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Inactivity-induced motor nerve terminal sprouting in amphibian skeletal muscles chronically blocked by α-bungarotoxin
EXPERIMENTAL
NEUROLOGY
111,115-122
(1991)
Inactivity-Induced Motor Nerve Terminal Sprouting in Amphibian Skeletal Muscles Chronically Blocked by a-Bungarotoxin M. M. WINES’ Department
of Physiology, University
Jerry Lewis of California,
Neuromuscular Los Angeles,
AND M. S. LETINSKY’ Research Center, School of Medicine,
There is a positive correlation between contractile inactivity and the initiation of motor neuron sprouting. However, the exact mechanism responsible for this neuronal growth remains obscure. In a previous study (M. M. Wines and M. S. Letinsky, 1988, J. Neurosci. 8: 3909-3919) we investigated this phenomenon by inducing chronic contractile inactivity of an amphibian muscle by exposure to formamide and found that motor neuron sprouting occurs in the presence of normal presynaptic transmitter release and propagated muscle fiber action potentials. The present study investigates motor neuron sprouting in response to inactivity produced when neuromuscular transmission is blocked by chronic exposure to a-bungarotoxin ((Y-BTX). The CP BTX-induced muscle paralysis was maintained for l-63 days by repetitive application of the toxin to the cutaneous pectoris muscle of adult Rana pipiens. During the chronic (Y-BTX treatment end-plate potentials were reduced below threshold, which therefore removed both muscle fiber action potentials and contractile activity. Our findings showed only terminal sprouting. Also, higher sprouting frequencies (up to 100% of the observed terminals) were observed after chronic (YBTX treatment, compared to the sprouting response induced by formamide treatment. In view of our earlier formamide results, these observations suggest that the inhibition of the postsynaptic acetylcholine response, and consequently inhibition of muscle fiber electrical and contractile activity, produces a stronger stimulus to motor neuron sprouting than the presence of contractile inactivity alone coupled with normal synaptic transmission and muscle electrical activity. o 1991 Academic
Press,
Inc.
INTRODUCTION Two of the hallmarks of the vertebrate nervous system are seen in the precision of its synaptic connections
’ Current address: California State University, Long Beach, ment of Anatomy and Physiology, Long Beach, CA 90410. ’ To whom correspondence should be addressed.
Depart-
and Ahmanson Laboratories of Neurobiology, Los Angeles, California 90024
and in its ability to modify these connections in response to a variety of naturally occurring and artificially imposed stimuli. One example of such plasticity is motor neuron sprouting. Much of our understanding of this process has come from studies in lower vertebrates where sprouting was induced by partial denervation or by inactivity resulting from treatment with either preor postsynaptic neuromuscular blocking agents (1,3,18, 35,39). Since each of these experimental interventions causes some degree of muscle fiber contractile inactivity, it is generally thought that muscle fiber inactivity is the source of the sprouting stimulus (1, 3, 18). An appealing explanation for the link between inactivity and sprouting is that a diffusible sprouting factor is released from inactive skeletal muscle fibers following partial denervation or procedures which inhibit contractile activity (( 1,3,4) but see (33) also). Evidence for such a factor and it’s putative role in sprouting has come from experiments in which normal neuromuscular function was interrupted. For example, terminal sprouting has been observed after presynaptic blockage of nerve conduction with tetrodotoxin (7) and after interruption of synaptic transmission by either botulinum toxin or tetanus toxin (6, 11, 12, 13, 23). These studies showed that perturbations in the presynaptic release of acetylcholine (ACh) or in ACh binding to its receptor can induce sprouting. However, recently we have shown that terminal sprouting can also occur in amphibians when only muscle contractions are blocked with formamide, which leaves both of these parameters unaltered (41). In these experiments (41) the cutaneous pectoris (CP) muscle was chronically treated with formamide which produces contractile paralysis but does not affect either presynaptic release of ACh or propagation of muscle fiber action potentials (see (X5,16)). The degree of formamide-induced muscle paralysis was analogous to what others have seen following toxin administration; but interestingly, these formamide-treated preparations demonstrated relatively small amounts of terminal sprouting as compared to the amount seen in mammals following neurotoxin-induced paralysis. Thus, at least in amphibians, the presence of contractile inactivity coupled with functional synaptic transmission and
115 All
C opyright Q 1991 rights of reproduction
0014.4886/91 $3.00 by Academic Press. Inc. in any form reserved.
