Comparison of the 3-D patterns of the parasympathetic nervous system in the lung at late developmental stages between mouse and chicken.

Although the basic schema of the body plan is similar among different species of amniotes (mammals, birds, and reptiles), the lung is an exception. Here, anatomy and physiology are considerably different, particularly between mammals and birds. In mammals, inhaled and exhaled airs mix in the airways, whereas in birds the inspired air flows unidirectionally without mixing with the expired air. This bird-specific respiration system is enabled by the complex tubular structures called parabronchi where gas exchange takes place, and also by the bellow-like air sacs appended to the main part of the lung. That the lung is predominantly governed by the parasympathetic nervous system has been shown mostly by physiological studies in mammals. However, how the parasympathetic nervous system in the lung is established during late development has largely been unexplored both in mammals and birds. In this study, by combining immunocytochemistry, the tissue-clearing CUBIC method, and ink-injection to airways, we have visualized the 3-D distribution patterns of parasympathetic nerves and ganglia in the lung at late developmental stages of mice and chickens. These patterns were further compared between these species, and three prominent similarities emerged: (1) parasympathetic postganglionic fibers and ganglia are widely distributed in the lung covering the proximal and distal portions, (2) the gas exchange units, alveoli in mice and parabronchi in chickens, are devoid of parasympathetic nerves, (3) parasympathetic nerves are in close association with smooth muscle cells, particularly at the base of the gas exchange units. These observations suggest that despite gross differences in anatomy, the basic mechanisms underlying parasympathetic control of smooth muscles and gas exchange might be conserved between mammals and birds.

Fetal Heart-Rate Variability: Validation of a New Continuous, Noninvasive Computerized Analysis.

OBJECTIVE: We developed a computerized heart-rate variability index related to the fetal parasympathetic activity: the Fetal Stress Index (FSI). The objective was to determine whether the FSI is related to the visual analysis of the fetal heart rate (FHR). METHODS: Thirty tracings recorded at a labor ward were classified according to the NICHD categories: (I) normal FHR tracing, (II) intermediate risk of acidosis, and (III) high risk. FSI was calculated as minimum, maximum, and mean, and was evaluated before the onset of the FHR pattern, during the 10 min following, and between 10 and 20 min after that. RESULTS: The FSI for categories II and III was similar to that of category I before the onset of the FHR pattern. FSI min was lower just after the onset of the abnormal FHR in category III, compared with that of category I (33 vs. 43, p < 0.001). Between 10 and 20 min after the onset of the abnormal FHR, we observed a significant reduction in FSI min in categories II and III (44 vs. 39 vs. 29.7, p < 0.0001). CONCLUSION: Although further studies are necessary for the sake of clinical validation, FSI could constitute an interesting method for the evaluation of fetal well-being.

GDNF is required for neural colonization of the pancreas.

The mammalian pancreas is densely innervated by both the sympathetic and parasympathetic nervous systems, which control exocrine and endocrine secretion. During embryonic development, neural crest cells migrating in a rostrocaudal direction populate the gut, giving rise to neural progenitor cells. Recent studies in mice have shown that neural crest cells enter the pancreatic epithelium at E11.5. However, the cues that guide the migration of neural progenitors into the pancreas are poorly defined. In this study we identify glial cell line-derived neurotrophic factor (GDNF) as a key player in this process. GDNF displays a dynamic expression pattern during embryonic development that parallels the chronology of migration and differentiation of neural crest derivatives in the pancreas. Conditional inactivation of Gdnf in the pancreatic epithelium results in a dramatic loss of neuronal and glial cells and in reduced parasympathetic innervation in the pancreas. Importantly, the innervation of other regions of the gut remains unaffected. Analysis of Gdnf mutant mouse embryos and ex vivo experiments indicate that GDNF produced in the pancreas acts as a neurotrophic factor for gut-resident neural progenitor cells. Our data further show that exogenous GDNF promotes the proliferation of pancreatic progenitor cells in organ culture. In summary, our results point to GDNF as crucial for the development of the intrinsic innervation of the pancreas.

Defective parasympathetic innervation is associated with airway branching abnormalities in experimental CDH.

