Differential age-, gender-, and side-dependency of vagus, spinal accessory, and phrenic nerve calibers detected with precise ultrasonography measures.

INTRODUCTION: The standard ultrasonographic measurement tools (trace, ellipse) of cross-sectional areas (CSAs) of very small nerves typically yield rough measures in full square millimeters. METHODS: In 70 volunteers, the elliptically shaped CSAs of mid-cervical vagus, accessory, and phrenic nerves were estimated with three methods: 2 on-board tools (area tracing, ellipse fitting) and an off-line calculation of the CSA after on-board measuring of its long-axis and short-axis diameters both displayed with 1-2 digits following the decimal point. RESULTS: CSA measures of all mid-cervical nerves obtained with the precise approach were smaller than the two standard measures (each P < 0.001). Larger CSA of right compared to left vagus nerve was detected with all methods. However, decrease of accessory and phrenic nerve CSAs with increasing age and larger size of vagus nerve CSA in women vs. men were evident only with precise measures. DISCUSSION: Small nerve CSA should preferably be estimated with precise measures. Muscle Nerve 59:486-491, 2019.

Evidence of vagus nerve sprouting to innervate the urinary bladder and clitoris in a canine model of lower motoneuron lesioned bladder.

AIMS: Complete spinal cord injury does not block perceptual responses or inferior solitary nucleus activation after genital self-stimulation, even though the vagus is not thought to innervate pelvic structures. We tested if vagus nerve endings sprout after bladder decentralization to innervate genitourinary structures in canines with decentralized bladders. METHODS: Four reinnervation surgeries were performed in female hounds: bilateral genitofemoral nerve transfer to pelvic nerve with vesicostomy (GNF-V) or without (GFN-NV); and left femoral nerve transfer (FNT-V and FNT-NV). After 8 months, retrograde dyes were injected into genitourinary structures. Three weeks later, at euthanasia, reinnervation was evaluated as increased detrusor pressure induced by functional electrical stimulation (FES). Controls included un-operated, sham-operated, and decentralized animals. RESULTS: Increased detrusor pressure was seen in 8/12 GFNT-V, 4/5 GFNT-NV, 5/5 FNT-V, and 4/5 FNT-NV animals after FES, but not decentralized controls. Lumbar cord segments contained cells labeled from the bladder in all nerve transfer animals with FES-induced increased detrusor pressure. Nodose ganglia cells labeled from the bladder were observed in 5/7 nerve transfer animals (1/2 GNT-NV; 4/5 FNT-V), and from the clitoris were in 6/7 nerve transfer animals (2/2 GFNT-NV; 4/5 FNT-V). Dorsal motor nucleus vagus cells labeled from the bladder were observed in 3/5 nerve transfer animals (1/2 GFNT-NV; 2/3 FNT-V), and from the clitoris in 4/5 nerve transfer animals (1/2 GFNT-NV; 3/3 FNT-V). Controls lacked this labeling. CONCLUSIONS: Evidence of vagal nerve sprouting to the bladder and clitoris was observed in canines with lower motoneuron lesioned bladders. Neurourol. Urodynam. 36:91-97, 2017. © 2015 Wiley Periodicals, Inc.

Developmental regulation of inhibitory synaptic currents in the dorsal motor nucleus of the vagus in the rat.

Prior immunohistochemical studies have demonstrated that at early postnatal time points, central vagal neurons receive both glycinergic and GABAergic inhibitory inputs. Functional studies have demonstrated, however, that adult vagal efferent motoneurons receive only inhibitory GABAergic synaptic inputs, suggesting loss of glycinergic inhibitory neurotransmission during postnatal development. The purpose of the present study was to test the hypothesis that the loss of glycinergic inhibitory synapses occurs in the immediate postnatal period. Whole cell patch-clamp recordings were made from dorsal motor nucleus of the vagus (DMV) neurons from postnatal days 1-30, and the effects of the GABAA receptor antagonist bicuculline (1-10 µM) and the glycine receptor antagonist strychnine (1 µM) on miniature inhibitory postsynaptic current (mIPSC) properties were examined. While the baseline frequency of mIPSCs was not altered by maturation, perfusion with bicuculline either abolished mIPSCs altogether or decreased mIPSC frequency and decay constant in the majority of neurons at all time points. In contrast, while strychnine had no effect on mIPSC frequency, its actions to increase current decay time declined during postnatal maturation. These data suggest that in early postnatal development, DMV neurons receive both GABAergic and glycinergic synaptic inputs. Glycinergic neurotransmission appears to decline by the second postnatal week, and adult neurons receive principally GABAergic inhibitory inputs. Disruption of this developmental switch from GABA-glycine to purely GABAergic transmission in response to early life events may, therefore, lead to adverse consequences in vagal efferent control of visceral functions.

