Central integration of swallow and airway-protective reflexes.

The relationship between the timing of respiration and swallowing has been proven not to be random. Using pseudorabies virus (PRV) as a transsynaptic neural tracer, a basis for the central integration of swallowing and airway-protective reflexes can be located in the neural circuits projecting to swallowing-related muscles. The premotor neurons (PMNs) that constitute the swallowing central pattern generators, interneuronal networks able to initiate repetitive rhythmic muscle activity independent of sensory feedback, connect with multiple areas of the brainstem and other areas of the central nervous system. Those PMNs that project to muscles used in swallowing have been localized within the nucleus of the solitary tract (NTS) and its adjacent reticular formation, and they are synaptically linked both to peripheral afferents and to cortical swallowing areas. Bartha PRV, an attenuated vaccine strain of swine alpha-herpesvirus with a long postinjection survival rate and the ability to produce controlled infections that spread in a hierarchical manner within synaptically linked neurons, can specifically label neurons projecting to PMNs of a given circuit. Thus, it has been used to isolate two neuroanatomically distinct subnetworks of PMNs involved in the buccopharyngeal and esophageal phases of swallowing. Use of PRV as a neural tracer shows that during the buccopharyngeal phase of swallowing, vagal afferents from the pharynx and larynx and from the superior laryngeal nerve terminate in the intermediate and interstitial subnuclei of the NTS. Motoneurons projecting to the pharynx and larynx are located in the semicompact and loose formations of the nucleus ambiguus (NA). Neural tracing with PRV also shows that esophageal PMNs have direct synaptic contact with esophageal motoneurons in the compact formation of the NA. Moreover, esophageal PMNs are localized exclusively to the central subnucleus of the NTS, a site that also is the sole point of termination of esophageal vagal afferents. Using PRV, one can identify third-order (neurons projecting to PMNs) esophageal neurons in sites where pharyngeal PMNs have been noted. Injection of PRV into the esophagus and subsequent detection using immunofluorescence found a subpopulation of neurons in the intermediate and interstitial subnuclei of the NTS. This subpopulation projects to pharyngeal motoneurons and buccopharyngeal PMNs, and it is synaptically linked to esophageal PMNs. The synaptic link between buccopharyngeal and esophageal PMNs provides a potential anatomic substrate within the NTS for the central integration of esophageal peristalsis with the pharyngeal phase of swallowing and airway-protective reflexes. Human studies and animal models investigating esophagoglottal closure and pharyngo-upper esophageal sphincter (pharyngo-UES) contractile reflexes have located the neural pathways that mediate airway-protective reflexes. Similar studies and models using two PRV strains injected simultaneously into different swallowing and respiration-related muscle groups may identify synaptic connectivity between laryngeal, esophageal, and pharyngeal PMNs and, thus, may help to demonstrate the central integration of swallowing and airway-protective reflexes.

Brainstem viscerotopic organization of afferents and efferents involved in the control of swallowing.

