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Background The enteric nervous system (ENS) possesses extensive synaptic connections which integrate information and provide appropriate outputs to coordinate the activity of the gastrointestinal tract. The regulation of enteric synapses is not well understood. Cannabinoid (CB)1 receptors inhibit the release of acetylcholine (ACh) in the ENS, but their role in the synapse is not understood. We tested the hypothesis that enteric CB1 receptors provide inhibitory control of excitatory neurotransmission in the ENS.
Methods Intracellular microelectrode recordings were obtained from mouse myenteric plexus neurons. Interganglionic fibers were stimulated with a concentric stimulating electrode to elicit synaptic events on to the recorded neuron. Differences between spontaneous and evoked fast synaptic transmission was examined within preparations from CB1 deficient mice (CB1−/−) and wild-type (WT) littermate controls.
Key Results Cannabinoid receptors were colocalized on terminals expressing the vesicular ACh transporter and the synaptic protein synaptotagmin. A greater proportion of CB1−/− neurons received spontaneous fast excitatory postsynaptic potentials than neurons from WT preparations. The CB1 agonist WIN55,212 depressed WT synapses without any effect on CB1−/− synapses. Synaptic activity in response to depolarization was markedly enhanced at CB1−/− synapses and after treatment with a CB1 antagonist in WT preparations. Activity-dependent liberation of a retrograde purine messenger was demonstrated to facilitate synaptic transmission in CB1−/− mice.
Conclusions & Inferences Cannabinoid receptors inhibit transmitter release at enteric synapses and depress synaptic strength basally and in an activity-dependent manner. These actions help explain accelerated intestinal transit observed in the absence of CB1 receptors.
The myenteric and submucosal plexuses of the enteric nervous system (ENS) exist as interconnected integrative nerve networks within the wall of the gastrointestinal (GI) tract. Activity of the ENS is responsible for the control of the digestive and protective functions of the gut.1 Synaptic transmission between enteric neurons propagates information from intrinsic primary afferent neurons to interneurons, and then from interneurons to motor neurons that control final effectors, such as smooth muscle and the secretory epithelium. Acetylcholine (ACh) is the major excitatory neurotransmitter in the myenteric plexus, acting on nicotinic receptors at synapses between neurons and on muscarinic receptors at neuromuscular junctions.1,2 Unlike the central nervous system (CNS), fast inhibitory neurotransmission is not commonly observed in the myenteric plexus. Noradrenaline released from extrinsic sympathetic terminals generates inhibitory postsynaptic potentials of an intermediate duration in the guinea pig. However, the role of noradrenaline in regulating the moment-to-moment function of the GI tract remains unclear, as under physiological conditions sympathetic denervation has little effect.3 Thus, other mechanisms are likely to exist that could more specifically control synaptic strength in the ENS, through the graded control of ACh release from the presynaptic neuron.
In the CNS, the discovery of retrograde endocannabinoid signaling provided an explanation for some forms of postsynaptic activity-dependent changes in presynaptic neurotransmitter release.4,5 Presynaptic activation of the cannabinoid (CB)1 receptor causes both transient and sustained inhibition of neurotransmitter release, allowing for several forms of synaptic plasticity.4,6,7 Furthermore, CB1 receptor-mediated plasticity at the synapse can itself be regulated, a phenomenon known as metaplasticity.8,9 If a similar system is functioning at enteric synapses, then activity-dependent endocannabinoid signaling could exert homeostatic inhibitory control of excitatory neurotransmission in the ENS.
Cannabinoid receptors are expressed in the ENS, and in this system, exogenous CB1 receptor agonists inhibit ACh release and reduce the amplitude of fast excitatory postsynaptic potentials (fast EPSPs).10–12 The key elements of the endocannabinoid signaling system are present in the ENS, including receptors13–15 and putative transporters,16 and the enzymes responsible for the synthesis and degradation of endocannabinoids.13,17,18 Through the use of drugs that target individual components of this system it is apparent that CB1 signaling influences motor and other gut functions,13 but its role in enteric neurotransmission remains to be determined. In the gut, there also appears to be a basal level of endocannabinoid tone.12,13,19 We tested the hypothesis that enteric CB1 receptors provide inhibitory control of excitatory neurotransmission in the ENS. Our data support this hypothesis, and we have also discovered a new form of metaplasticity in the ENS, that has not been previously been reported in the peripheral or CNS.
