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Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA

	Abstract

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A controversy currently exists as to the mechanism of action by which adenosine, an endogenous mediator of neurotransmitter depression, reduces the evoked release of the neurotransmitter acetylcholine (ACh) at the skelelal neuromuscular junction. Specifically, it is uncertain whether adenosine inhibits ACh release from mammalian motor nerve endings by reducing Ca²⁺ calcium entry through voltage-gated calcium channels or, as is the case at amphibian motor nerve endings, by an effect downstream of Ca²⁺ entry. In an attempt to address this controversy, the effects of adenosine on membrane ionic currents and neurotransmitter release were studied at neuromuscular junctions in adult mouse phrenic nerve hemidiaphragm preparations. In wild-type mice, adenosine (500 µM–1 mM) reduced prejunctional Ca²⁺ currents simultaneously with a reduction in evoked ACh release. In Rab3A knockout mice, which have been shown to have an increased sensitivity to adenosine, the simultaneous reduction in Ca²⁺ currents and ACh secretion occurred at significantly lower adenosine concentrations ( 50 µM). Measurements of nerve terminal Na⁺ and K⁺ currents made simultaneously with evoked ACh release demonstrated that the decreases in Ca²⁺ currents were not attributable to changes in cation entry through voltage-gated Na⁺ or K⁺ channels. Furthermore, no effects of adenosine on residual ionic currents were observed when P/Q-type calcium channels were blocked by Cd²⁺ or -agatoxin-IVA. The results demonstrate that inhibition of evoked neurotransmitter secretion by adenosine is associated with a reduction in Ca²⁺ calcium entry through voltage-gated P/Q Ca²⁺ channels at the mouse neuromuscular junction. Whilst it may be that adenosine inhibits ACh release by different mechanisms at amphibia and mammalian neuromuscular junctions, it is also possible that the secretory apparatus is more intimately coupled to the Ca²⁺ channels in the mouse such that an effect on the secretory machinery is reflected as changes in Ca²⁺ currents.

(Received 16 January 2004; accepted after revision 7 May 2004; first published online 14 May 2004)
Correspondence E. M. Silinsky: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA. Email: e-silinsky{at}northwestern.edu

	Introduction

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The skeletal neuromuscular junction has been an important resource for comprehensive studies on the modulation of neurotransmitter release by G-protein-coupled receptors (Ginsborg & Hirst, 1972; Silinsky, 1980, 1981, 1984; Meriney & Grinnell, 1991; Hamilton & Smith, 1991; Nagano et al. 1992; Slutsky et al. 2003; for reviews see Silinsky, 1985; Miller, 1998). At the amphibian neuromuscular junction, activation of A₁ adenosine receptors inhibits neurotransmitter release when A₁ agonists are applied exogenously to non-depressed preparations (Ginsborg & Hirst, 1972; Silinsky, 1980, 1984; Redman & Silinsky, 1995). In addition, ATP after hydrolysis to adenosine appears to be the predominant mediator of prejunctional neuromuscular depression at low frequencies of nerve stimulation in amphibia (Redman & Silinsky, 1994), with depletion of the immediately available pool of neurotransmitter occurring only later or with higher frequencies of stimulation (Meriney & Grinnell, 1991; Redman & Silinsky, 1994). Adenosine also plays a role in the prejunctional depression of evoked neurotransmitter secretion in rat (Hamilton & Smith, 1991) and mouse (Nagano et al. 1992; Hirsh et al. 2002) motor nerve endings as well.

Studies at the amphibian neuromuscular junction provided the first evidence that modulation of neurotransmitter release by G-protein-coupled receptors can occur downstream of Ca²⁺ entry (Silinsky, 1981, 1984, 1986; Silinsky & Solsona, 1992; Robitaille et al. 1999; for review see Miller, 1998). Briefly, both evoked and spontaneous ACh release activated by methods that bypass membrane ionic channels are inhibited by adenosine with similar potency and efficacy to evoked release mediated by Ca²⁺ entry through voltage-gated Ca²⁺ channels (Silinsky, 1984; Hunt & Silinsky, 1993; Hunt et al. 1994; for review see Silinsky et al. 1999, 2001). Furthermore, Ca²⁺ currents measured using the perineural recording technique (Silinsky & Solsona, 1992; Redman & Silinsky, 1994, 1995) or by confocal measurements of Ca²⁺ concentrations using Ca²⁺ sensitive dyes (Robitaille et al. 1999) are not affected by either exogenous or endogenous adenosine. In contrast, some studies in the rat suggest that evoked neurotransmitter release is inhibited by adenosine as a consequence of a reduction in Ca²⁺ entry through voltage-gated calcium channels (Hamilton & Smith, 1991; but see Ginsborg & Hirst, 1972 for an alternative interpretation). Despite the importance of the mouse as a tool for correlative genetic studies of synaptic transmission, and the role of A₁ adenosine receptors in mediating presynaptic depression in the mouse diaphragm (Nagano et al. 1992), a comprehensive study on the mechanisms by which adenosine inhibits neurally evoked neurotransmitter release is absent at mouse neuromuscular junctions. It would thus be of interest to apply the tools developed at other neuromuscular junctions (see, e.g. Gunderson et al. 1982; Brigant & Mallart, 1982; Mallart, 1985a,b; Anderson et al. 1988; Redman & Silinsky, 1995; Xu & Atchison, 1996) to the widely studied mouse phrenic nerve hemidiaphragm preparation as a means to determine the mode of action of adenosine at mouse motor nerve endings. An additional advantage to further characterizing this preparation electrophysiologically is that the data can serve as a baseline for studies of the microphysiological effects of mutating strategic components of the secretory apparatus using molecular genetics techniques, as the diaphragms of newborn mice are electrophysiologically accessible.

