BChE inhibition decreases the quantal content of EPP when AChE is active

We next addressed the question of the function of BChE anchored at the surface of the TSC. We have previously shown that, when AChE is absent or inhibited, inhibition of BChE reduces the probability of ACh release (Minic et al., 2003). If a similar modulation exists when AChE is active, one expects to observe a reduction the EPP quantal content (QC) after inhibition of BChE. The EPP QC at low frequency of nerve stimulation varies significantly between the NMJs of the diaphragm muscle, but little for a given NMJ during experiments performed ex vivo. Therefore, we analyzed the effect of BChE inhibition on the EPP QC at the level of a single NMJ (Fig. 3A). We found that the EPP QC was reduced by 18% after 30 min application of 50 μm iso-OMPA, a specific, irreversible BChE inhibitor. To validate the selectivity of iso-OMPA toward BChE in our experimental conditions, we measured the EPP QC in BChE KO mice and found that it did not change significantly after application of iso-OMPA (Fig. 3B); then, we compared the mean QC in WT and BChE KO mice and found a reduction of 30% of the mean QC in BChE KO mice compared with WT mice (Fig. 3C). Therefore, the EPP QC is reduced both in BChE KO mice and after the specific pharmacological inhibition of BChE, likely through the action of residual ACh on AChRs.

Open in a separate window

Figure 3.

BChE inhibition depresses ACh release at the NMJ. A, Experimental protocol. The muscle contraction was blocked by inhibition of the muscle voltage-gated sodium channels with μ-conotoxin GIIIB. A sharp electrode was inserted close to a NMJ to record focal mEPPs and EPPs. A first file was obtained for the mEPPs (no nerve stimulation), then a second for the EPP (0.5 Hz nerve stimulations), and then a third for the mEPPs (no stimulation). Iso-OMPA (50 μm) was added to the Ringer's buffer and the muscle was stimulated indirectly at 0.1 Hz during the incubation. The electrode was maintained in place. All of the records were obtained in 30–40 min. The QC was obtained by dividing the mean amplitude of the EPPs by the mean amplitude of the mEPPs recorded before and after the EPPs if the resting membrane potential (RMP) did not change >1 mV during mEPP and EPP recording. B, Quantification of the QC in the same muscle fiber after inhibition of BChE with iso-OMPA. The percentage of decrease of the QC in WT mice is higher than in BChE KO mice (n = 5, *p < 0.05, paired Student's t test). C, The mean QC in the NMJs of WT mice is higher than in BChE KO mice (n = 20, *p < 0.05, and n = 5, *p < 0.05, respectively, unpaired Student's t test). D, Quantification of the QC in the same muscle fiber after inhibition of BChE with iso-OMPA when AChRs are absent or blocked. The graph presents the quantification of the QC in the same muscle fiber after iso-OMPA. All of the muscarinic receptors were blocked with 1 μm atropine (n = 5, *p < 0.05, paired Student's t test).

Because BChE is localized on TSCs, we wanted to find out whether TSCs contribute to the reduction of ACh release after BChE inhibition or if they simply act as a scaffold localizing BChE close to, but outside of, the synaptic cleft. To address this question, we lowered specifically the metabolism of the TSC with fluorocitric acid, a Krebs' cycle blocker that affects mainly the glial cells (Bonansco et al., 2011). Incubation in fluorocitric acid (0.2 mm) during 1 h did not alter significantly the EPP QC (92 ± 5%, n = 5), but when fluorocitric acid was washed out and BChE was inhibited by iso-OMPA (50 μm, 30 min), the EPP QC was not significantly reduced (95 ± 6%, n = 5). This result suggests that TSCs may participate actively in the reduction of EPP QC after BChE inhibition.

Blockade of α7 receptors prevents the depression of ACh release after BChE inhibition

The effects of BChE inhibition suggest that the excess of ACh resulting from this inhibition depresses ACh release. To identify the ACh receptors mediating this depression, we estimated the EPP QC after blockade of muscarinic receptors (1 μm atropine) and in the absence of nicotinic β2, β4, or α7 receptors (in β2 KO, β4 KO, or α7 KO mice, respectively). The incubation of the ex vivo nerve–muscle preparations from WT mice with atropine did not prevent the reduction of the EPP QC observed after the specific BChE inhibition by iso-OMPA (Fig. 3C). EPP QC was also decreased after iso-OMPA incubation of ex vivo unitary nerve–muscle preparations from β2 nAChR KO and β4 nAChR KO mice, as in the WT mice (Fig. 3C). In stark contrast, the incubation with iso-OMPA of ex vivo diaphragms obtained from α7 nAChR KO mice did not change the EPP QC (Fig. 3C). To further explore the implication of α7 nAChR, we incubated the WT muscle preparation with 10 nm MLA, which is a specific inhibitor of α7 nAChR, and followed the EPP QC before and after iso-OMPA addition to the bath containing the ex vivo nerve–muscle preparations (Fig. 3C), QC was not changed by iso-OMPA. It is worthwhile to emphasize that MLA did not affect the EPP QC in α7 KO mice.

