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Nager syndrome is a rare human developmental disorder characterized by hypoplastic neural crest-derived craniofacial bones and limb defects. Mutations in SF3B4 gene, which encodes a component of the spliceosome, are a major cause for Nager. A review of the literature indicates that 45% of confirmed cases are also affected by conductive, sensorineural or mixed hearing loss. Conductive hearing loss is due to defective middle ear ossicles, which are neural crest derived, while sensorineural hearing loss typically results from defective inner ear or vestibulocochlear nerve, which are both derived from the otic placode. Animal model of Nager syndrome indicates that upon Sf3b4 knockdown cranial neural crest progenitors are depleted, which may account for the conductive hearing loss in these patients. To determine whether Sf3b4 plays a role in otic placode formation we analyzed the impact of Sf3b4 knockdown on otic development. Sf3b4-depleted Xenopus embryos exhibited reduced expression of several pan-placodal genes six1, dmrta1 and foxi4.1. We confirmed the dependence of placode genes expression on Sf3b4 function in animal cap explants expressing noggin, a BMP antagonist critical to induce placode fate in the ectoderm. Later in development, Sf3b4 morphant embryos had reduced expression of pax8, tbx2, otx2, bmp4 and wnt3a at the otic vesicle stage, and altered otic vesicle development. We propose that in addition to the neural crest, Sf3b4 is required for otic development, which may account for sensorineural hearing loss in Nager syndrome., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.) more...

The aim of this study was to determine the main stages of submandibular salivary gland development during the embryonic period in humans. In addition, we studied submandibular salivary gland development in rats on embryonic days 14-16 and expression in the submandibular salivary gland region with the monoclonal antibody HNK-1. Serial sections from 25 human embryos with a greatest length ranging from 10 to 31 mm (Carnegie stages 16-23; weeks 5.5-8 of development) and Wistar rats of embryonic days (E) 14-16 were analysed with light microscopy. Five stages of submandibular salivary gland development were identified. The prospective stage (1), between weeks 5.5 and early week 6, is characterized by a thickening of the epithelium of the medial paralingual groove in the floor of the mouth corresponding to the primordium of the submandibular salivary gland parenchyma. At this stage, the primordium of the parasympathetic ganglion lies below the lingual nerve. The primordium of the submandibular salivary gland parenchyma is observed in rats on E14 in the medial paralingual groove with mesenchymal cells, underlying the lingual nerve. These cells are HNK-1-positive, corresponding to the primordium of the parasympathetic ganglion. The bud stage (2), at the end of week 6 in humans and on E15 in rats, is characterized by the proliferation and invagination of the epithelial condensation, surrounded by an important condensation of the mesenchyme. The pseudoglandular stage (3) at week 6.5 is characterized by the beginning of the formation of lobes in the condensed mesenchyme. The canalicular stage (4), between week 7 and 7.5, is characterized by the appearance of a lumen in the proximal part of the submandibular duct. The innervation stage (5) occurs during week 8, with the innervation of the submandibular and interlobular ducts. Nervous branches arriving from the parasympathetic ganglion innervate the glandular parenchyma. Numerous blood vessels are observed nearby. Our results suggest that submandibular salivary gland development requires interactions among epithelium, mesenchyme, parasympathetic ganglion and blood vessels., (© 2019 Anatomical Society.) more...

Background: Naturally hypoxic conditions in amniote embryos play important roles in normal development. We previously showed that a hypoxic condition is required to produce a sufficient amount of neural crest cells (NCCs) during embryogenesis and that promoting a hypoxic response by prolyl-hydroxylase (PHD) inhibitors increases NCCs. Given that PHD inhibitors are considered as a potential treatment for anemia and ischemic diseases, we investigated the phenotypic effect of PHD inhibitors on embryonic development., Methods: Chick embryos were administered with PHD inhibitors prior to the induction of NCCs on day 1.5. Three main events relating to hypoxia, NCCs induction, vasculogenesis and chondrogenesis, were examined., Results: PHD inhibitors caused an increase of Sox10-positive NCCs in vivo. Vasculogenesis was promoted temporarily, although rapid vasculogenesis diminished the effect by day 5 in cephalic and pharyngeal regions. Studies on chondrogenesis at day 7 showed advanced development of the otic capsule, a cartilaginous structure encapsulating the inner ear. Analysis by X-ray micro-computed-tomography (μCT) revealed smaller otic capsule, suggesting premature differentiation. This in turn, deformed the developing semicircular canals within it. Other skeletal structures such as the palate and jaw were unaffected. The localized effect on the otic capsule was considered a result of the multiple effects from the hypoxic responses, increased NCCs and promoted chondrogenesis., Conclusion: Given the wide range of clinical applications being considered for PHD inhibitors, this study provides crucial information to caution and guide use of PHD inhibitors when treating women of childbearing age., (© 2018 Wiley Periodicals, Inc.) more...

The otic and pterygopalatine ganglia are located close to the greater wing (alisphenoid) of the sphenoid bone and many researchers have noted nerves connecting these ganglia in human embryos. The greater wing (alisphenoid) arises from the cartilaginous ala temporalis independently of the lesser wing, but no topographical changes between this cartilage and nerve elements have been demonstrated. We examined histological sections of 20 human embryos and fetuses from 6 to 15 weeks of development (WD). At 6 WD, the ala temporalis, the alar process and ganglia were all identified as a single, undifferentiated cell mass. Subsequently, the two ganglia became identifiable, but were continuous on the superior side of the initial ala temporalis. The temporal, superior spine of the ala temporalis was surrounded by the part that connected the ganglia. At 7 WD, the superior spine of the ala temporalis was reduced in size and the continuity of these ganglia was lost. At this point, a secondarily-formed communicating branch between the ganglia, the nervus sphenoidalis was first identifiable. At 9 WD, the ala temporalis and the alar process had clearly become cartilages, and the anterior end of the otic ganglion was separated from the ala temporalis. The nervus sphenoidalis became longer. At 15 WD, the otic and pterygopalatine ganglia were clear separated from the alisphenoid, which consisted of the cartilaginous ala temporalis and membranous bone. Consequently, the separation between the otic and pterygopalatine ganglia seemed to be due to the developing ala temporalis. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.) more...

Neurogenesis in embryonic sympathetic ganglia involves neuroblasts that resume proliferation following neuronal differentiation. As cell cycle exit is not associated with neuronal differentiation, the identity of proliferating neuroblasts is incompletely understood. Here, we use sympathetic ganglia of chick embryos to define the timing of neurogenesis and neuroblast identity focusing on the expression and function of the transcription factor Prox1. We show that a large fraction of neuroblasts has initially withdrawn from the cell cycle at embryonic day 3 (E3), which is reflected by a high proportion of p27(+)/Islet1(+) neuroblasts (63%) and low numbers of EdU(+)/Islet1(+) cells (12%). The proportion of proliferating Islet1(+) neuroblasts, identified by EdU pulse labeling and by the absence of the postmitotic marker p27 increases to reach maximal levels at E5, when virtually all neuroblasts are in the cell cycle (95%). Subsequently, the proportion of EdU-labeled and p27(-) neuroblasts is reduced to reach low levels at E11. Interestingly, the expression of the transcription factor Prox1 is restricted to the neuronal lineage, that is, Sox10(+)/Phox2b(+) neuron progenitors, proliferating p27(-)/Islet1(+) neuroblasts and nascent neurons but is rapidly lost in postmitotic neurons. In vitro and in vivo knockdown and overexpression experiments demonstrate effects of Prox1 in the support of neuroblast proliferation and survival. Taken together, these results define the neurogenesis period in the chick paravertebral sympathetic ganglia including an initial cell cycle withdrawal and identify Prox1 as a marker and regulator of proliferating sympathetic neuroblasts., (© 2015 Wiley Periodicals, Inc.) more...