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AND
muscle fiber action potentials may somehow be less of a stimulus for sprouting than if pre- and/or postsynaptic mechanisms were inhibited. The present studies were designed to specifically test whether treatment with (YBTX, which blocks synaptic transmission and therefore - interrupts both muscle electrical and mechanical activity, induces more sprouting than what occurs following formamide treatment. METHODS
Experimental Preparation Experiments were performed on CP muscles of adult (5 to 7-cm body lengths) frogs (Rana pipiens). To facilitate comparisons with our earlier formamide experiments (41), similar sized frogs were obtained from the same supplier (Amphibians of North America/Sullivan) and experiments were performed at the same times during the year. Chronic contractile inactivity was maintained by blocking postsynaptic ACh receptors with repeated applications of a-BTX (Biotoxins) using the same experimental approach as in our previous study using chronic formamide treatment (41). That paper describes the detailed protocol for the surgical exposure of the CP and the construction of a reservoir surrounding the CP. Briefly, frogs were anesthetized at room temperature by submersion in an aqueous solution containing 0.1% tricaine methane sulfonate (MS-222, Sigma) for 15-20 min. Fully anesthetized animals were placed on their backs on a bed of ice; then the right CP and portions of the adjoining lateral pectoralis muscle and the contralateral CP muscle were surgically exposed. A reservoir (volume, 0.5-1.0 ml) of orabase gel (Hoyt Laboratories) was constructed surrounding the fully exposed CP. This ensured that the entire end-plate region of the CP muscle was exposed to a-BTX. Experimental muscles were bathed for 30 min in normal frog Ringers (NFR) containing lo-100 pg/ml wBTX and 0.1% bovine serum albumin (BSA; Sigma). Following this incubation, the a-BTX solution was drawn off and the muscle was rinsed for 15 min in NFR with 0.1% BSA. The incision was closed using surgical glue (Histoacryl blau, B. Braun Melsungen AG), which allowed for repeated opening of the original suture line without extensive tissue damage. The entire surgical procedure required approximately 1.5 h. Control experiments were performed in which the CP was bathed in the same manner using NFR alone or NFR containing 0.1% BSA. In preliminary experiments, a-BTX-treated animals recovered functional synaptic transmission and contractile activity in approximately 4-5 days following the initial treatment. Chronic neuromuscular block was maintained therefore, by repeating the (Y-BTX incubation three times per week. Using this protocol, muscle
LETINSKY
fiber contractions were eliminated for periods up to 9 weeks. During this time the animals were housed in clear Lucite boxes and were fed mealworms weekly. Electrophysiology _ Frogs (N = 22) were sacrificed from 1 day to 9 weeks after the initial a-BTX treatment, and the experimental CP muscles were removed for electrophysiology (the contralateral CP muscles were also removed and used in the histological analysis described below). Indirect stimulation of the nerve supplying the CP was used initially to ascertain the degree of contractile block in the treated muscles. Concomitant viewing with a stereo microscope (6-50X) confirmed the presence or absence of nerve-evoked twitches (see (41)). Completely inactive experimental muscles were pinned out in a Sylgardlined plastic petri dish modified for electrophysiological recording (28) and continuously perfused with NFR solution (without any additional synaptic blocking agents). Nerve terminals were localized using Nomarski optics and conventional intracellular recording techniques were employed using glass microelectrodes containing 0.6 M K,SO,. Nerve-evoked end-plate potentials (epps) were elicited (square pulse stimulation, 0.33 Hz, 200 ps duration), and epps were collected, stored, and analyzed with on-line computer facilities (Digital Micro 11/23+). Histology After electrophysiological recording the treated and contralateral CP muscles were processed together for light microscopy. Axons and motor nerve terminals were stained with TNBT ((26, 27) and see (41)) and postsynaptic acetylcholinesterase (AChE) activity was simultaneously demonstrated using the Karnovsky method (25, 26, 27). Following staining, the intact CP muscles were mounted between two coverslips in Aquamount (Lerner Labs). Each preparation was then examined for sprouting using 40X, 63X, and 100X high-resolution plan apochromatic objectives to visualize even the finest sprouts (see (41)). Sprouts were defined as terminal outgrowths that extended more than l-2 pm beyond the original confines of the junctional AChE reaction product and which were themselves not associated with any AChE activity (see (41)). Unambiguous identification of sprouts was further facilitated by the contrast between the blue TNBT-stained nerve terminal processes and the brown AChE reaction product (26, 27). Sprouting was determined in these experiments as in our previous formamide experiments (41). Our measurements of terminal sprouting were conservative because we did not include as sprouts any thin neuritic extensions which were occasionally seen to be associated with AChE activity (i.e., ring fibers, see (40)).
MOTOR
NEURON
The morphology of 32-98 motor nerve terminals per muscle (25 experimental muscles) was assessed and a two-tailed Student t test was performed to test for significance. RESULTS
Extent
of a-BTX-Induced
Contractile Inactivity
The extent of the cY-BTX-induced contractile block was determined for each of the experimental muscles. At the time of dissection the contractile block was complete, with all 25 experimental muscles showing no discernible, gross, nerve-evoked contractions. However, when observed at higher magnifications (25-50X) eight of these cu-BTX-treated CP muscles had 2-3 fibers that twitched in response to nerve stimulation. The length of time that these fibers had been active is unknown. The remaining 17 muscles showed no nerve-evoked muscle fiber twitching when observed at these higher magnifications. There was no evidence of a difference in the sprouting frequencies of the few muscles demonstrating this negligible amount of activity. Electrophysiological recording from the motor end plates on superficial muscle fibers in 22 a-BTX-treated CP muscles was performed to determine the effectiveness of the synaptic block. As expected when using a postsynaptic blocking agent, no spontaneous miniature end-plate potentials were seen even with high-gain recording. End-plate potentials were recorded at each neuromuscular junction studied (15 to 50 per muscle). While epp amplitudes varied, they were always subthreshold; no nerve-evoked muscle fiber action potentials were observed. Table 1 shows the distribution of epp amplitudes recorded from experimental muscles after different periods of a-BTX treatment. The average epp amplitudes were less than 10 mV and well below the 20-25 mV depolarization needed to exceed threshold for action potential initiation in the CP. Nerve Terminal and Sprout Morphology Lu-BTX-induced contractile inactivity resulted in only terminal sprouting; nodal sprouting was not observed. The morphology of these terminal sprouts was similar to that seen following formamide-induced muscle inactivity (41). Figure 1 shows that after short periods of a-BTX treatment, rudimentary terminal sprouts extended from the distal ends of nerve terminal processes beyond the confines of junctional AChE reaction product. With longer periods of cu-BTX-induced contractile inactivity, the terminal sprouts increased in both length and complexity. However, generally sprouts were short and remained on the same muscle fiber as the nerve terminal they emerged from; but, there were a few examples (approximately 1.1%; 15 of 1352 experimental end
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SPROUTING
TABLE End-Plate Days” 1 1 1 1 2 2 3 4 4 6 I 7 8 14 14 21 35 35 49 49 56 63 ’ Days
Minimum
(mV)
Sizes
Potential Maximum
2.0 0.5 2.0 0.5 1.0 1.0 0.5 4.0 4.0 1.0 2.5 1.0 0.5 0.3 0.5 0.5 0.5 1.0 1.0 1.0 0.3 1.0 of chronic
1
(mV)
12.0 10.0 15.0 1.5 12.0 14.0 15.0 20.0 17.0 12.0 18.0 18.0 9.0 4.0 17.0 3.0 12.0 3.0 10.0 13.0 1.0 4.0 cu-BTX-induced
contractile
Average 4.5 3.6 6.8 0.8 4.8 6.1 4.0 10.2 9.4 5.1 9.4 3.7 2.9 1.0 5.2 1.2 3.1 1.7 3.9 5.4 0.5 2.1
(mV
f SD)
* 2.1 f 3.9 + 3.7 2 0.3 f 2.3 -+ 2.9 + 4.8 + 5.4 T!T3.7 f 2.7 f 3.8 f 3.4 f 2.8 f 0.7 f 4.1 f 0.6 f 2.8 f 0.6 * 2.0 f 2.7 + 0.2 f 1.0
inactivity.
