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Previous Article | Next Article
The Journal of Neuroscience, December 1, 2000, 20(23):8771-8779
Disparity in Neurotransmitter Release Probability among Competing Inputs during Neuromuscular Synapse Elimination
Diane M. Kopp, David J. Perkel, and Rita J. Balice-Gordon Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6074
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Competition among the several motor axons transiently innervating neonatal muscle fibers results in an increasing disparity in the quantal content and synaptic territory of each competitor, culminating in the permanent loss of all but one axon from neuromuscular junctions. We asked whether differences in the probability of neurotransmitter release also contribute to the increasing disparity in quantal content among competing inputs, and when in the process of competition changes in release probability become apparent. To address these questions, intracellular recordings were made from dually innervated neonatal mouse soleus muscle fibers, and quantal content and paired-pulse facilitation were evaluated for each input. At short interpulse intervals, paired-pulse facilitation was significantly higher for the weaker input with the smaller quantal content than the stronger input with the larger quantal content. Because neurotransmitter release probability across all release sites is inversely related to the extent of facilitation observed after paired-pulse stimulation, this result suggests that release probability is lower for weak compared with strong inputs innervating the same junction. A disparity in the probability of neurotransmitter release thus contributes to the disparity in quantal content that occurs during synaptic competition. Together, this work suggests that an input incapable of sustaining a high release probability may be at a competitive disadvantage for synaptic maintenance.
Key words: synapse elimination; nerve terminal; quantal content; paired-pulse facilitation; motor neuron; synaptic transmission
INTRODUCTION
Experience-dependent editing shapes synaptic connections throughout the developing nervous system, but the underlying mechanisms are poorly understood. At developing neuromuscular junctions transiently innervated by multiple motor axons, changes in the structure and strength of the synapses of each input occur in parallel. Previous work has shown that, as multiple innervation of muscle fibers is established, each input has relatively equal presynaptic terminal area (Balice-Gordon et al., 1993
; Gan and Lichtman, 1998
As competition begins, a progressive strengthening of some inputs and weakening of others is evident functionally as well as structurally within individual junctions. Quantal content becomes increasingly disparate among competing inputs (Colman et al., 1997
Although a reduction in the postsynaptic AChR density and presynaptic terminal area contribute to the weakening of inputs, neurotransmitter release probability could also affect synaptic strength. To address whether differences in the probability of neurotransmitter release across all release sites contribute to the increasing disparity in quantal content among competing inputs, intracellular recordings were made from dually innervated neonatal mouse soleus muscle fibers, and quantal content and paired-pulse facilitation were evaluated for each input. Although measuring release probability directly is impossible in this preparation, even with optical approaches (Betz and Bewick, 1992
Parts of this work have been published previously in abstract form (Kopp and Balice-Gordon, 1999
MATERIALS AND METHODS
Immunohistochemical analysis of multiply innervated neuromuscular junctions. The soleus muscle was chosen for these experiments because of the following: junctions are located in a single band in the middle of the muscle belly; the muscle and its innervation can be easily dissected back to ventral root filaments; axons are located in two or three ventral roots that can be easily separated for stimulation; and this muscle has been widely used for structural and functional studies of synapse elimination. Whole mounts of postnatal day 1 (P1) to P14 soleus muscles from CD1 mice (Harlan Sprague Dawley, Indianapolis, IN) were immunostained for motor axons, nerve terminals, and AChRs as described previously (Gonzalez et al., 1999
Electrophysiological analyses of synaptic transmission. P7-P9 mice were anesthetized by intraperitoneal injection of 0.05 cc of a mixture of 17.4 mg/ml ketamine and 2.6 mg/ml xylazine (Phoenix Pharmaceuticals, St. Joseph, MO). The soleus muscle and its innervation to the ventral roots were dissected under oxygenated (95% O2, 5% CO2) Rees' Ringer's solution (Rees, 1978
Intracellular recording from skeletal muscle fibers was performed using glass microelectrodes filled with 3 M KCl (50-70 M
resistance). Contractions were prevented by adjusting the Ca2+/Mg2+ ratio of the Ringer's solution, and recordings were made under one of three conditions: low Ca2+ (1 mM Ca2+, 8-10 mM Mg2+), normal Ca2+ (2 mM Ca2+, 13-15 mM Mg2+), or high Ca2+ (4 mM Ca2+, 24-27 mM Mg2+). Magnesium concentration was adjusted using MgSO4. The exact Mg2+ required to block muscle contractions and to allow stable intracellular recordings varied somewhat from muscle to muscle, even in muscles from pups of the same age and in muscles from pups from the same litter. The minimal Mg2+ concentration that abolished contractions was used. Measurements of paired-pulse facilitation (F) and quantal content (m) of weak and strong inputs to the same junction were different regardless of Mg2+ concentration.
