Venous endothelin guides sympathetic innervation of the developing mouse heart.

The mechanisms responsible for establishing correct target innervation during organ development are largely unknown. Sympathetic nerves follow blood vessels--typically arteries--to reach their endorgans, suggesting the existence of vascular guidance cues that direct axonal extension. The sinoatrial node and the ventricle of the heart receive sympathetic innervation from the stellate ganglia (STG). Here we show that STG axons follow veins, specifically the superior vena cavae and sinus venosus, to reach these targets. We find that election of these routes is determined by venous endothelium-derived endothelin-1, acting through its specific receptor Ednra expressed within a subpopulation of STG neurons. Furthermore, we demonstrate that Edn1-Ednra signalling is essential for functional regulation of the heart by sympathetic nerves. Our findings present venous Edn1 as a sympathetic guidance cue, and show how axon guidance mechanisms are coordinated with endorgan morphogenesis.

Proliferation and cell cycle dynamics in the developing stellate ganglion.

Cell proliferation during nervous system development is poorly understood outside the mouse neocortex. We measured cell cycle dynamics in the embryonic mouse sympathetic stellate ganglion, where neuroblasts continue to proliferate following neuronal differentiation. At embryonic day (E) 9.5, when neural crest-derived cells were migrating and coalescing into the ganglion primordium, all cells were cycling, cell cycle length was only 10.6 h, and S-phase comprised over 65% of the cell cycle; these values are similar to those previously reported for embryonic stem cells. At E10.5, Sox10(+) cells lengthened their cell cycle to 38 h and reduced the length of S-phase. As cells started to express the neuronal markers Tuj1 and tyrosine hydroxylase (TH) at E10.5, they exited the cell cycle. At E11.5, when >80% of cells in the ganglion were Tuj1(+)/TH(+) neuroblasts, all cells were again cycling. Neuroblast cell cycle length did not change significantly after E11.5, and 98% of Sox10(-)/TH(+) cells had exited the cell cycle by E18.5. The cell cycle length of Sox10(+)/TH(-) cells increased during late embryonic development, and ∼25% were still cycling at E18.5. Loss of Ret increased neuroblast cell cycle length at E16.5 and decreased the number of neuroblasts at E18.5. A mathematical model generated from our data successfully predicted the relative change in proportions of neuroblasts and non-neuroblasts in wild-type mice. Our results show that, like other neurons, sympathetic neuron differentiation is associated with exit from the cell cycle; sympathetic neurons are unusual in that they then re-enter the cell cycle before later permanently exiting.

Cervico-thoracic ganglion: its clinical implications.

Lesions of the cervicothoracic ganglion (CTG) result in interruption of sympathetic fibers to the head, neck, upper limb, and thoracic viscera. The accurate understanding of the anatomy of the CTG is relevant to sympathectomy procedures that may be prescribed in cases where conventional intervention has failed. This study documents the incidence and distribution of the CTG to avoid potential complications such as Horner's syndrome and cardiac arrhythmias. This study utilized 48 cadavers, in which a total of 89 sympathetic chains were dissected. The inferior cervical ganglion (ICG) and the first thoracic ganglion was fused in 75 cases (84.3%) to form the CTG. It was present bilaterally in 48 of these specimens (65.3%). Three different shapes of CTG were differentiated, viz. spindle, dumbbell, and an inverted "L" shape. The dumbbell and inverted "L" shapes demonstrated a definite "waist" (i.e., a macroscopically visible union of the ICG and T1 components of the CTG). Rami from the CTG was distributed to the brachial plexus, the subclavian and vertebral arteries, the brachiocephalic trunk, and the cardiac plexus. This study demonstrates a high incidence of a double cardiac sympathetic nerve arising from CTG. It is therefore imperative that in the technique of sympathectomy, for intractable anginal pain, the surgeon excises both these rami but does not destroy the ganglion itself. The ever-improving technology in endoscopic surgery has made investigations into the nuances of the anatomy of the sympathetic chain essential.

c-ret regulates cholinergic properties in mouse sympathetic neurons: evidence from mutant mice.

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.

Long-lasting adverse effects of prenatal hypoxia on developing autonomic nervous system and cardiovascular parameters in rats.