	Methods

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General

Experiments were performed on the isolated phrenic nerve hemidiaphragm preparation. Both wild-type mice (B6129F2/J, stock no. 101045) and the Rab3A^–/– mice (B6,129-Rab3Atm1Sud; stock no. JR2443) were obtained from The Jackson Laboratory (Bar Harbour, ME, USA). This particular strain of wild-type mice is of similar genetic background to the Rab3A^–/– mutant. Studies were made in accordance with the guidelines of the Northwestern University Animal Care and Use Committee and the National Institutes of Health of the USPHS. Mice (20–30 g) were humanely anaesthetized with 5–10 ml of diethyl ether for 3–5 min. Once the animals were unresponsive to touch, they were exsanguinated. The isolated phrenic nerve-hemidiaphragm was pinned in a Perspex recording chamber. The preparation was continuously superfused with physiological saline solution at room temperature (21–23°C). A₁ adenosine receptor activation has been shown to inhibit evoked ACh release in mouse phrenic nerve hemidiaphragm preparations at both 37°C (Nagano et al. 1992) and at room temperature (Hirsh et al. 2002). Solutions were delivered using a peristaltic pump. The flow rate was generally 3.0 ml min^–1 but on some experiments it was increased to 10–20 ml min^–1 to obtain rapid solution changes (see, e.g. Figs 2B and 7). Solutions were removed from the bath by vacuum suction.

View larger version (17K): [in this window] [in a new window]   Figure 2.  Antagonism of calcium currents by adenosine in wild-type and Rab3A–/– mutant mice Traces in A show the effects of 500 µM adenosine (averaged data from a wild-type mouse). B shows the effect of 1 mM adenosine in another experiment made on a wild-type mouse. The raw data in B were obtained using the protocol for rapid solution changes as described in the Methods. C shows averaged traces before and after application of 50 µM adenosine to a preparation from a Rab3A–/– mutant mouse. The phrenic nerve was stimulated at 0.016 Hz.

View larger version (15K): [in this window] [in a new window]   Figure 7.  Comparison of the relationship between the simultaneous inhibition of EPPs and perineural Ca2+ currents by adenosine and by equi-effective concentrations of the Ca2+ channel blocker Cd2+ For each of the 5 experiments that contributed to the averaged data in Fig. 7, simultaneous measurements of EPPs and perineural Ca2+ currents were made from the same site for the duration of the experiments. It should be noted that these results with Cd2+ on both EPPs and perineural Ca2+ currents are submaximal as higher concentrations of Cd2+ (100 µM) produce complete inhibition of the EPPs and further reductions in the Ca2+ current (see Figs 1 and 5). Due to cost and the experimental impracticality of the long incubation times required for after -agatoxin-IVA (see, e.g. Fig. 1B), Cd2+ was used as the Ca2+ channel blocker in these experiments. Error bars are +1 S.E.M. 0.017 Hz, frequency of stimulation.

Specific solutions and their electrophysiological correlates

Control physiological saline solution consisted of (mM): NaCl 137, KCl 5, CaCl₂ 2, MgCl₂ 2, NaH₂PO₄ 1, NaHCO₃ 24, dextrose 11 (pH 7.2–7.4 when gassed with a 95% O₂–5% CO₂ mixture) and was used in most experiments (Gage & Hubbard, 1966). In many experiments, this solution with varying concentrations of tubocurarine was used to make perineural recordings at the beginning of the experiments (e.g. Fig. 1A). In experiments in which divalent cation concentrations were increased (e.g. when Cd²⁺ was employed), the solutions were buffered with 30 mM Hepes (pH 7.2–7.4) and gassed with 100% oxygen to avoid the precipitation of divalent cations that can occur with phosphate–bicarbonate solutions. Specifically, the Hepes-buffered solutions contained 137 mM NaCl, 5 mM KCl, 11 mM dextrose, varying concentrations of CaCl₂ and MgCl₂ but no NaH₂PO₄ or NaHCO₃. For measurements of perineural Ca²⁺ currents, the K⁺ channel blockers 3,4,-diaminopyridine (DAP) and tetraethylammonium (TEA) were added to the control solutions to expose the underlying Ca²⁺ current. The standard solution used for such recording when EPPs were not measured was normal physiological saline solution with the addition of 10 mM TEA, 300 µM DAP and 50 µM tubocurarine to block postjunctional nicotinic receptors completely (standard Ca²⁺ current solution; see Xu & Atchison, 1996). To make simultaneous measurements of perineural Ca²⁺ currents and EPPs, the ionic composition of the solution was initially changed to contain a lower Ca²⁺ concentration (0.7 mM Ca²⁺, 2 mM Mg²⁺) and reduced concentrations of DAP (100 µM), TEA (250 µM), and d-tubocurarine (7–20 µM) (low Ca²⁺ solution). Preliminary experiments revealed that concentrations of Ca²⁺ lower than 0.7 mM in the mouse did not give sufficient resolution in the Ca²⁺ component of the perineural waveform to perform these studies. In low Ca²⁺ solution, the lower concentrations of potassium channel blockers still allowed for reliable measurements of peak Ca²⁺ currents, but also permitted stable measurements of the electrophysiological correlate of evoked ACh release (i.e. EPPs) to be made simultaneously with an intracellular microelectrode (see, e.g. Redman & Silinsky, 1995). Because of the complications due to twitches in the deep muscle fibres, concentrations of tubocurarine were adjusted to produce small EPPs (generally < 1 mV). These EPPs, because they reflected ACh secretion of high probability, were fairly constant during the course of the experiments reported in this paper (see, e.g. Figs 1B and 3).