plates examined) of terminal sprouts that extended to neighboring muscle fibers and formed apparent synaptic contacts (Fig. 2). The number of sprouting nerve terminals in each muscle (i.e., sprouting frequency) increased as the time of a-BTX block was extended (Fig. 3). The sprouting frequency during the first 8 days of treatment was not different than the ongoing sprouting normally associated with the synaptic remodelling seen in untreated CP muscles (i.e., approximately 8% maximally following cx-BTX treatment versus 9% maximum sprouting in untreated normal CP muscles; see (41)). From 14 days of a-BTX treatment and continuing throughout the 9week duration of the experiment (Fig. 3) there was a progressive increase in the number of sprouting nerve terminals in each muscle examined. One muscle even showed 100% of its terminals possessing sprouts after 7 weeks of inactivity. This progressive increase in sprouting frequency was supported by a strong correlation between the duration of contractile inactivity and the frequency of sprouting (R = 0.96; P < 0.001). Sprouting frequencies between 0 and 13% were observed in the contralateral CP during the initial 2 weeks of a-BTX treatment, and, due to toxin spread, subsequent ol-BTX treatments past this time point resulted in progressive paralysis of the untreated contralateral muscle as well. This produced a delayed sprouting response in the contralateral CP, which was morphologi-
118
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AND
LETINSKY
FIG. 1. Examples of TNBT-stained nerve terminal sprouting following 1 (A), 3 (B, C), 5 (D, E), and 7 (F, G, H) weeks of chronic a-BTX-induced contractile inactivity are shown. The arrowheads mark the point of origin of each sprout. The region of AChE reaction product is lightly stained and faintly visible in A and F (small arrows); it is below the plane of focus in the remainder of the panels. Scale bar equals 30 pm.
MOTOR
NEURON
119
SPROUTING
methodology as with or-BTX treatment. These muscles were examined after I4 days when the expected level of inactivity-induced sprouting would have been greater than 20%; in each case muscle contractions were normal and motor nerve terminal sprouting was minimal. Repeated exposures to NFR for 14 days resulted in 6.3 and 6.7% sprouting, and the application of NFR and serum albumin caused equally small frequencies of sprouting (7.5 + 4% + SD). Moreover, all of these sprouting frequencies were not appreciably different from the normal control levels of sprouting seen without treatment (see (41)). The morphology of the terminal sprouts in these control muscles was identical to that seen in both the untreated muscles and the experimental muscles. DISCUSSION
FIG. 2. TNBT-stained terminal sprouts are shown extending to adjacent muscle fibers. The (*) marks the possible location of junctional sites. The small arrows indicate the path of sprout growth following 7 (A) and 8 (B) weeks of ol-BTX-induced contractile inactivity. Scale bar equals 30 grn.
tally similar to, but slightly less frequent than that seen in the treated CP. The 2-week delay in the onset of contraction block in the contralateral CP, plus the 4-8 weeks normally needed for the appearance of a significant contralateral sprouting response (34), suggests that this contractile inactivity in the contralateral CP probably had little influence on the amount of sprouting we saw in the a-BTX treated muscles (for a review of such contralateral effects see (35)).
The results presented here describe the sprouting response seen at amphibian motor nerve terminals following inactivity produced by postsynaptic block with czBTX. While similar results have been seen in mammals following application of cr-BTX (22) and in frogs using curare (40), our experiments were unique in several ways. In contrast to these other experiments, we were able to produce and maintain complete muscle electrical and mechanical inactivity for extended periods of time. In addition, we can compare the present sprouting response induced by electrical and mechanical inactivity to our earlier work in which terminal sprouting was produced by selectively blocking only muscle contractile activity with formamide (41). Comparing the results from these experiments in which inactivity was caused by different means makes it possible to begin to determine the specific regulatory roles of muscle fiber electrical and mechanical activity on motor nerve terminal growth. Our experimental results show a greater sprouting response than has been seen in previous mammalian and
Control Studies Control studies were performed to rule out the possibility that sprouting in a-BTX-treated muscles was caused by the experimental procedures or solutions. As in our previous work (41), the creation of the orabase well and subsequent experimental procedures did not cause terminal sprouting. CP muscles were repeatedly exposed to chilled NFR (N = 2) or to stock solutions of NFR and bovine serum albumin (N = 7) using the same
FIG. 3. Graph showing the individual frequencies (triangles) of nerve terminal sprouting in cutaneous pectoris muscles chronically treated with a-BTX for varying periods of time. The regression line is plotted for clarity.