Muscle membrane potentials were amplified using an Axoprobe 1A amplifier (Axon Instruments, Foster City, CA), low-pass filtered at 1 kHz, and digitized at 10 kHz using an analog-to-digital converter (DigiData; Axon Instruments) and interactive software (Axoscope; Axon Instruments).
By recording from a muscle fiber while independently altering the stimulation voltage to each ventral root bundle, the number of inputs to each fiber was counted and recorded. In initial experiments to count the number of inputs to junctions and choose the appropriate age range of animals for future studies, a total of 50 junctions was sampled from at least three P7, P8, and P9 muscles. Independent stimulation of each input resulted in an endplate potential (epp) that had a single amplitude component as well as constant rise time and duration. Muscle fibers that were innervated by only two axons, one from each of two different suction electrodes, were selected for further study. Stimulus intensity was set at 1.5 times threshold for eliciting an epp, stimulus frequency was 1 Hz, and these generally remained constant for the duration of the experiment. Preparations that contained denervated muscle fibers, indicative of possible damage to axons during the dissection, were not studied further.
All experiments were performed at room temperature. The resting membrane potential was continuously monitored, and only fibers with resting potentials more hyperpolarized than
55 mV, and in which the resting potential did not change by >5 mV during the course of the experiment, were studied further. In contrast to recordings from adult muscle fibers in which resting potentials of
Quantal content was determined from 300-1000 stimuli (mean of 862) using the method of failures (Del Castillo and Katz, 1954
To determine the amount of paired-pulse facilitation (F) (Mallart and Martin, 1968
In some experiments, continuous recordings and evaluation of F were made from the same muscle fiber while altering the Ca2+/Mg2+ composition of the Ringer's solution. The same fibers were recorded in at least two solutions. In these experiments, each Ringer's solution was allowed to perfuse over the muscle for at least 10 min before stimulation, a time sufficient to ensure both complete exchange of the solution and stable recording conditions.
In one set of experiments, intracellular recordings from skeletal muscle fibers were obtained in 4 mM Ca2+/1 mM Mg2+ in the presence of 7-10 µM curare (Sigma, St. Louis, MO). In these experiments, 4 mM Ca2+ allowed stable recordings to be made over relatively long times from neonatal muscle fibers. Facilitation was examined at 10, 20, 50, and 100 msec and 1 sec i.p.i.
Statistical analyses were done using Sigma Plot 4.0, Sigma Stat 2.03 (SPSS Inc., Chicago, IL) and Prism (GraphPad Software, San Diego, CA). Data are presented as mean ± SEM (n = number of junctions).
RESULTS
Differences in quantal content among competing inputs at dually innervated soleus neuromuscular junctions
Intracellular recording of epps from P7-P9 soleus muscle fibers showed that ~50% of junctions were innervated by more than one motor axon, consistent with anatomical measures of multiple innervation from immunostaining experiments (Fig. 1A,B). At P7-P9, physiological and anatomical measures showed that a large proportion of multiply innervated junctions were innervated by only two axons (47 ± 2 and 45 ± 2%, respectively).