[Age-related changes in sensory neurons containing calcitonin gene-related peptide under conditions of afferentation deficit in rats].

Morphological features of calcitonin gene-related peptide (CGRP)-immunoreactive neurons were studied in the sensory ganglia of the vagus and thoracic nerves in 3-, 10-, 20-, 30-, 60-, 90-, and 120-day-old rats under conditions of chemically-induced deafferentation. We found that, in rats, CGRP-containing neurons appeared in both ganglia immediately after they were born and their number decreased with aging. Most of CGRP-immunoreactive neurons were small in size, i.e., up to 600 microm2. Administration of capsaicin modified age-related changes in the number of CGRP-immunopositive neurons. In the thoracic nerve ganglion, the mean square of these cells and their number substantially decreased, whereas, in the vagus nerve ganglion, positive cells were not observed.

Developmental changes in brainstem neurons regulating lower airway caliber.

Premature infants are at risk for lower airway obstruction; however, maturation of reflex pathways regulating lower airway patency is inadequately studied. We hypothesized that postnatal maturation causes developmental change in brainstem efferent airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) that project to the airways and in pulmonary afferent fibers that terminate in the nucleus tractus solitarius (NTS). Ferrets aged 7, 14, 21, and 42 d received intrapulmonary injection of cholera toxin (CT)-beta subunit, a transganglionic retrograde tracer. Five days later, their brainstem was processed for dual immunolabeling of CT-beta and the cholinergic marker, choline acetyl transferase. CT-beta-labeled AVPNs and CT-beta-labeled afferent fiber optical density (OD) were analyzed. There was a significantly higher CT-beta-labeled cell number within the rNA at the youngest compared with older ages. All efferent CT-beta-labeled cells expressed choline acetyl transferase. OD of CT-beta-labeled afferent fibers was also higher at 7 d compared with 14 d. We conclude that the number of efferent AVPNs and afferent fiber OD both diminish over the second postnatal week. We speculate that exposure to injurious agents in early postnatal life may inhibit natural remodeling and thereby enhance later vulnerability to airway hyperreactivity.

Fetal growth restriction: From Polyvagal theory to developmental impairments?

BACKGROUND: The Polyvagal theory argues that behavioral modulation is a fundamental neurodevelopmental process that depends on autonomic regulation. OBJECTIVE: The present study aimed to assess sleep architecture in newborns with fetal growth restriction (FGR) using polysomnography as an indicator of Polyvagal theory. METHODS: We studied polysomnography recordings from 68 preterm infants, 34 with FGR and 34 born with appropriate growth for gestational age (AGA), who were matched according to the corrected age for prematurity (CA). Total sleep time, arousals, the percentage of quiet sleep, active sleep, indeterminate sleep, and heart rate were compared between the groups. Linear multiple regression analyses were used to evaluate polysomnography data for the FGR and AGA groups. RESULTS: Average heart rate was significantly lower in most FGR groups compared with AGA groups, and small to large effect sizes were observed in several sleep responses when comparing these groups. In the lineal regression model the CA explains significantly the differences in heart rate, controlled by FGR (p = .012). Additionally, there was evidence that sleeping states show similar trends, that is, increases in quiet and indeterminate sleep, as well as decreases in active sleep when CA was controlled by FGR. CONCLUSION: FGR probably intensifies the unfavorable effect of preterm birth in the responses evaluated by polysomnography. It seems that FGR is associated with alteration in sleep regulation and with differences in heart rate modulation, which may serve as a strategy to preserve energy and such differences likely underlie neurodevelopmental impairments in affected newborns.

Factors regulating vagal sensory development: potential role in obesities of developmental origin.

