Changing interactions between astrocytes and neurons during CNS maturation

The environments of the developing brain and injured adult brain differ in their abilities to support axonal growth. To determine if astrocytes contribute to this difference, neurons were plated onto astrocytes cultured from the neonatal rat cortex and from the injured adult brain. Two patterns of neurite growth were observed in these two astrocyte culture systems. Neurons contacting the neonatal astrocytes had neurites that were twice as long as those contacting the injured adult astrocytes. Furthermore, in cultures with neonatal astrocytes, neurites faithfully followed the astrocytic processes, maximizing their contact, while in cultures of injured adult astrocytes, the neurites had a tendency to cross the processes orthogonally, minimizing their interaction with the astrocytes. When neurons were grown suspended over either neonatal or injured adult astrocytes, no difference in neurite length or the pattern of neurite growth was observed, indicating that neurite growth was not differentially affected by soluble factors released from the two populations of astrocytes. The addition of fetal calf serum, which is known to contain protease inhibitors, did not alter neurite growth when compared to serum-free medium, suggesting that a substantial difference in protease activity does not account for the variations in neurite length observed. Based on these results, it appears that the molecular components of the external surface of injured adult astrocytes do not support neurite growth to the same extent as those found on neonatal astrocytes. The differing abilities of these two populations of cultured astrocytes to support neurite growth in culture may reflect a change in the functional role of these cells that occurs during the development of the central nervous system.     

Forskolin Mediates the Survival of Nerve Growth Factor-Dependent Sympathetic Neurons of Chick Embryo by a Cyclic AMP-Independent Mechanism

Forskolin has become an invaluable tool for exploring the involvement of cyclic AMP in a variety of cellular functions. The diterpine directly activates the catalytic subunit of adenylate cyclase, causing a marked increase in cyclic AMP content. Because of this well-characterized action, practically all the observed effects of forskolin are commonly attributed to cyclic AMP-dependent processes. We show here that forskolin exerts a neurotrophic action that is almost identical to that of nerve growth factor (NGF) and phorbol 12,13-dibutyrate (PDB) but independent of cyclic AMP. Sympathetic neurons of the chick embryo supported in culture for 2 days by NGF, forskolin plus 3-isobutyl-1-methylxanthine (IBMX), or PDB had almost identical levels of cyclic AMP (between 9 and 12 pmol/mg protein). Neurons supported in culture for 2 days by NGF or PDB when challenged with forskolin plus IBMX showed almost a 15-fold increase in cyclic AMP, but those supported by forskolin plus IBMX and then exposed to the same combination of drugs did not show an increase in cyclic AMP, exhibiting a marked down-regulation. Exposure of neurons to forskolin for 2 h was ineffective in supporting long-term survival, suggesting that an initial increase in cyclic AMP formation is not sufficient but the continued presence of the drug is essential for survival. Effects of forskolin on the survival of these neurons could be observed even in the presence of dideoxyadenosine, and inhibitor of adenylate cyclase. Neurons supported by PDB for 2 days in culture when exposed to NGF for the first time did not show any increase in cyclic AMP, providing clear evidence that NGF does not affect this second messenger in its target cells. Similarly, neurons supported by NGF for 2 days when exposed to PDB did not show an increase in cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of human fetal hippocampal neurons by flow cytometry for transplantation

We have established primary cultures, highly enriched in neurons, from the hippocampus of human fetal brains at 20-23 gestational weeks. More than 80% of cells were viable when seeded. Neurons were isolated from primary cultures by flow cytometry to a high degree of purity, as demonstrated by immunocytochemical staining. FACS scanning analysis using a DNA-staining dye showed that hippocampal neurons did not divide in culture. To demonstrate that FACS-sorted neurons can be transplanted and integrated into the host brain, neuron-enriched primary culture from human fetal striatum was infected with a viral-mediated vector containing a reporter gene, beta-galactosidase. Striatal neurons were subsequently purified by flow cytometry and transplanted into the striatum of rats. Following transplantation, the rat brains were processed for beta-galactosidase histochemistry and electron microscopy. Beta-galactosidase expression indicates that transplanted human neurons survived in the host and were metabolically active. The transplanted neurons received synaptic inputs, as judged from the presence of presynaptic terminals on their surface. Our study demonstrates connectivity between transplanted human fetal primary neurons and host tissue at the ultrastructural level. Our results support the feasibility of ultimately transplanting neurons into humans as a possible treatment for recovery of the nervous system (e.g., neurodegenerative diseases).     

