(2002), 545.2, pp. 337 343 DOI: 10.1113/jphysiol.2002.032516 The Physiological Society 2002 www.jphysiol.org Rapid Report Regulation by Rab3A of an endogenous modulator of neurotransmitter release at mouse motor nerve endings Jody K. Hirsh, Timothy J. Searl and Eugene M. Silinsky Department of Molecular Pharmacology and Biological Chemistry S-215, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA Rab3A, a small GTP-binding protein attached to synaptic vesicles, has been implicated in several stages in the process of neurosecretion, including a late stage occurring just prior to the actual release of neurotransmitter. The inhibitory neuromodulator adenosine also targets a late step in the neurosecretory pathway. We thus compared neuromuscular junctions from adult Rab3A _/_ mutant mice with those from wild-type mice with respect to: (a) the basic electrophysiological correlates of neurotransmitter release at different stimulation frequencies, and (b) the actions of exogenous and endogenous adenosine on neurotransmitter release in normal calcium solutions. Neither the spontaneous quantal release of acetylcholine (ACh) nor basal evoked ACh release (0.05 Hz) differed between the mutant and wild-type mice. At 50 100 Hz stimulation (10 19 stimuli), facilitation of release was observed in the mutant mice but not in wild-type, followed by a depression of ACh release in both strains. ACh release at the end of the stimulus train in the mutant mouse was approximately double that of the wild-type mouse. The threshold concentration for inhibition of ACh release by exogenous adenosine was over 20-fold lower in the mutant mouse than in the wildtype mouse. The adenosine A 1 receptor antagonist 8-cyclopentyltheophylline (CPT) increased ACh release (0.05 1 Hz stimulation) in the mutant mouse under conditions in which it had no effect in the wild-type mouse. CPT had no effect on the pattern of responses recorded during repetitive stimulation in either strain. The results suggest that Rab3A reduces the potency of adenosine as an endogenous mediator of neuromuscular depression. (Resubmitted 13 September 2002; accepted 21 October 2002; first published online 1 November 2002) Corresponding author E. M. Silinsky: Department of Molecular Pharmacology and Biological Chemistry S-215, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL 60611, USA. Email: e-silinsky@northwestern.edu Low molecular weight GTP-binding proteins of the Rab family participate in several important transport processes that ensue between the maturation of a secretory organelle and the exocytosis of encapsulated secretory product (Hess et al. 1993; Bennett & Scheller, 1993; Fischer von Mollard et al. 1994). Studies on knockout mice have implicated the neuron-specific Rab, Rab3A, in the targeting of synaptic vesicles to the plasma membrane and the docking of such vesicles at the active zones of secretion (Geppert et al. 1994, 1997). More recently it was suggested that this GTP-binding protein and its associated effectors, RIM and rabphilin, may be implicated in a late step in the exocytosis process (Geppert et al. 1997; Geppert & Südhof, 1998), as well as in certain forms of synaptic plasticity (Castillo et al. 1997). Specifically, in embryonic hippocampal neurons cultured from the Rab3A _/_ mouse, Geppert et al. (1997) found that both neurotransmitter release per impulse and facilitation of release is increased without a concomitant depletion of the immediately available store of neurotransmitter (Hubbard, 1973) at high frequencies of nerve stimulation. This is an intriguing result, as increases in basal evoked neurotransmitter release are generally not associated with increases in facilitation or decreases in the rundown of neurotransmitter release during such high-frequency stimulation. It would be of interest to determine whether similar effects are observed at adult motor nerve endings in Rab3A _/_ mutant mice. Adenosine is an endogenously released neuromodulator that mediates presynaptic depression at very low stimulation frequencies at the skeletal neuromuscular junction (Redman & Silinsky, 1994). In addition, the presynaptic target for this inhibitory feedback modulator appears to be a neuronal substrate that controls a late step in the process of neurotransmitter release (Redman & Silinsky, 1994; Robitaille et al. 1999). Given this overlap of characteristics between the sites of action of Rab3A and the proposed targets for adenosine, it would also be of interest to examine if the actions of both exogenously applied and endogenously released adenosine are altered in the Rab3A