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amphibian experiments. This variability might have resulted from a more complete block of ACh receptors in the CP compared to previous paralytic models. For example, cz-BTX studies by Holland and Brown (22) and Rachel and Robbins (33) using repetitive toxin injections successfully inhibited up to 94% of the muscle’s contractile activity; but substantial recovery of muscle contractile activity was noted in the majority of fibers within the first postinjection day. A similar problem was noted in frogs by Wernig and others (40) in their longterm immobilization experiments in which they repeatedly injected curare into the dorsal lymph sac. While the muscles in each of the above studies were surely inactive for relatively long periods of time, there were obviously varying intervals of contractile activity. Since it was not possible to determine the extent of the postsynaptic receptor block in any of these experiments, it was not possible to know the degree of muscle inactivity throughout the entirety of these experiments. It is possible that even small amounts of muscle activity may be sufficient to inhibit or reduce the sprouting response (as has been shown for low levels of activity which are able to suppress the development of acetylcholine sensitivity; e.g., (29)). If this is the case, then the recovery of even a portion of contractile activity could suppress the frequency of sprouting seen by these researchers. The possibility of contractile activity reducing sprouting was diminished in our experiments. While in preliminary studies we too observed a functional recovery from the (Y-BTX treatment in 4 to 5 days, we ensured complete contractile block by repeating the toxin treatments 3 days per week. The effectiveness of the contractile block was further demonstrated by the presence of only subthreshold epps and the absence of twitching fibers at the time of biopsy. These results strongly suggest that muscle electrical and contractile activity were completely blocked in our studies. Many researchers (e.g., (10, 14, 19, 20, 21, 32, 38)) have suggested that muscle fibers can release a factor which either directly or indirectly regulates motor nerve terminal sprouting. Two general mechanisms have been proposed: first, that inactive muscle fibers release a factor which induces terminal sprouting (see below); or second, that active muscle fibers release a sprout-inhibiting factor which prevents or restrains nerve terminals from a normal propensity to grow (see (9, 24)). If one assumes that a sprout-inducing factor is released whenever muscle fibers are rendered mechanically inactive, then the amount of sprouting should be the same in a given muscle regardless of the means by which inactivity is produced. However, this was not the case in our experiments using the frog CP. Contractile inactivity in the presence of synaptic transmission and muscle fiber action potentials (as after formamide treatment, see (41)) causes less terminal sprouting than the sprouting
LETINSKY
seen following a-BTX-induced interruption of both muscle fiber electrical and mechanical activities. These results suggest that in addition to contractile inactivity, the removal of muscle fiber electrical and/or the postsynaptic ACh receptor response must also be involved in the sprouting response. A specific role for the acetylcholine receptor in terminal sprouting seems unlikely since there was no apparent correlation between epp size, which is proportional to the amount of ACh receptors blocked, and the frequency of terminal sprouting. Thus, it seems apparent that the comparatively larger sprouting frequencies seen after toxin treatment are related to the absence of muscle fiber action potentials. This is consistent with other results which have shown that contractions elicited by direct stimulation of muscle fibers (which elicits propagated muscle fiber action potentials) can reduce the terminal sprouting typically seen in botulinurn-poisoned muscles (4). The question which remains is how does the interruption of muscle fiber action potentials and mechanical contractile activity lead to terminal sprouting? A possible explanation for the differential effects of muscle fiber electrical and contractile inactivity on terminal sprouting could be related to changes in the normal fluctuations of sarcoplasmic levels of calcium. In normally functioning muscle there are two potential pathways for changing sarcoplasmic calcium: (i) by release of calcium from the sarcoplasmic reticulum during the normal excitation-contraction sequence, and (ii) by an influx of calcium through voltage-dependent channels (8, 37). Two types of voltage-dependent calcium channels are present in intact skeletal muscles. One is a slow activating channel that is thought to be present in the transverse tubular system (31). The other is a fast activating channel whose kinetic properties suggest that it may be activated during a single twitch (8). Although muscle calcium levels increase several orders of magnitude with every twitch, this is primarily due to the release of sequestered calcium from the sarcoplasmic reticulum, and not the result of calcium entering through membrane channels (37). While the role of calcium influx through the sarcolemma on sprouting is unknown, this intriguing possibility should be considered. For example, since internal calcium has been shown to be involved in the regulation of ACh receptors and junctional AChE (36), it is conceivable that a reduction of calcium within the fiber may cause the production or release of a sprout-inducing factor. This could account for the differences in sprouting noted after formamideinduced interruption of only mechanical activity (41) as compared to complete muscle inactivity produced by cyBTX. With formamide-induced inactivity calcium is not released from the sarcoplasmic reticulum, but presumably some calcium ions continue to enter the muscle fiber through calcium channels that have been activated
MOTOR
NEURON
by the muscle fiber action potentials (8). However, in the a-BTX-treated muscles a more severe reduction in intramuscular calcium concentration may occur as the absence of propagated action potentials further reduces calcium entry through even these voltage-dependent calcium channels. This more pronounced decrease in calcium entry may trigger the muscle fiber to produce an enhanced amount of sprout-inducing factor as compared to that seen following formamide treatment. Hence, (u-BTX produces a more robust sprouting response. While calcium’s influence as a second messenger on a number of other physiological processes has been well established, a particular role for calcium in the sprouting mechanism awaits further experimental confirmation.
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L. W., AND S. J. STRICH. 1968. The effects of botulinum toxin on the pattern of innervation of skeletal muscle in the mouse. Q. J. Exp. Physiol. 53: 84-89.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the expert technical assistance of both D. Guy Garrett and H. Garcia. Our thanks are further extended to Drs. Alan Grinnell, Yoshi Kidokoro, and Diana Linden for their suggestions regarding the manuscript. This work was supported by a grant to M.S.L. from the USPHS. M.M.W. was supported by a USPHS training grant.