Both nerve growth factor and high K concentrations support the survival of chick embryo sympathetic neurons : Evidence for a common mechanism of action

Neurons were dissociated from the sympathetic ganglia of embryonic chicks, and cultured in the absence of non-neuronal cells. Both nerve growth factor (NGF) and high concentrations of extracellular K⁺ supported neuronal survival, and these effects were independent of the presence of serum in the culture medium. Only 60% of the neurons survived in response to 35 mM K⁺, and survival was not increased when both NGF and K⁺ were present together. It was, however, possible to maintain essentially all the neurons in culture with either NGF or high K⁺ concentrations if the culture substrate had been pretreated with heart cell-conditioned medium (which did not itself support neuronal survival). These observations are consistent with a common mechanism of action of both K⁺ and NGF for the survival of cultured embryonic neurons.     

The effect of nerve growth factor on cholinergic neurons in dissociated cultures of the septum pellucidum

Neurons dissociated from septal area of fetal (E 18-19) rat brain were grown for 7 days in culture. Cholinergic neurons were identified by cytochemical demonstration of acetyl cholinesterase. Addition of the nerve growth factor to the culture medium (200 u/ml) increases the number of AChE-positive neurons, the size of the cell body and activity of AChE in these neurons.     

The culture of chick embryo dorsal root ganglionic cells on polylysine-coated plastic

Polylysine-coated culture surfaces are strongly adhesive for neural cells, restrict locomotion on nonneuronal elements, but do not inhibit neurite elongation. In the present study, culture dishes were pre-treated with poly-d-lysine (PDL) at various concentrations, seeded with dissociates from 8-day chick embryo dorsal root ganglia, and incubated under conditions that normally support both neuronal survival and nonneuronal proliferation. Pretreatment with low (0.1 mg/ml) PDL concentrations had no effect on neuronal survival and neuritic growth, but entirely prevented an increase in ganglionic nonneurons, yielding a numerically stable culture greatly enriched in neurons. Higher PDL concentrations caused increasing losses in both cell classes. The 50% levels of cell loss were achieved at about the same PDL dose, but earlier for neurons than nonneurons and still with no impairment of neuritic growth from the surviving neurons. A procedure was developed to compare acid-soluble and acid-precipitable accumulation of radioactivity under 1-hr pulses of [³H]uridine, which was applicable even to poorly attached cells. The cytotoxic effects of higher PDL pretreatments was revealed as early as 6 hr after seeding by 2- to 4-fold lower radioaccumulation. The data are discussed in terms of possible regulations of cell permeability and metabolism by adhesive interactions between cells and their substratum, or other cells.     

Excess K+ and Phorbol Ester Activate Protein Kinase C and Support the Survival of Chick Sympathetic Neurons in Culture

The effects of phorbol esters were investigated on the survival of chick sympathetic neurons in a serum-free culture medium. The protein kinase C activator phorbol 12,13-dibutyrate (PDB) supported about 40% of the plated sympathetic neurons. This number was comparable to that supported by nerve growth factor (NGF). A combination of phorbol ester and NGF did not significantly increase the number of surviving neurons. Phorbol ester-supported sympathetic neurons possessed desipramine-sensitive [3H]-norepinephrine uptake mechanism, and therefore were noradrenegic in character. Two days after the start of cultures, if NGF was replaced by phorbol ester, or phorbol ester was replaced by NGF, the number of surviving sympathetic neurons was essentially the same in both groups, and the uptake of [3H]norepinephrine was also comparable when examined 2 days after the switchover. Interchangeability between phorbol ester and NGF in the survival of sympathetic neurons suggests that both agents act on the same subpopulation of neurons of the chick sympathetic ganglia. The protein kinase C activity of cytosol and particulate fractions of NGF-supported neurons was 0.14 and 0.09 pmol/min/mg protein, respectively. In phorbol ester-supported neurons the activity in the particulate fraction increased by about fivefold. Removal of the phorbol ester after 2 days resulted in restoration of the enzyme activity in less than 1 h, and readdition of the phorbol ester again increased the activity by fivefold. When NGF was added to these neurons (1 microgram for 15 min), there was no change in the enzyme activity. Phorbol 13-acetate was ineffective in supporting sympathetic neurons in culture, as well as in enhancing protein kinase C activity. We also compared the protein kinase C activity of sympathetic neurons supported in culture by NGF and excess potassium (35 mM K+) Neurons supported in culture by 35 mM K+ for 2 days had almost eightfold more protein kinase C activity in their particulate fraction than in cytosol fraction. In NGF-supported neurons were acutely treated with excess K+, the protein kinase C activity was increased in the particulate fraction by about sevenfold in a concentration- and time-dependent manner. Excess K+ plus phorbol ester did not produce an additive effect on protein kinase C activity. PDB and excess K+ had no effect on cyclic AMP content of sympathetic neurons. In summary, the present data suggest that the neurotrophic action of PDB and excess K+ is probably mediated through protein kinase C.     

