Inhibitory effects of endomorphin-2 on excitatory synaptic transmission and the neuronal excitability of sacral parasympathetic preganglionic neurons in young rats

Abstract

The function of the urinary bladder is partly controlled by parasympathetic preganglionic neurons (PPNs) of the sacral parasympathetic nucleus (SPN). Our recent work demonstrated that endomorphin-2 (EM-2)-immunoreactive (IR) terminals form synapses with μ-opioid receptor (MOR)-expressing PPNs in the rat SPN. Here, we examined the effects of EM-2 on excitatory synaptic transmission and the neuronal excitability of the PPNs in young rats (24–30 days old) using a whole-cell patch-clamp approach. PPNs were identified by retrograde labeling with the fluorescent tracer tetramethylrhodamine-dextran (TMR). EM-2 (3 μM) markedly decreased both the amplitude and the frequency of the spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) of PPNs. EM-2 not only decreased the resting membrane potentials (RMPs) in 61.1% of the examined PPNs with half-maximal response at the concentration of 0.282 μM, but also increased the rheobase current and reduced the repetitive action potential firing of PPNs. Analysis of the current–voltage relationship revealed that the EM-2-induced current was reversed at −95 ± 2.5 mV and was suppressed by perfusion of the potassium channel blockers 4-aminopyridine (4-AP) or BaCl2 or by the addition of guanosine 5′-[β-thio]diphosphate trilithium salt (GDP-β-S) to the pipette solution, suggesting the involvement of the G-protein-coupled inwardly rectifying potassium (GIRK) channel. The above EM-2-invoked inhibitory effects were abolished by the MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), indicating that the effects of EM-2 on PPNs were mediated by MOR via pre- and/or post-synaptic mechanisms. EM-2 activated pre- and post-synaptic MORs, inhibiting excitatory neurotransmitter release from the presynaptic terminals and decreasing the excitability of PPNs due to hyperpolarization of their membrane potentials, respectively. These inhibitory effects of EM-2 on PPNs at the spinal cord level may explain the mechanism of action of morphine treatment and morphine-induced bladder dysfunction in the clinic.

Endomorphin-2 Inhibits the Activity of the Spinoparabrachial Projection Neuron through Presynaptic Mechanisms in the Spinal Dorsal Horn in Rats

“…To investigate whether EM2 could change the intrinsic properties of TMR-labeled PNs, the single action potentials (APs) at the threshold and firing patterns (Fig. 5), we use EM2 (3 µM) bath application into the ACSF following our previous studies [20, 31]. No changes were found in the firing pattern test, where EM2 did not change the spike number in most of the recorded TMR-labeled PNs (n = 15, from 8 rats, F (1,70) =1.64, P =0.16, two-way repeated measures ANOVA).…”

“…Although EM2 is known to exert inhibitory effects in the SDH [23, 31, 50, 52], EM2-ir axon terminals mainly exhibited asymmetric synaptic connections with PNs, and thus it is likely that the synapses are excitatory. In the SDH, periaqueductal gray (PAG) and dorsal raphe nucleus, opioid peptide-ir axon terminals have been reported to mainly establish asymmetric synapses on neurons [6, 12, 22, 53, 54].…”

“…Transverse spinal cord slices at the level of the lumbar enlargement were prepared following our previous method [31]. Fifteen rats were perfused transcardially with 100 ml of 4°C sucrose artificial cerebrospinal fluid (sucrose-aCSF) containing the following reagents: 220 mM sucrose, 2.5 mM KCl, 26 mM NaHCO 3 , 6.0 mM MgSO4, 1.2 mM KH 2 PO 4 , 0.5 mM CaCl 2 , 10 mM glucose, 1 mM ascorbate, and 3 mM sodium pyruvate.…”

“…For electron microscopy analysis, 0.1 µl of 2% (w/v) wheat germagglutinin-horseradish peroxidase (WGA-HRP) conjugate (Toyobo, Osaka, Japan) dissolved in normal saline was injected into the LPB with the same coordinates used as the FG injection. For whole-cell patch clamp recording, 0.1 µl of 10% tetramethylrhodamine (TMR, 3000 MW, Molecular Probe, Eugene, OR, USA) distilled in 0.1 M citrate-NaOH (pH 3.0) [31] was injected into the LPB (7.8 mm posterior to Bregma, 1.7 mm lateral to the midline, and 6.1 mm ventral to the surface of the cranium) of young rats. Each injection was made slowly over 10 min and the injection needle was then kept in place for another 10 min.…”

Endomorphins: Promising Endogenous Opioid Peptides for the Development of Novel Analgesics

“…EM2 activated pre-and post-synaptic MORs, thereby inhibiting excitatory neurotransmitter release from the presynaptic terminals and decreasing the excitability of PPNs due to the hyperpolarization of their membrane potentials. These inhibitory effects of EM2 on PPNs in the spinal cord may explain the mechanism of action of morphine treatment and morphine-induced bladder dysfunction in the clinic [19,114].…”