Developmental mechanisms leading to lung hypoplasia in congenital diaphragmatic hernia (CDH) remain poorly defined. Pulmonary innervation is defective in the human disease and in the rodent models of CDH. We hypothesize that defective parasympathetic innervation may contribute to airway branching abnormalities and, therefore, lung hypoplasia, during lung development in CDH. The murine nitrofen model of CDH was utilized to study the effect of the cholinergic agonist carbachol on embryonic day 11.5 (E11.5) lung explant cultures. Airway branching and contractions were quantified. In a subset of experiments, verapamil was added to inhibit airway contractions. Sox9 immunostaining and 5-bromo-2-deoxyuridine incorporation were used to identify and quantify the number and proliferation of distal airway epithelial progenitor cells. Intra-amniotic injections were used to determine the in vivo effect of carbachol. Airway branching and airway contractions were significantly decreased in nitrofen-treated lungs compared with controls. Carbachol resulted in increased airway contractions and branching in nitrofen-treated lungs. Nitrofen-treated lungs exhibited an increased number of proliferating Sox9-positive distal epithelial progenitor cells, which were decreased and normalized by treatment with carbachol. Verapamil inhibited the carbachol-induced airway contractions in nitrofen-treated lungs but had no effect on the carbachol-induced increase in airway branching, suggesting a direct carbachol effect independent of airway contractions. In vivo treatment of nitrofen-treated embryos via amniotic injection of carbachol at E10.5 resulted in modest increases in lung size and branching at E17.5. These results suggest that defective parasympathetic innervation may contribute to airway branching abnormalities in CDH.

Defective pulmonary innervation and autonomic imbalance in congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is associated with significant mortality due to lung hypoplasia and pulmonary hypertension. The role of embryonic pulmonary innervation in normal lung development and lung maldevelopment in CDH has not been defined. We hypothesize that developmental defects of intrapulmonary innervation, in particular autonomic innervation, occur in CDH. This abnormal embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH. To define patterns of pulmonary innervation in CDH, human CDH and control lung autopsy specimens were stained with the pan-neural marker S-100. To further characterize patterns of overall and autonomic pulmonary innervation during lung development in CDH, the murine nitrofen model of CDH was utilized. Immunostaining for protein gene product 9.5 (a pan-neuronal marker), tyrosine hydroxylase (a sympathetic marker), vesicular acetylcholine transporter (a parasympathetic marker), or VIP (a parasympathetic marker) was performed on lung whole mounts and analyzed via confocal microscopy and three-dimensional reconstruction. Peribronchial and perivascular neuronal staining pattern is less complex in human CDH than control lung. In mice, protein gene product 9.5 staining reveals less complex neuronal branching and decreased neural tissue in nitrofen-treated lungs from embryonic day 12.5 to 16.5 compared with controls. Furthermore, nitrofen-treated embryonic lungs exhibited altered autonomic innervation, with a relative increase in sympathetic nerve staining and a decrease in parasympathetic nerve staining compared with controls. These results suggest a primary defect in pulmonary neural developmental in CDH, resulting in less complex neural innervation and autonomic imbalance. Defective embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH.

Cardiac response to startle stimuli in larval zebrafish: sympathetic and parasympathetic components.

Central regulation of cardiac output via the sympathetic and parasympathetic branches of the autonomic nervous system allows the organism to respond to environmental changes. Sudden onset stimuli, startle stimuli, are useful probes to study central regulatory responses to the environment. In mammals, startle stimuli induce a transient bradycardia that habituates with repeated stimulation. Repeated presentation of the stimulus results in tachycardia. In this study, we investigate the behavioral regulation of heart rate in response to sudden stimuli in the zebrafish. Larval zebrafish show a stereotyped heart rate response to mild electrical shock. Naïve fish show a significant increase in interbeat interval that resolves in the 2 s following stimulation. This transient bradycardia decreases on repeated exposure to the stimulus. Following repeated stimulation, the fish become tachycardic within 1 min of stimulation. Both the transient bradycardia and following tachycardia responses are blocked with administration of the ganglionic blocker hexamethonium, demonstrating that these responses are mediated centrally. The transient bradycardia is blocked by the muscarinic antagonist atropine, suggesting that this response is mediated by the parasympathetic system, while the following tachycardia is specifically blocked by the beta-adrenergic antagonist propranolol, suggesting that this response is mediated by the sympathetic nervous system. Together, these results demonstrate that at the larval stage, zebrafish actively regulate cardiac output to changes in their environment using both the parasympathetic and sympathetic branches of the autonomic nervous system, a behavioral response that is markedly similar to that observed in mammals to similar sudden onset stimuli.