From these experiments, it can be concluded that the decrease of EPP QC induced by BChE inhibition is mediated by α7 nAChR.

α7 nAChRs are localized and functional at the TSC of the NMJ

To analyze how activation of α7 nAChRs may depress the release of ACh, we attempted to localize them at the NMJ using polyclonal antibodies directed against mouse α7 nAChR (Kalashnyk et al., 2012). As shown in Figure 4A, we obtained a reproducible weak signal at the NMJ identified by α-bungarotoxin labeling. α-Bungarotoxin is an α-neurotoxin known to bind irreversibly and competitively to the nAChRs found at the NMJ and to α7 nAChRs in the brain. The specificity of α7 nAChR labeling observed with the polyclonal antibodies used is substantiated by the absence of any detectable immunolabeling in α7 KO mice with the same polyclonal antibodies. The weak α7 nAChR immunolabeling observed did not overlap with the strong labeling obtained with fluorescent α-bungarotoxin. At the EM level, we also found a dense deposit of gold particles along the postsynaptic membrane labeled with α-bungarotoxin. However, we did not find gold particles along the plasma membranes of the TSC or the nerve terminal. Moreover, immunoparticles that were not associated with the muscle membranes appeared equally distributed in WT and α7 KO muscle preparation. Altogether, these observations failed to reveal α7 nAChR by using labeled α-bungarotoxin, presumably because the density of α7 nAChR on the TSC is much lower than that of the muscle postsynaptic receptors. A low density of α7 nAChR was previously observed in brain astrocytes (Vélez-Fort et al., 2009). To further analyze the distribution of α7 nAChR, we looked for colocalization labeling with S100, a specific marker of TSCs. We found a colabeling of α7 nAChR with S100, but not with α-bungarotoxin (Fig. 3B). These observations suggest that the α7 nAChRs detectable with the polyclonal antibodies are mainly on the TSCs.

TSCs are close to the nerve terminal and sensitive to the synaptic transmission (Auld and Robitaille, 2003). The hallmarks of TSC sensitivity to the synaptic activity are calcium waves recorded specifically in the TSCs after long bursts of nerve stimulations (Todd et al., 2010). We generated calcium waves at the TSC by 120 s nerve stimulations (20 Hz) in presence of μ-conotoxin GIIIB (a specific muscle voltage-gated sodium channel blocker used to prevent muscle contraction). After 30 min of recovery, the calcium waves were again generated by 120 s nerve stimulations (20 Hz). To evaluate whether α7 nAChRs mediate this signaling, the muscle preparation was incubated during the recovery period (30 min) with 10 nm MLA (n = 5 experiments). We found that the second calcium waves were totally abolished after MLA, but not in control (Fig. 4C). This result supports that α7 nAChRs are functional at the TSC, where they are activated by the spillover of ACh. This can actually occur for prolonged stimulations, even when both AChE and BChE are active, but is likely to be much larger in extreme physiological circumstances and/or medical or pathological conditions.

AChE and BChE coinhibition decreases the mean amplitude of EPP

The previous experiments revealed that the selective inhibition of BChE anchored at the surface of TSCs reduces ACh release. This means that, in the presence of AChE, some ACh must escape the cleavage by AChE clustered in the synaptic cleft to reach BChE. This spillover is likely to be moderate, which might explain the modest inhibition of the synaptic transmission observed after BChE specific inhibition at low frequency of nerve stimulation. The inhibition of both AChE and BChE is expected to increase the spillover of ACh and should increase the depression of the EPP QC. Unfortunately, after AChE inhibition, the direct estimation of the QC as the summation of the individual quanta (mEPP) is not possible. In normal conditions, EPP is the simple sum of individual mEPPs. After AChE inhibition, the surfaces of the postsynaptic area ultimately reached by individual quanta overlap, so the number of quanta released cannot be calculated as the quotient of EPP by the mEPP (Hartzell et al., 1975). We thus calculated the mean value of EPP amplitude recorded in the same area of the diaphragm. The amplitude of EPPs is the key parameter for neuromuscular synaptic transmission. The muscle contraction occurs only if the EPPs pass the threshold of AP generation. To record the full EPP, the APs were blocked with μ-conotoxin GIIIB. The resting membrane potential was constant. The mean EPP amplitude was 31.5 ± 0.95 mV in control diaphragm and, after AChE and BChE inhibition (3 μm neostigmine, 20 min), it decreased to 14.2 ± 0.75 mV (Fig. 5D). It is important to note that the amplitude of mEPCs recorded in the interstimuli intervals after ChE inhibition by neostigmine was higher than in the untreated intact preparation (Fig. 5B). Therefore, after the inhibition of AChE and BChE, the reduction of the number of quanta released was likely larger than the reduction of the EPP amplitude.

