Ciliary Neurons Research Articles

Neurogenesis in directly and indirectly developing enteropneusts: of nets and cords.

Concerning the evolution of deuterostomes, enteropneusts (acorn worms) occupy a pivotal role as they share some characteristics with chordates (e.g., tunicates and vertebrates) but are also closely related to echinoderms (e.g., sea urchin). The nervous system in particular can be a highly informative organ system for evolutionary inferences, and advances in fluorescent microscopy have revealed overwhelming data sets on neurogenesis in various clades. However, immunocytochemical descriptions of neurogenesis of juvenile enteropneusts are particularly scarce, impeding the reconstruction of nervous system evolution in this group. We followed morphogenesis of the nervous system in two enteropneust species, one with direct (Saccoglossus kowalevskii) and the other with indirect development (Balanoglossus misakiensis), using an antibody against serotonin and electron microscopy. We found that all serotonin-like immunoreactive (LIR) neurons in both species are bipolar ciliary neurons that are intercalated between other epidermal cells. Unlike the tornaria larva of B. misakiensis, the embryonic nervous system of S. kowalevskii lacks serotonin-LIR neurons in the apical region as well as an opisthotroch neurite ring. Comparative analysis of both species shows that the projections of the serotonin-LIR somata initially form a basiepidermal plexus throughout the body that disappears within the trunk region soon after settlement before the concentrated dorsal and ventral neurite bundles emerge. Our data reveal a highly conserved mode of neurogenesis in enteropneusts that is independent of the developing mode and is inferred to be a common feature for Enteropneusta. Moreover, all detected serotonin-LIR neurons are presumably receptor cells, and the absence of serotonin-LIR interneurons from the enteropneust nervous system, which are otherwise common in various bilaterian central nervous systems, is interpreted as a loss that might have occurred already in the last common ancestor of Ambulacraria.

New mechanisms and treatment modalities for retinal degenerative diseases

R and macular degenerative diseases due to dysfunction of photoreceptors are a leading cause of blindness worldwide. Photoreceptors are highly polar and metabolically active ciliary neurons. They have a distinct inner segment and outer segment (OS). The OS is considered is an extension of a primary cilium and boasts of being a privileged compartment. The composition of the OS, with regards to the proteins and lipids, is distinct from the rest of the neuron. The OS is also composed of a coin-stack shaped arrangement of thousands of membranous discs loaded with the chromophore rhodopsin and other proteins involved in phototransduction cascade. Intriguingly, ~10% of the tip of the OS discs is shed every day and new discs are added at the base of the OS. The shedding is mediated by the interaction of the OS with the overlying retinal pigment epithelium (RPE). Owing to their involvement in development and maintenance of visual cascade, defects in the formation or composition of the OS or in the shedding by the RPE result in photoreceptor degenerative diseases. In this session, we will focus on recent advances in delineating the underlying mechanism of photoreceptor OS development and shedding pathways. Specifically, this session will present evidence for involvement of ciliary proteins in modulating OS composition and disc maintenance as well as ascertaining the role of potassium channel proteins in the RPE in photoreceptor survival and function. These studies will also assist in the development of rational treatment modalities for these blinding disorders.

Differential effects of ret and TRKB on axonal branching and survival of parasympathetic neurons

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.

HoxB8 in noradrenergic specification and differentiation of the autonomic nervous system

Different prespecification of mesencephalic and trunk neural crest cells determines their response to environmental differentiation signals and contributes to the generation of different autonomic neuron subtypes, parasympathetic ciliary neurons in the head and trunk noradrenergic sympathetic neurons. The differentiation of ciliary and sympathetic neurons shares many features, including the initial BMP-induced expression of noradrenergic characteristics that is, however, subsequently lost in ciliary but maintained in sympathetic neurons. The molecular basis of specific prespecification and differentiation patterns has remained unclear. We show here that HoxB gene expression in trunk neural crest is maintained in sympathetic neurons. Ectopic expression of a single HoxB gene, HoxB8, in mesencephalic neural crest results in a strongly increased expression of sympathetic neuron characteristics like the transcription factor Hand2, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) in ciliary neurons. Other subtype-specific properties like RGS4 and RCad are not induced. HoxB8 has only minor effects in postmitotic ciliary neurons and is unable to induce TH and DBH in the enteric nervous system. Thus, we conclude that HoxB8 acts by maintaining noradrenergic properties transiently expressed in ciliary neuron progenitors during normal development. HoxC8, HoxB9, HoxB1 and HoxD10 elicit either small and transient or no effects on noradrenergic differentiation, suggesting a selective effect of HoxB8. These results implicate that Hox genes contribute to the differential development of autonomic neuron precursors by maintaining noradrenergic properties in the trunk sympathetic neuron lineage.