Fetal heart rate variability reveals differential dynamics in the intrauterine development of the sympathetic and parasympathetic branches of the autonomic nervous system.

The aim of this study was to investigate the hypothesis that fetal beat-to-beat heart rate variability (fHRV) displays the different time scales of sympatho-vagal development prior to and after 32 weeks of gestation (wks GA). Ninety-two magnetocardiograms of singletons with normal courses of pregnancy between 24 + 1 and 41 + 6 wks GA were studied. Heart rate patterns were either quiet/non-accelerative (fHRP I) or active/accelerative (fHRP II) and recording quality sufficient for fHRV. The sample was divided into the GA groups <32 wks GA/>32 wks GA. Linear parameters of fHRV were calculated: mean heart rate (mHR), SDNN and RMSSD of normal-to-normal interbeat intervals, power in the low (0.04-0.15 Hz) and high frequency range (0.15-0.4 Hz) and the ratios SDNN/RMSSD and LF/HF as markers for sympatho-vagal balance. fHRP I is characterized by decreasing SDNN/RMSSD, LF/HF and mHR. The decrease is more pronounced <32 wks GA. Beyond that GA SDNN/RMSSD is predominantly determined by RMSSD during fHRP I and by SDNN during fHRP II. In contrast to fHRP I, during fHRP II, mHR is positively correlated to SDNN/RMSSD instead of SDNN >32 wks GA. LF/HF increases in fHRP II during the first half of the third trimester. Non-accelerative fHRP are indicative of parasympathetic dominance >32 wks GA. In contrast, the sympathetic accentuation during accelerative fHRP is displayed in the interrelations between mHR, SDNN and SDNN/RMSSD. Prior to 32 wks GA, fHRV reveals the increasing activity of the respective branches of the autonomic nervous system differentiating the types of fHRP.

Differential expression of the neuronal acetylcholine receptor alpha 2 subunit gene during chick brain development.

In situ hybridization histochemistry reveals localized expression of the nicotinic acetylcholine receptor (nAChR) alpha 2 subunit mRNA restricted to the lateral spiriform nucleus (SpL) of the chick diencephalon. The alpha 2 nAChR transcripts are not detected in immature SpL neurons at 4.5-5 days of embryonic development. They begin to accumulate in the SpL at embryonic day 11 and increase until the newborn stage. Specific alpha 2 cDNA amplification by the polymerase chain reaction shows that during this period, the absolute content of alpha 2 mRNA increases about 20-fold. The expression of the alpha 2 nAChR gene is thus developmentally regulated and appears concomitant with the entry of cholinergic fibers into the SpL, as demonstrated by choline acetyltransferase immunohistochemistry.

Neural crest stem cells undergo cell-intrinsic developmental changes in sensitivity to instructive differentiation signals.

Rat neural crest stem cells (NCSCs) prospectively isolated from uncultured E14.5 sciatic nerve and transplanted into chick embryos generate fewer neurons than do NCSCs isolated from E10.5 neural tube explants. In addition, they differentiate primarily to cholinergic parasympathetic neurons, although in culture they can also generate noradrenergic sympathetic neurons. This in vivo behavior can be explained, at least in part, by a reduced sensitivity of sciatic nerve-derived NCSCs to the neurogenic signal BMP2 and by the observation that cholinergic neurons differentiate at a lower BMP2 concentration than do noradrenergic neurons in vitro. These results demonstrate that neural stem cells can undergo cell-intrinsic changes in their sensitivity to instructive signals, while maintaining multipotency and self-renewal capacity. They also suggest that the choice between sympathetic and parasympathetic fates may be determined by the local concentration of BMP2.

Cells migrating from the neural crest contribute to the innervation of the venous pole of the heart.