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Figure 1. Characterization of synapse elimination in the neonatal mouse soleus muscle. A, Immunostaining of motor axons and nerve terminals (green) and AChR clusters (red) at neuromuscular junctions from P7 mouse soleus muscle. At this age, most junctions are innervated by two or more axons. In this field, there are four examples (unlabeled). The terminals of each axon are extensively intermingled over AChR clusters in the postsynaptic muscle fiber membrane. Competition results in the elimination of all but one axon, resulting in the mature pattern of single innervation of each junction. In this field, there are two singly innervated junctions (arrows). Near the single axon innervating the AChR cluster at the bottom left, there is an enlarged bulb-like ending attached to an atrophied axonal branch (arrowhead). These are remnants of the losing axon that withdraws from the junction and are resorbed back into a parent axon in the intramuscular nerve (cf. Balice-Gordon et al., 1993
The quantal content of each input to dually innervated neuromuscular junctions was measured in 2 mM Ca2+/13-15 mM Mg2+ saline. The quantal content of the weaker input was on average 0.4 ± 0.1, whereas the quantal content of the stronger input was 0.9 ± 0.1 (n = 22). The quantal content of the inputs to dually innervated junctions differed from 1-fold to 5.5-fold (mean of 2.7 ± 0.3-fold; n = 22) (Fig. 1B), similar to the range of quantal content ratios observed at trapezius neuromuscular junctions at comparable stages (Colman et al., 1997
Differences in paired-pulse facilitation between weak and strong inputs to dually innervated neuromuscular junctions
Previous work suggested that the divergence in the synaptic strength of competing inputs could be explained by the progressive reduction in the strength of losing inputs. Because synaptic area approximately correlates with the amount of neurotransmitter released (Kuno et al., 1971
Intracellular recordings, made in 2 mM Ca2+/13-15 mM Mg2+ to prevent contractions, during paired-pulse stimulation at 10 msec i.p.i., showed that F was significantly greater for the weaker compared with the stronger input. A representative example from a dually innervated P8 neuromuscular junction is shown in Figure 2. Input 1 had a smaller epp amplitude (0.23 mV) (Fig. 2A), a lower quantal content (m = 0.5) (Fig. 2B), and a higher F at 10 msec i.p.i. (F = 1.4) (Fig. 2C) compared with input 2 (epp amplitude = 0.37 mV; m = 1.4; F = 1.0). As expected, no facilitation of either input was observed at 1 sec i.p.i. (Fig. 2D).
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Figure 2. Paired-pulse facilitation differs between strong and weak inputs to dually innervated neuromuscular junctions. Representative characterization of a dually innervated neuromuscular junction from a P8 soleus muscle in 2 mM Ca2+/13-15 mM Mg2+ Ringer's solution. A, Sample traces of epp (average of ~1000 consecutive traces). Amplitude: input 1 (weak), 0.23 mV; input 2 (strong), 0.37 mV. B, epp amplitude histogram, including failures. Quantal content: input 1, 0.5; input 2, 1.4. C, Responses to paired-pulse stimulation at 10 msec i.p.i. Facilitation ratio: input 1, 1.4; input 2, 1.0. D, Responses to paired-pulse stimulation at 1 sec i.p.i. Facilitation ratio: input 1, 1.0; input 2, 0.9. This depression was not consistently observed. Calibration: A, C, D, 0.1 mV, 5.0 msec. Thus, in this case, input 1 had a smaller epp amplitude, a lower quantal content, and a higher F at 10 msec i.p.i. compared with input 2.
The observation that F was significantly greater for the weaker input compared with the stronger input at 10 msec but not at 1 sec i.p.i. held for 17 of 22 junctions examined in 2 mM Ca2+/13-15 mM Mg2+ (Fig. 3A). Fweak was significantly greater than Fstrong (Fig. 3B) when examined at 10 msec i.p.i. (Fweak = 1.7 ± 0.1; Fstrong = 1.3 ± 0.1; n = 22; p < 0.001, Student's t test) but not at 1 sec i.p.i. (Fweak = 1.0 ± 0.1; Fstrong = 1.0 ± 0.1; n = 22; p > 0.1, Student's t test).