338 J. K. Hirsh, T. J. Searl and E. M. Silinsky J. Physiol. 545.2 knockout mouse. The healthy phenotype of the Rab3A knockout mice (Geppert et al. 1994, 1997) makes them ideal targets for studies on the mature, differentiated skeletal neuromuscular junction in vitro. Indeed, this synapse allows one to make accurate assessments of the electrophysiological correlates of the release of the neurotransmitter acetylcholine (ACh; Silinsky, 1987) without the need for culturing motor neurons with skeletal muscle fibres. In this study, we focused our efforts on the evoked release of ACh from phrenic nerve hemidiaphragm preparations in normal calcium solutions and thus at normal levels of neurotransmitter release. METHODS Isolation of the phrenic nerve hemidiaphragm from Rab3A _/_ and wild-type mice Rab3A _/_ mice (B6, 129-Rab3Atm1Sud; stock no. JR2443) and a wild-type strain of similar genetic background (B6129F2/J; stock no. 101045) were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). Studies were done in accordance with the guidelines of the Northwestern University Animal Care and Use Committee and the NIH. Male (wild-type) or either sex (Rab3A _/_ ) mice of 20 30 g weight were anaesthetized by exposure to diethyl ether by inhalation for 3 5 min, until unresponsive to touch, and killed by exsanguination. The entire rib cage was excised and rinsed in physiological saline solution to remove extraneous blood. Electrophysiology The phrenic nerve hemidiaphragm was isolated and pinned out in a recording chamber whilst being continuously superfused with gassed physiological saline solution at room temperature (21 23 C) at a flow rate of 3.0 ml min _1. Drugs were added to this superfusion solution. In all experiments in which the effect of a drug was studied, electrophysiological recordings were made continuously from single endplates with each fibre serving as its own control. Intracellular electrodes were prepared using borosilicate glass (WPI) and filled with 3 M KCl. Electrode resistance ranged from 40 to 70 MV. Evoked ACh release, reflected as endplate potential (EPP) amplitude, was recorded on a PC using the Digidata 1200 interface and the program pclamp, and analysed using the program Clampan (Axon Instruments). Control physiological saline solution consisting of (mm): NaCl 137, KCl 5, CaCl 2 2, MgCl 2 2, NaH 2 PO 4 1, NaH 2 HCO 3 24, dextrose 11, ph 7.2 7.4 when gassed with 95 % O 2 5 % CO 2, was used in all experiments. d-tubocurarine chloride (TC) was added to reduce the threshold for action potential firing. Estimates of the mean number of ACh quanta released synchronously in response to an action potential (M, mean quantal content) were made using the curare method as follows. M was calculated using the formula: M = {(EPP in TC solution)/(mepp in normal solution)}(1+ K TC [TC]), where EPP is mean endplate potential amplitude (the responses to 16 stimuli were averaged), MEPP is the mean miniature endplate potential amplitude from the same muscle fibre (n = 100 MEPPs) prior to adding TC, [TC] is the concentration of d-tubocurarine (3.0 4.0 mm), and K TC is the equilibrium affinity constant for TC (2.6 mm _1 ) (see Silinsky, 1987). Statistical methods For comparison of two groups of normally distributed data (i.e. when experiments performed under the same conditions on wild-type and Rab3A _/_ mice were compared), Student s unpaired t test was used to determine whether statistically significant differences occurred between the mean values. When comparing three or more groups (i.e. when more than one experimental condition was used), a one-way repeated measures analysis of variance (RM ANOVA) was used to determine whether statistically significant differences occurred between the mean values of the treatment groups. Bonferroni s method of multiple comparison procedures was utilized to isolate the groups that differed from the others. The data are expressed as means ± 1 S.E.M. from n observations. Statistical significance was determined at the P 0.05 level. Data were analysed using SigmaStat and results were plotted using SigmaPlot (SPSS, Inc.). RESULTS No differences in basal levels of spontaneous and evoked ACh release between Rab3A _/_ and wild-type mice The electrophysiological properties of skeletal neuromuscular junctions in wild-type and mutant mice under basal release conditions were comparable. Specifically, no differences in the rate of spontaneous ACh release, reflected as the frequency of occurrence of spontaneous quantal events (miniature endplate potentials, MEPPs), were observed between wild-type mice (MEPP frequency = 0.75 ± 0.11 s _1, n = 13 experiments) and Rab3A _/_ mice (MEPP frequency = 0.52 ± 0.05 s _1, n =10 experiments; P = 0.1). Furthermore, there were no differences