Neuromuscular Junction (NMJ): Presynaptic Stretch Effects on Neuromuscular Transmission

Neuromuscular Junction (NMJ): Presynaptic Stretch Effects on Neuromuscular Transmission 635

Neuromuscular Junction (NMJ): Presynaptic Stretch Effects on Neuromuscular Transmission A D Grinnell, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA ã 2009 Elsevier Ltd. All rights reserved.

How Muscle Stretch Influences Neuromuscular Transmission One of the most robust forms of synaptic plasticity described to date is the modulation of neurotransmitter release from frog motor nerve terminals by muscle stretch. Fatt and Katz, in their classic paper first describing the quantal nature of neurotransmission, noted that ‘‘Stretching a muscle 10–15% beyond its resting length reversibly increased the rate by a factor of 2.5–3, and pulling on the motor nerve sometimes caused a similar ‘speeding up’ of the discharge.’’ Indeed, subsequent research has shown that over the physiological range of muscle length, from about 5–10% below rest length to 15–20% muscle stretch, miniature endplate potential (mEPP) frequency increases by about 10% for each 1% change in length. The effect is fully reversible. Following partial curarization, or at a reduced [Ca2þ] where quantal content can be measured, EPP amplitude shows comparable enhancement (Figure 1). The Kinetics of Stretch-Induced Enhancement of Release

In the 1990s, Grinnell and his associates turned their attention to the kinetics and possible mechanisms of the stretch effect. Using ‘floating electrodes’ and a computer-driven stretching/shortening apparatus that could change the length of a 30 mm-long frog sartorious muscle linearly by as much as 4 mm at rates up to 1 mm/5 ms, they confirmed that the enhancement of EPP amplitude is linear with length, maintained constant at a new length, and fully reversible. Remarkably, there is virtually no delay between the change in length and the corresponding change in EPP amplitude (Figure 2). Lack of Dependence on Ca2þ Influx or Release of Ca2þ from Internal Stores

Given the importance of Ca2þ in triggering neurotransmitter release, an obvious hypothesis is that stretch increases Ca2þ influx or causes release of Ca2þ from internal stores. Neither appears to be the case. The enhancement of spontaneous release is not blocked by o-conotoxin GVIA, which blocks N-type Ca2þ channels and EPPs in frog muscle, and it persists

in a Ringer containing only 0.1 mM Ca2þ and 10 mM Mg2þ. Even after an hour’s incubation in a Ringer containing 0 mM Ca2þ, 2 mM Mg2þ and 1 mM ethylene glycol tetracetic acid (EGTA), which blocked EPPs and reduced the mean resting mEPP frequency by about 50%, the stretch effect was not blocked, although it too was reduced by about 50%. Thus, Ca2þ influx through stretch-activated or any other kind of channel is not required for the stretch enhancement of spontaneous release, although some Ca2þ in the external environment appears to enhance the effect. Several experiments also ruled out the possibility that nerve terminal stretch somehow causes the release of Ca2þ from internal stores. When junctions were incubated in 25 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid tetraacetoxy methyl ester (BAPTA-AM) to clamp the intraterminal [Ca2þ] at 100 nM and further treated with 10 mM thapsigargin to block the endoplasmic reticulum Ca2þ-ATPase, disabling a major compartment for sequestering or releasing Ca2þ, stretch still produced about half the normal enhancement of release. Moreover, the rapidity and linearity of the modulation, the lack of ‘overshoot’, and the equally rapid and linear reversal of the phenomenon are inconsistent with internal Ca2þ release, or indeed with any mechanism on involving biochemical reactions or a second messenger cascade. Finally, when the effects of stretch on mEPP frequency and EPP amplitude were compared at 13 and 22 , the degree of modulation was the same, that is, the temperature effect (Q10) of the effect is 1. These findings are compelling evidence that the modulation is a purely mechanical phenomenon. Several hypotheses have been proposed to explain the modulation on the basis of physical phenomena. Frog motor terminals consist of multiple branches, often up to 100–200 mm in length and running parallel to the muscle fiber, slightly invaginated into gutters in the muscle fiber surface. As a muscle fiber is stretched, the nerve terminal branches are proportionately elongated. This could slightly decrease the terminal diameter, arguably forcing water out and causing an increase in the internal Ca2þ concentration. However, calculations suggest that a possible change in [Ca2þ] due to decrease in volume per unit length of terminal, if it occurs, would be much too small to explain the large change in release efficacy. Alternatively, stretch of the terminal might expose release sites that, at the shorter length, are cryptic, for example, hidden by folding of the presynaptic membrane or by Schwann cell processes and exposed by