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terminal skeletal
NEUROLOGY
111,115-122
(1991)
Inactivity-Induced Motor Nerve Terminal Sprouting in Amphibian Skeletal Muscles Chronically Blocked by a-Bungarotoxin M. M. WINES’ Department
of Physiology, University
Jerry Lewis of California,
Neuromuscular Los Angeles,
AND M. S. LETINSKY’ Research Center, School of Medicine,
There is a positive correlation between contractile inactivity and the initiation of motor neuron sprouting. However, the exact mechanism responsible for this neuronal growth remains obscure. In a previous study (M. M. Wines and M. S. Letinsky, 1988, J. Neurosci. 8: 3909-3919) we investigated this phenomenon by inducing chronic contractile inactivity of an amphibian muscle by exposure to formamide and found that motor neuron sprouting occurs in the presence of normal presynaptic transmitter release and propagated muscle fiber action potentials. The present study investigates motor neuron sprouting in response to inactivity produced when neuromuscular transmission is blocked by chronic exposure to a-bungarotoxin ((Y-BTX). The CP BTX-induced muscle paralysis was maintained for l-63 days by repetitive application of the toxin to the cutaneous pectoris muscle of adult Rana pipiens. During the chronic (Y-BTX treatment end-plate potentials were reduced below threshold, which therefore removed both muscle fiber action potentials and contractile activity. Our findings showed only terminal sprouting. Also, higher sprouting frequencies (up to 100% of the observed terminals) were observed after chronic (YBTX treatment, compared to the sprouting response induced by formamide treatment. In view of our earlier formamide results, these observations suggest that the inhibition of the postsynaptic acetylcholine response, and consequently inhibition of muscle fiber electrical and contractile activity, produces a stronger stimulus to motor neuron sprouting than the presence of contractile inactivity alone coupled with normal synaptic transmission and muscle electrical activity. o 1991 Academic
Press,
Inc.
INTRODUCTION Two of the hallmarks of the vertebrate nervous system are seen in the precision of its synaptic connections
’ Current address: California State University, Long Beach, ment of Anatomy and Physiology, Long Beach, CA 90410. ’ To whom correspondence should be addressed.
Depart-
and Ahmanson Laboratories of Neurobiology, Los Angeles, California 90024
and in its ability to modify these connections in response to a variety of naturally occurring and artificially imposed stimuli. One example of such plasticity is motor neuron sprouting. Much of our understanding of this process has come from studies in lower vertebrates where sprouting was induced by partial denervation or by inactivity resulting from treatment with either preor postsynaptic neuromuscular blocking agents (1,3,18, 35,39). Since each of these experimental interventions causes some degree of muscle fiber contractile inactivity, it is generally thought that muscle fiber inactivity is the source of the sprouting stimulus (1, 3, 18). An appealing explanation for the link between inactivity and sprouting is that a diffusible sprouting factor is released from inactive skeletal muscle fibers following partial denervation or procedures which inhibit contractile activity (( 1,3,4) but see (33) also). Evidence for such a factor and it’s putative role in sprouting has come from experiments in which normal neuromuscular function was interrupted. For example, terminal sprouting has been observed after presynaptic blockage of nerve conduction with tetrodotoxin (7) and after interruption of synaptic transmission by either botulinum toxin or tetanus toxin (6, 11, 12, 13, 23). These studies showed that perturbations in the presynaptic release of acetylcholine (ACh) or in ACh binding to its receptor can induce sprouting. However, recently we have shown that terminal sprouting can also occur in amphibians when only muscle contractions are blocked with formamide, which leaves both of these parameters unaltered (41). In these experiments (41) the cutaneous pectoris (CP) muscle was chronically treated with formamide which produces contractile paralysis but does not affect either presynaptic release of ACh or propagation of muscle fiber action potentials (see (X5,16)). The degree of formamide-induced muscle paralysis was analogous to what others have seen following toxin administration; but interestingly, these formamide-treated preparations demonstrated relatively small amounts of terminal sprouting as compared to the amount seen in mammals following neurotoxin-induced paralysis. Thus, at least in amphibians, the presence of contractile inactivity coupled with functional synaptic transmission and
115 All
C opyright Q 1991 rights of reproduction
0014.4886/91 $3.00 by Academic Press. Inc. in any form reserved.
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muscle fiber action potentials may somehow be less of a stimulus for sprouting than if pre- and/or postsynaptic mechanisms were inhibited. The present studies were designed to specifically test whether treatment with (YBTX, which blocks synaptic transmission and therefore - interrupts both muscle electrical and mechanical activity, induces more sprouting than what occurs following formamide treatment. METHODS
Experimental Preparation Experiments were performed on CP muscles of adult (5 to 7-cm body lengths) frogs (Rana pipiens). To facilitate comparisons with our earlier formamide experiments (41), similar sized frogs were obtained from the same supplier (Amphibians of North America/Sullivan) and experiments were performed at the same times during the year. Chronic contractile inactivity was maintained by blocking postsynaptic ACh receptors with repeated applications of a-BTX (Biotoxins) using the same experimental approach as in our previous study using chronic formamide treatment (41). That paper describes the detailed protocol for the surgical exposure of the CP and the construction of a reservoir surrounding the CP. Briefly, frogs were anesthetized at room temperature by submersion in an aqueous solution containing 0.1% tricaine methane sulfonate (MS-222, Sigma) for 15-20 min. Fully anesthetized animals were placed on their backs on a bed of ice; then the right CP and portions of the adjoining lateral pectoralis muscle and the contralateral CP muscle were surgically exposed. A reservoir (volume, 0.5-1.0 ml) of orabase gel (Hoyt Laboratories) was constructed surrounding the fully exposed CP. This ensured that the entire end-plate region of the CP muscle was exposed to a-BTX. Experimental muscles were bathed for 30 min in normal frog Ringers (NFR) containing lo-100 pg/ml wBTX and 0.1% bovine serum albumin (BSA; Sigma). Following this incubation, the a-BTX solution was drawn off and the muscle was rinsed for 15 min in NFR with 0.1% BSA. The incision was closed using surgical glue (Histoacryl blau, B. Braun Melsungen AG), which allowed for repeated opening of the original suture line without extensive tissue damage. The entire surgical procedure required approximately 1.5 h. Control experiments were performed in which the CP was bathed in the same manner using NFR alone or NFR containing 0.1% BSA. In preliminary experiments, a-BTX-treated animals recovered functional synaptic transmission and contractile activity in approximately 4-5 days following the initial treatment. Chronic neuromuscular block was maintained therefore, by repeating the (Y-BTX incubation three times per week. Using this protocol, muscle
