e of these p h e n o m e n a contribute to the alterations in m o t o n e u r o n p h e n o t y p e o b s e r v e d in the reinnervated uropod musculature.
Acknowledgements This w o r k was supported by a Bureau o f B i o l o g i c a l Research, B u s c h M e m o r i a l Research Grant to C . H . P . H . J.L. w a s supported by a grant f r o m the National S c i e n c e Council, R.O.C.
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