Morphological And Functional Studies On Inhibitory Effects Of Endomorphin-2 On Activities Of The Preganglionic Neurons In The Sacral Parasympathetic Nucleus

Background: The sacral parasympathetic nucleus(SPN) is one of the spinal autonomic nervous centers that control various functions of pelvic organs, including micturition, defecation and penile erection. The parasympathetic preganglionic neurons(PPNs) in the SPN play a critical role in the processes of micturition reflex. In the intact spinal cord of adult animals, micturition is fulfilled mainly by multisynaptic reflexes that consist of micturition-related nuclei at the supraspinal level. However, in chronically spinalized animals after spinal cord injury at cervical or thoracic levels, the primary sensory afferents make direct synaptic connections with the processes of the PPNs. Endomorphins, including endomorphin 1(EM-1) and endomorphin 2(EM-2), have high affinity and selectivity for μ-opioid receptor(MOR) and are considered as their endogenous ligands. Morphine, the exogenous ligand of MOR, is involved in the neurogenic inhibition of bladder motility via spinal mechanisms. Our recent study has revealed that EM-2-immunoreactive(IR) terminals form synapses with MOR-expressing PPNs in the rat SPN. Based on the results of these previous findings, we speculate that urinary retention which is the most common clinically relevant side effects of the intrathecal administration of morphine is related to the inhibition of PPNs and the inhibition of micturition reflex by morphine. However, it remains unclear whether endogenous opioids could affect on the activities of the PPNs at cellular levels.Objects: In order to further investigate the morphological features of the PPNs in the SPN and to further reveal the mechanisms of the inhibitory effects of endogenous opioids peptides on PPNs, both morphological and electrophysiological methods were used in the present study. These results will provide experimental basis for understanding and treating morphine-induced urinary retention.Methods: The PPNs were identified by retrograde tracing with tetramethylrhodamine-dextran(TMR). GAD67-GFP knock in mice were used to investigate the morphological features of the PPNs in the SPN and the expression of MOR in the PPNs of SPN. The effects of EM-2 on excitatory and inhibitory synaptic transmissions and the neuronal excitability of PPNs in young rats(24-30 days old) were studied using whole-cell patch-clamp recording.Results:1. In the GAD67-GFP knock in mice, it was confirmed that there were GABAergic inhibitory interneurons within the SPN and in its vicinity. There were also non-PPNs and non-GABAergic neurons. These non-PPNs and non-GABAergic type of neurons might be excitatory interneurons, glyergic inhibatory interneuron or spinal tract neurons which project to other regions of the spinal cord and, even to the brain.2. Most of the PPNs were expression for MOR. Only a few of GABAergic inhibitory interneurons were weakly expression for MOR. Most of the non-PPNs and non-GABAergic neurons were also expression for MOR.3. EM-2 not only decreased the resting membrane potential(RMP) 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. These results indicate that EM-2 decreased the excitability of PPNs by hyperpolarization of their membrane potentials.4. Analysis of the current–voltage relationship revealed that the EM-2-induced current was reversed at-95 ± 2.5 m V and was suppressed by perfusion of potassium channel blocker 4-aminopyridine(4-AP) or Ba Cl2; EM-2-induced current was blocked by the addition of guanosine 5′-[β-thio]diphosphate trilithium salt(GDP-β-S) to the pipette solution and by perfusion of MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2(CTOP). These results suggest that the membrane hyperpolarization of PPNs involved opening the G-protein-coupled inwardly rectifying potassium(GIRK) channel after EM-2 binding to the MOR.5. EM-2(3 μM) markedly decreased both the amplitude and the frequency of the miniature excitatory postsynaptic currents(m EPSCs) of PPNs. EM-2(3 μM) decreased both the amplitude and the frequency of the spontaneous inhibitory postsynaptic currents(s IPSCs) of PPNs. However, EM-2(3 μM) did not decrease both the amplitude and the frequency of the miniature inhibitory postsynaptic currents(m IPSCs) of PPNs. The EM-2-invoked inhibitory effects as mentioned above were abolished by CTOP. These results suggest that the activation of presynaptic MORs by EM-2 caused the inhibition of excitatory but not inhibitory neurotransmitter release from presynaptic terminals.Conclusions:1. The neuronal component of SPN consists of PPNs, GABAergic inhibitory interneurons and other type of neurons.2. The PPNs and other type of neurons in the SPN were expression for MOR, only a few of GABAergic neuron weakly expression for MOR.3. EM-2 hyperpolarized the resting membrane potentials of the PPNs by the activation of their postsynaptic MORs resulting in decreases in the excitability of the PPNs. Activation of presynaptic MORs by EM-2 which caused the inhibition of excitatory but not inhibitory neurotransmitter release from presynaptic terminals. EM-2 not only has direct inhibitory effects on PPNs at spinal cord levels, but also inhibits excitatory neurotransmitter release from presynaptic terminals onto PPNs, both of these contribute to inhibition of micturition reflex formed by the synaptic connections between primary afferent terminals and PPNs. These results might reveal the mechanisms underlying morphine-induced bladder dysfunction caused by intrathecal administration of morphine in the clinic.