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Regulation of synaptic activity in the ENS is a rapidly evolving area of study in enteric neurobiology. Here, we tested the hypothesis that enteric CB1 receptors provide a mechanism of dampening excitatory neurotransmission and maintaining network stability through the regulation of synaptic strength in the myenteric plexus. Collectively, our results indicate that endocannabinoids acting via the CB1 receptor modulate cholinergic transmission in myenteric neurons through presynaptic mechanisms. This modulation includes activity-independent inhibition of synapses and activity-dependent inhibition. This finding provides an explanation for previous reports that endocannabinoid tone inhibits neurogenic spontaneous contractility in the mouse ileum.36
The increased spontaneous and fast EPSPs evoked by prolonged presynaptic stimulation in CB1−/− mice, combined with the CB1 mediated depression of fast EPSPs and paired-pulse facilitation in WT mice, implicate a presynaptic site of action of endocannabinoids. This conclusion is further supported by the observation in CB1−/− mice that postsynaptic depolarization to applied ACh remains unchanged. In the cerebellum, CB1 coupling via Gβγ decreases Ca2+ entry into the presynaptic bouton, and hyperpolarizes the bouton by increasing K+ conductances.5 Both actions decrease quantal release and thus reduce the fast EPSP amplitude and increase the PPR of fast postsynaptic currents.37 A similar mechanism may be active at the enteric synapse to inhibit vesicular neurotransmitter release. It should be noted that others have demonstrated the opposite effect of CB1 receptor agonists on vesicular release;19 however, as those authors state, prolonged incubation with CB1 compounds (48 h) may have led to CB1 receptor desensitization. In our study, we have demonstrated CB1 receptor mediated inhibition of vesicular release on a shorter time scale minimizing the confound of receptor desensitization.
Other possible sites of action for endocannabinoid mediated inhibition of vesicular release include blockade of axonal conductance, and interference with the vesicular release machinery.37 Both could account for the observed effects. However, uniform Ca2+ waves were seen in climbing fibers to Purkinje cells following a protocol to elicit depolarization suppression of excitation,5 arguing against axonal conductance failure as a mechanism. CB1 receptor activity may also interfere with the proteins responsible for vesicular fusion and neurotransmitter release. However, the fact that the frequency of spontaneous events remains unchanged between WT and CB1−/− S neurons suggests that sensitivity of the vesicular release machinery is the same in the presence of an intact CB1/endocannabinoid system. Thus, direct interference with the vesicular release machinery by endocannabinoids acting at CB1 may not be involved. Indeed, this mechanism of inhibition has been ruled out in other regions, such as the hippocampus.38
Another possible explanation is the direct inhibition of the firing of interneurons within the preparation. However, the S neurons investigated in this study will have included a population of interneurons, and spontaneous action potential firing was never detected. Furthermore, spontaneous release of neurotransmitter was not affected by the application of TTX to block interneuron action potentials. Boesmans et al.19 demonstrated in enteric neurons that CB1 antagonism increased the percentage of neurons with spontaneous Ca2+ waves within the soma. This result parallels well with our observation of an increased percentage of neurons receiving spontaneous EPSPs in CB1−/− myenteric plexus. It seems most likely that the CB1 mediated inhibition of synaptic transmission is due to changes in Ca2+ influx to the presynaptic neuron. In addition, prolonged synaptic stimulation, such as occurs during the slow EPSP stimulus, would lead to a large increase in presynaptic Ca2+, possibly further potentiated by calcium-induced calcium release. The occurrence of a prolonged train of fast EPSPs superimposed on the slow EPSP of CB1−/− S neurons (Fig. 5) may result from the unopposed presynaptic influx of Ca2+ triggering late neurotransmitter release. In the WT mice with an intact inhibitory endocannabinoid signaling system, the Ca2+ influx may be inhibited in a CB1-dependent fashion. Hence, by a process of elimination we propose that at the enteric synapse, CB1 receptors act to inhibit the Ca2+ influx driving vesicular release. It is interesting that the CB1 agonist WIN55,212-2 is able to depress WT synapses globally while spontaneous activity is apparently regulated by CB1 receptors in only a subset of these synapses. It is possible that endocannabinoid signaling is not uniform across all synapses in the ENS. Additionally synapses originating from different subtypes of neurons may be regulated by endocannabinoids to differing degrees.