View larger version (16K): [in this window] [in a new window]   Figure 1.  General observations on the development (A) and antagonism (B) of perineural Ca2+ currents in mouse phrenic nerve hemidiaphragm A shows the Na+ currents and K+ currents as downward deflections prior to application of K + channel blockers (5 superimposed sweeps, 0.1 Hz). Superfusion with solution containing potassium channel blockers (standard Ca2+ current solution; see Methods) produces the blockade of K+ currents and the progressive development of Ca2+ currents (1–4) during stimulation of the phrenic nerve (0.016 Hz). For details of current polarities and description of the perineural currents, which reflect voltage changes in the extracellular space and are thus calibrated in mV, see Methods. A shows the typical result from n= 10 preparations. B shows averaged perineural Ca2+ currents (Ca2+) and EPPs recorded simultaneously in standard Ca2+ current solution. In the absence of agatoxin, both the Ca2+ currents and EPPs in B were stable for a period of approximately 23 min. The fast and slow phases of the Ca2+ current are shown (for discussion see Methods and Xu & Atchison, 1996). The lower two traces in B show the progressive decreases in both the averaged Ca2+ currents and EPPs (average of 5–15 stimuli, 0.012 Hz) in the presence of the P/Q-type Ca2+ channel blocker -agatoxin-IVA (100 nM). Intervals of approximately 14 min elapsed between the traces in B (B shows the typical result from n= 4 preparations).

View larger version (33K): [in this window] [in a new window]   Figure 3.  Simultaneous reduction in ACh release (EPPs) and Ca2+ currents produced by adenosine A shows the reversible inhibition of ACh release and perineural Ca2+ currents in low Ca2+ solutions (0.7 mM Ca2+, 2 mM Mg2+ and reduced concentrations of K+ channel blockers; see Methods). B and C show experiments in the standard perineural solution (Xu & Atchison, 1996). B shows raw data from another experiment that illustrate the onset of inhibition by adenosine. The phrenic nerve was stimulated at 0.015 Hz. C shows averaged data (n= 4–5 stimuli) from a Rab3A–/– mouse depicting the inhibitory effect adenosine (50 µM) on both the EPPs and perineural currents.

Finally, in some experiments (e.g. Fig. 4B) recordings of focal nerve terminal currents (NTCs) and end-plate currents (EPCs) were made using a patch electrode at the nerve ending (see, e.g. Hubbard & Schmidt, 1963; Katz & Miledi, 1965; Silinsky, 1984). Localization of the nerve ending was ascertained visually under 200–400 x magnification at the edge fibres where transillumination is feasible using phase contrast optics and/or by recording focal miniature end-plate currents (mEPCs) prior to treating the preparation with tubocurarine. Brief application of solutions containing 100 mM sucrose was employed to accelerate mEPC frequencies to assist in their detection with the patch electrode (see Fig. 4B).

View larger version (20K): [in this window] [in a new window]   Figure 4.  Absence of effects of adenosine on nerve terminal Na+ and K+ currents In A, currents (lower traces) were measured from the perineurium at the junction of the myelinated and non-myelinated axon (the heminode). Upper traces show averaged EPPs. Note the absence of effects of 50 µM adenosine in this experiment on a Rab3A–/– mouse on Na+ or K+ currents under conditions in which the averaged EPPs are decreased to approximately 58% of the control level by this concentration of adenosine. The average responses to 5 stimuli (0.1 Hz) are depicted. B shows experiments made using a loose patch electrode at the nerve terminal from a wild-type mouse. Spontaneous focal mEPCs, were recorded first, the preparation curarized, the motor nerve stimulated (arrow marks the stimulus artifact) and the nerve terminal currents (NTCs) and end-plate currents (EPCs) measured. Note the absence of effects of adenosine (5 mM) on NTCs under conditions in which it produces a reversible inhibition of end-plate currents (EPCs) to approximately 60% of the control level. Each trace is the averaged response to 256 stimuli (0.25 Hz). C shows recording of Na+ and K+ currents from the nerve terminal proper using the perineural recording method. The currents are mirror-images of those recorded from the heminode, and represent a higher resolution image of the NTCs recorded with a patch electrode in B. Again, no effects of adenosine were observed on the Na+ or K+ currents in this wild-type mouse. Traces in C are the average response to 20 stimuli (0.25 Hz).