Programmed cell death during chicken ciliary ganglion (CG) development is mostly discussed as an extrinsically regulated process, guided either by the establishment of a functional balance between preganglionic and postganglionic activity or the availability of target-derived neurotrophic factors. We found that the expression of the gene coding for the nuclear retinoic acid receptor β (RARB) is transiently upregulated prior to and during the execution phase of cell death in the CG. Using retroviral vectors, the expression of RARB was knocked down during embryonic development in ovo. The knockdown led to a significant increase in CG neuron number after the cell death phase. BrdU injections and active caspase-3 staining revealed that this increase in neuron number was due to an inhibition of apoptosis during the normal cell death phase. Furthermore, apoptotic neuron numbers were significantly increased at a stage when cell death is normally completed. While the cholinergic phenotype of the neurons remained unchanged after RARB knockdown, the expression of the proneural gene Cash1 was increased, but somatostatin-like immunoreactivity, a hallmark of the mature choroid neuron population, was decreased. Taken together, these results point toward a delay in neuronal differentiation as well as cell death. The availability of nuclear retinoic acid receptor β (RARβ) and RARβ-induced transcription of genes could therefore be a new intrinsic cue for the maturation of CG neurons and their predisposition to undergo cell death., (© 2015 The Authors Developmental Neurobiology Published by Wiley Periodicals, Inc.) more...

The TGF-β ligand superfamily members activin A and BMP control important aspects of embryonic neuronal development and differentiation. Both are known to bind to activin receptor subtypes IIA (ActRIIA) and IIB, while in the avian ciliary ganglion (CG), so far only ActRIIA-expression has been described. We show that the expression of ACVR2B, coding for the ActRIIB, is tightly regulated during CG development and the knockdown of ACVR2B expression leads to a deregulation in the execution of neuronal apoptosis and therefore affects ontogenetic programmed cell death in vivo. While the differentiation of choroid neurons was impeded in the knockdown, pointing toward a reduction in activin A-mediated neural differentiation signaling, naturally occurring neuronal cell death in the CG was not prevented by follistatin treatment. Systemic injections of the BMP antagonist noggin, on the other hand, reduced the number of apoptotic neurons to a similar extent as ACVR2B knockdown. We therefore propose a novel pathway in the regulation of CG neuron ontogenetic programmed cell death, which could be mediated by BMP and signals via the ActRIIB., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.) more...

Parasympathetic innervation is critical for submandibular gland (SMG) development and regeneration. Parasympathetic ganglia (PSG) are derived from Schwann cell precursors that migrate along nerves, differentiate into neurons, and coalesce within their target tissue to form ganglia. However, signals that initiate gangliogenesis after the precursors differentiate into neurons are unknown. We found that deleting negative regulators of FGF signaling, Sprouty1 and Sprouty2 (Spry1/2DKO), resulted in a striking loss of gangliogenesis, innervation, and keratin 5-positive (K5+) epithelial progenitors in the SMG. Here we identify Wnts produced by K5+ progenitors in the SMG as key mediators of gangliogenesis. Wnt signaling increases survival and proliferation of PSG neurons, and inhibiting Wnt signaling disrupts gangliogenesis and organ innervation. Activating Wnt signaling and reducing FGF gene dosage rescues gangliogenesis and innervation in both the Spry1/2DKO SMG and pancreas. Thus, K5+ progenitors produce Wnt signals to establish the PSG-epithelial communication required for organ innervation and progenitor cell maintenance., (Copyright © 2015 Elsevier Inc. All rights reserved.) more...

Fibroblast growth factors (Fgfs) play important roles in developmental processes of the inner ear, including the ontogeny of the statoacoustic ganglia (SAG) and hair cells. However, the detailed genetic mechanism(s) underlying Fgf/Fgfr-dependent otic neural development remains elusive. Using conditional genetic approaches and inhibitory small molecules, we have revealed that Fgfr-PI3K/Akt signaling is mainly responsible for zebrafish SAG development and have determined that Sox9a and Atoh1a act downstream of Fgfr-Akt signaling to specify and/or maintain the otic neuron fate during the early segmentation stage. Sox9a and Atoh1a coregulate numerous downstream factors identified through our ChIP-seq analyses, including Tlx2 and Eya2. Fgfr-Erk1/2 signaling contributes to ultricular hair cell development during a critical period between 9 and 15 hours postfertilization. Our work reveals that a genetic network of the previously known sensory determinant Atoh1 and the neural crest determinant Sox9 plays critical roles in SAG development. These newly uncovered roles for Atoh1and Sox9 in zebrafish otic development may be relevant to study in other species., (Copyright © 2015 the authors 0270-6474/15/350234-11$15.00/0.) more...

The peripheral autonomic nervous system reaches far throughout the body and includes neurons of diverse functions, such as sympathetic and parasympathetic. We show that the parasympathetic system in mice--including trunk ganglia and the cranial ciliary, pterygopalatine, lingual, submandibular, and otic ganglia--arise from glial cells in nerves, not neural crest cells. The parasympathetic fate is induced in nerve-associated Schwann cell precursors at distal peripheral sites. We used multicolor Cre-reporter lineage tracing to show that most of these neurons arise from bi-potent progenitors that generate both glia and neurons. This nerve origin places cellular elements for generating parasympathetic neurons in diverse tissues and organs, which may enable wiring of the developing parasympathetic nervous system., (Copyright © 2014, American Association for the Advancement of Science.) more...

Neural crest cells migrate extensively and give rise to most of the peripheral nervous system, including sympathetic, parasympathetic, enteric, and dorsal root ganglia. We studied how parasympathetic ganglia form close to visceral organs and what their precursors are. We find that many cranial nerve-associated crest cells coexpress the pan-autonomic determinant Paired-like homeodomain 2b (Phox2b) together with markers of Schwann cell precursors. Some give rise to Schwann cells after down-regulation of PHOX2b. Others form parasympathetic ganglia after being guided to the site of ganglion formation by the nerves that carry preganglionic fibers, a parsimonious way of wiring the pathway. Thus, cranial Schwann cell precursors are the source of parasympathetic neurons during normal development., (Copyright © 2014, American Association for the Advancement of Science.) more...

Interactions between neurons and their targets of innervation influence many aspects of neural development. To examine how synaptic activity interacts with neurotrophic signaling, we determined the effects of blocking neuromuscular transmission on survival and axonal outgrowth of ciliary neurons from the embryonic chicken ciliary ganglion. Ciliary neurons undergo a period of cell loss due to programmed cell death between embryonic Days (E) 8 and 14 and they innervate the striated muscle of the iris. The nicotinic antagonist d-tubocurarine (dTC) induces an increase in branching measured by counting neurofilament-positive voxels (NF-VU) in the iris between E14-17 while reducing ciliary neuron survival. Blocking ganglionic transmission with dihyro-β-erythroidin and α-methyllycacontine does not mimic dTC. At E8, many trophic factors stimulate neurite outgrowth and branching of neurons placed in cell culture; however, at E13, only GDNF stimulates branching selectively in cultured ciliary neurons. The GDNF-induced branching at E13 could be inhibited by BDNF. Blocking ret signaling in vivo with a dominant negative (dn)ret decreases survival of ciliary and choroid neurons at E14 and prevents dTC induced increases in NF-VU in the iris at E17. Blocking TRKB signaling with dn TRKB increases NF-VU in the iris at E17 and decreases neuronal survival at E17, but not at E14. Thus, RET promotes survival during programmed cell death in the ciliary ganglion and contributes to promoting branching when synaptic transmission is blocked while TRKB inhibits branching and promotes maintenance of neuronal survival. These studies highlight the multifunctional nature of trophic molecule function during neuronal development., (Copyright © 2012 Wiley Periodicals, Inc.) more...