Contributors to increased obesity in children may include perinatal under- or overnutrition. Humans and rodents raised under these conditions develop obesity, which like obesities of other etiologies has been associated with increased meal size. Since vagal sensory innervation of the gastrointestinal (GI) tract transmits satiation signals that regulate meal size, one mechanism through which abnormal perinatal nutrition could increase meal size is by altering vagal development, possibly by causing changes in the expression of factors that control it. Therefore, we have begun to characterize development of vagal innervation of the GI tract and the expression patterns and functions of the genes involved in this process. Important events in development of mouse vagal GI innervation occurred between midgestation and the second postnatal week, suggesting they could be vulnerable to effects of abnormal nutrition pre- or postnatally. One gene investigated was brain- derived neurotrophic factor (BDNF), which regulates survival of a subpopulation of vagal sensory neurons. BDNF was expressed in some developing stomach wall tissues innervated by vagal afferents. At birth, mice deficient in BDNF exhibited a 50% reduction of putative intraganglionic laminar ending mechanoreceptor precursors, and a 50% increase in axons that had exited fiber bundles. Additionally, BDNF was required for patterning of individual axons and fiber bundles in the antrum and differentiation of intramuscular array mechanoreceptors in the forestomach. It will be important to determine whether abnormal perinatal environments alter development of vagal sensory innervation of the GI tract, involving effects on expression of BDNF, or other factors regulating vagal development.

Development of the human dorsal nucleus of the vagus.

The dorsal nucleus of the vagus nerve plays an integral part in the control of visceral function. The aim of the present study was to correlate structural and chemical changes in the developing nucleus with available data concerning functional maturation of human viscera and reflexes. The fetal development (ages 9 to 26 weeks) of the human dorsal nucleus of the vagus nerve has been examined with the aid of Nissl staining and immunocytochemistry for calbindin and tyrosine hydroxylase. By 13 weeks, the dorsal vagal nucleus emerges as a distinct structure with at least two subnuclei visible in Nissl stained preparations. By 15 weeks, three subnuclei (dorsal intermediate, centrointermediate and ventrointermediate) were clearly discernible at the open medulla level with caudal and caudointermediate subnuclei visible at the level of the area postrema. All subnuclei known to exist in the adult were visible by 21 weeks and cytoarchitectonic differentiation of the nucleus was largely completed by 25 weeks. The adult distribution pattern of calbindin and tyrosine hydroxylase immunoreactive neurons was also largely completed by 21 weeks, although morphological differentiation of labeled neurons continued until the last age examined (26 weeks). The structural development of the dorsal nucleus of the vagus nerve appears to occur in parallel with functional maturation of the cardiovascular and gastric movements, which the nucleus controls.

Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract.

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations or pharmacological manipulations. Therefore, a method using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development.

Gastric preloads of corn oil and mineral oil produce different patterns of increases of c-Fos-like immunoreacitve cells in the brain of 9-12 day-old rats.

Equivolumetric gastric preloads of corn oil and mineral oil administered to rats on postnatal day 12 (P12) inhibited intake equally during a 30-min test of independent ingestion (II), but preloads of corn oil inhibited intake significantly more than preloads of mineral oil on P15 and P18 [Weller, A., Gispan, I.H., Armony-Sivan, R., Ritter, R.C., Smith, G.P., 1997. Preloads of corn oil inhibit independent ingestion on postnatal day 15 in rats. Physiol. Behav. 62, 871-874]. It is possible that the equivalent inhibition of intake by the oil preloads on P12 resulted from the failure of the preabsorptive sensory properties of the preloads to be discriminated by peripheral or central sensory mechanisms. To investigate this possibility, we administered equivolumetric gastric preloads of 25% corn oil and 25% mineral oil to pups on P9-12 and counted the number of c-Fos-like immunoreactive (CFLI) cells in central sites that are activated by food intake and postingestive preabsortive mechanisms in adult rats and in pups on P10-11. The major result was that preloads of 25% corn oil and 25% mineral oil that produced equivalent inhibition of II intake produced differential increases of CFLI cells in the forebrain and hindbrain. Specifically, preloads of corn oil increased the number of CFLI cells in the caudal Nucleus Tractus Solitarius significantly more than preloads of mineral oil. Furthermore, preloads of corn oil increased the number of CFLI cells in the Paraventricular and Supraoptic nuclei, but preloads of mineral oil did not. This differential pattern of increases of CFLI cells is evidence that the brain discriminates the preabsorptive sensory properties of preloads of corn oil and mineral oil on P9-12.

There is no relation between the fascicular and the endoneural blood vessel areas on the cervical vagus nerve of normotensive rats.