The perineural electrical waveforms

The perineural waveforms are voltage changes across the resistance of the perineural sheath produced by currents flowing between the myelinated portion of the axons and the nerve terminals at the junction of the myelinated and non-myelinated regions (the heminode). These extracellular currents are proportional to the difference in potential between the nodes of Ranvier and the nerve endings. These waveforms are not, in the rigorous sense, membrane ionic currents; they are, however, highly related to membrane conductance changes that occur both at the final node of Ranvier and at the nerve endings. With respect to the electophysiological correlate of the P/Q-type Ca²⁺ current that mediates evoked neurotransmitter secretion, the conductance increase that underlies this Ca²⁺ current is localized to the nerve terminals (Mallart, 1985a,b; Xu & Atchison, 1996). The inward Ca²⁺ current in the nerve terminals produces a proportional current in the perineural space; this current flows back to the recording site from the nerve ending where it is detected as an upward-going (outward) waveform. For the sake of brevity, the upward-going perineural voltage change that is antagonized by P/Q-type Ca²⁺ channel blockers will be referred to as the ‘prejunctional Ca²⁺ current’ or the ‘perineural calcium current’ as this voltage change is generated by Ca²⁺ currents emanating from the nerve ending flowing across the perineurial resistance. The calibration bars will thus be in units of mV. For similar reasons, perineural voltage changes attributable to Na⁺ and K⁺ currents in the presynaptic element will be termed ‘perineural Na⁺ currents’ and ‘perineural K⁺ currents’, respectively, as they are reflective of the membrane permeability changes associated with Na⁺ and K⁺ currents.

Depending upon the concentrations of TEA and DAP, Ca²⁺ currents may have either one or two distinct phases (see Fig. 1B where the arrows show a slow and a fast phase). Biphasic Ca²⁺ currents are often observed in the presence of procaine (used to prevent repetitive firing; see Xu & Atchison, 1996 for discussion). Procaine was not used in these experiments to preclude the possibility of an interaction between adenosine receptor activation and procaine (see, e.g. Anderson et al. 1988). Consequently, the quantitative aspects of this study will focus on the early phasic component of the Ca²⁺ current, as it is present under all conditions employed in this study. In addition, the time course of the EPPs recorded simultaneously with Ca²⁺ currents suggests that this early phasic component is the relevant Ca²⁺ current that mediates the synchronous release of ACh. Hence, perineural deflections are quantified as the magnitude of the extracellular voltage change as measured from the baseline to the peak of the current (see Silinsky & Solsona, 1992; Redman & Silinsky, 1995 for further details).

Adenosine concentrations and statistical methods

The important aspects of the adenosine dose–response relationships for neurotransmitter release in both normal and Rab3A^–/– deletion mutants have been published previously from this laboratory (Hirsh et al. 2002). Consequently, concentration–response curves were performed in this study only to determine the threshold concentrations of agents that produced effects on Ca²⁺ currents in both wild-type and Rab3A^–/– mutant mice under similar conditions (see Figs 2D and 6). Appropriate concentrations of adenosine were then chosen to study the effect of adenosine on both evoked release and Ca²⁺ currents with the intent of optimizing the concentration to obtain rapid effects and reduce the likelihood of depletion of ACh stores. In some experiments, high concentrations of adenosine were employed to compare the maximal effects of adenosine with equiactive concentrations of Cd²⁺ in the same experiment (e.g. Fig. 7), or, to determine non-selective effects of adenosine on ionic currents other than those through P/Q-type Ca²⁺ channels (Figs 4 and 5). It should be stressed that changes in EPP amplitudes after adenosine treatment are due to changes in ACh release and not to changes in postjunctional sensitivity to ACh as MEPP amplitudes are not affected by adenosine in any species studied (Ginsborg & Hirst, 1972; Silinsky, 1980, 1984; Nagano et al. 1992; Hirsh & Silinsky, 2002; Hirsh et al. 2003).

View larger version (10K): [in this window] [in a new window]   Figure 6.  Dose–response curves for the inhibition of Ca2+ currents (A) and EPPs (B) by adenosine in wild-type () and Rab3A–/– mutant mice (•) Note the differences in potency for adenosine in the two species of mouse for both Ca2+ currents (A) and EPPs (B) under the same conditions. Each data point represents the mean of 4–9 experiments. Error bars are ± 1 S.E.M. 0.016 Hz stimulation rate.

View larger version (18K): [in this window] [in a new window]   Figure 5.  Absence of effects of adenosine on the residual ionic currents after treatment with Cd2+ (B and C) or -agatoxin-IVA (D and E) A shows progressive antagonism of the perineural Ca2+ current by Cd2+ (0.017 Hz). B shows the averaged perineural waveform after 1 mM Cd2+ treatment (1.60 mV, n= 6 stimuli). C shows that the residual current in the presence of Cd2+ is not affected by the application of 1 mM adenosine (1.56 mV, n= 6 stimuli). D shows the averaged perineural waveform after -agatoxin-IVA (100 nM) in another experiment (1.65 mV, n= 5 stimuli). E shows that the residual current in the presence of -agatoxin-IVA is not affected by the application of 10 mM adenosine (1.60 mV, n= 6 stimuli). Prior to treatment with -agatoxin-IVA the control Ca2+ currents for traces D and E resembled that of A but with an average peak of 2.74 mV (n= 6 stimuli).

	Results

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General observations on perineural currents and EPPs in mouse phrenic nerve–hemidiaphragm preparations

Figure 1A depicts the raw data from a typical recording made using a perineural electrode in the mouse phrenic nerve–hemidiaphragm preparation (sufficient concentrations of tubocurarine were used to block all postsynaptic electrical activity). The downward deflections that appear after the stimulus artifact in the lower part of the figure depict the Na⁺ and K⁺ currents in the presynaptic terminal in normal extracellular bathing solution prior to treatment with potassium channel blockers. The Na⁺ current is due to sodium influx in the nodes of Ranvier and heminodes near the recording electrode and the K⁺ current is a fast voltage-dependent current in the nerve terminal proper, namely the delayed rectifier (see Mallart, 1985a,b; Anderson et al. 1988; Deist et al. 1992; Redman & Silinsky, 1995). The traces depicting the Na⁺ and K⁺ currents reflect five superimposed responses to nerve stimulation (0.1 Hz). Treatment with the K⁺ channel blockers 3,4 diaminopyridine (DAP, 300 µM) and tetraethylammonium (TEA 10 mM) eliminated the K⁺ current and revealed the progressive development of the Ca²⁺ current (Fig. 1A, traces 1–4, Ca²⁺) associated with evoked ACh release (0.016 Hz; for details of current polarities and other technical aspects of these recordings, see Methods).