636 Neuromuscular Junction (NMJ): Presynaptic Stretch Effects on Neuromuscular Transmission

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Figure 1 Modulation of neurotransmitter release by stretch. (a) mEPP frequency in a sartorius muscle fiber before, during, and after a 22% stretch of the muscle from a rest length of 2.59 mm/sarcomere. Each point represents the mean  SE of frequency calculated from the time required for occurrence of 100 mEPPs. (b) Histograms of mEPP amplitude during the prestretch, stretched, and poststretch periods in the same preparation. There was no change in mean mEPP amplitude. (c) Average change in EPP amplitude as a function of changes in muscle length in six sartorius muscle junctions (mean  SE). The EPP quantal content was reduced in a low-Ca2þ frog Ringer containing 0.56 mM Ca2þ and 4 mM Mg2þ. Inset: EPPs obtained in one preparation at 95%, 100%, 110%, and 120% of rest length. (At rest length, the sarcomere spacing was 2.25 mm.) (a, b) Reproduced from Turkanis SA (1973) Effects of muscle stretch on transmitter release at end-plates of rat diaphragm and frog sartorius muscle. Journal of Physiology (London) 230: 391–403, with permission from Blackwell publishing (c) From Chen B-M and Grinnell AD (1997) Kinetics, Ca2þ dependence, and biophysical properties of integrinmediated mechanical modulation of transmitter release from frog motor nerve terminals. Journal of Neuroscience 17(3): 904–916.

stretch. However, many studies of the ultrastructure of frog motor terminals have failed to find any evidence of significant Schwann cell occlusion or folding of the presynaptic membrane that could account for a large increase in exposed active zones with stretch. Thus, the likely mechanism is one that affects the configuration or distance between interacting molecules (or membranes) involved in release, thereby altering release probability. Integrins as Mediators of the Stretch Modulation

The magnitude and rapidity of the stretch modulation of release suggest that release is influenced by stretchimposed mechanical stress on the presynaptic membrane near active zones. Because Ca2þ influx is not necessary but the effect is reduced in zero-Ca2þ Ringer, Ca2þ-dependent adhesion interactions with one or more ligands in the extracellular matrix (ECM) are implicated. Obvious candidate adhesion molecules are cadherins and integrins, both of which occur widely at synapses throughout the nervous system. The role of

cadherins, if any, has not been investigated, but integrins play a major role in the stretch effect. Integrins are nearly ubiquitous heterodimeric transmembrane molecules that are the principal receptors connecting the cytoskeleton, intracellularly, to different ligands in the ECM. Ligand bonding can trigger profound changes in cells, including activation of protein kinase C (PKC) and phospholipase C, initiation of tyrosine phosphorylation cascades, induction of nitric oxide (NO) synthase, cytoplasmic alkalinization, and changes in gene expression. Integrins are also capable of transducing mechanical tension in both directions across the cell membrane, potentially bringing reactant molecules closer together or changing reaction equilibria by altering protein–protein conformation or stress on noncovalent bonds. A large fraction of the extracellular binding by integrins is to ligands containing a specific triplet of amino acids: arginine, glycine, and aspartic acid (RGD). Short peptides that contain the RGD sequence reduce the stretch enhancement of mEPP frequency and EPP amplitude by 40–50%, presumably by displacing

Neuromuscular Junction (NMJ): Presynaptic Stretch Effects on Neuromuscular Transmission 637 80