LETINSKY
fiber contractions were eliminated for periods up to 9 weeks. During this time the animals were housed in clear Lucite boxes and were fed mealworms weekly. Electrophysiology _ Frogs (N = 22) were sacrificed from 1 day to 9 weeks after the initial a-BTX treatment, and the experimental CP muscles were removed for electrophysiology (the contralateral CP muscles were also removed and used in the histological analysis described below). Indirect stimulation of the nerve supplying the CP was used initially to ascertain the degree of contractile block in the treated muscles. Concomitant viewing with a stereo microscope (6-50X) confirmed the presence or absence of nerve-evoked twitches (see (41)). Completely inactive experimental muscles were pinned out in a Sylgardlined plastic petri dish modified for electrophysiological recording (28) and continuously perfused with NFR solution (without any additional synaptic blocking agents). Nerve terminals were localized using Nomarski optics and conventional intracellular recording techniques were employed using glass microelectrodes containing 0.6 M K,SO,. Nerve-evoked end-plate potentials (epps) were elicited (square pulse stimulation, 0.33 Hz, 200 ps duration), and epps were collected, stored, and analyzed with on-line computer facilities (Digital Micro 11/23+). Histology After electrophysiological recording the treated and contralateral CP muscles were processed together for light microscopy. Axons and motor nerve terminals were stained with TNBT ((26, 27) and see (41)) and postsynaptic acetylcholinesterase (AChE) activity was simultaneously demonstrated using the Karnovsky method (25, 26, 27). Following staining, the intact CP muscles were mounted between two coverslips in Aquamount (Lerner Labs). Each preparation was then examined for sprouting using 40X, 63X, and 100X high-resolution plan apochromatic objectives to visualize even the finest sprouts (see (41)). Sprouts were defined as terminal outgrowths that extended more than l-2 pm beyond the original confines of the junctional AChE reaction product and which were themselves not associated with any AChE activity (see (41)). Unambiguous identification of sprouts was further facilitated by the contrast between the blue TNBT-stained nerve terminal processes and the brown AChE reaction product (26, 27). Sprouting was determined in these experiments as in our previous formamide experiments (41). Our measurements of terminal sprouting were conservative because we did not include as sprouts any thin neuritic extensions which were occasionally seen to be associated with AChE activity (i.e., ring fibers, see (40)).
MOTOR
NEURON
The morphology of 32-98 motor nerve terminals per muscle (25 experimental muscles) was assessed and a two-tailed Student t test was performed to test for significance. RESULTS
Extent
of a-BTX-Induced
Contractile Inactivity
The extent of the cY-BTX-induced contractile block was determined for each of the experimental muscles. At the time of dissection the contractile block was complete, with all 25 experimental muscles showing no discernible, gross, nerve-evoked contractions. However, when observed at higher magnifications (25-50X) eight of these cu-BTX-treated CP muscles had 2-3 fibers that twitched in response to nerve stimulation. The length of time that these fibers had been active is unknown. The remaining 17 muscles showed no nerve-evoked muscle fiber twitching when observed at these higher magnifications. There was no evidence of a difference in the sprouting frequencies of the few muscles demonstrating this negligible amount of activity. Electrophysiological recording from the motor end plates on superficial muscle fibers in 22 a-BTX-treated CP muscles was performed to determine the effectiveness of the synaptic block. As expected when using a postsynaptic blocking agent, no spontaneous miniature end-plate potentials were seen even with high-gain recording. End-plate potentials were recorded at each neuromuscular junction studied (15 to 50 per muscle). While epp amplitudes varied, they were always subthreshold; no nerve-evoked muscle fiber action potentials were observed. Table 1 shows the distribution of epp amplitudes recorded from experimental muscles after different periods of a-BTX treatment. The average epp amplitudes were less than 10 mV and well below the 20-25 mV depolarization needed to exceed threshold for action potential initiation in the CP. Nerve Terminal and Sprout Morphology Lu-BTX-induced contractile inactivity resulted in only terminal sprouting; nodal sprouting was not observed. The morphology of these terminal sprouts was similar to that seen following formamide-induced muscle inactivity (41). Figure 1 shows that after short periods of a-BTX treatment, rudimentary terminal sprouts extended from the distal ends of nerve terminal processes beyond the confines of junctional AChE reaction product. With longer periods of cu-BTX-induced contractile inactivity, the terminal sprouts increased in both length and complexity. However, generally sprouts were short and remained on the same muscle fiber as the nerve terminal they emerged from; but, there were a few examples (approximately 1.1%; 15 of 1352 experimental end
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TABLE End-Plate Days” 1 1 1 1 2 2 3 4 4 6 I 7 8 14 14 21 35 35 49 49 56 63 ’ Days
Minimum
(mV)
Sizes
Potential Maximum
2.0 0.5 2.0 0.5 1.0 1.0 0.5 4.0 4.0 1.0 2.5 1.0 0.5 0.3 0.5 0.5 0.5 1.0 1.0 1.0 0.3 1.0 of chronic
1
(mV)
12.0 10.0 15.0 1.5 12.0 14.0 15.0 20.0 17.0 12.0 18.0 18.0 9.0 4.0 17.0 3.0 12.0 3.0 10.0 13.0 1.0 4.0 cu-BTX-induced
contractile
Average 4.5 3.6 6.8 0.8 4.8 6.1 4.0 10.2 9.4 5.1 9.4 3.7 2.9 1.0 5.2 1.2 3.1 1.7 3.9 5.4 0.5 2.1
(mV
f SD)
* 2.1 f 3.9 + 3.7 2 0.3 f 2.3 -+ 2.9 + 4.8 + 5.4 T!T3.7 f 2.7 f 3.8 f 3.4 f 2.8 f 0.7 f 4.1 f 0.6 f 2.8 f 0.6 * 2.0 f 2.7 + 0.2 f 1.0
inactivity.