The aim of this study was to determine the existence of a relation between the fascicular area and total vessel area on the cervical vagus nerve of Wistar-Kyoto female rats. Animals aged 30, 180 or 360 days had their right vagus nerves prepared for epoxy resin embedding and light microscopy study. Semithin serial sections were studied every 100 microm of the longitudinal extension of the nerve. The fascicular area and the total endoneural vessel area were obtained for each nerve segment (proximal, middle and distal) and compared between segments and ages. The relation between fascicular area and endoneural vessel area was accessed by linear regression analysis and correlation coefficient calculations. No significant relationship between the two variables was detected in all nerves studied. This suggests that the entrance of blood vessels to the endoneural space is not accompanied by an increase in the fascicular area and this may have implications on the fiber density calculations since, in the presence of large vessels, this density can be underestimated if the vessel area is not discounted. The endoneural vessel area increased from proximal to distal in all groups but got smaller with ageing, suggesting that older animals may be more susceptible to nerve ischemia.

Comparative postnatal development of spinal, trigeminal and vagal sensory root entry zones.

Somatic and visceral sensory information enters the central nervous system (CNS) via root entry zones where sensory axons span an environment consisting of Schwann cells in the peripheral nervous system (PNS) and astrocytes and oligodendrocytes in the CNS. While the embryonic extension of these sensory axons into the CNS has been well-characterized, little is known about the subsequent, largely postnatal development of the glial elements of the root entry zones. Here we sought to establish a comparative developmental timecourse of the glial elements in the postnatal (P0, P3, P7, P14) and adult rat of three root entry zones: the spinal nerve dorsal root entry zone, the trigeminal root entry zone, and the vagal dorsal root entry zone. We compared entry zone development based on the expression of antigens known to be expressed in astrocytes, oligodendrocytes, oligodendrocyte precursor cells, Schwann cells, radial glial fibres and the PNS extracellular matrix. These studies revealed an unexpected distribution among glial cells of several antigens. In particular, antibodies used to label mature oligodendrocytes (RIP) transiently labelled immature Schwann cell cytoplasm, and a radial glial antigen (recognized by the 3CB2 antibody) initially decreased, and then increased in postnatal astrocytes. While all three root entry zones had reached morphological and antigenic maturity by P14, the glial elements comprising the PNS-CNS interface of cranial root entry zones (the trigeminal root entry zone and the vagal dorsal root entry zone) matured earlier than those of the spinal nerve dorsal root entry zone.

Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer.

We generated germ line-transmitting transgenic zebrafish that express green fluorescent protein (GFP) in the cranial motor neurons. This was accomplished by fusing GFP sequences to Islet-1 promoter/enhancer sequences that were sufficient for neural-specific expression. The expression of GFP by the motor neurons in the transgenic fish enabled visualization of the cell bodies, main axons, and the peripheral branches within the muscles. GFP-labeled motor neurons could be followed at high resolution for at least up to day four, when most larval neural circuits become functional, and larvae begin to swim and capture prey. Using this line, we analyzed axonal outgrowth by the cranial motor neurons. Furthermore, by selective application of DiI to specific GFP-positive nerve branches, we showed that the two clusters of trigeminal motor neurons in rhombomeres 2 and 3 innervate different peripheral targets. This finding suggests that the trigeminal motor neurons in the two clusters adopt distinct fates. In future experiments, this transgenic line of zebrafish will allow for a genetic analysis of cranial motor neuron development.

Maturation of cardiovagal autonomic function from childhood to young adult age.

BACKGROUND: Cardiovagal autonomic control declines with age in adult subjects, which is related in part to increasing stiffness of the barosensory vessel wall. It is not known, however, whether autonomic function changes with age in children. METHODS AND RESULTS: We studied 137 healthy subjects divided into 4 age groups: group 1, 7 to 14 years; group 2, 11 to 14 years; group 3, 15 to 18 years; and group 4, 19 to 22 years. Brachial artery pressure was measured by sphygmomanometry and continuous radial artery pressure and carotid artery pulse pressure (DeltaP) by applanation tonometry. The R-R interval was derived from the ECG. Autonomic function was assessed by spontaneous sequence and frequency-domain indices, which indicate the extent of coupling between fluctuations in heart rate and systolic pressure. Carotid artery diastolic diameter (DD) and pulsatile distension (DeltaD) were measured by echo wall tracking; carotid compliance coefficient (CC) was defined as DeltaD/DeltaP and distensibility coefficient as 2DeltaD/DD . DeltaP. From group 1 to group 3, spontaneous indices increased significantly (18.1+/-1.7 versus 33.3+/-4.0; 14.4+/-1.1 versus 25.5+/-22; 12.9+/-1.1 versus 20.8+/-2.0; and 6.4+/-0.6 versus 16.2+/-1.4 ms/mm Hg [mean+/-SEM] for Seq+, Seq-, LFalpha, and LF(gain), respectively), with no significant changes afterward. CC and DC were inversely proportional to age (r=-0.49 and -0.62, respectively, P<0.001). The efficiency of neural integrative mechanisms, estimated as the ratio of spontaneous indices and CC, more than doubled from group 1 to group 3. Spontaneous indices were linearly related to measures of cardiac vagal activity. CONCLUSIONS: The increase in spontaneous indices from early childhood to adolescence, despite gradual stiffening of the carotid artery, may indicate improved cardiovagal autonomic function, which is most likely a result of maturation of neural mechanisms, attaining peak level at adolescence.