Caenorhabditis elegans ciliary protein NPHP-8, the homologue of human RPGRIP1L, is required for ciliogenesis and chemosensation

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage renal failure in children and young adults. NPHP8/ RPGRIP1L is a novel ciliary gene that, when mutated, in addition to causing NPHP, also causes Joubert syndrome (JBTS) and Meckel syndrome (MKS). The exact function of NPHP8 and how defects in NPHP8 lead to human diseases are poorly understood. Here, we studied the Caenorhabditis elegans homolog nphp-8 ( C09G5.8) and explored the possible function of NPHP-8 in ciliated sensory neurons. We determined the gene structure of nphp-8 through rapid amplification of cDNA ends (RACE) analysis and discovered an X-box motif that had been previously overlooked. Moreover, NPHP-8 co-localized with NPHP-4 at the transition zone at the base of cilia. Mutation of nphp-8 led to abnormal dye filling (Dyf) and shorter cilia lengths in a subset of ciliary neurons. In addition, chemotaxis to several volatile attractants was significantly impaired in nphp-8 mutants. Our data suggest that NPHP-8/ RPGRIP1L plays an important role in cilia formation and cilia-mediated chemosensation in a cell type-specific manner.

Differential bulbar and extrabulbar projections of diverse olfactory receptor neuron populations in the adult zebrafish (Danio rerio)

Immunohistochemical methods were used to characterize the expression of two calcium-binding proteins, calretinin (CR) and S100, in the olfactory rosette of the adult zebrafish. These proteins are expressed in different sets of sensory neurons, and together represent a large proportion of these cells. Double immunofluorescence for CR and Gα(olf) protein, and CR immunoelectron microscopy, indicated that most CR-immunoreactive (ir) cells were ciliary neurons. Differential S100- and CR-ir projections to glomerular fields of the olfactory bulb were also observed, although these projections overlap in some glomeruli. Application of the carbocyanine dye DiI to either S100-ir or CR-ir glomerular regions led to labeling of cells mostly similar to S100-ir and CR-ir neurons, respectively. Instead, these bulbar regions project to similar telencephalic targets. On the other hand, antibodies against keyhole limpet hemocyanin (KLH)-stained numerous sensory cells in the olfactory rosette, including cells that were CR- and S100-negative. This antiserum also stained most primary bulbar projections and revealed extrabulbar olfactory primary projections coursing to the ventral area of the telencephalon through the medial olfactory tract. This extrabulbar projection was confirmed by tract-tracing with DiI. A loose association of this extrabulbar primary olfactory projection and the catecholaminergic populations of the ventral area was also observed with double tyrosine hydroxylase/KLH-like immunohistochemistry. Comparison between KLH-like-ir pathways and the structures revealed by FMRFamide immunohistochemistry (a marker of terminal ganglion cells and fibers) indicated that the KLH-like-ir extrabulbar projection was different from the terminal nerve system. The significance of the extrabulbar olfactory projection of zebrafish is discussed.

Network model for visually mediated ciliary locomotion in Hermissenda

The overall goal of this study is to investigate the ways in which learning modifies behavior. A combination of computational and empirical studies is being used to address this issue. Empirical studies investigate learning from a cellular and synaptic perspective in the relatively simple nervous system of the nudibranch mollusk Hermissenda [1-3]. Pavlovian conditioning produces light-elicited inhibition of normal positive phototaxis in Hermissenda. Learning changes both cellular excitability and synaptic strength in the neural circuit that supports phototaxis. In the present study, a model of the circuit that supports visually mediated locomotion (Fig. ​(Fig.1A)1A) was developed. Consistent with empirical observations, simulated responses to light increased the level of VP1 spike activity (Fig. 1B1), which is equivalent to positive phototaxis. Simulations indicated that phototaxis resulted from disinhibition of VP1. Light increased activity in Ie and decreased activity in Ii (Fig. 1B2). The net result was less activity in IIIi and disinhibition of VP1 (Fig. 1B2). Simulations also indicated that disinhibition produced phototaxis only if VP1 had a high level of tonic firing. The model is being refined and expanded, and will be used to investigate the generation of other behaviors (e.g., foot contraction), the responses to other sensory inputs (e.g., gravity), and the influence of learning-induced plasticity (e.g., increased Ie excitability and decreased VP1 tonic firing). Simulations also will help identify features of the model that warrant further empirical investigation. Figure 1 Network model for visually mediated ciliary locomotion. A: The model had six cells: LB, lateral B-type photoreceptor; Ib, Ie, Ii and IIIi, interneurons; VP1, ciliary motor neuron. The cells were Hodgkin-Huxley-like neurons. Membrane conductances also ...

