Synaptic Connections Between EM2-immunoreactive Terminals And MOR-expressing Neurons In The Sacral Parasympathetic Nucleus Of The Rat

Sacral parasympathetic nucleus (SPN) is one of an important nucleus in the spinalcord involved in bladder activity. In rats, neurons in the SPN are located at L6-S1segments in the sacral intermediolateral gray matter lamellar VII and are known asparasympathetic preganglionic neurons (PPNs), the function of which may be completeddirectly by the primary afferents form monosynaptic reflex or polysynaptic reflex withintermediate neurons upper micturition nucleus, Onuf’s nuclear or pelvic ganglianeurons. Morphine has been widely used for postoperative analgesia, although the effectworks well, it prone to uroschesis and other adverse reactions. In vivio, morphine willhave analgesic effect only if it binds to MOR. Some scholars believed that morphinebinding to MOR will increase ureter tension, lessen the bladder detrusor contraction andincreased pressure within the urethra. However, the real mechanism is not very clear sofar. Studies have shown that MOR are widely distributed in the spinal cord, includingdorsal horn, dorsal commsssural nucleus (DCN), onuf’s nucleus and SPN. EM2is arecently discovered endogenous opioid peptide, which is the specific and high-affinityendogenous ligand of MOR. EM2is mainly distribution in the spinal cord and peripheralnerves. Its analgesic effects similar to morphine, but the side effects are far less thanmorphine, so it has broad application prospects in clinical analgesic treatment.Purpose To observe synaptic connections between MOR positive PPNs in the SPN andEM2positive primary afferent fibers and terminals. In order to confirm MOR and EM2are involved in the bladder control of the rat in spinal cord level.Methods (1) The pelvic nerve of Sprague-Dawley rats were dissected, Alexa594bindingcholera toxin b subunit (CTb) was inject to the central end of pelvic nerve for retrogradetract-tracing, then the PPNs in the SPN were marked out.(2) Immunohistochemistry wasused to confirme that MOR-positive neurons and EM2-positive primary afferent fibersand terminal were found in the SPN area.(3) CTb retrograde labeling combined withimmunofluorescence staining for EM2and MOR to observe the co-localization betweenMOR positive PPNs and EM2-positive terminally.(4) The methods of wheat germagglutinin-horseradish peroxidase (WGA-HRP) retrograde tract-tracing combined withMOR and EM2the immune electron microscopy were used to confirm the synapticconnections between MOR positive PPNs and EM2positive fiber and their type.Results (1) CTb retrograde labeled PPNs are located at L6-S1segments in the sacralintermediolateral gray matter of the lamellar VII.(2) MOR-positive neurons andEM2-positive primary afferent fibers and terminals were found within the SPN byimmunohistochemical staining.(3) All of CTb marked PPNs cell bodies and processeswere MOR-positive, and the co-localization between EM2positive terminal and MOR-positive PPNs can be observed in the SPN area.(4) The synaptic connectionsbetween MOR-positive PPNs and EM2positive terminal were further confirmed byimmune electron microscopy.Conclusions Taken together, the results of the present study show that there are directsymmetric synaptic connections between EM2-containing primary afferent terminals andMOR-expressing PPNs in the SPN. These synaptic connections further confirm ourhypothesis that EM2might act via MOR-expressing PPNs within the SPN andcontributes to the inhibitory effects on bladder control. Thus, we infer that the inhibitionof EM2-containing primary afferent terminals to MOR-expressing PPNs is partiallyderived from the synaptic mechanisms.