The present study was to investigate the localization of preganglionic parasympathetic neurons of gallbladder in brain stem by anatomical and functional approaches. Male or female rabbits (n = 11) were anesthetized with sodium pentobarbital (30 mg/kg, i.v.). Cholera toxin B conjugated to horseradish peroxidase (CB-HRP) was injected into the gallbladder wall. Four days later, animals were re-anesthetized and perfused transcardially with paraformaldehyde solution in a 0.1 M phosphate buffer. The rabbit brain was then frozenly sectioned. The sections were processed for HRP label and stained with neutral red. Another group of rabbits (n = 54) were anesthetized by urethane (1 g/kg) after fasting for 18-24 hours, Gallbladder pressure (GP) was measured by inserting a frog bladder filled with normal saline into the gallbladder. Myoelectrical activity of the sphincter of Oddi (SO) was induced by a pair of copper electrodes. A glass tube (30 microm tip diameter) connected with a microsyringe was directed to the dorsal vagal complex (DVC) for microinjection. Majority of retrogradely labeled cells was found bilaterally in dorsal motor nucleus of the vagus nerve (DMV) throughout the length, except the rostral and caudal part. These cells were distributed in subnuclei parvicellularis or mediocellularis of DMV. Some labeled perikarya located in the medial subnucleus of the solitary tract (mNTS). Thyrotropin-releasing hormone (TRH, 1.3 mmol/L, 0.2 microl) microinjected into the rostral portion of the DVC (including DMV and NTS) enhanced the motility of gallbladder and SO. Microinjection of TRH at the middle part of DVC seldom induces excitatory effects on the gallbladder or SO. TRH microinjected into the caudal portion of the DVC elicited weaker response of gallbladder and SO than rostral portion. Our results indicated that DMV is one of the most important original nuclei of gallbladder's vagus nerves and mNTS may be also involved in the control of gallbladder's parasympathetic activity. Neurons that innervate the gallbladder distribute at most part of DVC, and are relatively dense at rostral and caudal position of DMV.

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