Autonomic nervous control of heart rate: sympathetic-parasympathetic interactions and age related differences.

A factorial experimental design was used to quantify the changes in heart rate produced by stimulation of the cardiac sympathetic and vagal nerves in eleven adult dogs and four puppies, and to quantify the extent of the peripheral sympathetic-vagal interactions. The chronotropic responses to autonomic stimulation were significantly less in the puppies than in the adult dogs, which suggests that autonomic regulation is functionally incomplete in the puppies. In both adult dogs and puppies, the chronotropic responses to autonomic nerve stimulation were bilaterally asymmetrical. The heart rate responses to a given level of right-sided stimulation of either the sympathetic or vagal nerves were greater than those to comparable left-sided stimulation. In both adult dogs and puppies, there were significant sympathetic-vagal interactions, such that the sympathetic enhancement of heart rate was less effective the higher the background level of vagal activity. The sympathetic-vagal interactions were prominent in the puppies as well as in the adult animals, regardless of whether the stimulated sympathetic and vagal nerves were located ipsilaterally or contralaterally to one another. Thus, the mechanisms responsible for the sympathetic-vagal interactions appear to be fully developed in puppies. Also, the cardiac sympathetic nerve endings that originate from one side of the body must lie in close apposition to the cardiac vagal nerve endings that originate from either the same side or from the opposite side of the body.

Vagal Fibers Form Associations With Interstitial Cells of Cajal During Fetal Development.

Vagal intramuscular arrays (IMAs) have been shown to form complexes with intramuscular interstitial cells of Cajal (ICC). We tested the hypothesis that associations between vagal nerve endings and ICC arise in fetal development. Intraganglionic laminar endings (IGLEs) and IMAs were identified by applying 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanineperchlorate (DiI) to vagal nerve trunks and myenteric plexus (MP) and intramuscular (IM) ICC were immunolabeled with antibodies to c-Kit in fetal and early postnatal mice (E16-P7). At E16, c-Kit immunoreactive cells were abundant in the primordial smooth muscle, with early ICC networks discernable by E18 and ongoing organization at P1 and P7. The distribution of vagal endings was found to change during the course of development, with significantly more putative IGLEs in the prenatal compared to the postnatal period and less IMAs in the prenatal compared to postnatal period. Associations of ICC with both IGLEs and IMAs were detected as early as E16 and were maintained into postnatal life. These findings suggest that vagal fibers begin to associate with ICC during prenatal development. Future studies will be needed to determine the mechanisms through which vagal endings and ICC interact.

Myelination of the human vagus nerve from 24 weeks postconceptional age to adolescence.

Significant changes in respiratory reflexes occur with maturation. The vagus nerve, the pathway for the Hering-Breuer and irritant-receptor reflexes, was studied quantitatively in 33 infants and 5 adolescents. In the infants, total myelinated vagus fibers increased linearly (r m0.682, p less than 0.001) with postconceptional age (PCA), and by 40 weeks after conception, total counts were comparable to those of adolescent group. Counts of total myelinated vagus fibers in 16 term infants (greater than 41 weeks PCA) were comparable to those in the adolescent group (p less than 0.40), whereas 17 preterm infants (less than 38 weeks PCA) showed significantly fewer total myelinated vagus fibers than term or adolescent groups (p less than 0.001). Smaller-diameter (less than 2 micrometer) myelinated vagus fibers depended upon PCA in the preterm group (p less than 0.005), but were independent of PCA in the term group (p less than 0.5). Preterm infants have a higher percentage of small to total myelinated vagus fibers than term infants (p less than 0.1).

Formation of ganglia in the gut of the chick embryo.