The absence of the inhibitory influence of the CB1 receptor in CB1−/− animals reveals a potent activity-dependent purinergic facilitation of fast EPSPs. A synergistic postsynaptic relationship between ATP and ACh has been demonstrated in pancreatic islets;39 here we show that a similar relationship exists in the ENS involving a presynaptic mechanism. Our data indicate that retrograde endocannabinoid and purinergic transmitters interact to regulate vesicle release probability and control synaptic communication in the myenteric plexus (Fig. 6F). Removal of endocannabinoid signaling results in unopposed purinergic synaptic facilitation and increased vesicle release, ultimately culminating in the increased excitatory neurotransmission in the ENS, and this likely leads to accelerated GI transit as shown here (Fig. 3D) and by others.13 The P2 receptor isotype that mediates the response described here has not been identified. The rapid onset and transient nature of the observed phenomenon suggests a ligand-gated ion channel, such as the P2X receptors which are ligand-gated non-specific cation channels. When open, these channels would allow for the permeation of calcium into the presynaptic neuron, a mechanism by which a purine messenger could potentiate synaptic transmission. However, the concentration of PPADS used was sufficient to block P2Y1 receptors40 and activation of these receptors can excite a relatively rapid (50 ms rise, 250 ms duration) depolarization in enteric neurons,40,41 albeit in guinea pig. Thus, the data do not exclude the possibility of the involvement of P2Y1 receptors. These receptors are coupled by inositol trisphosphate to intracellular calcium stores, so that they might be expected to enhance transmitter release.
We have established that purinergic–endocannabinoid interactions in the ENS regulate synaptic homeostasis. An intimate link between purine liberation and endocannabinoid action is suggested by our observation that P2 antagonism with PPADs did not reveal potent synaptic inhibition in WT animals. Autocrine signaling by purines may be required to generate a burst of endocannabinoid production. Indeed purines have been shown to stimulate endocannabinoid production in the cerebellar cortex.42 Alternatively, the inability to reveal synaptic depression with a P2 antagonist in the WT preparations may be due to a dosage effect of liberated endocannabinoid. Coincident stimulation would lead to activation of metabotopic receptor activation and potentiation of endocannabinoid release.6 Indeed, using this experimental paradigm of coincident stimulation we revealed synaptic depression (Fig. 6E). Unfortunately, it is difficult to test if P2 antagonism could potentiate this phenomenon as activity of PPADS on postsynaptic P2Y receptors would inhibit part of the postsynaptic metabotopic stimulation that this protocol would elicit.43
Our model does not account for the contribution of other cell types in the ENS. Enteric glia can participate in the metabolism of neurotransmitters,44 and neuron – glia purinergic communication is a feature of the murine ENS.45 Our model also does not address the possibility of polysynaptic contributions from downstream neurons excited by our depolarization protocol. Myenteric S neurons project to longitudinal or circular smooth muscle, or extend in a oral or aboral direction to distant enteric ganglion.21 The projections are uniaxonal and highly polarized to allow the propagation of peristaltic patterns of motility. We cannot rule out the contribution of feedback mechanisms from downstream enteric neurons, which have been demonstrated in the ENS.46–48 The route traveled by the trans-synaptic signal may be tortuous; however, the ability of postsynaptic calcium chelation to block synaptic potentiation indicates that the signal originates from the postsynaptic neuron. Furthermore, if the retrograde purine signal also originates from the postsynaptic neuron this implicates a calcium sensitive release mechanism for ATP and related purines from the cell soma of neurons within the ENS.
These results are the first to identify presynaptic endocannabinoid modulation of enteric synapses. In the absence of endocannabinoid modulation, a purinergic signal is also involved in synaptic regulation by facilitating transmitter release in an activity-dependent manner. In the postsynaptic depolarization model we employed, we were unable to conclusively demonstrate activity and CB1 receptor dependent inhibition of synapses. However, the absence of apparent synaptic plasticity when the CB1 receptor is intact suggests an endocannabinoid effect that opposes activity dependent purinergic facilitation on an identical time course. These findings suggest a novel form of metaplasticity through the balance of endocannabinoid and purinergic signaling at the enteric synapse, and have important implications for our understanding of enteric physiology. These findings demonstrate that the enteric synapse can respond to high levels of network activity and that at least two signaling molecules are involved in manipulating synaptic strength in response to this activity. Clearly computations that occur at the enteric synapse are an important feature in maintaining ENS network homeostasis.