Distribution of aromatase activity in cultured neurons and glia cells

Aromatase plays a crucial role in the mechanism of action of testosterone in the central nervous system. Nevertheless, the exact cellular localization of this enzymatic complex within the different cell populations of the brain is still uncertain. In the experiments described here the presence of aromatase (assayed by the tritiated water method) has been evaluated in the two main cellular components of the brain: neurons and glia. Neurons, mixed glial cells, type 1 astrocytes, were obtained in cultures; oligodendrocytes were prepared by gradient ultracentrigugation. The results indicate that, among the different cells tested, only neurons possess a significant degree of aromatase activity, while the enzymatic activity is extremely low in mixed glial cell and in astrocyte preparations. Oligodendrocytes seem to be completely inactive in this respect.     

Effects of nerve growth factor on the development of the dendritic system of cholinergic neurons in dissociated culture of the rat septum]

Neurons dissociated from septal area of fetal (E18-19) rat brain were grown 14-days in culture. Cholinergic neurons were identified by cytochemical demonstration of acetyl cholinesterase. It was shown that the nerve growth factor added to the culture medium (50 u/ml) has increased the size of cell body of AchE-positive neurons, mean total length and arborization of dendrites and also the dendritic tree area.     

Effects of pineal factors on the action potentials of sympathetic neurons

Neurons from rat superior cervical ganglia were grown in coculture with pineal cells. Action potentials of neurons in cocultures were 25% longer and were 25% greater in amplitude than those recorded from neurons grown in the presence of ganglionic nonneuronal cells alone. Neurons showed an increase in action potential duration with increasing time in culture. This may have been related to the recovery of nonneuronal cell populations after an initial exposure to the antimitotic agent Ara-C. In cultures not initially exposed to Ara-C, a subsequent exposure after 7 days in culture resulted in a shortening of the action-potential duration. Neuronal cultures were exposed to gel slabs containing the pineal indolamines, serotonin, N-acetylserotonin, and melatonin. Serotonin and N-acetylserotonin showed no effect on the neuronal action potentials at the concentrations tested. Melatonin caused an increase in action-potential duration that was associated not with an increase in action-potential amplitude, but with a decrease in action-potential rise rates. The effects of long-term exposure in melatonin appeared to be reversible in some cells but not in all. Short-term effects of melatonin were observed in older cultures and in younger cultures after the cells were stimulated repeatedly. Older cultures also had higher levels of spontaneous activity. The dependence of the short-term effects of melatonin on electrical activity may suggest a role for melatonin as a neuromodulator.     

Pathway-specific patterns of the co-existence of substance P,calcitonin gene-related peptide,cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig

Summary The co-existence of immunoreactivities to substance P (SP), calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and dynorphin (DYN) in neurons of the dorsal root ganglion (DRG) of guinea-pigs has been investigated with a double-labelling immunofluorescence procedure. Four main populations of neurons could be identified that contained different combinations of these peptides and had distinctive peripheral projections: (1) Neurons that contained immunoreactivity to SP, CGRP, CCK and DYN were distributed mainly to the skin. (2) Neurons with immunoreactivity to SP, CGPR and CCK, but not DYN, were distributed mainly to the small blood vessels of skeletal muscles. (3) Neurons with immunoreactivity to SP, CGRP and DYN, but not CCK, were distributed mainly to pelvic viscera and airways. (4) Neurons containing immunoreactivity to SP and CGRP, but not CCK and DYN, were distributed mainly to the heart, systemic blood vessels, blood vessels of the abdominal viscera, airways and sympathetic ganglia. Other small populations of DRG neurons containing SP, CGRP or CCK alone also were detected. Perikarya containing these combinations of neuropeptides were not found in autonomic ganglia. The peripheral axons of neurons containing immunoreactivity to at least SP and CGRP were damaged by chronic treatment with capsaicin. However, some sensory neurons containing CCK alone were not affected morphologically by capsaicin.These results clearly show that individual DRG neurons can contain many different neuropeptides. Furthermore, the combination of neuropeptides found in any particular neuron is related to its peripheral projection.     