It is also possible to make simultaneous measurements of both evoked ACh release (i.e. EPPs) and Ca²⁺ currents in the presence of DAP and TEA (see, e.g. Redman & Silinsky, 1995). Figure 1B (top) shows averaged control Ca²⁺ currents (left) and control EPPs (right), which were both stable in this experiment for approximately 23 min. Specifically for evoked ACh release, EPPs were 0.79 ± 0.04 mV (n= 8 EPPs recorded over a time course of approximately 11 min) and 0.70 ± 0.04 mV (n= 9 EPPs, recording completed 12.5 min later, 0.012 Hz stimulation, P= 0.15). The Ca²⁺ current peak during that period was 2.30 ± 0.01 mV (first 11 min) and 2.40 ± 0.05 mV for the next 12.5 min (P= 0.09). Application of the P/Q-type Ca²⁺ channel blocker -agatoxin-IVA (100 nM; see Xu & Atchison, 1996) caused a progressive decline of both the averaged Ca²⁺ currents and EPPs (Fig. 1B, lower traces). These data confirm previous observations that P/Q-type calcium channels exclusively mediate evoked ACh release at adult mouse neuromuscular junctions (Protti & Uchitel, 1993; Xu & Atchison, 1996). The ability to record stable EPPs simultaneously with perineural Ca²⁺ currents demonstrated that this method may be used to determine whether the inhibition of ACh release by adenosine is associated with an inhibition of Ca²⁺ entry in the mouse.

In most experiments, residual outward ionic currents are detectable after blockade of Ca²⁺ channels with conventional Ca²⁺ channel blockers (for review see Redman & Silinsky, 1995). The upward-going residual deflections will be discussed again later (see Fig. 5). This residual current unfortunately prevents accurate assessments of the quantitative relationship between the magnitude of the P/Q-type Ca²⁺ currents and ACh release.

Effects of adenosine on perineural calcium currents in the absence of postjunctional electrical activity

Studies using selective adenosine agonists and antagonists have demonstrated that the presynaptic inhibitory effects of adenosine are mediated exclusively by A₁ receptors at the mouse neuromuscular junction (Nagano et al. 1992; Hirsh & Silinsky, 2002). In experiments from our laboratory, the threshold for inhibition of ACh release by adenosine in wild-type mice was 100 µM (Hirsh et al. 2002). In these present experiments, no statistically significant effects of adenosine on Ca²⁺ currents were observed in 100 µM adenosine (n= 4 experiments) in the wild-type mice. When higher concentrations of adenosine were employed (500 µM–1 mM), inhibitory effects of adenosine on perineural Ca²⁺ currents were observed (P < 0.05). Figure 2A depicts the threshold effect of 500 µM adenosine on the perineural Ca²⁺ current. Note that the mean peak of the perineural waveform (4.0 ± 0.09 mV, n= 4 stimuli) is decreased to approximately 78% of the control value (3.1 ± 0.07 mV, n= 4 stimuli, P < 0.001) in the presence of 500 µM adenosine. The effect was reversible upon washout of adenosine (the wash is shown for clarity as a lightly dotted line = 3.7 ± 0.2 mV, n= 5 stimuli). There was no statistically significant difference between the control and wash periods (P= 0.13).

In consort with the results on evoked neurotransmitter release (Hirsh et al. 2002), higher concentrations of adenosine produced more profound effects on Ca²⁺ currents; this is shown in Fig. 2B which depicts the raw data from another experiment. In this experiment, the preparation was rapidly superfused with 1 mM adenosine between the 8th and 9th stimulus (an interval of 90 s) and a rapid, stable inhibition of the perineural Ca²⁺ current to 61% of the control level was observed (P < 0.001). Specifically the mean control peak calcium component of the perineural waveform, 1.89 ± 0.03 mV (n= 8 responses), was reduced by adenosine to 1.17 ± 0.01 mV (n= 7 responses). In a total of eight experiments, Ca²⁺ currents were decreased by concentrations of adenosine ranging from 500 µM to 1 mM in wild-type mice (P < 0.05). As expected from previous results in mouse phrenic nerve–hemidiaphragm preparations (Nagano et al. 1992; Hirsh et al. 2002), this action of adenosine on perineural Ca²⁺ currents is mediated by prejunctional A₁ adenosine receptors: inhibition is prevented by the selective A₁ receptor antagonist 8-cyclopentyl theophylline (1 µM, n= 3 experiments, data not shown).