Open in a separate window

Figure 5.

The amplitudes of the EPP are rescued partially by the block of α7 nAChR when both cholinesterases were inhibited. A, Experimental protocol. The muscle contraction in diaphragm was blocked by μ-conotoxin GIIIB. Fifty EPPs were recorded from each muscle fiber. Only data from the fibers in which the resting membrane potential was >70 mV at the end of the recording have been used. Mean amplitude of EPPs was obtained from 20–30 fibers recorded in control conditions and after application of the drugs (neostigmine, MLA). B, Examples of EPPs recorded before and after cholinesterase inhibition with 3 μm neostigmine. Note the difference of scale between the two conditions. In each of the selected traces, one EPP and one mEPP are visible. C, Evaluation of the threshold of nerve-evoked muscle fiber APs. The phrenic nerve was stimulated once for recording an AP in each muscle fiber. The resting membrane potential was −70 to 75 mV. Records of APs were differentiated (lower profile, dV/dt) and the time point on the rising phase of the nerve-evoked AP when the rate of change of voltage with time was closest to zero was determined. The threshold was defined as the voltage at this time. D, Mean amplitude of EPPs recorded in the phrenic-diaphragm preparations of WT mice and α7 nAChR KO mice in control conditions and after application of the drugs (neostigmine 3 μm, MLA 10 nm; n = 20–30, *p < 0.05, unpaired Student's t test).

To test the involvement of α7 nAChR, we used both MLA, a specific blocker of α7 nAChR, and α7 KO mice. After 30 min incubation of the diaphragms of WT mice with MLA (10 nm), the mean EPP was unchanged at 33.4 ± 0.6 mV. Then, after 20 min incubation with neostigmine (3 μm), the mean EPP decreased only to a value of 24.5 ± 1.3 mV. The mean EPP amplitude was 33.3 ± 0.9 mV in the diaphragms of α7 KO mice and 23.4 ± 1.4 mV after 20 min of incubation with neostigmine. We also controlled the specificity of MLA by treating muscles of α7 KO mice. The mean EPP amplitude in α7 KO mice treated by MLA (10 nm) was 32.5 ± 1.1 mV before and 25.2 ± 1.0 mV after neostigmine. These results unambiguously reveal that the activation of α7 nAChR significantly reduces the amplitude of EPP after inhibition of cholinesterase by neostigmine.

The amplitude of EPP after inhibition of AChE and BChE was found to be close to the threshold of AP generation evaluated in extensor digitorum longus and soleus muscles of rat (13 mV and 14 mV, respectively; Wood and Slater, 1995). To perform a direct comparison with the mouse diaphragm, we estimated the threshold of AP generation in mouse diaphragm when both cholinesterases were inhibited by 3 μm neostigmine (Fig. 5C). The average level of threshold of AP generation was 13 ± 3 mV (n = 20), a value close to the mean EPP amplitude (14.2 ± 0.75 mV) obtained after complete inhibition of all cholinesterases with neostigmine. In contrast, the amplitudes of the EPP appear higher after inhibition of cholinesterases when α7 nAChR are absent or inactivated.