Cholera toxin horseradish peroxidase (CT-HRP), a sensitive antegrade and retrograde tracer, is effective at labeling swallowing motoneurons and their dendritic fields within the nucleus ambiguus (NA), nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus nerve, and hypoglossal nucleus. Using this tracer to label motoneurons within the NTS demonstrates that palatal, pharyngeal, and laryngeal afferents overlap considerably within the interstitial and intermediate subnuclei. These afferents have a pattern of distribution within the NTS similar to the labeling observed after application of the same tracer to the superior laryngeal nerve. Esophageal afferents, however, terminate entirely within the central (NTScen) subnucleus and do not overlap their distribution with palatal, pharyngeal, or laryngeal afferents. Within the nodose ganglion (NG), sensory neurons projecting to the soft palate and pharynx are located superiorly, and those projecting to the esophagus and stomach are located inferiorly, an organization that indicates rostrocaudal positioning along the alimentary tract. Sensory neurons within the NG and NTS contain, among others, the major excitatory and inhibitory amino acid neurotransmitters glutamate (Glu) and gamma-aminobutyric-acid (GABA). Both Glu and GABA help to coordinate esophageal peristalsis. Using pseudorabies virus as a transsynaptic tracer demonstrates the role of GABA and Glu as mediators of synaptic transmission within the swallowing central pattern generator, a fact further supported by the presence of specific receptors for each neurotransmitter within the NTScen. Anatomic studies using CT-HRP have been effective in revealing the total extent of extranuclear dendritic projections and the organization of dendrites within the confines of a nucleus; further studies have produced the following data. Motoneurons innervating the soft palate, pharynx, larynx, and cervical esophagus have extensive dendrites that extend into the adjacent reticular formation with a distinct pattern for each muscle group. Motoneurons of the musculature active during the buccopharyngeal phase of swallowing (soft palate, pharynx, cricothyroid, and cervical esophagus) have extensive dendritic arborizations that terminate within the adjacent reticular formation of the NA. Swallowing premotor neurons located in the reticular formation surrounding the NA are active during the buccopharyngeal phase of swallowing. These data provide an anatomic basis for interaction of swallowing motoneurons with premotor neurons located in this area. Motoneurons innervating all levels of the esophagus are confined to the compact formation (NAc), whereas those motoneurons projecting to the pharynx and cricothyroid muscle are located in the semicompact formation (NAsc). The intrinsic laryngeal muscles were represented within the loose formation (NAI) and the heart within the external formation. In contrast, the dendrites of motoneurons projecting to the thoracic and subdiaphragmatic esophagus are confined to the NAc. Both the NAsc and NAc have extensive longitudinal bundling of dendrites within the confines of the nucleus, resulting in the formation of a rostrocaudal dendritic plexus where dendrites crisscross between bundles. Intranuclear bundling of dendrites is evident in the soft palate, pharynx, and esophagus and is lacking only for the cricothyroid muscle. Moreover, ventrolateral- and dorsomedial-oriented dendritic bundles are present within the NAsc. In contrast to the longitudinal dendritic bundles, the ventrolateral- and dorsomedial-oriented dendritic bundles exit the NAsc and penetrate the adjacent reticular formation. The extensive bundling of motoneuronal dendrites within the NA supports the hypothesis that these structures serve as networks for the generation of complex motor activities, such as swallowing.

Action of substance P and interaction of calcitonin gene-related peptide and substance P on the cat antral circular muscle.

The actions of substance P (SP) and calcitonin gene-related peptide (CGRP) and their interaction were examined in vitro in the feline antrum and colon. Circular muscle contraction was seen in the antrum to both peptides, but only to SP in the proximal colon. Antral contraction was enhanced when both peptides were given together. This interaction was inhibited by tetrodotoxin or atropine. SP acted at the antrum via a smooth muscle neurokinin receptor which is not a (NK)-1 receptor. SP binding was displaced by neurokinin A but not by the NK-1 receptor antagonist, CP-96345. The colonic response was inhibited by CP-96345. Immunohistochemistry revealed SP-like immunoreactivity (SP-LI) in fibers in the antral myenteric plexus and circular muscle, while CGRP-like immunoreactivity (CGRP-LI) was seen in the myenteric plexus only, without co-localization. These studies supported the hypothesis that SP acted via the NK-2 receptor at the feline circular muscle in the antrum to induce contraction and at the NK-1 receptor in the proximal colon. CGRP enhanced the effect of SP via a cholinergic pathway.

Somatostatin immunoreactivity in esophageal premotor neurons of the rat.

Esophageal peristalsis is coordinated by premotor neurons localized to the central subnucleus of the nucleus of the solitary tract (NTScen). These premotor neurons project directly to motoneurons within the compact formation of the nucleus ambiguus (NAc). Somatostatin immunoreactive terminals have been previously demonstrated encircling motoneurons in the (NAc) (Cunningham, E.T., Jr. and Sawchenko, P.E., J. Neurosci., 9 (1989) 1668-1682). We combined transsynaptic tracing with pseudorabies virus and immunohistochemistry to localize somatostatin to premotor neurons within the NTScen.

Solitarial premotor neuron projections to the rat esophagus and pharynx: implications for control of swallowing.