between the mean levels of evoked ACh release in wild-type mice (34.8 ± 8.9 quanta per impulse, n = 8 experiments) or Rab3A _/_ mutant mice (29.0 ± 5.7 quanta per impulse, n = 7 experiments; P = 0.6) at a very low stimulation frequency (0.05 Hz). High-frequency stimulation reveals differences between Rab3A _/_ and wild-type mice The phrenic nerve generally fires in brief high-frequency bursts during respiration. We thus decided to compare the effects of higher frequencies of stimulation in the two strains. We chose stimulation frequencies of 50 and 100 Hz, as these rates are representative of the normal firing frequencies in this preparation and also revealed the electrophysiological differences between hippocampal neurons cultured from wild-type and Rab3A _/_ mice (Geppert et al. 1997). Figure 1 shows representative experiments in which phrenic nerves from wild-type (A) and Rab3A _/_ mutants (B) were stimulated at 100 Hz and EPPs (which are a reliable electrophysiological assay for evoked neurotransmitter release) were recorded from the skeletal muscle fibres (mean data are depicted in Fig. 1C and D). Note that facilitation of the second impulse occurred only in the mutant mouse (see legend to Fig. 1 for details). In addition, despite the decline in ACh release during stimulation in both the wild-type and the mutant,
J. Physiol. 545.2 Rab3A in mouse motor neurons 339 the higher level of neurotransmitter release was maintained throughout the train, with the response to the last impulse being approximately 2-fold higher in the mutant than in the wild-type mouse (see legend to Fig. 1). Qualitatively similar results were observed at 50 Hz (Fig. 1D; see legend for details). The changes in facilitation and the higher level of secretion at the end of the 50 or 100 Hz trains at mouse neuromuscular junctions are similar to the results previously observed for cultured hippocampal neurons (Geppert et al. 1997), thus suggesting that Rab3A has a common role in the neurotransmitter release process at these different synapses. Enhanced effects of adenosine in Rab3A _/_ mice As noted above, adenosine plays an important role in controlling the release of ACh from the neuromuscular junction and may share common presynaptic targets with Rab3A. Figure 2A shows the typical relationship for the inhibition of ACh release by adenosine in the wild-type mouse strain. As shown in the averaged EPPs, the threshold for inhibition of ACh release was generally >100mM adenosine, with maximal inhibitory effects (approximately 50 % inhibition) being observed at concentrations of adenosine near 1mM. Indeed, no statistically significant effect was produced by 100 mm adenosine in wild-type mice (n = 5 experiments). In contrast to this relative insensitivity to adenosine in the wild-type mouse, inhibition of ACh release by adenosine in the Rab3A _/_ mutant occurred in the micromolar range. In the experiment depicted in Fig. 2B (n = 4 total experiments), 20 mm adenosine produced a highly statistically significant reduction in the averaged EPP amplitude (P < 0.001), which was fully reversible following washout to a lower concentration of adenosine (5 mm). A typical concentration response relationship for Figure 1. ACh release evoked at high stimulation frequencies is increased in Rab3A _/_ mutant mice A and B show representative experiments in which phrenic nerves from wild-type (A) and Rab3A _/_ (B) mice were stimulated with a train of stimuli delivered at a frequency of 100 Hz (each trace represents the average EPP in response to 8 stimuli). For the data depicted in C, the EPP amplitudes in the train were normalized to the amplitude of the first EPP and then plotted for the combined experiments. Specifically, the EPP amplitudes of the wild-type (filled bars, n = 6 preparations) and Rab3A _/_ (open bars, n = 8) strains were plotted vs. pulse number in the train of phrenic nerve stimuli elicited at 100 Hz for 200 ms. Trains of stimuli were delivered every 20 s. ACh release in the Rab3A _/_ strain was increased by 1.14 ± 0.04-fold in the second EPP (n = 8, P < 0.01). The wild-type did not show this effect as the mean amplitude of the second EPP was 1.07 ± 0.03 vs. the first EPP (n = 6, P = 0.06). In addition, at the end of the train the level of ACh release in the Rab3A _/_ strain was approximately double that of the wild-type (normalized amplitude of 0.32 ± 0.04 (n =8) for the final EPP of Rab3A _/_ vs. 0.15 ± 0.01 (n = 6) for the wild-type; P = 0.002). D shows that similar results were observed using the same protocol and stimulating the phrenic nerve at 50 Hz (wild-type: filled bars, n = 6 preparations; mutant: open bars, n = 8 preparations).