AChE and BChE coinhibition and muscle contraction

The decrease of EPP amplitude close or below to the threshold of AP after AChE and BChE inhibition should be translated at the physiological level into a reduction of the muscle contraction triggered by the nerve. The anesthetists routinely assess via neuromuscular monitors the decrease of the EPP amplitude during muscle relaxation. They follow the modification of the muscle contractions triggered by the nerve stimulations. A sensitive method to monitor the neuromuscular blockade is used. The TOF stimulation method consisted of recording the four muscle contractions elicited by four nerve stimulations at 2 Hz (Ali et al., 1975; Srivastava and Hunter, 2009; Plaud et al., 2010). Next, the ratio of the fourth over the first response, known as the TOF ration (TOFR), which reflects the degree of neuromuscular blockade during onset and recovery after nondepolarizing muscle relaxant administration, can be calculated. When this pattern of stimulation was applied to the phrenic nerve diaphragm preparation ex vivo, the amplitudes of the four twitches were identical during hours if TOFs were repeated every 30 s (Fig. 6A). We then examined the effects of neostigmine on the first twitch (T1) and on T2-T4. After 20 min of incubation with 3 μm neostigmine, the mean amplitude of T1 decreased to 60% of the initial amplitude in WT muscle (Fig. 6B,C). A concentration of 0.1 μm atropine (to block all of the muscarinic receptors) did not change the reduction of the T1. In contrast, after blockade of α7 nAChR (MLA treatment, 10 nm) or in α7 KO mice, the mean amplitudes of T1 did not decrease in neostigmine (Fig. 6B,C). Therefore, as expected, the partial recovery of the amplitude of the EPP after α7 nAChR block following AChE and BChE inhibition described in the preceding section is translated in the recovery of T1 amplitude during supramaximal stimulation. These changes required 20 min to be effective and were maintained over time.

Open in a separate window

Figure 6.

The reduction of the muscle contraction after inhibition of AChE and BChE is partially reversed by the block of α7 nAChR. A, Experimental protocol. The phrenic-diaphragm preparation was mounted in the organ bath. The muscle is indirectly stimulated by the nerve and the twitch was quantified with a tension sensor. The muscle was stimulated each 30 s by 4 stimulations at 2 Hz. B, Example of the representative traces recorded before and 20 min after incubation with neostigmine (3 μm). C, Amplitude of 6 TOFs obtained from in control condition, in atropine (0.1 μm), in MLA (10 nm), and in α7 nAChR KO mice. All values correspond to the mean of six successive TOFs in six different animals.

The analysis of T2-T4 revealed an additional process. When cholinesterases were inhibited in WT, the amplitudes of T4 were smaller than T1 (TOFR ~0.7). These events occur during the range of few hundred milliseconds to seconds after T1 and are cumulative with T2 and T3. These events are reversible: after 28 s, the amplitude of T1 is returned to the value of T1 of the TOFn-1. A similar ratio was observed after inhibition of muscarinic receptors by atropine. Unexpectedly, the TOFR was also ~0.6–0.7 when α7 nAChR was not functional, even when T1 mean amplitudes were maintained. To ensure that the decrease of the contraction did not result from the failure of the muscle function, but rather from a decrease of the synaptic transmission, we stimulated the muscles directly. Synaptic transmission was blocked with d-tubocurarine (a nAChR blocker). In these conditions, the four responses after TOF stimulation were identical to the control responses (Fig. 6B,C). Therefore, the reduction of the contraction was due to an alteration in synaptic transmission. The interpretation of this second process needs more investigation and is outside of the scope of this study.

α7 nAChR at the TSC modulate the QC at the NMJ

Numerous experiments have shown that ACh acts on the nerve terminal and modulates its own release (Ciani and Edwards, 1963; Okamoto and Riker, 1969; Duncan and Publicover, 1979; Dudel, 2007; Kupchik et al., 2011). Many investigators have applied exogenous agonists of AChR to reveal the presence of muscarinic and nicotinic AChRs (Miyamoto and Volle, 1974; Nikolsky et al., 1991, 2004; Vizi and Lendvai, 1997; Slutsky et al., 2001; Minic et al., 2002). In some experiments, muscarinic agonists have inhibited ACh release from motor nerve terminals, whereas in others, ACh release was increased (Abbs and Joseph, 1981; Wessler et al., 1987; Arenson, 1991; Robitaille et al., 1997; Santafé et al., 2007). At the vertebrate NMJ, the activation of m2 AChR reduced the release, whereas the activation of m1/m3 AChR facilitated the release (Slutsky et al., 2001; Minic et al., 2002; Oliveira et al., 2002, 2009; Dudel, 2007).

The regulation of ACh release at NMJs by nAChRs is less documented. The differential effects of nAChR blockers used by the anesthesiologists have been interpreted so far by their potential action on nAChRs. It was assumed that these receptors are localized at the nerve terminal (for review, see Vizi and Lendvai, 1999). Nicotine, a potent agonist of nAChRs, inhibits rhythmic activity of mouse NMJs (Balezina et al., 2006). This effect is prevented by α-cobratoxin (a blocker of muscle and α7 nAChRs) and by apamin (a blocker of Ca²⁺-activated potassium channels). It was thus proposed that nicotine stimulates presynaptic nAChRs (presumably α7) that activate Ca²⁺-activated potassium channels. However, previous experiments failed to find α-bungarotoxin (α7 nAChRs ligand in neurons) on the nerve terminal (Jones and Salpeter, 1983) and our results suggest that α7 nAChRs are localized at the TSC. We found a weak labeling with specific antibodies colocalized with S100 (a well established TSC marker). This labeling was absent in α7 KO mice. Calcium waves in the TSC triggered by long bursts of nerve stimulations were abolished by 10 nm MLA (a specific blocker of α7 nAChR). These observations do not exclude that α7 nAChRs are also present at the postsynaptic membrane, where they were found in pathological conditions such as burning or immobilization (Lee et al., 2014).

