Laryngeal and respiratory protective reflexes.

Swallowing is a complex motor behavior that relies on an interneuronal network of premotor neurons (PMNs) to organize the sequential activity of motor neurons that are active during the buccopharyngeal and esophageal phases. Swallowing PMNs are highly interconnected to multiple areas of the brain stem and the central nervous system and provide a potential anatomic substrate integration of swallowing activity with airway protective reflexes. Because these neurons have synaptic contact with both afferent inputs and motor neurons and exhibit a true central activity, they appear to constitute the swallowing central pattern generator. We studied the viscerotopic organization of the nucleus of the solitary tract (NTS), the nucleus ambiguus (NA), the dorsal motor nucleus (DMN), and the hypoglossal nucleus (XII) using cholera toxin horseradish peroxidase (CT-HRP), a sensitive antegrade and retrograde tracer that effectively labels afferent terminal fields within the NTS as well as swallowing motor neurons and their dendritic fields within the NA, DMN, and XII. We used CT-HRP to provide a comprehensive description of the dendritic architecture of NA motor neurons innervating swallowing muscles. We also conducted studies using pseudorabies virus (PRV), a swine alpha-herpesvirus, to map central neural circuits after injection in the peripheral or central nervous systems. One attenuated vaccine strain, Bartha PRV, has preferential affinity for sites of afferent synaptic contact on the cell body and dendrites and a reactive gliosis that effectively isolates the infected neurons and provides a barrier to the nonspecific spread to adjacent neurons. The findings provide a basis for the central integration of swallowing and respiratory protective reflexes.

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.

CFTR is functionally active in GnRH-expressing GT1-7 hypothalamic neurons.

We have demonstrated the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, mRNA, and protein within the rat and human brains, in areas regulating sexual differentiation and function. We have found that GT1-7, a gonadotropin-releasing hormone (GnRH)-secreting hypothalamic neuronal cell line, expresses the CFTR gene, mRNA, and protein and cAMP-dependent (36)Cl efflux. A linear 7-pS Cl- conductance, which is stimulated by ATP and cAMP analogs and inhibited by glibenclamide, consistent with CFTR activity, has been identified in GT1-7 cells. Antisense oligo(dN) generated against exon 10 of the CFTR gene transcript (mRNA) inhibit GnRH secretion into media [312 +/- 73, 850 +/- 150, 963 +/- 304, and 912 +/- 74 pg GnRH/4 x 10(6) cells for antisense, sense, missense, and no oligo(dN), respectively; P < 0. 029 for antisense oligo(dN)-treated vs. normal cells]. No changes in intracellular synthesis of GnRH were noted [1,400 +/- 371 and 1,395 +/- 384 pg GnRH/4 x 10(6) cells for antisense and sense oligo(dN), respectively]. Antisense oligo(dN), but not sense or missense oligo(dN), inhibited cAMP-dependent 36Cl efflux. The expression of CFTR protein, detected by Western blotting, was also inhibited 68% by preincubation of cells with antisense oligo(dN). GT1-7 hypothalamic neurons express the CFTR gene, mRNA, and protein, which modulate neurosecretion. Abnormal neuropeptide vesicle trafficking by mutant CFTR may help to explain some of the diverse manifestations of cystic fibrosis.

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.

Cystic fibrosis transmembrane conductance regulator expression in human hypothalamus.

We have previously characterized the expression of the cystic fibrosis transmembrane conductance regulator protein (CFTR) gene, mRNA and protein in rat brain with reverse transcriptase (RT)-PCR amplification, in situ hybridization and immunocytochemistry. We now report that the CFTR mRNA is expressed in the human anterior hypothalamus, an area involved in regulation of appetite, resting energy expenditure and sexual differentiation. Expression of CFTR in neurons localized to this region may elucidate the pathogenesis of other non-pulmonary manifestations of cystic fibrosis which commonly are observed in children with CF, including congenital absence of the vas deferens. Neuron-specific expression of CFTR in brain may be involved in the regulation of homeostatic functions including reproductive function and fertility through effects on neurosecretion, i.e. GnRH release. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain my lead to alteration in physiological function.

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.

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.

Oral inoculation of SCID mice with an attenuated herpes simplex virus-1 strain causes persistent enteric nervous system infection and gastric ulcers without direct mucosal infection.

BACKGROUND: After placement of herpes simplex virus type 1 (HSV-1) into the esophageal lumen of BALB/c mice, the virus replicated in enteric neurons within the esophagus and stomach and was transported to the sensory ganglia of the vagus nerve (nodose ganglia), where viral replication also occurs and where ultimately a long term latent infection is established. This described infection of immunocompetent mice primarily involved neuronal cells and associated satellite cells. EXPERIMENTAL DESIGN: Severe combined immunodeficient (SCID) mice were orally infected with an attenuated strain of HSV-1 to better identify sites of viral involvement in the gastrointestinal tract, particularly the mucosa. RESULTS: Three to five weeks after oral inoculation of SCID mice with HSV-1 strain in1814, a persistent viral infection of the gastrointestinal tract was established in most of the mice. Extensive viral replication was detected by immunohistochemistry throughout pathways of the vagus nerve and within the intrinsic enteric nervous system. Despite this ultimately fatal infection, viral replication in the gut occurred almost exclusively in enteric neurons and their processes; viral proteins were occasionally seen in smooth muscle cells immediately adjacent to heavily infected enteric ganglia. More than 50% of these persistently infected mice, when killed 18 to 31 days postinoculation, had gastric ulcers that were identified grossly and histologically. Only one of the 40 gastric ulcers was found to contain viral Ag. The remaining ulcers, although devoid of viral proteins, were found adjacent to virus-infected ganglia. CONCLUSIONS: HSV-1 can enter enteric neurons with minimal initial mucosal involvement, and once inside the nervous system, the virus is contained there despite the absence of a specific host immune response. Furthermore, chronically infected enteric neurons may provide an indirect mechanism for the pathogenesis of gastric ulcers in these immune-deficient mice.