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Figure 3. Paired-pulse facilitation is greater for the weak input than the strong input at dually innervated neonatal neuromuscular junctions. A, F at 10 msec i.p.i. (filled circles) and 1 sec i.p.i. (open circles) for weak (smaller quantal content) plotted against F for strong (higher quantal content) inputs to dually innervated neuromuscular junctions examined in 2 mM Ca2+/13-15 mM Mg2+ (n = 22 junctions). At 10 msec i.p.i., in 17 of 22 junctions, Fweak was larger than Fstrong, as evidenced by most points clustering above the thin line that indicates a slope of 1. At 1 sec i.p.i., Fweak was similar to Fstrong. B, On average, Fweak (open bars) was significantly greater than Fstrong (filled bars) at 10 msec i.p.i. (p < 0.001, Student's t test) but not 1 sec i.p.i. (p > 0.09, Student's t test). C, F was determined at 10 msec i.p.i. and plotted against quantal content (m) for weak (open circles) and strong (filled circles) inputs to dually innervated junctions. The two inputs that innervated the same junction are connected by a line. For most pairs of inputs, there is an inverse relationship between F and m. Asterisk indicates a pair of inputs in which m was approximately equal for both, but F was different. D, At 1 sec i.p.i., similar F was observed between weak and strong inputs. E, Mean F at 10 msec i.p.i. is plotted against mean m for all weak (open circle) and all strong (filled circle) inputs. The mean quantal content ratio for all strong and weak inputs to dually innervated junctions examined in 2 mM Ca2+/13-15 mM Mg2+ was 2.7 ± 0.3 (see also Fig. 1B), and the mean mstrong (0.9 ± 0.1) was significantly greater than the mean mweak (0.4 ± 0.1; p < 0.001, Student's t test). F, At 1 sec i.p.i., no significant difference in F was observed between weak and strong inputs to dually innervated junctions (see also B).
Facilitation was also significantly greater for weaker compared with stronger inputs to the same muscle fiber when measurements were made in 4 mM Ca2+/1 mM Mg2+ in the presence of 7-10 µM curare (Sigma). Fweak was significantly greater than Fstrong when examined at 10 msec i.p.i. (Fweak = 1.3 ± 0.07; Fstrong = 1.0 ± 0.03; n = 9; p < 0.003, Student's t test) but not at 1 sec i.p.i. (Fweak = 0.88 ± 0.05; Fstrong = 0.80 ± 0.04; n = 9; p > 0.1, Student's t test). Depression at 1 sec was observed under these conditions for eight of nine cells. In this series of experiments, facilitation was also examined at 20, 50, and 100 msec i.p.i. Fweak and Fstrong were significantly different at 20 msec (Fweak = 1.1 ± 0.06; Fstrong = 0.93 ± 0.05; n = 9; p < 0.005, Student's t test), but no facilitation was observed at 50 and 100 msec, and F was similar for weak and strong inputs at 50 and 100 msec (data not shown). Whereas Fweak and Fstrong were different at 10 and 20 msec, the time course of facilitation across i.p.i was similar between weak and strong inputs (data not shown). Thus, the difference in facilitation between weak and strong inputs to the same junction is also apparent in more physiological concentrations of Ca2+ and Mg2+, albeit in the presence of curare to prevent muscle contractions.
For most neuromuscular junctions examined, F and quantal content were inversely related for weak and strong inputs examined at 10 msec i.p.i. (Fig. 3C) but not at 1 sec i.p.i. (Fig. 3D). F was greater for the weaker input regardless of the absolute value of quantal content for either input (Fig. 3C). On average, Fweak was significantly greater than Fstrong at 10 msec i.p.i. but not at 1 sec i.p.i. (Fig. 3B,E,F). The relationship between F and quantal content was different for each pair of inputs that coinnervated the same neuromuscular junction, consistent with each pair being at a different stage in the competitive process. Together, these data suggest that the disparity in quantal content between competing inputs is attributable, at least in part, to a disparity in the probability of neurotransmitter release.