Cells migrating from the neural crest are known to septate the outflow tract of the developing heart, and to contribute to the formation of the arterial valves, their supporting sinuses, the coronary arteries and cardiac neural ganglia. Neural crest cells have also been suggested to contribute to development of the venous pole of the heart, but the extent and fate of such cells remains unclear. In this study, in the mouse, it is shown that cells from the neural crest contribute to the parasympathetic and, to a lesser extent, the sympathetic innervation of the venous pole of the heart. Nerves within the venous pole of the heart are shown to be of mixed origin, with some being derived from the neural crest, while others have an alternative origin, presumably placodal. The neurons innervating the nodal tissue, which can exert chronotropic effects on cardiac conduction, are shown not to be derived from the neural crest. In particular, no evidence was found to support previous suggestions that cells from the neural crest make a direct contribution to the myocardial atrioventricular conduction axis, although a small subset of these cells do co-localize with the developing left bundle branch. We have therefore confirmed that cells from the neural crest migrate to the venous pole of the heart, and that their major role is in the development of the parasympathetic innervation. In addition, in some embryos, a population of cells derived from the neural crest persist in the leaflets of the atrioventricular valves, but their role in subsequent development remains unknown.

Pancreatic innervation in mouse development and beta-cell regeneration.

Pancreatic innervation is being viewed with increasing interest with respect to pancreatic disease. At the same time, relatively little is currently known about innervation dynamics during development and disease. The present study employs confocal microscopy to analyze the growth and development of sympathetic and sensory neurons and astroglia during pancreatic organogenesis and maturation. Our research reveals that islet innervation is closely linked to the process of islet maturation-neural cell bodies undergo intrapancreatic migration/shuffling in tandem with endocrine cells, and close neuro-endocrine contacts are established quite early in pancreatic development. In addition, we have assayed the effects of large-scale beta-cell loss and repopulation on the maintenance of islet innervation with respect to particular neuron types. We demonstrate that depletion of the beta-cell population in the rat insulin promoter (RIP)-cmyc(ER) mouse line has cell-type-specific effects on postganglionic sympathetic neurons and pancreatic astroglia. This study contributes to a greater understanding of how cooperating physiological systems develop together and coordinate their functions, and also helps to elucidate how permutation of one organ system through stress or disease can specifically affect parallel systems in an organism.

The GDNF family ligands and receptors - implications for neural development.

The glial cell line derived neurotrophic factor (GDNF) family has recently been expanded to include four members, and the interactions between these neurotrophic factors and their unique receptor system is now beginning to be understood. Furthermore, analysis of mice lacking the genes for GDNF, neurturin, and their related receptors has confirmed the importance of these factors in neurodevelopment. The results of such analyses reveal numerous similarities and potential overlaps in the way the GDNF and the nerve growth factor (NGF) families regulate development of the peripheral nervous system.

Respiratory sinus arrhythmia: endogenous activation of nicotinic receptors mediates respiratory modulation of brainstem cardioinhibitory parasympathetic neurons.

The heart rate increases during inspiration and decreases during expiration. This respiratory sinus arrhythmia (RSA) occurs by modulation of premotor cardioinhibitory parasympathetic neuron (CPN) activity. However, RSA has not been fully characterized in rats, and despite the critical role of CPNs in the generation of RSA, little is known about the mechanisms that mediate this cardiorespiratory interaction. This study demonstrates that RSA in conscious rats is similar to that in other species. The mechanism of RSA was then examined in vitro. Rhythmic inspiratory-related activity was recorded from the hypoglossal rootlet of 700- to 800-microm medullary sections. CPNs were identified by retrograde fluorescent labeling, and neurotransmission to CPNs was examined using patch-clamp electrophysiological techniques. During inspiratory bursts, the frequency of both spontaneous gamma-aminobutyric acidergic (GABAergic) and spontaneous glycinergic synaptic events in CPNs was significantly increased. Focal application of the nicotinic antagonist dihydro-beta-erythroidine in an alpha4beta2-selective concentration (3 micromol/L) abolished the respiratory-evoked increase in GABAergic frequency. In contrast, the increase in glycinergic frequency during inspiration was not altered by nicotinic antagonists. Prenatal nicotine exposure exaggerated the increase in GABAergic frequency during inspiration and enhanced GABAergic synaptic amplitude both between and during inspiratory events. Glycinergic synaptic frequency and amplitude were unchanged by prenatal nicotine exposure. This study establishes a neurochemical link between neurons essential for respiration and CPNs, reveals a functional role for endogenous acetylcholine release and the activation of nicotinic receptors in the generation of RSA, and demonstrates that this cardiorespiratory interaction is exaggerated in rats prenatally exposed to nicotine.

