Brainstem sites controlling the lower esophageal sphincter and crural diaphragm in the ferret: A neuroanatomical study Mark Niedringhaus a , Patrick G. Jackson b , Rebecca Pearson a , Min Shi c , Kenneth Dretchen a , Richard A. Gillis a , Niaz Sahibzada a, ⁎ a Departments of Pharmacology, Georgetown University Medical Center, Washington, DC, United States b Departments of Surgery, Georgetown University Medical Center, Washington, DC, United States c Departments of Medicine, Georgetown University Medical Center, Washington, DC, United States abstract article info Article history: Received 16 April 2008 Received in revised form 24 September 2008 Accepted 29 September 2008 Keywords: CD DMV LES Brainstem The lower esophageal sphincter (LES) and the crural diaphragm (CD) surrounding the esophagogastric junction are key components of the gastroesophageal reflex mechanism, which engages the vago-vagal brainstem circuitry. Although both components work in conjunction to prevent gastroesophageal reflux, little is known about the brain area(s) where this integration takes place. The aims of this study were to: (1) trace the brainstem circuitry associated with the CD and the LES, and (2) determine possible sites of convergence. Experiments were done in adult male ferrets. Under isoflurane anesthesia, recombinant strains of the transneuronal pseudorabies virus (PRV-151 or PRV-Bablu) or the monosynaptic retrograde tracer cholera toxin beta-subunit (CTb) were injected into either the CD or the LES. Following a survival period of 5–7 days, animals were euthanized, perfused and their brains removed for dual-labeling immunofluorescence processing. In animals injected with recombinants of PRV into the CD and the LES, distinct labeling was found in various brainstem nuclei including: area postrema, DMV, nucleus tractus solitarius (NTS), medial reticular formation (MRF) and nucleus ambiguous (NA). Double-labeled cells were only evident in the DMV, NTS and MRF. Injections of CTb into the CD or the LES resulted in retrograde labeling only in the DMV. These findings demonstrate the presence of a direct projection from the DMV to the CD. They further suggest that the neuronal connections responsible for CD or LES function are contained in circuitries that, though largely independent, may converge at the level of DMV, NTS and MRF. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The esophagogastric junction contains an area of high-pressure, which serves as an effective barrier against the retropulsion of gastric contents into the esophagus. This high pressure zone is maintained in humans and some animals by two superimposed muscles: the internal smooth muscle of the lower esophagus and the external skeletal muscle of the crural diaphragm (CD) (Mittal and Balaban, 1997). Both muscles act as sphincters and together are largely responsible for creating the high pressure zone. Intermittently, these sphincters undergo concurrent transient relaxations that result in a breach of the high pressure zone. These transient lower esophageal sphincter relaxations (tLESRs) have been proposed as the main mechanism associated with gastroesophageal reflux disease (GERD) (Dent et al., 1980; Dodds et al., 1982; Mittal et al., 1995; Iwakiri et al., 2005). During a tLESR, the CD and the intrinsic muscle of the lower esophagus simultaneously relax (Mittal and Balaban, 1997; Pandolfino et al., 2002). Concomitant relaxation of these sphincters also occur in response to conditions such as swallowing, eructation (Monges et al., 1978), esophageal stretch and gastric distension (Monges et al., 1978; Altschuler et al., 1985; Oyer et al., 1989; Martin et al., 1992; Liu et al., 2000). These simultaneous sphincter relaxations are thought to involve CNS hindbrain circuitry (Mittal and Balaban, 1997). However, in the hindbrain, only the origin of CNS control of the LES can be delineated with any certainty. LES relaxation has been shown to be vagally mediated by neurons in the dorsal motor nucleus of the vagus (DMV) (Barone et al., 1984; Rossiter et al., 1990; Abrahams et al., 2002; Niedringhaus et al., 2008). Furthermore, this relaxation is site-specific as it is elicited only from the intermediate (Abrahams et al., 2002; Niedringhaus et al., 2008) and the caudal areas of the DMV (Rossiter et al., 1990; Abrahams et al., 2002; Niedringhaus et al., 2008). Activation of the rostral area of the DMV leads to contraction of the LES (Rossiter et al., 1990; Abrahams et al., 2002; Niedringhaus et al., 2008). All these changes in LES pressure are prevented by either ipsilateral (Rossiter et al., 1990; Niedringhaus et al., 2008) or bilateral vagotomy (Abrahams et al., Autonomic Neuroscience: Basic and Clinical 144 (2008) 50–60 ⁎ Corresponding author. Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Rd., NW, Washington, DC 20007, United States. E-mail address: sahibzan@georgetown.edu (N. Sahibzada). 1566-0702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2008.09.007 Contents lists available at ScienceDirect Autonomic Neuroscience: Basic and Clinical journal homepage: www.elsevier.com/locate/autneu