The establishment of polarity by hippocampal neurons: the relationship between the stage of a cell's development in situ and its subsequent development in culture

Neurons removed from the embryonic hippocampus and placed into culture develop structurally and functionally distinct axonal and dendritic processes. The central issue addressed in this study concerns the extent to which the sequence of events which results in the differentiation of neurites by hippocampal neurons in culture is influenced by the cell's state of development in situ. [3H]thymidine was administered to pregnant rats either on Embryonic Day 15 (E15) or on E18.5 to label hippocampal neurons at known stages of their development. All fetuses were sacrificed on E19. Some of the fetal brains were sectioned and examined by autoradiography to determine the location of labeled cells in the hippocampus. The remaining brains were used to prepare hippocampal cell cultures. Neurons labeled at E18.5 remained confined to the ventricular zone at E19. Those labeled at E15 had completed their migration to the cortical plate. Other data suggest that the former cells had not yet initiated process outgrowth, while the latter cells had begun to elaborate both axons and dendrites. When introduced into culture, both populations of cells developed axons and dendrites and both compartmentalized MAP2 to the dendritic domain. Moreover, despite marked differences in their developmental state at the time of introduction into culture, both underwent the same sequence of developmental events leading to axonal and dendritic development. In a few cases cells that incorporated [3H]thymidine in situ at E18.5 apparently underwent mitosis in culture. These neurons also developed axons and dendrites appropriately. These results indicate that hippocampal neurons become polarized in culture, even if they have never developed axons or dendrites in situ, and do so as efficiently as cells that have become polarized before being placed into culture. Moreover, they indicate that the same sequence of events leading to the establishment of polarity occurs for hippocampal neurons with different developmental histories prior to culturing.     

The fine structure of the parietal retinas of Anolis carolinensis and Iguana iguana

An electron microscopic examination of the parietal retinas of Anolis carolinensis and Iguana iguana demonstrated within each retina (1) two distinct populations of neurons, (2) two populations of glia, and (3) a population of photoreceptors which could not be subdivided. A small population of very electron-dense cells, in many respects similar to photoreceptors, was also found in the iguana. Correspondingly dark processes were found in the plexiform layer of each retina. Parietal photoreceptors generally resemble cones of the lateral eye. Glial cells were sub-classified on the basis of the location of their somata and the disposition of their processes. Neurons were identified by virtue of their cytology and their reception of axosomatic ribbon synapses from unidentified plexiform layer processes. Neuronal subtypes were located on opposite sides of the plexiform layer. Neurons distal to that layer were found to project the initial segments of their processes into the plexiform layer parallel to its long axis, while neurons central to the plexiform layer projected axons centrally and dendrites radially into the plexiform layer. The existence of at least two neuronal populations and of interphotoreceptor synapses suggests that photosensory processing within the parietal retina may be more complex than previously assumed.     

Matrices with compliance comparable to that of brain tissue select neuronal over glial growth in mixed cortical cultures

Cortical neurons and astrocytes respond strongly to changes in matrix rigidity when cultured on flexible substrates. In this study, existing polyacrylamide gel polymerization methods were modified into a novel method for making substrates capable of engaging specific cell-adhesion receptors. Embryonic cortical dissociations were cultured on polyacrylamide or fibrin gel scaffolds of varying compliance. On soft gels, astrocytes do not spread and have disorganized F-actin compared to the cytoskeletons of astrocytes on hard surfaces. Neurons, however, extend long neurites and polymerize actin filaments on both soft and hard gels. Compared to tissue culture plastic or stiff gel substrates coated with laminin, on which astrocytes overgrow neurons in mixed cultures, laminin-coated soft gels encourage attachment and growth of neurons while suppressing astrocyte growth. The number of astrocytes on soft gels is lower than on hard even in the absence of mitotic inhibitors normally used to temper the astrocyte population. Dissociated embryonic rat cortices grown on flexible fibrin gels, a biomaterial with potential use as an implant material, display a similar mechano-dependent difference in cell population. The stiffness of materials required for optimal neuronal growth, characterized by an elastic modulus of several hundred Pa, is in the range measured for intact rat brain. Together, these data emphasize the potential importance of material substrate stiffness as a design feature in the next generation of biomaterials intended to promote neuronal regeneration across a lesion in the central nervous system while simultaneously minimizing the ingrowth of astrocytes into the lesion area.     