Very little is known about the development of cardiac parasympathetic ganglia and cholinergic innervation of the mouse heart. Accordingly, we evaluated the growth of cholinergic neurons and nerve fibers in mouse hearts from embryonic day 18.5 (E18.5) through postnatal day 21(P21). Cholinergic perikarya and varicose nerve fibers were identified in paraffin sections immunostained for the vesicular acetylcholine transporter (VAChT). Satellite cells and Schwann cells in adjacent sections were identified by immunostaining for S100β calcium binding protein (S100) and brain-fatty acid binding protein (B-FABP). We found that cardiac ganglia had formed in close association to the atria and cholinergic innervation of the atrioventricular junction had already begun by E18.5. However, most cholinergic innervation of the heart, including the sinoatrial node, developed postnatally (P0.5-P21) along with a doubling of the cross-sectional area of cholinergic perikarya. Satellite cells were present throughout neonatal cardiac ganglia and expressed primarily B-FABP. As they became more mature at P21, satellite cells stained strongly for both B-FABP and S100. Satellite cells appeared to surround most cardiac parasympathetic neurons, even in neonatal hearts. Mature Schwann cells, identified by morphology and strong staining for S100, were already present at E18.5 in atrial regions that receive cholinergic innervation at later developmental times. The abundance and distribution of S100-positive Schwann cells increased postnatally along with nerve density. While S100 staining of cardiac Schwann cells was maintained in P21 and older mice, Schwann cells did not show B-FABP staining at these times. Parallel development of satellite cells and cholinergic perikarya in the cardiac ganglia and the increase in abundance of Schwann cells and varicose cholinergic nerve fibers in the atria suggest that neuronal-glial interactions could be important for development of the parasympathetic nervous system in the heart., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.) more...

Background: The neural crest (NC) and placode are transient neurogenic cell populations that give rise to cranial ganglia of the vertebrate head. The formation of the anterior NC- and placode-derived ganglia has been shown to depend on the single activity of either Neurog1 or Neurog2. The requirement of the more posterior cranial ganglia on Neurog1 and Neurog2 is unknown., Results: Here we show that the formation of the NC-derived parasympathetic otic ganglia and placode-derived visceral sensory petrosal and nodose ganglia are dependent on the redundant activities of Neurog1 and Neurog2. Tamoxifen-inducible Cre lineage labeling of Neurog1 and Neurog2 show a dynamic spatiotemporal expression profile in both NC and epibranchial placode that correlates with the phenotypes of the Neurog-mutant embryos., Conclusion: Our data, together with previous studies, suggest that the formation of cranial ganglia along the anterior-posterior axis is dependent on the dynamic spatiotemporal activities of Neurog1 and/or Neurog2 in both NC and epibranchial placode., (Copyright © 2011 Wiley-Liss, Inc.) more...

The cranial parasympathetic ganglia have been reported to paradoxically contain the sympathetic nerve marker, tyrosine hydroxylase (TH), in addition to neurons expressing parasympathetic markers such as vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS). However, the distribution of these molecules in the cranial ganglia of human fetuses has not yet been examined. Using paraffin sections from 10 mid-term human fetuses (12-15 weeks), we performed immunohistochemistry for TH, VIP, and nNOS in the parasympathetic ciliary, pterygopalatine, otic, and submandibular ganglia, and for comparison, the sensory inferior vagal ganglion. The ciliary and submandibular ganglia contained abundant TH-positive neurons. In the former, TH-positive neurons were much more numerous than nNOS-positive neurons, whereas in the latter, nNOS immunoreactivity was extremely strong. No or a few cells in the pterygopalatine, otic, and inferior vagal ganglia expressed TH. Ciliary TH neurons appeared to compensate for classically described sympathetic fibers arising from the superior cervical ganglion, whereas in the submandibular ganglion, nNOS-positive neurons as well as TH neurons might innervate the lingual artery in addition to the salivary glands. Significant individual variations in the density of all these markers suggested differences in sensitivity to medicine affecting autonomic nerve function. Consequently, in the human cranial autonomic ganglia, it appears that there is no simple dichotomy between sympathetic and parasympathetic function., (Copyright © 2011 Wiley Periodicals, Inc.) more...

The extracellular membrane-associated protein F-spondin has been implicated in cell-matrix and cell-cell adhesion and plays an important role in axonal pathfinding. We report here that F-spondin is expressed in non-neuronal cells in the embryonic chicken ciliary ganglion (CG) and robustly promotes survival of cultured CG neurons. Using deletion constructs of F-spondin we found that the amino-terminal Reelin/Spondin domain cooperates with thrombospondin type 1 repeat (TSR) 6, a functional TGFβ-activation domain. In ovo treatment with blocking antibodies raised against the Reelin/Spondin domain or the TSR-domains caused increased apoptosis of CG neurons during the phase of programmed cell death and loss of about 30% of the neurons compared to controls. The Reelin/Spondin domain receptor - APP and its downstream signalling molecule disabled-1 are expressed in CG neurons. F-spondin induced rapid phosphorylation of disabled-1. Moreover, both blocking the central APP domain and interference with disabled-1 signalling disrupted the survival promoting effect of F-spondin. Taken together, our data suggest that F-spondin can promote neuron survival by a mechanism involving the Reelin/Spondin and the TSR domains., (Copyright © 2010 Elsevier Inc. All rights reserved.) more...

The maintenance of a progenitor cell population as a reservoir of undifferentiated cells is required for organ development and regeneration. However, the mechanisms by which epithelial progenitor cells are maintained during organogenesis are poorly understood. We report that removal of the parasympathetic ganglion in mouse explant organ culture decreased the number and morphogenesis of keratin 5-positive epithelial progenitor cells. These effects were rescued with an acetylcholine analog. We demonstrate that acetylcholine signaling, via the muscarinic M1 receptor and epidermal growth factor receptor, increased epithelial morphogenesis and proliferation of the keratin 5-positive progenitor cells. Parasympathetic innervation maintained the epithelial progenitor cell population in an undifferentiated state, which was required for organogenesis. This mechanism for epithelial progenitor cell maintenance may be targeted for organ repair or regeneration. more...

The wiring of the nervous system arises from extensive directional migration of neuronal cell bodies and growth of processes that, somehow, end up forming functional circuits. Thus far, this feat of biological engineering appears to rely on sequences of pathfinding decisions upon local cues, each with little relationship to the anatomical and physiological outcome. Here, we uncover a straightforward cellular mechanism for circuit building whereby a neuronal type directs the development of its future partners. We show that visceral afferents of the head (that innervate taste buds) provide a scaffold for the establishment of visceral efferents (that innervate salivatory glands and blood vessels). In embryological terms, sensory neurons derived from an epibranchial placode--that we show to develop largely independently from the neural crest--guide the directional outgrowth of hindbrain visceral motoneurons and control the formation of neural crest-derived parasympathetic ganglia. more...