How the TSCs may affect the release on the nerve terminal is not clear. There are many steps between the activation of α7 nAChR by the spillover of ACh and the reduction of ACh release at the nerve terminal. The initial step is likely to be the entry of calcium in the TSCs and the last one could be a decrease of calcium entry through inhibition of P/Q-type calcium channels of the nerve terminal. Between these two steps, a plausible hypothesis is the release by the TSCs of “gliotransmitters” such as glutamate, ATP, or adenosine because, in the CNS, these compounds have been shown to be released by glial cells, to have receptors on nerve terminals, and to interfere with voltage-dependent calcium channels (Araque et al., 2014), and there is evidence for similar phenomena in the peripheral nervous system (Castonguay et al., 2001). However, our main observations have been made minutes after the activation of α7 nAChRs so one cannot exclude slower cellular mechanisms, including the transcription and/or translation of new proteins or peptides.

The observation that long bursts of nerve stimulations (30 s, 50 Hz) can trigger calcium waves at the TSCs of mammalian NMJ was first made by Rochon et al. (2001). These calcium waves were blocked by 20 μm atropine and were therefore attributed to the activation of muscarinic receptors (mAChRs). However, after 25 min of recovery, long bursts of nerve stimulations were not able to generate new calcium waves (Rochon et al., 2001). In contrast, with a similar pattern of nerve stimulation (120 s, 20 Hz), we triggered repetitive calcium waves in the TSCs when 30 min recovery periods were allowed. Moreover, we found that the second waves were abolished by MLA. The design of the experiments likely explains this discrepancy. Calcium imaging at the NMJ requires that muscle contraction be abolished. To prevent muscle contraction, we used μ-conotoxin GIIIB, a blocker of the muscle voltage-gated sodium channel that does not interfere with α7 nAChRs and muscle nAChRs. In contrast, Rochon et al. (2001) (see also Todd et al., 2007; Rousse et al., 2010) blocked the contraction using d-tubocurarine or α-bungarotoxin, which are known to block α7 nAChRs (Alexander et al., 2013), and applied atropine at 20 μm, a concentration that blocks mAChRs, but also α7 nAChRs (Zwart and Vijverberg, 1997; González-Rubio et al., 2006). α7 nAChRs were thus likely to be blocked in these experiments and thus prevented the generation of a second wave of calcium.

The presence of functional α7 nAChRs was previously shown on astrocytes in cell culture (Sharma and Vijayaraghavan, 2001) and on NG2 cells in brain slices (Vélez-Fort et al., 2009). When α7 nAChRs are activated by ACh they induce calcium entry, but their function remains elusive. We found for the first time here that activation of extrasynaptic α7 nAChRs depresses ACh release. It remains to be determined in which physiological conditions ACh activates these α7 nAChRs. It is tempting to propose that α7 nAChRs play the role of ACh sensor outside of the synaptic cleft at the NMJ. In the context of synaptic homeostasis, it represents the first sensor that detects the excess of neurotransmitter (Davis, 2013).

BChE, a forgotten enzyme

The mammalian cholinesterases include two well known closely related enzymes, AChE (E.C. 3.1.1.7) and BChE (E.C. 3.1.1.8), that hydrolyze ACh efficiently. The participation of BChE in synaptic transmission has been completely ignored until now (Johnson and Moore, 2012). We have now revealed that BChE localized on the TSC regulates the activation of α7 nAChRs, but not the activation of the muscle nAChRs. We found that BChE is anchored by PRiMA, the small transmembrane protein required for the maturation of AChE in the brain (Perrier et al., 2002; Dobbertin et al., 2009). This localization of BChE/PRiMA at the TSC contrasts with the localization of AChE mainly anchored by ColQ in the extracellular matrix of the synaptic cleft (Younkin et al., 1982; Bernard et al., 2011). We can speculate that one of the physiological functions of BChE is to control the spillover of ACh around the TSC and thus the activation of AChRs localized outside of the synaptic cleft. Our results suggest that the spillover of ACh in the CNS described previously (Lamotte d'Incamps et al., 2012) also exists at the NMJ despite the high density of AChE.