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Figure 2 Kinetics of stretch/shortening modulation of EPP amplitude. Average of six experiments in low-Ca2þ Ringer containing 0.56 mM Ca2þ and 4 mM Mg2þ. The muscles were attached at either end to a pair of modified chart recorder arms that moved in opposite directions in response to a command signal from a computer. The stretch began at 0 ms, reached 2 mm (6%) stretch at 50 ms, was held at that length for 45 ms, and shortened in another 50 ms (thin solid line at bottom). Upper records show the actual measured change in length of this muscle (noisy solid line from phototransistor) and the EPP amplitude evoked by nerve stimulation timed to generate EPPs at 36 different times during the lengthening/shortening cycle. Each point is the average of 50 or more EPPs, each evoked in a single lengthening/shortening cycle. There was virtually no delay between change in muscle length and change in release efficacy. From Chen B-M and Grinnell AD (1997) Kinetics, Ca2þ dependence, and biophysical properties of integrin-mediated mechanical modulation of transmitter release from frog motor nerve terminals. Journal of Neuroscience 17(3): 904–916.

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many of the integrin–ECM bonds. Inactive control peptides containing the sequence RGE have no effect (Figure 3). Moreover, a function-blocking polyclonal antibody against the b1 subunit of Xenopus integrin also reduces the stretch enhancement, while the preimmune serum has no effect. The remaining stretch effect in the presence of RGD peptides might indicate that integrins mediate only part of the phenomenon. However, integrin binding is dependent on the presence of Ca2þ. By lowering the concentration of Ca2þ to 50 mM or less, integrin bonds to native ligands are broken and the suppression of the stretch effect by RGD-containing peptides is much greater. Although the effect of reducing integrin binding to native ligands with RGD-containing peptides in a low-Ca2þ Ringer did not totally block the stretch effect, this can be explained by the probability that many of the native ligand bonds survived the treatment. Other experiments, moreover, support the conclusion that virtually all of the stretch effect can be attributed to integrins. These experiments also help

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Figure 3 Suppression of the stretch enhancement by RGD peptides, but not the inactive control RGE peptide, at frog sartorius neuromuscular junctions. mEPP frequency (a) was recorded in normal frog Ringer and EPP amplitude in 3–6 mM D-tubocurarine chloride. (b) Muscles were incubated for 1 h or more in 0.2 mM peptide before commencing recording. From Chen B-M and Grinnell AD (1997) Kinetics, Ca2þ dependence, and biophysical properties of integrin-mediated mechanical modulation of transmitter release from frog motor nerve terminals. Journal of Neuroscience 17(3): 904–916.

explain the reduction in the stretch enhancement in zero-Ca2þ Ringer. When frog nerve–muscle preparations are treated with Ringer containing zero-Ca2þ and 0.2 mM Mn2þ, the effect of stretch on mEPP frequency is close to that in normal Ringer. Manganese is a potent Ca2þ channel blocker but equally potent stabilizer of integrin–ligand bonds. Hence, Mn2þ apparently stabilizes integrin bonds that would otherwise be broken in the absence of extracellular Ca2þ. On the other hand, in the presence of Mn2þ integrins have been shown to bind more avidly to small RGD-containing peptides than to native ligands. Under these conditions,

638 Neuromuscular Junction (NMJ): Presynaptic Stretch Effects on Neuromuscular Transmission

RGD-containing peptides virtually totally block the stretch modulation of release at the frog neuromuscular junctions (Figure 4). It appears, therefore, that integrin binding to native ligands in the ECM can explain most or all of the stretch effect. Interfering with integrin–ECM binding does not alter the appearance of frog motor nerve terminals or their elongation with muscle stretch; other connective tissue components are still intact and attach the terminal tightly to the muscle fiber surface. Thus, the effect of reducing integrin–ligand binding appears to be specific to a subset of connections, probably attaching close to the presynaptic release sites, that are not needed for terminal elongation as a muscle is stretched but which profoundly regulate release probability.