An age-related increase in resistance to DNA damage-induced apoptotic cell death is associated with development of DNA repair mechanisms

Neurons in the developing brain die via apoptosis after DNA damage, while neurons in the adult brain are generally resistant to these insults. The basis for this resistance is a matter of conjecture. We report here that cerebellar granule neurons (CGNs) in culture lose their competence to die in response to DNA damage as a function of time in culture. CGNs at either 1 day in vitro (DIV) or 7 DIV were treated with the DNA damaging agents camptothecin, UV or gamma-irradiation and neuronal survival measured. The younger neurons were effectively killed by these agents, while the older neurons displayed a significant resistance to killing. Neuronal survival did not change with time in culture when cells were treated with C2-ceramide or staurosporine, agents which do not target DNA. The resistance to UV irradiation developed over time in culture and was not due to changes in mitotic rate. Increases in DNA strand breakage, up-regulation of the levels of both p53 and its phosphorylated form and nuclear translocation of p53 were equivalent in both older and younger neurons, indicating a comparable p53 stress response. In addition, we show that treatment of older neurons with pharmacological inhibitors of distinct components of the DNA repair machinery promotes the accumulation of DNA damage and sensitizes these cells to the toxic effects of UV exposure. These data demonstrate that older neurons appear to be more proficient in DNA repair in comparison to their younger counterparts, and that this leads to increased survival after DNA damage.     

Quantitative morphological analysis of embryonic cockroach (Periplaneta americana) brain neurons developing in vitro

Neurons dissociated from the brain of embryonic cockroaches (Periplaneta americana) can be maintained in culture for several weeks. The survival as well as the progressive organization of the neurons into a complex network was studied during a 5-week period under different culture conditions. About 10% of the dissociated cells adhered to the culture dish. This figure remained constant throughout the culture. The cell diameter ranged from 10 to 20 microns and did not change significantly over time in culture. Whereas only a few cells exhibited neurites at the start of the culture, the number of cells exhibiting neurites increased to reach about 99% after 2 weeks. The different cells were then connected to each other, forming a network, which became more and more complex. The number of cells per cluster as well as the length and the diameter of the "connectives" that linked the different clusters were found to increase with time. The morphology of individual neurons within the network was visualized after intracellular injection of biocytin. Labeling with antibodies raised against serotonin or GABA indicated that neurons were able to differentiate and to acquire specific neurotransmitter fates. The serotonergic phenotype was found to appear progressively throughout the culture, in parallel with the formation of the network. Cell density, addition of fetal calf serum, and ecdysone were shown to influence the development of the network.     

骨髓间充质干细胞源神经细胞移植治疗帕金森病大鼠模型

目的探讨骨髓间充质干细胞（mesenchymal stemcells,MSCs）源神经细胞脑内移植对帕金森病（Parkinson s disease,PD）大鼠的治疗作用。方法贴壁培养法分离、培养大鼠骨髓MSCs,脑匀浆上清诱导第3代MSCs向神经细胞分化,采用免疫细胞化学法鉴定诱导分化后细胞的性质,激光共聚焦显微镜检测诱导前后细胞Ca2＋浓度变化,6只PD大鼠行纹状体内MSCs源神经细胞移植作为细胞移植组,6只PD大鼠作为对照组。细胞移植术后4周检测PD大鼠的行为变化,观察移植细胞在脑内的分布情况。结果倒置显微镜下可见MSCs呈纺锤形和多角形,有1～2个核仁,MSCs经脑匀浆上清诱导后其胞体折光性增强,发出数个细长突起,互相交织成网,有的似轴突。诱导后细胞表达神经元特异性标志物神经元特异性烯醇化酶（NSE）和神经丝蛋白（NF）,胞质Ca2＋荧光强度显著增强,可推测诱导后的细胞为MSCs源神经细胞,将BrdU标记的MSCs源神经细胞移植到PD大鼠纹状体治疗4周后,可见细胞散在分布于注射侧脑组织,有少量细胞可迁移到对侧脑组织,PD大鼠的旋转行为得到显著改善。结论MSCs源神经细胞移植治疗帕金森病大鼠可使其旋转行为得到改善。     