BACKGROUND & AIMS: The buccopharyngeal and esophageal phases of swallowing are controlled by distinct networks of premotor neurons localized in the nucleus tractus solitarius. The neuronal circuitry coordinating the two phases was investigated using a combination of central and peripheral tracing techniques. METHODS: Using pseudorabies virus, a transsynaptic tracer, in anesthetized rats, third-order esophageal neurons (neurons projecting to premotor neurons) were identified. In a separate protocol that combined transsynaptic and retrograde fluorescent tracing, third-order esophageal neurons projecting to pharyngeal motoneurons (buccopharyngeal premotor neurons) were then identified. RESULTS: Third-order esophageal neurons were identified in the interstitial and intermediate subnuclei of the nucleus tractus solitarius and in other medullary, pontine, midbrain, and forebrain nuclei. A subpopulation of these neurons (double labeled) in the interstitial and intermediate subnuclei were found to project to pharyngeal motoneurons (buccopharyngeal premotor neurons) and to be linked synaptically to esophageal premotor neurons. CONCLUSIONS: The synaptic link between buccopharyngeal and esophageal premotor neurons provides an anatomic pathway for the central initiation of esophageal peristalsis and its coordination with the pharyngeal phase of swallowing. This neural circuitry within the nucleus tractus solitarius is consistent with a complex central control mechanism for the swallowing motor sequence that can function independently of afferent feedback.

Colocalization of GABA(A) and NMDA receptors within the dorsal motor nucleus of the vagus nerve (DMV) of the rat.

Changes in gastric motor activity are observed in response to glutamate and GABA in the DMV. We investigated the expression of GABA(A) and NMDA receptors within DMV neurons projecting to the stomach using pseudorabies virus (PRV). PRV immunoreactive (PRV-IR) cells expressing GABA(A) alpha1-IR, also expressed NMDAR1 suggesting that NMDA and GABA(A) receptors are colocalized. These results provide a neuroanatomical basis for these receptors jointly playing a role in gastric motor functions.

An uncommon presentation of Salmonella.

Salmonella infection can cause appendicitis by direct invasion of the appendix, or can mimic appendicitis by causing mild inflammation of the appendix, ileum, or lymph nodes. Clinical presentation and radiologic and laboratory evaluation may not distinguish the extent of underlying pathology. This case of a child with an atypical presentation of Salmonella who underwent diagnostic laparotomy illustrates the overlap of enteric infections and acute appendicitis. A literature review confirms the variety of clinical scenarios of patients with suspected appendicitis and Salmonella-positive cultures. We conclude that enteric infection should be considered in children with atypical presentations of appendicitis, and that the knowledge that Salmonella can progress to appendicitis should guide management if signs and symptoms of appendicitis develop.

Localization of GABAA alpha 1 mRNA subunit in the brainstem nuclei controlling esophageal peristalsis.

The nucleus of the solitary tract, the site of esophageal premotor neurons (PMN), is tonically inhibited by GABAergic neurons via the GABAA receptor. We investigated the expression of GABAA alpha 1 subunit mRNA within esophageal PMNs of the NTS utilizing transynaptic tracing with pseudorabies virus and nonisotopic in-situ hybridization. Double-labeling studies revealed that the majority of PRV-immunoreactive cells also expressed GABAA alpha 1 mRNA. The expression of GABAA subunits supports a role for GABA in the brainstem circuit controlling esophageal peristalsis.

Identification of the GABAA receptor alpha-subunit mRNA in rat intestine.

We investigated the expression of gamma-aminobutyric acid type A (GABAA) receptor alpha 1-, alpha 2- and alpha 3-subunit mRNAs in the rat intestine using reverse transcription and polymerase chain reaction. alpha 1- and alpha 3-, but not alpha 2-, subunit mRNAs were amplified from the proximal intestine, ileum, and colon. In-situ hybridization studies demonstrated the expression of alpha 1-subunit mRNA by myenteric neurons. GABA may be active via the GABAA receptor in the enteric nervous system.

Co-localization of NOS and NMDA receptor in esophageal premotor neurons of the rat.

Nitric oxide (NO) production following NMDA receptor stimulation plays a role in signaling between neurons. Using trans-synaptic tracing with pseudorabies virus (PRV), immunocytochemistry and histochemistry, we have demonstrated the expression of NMDAR1 and nitric oxide synthase (NOS) within brain stem neurons controlling esophageal peristalsis. PRV-immunoreactive second order esophageal premotor neurons of the central subnucleus of the nucleus of the solitary (NTScen) expressed NMDAR1 and NOS. First order motoneurons of the compact formation of the nucleus ambiguus (NAc) expressed NMDAR1, but did not contain NOS. NTScen neurons may synthesize and release NO in response to NMDA activation, suggesting a role for NO in the coordination of esophageal motility.