plates examined) of terminal sprouts that extended to neighboring muscle fibers and formed apparent synaptic contacts (Fig. 2). The number of sprouting nerve terminals in each muscle (i.e., sprouting frequency) increased as the time of a-BTX block was extended (Fig. 3). The sprouting frequency during the first 8 days of treatment was not different than the ongoing sprouting normally associated with the synaptic remodelling seen in untreated CP muscles (i.e., approximately 8% maximally following cx-BTX treatment versus 9% maximum sprouting in untreated normal CP muscles; see (41)). From 14 days of a-BTX treatment and continuing throughout the 9week duration of the experiment (Fig. 3) there was a progressive increase in the number of sprouting nerve terminals in each muscle examined. One muscle even showed 100% of its terminals possessing sprouts after 7 weeks of inactivity. This progressive increase in sprouting frequency was supported by a strong correlation between the duration of contractile inactivity and the frequency of sprouting (R = 0.96; P < 0.001). Sprouting frequencies between 0 and 13% were observed in the contralateral CP during the initial 2 weeks of a-BTX treatment, and, due to toxin spread, subsequent ol-BTX treatments past this time point resulted in progressive paralysis of the untreated contralateral muscle as well. This produced a delayed sprouting response in the contralateral CP, which was morphologi-
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LETINSKY
FIG. 1. Examples of TNBT-stained nerve terminal sprouting following 1 (A), 3 (B, C), 5 (D, E), and 7 (F, G, H) weeks of chronic a-BTX-induced contractile inactivity are shown. The arrowheads mark the point of origin of each sprout. The region of AChE reaction product is lightly stained and faintly visible in A and F (small arrows); it is below the plane of focus in the remainder of the panels. Scale bar equals 30 pm.
MOTOR
NEURON
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SPROUTING
methodology as with or-BTX treatment. These muscles were examined after I4 days when the expected level of inactivity-induced sprouting would have been greater than 20%; in each case muscle contractions were normal and motor nerve terminal sprouting was minimal. Repeated exposures to NFR for 14 days resulted in 6.3 and 6.7% sprouting, and the application of NFR and serum albumin caused equally small frequencies of sprouting (7.5 + 4% + SD). Moreover, all of these sprouting frequencies were not appreciably different from the normal control levels of sprouting seen without treatment (see (41)). The morphology of the terminal sprouts in these control muscles was identical to that seen in both the untreated muscles and the experimental muscles. DISCUSSION
FIG. 2. TNBT-stained terminal sprouts are shown extending to adjacent muscle fibers. The (*) marks the possible location of junctional sites. The small arrows indicate the path of sprout growth following 7 (A) and 8 (B) weeks of ol-BTX-induced contractile inactivity. Scale bar equals 30 grn.
tally similar to, but slightly less frequent than that seen in the treated CP. The 2-week delay in the onset of contraction block in the contralateral CP, plus the 4-8 weeks normally needed for the appearance of a significant contralateral sprouting response (34), suggests that this contractile inactivity in the contralateral CP probably had little influence on the amount of sprouting we saw in the a-BTX treated muscles (for a review of such contralateral effects see (35)).
The results presented here describe the sprouting response seen at amphibian motor nerve terminals following inactivity produced by postsynaptic block with czBTX. While similar results have been seen in mammals following application of cr-BTX (22) and in frogs using curare (40), our experiments were unique in several ways. In contrast to these other experiments, we were able to produce and maintain complete muscle electrical and mechanical inactivity for extended periods of time. In addition, we can compare the present sprouting response induced by electrical and mechanical inactivity to our earlier work in which terminal sprouting was produced by selectively blocking only muscle contractile activity with formamide (41). Comparing the results from these experiments in which inactivity was caused by different means makes it possible to begin to determine the specific regulatory roles of muscle fiber electrical and mechanical activity on motor nerve terminal growth. Our experimental results show a greater sprouting response than has been seen in previous mammalian and
Control Studies Control studies were performed to rule out the possibility that sprouting in a-BTX-treated muscles was caused by the experimental procedures or solutions. As in our previous work (41), the creation of the orabase well and subsequent experimental procedures did not cause terminal sprouting. CP muscles were repeatedly exposed to chilled NFR (N = 2) or to stock solutions of NFR and bovine serum albumin (N = 7) using the same
FIG. 3. Graph showing the individual frequencies (triangles) of nerve terminal sprouting in cutaneous pectoris muscles chronically treated with a-BTX for varying periods of time. The regression line is plotted for clarity.