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels encoded by the Slo1 gene are often components of large multiprotein complexes in excitable and nonexcitable cells. Here we show that Slo1 proteins interact with Neph1, a member of the immunoglobulin superfamily expressed in slit diaphragm domains of podocytes and in vertebrate and invertebrate nervous systems. This interaction was established by reciprocal coimmunoprecipitation of endogenous proteins from differentiated cells of a podocyte cell line, from parasympathetic neurons of the embryonic chick ciliary ganglion, and from HEK293T cells heterologously expressing both proteins. Neph1 can interact with all three extreme COOH-terminal variants of Slo1 (Slo1(VEDEC), Slo1(QEERL), and Slo1(EMVYR)) as ascertained by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation. Neph1 is partially colocalized in intracellular compartments with endogenous Slo1 in podocytes and ciliary ganglion neurons. Coexpression in HEK293T cells of Neph1 with any of the Slo1 extreme COOH-terminal splice variants suppresses their steady-state expression on the cell surface, as assessed by cell surface biotinylation assays, confocal microscopy, and whole cell recordings. Consistent with this, small interfering RNA (siRNA) knockdown of endogenous Neph1 in embryonic day 10 ciliary ganglion neurons causes an increase in steady-state surface expression of Slo1 and an increase in whole cell Ca(2+)-dependent K(+) current. Surprisingly, a comparable Neph1 knockdown in podocytes causes a decrease in surface expression of Slo1 and a decrease in whole cell BK(Ca) currents. In podocytes, Neph1 siRNA also caused a decrease in nephrin, even though the Neph1 siRNA had no sequence homology with nephrin. However, we could not detect nephrin in ciliary ganglion neurons. more...

Vertebrate alpha-bungarotoxin-like molecules of the Ly-6 superfamily have been implicated as balancers of activity and survival in the adult nervous system. To determine whether a member of this family could be involved in the development of the avian ciliary ganglion, we identified 6 Gallus genes by their homology in structure to mouse lynx1 and lynx2. One of these genes, an ortholog of prostate stem cell antigen (psca), is barely detectable at embryonic day (E) 8, before neuronal cell loss in the ciliary ganglion, but increases >100-fold as the number of neurons begins to decline between E9 and E14. PSCA is highly expressed in chicken and mouse telencephalon and peripheral ganglia and correlates with expression of alpha7-containing nicotinic acetylcholine receptors (alpha7-nAChRs). Misexpressing PSCA before cell death in the ciliary ganglion blocks alpha7-nAChR activation by nicotine and rescues the choroid subpopulation from dying. Thus, PSCA, a molecule previously identified as a marker of prostate cancer, is a member of the Ly-6 neurotoxin-like family in the nervous system, and is likely to play a role as a modulator of alpha7 signaling-induced cell death during development. more...

Onuf's nucleus in the lumbosacral spinal cord, comprising somatic motoneurons that innervate the pelvic floor muscles via the pudendal nerve, shares some characteristics with the autonomic preganglionic neurons and functions in coordination with the autonomic nervous system. In mouse, neurons projecting to the urethral sphincter and ischiocavernosus muscles form the dorsolateral (DL) nucleus at the caudal lumbar levels, whereas neurons projecting to the limb and hip joint muscles comprise the retrodorsolateral and ventral nucleus, as well as the DL nucleus at the rostral lumbar levels. The results of the present study in mouse revealed that the expression pattern of a LIM homeodomain protein Islet-1, an embryonic marker for motoneurons in the spinal cord, was different among motoneuronal groups at the prenatal stage (embryonic days 13.5-15.5); the highest expression was observed in the DL at the caudal lumbar cord, whereas there was little expression in the lateral part of the rostral DL. Islet-1 expression was also observed in the parasympathetic preganglionic neurons at the sacral spinal cord. These findings provide evidence that the DL neurons at the caudal lumbar cord, corresponding to Onuf's nucleus, are chemically distinct among the motoneuronal groups at the prenatal stages. This differential Islet-1 expression among the motoneuronal groups suggests that Islet-1 not only leads to a motoneuronal lineage, but also to the differentiation of motoneuronal subsets in the lumbosacral spinal cord. more...

Background: Pelvic ganglia are derived from the sacral neural crest and contain both sympathetic and parasympathetic neurons. Various members of the neurotrophin and GDNF families of neurotrophic factors have been shown to play important roles in the development of a variety of peripheral sympathetic and parasympathetic neurons; however, to date, the role of these factors in the development of pelvic ganglia has been limited to postnatal and older ages. We examined the effects of NGF, NT-3, GDNF, neurturin and artemin on cell migration and neurite outgrowth from explants of the pelvic ganglia from embryonic and newborn mice grown on collagen gels, and correlated the responses with the immunohistochemical localization of the relevant receptors in fixed tissue., Results: Cell migration assays showed that GDNF strongly stimulated migration of tyrosine hydroxylase (TH) cells of pelvic ganglia from E11.5, E14.5 and P0 mice. Other factors also promoted TH cell migration, although to a lesser extent and only at discrete developmental stages. The cells and neurites of the pelvic ganglia were responsive to each of the GDNF family ligands--GDNF, neurturin and artemin--from E11.5 onwards. In contrast, NGF and NT-3 did not elicit a significant neurite outgrowth effect until E14.5 onwards. Artemin and NGF promoted significant outgrowth of sympathetic (TH+) neurites only, whereas neurturin affected primarily parasympathetic (TH-negative) neurite outgrowth, and GDNF and NT-3 enhanced both sympathetic and parasympathetic neurite outgrowth. In comparison, collagen gel assays using gut explants from E11.5 and E14.5 mice showed neurite outgrowth only in response to GDNF at E11.5 and to neurturin only in E14.5 mice., Conclusion: Our data show that there are both age-dependent and neuron type-dependent differences in the responsiveness of embryonic and neo-natal pelvic ganglion neurons to growth factors. more...

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) are neurotrophic factors for parasympathetic neurons including ciliary ganglion (CG) neurons. Recently, we have shown that survival and signaling mediated by GDNF in CG neurons essentially requires transforming growth factor beta (TGFbeta). We have provided evidence that TGFbeta regulates the availability of the glycosyl phosphatidylinositol (GPI)-anchored GDNF receptor alpha 1 (GFRalpha1) by promoting the recruitment of the receptor to the plasma membrane. We report now that in addition to GDNF, NRTN, but not persephin (PSPN) or artemin (ARTN), is able to promote survival of CG neurons. Interestingly, in contrast to GDNF, NRTN is not dependent on cooperation with TGFbeta, but efficiently promotes neuronal survival and intracellular signaling in the absence of TGFbeta. Additional treatment with TGFbeta does not further increase the NRTN response. Both NRTN and GDNF exclusively bind to and activate their cognate receptors, GFRalpha2 and GFRalpha1, respectively, as shown by the use of receptor-specific neutralizing antibodies. Immunocytochemical staining for the two receptors on the surface of CG neurons reveals that, in contrast to the effect on GFRalpha1, TGFbeta is not required for recruitment of GFRalpha2 to the plasma membrane. Moreover, binding of radioactively labeled GDNF but not NRTN is increased upon treatment of CG neurons with TGFbeta. Disruption of TGFbeta signaling does interfere with GDNF-, but not NRTN-mediated signaling and survival. We propose a model taking into account data from GFRalpha1 crystallization and ontogenetic development of the CG that may explain the differences in TGFbeta-dependence of GDNF and NRTN. more...

