Steven M. Altschuler

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.

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 36Cl 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 × 106 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 × 106 cells for antisense and sense oligo(dN), respectively]. Antisense oligo(dN), but not sense or missense oligo(dN), inhibited cAMP-dependent36Cl 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.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.

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.

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.

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.

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.

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.

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.