It has been shown that the potency by which adenosine inhibits evoked neurotransmitter release is increased in mice in which the Rab3A gene has been deleted (Hirsh et al. 2002). Rab3A is associated both with the trafficking of synaptic vesicles and with an effect on a late stage of the Ca²⁺-dependent neurotransmitter release process (for review see Geppert & Sudhof, 1998). It would thus be of interest to determine if the increased sensitivity of the secretion process to adenosine in the Rab3A^–/– mouse is accompanied by a corresponding increase in the sensitivity of the perineural Ca²⁺ currents to inhibition by adenosine. Figure 2C shows that this is indeed the case (representative data from n= 9 preparations). Note that 50 µM adenosine, which has no effect on perineural Ca²⁺ currents in the wild-type mouse, reduces the averaged perineural Ca²⁺ current in the mutant mouse from 1.4 to 1.1 mV (n= 7 stimuli; P < 0.05); the effect was reversible (averaged perineural current after the wash = 1.3 mV).

Effects of adenosine on membrane ionic currents measured simultaneously with neurotransmitter release (EPPs)

The results thus far suggest that under recording conditions in which K⁺ channels are blocked and evoked release cannot be measured, adenosine reduces P/Q-type Ca²⁺ currents in mouse phrenic nerve endings. The data illustrated in Fig. 1B suggest that stable EPPs may be measured under conditions in which perineural Ca²⁺ currents are also stable. It is thus of interest to determine if the effects of adenosine on perineural currents are relevant to effects on evoked ACh release by making simultaneous measurements of both perineural Ca²⁺ currents and EPPs.

Figure 3A shows the reversible effects of adenosine (500 µM) on both the averaged calcium currents and EPPs in low Ca²⁺ with reduced concentrations of potassium channel blockers, conditions found to be suitable for detection of changes in both EPPs and perineural calcium currents (low calcium solutions; see Methods). Note the reversible, reduction in both EPPs and perineural Ca²⁺ currents by adenosine. Specifically, in the experiment shown in Fig, 3A, the mean peak values for the perineural currents were: control = 1.0 ± 0.02 mV (n= 5 stimuli); adenosine = 0.71 ± 0.02 mV (n= 3 stimuli), and wash = 0.83 ± 0.01 mV (n= 3 stimuli); whilst the mean value for EPPs were: control = 1.1 ± 0.07 mV; adenosine (500 µM) = 0.79 ± 0.07 mV, wash = 1.1 ± 0.04 mV (P < 0.05). Similar results were obtained in three other experiments in low calcium solutions.

It was important to determine if similar results could be obtained in experiments made in solutions containing normal calcium concentrations and the usual high concentration of potassium channel blockers (standard Ca²⁺ current solution; see Fig. 1B). Figure 3B and C depicts the results of two such experiments. In the raw data shown in Fig. 3B, prior to the addition of adenosine, stable EPPs (3.1 ± 0.02 mV, n= 5 stimuli) and perineural Ca²⁺ currents (2.64 ± 0.06 mV, n= 5 stimuli) were recorded for approximately 6 min. Rapid application of 1 mM adenosine reduced the peak perineural Ca²⁺ currents to 2.2 ± 0.08 mV (n= 4 stimuli) and EPPs to a new stable level (0.91 ± 0.13 mV, n= 5 stimuli, P < 0.05). Figure 3C presents averaged responses in a preparation from a Rab3A^–/– mutant mouse (n= 5 stimuli). Again, decreases in both Ca²⁺ currents and EPPs were produced by 50 µM adenosine in the mutant mouse (P < 0.05). It thus appears that the inhibition of Ca²⁺ currents by adenosine is associated with a reduction in EPP amplitudes, and that the differences in the potency of adenosine for evoked ACh release between wild-type and mutant mice are preserved in solutions in which calcium currents may be measured simultaneously.

Adenosine does not affect Na⁺ currents, K⁺ currents or the residual outward currents observed after Ca²⁺ channel block

The correlation between the effects of adenosine on Ca²⁺ currents and ACh release suggests that the inhibition of ACh release by adenosine may be related to a reduction in Ca²⁺ entry through P/Q-type Ca²⁺ channels. It is possible, however, that the P/Q-type Ca²⁺ channels are not the direct targets for the effects of adenosine. Rather, the membrane ionic currents that precede Ca²⁺ entry might be the major target for the action of adenosine and this in turn could affect the voltage-gated P/Q-type Ca²⁺ channels. To investigate the possibility of such an indirect action of adenosine on P/Q-type Ca²⁺ channels, the effects of this nucleoside on both the nerve terminal Na⁺ and K⁺ currents, and in separate experiments, on the residual outward current that remained after Ca²⁺ and K⁺ channel block were examined in more detail. Figure 4A shows Na⁺ and K⁺ currents (lower traces) and EPPs (upper traces) recorded simultaneously in the absence of K⁺ channel blockade. Note that under conditions in which adenosine produces a reduction in evoked ACh release (EPPs) to 58% of the control level, no effect on the averaged Na⁺ and K⁺ currents was observed.