Neurotrophic molecules are key retrograde influences of cell survival in the developing nervous system, but other influences such as activity are also emerging as important factors. In the avian ciliary ganglion, half the neurons are eliminated between embryonic day 8 (E8) and E14, but it is not known how cell death is initiated. Because systemic application of alpha7-nicotinic acetylcholine receptor (nAChR) antagonists prevents this cell loss, we examined differences in receptor densities and responses of intracellular calcium to nicotine using the calcium-sensitive dye fura-2. In addition, we determined whether cell-autonomous inhibition of alpha7 activation in neurons prevented cell death. E8 neurons are heterogeneous with respect to alpha7-nAChR density, which leads to large increases in [Ca2+]i in some neurons; E8 neurons also exhibit a slower rate of Ca2+ decay after nicotinic stimulation than E13 neurons. Expressing alpha-bungarotoxin that is tethered to the membrane by a glycosylphosphatidylinositol linkage (GPIalpha btx) in ciliary ganglion neurons with the retroviral vector RCASBP(A) blocks increases in intracellular calcium induced by nicotine through alpha7-nAChRs and prevents neurons from dying. Expression of GPIalpha btx in surrounding non-neural tissues, but not in neurons, does not prevent cell loss. Furthermore, the GPIalpha btx is not efficiently expressed in the accessory oculomotor neurons, eliminating preganglionic inputs as another site for action of the antagonist. These results support the hypothesis that cholinergic inputs facilitate cell death in the developing autonomic nervous system by activating alpha7-nAChRs, possibly by leading to increases in intracellular calcium that exceed the threshold for cell survival. more...

GABA (gamma-aminobutyric acid), the major inhibitory transmitter in the brain, goes through a transitory phase of excitation during development. The excitatory phase promotes neuronal growth and integration into circuits. We show here that spontaneous nicotinic cholinergic activity is responsible for terminating GABAergic excitation and initiating inhibition. It does so by changing chloride transporter levels, shifting the driving force on GABA-induced currents. The timing of the transition is critical, because the two phases of GABAergic signaling provide contrasting developmental instructions. Synergistic with nicotinic excitation, GABAergic inhibition constrains neuronal morphology and innervation. The results reveal a multitiered activity-dependent strategy controlling neuronal development. more...

Basic fibroblast growth factor (bFGF) is a potent and multifunctional neurotrophic factor that can influence neuronal survival and differentiation. It has been shown to modulate growth and orientation of neuritic processes both in intact organs and in neuronal cultures, with a wide spectrum of effects on different preparations. Here we report that it promotes neurite growth in developing parasympathetic neurons from the chick ciliary ganglion. We have used both organotypic cultures and dissociated neurons, and we have combined assessment of global neurite growth by immunocytochemical techniques with evaluation of dynamic parameters of single neurites via time-lapse microscopy. We show that laminin, a molecule of the extracellular matrix that has been associated with stimulation of neurite extension, has only a limited and short-lived effect on neurite outgrowth. In contrast, bFGF can promote global growth of the neuritic network both in whole ganglia and in dissociated cultures for times up to 48 hr, and this effect is related to an increase in the growth rate of single neurites. Moreover, the effect can be observed even in enriched neuronal cultures, pointing to a direct action of bFGF on neurons. more...

Pituitary adenylate cyclase-activating polypeptide (PACAP) is expressed in the parasympathetic ciliary ganglion (CG) and modulates nicotinic acetylcholine receptor function. PACAP also provides trophic support, promoting partial survival of CG neurons in culture and full survival when accompanied by membrane depolarization. We probed the adenylate cyclase (AC) and phospholipase-C (PLC) transduction cascades stimulated by PACAP to determine their respective roles in supporting neuronal survival and examined their interaction with signals generated by membrane activity. While PLC-dependent signaling was dispensable, AC-generated signals proved critical for PACAP to support neuronal survival. Specifically, PACAP-supported survival was mimicked by 8Br-cAMP and blocked by inhibiting either PKA or the phosphorylation of mitogen-activated protein kinase (MAPK). The ability of PACAP to promote survival was additionally dependent on spontaneous activity as blocking Na+ or Ca2+ channel currents completely abrogated trophic effects. Our results underscore the importance of coordinated MAPK- and activity-generated signals in transducing neuropeptide-mediated parasympathetic neuronal survival. more...

In developing ciliary ganglion (CG) neurons, movement of functional large-conductance (BK type) Ca(2+)-activated K+ (K(Ca)) channels to the cell surface is stimulated by the endogenous growth factors TGF(beta)1 and beta-neuregulin-1 (NRG1). Here we show that a brief NRG1 treatment (0.5-1.5 h) mobilizes K(Ca) channels in a post-Golgi compartment, but longer treatments (>3.5 h) mobilize K(Ca) channels located in the endoplasmic reticulum or Golgi apparatus. Specifically, the effects of 3.5 h NRG1 treatment were completely blocked by treatments that disrupt Golgi apparatus function. These include inhibition of microtubules, or inhibition of the ADP-ribosylation factor-1 (ARF1) system by brefeldin A, by over-expression of dominant-negative ARF1, or over-expression of an ARF1 GTPase-activating protein that blocks ARF1 cycling between GTP- and GDP-bound states. These treatments had no effect on stimulation of K(Ca) evoked by 1.5 h treatment with NRG1, indicating that short-term responses to NRG1 do not require an intact Golgi apparatus. By contrast, both the acute and sustained effects of NRG1 were inhibited by treatments that block trafficking processes that occur close to the plasma membrane. Thus mobilization of K(Ca) was blocked by treatments than inhibit ADP-ribosylation factor-6 (ARF6) signaling, including overexpression of dominant-negative ARF6, dominant-negative ARNO, or dominant-negative phospholipase D1. TGF(beta)1, the effects of which on K(Ca) are much slower in onset, is unable to selectively mobilize channels in the post-Golgi pool, and its effects on K(Ca) are completely blocked by inhibition of microtubules, Golgi function and also by plasma membrane ARF6 and phospholipase D1 signaling. more...

Purpose: In the current study, the appearance and development of choroidal ganglion cells (CGCs) was investigated in eyes of 18 human donors between the 13th week of gestation (wog) and 42 years of age., Methods: The number and diameter of CGCs was evaluated in scleral and choroidal whole mounts stained for NADPH diaphorase. To demonstrate the synaptic input of the CGCs, sections were stained with antibodies against synaptophysin, vesicular acetylcholin transporter, tyrosin hydroxylase, and vesicular monoaminergic transporter 2., Results: Clusters of small CGCs were first seen in the 18th wog next to the nasal and temporal long ciliary nerves. Immunohistochemistry in the 25th wog revealed 3298 and 5429 nitric oxide synthase/NADPH diaphorase positive CGCs, surrounded by numerous cholinergic and aminergic boutons. The number of CGCs decreased to 1000-2500 after birth. During postnatal development, the CGCs spread into small groups, distributed all over the choroid. The size of CGCs increased markedly up to adulthood., Conclusions: CGCs appear late during choroidal development, in parallel with the differentiation of the outer vascular layers. This coincidence might point to the primary role of the CGCs as specific vasoregulators in species with a well-developed fovea centralis. more...