HAND transcription factors are required for neonatal sympathetic neuron survival

Expression of the basic helix-loop-helix transcription factor HAND2 begins early in sympathetic neuron development and is essential for the differentiation of noradrenergic neurons. Here, we show that the expression of HAND2 and related HAND1 are maintained in sympathetic neurons throughout fetal and postnatal development when these neurons depend on target-derived nerve growth factor (NGF) for survival. Short interfering RNA knockdown of endogenous HAND2 and, to a lesser extent, HAND1 in neonatal sympathetic neurons cultured with NGF, reduced the expression of the NGF receptor tyrosine kinase TrkA (tropomyosin-related kinase A), as well as neuronal survival. Chromatin immunoprecipitation analysis showed that NGF promotes HAND2 binding to the TrkA minimal enhancer and that transfection of sympathetic neurons with a TrkA expression plasmid rescued the neurons from HAND knockdown. These findings show that HAND transcription factors have a crucial function in sustaining the survival of neonatal sympathetic neurons with NGF by a feed-forward loop that maintains the expression of TrkA.

Capabilities of neurexins in the chick ciliary ganglion

Transcellular interactions between neuroligins (NL) and beta-neurexin have been widely documented to promote maturation and function of both glutamatergic and GABAergic synapses. Recently it has been shown that neuroligin-1 plays a similar role at nicotinic synapses on chick ciliary ganglion neurons in culture, acting from the postsynaptic side to enhance transmitter release from adjacent cholinergic terminals and boost nicotinic input to the cells. We show here that the ciliary ganglion expresses three forms of neuroligin as well as two beta-neurexins and an alpha-neurexin. Overexpression of the beta-neurexins, but not the alpha-neurexin, can induce clustering of endogenous PSD-95 in adjacent neurons, presumably engaging neuroligin in the postsynaptic cell. The trans effects of beta-neurexins are selective; though both alpha3- and alpha7-containing nicotinic receptors are available on opposing cells, beta-neurexins induce coclustering of alpha3- but not alpha7-containing nicotinic receptors. Overexpression of other putative synaptogenic molecules, including SynCAM and L1, are ineffective at trans-clustering of PSD-95 on adjacent neurons. The beta-neurexins also exert a cis effect, coclustering presynaptic markers along with beta-neurexin in neurites juxtaposed to postsynaptic proteins, consistent with organizing presynaptic components as well. Striated muscle, the synaptic target of ciliary neurons in vivo, also expresses neuroligin. The results demonstrate that NL and neurexins are present at multiple sites in nicotinic cholinergic pathways and suggest the possibility of both cis- and trans-interactions to influence nicotinic signaling.

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.

Continued Administration of Ciliary Neurotrophic Factor Protects Mice from Inflammatory Pathology in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis is an inflammatory disease of the central nervous system that leads to loss of myelin and oligodendrocytes and damage to axons. We show that daily administration (days 8 to 24) of murine ciliary neurotrophic factor (CNTF), a neurotrophic factor that has been described as a survival and differentiation factor for neurons and oligodendrocytes, significantly ameliorates the clinical course of a mouse model of multiple sclerosis. In the acute phase of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide 35-55, treatment with CNTF did not change the peripheral immune response but did reduce the number of perivascular infiltrates and T cells and the level of diffuse microglial activation in spinal cord. Blood brain barrier permeability was significantly reduced in CNTF-treated animals. Beneficial effects of CNTF did not persist after it was withdrawn. After cessation of CNTF treatment, inflammation and symptoms returned to control levels. However, slight but significantly higher numbers of oligodendrocytes, NG2-positive cells, axons, and neurons were observed in mice that had been treated with high concentrations of CNTF. Our results show that CNTF inhibits inflammation in the spinal cord, resulting in amelioration of the clinical course of experimental autoimmune encephalomyelitis during time of treatment.