340 J. K. Hirsh, T. J. Searl and E. M. Silinsky J. Physiol. 545.2 the Rab3A _/_ mutant is shown in Fig. 2C. Mean data comparing the threshold for inhibition of ACh release by adenosine in wild-type and Rab3A _/_ mice are shown in Fig. 2D. Note that concentrations of adenosine that were devoid of effects in the wild-type mouse (open circles) had highly statistically significant effects in the Rab3A _/_ mutant mouse (filled circles) when compared with the control level of release in the absence of adenosine (shaded circle). The results thus demonstrate that Rab3A _/_ mutants are much more sensitive to exogenous adenosine than wild-type mice. Endogenously released adenosine derivatives can achieve substantial concentrations after a single nerve impulse at amphibian neuromuscular junctions (Silinsky & Redman, 1996). Given the increased sensitivity to adenosine in the Rab3A _/_ mutant mouse, it was of interest to determine whether endogenous adenosine plays a greater role in the control of evoked ACh release in the mutant mouse than in the wild-type strain. To this end, we examined the effect of the selective A 1 adenosine receptor antagonist 8-cyclopentyltheophylline (CPT, 1 mm; see Redman & Silinsky, 1994) on evoked ACh release in both mouse strains. As shown in Fig. 3A and B, CPT increased ACh release at a very low frequency of nerve stimulation in the Rab3A _/_ mutant mouse (0.05 Hz); Fig. 3A shows a typical experiment and Fig. 3B shows the mean data from five experiments in the mutant. Note the statistically significant increase in ACh release at either 0.05 or 1Hz in the presence of CPT (Fig. 3B, P < 0.001). When experiments Figure 2. Sensitivity to inhibition of ACh release by exogenous adenosine is increased in Rab3A _/_ mice A, a typical example of a dose response relationship for exogenously applied adenosine in a wild-type mouse. Each trace represents the average of 16 stimuli (0.05 Hz). Note the absence of effect of adenosine at concentrations of 50 and 100 mm in the wild-type mouse. B, a representative experiment in a Rab3A _/_ mouse in which 20 mm adenosine produced a statistically significant inhibition of ACh release (n = 4 experiments), an effect that was reversible following washout to a sub-threshold concentration of adenosine (5 mm). C, a representative dose response relationship for exogenous adenosine in the Rab3A _/_ mouse. D, mean data from experiments depicting the effect of low concentrations of adenosine on ACh release in the wild-type and mutant mice. Each data point represents the mean data from n = 3 6 experiments. Whilst 100 mm adenosine had no effect in the wild-type mouse, a statistically significant inhibition was produced by 100 mm adenosine in the Rab3A _/_ strain (0.80 ± 0.05, n = 5 experiments; P = 0.009). The maximal extent of the initial ACh release in the presence of 1mM adenosine was similar in both strains (0.69 ± 0.12 (n = 3) for wildtype and 0.56 ± 0.09 (n = 4) for Rab3A _/_ ; P = 0.3). The shaded circle (without error bars) indicates the control level of release in the absence of adenosine.
J. Physiol. 545.2 Rab3A in mouse motor neurons 341 were performed under similar conditions in the wild-type mouse, no statistically significant change in evoked ACh release was observed (P = 0.312, n = 5 experiments). Figure 3C shows a representative experiment in the wildtype mouse. The different patterns of ACh release between Rab3A _/_ and wild-type mice at high frequencies of nerve stimulation are not due to endogenous adenosine To determine whether differences in the effects of 50 100 Hz stimulation between the two strains are due to the differing influences of endogenous adenosine, we repeated the experiments of Fig. 1 in the presence of the A 1 adenosine receptor antagonist CPT (1 mm). Figure 4A and B provides an illustrative experiment performed at 100 Hz in the Rab3A _/_ mutant mouse. Figure 4A shows the data prior to CPT and Fig. 4B depicts the increased level of secretion observed after application of the adenosine antagonist in the mutant. When the increased EPP in CPT was normalized to the level observed in the absence of CPT (Fig. 4C), there was no difference in the pattern of secretion in response to 100 Hz stimulation in the presence of CPT (Fig. 4C depicts the mean data from 6 different experiments). When experiments were conducted in the wild-type mice, no differences were observed in the presence of CPT at 100 Hz (Fig. 4D, n = 6 experiments). Qualitatively similar results were observed at 50 Hz stimulation (data not shown). It thus appears that the differences between the wild-type and Rab3A _/_ mutant mice at these high stimulation frequencies are not due to the differential effects of endogenous adenosine. DISCUSSION These results demonstrate two intriguing aspects of neuromuscular transmission in the adult Rab3A _/_ mutant mouse. The first aspect relates to the effects of repetitive stimulation at 50 100 Hz. Specifically, in the Rab3A _/_ mutant mouse, facilitation of release occurs after a single impulse and this is associated with a reduced depression of EPPs during stimulation when compared with the wildtype mouse (Fig. 1A D). Similar effects of repetitive stimulation were observed at glutamatergic synapses between cultured hippocampal neurons from Rab3A _/_ mice (Geppert et al. 1997), providing evidence in support of similar roles for Rab3A at both of these excitatory synapses. The second result is that adenosine produces a potent inhibition of secretion in the Rab3A _/_ mutant mouse, such that endogenous adenosine inhibits release Figure 3. Endogenous adenosine has a greater potency in Rab3A _/_ mice than in the wildtype mice The adenosine A 1 receptor-selective antagonist 8-cyclopentyltheophylline (CPT, 1 mm) was applied to the Rab3A _/_ mutant (A and B) and the wild-type mouse (C). In A and C, preparations were stimulated at 0.05 Hz. Only preparations from Rab3A _/_ mice showed a significant (37 %) increase in ACh release in the presence of CPT (A and B). Similar results were obtained at the higher stimulation frequency of 1Hz in the mutant mice (B). Hence CPT increased release by 1.40 ± 0.08-fold (n = 5, * P 0.001 vs. control). For wild-type mice in the presence of CPT, no statistically significant increase in ACh release was observed (n = 5 experiments, P = 0.31). C depicts a typical experiment in the wild-type mouse (each trace is the average of 16 EPPs).