The trafficking of large-conductance Ca2+-activated K+ channels (K(Ca)) in chick ciliary ganglion neurons is regulated by growth factors. Here we show that a canonical p38 cascade inhibits K(Ca) trafficking in ciliary ganglion neurons. Two different p38 inhibitors (SB202190 or SB203580) or over-expression of dominant-negative forms of several components of the p38 cascade increased K(Ca) in ciliary neurons. Inhibition of protein synthesis or Golgi processing had no effect on this phenomenon, suggesting that p38 is acting at a distal step of the trafficking pathway. Depolymerization of filamentous actin (F-actin) increased functional expression of K(Ca), whereas stabilization of F-actin inhibited the effect of SB202190 on K(Ca) trafficking. SB202190 also caused an immunochemically detectable increase in K(Ca) on the plasma membrane. Inhibition of p38 decreased the extent of cortical F-actin in ciliary neurons. Macroscopic K(Ca) is suppressed by transforming growth factor (TGF) beta3. Application of TGFbeta3 increased the phosphorylation of p38 in ciliary neurons and increased cortical F-actin. Thus, the p38 signaling cascade endogenously suppresses development of functional K(Ca), in part by stabilizing an F-actin barrier that prevents plasma membrane insertion of functional channel complexes. This cascade also appears to mediate inhibitory effects of TGFbeta3 on the expression of K(Ca). more...

The cloaca, the caudal limit of the avian gastrointestinal tract, acts as a collecting chamber into which the gastrointestinal, urinary, and genital tracts discharge. It is intrinsically innervated by the enteric nervous system, which is derived from neural crest emigres that migrate from the vagal and sacral regions of the neural tube. Abnormal cloacal development can cause a number of anorectal anomalies, including persistent cloaca. Ablation of the vagal neural crest has previously been shown to result in an aganglionic hindgut to the extent of the colorectum. The aim of our study was to investigate the effect of vagal neural crest ablation on the cloaca, the limit of the hindgut in the developing chick embryo. Chick embryos were incubated until the 10-12 somite stage. The vagal neural tube corresponding to the level of somites 3-6 was then ablated, and eggs were incubated until harvested on embryonic day 11 (E11). Whole chick embryos were fixed, embedded in paraffin, and sectioned. Immunohistochemistry was then carried out using the HNK-1 monoclonal antibody to label neural crest cells, and results were assessed by light microscopy. Vagal neural crest ablation resulted in a dramatic decrease in the number of neural crest cells colonizing the chick embryo cloaca compared with control embryos. Ablated embryos contained only a small number of HNK-1-positive neural crest cells, which were scattered within the myenteric plexus in a disorganised pattern. Hypoganglionosis was also evident in other regions of the hindgut in ablated embryos. Ablation of the vagal neural crest results in a hypoganglionic cloaca in addition to hypoganglionosis of the hindgut. These results suggest that the cloaca is largely innervated by vagal neural crest emigres. Further studies involving quail-chick chimeras to investigate the exact contribution provided by both vagal and sacral neural crest cells to the cloaca should increase our understanding of the pathophysiology of conditions like persistent cloaca. more...

The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance K(Ca) channels in ciliary ganglion (CG) neurons evoked by beta-neuregulin-1 (NRG1) and transforming growth factor-beta1 (TGFbeta1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGF(beta)1. NRG1- and TGF(beta)1-evoked stimulation of K(Ca) is blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional K(Ca) channels. NRG1 and TGF(beta)1 evoked an Akt-dependent increase in cell-surface SLO alpha-subunits. These procedures have no effect on voltage-activated Ca2+ currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons. more...

Rapid trafficking of glutamate receptors contributes importantly to synaptic plasticity, but whether similar trafficking extends to other ionotropic receptors is unknown. Nicotinic acetylcholine receptors containing alpha7 subunits are widely expressed in the nervous system and allow calcium influx. Because of this, alpha7-containing receptors regulate diverse events, depending on the signaling pathways available. We find that the receptors codistribute with target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) postsynaptically and that nicotinic stimulation rapidly induces SNARE-dependent vesicular endocytosis accompanied by receptor internalization. At the same time, a SNARE-dependent process recruits receptors to the cell surface from internal pools. Overall, the trafficking does not markedly change the number of surface receptors or their combined whole-cell response to nicotine. SNARE-dependent trafficking is needed, however, for the receptors to remain capable of activating the transcription factor cAMP response element-binding protein and attendant gene expression when repeatedly challenged. Thus, trafficking appears to be essential for maintaining functional coupling between alpha7-receptor responses and downstream signaling. more...

During neuromuscular development, "stop signals" present on the target myotube inhibit motor axon growth. Mice lacking either the neuronal form of agrin or the muscle-specific tyrosine kinase (MuSK) lose stop signal activity, suggesting that they are part of such signals or induce them in myotubes. To test whether MuSK complexes form stop signals in the absence of myotube signaling, we cultured ciliary ganglion (CG) neurons with nonmuscle cells expressing cell-surface MuSK. Expression of MuSK had no effect on neuronal adhesion. MuSK expression, however, inhibited neurite outgrowth from CG neurons, but not retinal ganglion cell neurons. The neurite-inhibitory effect could be completely reversed by an antibody to the MuSK extracellular domain, and partially reversed by an antibody to agrin, suggesting that inhibition is mediated by a complex of these proteins. Thus, an agrin/MuSK complex may form part of a motor neuron stop signal involved in "reverse signaling" to the motor neuron. more...

Arachidonic acid (AA, 20:4) has been reported to modulate a variety of calcium-permeable ionic channels, both in the plasma membrane and in the endoplasmic reticulum. We have studied the effects of AA on calcium signaling in a well-characterized model of developing peripheral neurons, embryonic chick ciliary ganglion neurons in culture. When given at low non-micellar concentrations (5 microM), in the majority of cells AA directly activated a delayed and long-lasting increase in [Ca2+]i, involving both the cytoplasm and the nucleoplasm, that was completely reversed by abolition of extracellular calcium. Other fatty acids (FAs), either saturated like arachidic acid (20:0), or unsaturated like linoleic (18:2) and docosahexaenoic acid (22:6), shared its ability to activate calcium influx. This entry was not suppressed by voltage-dependent calcium channel inhibitors omega-conotoxin and nifedipine, by the voltage-independent calcium channel antagonist LOE-908, by pre-treatment with blockers of AA metabolic pathways or with pertussis toxin. The arachidonate-activated calcium pathway was permeable to Mn2+ and blocked by La3+, Gd3+ and Ni2+. In a neuronal subpopulation, AA at the same concentration was also able to elicit calcium release from thapsigargin-sensitive intracellular stores; we provide evidence that cytochrome P450 epoxygenase is involved in this process., (Copyright 2005 S. Karger AG, Basel) more...