Relationship between paired-pulse facilitation and quantal content for weak compared with strong inputs to dually innervated neuromuscular junctions
Given the inverse relationship between F and the probability of neurotransmitter release, one interpretation of this result is that strong inputs have a higher probability of release than weak inputs at dually innervated neonatal junctions. This interpretation rests on the assumption that developing motor nerve terminals also have an inverse relationship between F and release probability, as has been demonstrated previously for many other synapses (Creager et al., 1980
To determine whether F and release probability were inversely related at developing motor nerve terminals, in some fibers, intracellular recordings were maintained while the extracellular Ca2+ concentration was changed from 1 mM Ca2+/8-10 mM Mg2+ to 4 mM Ca2+/24-27 mM Mg2+ (Fig. 4). Paired-pulse facilitation was examined at 10 msec i.p.i. for weak and strong inputs to dually innervated neonatal fibers, and single inputs to neonatal and adult fibers under conditions of low (1 mM Ca2+) and high (4 mM Ca2+) release probability. In each case, an inverse relationship between F and quantal content was observed at 10 msec i.p.i. (Fig. 4A-D) but not at 1 sec i.p.i. (data not shown). Thus, each individual input had an inverse relationship between F and extracellular Ca2+, despite differences in quantal content, developmental history, and degree of maturation. This was also true for populations of weak and strong inputs to dually innervated neonatal fibers, and single inputs to neonatal and adult fibers under conditions of low (1 mM Ca2+), normal (2 mM Ca2+), and high (4 mM Ca2+) release probability (Fig. 5A-D).
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Figure 4. Relationship among paired-pulse facilitation and quantal content at individual junctions as extracellular Ca2+ was varied. A-D, Continuous recordings were made from some individual neuromuscular junctions as extracellular Ca2+ was varied from 1 mM (left circle of each pair joined by a line) to 4 mM (right circle of each pair), and the extent of paired-pulse facilitation at 10 msec i.p.i. and quantal content were determined for weak (A) and strong (B) inputs to dually innervated P7-P9 junctions (n = 16), singly innervated P7-P9 (C; n = 28), and adult junctions (D; n = 12). In each case, an inverse relationship between F and m was observed. E, Summary of F and m for weak (open circles) and strong (filled circles) inputs to dually innervated junctions and single inputs to neonatal (open squares) and adult (filled squares) junctions. Left symbol of each pair represents mean ± SEM measured in low (1 mM) calcium, and right symbol represents mean ± SEM measured in high (4 mM) calcium. For weak inputs (open circles), in low Ca2+, Fweak = 2.9 ± 0.2, mweak = 0.3 ± 0.1; in high Ca2+, Fweak = 1.6 ± 0.2, mweak = 0.7 ± 0.1. For strong inputs (filled circles), in low Ca2+, Fstrong = 1.4 ± 0.1, mstrong = 0.8 ± 0.1; in high Ca2+, Fstrong = 1.0 ± 0.1, mstrong = 1.5 ± 0.1. For single inputs to neonatal junctions (open squares), in low Ca2+, Fsingle = 1.9 ± 0.2, msingle = 0.6 ± 0.1; in high Ca2+, Fsingle = 1.0 ± 0.1, msingle = 1.3 ± 0.1. For single inputs to adult junctions (filled squares), in low Ca2+, Fadult = 1.6 ± 0.1, madult = 0.6 ± 0.1; in high Ca2+, Fadult = 1.1 ± 0.1, madult = 1.2 ± 0.1.