The Intracellular Mechanism of Modulation of Release

The nature of the mechanical link between integrins and the release apparatus is still unknown. Integrins attach to the internal cytoskeletal via a number of linking proteins, and tension on integrins can transmit a mechanical stimulus almost instantly over many micrometers distance within the cell. To date, integrins have not been shown in any preparation to be connected, directly or indirectly, to any of the recognized vesicle or membrane soluble NSF attachment protein receptors (SNARES) or other docking/fusion apparatus. It is surely relevant, however, that treatment of frog junctions with okadaic acid, a broad-spectrum

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Figure 4 Stretch enhancement of mEPP frequency at sartorius neuromuscular junctions is not dependent on Ca2þ influx and is mostly blocked by 0.2 mM RGD. The stretch enhancement was reduced in 0 Ca2þ Ringer (column 2) and severely reduced by loading with BAPTA (column 5). 0.2 mM Mn2þ, which stabilizes integrin–ligand bonds, largely restored the stretch effect (columns 3 and 6). Further incubation with 0.2 mM RGD virtually eliminated the stretch effect (columns 4 and 7). From Chen B-M and Grinnell AD (1997) Kinetics, Ca2þ dependence, and biophysical properties of integrin-mediated mechanical modulation of transmitter release from frog motor nerve terminals. Journal of Neuroscience 17(3): 904–916.

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phosphatase inhibitor which causes ‘untethering’ of synaptic vesicles in the cloud around each active zone, also blocks the stretch effect, and that this effect of okadaic acid can be prevented by preincubation with the kinase inhibitor staurosporine (Figure 5). It would appear that one or more proteins in the mechanical chain between integrins and the release apparatus must be in the unphosphorylated state for the connection to be functional. Functional Importance and Generality of the Phenomenon

The stretch effect on release probability has been studied almost exclusively in the frog sartorius, in which it is clearly of functional significance. About 30% of the fibers in this muscle, at rest length in normal Ringer, do not generate suprathreshold EPPs to a single stimulation of the nerve. When a muscle is stretched, however, as it would be by contraction of an antagonist muscle, a single nerve impulse can excite most or all of the fibers, that is, the stretch effect is a strong peripheral amplifier of the spinal stretch reflex. Other frog muscles that have been tested, such as the cutaneous pectoris and some toe muscles, also exhibit a stretch effect, even though most or all of their fibers receive suprathreshold input at rest length. Integrins have been found at a wide variety of synapses and may be present in almost all. In Xenopus

motor terminals, active zones contain a3 b1 interins, while at rat neuromuscular junctions, the b1 subunit is associated with a1 presynaptically and with aV and with three different isoforms of a7 postsynaptically. Whether there is stretch enhancement of release in rat junctions is uncertain, however. Studies of rat diaphragm muscle have found no stretch effect, but the motor nerve boutons in most mammalian muscles are on top of distinct postsynaptic pads (‘endplates’) and might not undergo significant mechanical stretch or distortion when a muscle is stretched. Moreover, EPPs at mammalian neuromuscular junctions normally are suprathreshold at rest length, with a large safety margin, so there would be no obvious advantage in having peripheral amplification of release. Smooth muscle, with multiple innervation and contraction graded in response to EPP amplitude would seem a better candidate for this form of modulation. The widespread presence of integrins at synapses throughout the central nervous system (CNS) suggests that they may play important roles in nonneuromuscular connections. Evidence is accumulating that this is the case. Integrins are implicated in memory formation and in the consolidation of longterm potentiation (LTP) in hippocampal slices, perhaps stabilizing activity-related change in spine architecture; RGD peptides also cause abnormalities in kindling behavior in hippocampal slices, suggesting changes in

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Figure 5 Stretch enhancement of sartorius EPP amplitude and mEPP frequency are severely reduced by 1 h incubation at 0.3 mM okadaic acid (OA), a broad-spectrum phosphatase inhibitor. This effect of OA was largely prevented by 1–1.5 h preincubation in 0.5 mM staurosporine, a broad-spectrum kinase inhibitor. From Grinnell AD, Chen B-M, Kashani A, Lin J, Suzuki K, and Kidokoro Y (2003) The role of integrins in the modulation of neurotransmitter release from motor nerve terminals by stretch and hypertonicity. Journal of Neurocytology 32: 489–503.