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.

Transsynaptic localization of pharyngeal premotor neurons in rat.

We determined the anatomy and connectivity of the brainstem circuit controlling the buccopharyngeal phase of swallowing, using pseudorabies virus to identify linked circuits of neurons. Pharyngeal vagal afferents terminate on premotor neurons in the interstitial and intermediate subnuclei of the nucleus of the solitary tract, which in turn, innervate pharyngeal motoneurons in the semicompact subnucleus of the nucleus ambiguus. This circuit is separate and distinct from the esophageal swallowing circuit.

Intravenous sedation in pediatric upper gastrointestinal endoscopy.

To assess the safety and efficacy of intravenous sedation in pediatric upper endoscopy, all elective outpatient procedures performed during a 2-year period (January 1, 1991 through December 31, 1992) were retrospectively reviewed. Of 614 children, 553 received intravenous meperidine and midazolam; 61 received fentanyl and midazolam. The mean dose of meperidine was 1.5 +/- 0.7 mg/kg and of fentanyl 0.0031 +/- 0.0014 mg/kg. Less midazolam was needed for children receiving fentanyl than for those receiving meperidine (0.05 +/- 0.03 mg/kg versus 0.08 +/- 0.05 mg/kg, p < 002). Recovery time (minutes) was shorter for those receiving fentanyl (74.7 +/- 22.8 versus 95.1 +/- 23.0, p < .003). Side effects occurred in 117 patients (19.1%), of which the majority were mild (83%); all were transient with no residual sequelae. Inability to complete the procedure occurred in fewer than 1%. We conclude that both combinations of medication are safe and effective for children of all ages. The use of fentanyl/midazolam results in a shorter recovery time and a lower dose of midazolam.

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.

Localization of nitric oxide synthase in the brain stem neural circuit controlling esophageal peristalsis in rats.

BACKGROUND/AIMS: The central subnucleus of the nucleus of the solitary tract has been implicated in central reflex control of esophageal peristalsis. This study determined the presence of nitric oxide synthase in the brain stem circuit controlling esophageal peristalsis by combining transsynaptic retrograde tract tracing with pseudorabies virus and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH) histochemistry. METHODS: Virus was injected into the esophagus of 10 of 15 rats. After 60-63 hours, brain sections were processed for viral immunofluorescence and NADPH histochemistry. RESULTS: Fluorescent neuronal labeling was limited to the compact formation of the nucleus ambiguus and the central subnucleus of the nucleus of the solitary tract. Most fluorescence-labeled neurons in the central subnucleus stained positively for NADPH (double labeled). In the compact formation, there were almost no double-labeled neurons; however, NADPH-stained terminals surrounded fluorescence-labeled motoneurons. CONCLUSIONS: NO synthase is present in premotor neurons of the central subnucleus of the nucleus of the solitary tract that innervate esophageal motoneurons in the compact formation of the nucleus ambiguus. NADPH staining in both somata and terminals of esophageal premotor neurons suggests that NO is involved in neurotransmission in the central subnucleus and at the site of synaptic contact between esophageal premotor neurons and motoneurons in the compact formation of the nucleus ambiguus.

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.

Brain stem localization of rodent esophageal premotor neurons revealed by transneuronal passage of pseudorabies virus.

BACKGROUND/AIMS: Brain stem premotor neurons control swallowing through contacts with both afferent neurons and motoneurons. The location and connectivity of premotor neurons innervating the esophagus was determined using pseudorabies virus. METHODS: In 30 rats, viral injections were made into either the cervical or subdiaphragmatic esophagus, cricothyroid muscle, or stomach. After a 48-62-hour survival, brain sections were processed immunocytochemically for the virus. RESULTS: Neuronal labeling was limited to the compact formation of the nucleus ambiguus for survivals of 48-54 hours. At 57-62-hour survivals, virus-labeled second-order neurons (premotor neurons) were localized to the central subnucleus of nucleus of the solitary tract. Injections in the cricothyroid muscle and stomach resulted in distinct patterns of motoneuronal labeling in the nucleus ambiguus and dorsal motor nucleus and premotor neuronal labeling in the nucleus of the solitary tract. CONCLUSIONS: Virus-labeled premotor neurons in the nucleus of the solitary tract occurred as a result of retrograde transport of the virus from the nucleus ambiguus because no viral antigen was present in the tractus solitarius. The esophagus is controlled by a central circuit whereby esophageal vagal afferents terminate on premotor neurons in the central subnucleus that in turn innervate esophageal motoneurons in the nucleus ambiguus.