Sympathetic neurons are specified during their development from neural crest precursors by a network of crossregulatory transcription factors, which includes Mash1, Phox2b, Hand2 and Phox2a. Here, we have studied the function of Gata2 and Gata3 zinc-finger transcription factors in autonomic neuron development. In the chick, Gata2 but not Gata3 is expressed in developing sympathetic precursor cells. Gata2 expression starts after Mash1, Phox2b, Hand2 and Phox2a expression, but before the onset of the noradrenergic marker genes Th and Dbh, and is maintained throughout development. Gata2 expression is affected in the chick embryo by Bmp gain- and loss-of-function experiments, and by overexpression of Phox2b, Phox2a, Hand2 and Mash1. Together with the lack of Gata2/3 expression in Phox2b knockout mice, these results characterize Gata2 as member of the Bmp-induced cluster of transcription factors. Loss-of-function experiments resulted in a strong reduction in the size of the sympathetic chain and in decreased Th expression. Ectopic expression of Gata2 in chick neural crest precursors elicited the generation of neurons with a non-autonomic, Th-negative phenotype. This implies a function for Gata factors in autonomic neuron differentiation, which, however, depends on co-regulators present in the sympathetic lineage. The present data establish Gata2 and Gata3 in the chick and mouse, respectively, as essential members of the transcription factor network controlling sympathetic neuron development. more...

The protocadherin families pcdh-alpha, beta, and gamma have been proposed to mediate synaptic specificity via homophilic interactions. Here we report isolation of two pcdh-alpha family members from chick. We find pcdh-alpha mRNA in multiple regions of chick CNS including cerebellum, tectum, olfactory bulb, and forebrain, and in the autonomic nervous system. Immunoblots identify major components of 120 and 140 kDa both in brain and ciliary ganglion extracts. Immunohistochemistry reveals pcdh-alphas in axons and perisynaptically in preganglionic terminals, adjacent to transmitter release sites. Pcdh-alphas appear to be absent from postsynaptic sites: They are nonoverlapping with postsynaptic receptor clusters in the ganglion and are rapidly lost after ganglionic denervation. Similar pcdh-alpha patterns are found in motor axons and at neuromuscular junctions of birds and mammals, and persist into adulthood. The results indicate that pcdh-alphas are widely expressed in nicotinic cholinergic pathways and may engage in heterophilic interactions at synapses and on axons. more...

Normal cognitive and autonomic functions require nicotinic synaptic signaling. Despite the physiological importance of these synapses, little is known about molecular mechanisms that direct their assembly during development. We show here that the tumor-suppressor protein adenomatous polyposis coli (APC) functions in localizing alpha3-nicotinic acetylcholine receptors (nAChRs) to neuronal postsynaptic sites. Our quantitative confocal microscopy studies indicate that APC is selectively enriched at cholinergic synapses; APC surface clusters are juxtaposed to synaptic vesicle clusters and colocalize with alpha3-nAChRs but not with the neighboring synaptic glycine receptors or perisynaptic alpha7-nAChRs on chick ciliary ganglion (CG) neurons. We identify PSD (postsynaptic density)-93, beta-catenin, and microtubule end binding protein EB1 as APC binding partners. PSD-93 and beta-catenin are also enriched at alpha3-nAChR postsynaptic sites. EB1 shows close proximity to and partial overlap with alpha3-nAChR and APC surface clusters. We tested the role of APC in neuronal nicotinic synapse assembly by using retroviral-mediated in vivo overexpression of an APC dominant-negative (APC-dn) peptide to block the interaction of endogenous APC with both EB1 and PSD-93 during synapse formation in CG neurons. The overexpressed APC-dn led to dramatic decreases in alpha3-nAChR surface levels and clusters. Effects were specific to alpha3-nAChR postsynaptic sites; synaptic glycine receptor and perisynaptic alpha7-nAChR clusters were not altered. In addition, APC-dn also reduced surface membrane-associated clusters of PSD-93 and EB1. The results show that APC plays a key role in organizing excitatory cholinergic postsynaptic specializations in CG neurons. We identify APC as the first nonreceptor protein to function in localizing nAChRs to neuronal synapses in vivo. more...

Parasympathetic neurons do not require neurotrophins for survival and are thought to lack high-affinity neurotrophin receptors (i.e., trks). We report here, however, that mRNAs encoding both brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (trkB) are expressed in the parasympathetic chick ciliary ganglion (CG) and that BDNF-like protein is present in the ganglion and in the iris, an important peripheral target of ciliary neurons. Moreover, CG neurons express surface trkB and exogenous BDNF not only initiates trk-dependent signaling, but also alters nicotinic acetylcholine receptor (nAChR) expression and synaptic transmission. In particular, BDNF applied to CG neurons rapidly activates cAMP-dependent response element-binding protein (CREB), and over the long-term selectively upregulates expression of alpha7-subunit-containing, homomeric nAChRs (alpha7-nAChRs), increasing alpha7-subunit mRNA levels, alpha7-nAChR surface sites, and alpha7-nAChR-mediated whole-cell currents. At nicotinic synapses formed on CG neurons in culture, brief and long-term BDNF treatments also increase the frequency of spontaneous EPSCs, most of which are mediated by heteromeric nAChRs containing alpha3, alpha5, beta4, and beta2 subunits (alpha3*-nAChRs) with a minor contribution from alpha7-nAChRs. Our findings demonstrate unexpected roles for BDNF-induced, trk-dependent signaling in CG neurons, both in regulating expression of alpha7-nAChRs and in enhancing transmission at alpha3*-nAChR-mediated synapses. The presence of BDNF-like protein in CG and iris target coupled with that of functional trkB on CG neurons raise the possibility that signals generated by endogenous BDNF similarly influence alpha7-nAChRs and nicotinic synapses in vivo. more...

Nicotine's ability to enhance neurotransmitter release has implicated presynaptic nicotinic acetylcholine receptors (nAChRs) in synaptic modulation, but there aze few examples where presynaptic nAChRs are known to be activated by nerve-released transmitter. We searched for endogenous activation of presynaptic nAChRs in the calyceal nerve terminals of the chick ciliary ganglion by imaging presynaptic calcium transients using dextran-coupled indicator dyes. The amplitude of Ca(+)signals recorded in individual nerve terminals was frequency dependent over 2-50 Hz. Calcium transients evoked by stimulation of the preganglionic nerve were significantly reduced (approximately 10-15%) by the nonspecific nAChR antagonist d-tubocurarine (d-TC; 100 microM) and the alpha7-specific antagonist methyllycaconitine (20-50 nM) but were not affected by 10 microM dihydro-beta-erythroidine, which should inhibit several non-alpha7 nAChRs. Feedback was rapid and did not require a stimulation-dependent build-up of transmitter, as d-TC and MLA reduced the amplitude of the first calcium transient in a 2-Hz train. Choline is an agonist at alpha7 nAChRs but is not the sole agonist in this system, as inhibition of acetylcholinesterase by echothiophate failed to reduce calcium transients. These results show that nerve-released acetylcholine (ACh) feeds back onto presynaptic alpha7 nAChRs to enhance calcium signals within the terminal. This feedback may help maintain the high rate of transmission at this cholinergic synapse. more...

The chick ciliary ganglion is a neural crest-derived parasympathetic ganglion that innervates the eye. Here, we examine its axial level of origin and developmental relationship to other ganglia and nerves of the head. Using small, focal injections of DiI, we show that neural crest cells arising from both the caudal half of the midbrain and the rostral hindbrain contribute to the ciliary as well as the trigeminal ganglion. Precursors to both ganglia have overlapping migration patterns, moving first ventrolaterally and then rostrally toward the optic vesicle. At the level of the midbrain/forebrain junction, precursors to the ciliary ganglion separate from the main migratory stream, turn ventromedially, and condense in the vicinity of the rostral aorta and Rathke's pouch. Ciliary neuroblasts first exit the cell cycle at early E2, prior to and during ganglionic condensation, and neurogenesis continues through E5.5. By E3, markers of neuronal differentiation begin to appear in this population. By labeling the ectoderm with DiI, we discovered a new placode, caudal to the eye and possibly contiguous to the trigeminal placode, that contributes a few early differentiating neurons to the ciliary ganglion, oculomotor nerve, and connecting branches to the ophthalmic nerve. These results suggest for the first time a dual neural crest and placodal contribution to the ciliary ganglion and associated nerves. more...