640 Neuromuscular Junction (NMJ): Presynaptic Stretch Effects on Neuromuscular Transmission

excitability. Interference with integrin binding prevents a developmental increase in glutamate release and expression of N-methyl-D-aspartate (NMDA)-receptor subunits. In Drosophila, mutations in the volado gene, which encodes two forms of an a-integrin subunit, cause defective consolidation of olfactory memory. These mutants also exhibit abnormalities in neuromuscular neurotransmitter release and shortterm presynaptic plasticity, while mutations of the b-subunit lead to changes in motor nerve branching and neuromuscular morphology. Thus integrins may play a variety of important roles at synapses – roles that are only beginning to be elucidated. It is noteworthy that RGD peptides that disrupt integrin bonds sharply reduce the enhancement of spontaneous release by hypertonic solutions in both frog and Drosophila neuromuscular junctions. Since hypertonicity causes increased spontaneous release in almost all chemical synapses, integrins may play a role in regulating synaptic effectiveness at most synapses. See also: Calcium Channel Subtypes Involved in Neurotransmitter Release; Neuromuscular Junction (NMJ): Presynaptic Short-Term Plasticity of Neuromuscular Transmission; Neuromuscular Junction (NMJ): Presynaptic Schwann Cells and Modulation of Neuromuscular Transmission; Neuromuscular Junction (NMJ): Presynaptic Non-Quantal Release of Transmitter; Presynaptic Events in Neuromuscular Transmission; Presynaptic: Mitochondria and Presynaptic Function.

Further Reading Betz WJ and Henkel AW (1994) Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals. Journal of Cell Biology 124: 843–854.

Chavis P and Westbrook G (2001) Integrins mediate functional preand postsynaptic maturation of a hippocampal synapse. Nature 411: 317–321. Chen B-M and Grinnell AD (1995) Integrins and modulation of transmitter release from motor nerve terminals by stretch. Science 269: 1578–1580. Chen B-M and Grinnell AD (1997) Kinetics, Ca2þ dependence, and biophysical properties of integrin-mediated mechanical modulation of transmitter release from frog motor nerve terminals. Journal of Neuroscience 17(3): 904–916. Cohen MW, Hoffstrom BG, and Desimone DW (2000) Active zones on motor nerve terminals contain a3 b1 integrin. Journal of Neuroscience 20: 4912–4921. Fatt P and Katz B (1952) Spontaneous subthreshold activity at motor nerve endings. Journal of Physiology (London) 117: 109–128. Gailit J and Ruoslahti E (1988) Regulation of the fibronectin receptor affinity by divalent cations. Journal of Biological Chemistry 263: 12927–12932. Greensmith L and Vrbova G (1996) Motoneurone survival: A functional approach. Trends in Neuroscience 19: 450–455. Grinnell AD, Chen B-M, Kashani A, Lin J, Suzuki K, and Kidokoro Y (2003) The role of integrins in the modulation of neurotransmitter release from motor nerve terminals by stretch and hypertonicity. Journal of Neurocytology 32: 489–503. Grotewiel M, Beck C, Wu K, Zhu X, and Davis R (1998) Integrinmediated short-term memory in Drosophila. Nature 391: 455–460. Hutter OF and Trautwein W (1956) Neuromuscular facilitation by stretch of motor nerve endings. Journal of Physiology (London) 133: 610–625. Martin PT, Kaufman SJ, Kramer RH, and Sanes JR (1996) Synaptic integrins in developing, adult, and mutant muscle: Selective association of alpha1, a7A, and a7B integrins with the neuromuscular junction. Developmental Biology 174: 125–139. Martin PT and Sanes JR (1997) Integrins mediate adhesion to agrin and modulate agrin signaling. Development 124: 3909–3917. Staubli U, Chun D, and Lynch G (1998) Time dependent reversal of long-term potentiation by an integrin antagonist. Journal of Neuroscience 18: 3460–3469. Turkanis SA (1973) Effects of muscle stretch on transmitter release at end-plates of rat diaphragm and frog sartorius muscle. Journal of Physiology (London) 230: 391–403.