The sacral autonomic outflow is sympathetic.

A kinship between cranial and pelvic visceral nerves of vertebrates has been accepted for a century. Accordingly, sacral preganglionic neurons are considered parasympathetic, as are their targets in the pelvic ganglia that prominently control rectal, bladder, and genital functions. Here, we uncover 15 phenotypic and ontogenetic features that distinguish pre- and postganglionic neurons of the cranial parasympathetic outflow from those of the thoracolumbar sympathetic outflow in mice. By every single one, the sacral outflow is indistinguishable from the thoracolumbar outflow. Thus, the parasympathetic nervous system receives input from cranial nerves exclusively and the sympathetic nervous system from spinal nerves, thoracic to sacral inclusively. This simplified, bipartite architecture offers a new framework to understand pelvic neurophysiology as well as development and evolution of the autonomic nervous system.

Leptin Controls Parasympathetic Wiring of the Pancreas during Embryonic Life.

The autonomic nervous system plays a critical role in glucose metabolism through both its sympathetic and parasympathetic branches, but the mechanisms that underlie the development of the autonomic innervation of the pancreas remain poorly understood. Here, we report that cholinergic innervation of pancreatic islets develops during mid-gestation under the influence of leptin. Leptin-deficient mice display a greater cholinergic innervation of pancreatic islets beginning in embryonic life, and this increase persists into adulthood. Remarkably, a single intracerebroventricular injection of leptin in embryos caused a permanent reduction in parasympathetic innervation of pancreatic ß cells and long-term impairments in glucose homeostasis. These developmental effects of leptin involve a direct inhibitory effect on the outgrowth of preganglionic axons from the hindbrain. These studies reveal an unanticipated regulatory role of leptin on the parasympathetic nervous system during embryonic development and may have important implications for our understanding of the early mechanisms that contribute to diabetes.

The final stage of cholinergic differentiation occurs below inner hair cells during development of the rodent cochlea.

To gain further insights into the cholinergic differentiation of presynaptic efferent terminals in the inner ear, we investigated the expression of the high-affinity choline transporter (ChT1) in comparison to other presynaptic and cholinergic markers. In the adult mammalian cochlea, cholinergic axons from medial olivocochlear (OC) neurons form axosomatic synapses with outer hair cells (OHCs), whereas axons from lateral OC neurons form axodendritic synapses on afferent fibers below inner hair cells (IHCs). Mouse brain and cochlea homogenates reveal at least two ChT1 isoforms: a nonglycosylated approximately 73 kDa protein and a glycosylated approximately 45 kDa protein. In mouse brain, ChT1 is preferentially expressed by neurons in periolivary regions of the superior olive consistent with the location of medial OC neurons. In the adult mouse cochlea, ChT1-positive terminals are located almost exclusively below OHCs consistent with a medial OC innervation. Between postnatal day 2 (P2) and P4, ChT1-positive terminals are below IHCs and occur after the expression of growth-associated protein 43, synapsin, and the vesicular acetylcholine transporter. By P15, ChT1-positive terminals are mostly on OHCs. Accounting for differences in gestational age, the developmental expression of ChT1 in the rat cochlea is similar to the mouse. However, in older rats ChT1-positive terminals are below IHCs and OHCs. In both rat and mouse, our observations indicate that the onset of ChT1 expression occurs after efferent terminals are below IHCs and express other presynaptic and cholinergic markers. In the mouse, but not in the rat, ChT1 may preferentially identify medial OC neurons.

Rhombomere-specific origin of branchial and visceral motoneurons of the facial nerve in the rat embryo.