汉滩病毒感染体外培养神经元损伤及抗体对其保护作用

用体外培养的昆明小鼠胚胎脑皮质神经元,感染汉滩病毒后,　检测原癌基因ＦＯＳ的表达,加入ＨＦＲＳ病人恢复期血清,观察其对神经元的保护作用。将病毒　感染的神经元经免疫组化染色,检测原癌基因ＦＯＳ的快速表达;在病毒感染的神经元内加入Ｈ　ＦＲＳ病人恢复期血清,使用ＭＴＴ比色试验检测神经元存活情况。病毒感染的神经元ＦＯＳ表达明　显　增高,与对照组相比有显著性差异（Ｐ＜０．００１）;　ＨＦＲＳ病人恢复期血清保护病毒感染的神经元　组　与感染对照组神经元活性明显不同,有显著性差异（Ｐ＜０．００１）。　ＨＴＮＶ感染体外培养的神经元　,会使细胞内产生ＦＯＳ的快速表达,ＨＦＲＳ病人恢复期血清对病毒感染的神经元有一定的保护　作用。     

Selective attachment of neural cells to specific substrates including Cell-Tak, a new cellular adhesive

Adherence of embryonic hypothalamic cells and a homogeneous neuronal cell line was assessed on various substrates and compared to attachment to the new cellular and tissue adhesive, Cell-Tak. Cell-Tak provided the most advantageous surface with 100% of fetal brain cells attaching in 5 h. Attachment of hypothalamic cells to compounds such as poly-D-lysine or collagen within this time was increased by 45 and 25%, respectively, over tissue-culture plastic. All cells of the clonal cell line N2AB-1 attached to Cell-Tak in the presence or absence of fetal calf serum and were found to be resistant to trypsin removal. Conditioned medium from these cells enhanced attachment of N2AB-1 twofold when compared to adherence to tissue-culture plastic. Striking morphological changes were seen in N2AB-1 after culturing on Cell-Tak for 2 days. Thirty percent of the population extended long neurites when grown on Cell-Tak with serum. Without serum, 30 to 50% of the cells extended very broad neurites often branched at the end, which were morphological changes not seen on plastic surfaces. These findings indicate that Cell-Tak is an optimal adhesive for primary neural cell culture and maintenance. Moreover, this adhesive protein appears to induce neuritogenesis and cellular differentiation in a neuronal cell line.     

Differential control over postganglionic neurons in rat cardiac ganglia by NA and DmnX neurons: anatomical evidence

In previous single-labeling experiments, we showed that neurons in the nucleus ambiguous (NA) and the dorsal moto nucleus of the vagus (DmnX) project to intrinsic cardiac ganglia. Neurons in these two motor nuclei differ significantly in the size of their projection fields, axon caliber, and endings in cardiac ganglia. These differences in NA and DmnX axon cardiac projections raise the question as to whether they target the same, distinct, or overlapping populations of cardiac principal neurons. To address this issue, we examined vagal terminals in cardiac ganglia and trace injection sites in the brain stem using two different anterograde t ace s 1,1-dioleyl-3,3,3,3-tetramethylindocarbocyanine methanesulfonate and 4-[4-(dihexadecylamino)-styryl]-N-methylpyridinium iodide] and confocal microscopy in male Sprague-Dawley rats. We found that 1) NA and DmnX neurons innervate the same cardiac ganglia, but these axons target separate subpopulations of principal neurons and 2) axons arising from neurons in the NA and DmnX in the contralateral sides of the brain stem enter the cardiac ganglionic plexus through separate bundles and preferentially innervate principal neurons near their entry regions, providing topographic mapping of vagal motor neurons in left and right brain stem vagal nuclei. Because the NA and DmnX project to distinct populations of cardiac principal neurons, we propose that they may play different roles in controlling cardiac function.