Large-conductance Ca(2+)-activated K(+) channels (K(Ca)) in chick ciliary ganglion neurons are regulated by target-derived TGFbeta1. Here we show that TGFbeta1 stimulation of K(Ca) expression was blocked by the structurally dissimilar Ras protein farnesyl transferase inhibitors manumycin-A and FTI-277. A similar effect was produced in ciliary neurons overexpressing RasN17, a widely used dominant-negative form of Ras. Moreover, TGFbeta1-evoked increases in phosphorylation of SMAD2 were reduced by manumycin-A, suggesting that Ras-dependent transduction cascades activated by TGFbeta1 feed back onto SMAD signaling. Thus, Ras is a mediator of pleiotropic TGFbeta1 signaling in developing neurons. more...

Changes in the intrinsic spike discharge properties in one neuronal population can alter the functions and even the formation of an entire neuronal network. Therefore it is important to understand the factors that regulate acquisition of a mature electrophysiological phenotype. Here we focus on large-conductance K(Ca) channels, which shape the pattern of repetitive discharge and which are therefore likely to play a role in the refinement of neural networks during development. In the parasympathetic ciliary ganglion of chick, the developmental expression of K(Ca) channels coincides with stages at which ciliary cells form synapses with target tissues. Moreover, K(Ca) expression requires formation of synapses with target tissues, and with afferent preganglionic inputs. The trophic effect of targets is mediated by TGFbeta1, whereas the effect of the preganglionic input is mediated by an isoform of beta-neuregulin-1. These trophic factors act synergistically, and this appears to be a normal feature of their actions in vivo. The acute effects of TGFbeta1 entail translocation of preexisting K(Ca) channels from intracellular stores to the plasma membrane. This requires activation of the signaling enzymes Ras, Erk MAP kinase and PI3 kinase. TGFbeta1 also causes a more sustained increase in K(Ca) channels (i.e. for up to 2 weeks) that requires synthesis of new channel proteins. Inductive regulation of K(Ca) expression is also observed in CNS cells that form more complex networks. In lumbar motoneurons, the largest changes in K(Ca) expression coincide with the elimination of synapses with hindlimb targets. Interactions with target tissues play a key role in regulation of motoneuron K(Ca) expression, and this trophic effect of target muscle is mediated by GDNF or a closely related factor. In addition, K(Ca) expression in motoneurons is dependent on ongoing electrical activity both in vivo and in vitro. This provides an additional mechanism for use-dependent refinement of neural networks during embryonic development. more...

To investigate the effect of the N-terminal Slit2 protein on neuronal survival and development, recombinant human N-terminal Slit2 (N-Slit2) was assayed against isolated embryonic chick dorsal root ganglion sensory, ciliary ganglion and paravertebral sympathetic neurons. N-Slit2 promoted significant levels of neuronal survival and neurite extension in all of these populations. The protein was also assayed against postnatal mouse dorsal root ganglion neurons and found to promote neuronal survival in a similar manner. These findings suggest the Slit proteins may play an important role during development of the nervous system, mediating cellular survival in addition to the well documented role these proteins play in axonal and neuronal chemorepulsion. more...

Alpha7-nicotinic acetylcholine receptors (nAChRs) are widely expressed in the vertebrate nervous system. alpha7-nAChR functions include postsynaptic transmission, modulating neurotransmitter release, reinforcing nicotine addiction, and a role in neurological disorders, such as schizophrenia and Alzheimer's disease. In chick parasympathetic ciliary ganglion (CG) neurons, alpha7-nAChRs are excluded from the synapse and localize perisynaptically. Despite their extrasynaptic distribution, the highly Ca2+-permeable alpha7-nAChRs have important synapse-related Ca2+-dependent signaling functions in the CG. We show here that the synaptic partners regulate alpha7-nAChR expression during synapse formation in embryonic CG neurons in situ. The absence of inputs and target tissues cause reductions in alpha7-nAChR mRNA and protein levels that primarily resemble those seen for synaptic alpha3-nAChRs. However, there is a difference in their regulation. alpha7-nAChR levels are downregulated by reduced activity, whereas alpha3-nAChR levels are not. We propose that the activity-dependent regulation of extrasynaptic alpha7-nAChR levels may be an important mechanism for postsynaptic CG neurons to detect changes in presynaptic activity levels and respond with Ca2+-dependent plasticity changes in gene expression. more...

Rapid synaptic transmission in the embryonic chicken ciliary ganglion occurs through the activation of two distinct classes of nicotinic acetylcholine receptors (AChRs): those containing alpha3 subunits (alpha 3*-AChRs) and those containing alpha7 subunits (alpha 7*-AChRs). alpha3*-AChRs are found on ciliary neurons in clusters at synaptic sites on the cell body, whereas alpha7* -AChRs are found on somatic spines, which historically were thought not to have release sites in the embryo. However, Shoop et al. (Shoop et al. [1999] J. Neurosci. 19:692-704) recently described release sites having pre- and postsynaptic densities on somatic spines. We used transmission electron microscopy to compare the structure of synaptic sites on spines with those on the smooth surfaced part of the cell. We find that the two populations of sites are similar in active zone length, number of vesicles, and distance between vesicles and active zone. To study the functional properties of these sites, we examined their stimulation-dependent uptake and release of the extracellular tracer horseradish peroxidase (HRP). We found that each class of release sites both took up and released HRP in a stimulation- and calcium-dependent manner. The mean fraction of synaptic vesicles labeled with tracer was similar for the two populations, both after loading ( approximately 45%) and after unloading ( approximately 7%). Thus we detect no differences between these two anatomically distinct classes of release sites, other than their incidence: sites on spines occurred only 12% as often as those on the cell body. The release sites on somatic spines presumably underlie synaptic responses attributable to alpha7*-AChRs., (Copyright 2002 Wiley-Liss, Inc.) more...

The electrical responses elicited by the muscarinic cholinergic pathway have been studied in cultured embryonic chick ciliary ganglion (CG) neurons. Neurons obtained from E7-E8 ganglia were maintained in serum-free medium for 1 to 3 days. Stimulation with 50 microM muscarine induced depolarizing responses in about 30% of the cells tested. In voltage clamp experiments at a holding potential of -50 mV, an inward current could be recorded in the same percentage of cells in response to muscarinic stimulation. In single channel experiments, with standard physiological solution in the pipette, muscarine transiently activated an inward conducting channel. Cell-attached recordings with 100 mM CaCl(2) in the pipette provided evidence that muscarinic agonists can activate a cationic calcium-permeable channel. Two main conductance levels could be detected, of 2.3+/-0.6 and 5.6+/-0.6 pS, respectively. In excised patches, addition of 5-20 microM inositol 1,4,5-trisphosphate (InsP(3)) to the bath reactivated a channel that could be blocked by heparin and whose characteristics were very similar to those of the channel seen in response to muscarinic stimulation. A channel with similar properties has been previously shown to be activated by basic fibroblast growth factor (bFGF) and InsP(3) in the same preparation. more...