It might be argued that the nerve terminal currents differ from those measured from the heminodal region using a perineural recording electrode and that these nerve terminal currents could be affected by adenosine without a measurable effect on the perineural currents. To address this issue, experiments were made in which a loose patch electrode was placed on the nerve ending for focal recording of miniature end-plate currents (mEPCs), end-plate currents (EPCs) and nerve terminal currents (NTCs), and the effects of adenosine examined (Fig. 4B). The upper traces in Fig. 4B depict mEPCs recorded from the nerve terminal proper in the absence of postjunctional nicotinic receptor blockade (see Hubbard & Schmidt, 1963; Katz & Miledi, 1965; Silinsky, 1984). The preparation was then curarized and the motor nerve stimulated. Note that nerve stimulation under these conditions (arrow depicts the stimulus artifact) produces focal nerve terminal Na⁺ and K⁺ currents (NTCs), followed by the EPC. Application of adenosine produced a reversible inhibition of EPCs to 60% of the control level in this experiment without an effect on the nerve terminal currents. In other experiments, a perineural electrode was placed near the nerve terminal, revealing currents similar to the NTCs of Fig. 4B but with higher resolution. These currents are the inverted replica of those shown in Fig. 1A and 4A (see Fig. 1C in Brigant & Mallart, 1982; Konishi, 1985). As Fig. 4C shows, perineural Na⁺ and K⁺ currents in the nerve endings are unaffected by adenosine (10 mM). In a total of seven experiments, adenosine failed to affect the Na⁺ or K⁺ currents in motor nerve endings under conditions in which this nucleoside inhibited evoked ACh release.

As alluded to above, in many experiments, residual outward ionic currents are detectable after blockade of Ca²⁺ channels with conventional Ca²⁺ channel blockers. This net outward current may reflect the contribution of an outward Na⁺ current associated with a propagating action potential, a passive current that repolarizes the portion of nerve ending under the recording electrode (Hamilton & Smith, 1991), an unknown Ca²⁺ conductance (Xu & Atchison, 1996) or an as-yet uncharacterized ionic current. To determine if the effect of adenosine is exerted on the P/Q-type Ca²⁺ current or on the residual component of the outward current, the effects of adenosine were assessed after complete blockade of Ca²⁺ currents with Cd²⁺ or -agatoxin-IVA. Figure 5 illustrates the typical experiments. Figure 5A depicts the progressive inhibition of Ca²⁺ currents by Cd²⁺ (1 mM), leaving a residual outward current; the averaged residual outward current is shown in Fig. 5B. Figure 5C shows that adenosine (1 mM) has no effect on the residual outward current after blockade of Ca²⁺ currents with Cd²⁺(n= 3 experiments). As Cd²⁺ blocks a number of different Ca²⁺ channel types (Xu & Atchison, 1996), similar experiments were performed using the selective P/Q-type Ca²⁺ channel blocker -agatoxin-IVA (150 nM, Fig. 5D, Aga). Note that 10 mM adenosine had no effect on the residual outward currents observed after -agatoxin-IVA treatment (Fig. 5E, Aga + adenosine, n= 3 experiments).

Do the effects of adenosine on perineural Ca²⁺ currents account for the inhibition of ACh release?

From these results, it appears that adenosine receptor activation is associated with an inhibition of P/Q-type Ca²⁺ currents and not the membrane ionic currents that precede Ca²⁺ channel activation. To further evaluate the possibility of a causal relationship between the effects of adenosine on Ca²⁺ currents and ACh release, more complete concentration–response relationships were constructed to compare the threshold effects of adenosine on Ca²⁺ currents (Fig. 6A) and EPPs (Fig. 6B) in both the wild-type () and Rab3A^(–/–) mutant (•) under the same experimental conditions. With respect to Ca²⁺ currents, Fig. 6A shows that 50 µM adenosine has no effect on Ca²⁺ currents in wild-type mice (n= 4 preparations, P= 0.13) but a highly significant inhibitory effect in the mutant mice (P < 0.002, n= 9 preparations). The data in Fig. 6A also demonstrate that wild-type mice require a concentration of adenosine an order of magnitude higher (500 µM) to produce comparable levels of inhibition of Ca²⁺ currents (P= 0.32) to that produced by 50 µM adenosine in the mutant mice. As might be expected from previous results (Hirsh et al. 2002), when EPPs are recorded under similar conditions (Fig. 6B), the differential potency of adenosine is preserved. Whilst 10 µM adenosine had no effect on EPPs in the mutant mice (P= 0.54), 50 µM adenosine reduced EPPs to 77 ± 0.02%(P < 0.0001) in the mutants, but 100 µM adenosine had no effect on EPPs in the wild-type mouse (P= 0.82).

Given these results, it was of interest to determine whether the inhibition of Ca²⁺ currents by adenosine is quantitatively sufficient to account for changes in ACh release produced by this nucleoside. As mentioned earlier, the residual outward currents prevent accurate comparisons of the relationship between Ca²⁺ currents and ACh release and preclude comparisons between different preparations. An additional series of experiments were thus performed to compare the effects of adenosine and the Ca²⁺ channel blocker Cd²⁺ in the same experiments.