We have examined the formation of myenteric ganglia in the developing avian enteric nervous system. The monoclonal antibody HNK-1 was used to identify neural-crest-derived cells in whole mounts of fore- and midgut of chick embryos. We find that the crest-derived cells extend processes to their neighbors and form a complex network in the wall of the gut. Formation of this network is an unusual behavior of crest-derived cells and suggests the gut microenvironment is critical to this behavior. This cellular network disappears after ablation of the vagal neural crest, indicating the HNK-1-stained cellular network arises from crest-derived cells. The network is found in the gut wall before the vagal nerve fibers are present. This network is first found in the primordium of the proventriculus, distal to the evagination of the lung buds, and progresses just proximal to the yolk stalk at embryonic day (E) 3.5 and almost to the ileocecal junction at E5.5. The number of cells and the complexity of the network decrease in a rostral-caudal direction down the length of the gut at these stages. The leading edge of the network consists of cells serially arranged in longitudinally running strands. The organization of the network changes with increasing embryonic age; we have focused on network changes in the proventriculus. In the primordium of the proventriculus at E3.5, the network consists of a cluster of one or two adjacent crest-derived cells, which extend processes to a number of neighboring crest-derived cells. At E5.5 large increases in the number of cells per cluster and in the length of cellular connectives between clusters are apparent. At E6.5 a crude meshwork of clusters is seen. At E10.5 the arrangement of cell clusters resembles the pattern of ganglia found in the adult myenteric plexus. This network may provide the environmental cues for the differentiation of enteric neurons and a framework for the pattern of ganglia found in the adult enteric nervous system.

Development of neuromuscular junctions in the mouse esophagus: morphology suggests a role for enteric coinnervation during maturation of vagal myoneural contacts.

The time course of establishment of motor endplates and the subsequent developmental changes in their enteric and vagal innervation were examined in esophageal striated muscle of perinatal and adult C57/Bl6 mice by using immunocytochemistry and confocal laser scanning microscopy. Nicotinic acetylcholine receptors were visualized with alpha-bungarotoxin; vagal motor nerve terminals with antisera against vesicular acetylcholine transporter; and enteric nerve fibers with antisera against neuronal nitric oxide synthase, vasoactive intestinal peptide, and galanin. Because the various stages of esophageal striated myogenesis advance caudocranially, i.e., more mature stages are found cranial to immature stages, longitudinal cryosections through the esophagus were investigated. Synaptogenesis was divided into several distinct stages. 1) Mononucleated cells express acetylcholine receptors over their entire surface. 2) They start to cluster receptors without nerve fiber contacts. 3) The first nerve contact on a growing receptor cluster is made by a vagal nerve terminal, followed by an enteric terminal. 4) Vagal terminals grow until they match the size of endplate areas, and one to three enteric terminals intertwine with them on every receptor cluster. 5) After vagal terminals have covered the whole endplate area, enteric terminals are withdrawn from the majority of motor endplates. In a minority of endplates, enteric coinnervation persists through adulthood. The enteric innervation of all developing motor endplates, shortly after vagal terminals have contacted them, and the removal of enteric nerve fibers from the majority of mature motor endplates suggest a major role of enteric nerve fibers during maturation of esophageal neuromuscular junctions.

Oxytocinergic inputs to the nucleus of the solitary tract and dorsal motor nucleus of the vagus in neonatal rats.

The paraventricular nucleus of the hypothalamus (PVN) modulates vagal digestive motor functions via oxytocinergic projections to the nucleus of the solitary tract (NST) and dorsal motor nucleus of the vagus (DMV) in adult rats. Little is known regarding the structural or functional maturation of these projections. The present study examines the postnatal development of immunocytochemically identified oxytocinergic fibers in gastric subregions of the medial NST-DMV. For this purpose, a monoclonal antibody (PS36) that recognizes both oxytocin (OT)-neurophysin and its prohormone was used to identify oxytocinergic fibers. PS36-positive fibers already were present within the NST-DMV in rats on the day of birth. Retrograde transport of cholera toxin neural tracer from the NST-DMV in newborn rats confirmed that PVN neurons were the sole source of these oxytocinergic fibers. The cumulative length of PS36-positive fibers in sampled subregions of the medial NST and DMV increased approximately 23-fold and 94-fold, respectively, between birth and adulthood. The observed postnatal increases in PS36 immunolabeling could reflect increased delivery of immunoreactive antigen from hypothalamic perikarya to distal axons and/or increasing oxytocinergic innervation of the NST-DMV. Additional work will be needed to address these questions and to determine the time course during which central oxytocinergic pathways become mature in their ability to influence vagally mediated digestive functions.