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Figure 5. Relationship among paired-pulse facilitation, quantal content, release probability, and number of release sites. A-D, Scatter plot of F at 10 msec i.p.i. and m for individual junctions collected in 1, 2, and 4 mM Ca2+ for weak (A) and strong (B) inputs to dually innervated junctions (n = 72) and single inputs to neonatal (C; n = 89) and adult (D; n = 50) junctions. In each case, increasing Ca2+ decreased F and increased m (data not shown), resulting in an inverse relationship. The best-fit curve to each data set was defined by a simple hyperbola, y = y0 + a/x, shifted along the ordinate. E, Comparison of best-fit curves for each data set, made using a nonlinear, least-squares fitting algorithm. Best-fit parameters, y0 and a, were compared using a one-way ANOVA, followed by Tukey's multiple comparison post hoc test. The shape of curves, affected by the parameter a, was similar for weak and strong inputs to dually innervated junctions (weak a = 0.11 ± 0.03; strong a = 0.14 ± 0.04; not significantly different, p > 0.05). However, the curve for weak inputs was shifted upward, toward higher F values for the same range of m (weak y0 = 1.68 ± 0.12, strong y0 = 1.16 ± 0.07; p < 0.01). The curve for strong inputs was similar to that for singly innervated neonatal junctions (y0 = 1.12 ± 0.12; a = 0.25 ± 0.07; not significantly different, p > 0.05). However, the curve for single inputs to neonatal junctions was shifted to the right of that for strong inputs, although the shape parameter a was not significantly different. The curve for adult junctions had a similar y0 as that for single inputs (y0 = 0.77 ± 0.1; not significantly different, p > 0.05), but the shape of the curve was different (a = 0.46 ± 0.06; p < 0.05) and was shifted to the right, as would be expected if adult inputs had a higher number of release sites, n, than neonatal inputs. F, The relative contributions of n and p were evaluated by constructing a simple quantitative model of the relationship between F and m. We assumed that a reciprocal relationship exists between F and p, such that their product is a constant. 1, If the higher F of weak inputs were attributable primarily to changes in n, then when n is reduced twofold, the entire curve would be shifted to the left, because only m would be affected. 2, If the higher F of weak inputs were attributable primarily to a reduction in p, then when p is reduced twofold but n is unchanged, the relationship would be shifted along the same curve but toward higher values of F. 3, If the higher F of weak inputs were attributable to a reduction in both n and p, then when both n and p are reduced twofold, the observed relationship would be similar to that obtained for a twofold reduction in n only, but F would be shifted toward higher values. 4, If the higher F of weak inputs were attributable to a reduced p in weak inputs and a change the overall relationship between F and p, then the points would be shifted further toward the left along the curve, and the entire relationship would be shifted upward across all m values. Our experimental results (E) are consistent with this latter possibility.
We next asked why weak inputs become weak, assuming that an initial state exists when all inputs to a muscle fiber are structurally and functionally similar (Creager et al., 1980
The first possibility, consistent with previous work (Creager et al., 1980
However, the data did not fit any of these three possibilities. The experimentally determined relationship between F and m for weak inputs differed from that for strong inputs in two respects (Fig. 5E). First, it was shifted upward, toward higher asymptotic values of F across all values of m. This is not consistent with weak inputs being weak by virtue of decreased n (first possibility above) or a combination of decreased n with decreased p (third possibility above), which would instead have shifted values to the left of those for strong inputs. This upward shift suggests that there is a change in the process of facilitation itself (Fig. 5F4). As inputs become weak, there may be changes in the modulation of neurotransmitter release, or in release machinery itself, that affect facilitation.