Expression and localization of the cystic fibrosis transmembrane conductance regulator mRNA and its protein in rat brain.

In previous studies we have characterized the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in clathrin-coated vesicles derived from bovine brain and in neurons of rat brain. In this study we have further characterized the expression of the CFTR protein mRNA and protein in rat brain with reverse transcriptase polymerase chain reaction amplification (RT-PCR), in situ hybridization, and immunocytochemistry. The expression of CFTR mRNA and protein in discrete areas of brain, including the hypothalamus, thalamus, and amygdaloid nuclei, which are involved in regulation of appetite and resting energy expenditure, is identical. The presence of CFTR in neurons localized to these regions of brain controlling homeostasis and energy expenditure may elucidate the pathogenesis of other nonpulmonary and gastrointestinal manifestations which commonly are observed in children with cystic fibrosis. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain may serve as a pathogenic mechanism for disruption of homeostasis.

Gastrointestinal motility in the neonate.

Although patterns of gastrointestinal movements have been recognized for nearly a century, only in the past 10 years have we had the diagnostic tools to characterize both normal and abnormal motility in the neonate. Parallel with these advances, over the past 25 years, we have seen an explosion in our understanding of the ENS and its independent regulation of gut motility. It is expected that, as we come to a better understanding of the microregulation of intestinal motor activity, we can develop more effective means to treat many of these heretofore refractory disorders of gastrointestinal motility.

NMDAR1 mRNA expression in the brainstem circuit controlling esophageal peristalsis.

We investigated the expression of NMDA receptors within the brainstem circuit controlling esophageal swallowing using transneuronal viral labeling and in situ hybridization. Neurons of the central subnucleus of the nucleus solitary tract (NTScen) are interneurons linking vagal afferents with esophageal motoneurons in the compact formation of the nucleus ambiguus (NAc). Following injections of Pseudorabies virus (PRV) into rat esophagus and incubation with NMDAR1 cRNA, neurons infected with PRV localized to the NAc and NTScen expressed NMDAR1 mRNA.

The expression of a NMDA receptor gene in guinea-pig myenteric plexus.

Muscle tension studies of guinea-pig ileum longitudinal muscle-myenteric plexus preparations suggest that the N-methyl-D-aspartate (NMDA) receptor may be present in the enteric nervous system. Therefore, we investigated the expression of a gene for the NMDA receptor in guinea-pig taenia coli. The gene product was amplified using polymerase chain reaction (PCR) and its synthesis localized using in situ hybridization. A NMDA receptor PCR product from the myenteric plexus was demonstrated with nearly identical sequence characteristics to that in the brain. In situ hybridization studies identified myenteric neurons which express NMDA receptor messenger RNA. Demonstration of the genetic expression of the NMDA receptor supports a role for glutamate as a neurotransmitter in the enteric nervous system.

Electrophysiologic and molecular properties of cultured enteric glia.

Enteric glia, the support cells of myenteric ganglia, have been widely studied with respect to their morphology and immunohistochemical phenotype, but little is known about their functional properties. We developed a method for the amplification of enteric glia from newborn guinea pigs to further characterize these cells. Treatment with a combination of basic fibroblast growth factor and the adenylate cyclase activator, cholera toxin, permitted expansion of enteric glial cultures to confluence and serial passage for up to 8 months. The long-term cultured cells retained expression of 1) S100 protein, 2) GD3 ganglioside recognized by the monoclonal antibody LB1, and 3) the gene encoding glutamine synthetase. The electrophysiologic properties of cultured enteric glia were studied under whole-cell patch clamp conditions. Most cells expressed "delayed rectifier"-type potassium currents, and some also demonstrated tetrodotoxin-sensitive sodium currents. Other subsets of voltage-dependent potassium currents, calcium currents, and glutamate-gated currents were not demonstrable.

Japanese-style management: lessons for the American medical group administrator.

The Japanese have been very successful in developing and implementing a management style that has made Japan one of the most productive and affluent societies in the world today. It is a style of management based on a long-term investment in people and characterized by management techniques that emphasize simplicity, flexibility, and human values in the corporation. These techniques, when adapted to American medical group practices, can provide valuable lessons in management for the American medical group administrator.