342 J. K. Hirsh, T. J. Searl and E. M. Silinsky J. Physiol. 545.2 even at the lowest frequencies of stimulation studied (0.05 Hz). This is similar to the effect of endogenous adenosine at the amphibian neuromuscular junction, where the first detectable sign of prejunctional depression at low frequencies of stimulation is due to endogenous adenosine released from stimulated motor nerve endings (Redman & Silinsky, 1994). The present results do not rule out the possibility that levels of endogenous adenosine differ between the two mouse strains. Such differences would not, however, explain the increased sensitivity to adenosine of the mutant mouse, as increased endogenous adenosine in the mutant mouse would be predicted to produce an apparent decrease rather than an increase in the potency of exogenous adenosine (see e.g. Fig. 2D). In contrast, if endogenous adenosine release is greater in the wild-type mouse than in the mutant mouse, then the effects of the adenosine receptor antagonist CPT would be more pronounced in the wild-type mouse; Fig. 3 shows this not to be the case. Adenosine acts by inhibiting the secretion of primed vesicles (Redman & Silinsky, 1994; Silinsky et al. 2001). The increased potency of adenosine in the Rab3A _/_ mouse raises the possibility that a substrate that normally regulates the potency of adenosine is absent in this mutant. While the precise substrate responsible for this altered sensitivity is unknown, previous results suggest that adenosine may reduce the apparent affinity for calcium of a strategic component of the secretory apparatus. This effect is likely to occur with the brief latency usually reserved for interactions of the bg subunits of G proteins with a target effector in the plasma membrane (Silinsky et al. 2001; Blackmer et al. 2001). Figure 4. ACh release evoked at high stimulation frequencies is not altered by an adenosine A 1 antagonist Phrenic nerves from Rab3A _/_ (A C) and wild-type (D) mice were stimulated with a train of stimuli delivered at a frequency of 100 Hz (see legend to Fig. 1 for details). The raw data traces from an individual experiment at a single endplate are shown in A and B. The averaged responses to 8 trains of stimuli in Rab3A _/_ mice are shown before (A) and after (B) treatment with 1 mm CPT. Although CPT increased the absolute level of ACh release in the mutant, it did not change the relative amount of ACh release (C) when the amplitude was normalized to that of the first EPP (Control: filled bars; CPT: open bars; n = 6 experiments from 6 preparations.) In the wild-type mouse (D), CPT did not increase the absolute level of ACh release nor did it alter the relative ACh release pattern (n = 6 experiments from 6 preparations).
J. Physiol. 545.2 Rab3A in mouse motor neurons 343 One possible explanation for the increased sensitivity to adenosine is that Rab3A directly interacts with the adenosine receptor or its G proteins in the plasma membrane. However, this seems unlikely as the active, GTP-bound form of Rab3A resides in the vesicle membrane. Another possible explanation is that receptor activation modulates a plasma membrane effector of Rab3A such as RIM. For example, RIM1a possesses domains that bind to important calcium sensors (e.g. synaptotagmin I) and has been implicated in providing a scaffold to maintain the structural integrity of the active zones of secretion (Schoch et al. 2002). RIM levels are unchanged in the Rab3A _/_ mutant mouse (Geppert et al. 1997), raising the possibility that RIM, which is normally bound to Rab3A, is a more potent target for the inhibitory effect of adenosine once it is unencumbered from its interaction with Rab3A. 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