To determine whether prenatal hypoxia increases the risk of developing cardiovascular disorders as an adult and, if so, the identity of the cell mechanisms involved in such dysfunction, we evaluated the sympathoadrenal system and central areas related to cardiovascular events during development and the cardiovascular parameters in adults. Pregnant rats were exposed to hypoxia (10% oxygen) from embryonic day (E) 5 to E20 and the offspring studied at 1, 3, 9 and 12 weeks of age for neurochemistry and at 12 weeks of age for cardiovascular analysis. In the 1-, 3- and 9-week-old offspring, the levels and utilization of catecholamines were reduced in sympathetic ganglia, in target organs, in adrenals and in the rostral part of the A2 cell group in the nucleus tractus solitarius, but were increased in the locus coeruleus. In the 12-week-old adult offspring, the lowered autonomic nervous activity was restricted to cardiac-related structures, i.e. the stellate ganglion, heart and adrenals. In adult rats, prenatal hypoxia did not affect the cardiac parameters under resting conditions but increased blood pressure and the variability of blood pressure and heart rate under stress conditions. The altered metabolic activity of the sympathoadrenal system and related central areas during development and at adulthood for most structures might be part of the potential mechanisms contributing to cardiovascular disorders in adults.

The development of synaptophysin immunoreactivity in the human sympathetic ganglia.

Using an indirect immunohistochemical method, synaptophysin immunoreactivity (SYN-IR) has been studied in cryostat sections of stellate and thoracic ganglia in human fetuses, neonates, infants and adults. In the course of development, a progressive increase in SYN-IR in axonal terminals and around nerve cells was demonstrated. In contrast, large clusters of small intensely fluorescent (SIF) cells and paraganglionic cells increased in number in fetuses and premature neonates at 24-25 weeks. Such SIF cell clusters varied in form and often occurred at pole or subcapsular areas of sympathetic ganglia close to blood vessels or paraganglia. With increasing gestational age and during infancy, a decrease in sizes of SIF cell groups and paraganglionic cells as well as changes in their distribution were found. The results show that the amount and distribution of SYN-IR is temporally related to the maturation and functional activity of human sympathetic ganglia neurons. It was suggested that numerous SIF cells and paraganglia in human prenatal sympathetic ganglia were both indicative of incomplete cell migration and an important source of regulation of ganglionic microcirculation under the conditions of relative hypoxia and immature nervous regulation.

Distribution of vasoactive intestinal polypeptide-, calcitonin gene-related peptide-, somatostatin- and neurofilament-immunoreactivities in sympathetic ganglia of human fetuses and premature neonates.

The distribution patterns of vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), somatostatin (SOM) and neurofilament (NF) immunoreactivities (IR) were studied in the stellate ganglia of human fetuses and neonates at 24-26 weeks gestation. Sizeable populations with some quantitative variations of VIP-, CGRP- and SOM immunoreactive nerve cells were detected in all ganglia studied. In marked contrast, neurofilament expression was down-regulated. The upregulation of VIP, CGRP and SOM expression suggested their inductor effect on growth and differentiation neurons as well as on the development of their neurotransmitter properties. The main neuropeptides-inducing factor of sympathetic ganglia in human prenatal ontogenesis may be considered as a relative hypoxia.

NT-3, like NGF, is required for survival of sympathetic neurons, but not their precursors.

Superior cervical ganglia of postnatal mice with a targeted disruption of the gene for neurotrophin-3 have 50% fewer neurons than those of wild-type mice. In culture, neurotrophin-3 increases the survival of proliferating sympathetic precursors. Both precursor death (W. ElShamy et al., 1996, Development 122, 491-500) and, more recently, neuronal death (S. Wyatt et al., 1997, EMBO J. 16, 3115-3123) have been described in mice lacking NT-3. Consistent with the second report, we found that, in vivo, neurogenesis and precursor survival were unaffected by the absence of neurotrophin-3 but neuronal survival was compromised so that only 50% of the normal number of neurons survived to birth. At the time of neuron loss, neurotrophin-3 expression, assayed with a lacZ reporter, was detected in sympathetic target tissues and blood vessels, including those along which sympathetic axons grow, suggesting it may act as a retrograde neurotrophic factor, similar to nerve growth factor. To explore this possibility, we compared neuron loss in neurotrophin-3-deficient mice with that in nerve growth factor-deficient mice and found that neuronal losses occurred at approximately the same time in both mutants, but were less severe in mice lacking neurotrophin-3. Eliminating one or both neurotrophin-3 alleles in mice that lack nerve growth factor does not further reduce sympathetic neuron number in the superior cervical ganglion at E17.5 but does alter axon outgrowth and decrease salivary gland innervation. Taken together these results suggest that neurotrophin-3 is required for survival of some sympathetic neurons that also require nerve growth factor.