Second, values appeared to be shifted upward along the curve, toward lower values of m and higher values of F. This suggests that the higher F of weak inputs is attributable not only to a change in the process of facilitation itself but also to a reduction in p (second possibility above). To test this possibility, we calculated the F value for weak and strong inputs in the same range of m, from 0 to 0.5, measured in junctions in 1 mM Ca2+. Not only were more points from weak inputs in this range than from strong inputs, but the average F value was 2.4 ± 0.2 for weak inputs (n = 20), significantly greater than the value for strong inputs (1.6 ± 0.2; n = 7; Student's t test, p < 0.005). An additional test was performed to determine whether points from weak inputs are shifted along the curve toward lower values of m and higher values of F. The ratio of F/m was determined for each value. Inputs with large m and small F would have a small ratio, whereas those with small m and large F would have a large ratio. The ratio for strong inputs was 2.1 ± 0.3 (n = 72). Before calculating the ratio for weak inputs, we shifted their F value downward by the average difference in F, determined from the curve fits of the data from all muscle fibers (Fig. 5E). After this shift, the ratio for weak inputs was 6.1 ± 1.3, significantly larger than for strong inputs (p < 0.001; Mann-Whitney test). Thus, weak inputs had higher F values than strong inputs across all values of m, consistent with a change in the process of facilitation, and were also shifted to the left, upwards along the curve, consistent with a decreased release probability in weak inputs (Fig. 5F4).
The relationship between F and m for single inputs to neonatal junctions was observed to be shifted to the right of that for strong inputs to dually innervated junctions (although this was not significant; see legend to Fig. 5). The relationship for adult junctions was shifted to the right of that for strong inputs to dually innervated junctions and that for single inputs to neonatal junctions (Fig. 5E). This is consistent with a gradual increase in n, as well as with an increase in n and p, as inputs mature.
Progressive disparity in release probability among competitors
Previous work has shown that the quantal content ratio (mstrong/mweak) of competing inputs becomes progressively larger during postnatal life, as the transition from multiple to single innervation progresses (Colman and Lichtman, 1997
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Figure 6. Progressive disparity in F of weak compared with strong inputs compared with progressive disparity in quantal content. Previous work has shown that the quantal content ratio (mstrong/mweak) of competing inputs becomes progressively larger during postnatal life, as the transition from multiple to single innervation progresses (Colman and Lichtman, 1997
DISCUSSION
Intracellular recordings from dually innervated neonatal mouse soleus muscle fibers were used to characterize changes in synaptic function that occur during competition. Quantal content and paired-pulse facilitation, a measure of synaptic function that is inversely related to neurotransmitter release probability, were compared between weak and strong inputs to the same neuromuscular junction. Paired-pulse facilitation was greater for the weaker input compared with the stronger input, regardless of their absolute quantal content. This suggests that weaker inputs have a lower probability of neurotransmitter release than stronger inputs to the same junction. This difference in facilitation between weak and strong inputs to the same junction was apparent across a range of Ca2+ and Mg2+ concentrations and also when m was reduced by adding curare to the recording solution. This suggests that the differences in facilitation and quantal content may be functionally significant in vivo. Together, our results suggest that differences in presynaptic neurotransmitter release contribute to the increasing disparity in quantal content that is a hallmark of the competitive process.
How do inputs become weak during synaptic competition?