The goal of this study was to localize selectively the facial nerve branchial and visceral motoneurons in the rat embryo hindbrain. This was achieved by injecting dextran amines into the peripheral facial nerve on embryos maintained in an artificial cerebrospinal fluid. Sprague-Dawley rat embryos 13, 14, and 15 days old (E13, E14, E15) were obtained by cesarean section. Branchial motoneurons were first labeled at E13. They were close to the midline and migrated from rhombomere (r) 4 toward r5 and r6. By E15, they had migrated caudally and ventrolaterally into the former location of r6. Most of them had reached their "adult" position by E15. Another group of motoneurons, the accessory facial nucleus, was found in r4 at E13 and in corresponding regions at later stages. Visceral motoneurons were labeled from the periphery at all stages. At E13, they were mainly in r5 but also in r2, r3, r4, and r6. At E14, most of them had migrated laterally, and, by E15, they were in the prospective parvocellular reticular formation. They could be divided into two subgroups: a more rostral one with fibers that made loops close to the midline and a more caudal one with fibers that went directly to the exit. The findings presented here show that most branchial and visceral motoneurons of the facial nerve are born in different and specific rhombomeres. Interestingly, developmental genes are expressed specifically in these rhombomeres and could be involved in the genesis of the facial and superior salivatory nuclei.

Microglia from the developing rat medial septal area can affect cholinergic and GABAergic neuronal differentiation in vitro.

The normal development of the central nervous system is regulated by glia. In this regard, we have reported that astrocytes, stimulated by epidermal growth factor or transforming growth factor alpha, suppress the biochemical differentiation of rat medial septal cholinergic neurons in vitro, as evidenced by a decrease in choline acetyltransferase activity. In this study, we found that, in contrast to astrocytes, microglia enhance rather than suppress this aspect of cholinergic cell expression. When in excess, microglia can revert the effects of epidermal growth factor on the septal cholinergic neurons without altering the astroglial proliferative response to this growth factor. In the absence of growth factors or other glial cell types, microglia increase choline acetyltransferase activity above control levels and thus, may be a source of cholinergic differentiating activity. The increase in enzyme activity induced by microglia is rapid in onset, detected as early as 2 h after their addition to the septal neurons and maintained up to six or seven days in vitro. Furthermore, in the absence or presence of other glial cell types, microglia also influence septal GABAergic neurons by significantly increasing glutamate decarboxylase activity. As microglia affect neither septal cholinergic nor GABAergic neuronal cell survival, they appear to enhance the biochemical differentiation of these two neuronal cell types. Specific immunoneutralizing antibodies were used to identify the microglia-derived factors affecting these two neuronal types. In this regard, we found that the microglia-derived cholinergic differentiating activity is significantly suppressed by antibodies raised against interleukin-3. Furthermore, interleukin-3 was detected in both conditioned media and cell homogenates from septal neuronal-microglial co-cultures by western blotting. Finally, although basic fibroblast growth factor and interleukin-3 significantly increase septal glutamate decarboxylase activity, neither appears to be implicated in the GABAergic cell response to the microglia. In conclusion, these results demonstrate that microglia can enhance the biochemical differentiation of developing cholinergic and GABAergic neurons in vitro.

Early development of the nucleus basalis-cortical projection but late expression of its cholinergic function.

The aim of this study was to examine the development of the basalocortical pathway by using choline acetyltransferase and nerve growth factor receptor immunocytochemistry, acetylcholinesterase histochemistry and retrograde axonal transport. The observations were made in the ferret because in this species brain development occurs over a much more protracted period than in the rat. Staining for choline acetyltransferase immunoreactivity in the brain was minimal before birth. Adult levels of staining for the enzyme were not seen in cell bodies until three weeks after birth and in axons up to six weeks after birth. This, however, did not mean that presumptive cholinergic pathways are absent early in development. There was strong staining for nerve growth factor receptor in basal forebrain neurons from at least two weeks before birth. Positive staining for acetylcholinesterase was found in axons that begin to invade the cerebral cortex a week before birth. The retrograde axonal transport technique showed that the basalocortical pathway has a normal organization in the neonate. The conclusion is that cholinergic pathways form early in the prenatal period in the ferret but express their transmitter function late in postnatal development.