System to identify individual somites and their derivatives in the developing mouse embryo.

The identification of the axial levels of metameric elements along the rostro-caudal axis of vertebrates until now was not possible before late, fetal development, when the vertebral anlagen first appear. We developed a new system for the exact axial identification of somites and their derivatives from early, embryonic stages of mouse development on (Theiler stages (TS) 15 to TS18-19). The initial axial identification of the somites was performed by relating them to the rostral-most two cervical spinal ganglia (SG), that exhibited characteristic morphologies (SG-C1: bar-like, SG-C2: triangular). At all stages of somitic development, the most prominent somite along the rostro-caudal axis correlated with the bar-like SG-C1, and, therefore, we named it the first cervical somite (SO-C1). The next step, the axial identification of the somites independently from the SG, was based on the observation that after in situ hybridization to Myf5, Pax3, Pax1, and Mox1 riboprobes, a distinct and characteristic morphology of the last occipital somite (SO-O5) and the first two cervical somites (SO-C1, SO-C2) can be observed. From TS15 on, these three somites formed a triad of the most prominent somites along the rostro-caudal axis. Also, the dermomyotomal, myotomal, and sclerotomal derivatives of this somite triad were the most prominent in later somitic development. Furthermore, SG-C1 and SG-C2 exhibited a transient bipartite anlagen in their early development, suggesting a "resegmentation" during SG formation. Later, when somites started to dissolve, the caudal moiety of the bar-like SG-C1 anlagen fused to the anlagen of SG-C2.

The cervical sympathetic trunk--a new hypothesis.

There is a sympathetic ganglion, in relation to most if not all thoracic and lumbar segments whereas there are only 3 or 4 sympathetic ganglia in the cervical region. Thus it may be inferred that sympathetic ganglia are not directly related to spinal nerves. Therefore, the hypothesis is put forward that sympathetic ganglia are associated chiefly with intersegmental vessels such as the intercostal and lumbar arteries and that the differences seen in the neck region are due to the disappearance of most of the cervical intersegmental arteries and the subsequent modifications that follow during development. This results in the fusion of the upper 4 cervical ganglia to form the superior cervical sympathetic ganglion in relation to the developing external carotid artery which seems to provide the necessary inductive stimulus. Furthermore, the antero-inferior migration of the heart, its corresponding arch arteries and the dorsal aorta bring about the formation of the ansa subclavia around the subclavian artery and the positioning of the stellate ganglion behind the vertebral artery.

On the development of the cerebellum of the trout, Salmo gairdneri. IV. Development of the pattern of connectivity.

Synaptogenesis has been studied in the corpus cerebelli of the trout Salmo gairdneri, Richardson, 1836. The first synapses are observed in hatchlings and occur between parallel fibres and the shafts of Purkinje dendrites. Subsequently the axosomatic synapses of Purkinje axon collaterals on the neurons of the ganglionic layer appear, and finally the synapses made by climbing fibres and mossy fibres, and by stellate cell axons develop. Young synapses in the cerebellum of the trout resemble the mature structures so closely that the criteria for the identification of the latter can also be applied to the former. The number of parallel fibre synapses and of Purkinje axon collateral synapses increases considerably during development. Eurydendroid cells, the axons of which leave the cerebellum, receive an abundance of Purkinje axon collaterals on their somata and main dendritic trunks. Mossy fibre synapses are numerous in the granular layer. Climbing fibre contacts and synapses of stellate cell axons, both with Purkinje cells, are found occasionally. The following pattern of connectivity is proposed. The main input-output system is formed by the mossy fibres, the granule cells, the Purkinje cells and the eurydendroid cells. Additional pathways are formed by (1) the mossy fibres, granule cells and eurydendroid cells, and (2) the climbing fibres, Purkinje cells and eurydendroid cells. The afferent-efferent systems, mentioned above, are influenced by a number of internuncial elements: (1) The Golgi cells receive their input from the parallel fibres and contact with their axon collaterals the dendrites of granule cells. (2) Axon collaterals of Purkinje cells are in synaptic relation with Golgi cells. (3) Axon collaterals of Purkinje cells impinge upon the somata and main dendrites of other Purkinje cells. (4) Stellate cells, which derive their input from the parallel fibres, synapse with dendrites and somata of Purkinje cells. The possible functional roles of all of these neuronal elements are discussed.