Our results show that weak inputs had higher paired-pulse facilitation than strong inputs to dually innervated junctions, across similar ranges of quantal content. This difference was observed at 10 msec i.p.i. but not at 1 sec, in high Mg2+ Ringer's solution, and at 10 and 20 msec but not at 1 sec, in 4 mM Ca2+/1 mM Mg2+ with curare. The difference in F at short i.p.i. raises the issue of whether these intervals are physiologically relevant, given the firing rates of soleus motor neurons in vivo. We have measured the firing patterns and rates of single motor units from neonatal mouse soleus muscle (Personius and Balice-Gordon, 1999
Based on previous observations of a progressive disparity in the presynaptic terminal area of competitors (Balice-Gordon et al., 1993
A second possibility is that the higher F of weak inputs is attributable primarily to a reduction in p. If this were the case, the relationship between F and m for weak inputs would be on the same curve as that for strong inputs but shifted along that curve toward higher values of F and lower values of m (Fig. 5F2). This was in fact observed (Fig. 5), suggesting that weak inputs had a lower release probability than strong inputs. In addition, however, there was an upward shift in F across the same values of m for weak and strong inputs. The possibility that weak inputs could increase their facilitation, without reducing release probability, seems unlikely, because weak inputs had a significantly higher F than strong inputs across the same range of m, even when considering the upward shift in F. Moreover, there was no difference in the time course of facilitation across a range of i.p.i. between weak and strong inputs. Together, then, these data suggest that inputs may become progressively weak not only by a reduction in neurotransmitter release probability but also by a change in the process of facilitation itself. Whereas using paired-pulse facilitation to probe release probability is indirect, a more direct measure might be obtained by differential labeling of competing inputs with FM dyes, followed by optical measurement of destaining rates, for example (cf. Betz and Bewick, 1992
Surprisingly, an increase in the n of strong inputs to dually innervated junctions does not appear to contribute to the increasing disparity in m between weak and strong inputs. If this were the case, the relationship between F and m for strong inputs would be shifted to the right of the curve for weak inputs. The absence of such a rightward shift suggests that inputs become relatively stronger because other inputs are weakened. Thus, it appears that the weakening of one input, rather than the differential strengthening of competitors, is a major, early contributor to the increasing disparity in quantal content that is a consequence of the competitive process (Colman et al., 1997
A shift to the right in the relationship between F and m was detected for single inputs to neonatal and adult junctions (Fig. 5E), as would be expected for a gradual increase in n, or n as well as p, with maturation. Moreover, because strong inputs become single inputs, the shift to the right in the relationship between F and m of single inputs to neonatal and adult junctions, suggesting an increase in n (Fig. 5E and above), is also consistent with previous anatomical observations. The growth of winning inputs through adulthood occurs by the expansion of existing terminal regions (Balice-Gordon et al., 1993
What may underlie the reduction of neurotransmitter release probability as well as alter the facilitation process? Differences in Ca2+ buffering affect facilitation (cf. Hochner et al., 1991
Another possibility for the disparity in release probability and facilitation between competing inputs may be a difference in the maturation of vesicle release machinery, including structural maturation of active zones. Although proteins involved in neurotransmitter release continue to be elucidated, the developmental regulation of release machinery is poorly understood (Rosenmund and Stevens, 1996
Cascade of events underlying synaptic competition
The results presented here raise the question of whether the changes in F and m of weak compared with strong inputs are the cause, or the effect, of competition. Although a detailed molecular and cellular understanding of the mechanisms underlying competition is gradually emerging, our data suggest that a change in neurotransmitter release probability and the facilitation process is an early event in competition and that this change contributes to a progressive disparity in m that is the functional measure of the efficacy of an input. Collectively, the available data on competition at neuromuscular and most other synapses (Katz and Shatz, 1996
Active inputs, on the other hand, emerge as winners in the competitive process, by maintaining a high quantal content, in part by maintaining a higher release probability than their competitors. High release probability may, in turn, prevent the depletion of postsynaptic receptors, preserving synaptic area and strength. After single innervation is established, quantal content increases further until adulthood, probably by the gradual addition of release sites. A similar modulation of presynaptic release mechanisms, coupled to changes in postsynaptic neurotransmitter receptor density, may account for structural and functional plasticity commonly observed at neuron-neuron synapses in the developing and mature brain (Lissen et al., 1999
FOOTNOTES Received June 19, 2000; revised Aug. 21, 2000; accepted Sept. 7, 2000.
This work was supported by National Institutes of Health Grant NS38517 (to R. B.-G.) and National Institutes of Health National Research Service Award NS10624 (to D. M. K.). We thank Dr. T. Parsons for advice and comments on earlier versions of this manuscript, Dr. P. Nealen for help with statistical analyses, and Drs. M. Gonzalez, Q. Chang, and K. Personius for helpful discussions.
Correspondence should be addressed to Rita Balice-Gordon, Department of Neuroscience, University of Pennsylvania School of Medicine, 215 Stemmler Hall, Philadelphia, PA 19104-6074. E-mail: rbaliceg{at}mail.med.upenn.edu.
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