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amphibian experiments. This variability might have resulted from a more complete block of ACh receptors in the CP compared to previous paralytic models. For example, cz-BTX studies by Holland and Brown (22) and Rachel and Robbins (33) using repetitive toxin injections successfully inhibited up to 94% of the muscle’s contractile activity; but substantial recovery of muscle contractile activity was noted in the majority of fibers within the first postinjection day. A similar problem was noted in frogs by Wernig and others (40) in their longterm immobilization experiments in which they repeatedly injected curare into the dorsal lymph sac. While the muscles in each of the above studies were surely inactive for relatively long periods of time, there were obviously varying intervals of contractile activity. Since it was not possible to determine the extent of the postsynaptic receptor block in any of these experiments, it was not possible to know the degree of muscle inactivity throughout the entirety of these experiments. It is possible that even small amounts of muscle activity may be sufficient to inhibit or reduce the sprouting response (as has been shown for low levels of activity which are able to suppress the development of acetylcholine sensitivity; e.g., (29)). If this is the case, then the recovery of even a portion of contractile activity could suppress the frequency of sprouting seen by these researchers. The possibility of contractile activity reducing sprouting was diminished in our experiments. While in preliminary studies we too observed a functional recovery from the (Y-BTX treatment in 4 to 5 days, we ensured complete contractile block by repeating the toxin treatments 3 days per week. The effectiveness of the contractile block was further demonstrated by the presence of only subthreshold epps and the absence of twitching fibers at the time of biopsy. These results strongly suggest that muscle electrical and contractile activity were completely blocked in our studies. Many researchers (e.g., (10, 14, 19, 20, 21, 32, 38)) have suggested that muscle fibers can release a factor which either directly or indirectly regulates motor nerve terminal sprouting. Two general mechanisms have been proposed: first, that inactive muscle fibers release a factor which induces terminal sprouting (see below); or second, that active muscle fibers release a sprout-inhibiting factor which prevents or restrains nerve terminals from a normal propensity to grow (see (9, 24)). If one assumes that a sprout-inducing factor is released whenever muscle fibers are rendered mechanically inactive, then the amount of sprouting should be the same in a given muscle regardless of the means by which inactivity is produced. However, this was not the case in our experiments using the frog CP. Contractile inactivity in the presence of synaptic transmission and muscle fiber action potentials (as after formamide treatment, see (41)) causes less terminal sprouting than the sprouting
LETINSKY
seen following a-BTX-induced interruption of both muscle fiber electrical and mechanical activities. These results suggest that in addition to contractile inactivity, the removal of muscle fiber electrical and/or the postsynaptic ACh receptor response must also be involved in the sprouting response. A specific role for the acetylcholine receptor in terminal sprouting seems unlikely since there was no apparent correlation between epp size, which is proportional to the amount of ACh receptors blocked, and the frequency of terminal sprouting. Thus, it seems apparent that the comparatively larger sprouting frequencies seen after toxin treatment are related to the absence of muscle fiber action potentials. This is consistent with other results which have shown that contractions elicited by direct stimulation of muscle fibers (which elicits propagated muscle fiber action potentials) can reduce the terminal sprouting typically seen in botulinurn-poisoned muscles (4). The question which remains is how does the interruption of muscle fiber action potentials and mechanical contractile activity lead to terminal sprouting? A possible explanation for the differential effects of muscle fiber electrical and contractile inactivity on terminal sprouting could be related to changes in the normal fluctuations of sarcoplasmic levels of calcium. In normally functioning muscle there are two potential pathways for changing sarcoplasmic calcium: (i) by release of calcium from the sarcoplasmic reticulum during the normal excitation-contraction sequence, and (ii) by an influx of calcium through voltage-dependent channels (8, 37). Two types of voltage-dependent calcium channels are present in intact skeletal muscles. One is a slow activating channel that is thought to be present in the transverse tubular system (31). The other is a fast activating channel whose kinetic properties suggest that it may be activated during a single twitch (8). Although muscle calcium levels increase several orders of magnitude with every twitch, this is primarily due to the release of sequestered calcium from the sarcoplasmic reticulum, and not the result of calcium entering through membrane channels (37). While the role of calcium influx through the sarcolemma on sprouting is unknown, this intriguing possibility should be considered. For example, since internal calcium has been shown to be involved in the regulation of ACh receptors and junctional AChE (36), it is conceivable that a reduction of calcium within the fiber may cause the production or release of a sprout-inducing factor. This could account for the differences in sprouting noted after formamideinduced interruption of only mechanical activity (41) as compared to complete muscle inactivity produced by cyBTX. With formamide-induced inactivity calcium is not released from the sarcoplasmic reticulum, but presumably some calcium ions continue to enter the muscle fiber through calcium channels that have been activated
MOTOR
NEURON
by the muscle fiber action potentials (8). However, in the a-BTX-treated muscles a more severe reduction in intramuscular calcium concentration may occur as the absence of propagated action potentials further reduces calcium entry through even these voltage-dependent calcium channels. This more pronounced decrease in calcium entry may trigger the muscle fiber to produce an enhanced amount of sprout-inducing factor as compared to that seen following formamide treatment. Hence, (u-BTX produces a more robust sprouting response. While calcium’s influence as a second messenger on a number of other physiological processes has been well established, a particular role for calcium in the sprouting mechanism awaits further experimental confirmation.
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DUCHEN, L. W. 1973. The effects of tetanus toxin on the motor end-plates of the mouse-an electron microscopic study. J. Neural. Sci. 19: 153-167.
12.
DUCHEN, L. W., AND D. A. TONGE. 1973. The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor endplates in slow and fast skeletal muscle of the mouse. J. Physiol. 228: 157-172.
13. DUCHEN,
L. W., AND S. J. STRICH. 1968. The effects of botulinum toxin on the pattern of innervation of skeletal muscle in the mouse. Q. J. Exp. Physiol. 53: 84-89.
14. EDGAR, D., R. TIMPL, ing domain of laminin outgrowth and neuronal
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15. ESCALONA
DE MOTTA, G., D. S. SMITH, M. CAYER, AND J. DEL CASTILLO. 1982. Mechanism of the excitation-contraction uncoupling of frog skeletal muscle by formamide. Biol. Bull. 163:
276-286. 16. ESCALONA
DE MOWA, G., F. CORDOBA, M. DE LEON, AND J. DEL CASTILLO. 1982. Inhibitory actions of high formamide concentrations on excitation-contraction coupling in skeletal muscles. J. Neurosci. Res. 7: 1633178.
ACKNOWLEDGMENTS The authors gratefully acknowledge the expert technical assistance of both D. Guy Garrett and H. Garcia. Our thanks are further extended to Drs. Alan Grinnell, Yoshi Kidokoro, and Diana Linden for their suggestions regarding the manuscript. This work was supported by a grant to M.S.L. from the USPHS. M.M.W. was supported by a USPHS training grant.
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