Assembly of the N-cadherin complex during synapse formation involves uncoupling of p120-catenin and association with presenilin 1

N-cadherin is an adhesion receptor that participates in both interaction between immature pre- and postsynaptic neurons and in the stabilization and function of matured neuron–neuron synapses. To better understand how the N-cadherin complex contributes to synapse formation, we examined its distribution and composition during synapse formation in the chick ciliary neurons. It was found that at early phases of synaptogenesis, N-cadherin is distributed in small clusters on the cell surface and primarily associates with p120-catenin and β-catenin. In contrast, as synaptic contacts matured, larger N-cadherin clusters were found localized adjacent to the active zone and associated with PS1 and γ-catenin, while p120- and β-catenin were dispersed among other cell regions, including axons. As it is known that PS1 binds γ-catenin and that uncoupled p120-catenin can alter the cytoskeleton via its effect on Rho GTPases, these changes in the molecular composition of the N-cadherin complex (represented by the uncoupling of p120-catenin and association with PS1) may correspond to distinct functional states of the complex involved in synaptic maturation.

The p38 mitogen‐activated protein kinase pathway negatively regulates Ca2+‐activated K+ channel trafficking in developing parasympathetic neurons

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).

Regulation of neuronal K Ca channels by β-neuregulin-1 does not require activation of Ras-MEK-extracellular signal-regulated kinase signaling cascades

Endogenous β-neuregulin-1 is required for the plasma membrane expression of large-conductance (BK-type) Ca 2+-activated K + channels in developing chick ciliary neurons of the chick ciliary ganglion. During normal development, β-neuregulin-1 acts in concert with transforming growth factor-β1 to stimulate movement of large-conductance Ca 2+-activated K + channels from intracellular stores into the plasma membrane, although these two growth factors preferentially act on different intracellular pools. We have previously shown that actions of transforming growth factor-β1 on ciliary neurons require activation of phosphoinositol 3-kinase and Akt, as well as a parallel cascade composed of the small GTPase Ras and a mitogen-activated protein kinase (extracellular signal-regulated kinase). In addition, we have shown that the actions of β-neuregulin-1 require activation of phosphoinositol 3-kinase and the protein kinase Akt. Here we examine whether β-neuregulin-1-evoked mobilization of large-conductance Ca 2+-activated K + channels also requires activation of a Ras-extracellular signal-regulated kinase signaling cascade. We observed that application of β-neuregulin-1 caused a robust and MEK1/2-dependent increase in extracellular signal-regulated kinase diphosphorylation that indicates activation of this signaling cascade in ciliary ganglion neurons, similar to what we have previously observed for transforming growth factor-β1. However, activation of this cascade is not necessary for β-neuregulin-1-evoked mobilization because stimulation of macroscopic large-conductance Ca 2+-activated K + channels persisted in cells treated with the MEK1/2 inhibitors PD98059 or U0126, in cells over-expressing dominant-negative forms of extracellular signal-regulated kinase, and in cells treated with the Ras inhibitor FTI-277. These results indicate that the mechanisms that underlie β-neuregulin-1 and transforming growth factor-β1 mobilization of large-conductance Ca 2+-activated K + channels are only partly overlapping, possibly because they cause recruitment of spatially distinct signaling complexes.

N-Cadherin Juxtamembrane Domain Modulates Voltage-Gated Ca2+Current via RhoA GTPase and Rho-Associated Kinase

The juxtamembrane domain (JMD) of N-cadherin cytoplasmic tail is an important regulatory region of the clustering and adhesion activities of the protein. In addition, the JMD binds a diversity of proteins capable of modifying intracellular processes including cytoskeletal rearrangement mediated by Rho GTPases. These GTPases also function as regulators of voltage-activated calcium channels, which in turn modulate neuronal excitability. The present study was designed to determine whether there is a direct functional link, via Rho GTPase, between the N-cadherin JMD and these voltage-activated channels. It was found that the infusion of the soluble JMD into chick ciliary neurons causes a substantial decrease in the amplitude of the high-threshold voltage-activated (HVA) calcium current. The activation time is increased while the inactivation process is reduced, suggesting that the decreased current amplitude reflects a reduction in the number of channels available to open. This effect was reversed by inhibition of RhoA or its downstream effector, Rho-associated kinase (ROCK). Because ROCK determines the active state of myosin, these results suggest that the modulation of HVA by the JMD could be mediated by changes in the status of the actin-myosin cytoskeleton.