The protocol that allowed for such experiments to be performed was as follows. A supramaximal concentration of adenosine (10 mM) was rapidly superfused over the preparation and the maximal levels of inhibition of both Ca²⁺ currents and EPPs obtained (Fig. 7, black bars) (10 mM adenosine produces no secondary effects on other membrane ionic currents; see Figs 4 and 5). Next, the preparation was rapidly washed with adenosine-free solution and then superfused with concentrations of the Ca²⁺ channel blocker Cd²⁺ (12–20 µM) that produced similar levels of inhibition of ACh release to those produced by 10 mM adenosine (Fig. 7, grey bars; Xu & Atchison, 1996; Porter & Wray, 1996). Figure 7 depicts the average results from five experiments in which it was possible to record stable perineural Ca²⁺ currents and EPPs in normal Ca²⁺ solutions using this protocol. Note that matching levels of inhibition of EPPs produce by adenosine (to 51.8 ± 2.3% of control) and Cd²⁺ (to 53.2 ± 3.2% of control, P= 0.45) are associated with matching levels of inhibition of the Ca²⁺ current (with adenosine, to 75.4 ± 3.87% of control; with Cd²⁺ to 76.2 ± 4.1% of control, P= 0.48). Whilst precise quantitative relationships between Ca²⁺ currents and ACh release are not possible due to the residual outward currents (e.g. Fig. 5), the data shown in Fig. 7 suggest that the effect of adenosine on Ca²⁺ currents is sufficient to account for the inhibitory effects of adenosine on neurotransmitter release at the mouse neuromuscular junction.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
These results demonstrate that the inhibitory effect of adenosine on neurotransmitter release from mouse motor nerve endings is associated with a reduction in the amplitude of the P/Q-type Ca²⁺ currents that mediate evoked ACh release. They also show that the increased sensitivity of ACh release from motor nerve endings in the Rab3A^–/– mutant mouse to adenosine is associated with an increased sensitivity of P/Q-type Ca²⁺ channels to inhibition by adenosine. The action of adenosine is unlikely to be due to an effect on L-, N- or R-type Ca²⁺ channels as blockers of these channels do not affect evoked ACh release or perineural Ca²⁺ currents in the mouse under the conditions of these present experiments (see Protti & Uchitel, 1993). Furthermore, after blockade of P/Q-type channels with -agatoxin-IVA or Cd²⁺, no effects of adenosine on the residual outward currents were observed (Fig. 5).

The effects on perineural calcium currents are also unlikely to be due to effects on other major ionic channels in motor nerve endings. Both the delayed rectifier K⁺ channel and calcium-activated K⁺ channels in mouse motor nerve endings were blocked in this study and adenosine still inhibited both Ca²⁺ currents and evoked ACh release (Figs 3B and C; see also Anderson et al. 1988). Furthermore direct measurements of K⁺ currents both in the heminodal region and at the nerve endings failed to reveal an effect of adenosine. Finally, the effects of adenosine are not due to an action on ATP-dependent potassium channels as this would be detectable in the measurements of K⁺ currents such as those shown in Fig. 5A. In addition, alterations of ATP-dependent potassium channels produce only modest effects on the perineural potassium waveforms and these small effects are not associated with changes in evoked ACh release (see Deist et al. 1992).

There are two alternative explanations for the different effects of adenosine on neuromuscular transmission in mammalian and amphibian preparations. The first explanation is that the inhibition of evoked neurotransmitter release by adenosine occurs via different mechanisms in the two species: an interaction with Ca²⁺ channels but not with the with secretory apparatus for mammalian nerve endings and an effect on the secretory apparatus that is independent of Ca²⁺ channels in amphibia. The quantitative consistency between the effects of P/Q-type calcium channel blockers and adenosine on Ca²⁺ currents and evoked ACh release shown in Fig. 7 are in support of the first explanation. In addition, at mammalian motor nerve endings, P/Q-type Ca²⁺ channels mediate evoked ACh release whilst amphibia employ N-type Ca²⁺ channels. However, a selective action of adenosine on P/Q-type Ca²⁺ channels is an unlikely explanation for these present results as N-type Ca²⁺ currents have been shown to be inhibited by adenosine in both the soma of mouse motorneurones (Mynlieff & Beam, 1994) and in preganglionic nerve endings of chick ciliary ganglia (Yawo & Chuhma, 1993; see also Kimura et al. 2003).

The second explanation for these present results is that the target of inhibition by adenosine in both mouse and amphibian motor nerve endings may be a strategic component of the secretory apparatus, but the two species differ in the intimacy of the coupling between the presynaptic release machinery and the Ca²⁺ channels. At the frog neuromuscular junction, the effect of adenosine is targeted to a strategic component of the secretory apparatus without an effect on membrane ionic channels (Silinsky, 1984; Silinsky & Solsona, 1992; Redman & Silinsky, 1994a). Indeed, recent evidence has shown that a number of G-protein-linked receptors are able to modulate neurotransmitter secretion downstream of Ca²⁺ entry (Haydon et al. 1991; Blackmer et al. 2001; for review see Miller, 1998). Given the known abilities of the members of the core complex to affect the availability of Ca²⁺ channels in nerve endings (see Stanley & Mirotznik, 1997; Spafford & Zamponi, 2003), it is possible, that due to a more intimate coupling between Ca²⁺ channels and the core complex of presynaptic proteins (the SNAREs; see Sudhof, 1995) in the mouse, the effects of adenosine on mouse motor nerve endings are reflected as decreases in Ca²⁺ currents whilst those in amphibia are not (see Silinsky, 1986 for a discussion of this possibility). This unifying hypothesis that mouse and frog motor nerve endings differ in the intimacy of coupling between the between the Ca²⁺ channels and the SNAREs has some justification in the published literature. Specifically, G-protein modulation of calcium channels in cholinergic nerve endings is eliminated if the SNARE protein, syntaxin, is cleaved by botulinum toxin type C (chick ciliary ganglion; Stanley & Mirotznik, 1997). It would thus be of interest in future experiments to determine which of these two alternatives mechanisms best explains the presynaptic effects of adenosine at the mouse neuromuscular junction.

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	Acknowledgements

 
I wish to thank Ms Shirley Foster for her exceptional technical assistance with the dissections and making of solutions. I am also grateful to Drs Timothy Searl and Jody Hirsh for their comments on the manuscript. This work (CSPaper #2) was supported by a grant from the NIH (NS 12782).

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