Restricted response of mesencephalic neural crest to sympathetic differentiation signals in the trunk

Lineage diversification in the vertebrate neural crest may occur via instructive signals acting on pluripotent cells, and/or via early specification of subpopulations towards particular lineages. Mesencephalic neural crest cells normally form cholinergic parasympathetic neurons in the ciliary ganglion, while trunk neural crest cells normally form both catecholaminergic and cholinergic neurons in sympathetic ganglia. In contrast to trunk neural crest cells, mesencephalic neural crest cells apparently fail to express the catecholaminergic transcription factor dHAND in response to BMPs in the head environment. Here, we show that migrating quail mesencephalic neural crest cells grafted into the trunk of host chick embryos colonise the sympathetic ganglia. While many express dHAND and form tyrosine hydroxylase ( TH)-positive catecholaminergic neurons, the proportion that expresses either dHAND or TH is significantly smaller than that of quail trunk neural crest cells under the same conditions. Furthermore, the proportion of quail mesencephalic neural crest cells that is TH + in the sympathetic ganglia decreases with time, while the proportion of TH + quail trunk neural crest-derived cells increases. Thus, a subset of mesencephalic neural crest cells fails to express dHAND or TH in the sympathetic ganglia, while a further subset initiates but fails to maintain TH expression. Taken together, our results suggest that a subpopulation of migrating mesencephalic neural crest cells is refractory to catecholaminergic differentiation signals in the trunk. We suggest that this heterogeneity, together with local signals that repress catecholaminergic differentiation, may ensure that most ciliary neurons adopt a cholinergic fate.

Oestradiol rapidly inhibits Ca2+ signals in ciliary neurons through classical oestrogen receptors in cytoplasm.

Oestrogen plays a key role in a great variety of actions in the nervous system, either through classical or alternative pathways. The classical pathways are initiated after oestrogen binding to the oestrogen receptors ERalpha or ERbeta, which translocate from the cytoplasm to the nucleus and act there as transcription factors. Alternative pathways are initiated at the plasma membrane and cytoplasm, via binding to classical or non-classical ERs. Using isolated ciliary ganglion neurons from the chick embryo and Ca2+ imaging, we demonstrated that a 10-min exposure to 17beta-oestradiol reduces Ca2+ influx through the plasma membrane. This effect was not reproduced by oestradiol conjugated to bovine serum albumin, which does not cross the plasma membrane, indicating that 17beta-oestradiol was acting intracellularly. ERalpha was detected in the cytoplasm by immunostaining and its involvement in the regulation of Ca2+ influx by ICI182,780 inhibition. The phosphatidylinositol-3 kinase (Pi3-kinase) inhibitor wortmannin and the nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) both blocked the oestradiol effect. The oestradiol effect was reproduced by 8Br-cGMP and abolished in the presence of the cGMP-dependent protein kinase (PKG) inhibitor KT5823. Our study indicates that 17beta-oestradiol can regulate Ca2+ influx via PI3-kinase, NOS and PKG after activation of cytoplasmic ER.

Brain-derived neurotrophic factor and trkB signaling in parasympathetic neurons: relevance to regulating alpha7-containing nicotinic receptors and synaptic function.

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.

Ras is a mediator of TGFβ1 signaling in developing chick ciliary ganglion neurons

Large-conductance Ca 2+-activated K + channels (K Ca) in chick ciliary ganglion neurons are regulated by target-derived TGFβ1. Here we show that TGFβ1 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, TGFβ1-evoked increases in phosphorylation of SMAD2 were reduced by manumycin-A, suggesting that Ras-dependent transduction cascades activated by TGFβ1 feed back onto SMAD signaling. Thus, Ras is a mediator of pleiotropic TGFβ1 signaling in developing neurons.

Actin-ATP Hydrolysis Is a Major Energy Drain for Neurons

In cultured chick ciliary neurons, when ATP synthesis is inhibited, ATP depletion is reduced approximately 50% by slowing actin filament turnover with jasplakinolide or latrunculin A. Jasplakinolide inhibits actin disassembly, and latrunculin A prevents actin assembly by sequestering actin monomers. Cytochalasin D, which allows assembly-disassembly, but only at pointed ends, is less effective in conserving ATP. Ouabain, an Na(+)-K(+)-ATPase inhibitor, and jasplakinolide both prevent approximately 50% of the ATP loss. When applied together, they completely prevent ATP loss over a period of 20 min, suggesting that filament stabilization reduces ATP consumption by decreasing actin-ATP hydrolysis directly rather than indirectly by modulating the activity of Na(+)-K(+)-ATPase, a major energy consumer.