Laser ablation reveals regulation of ciliary activity by serotonergic neurons in molluscan embryos

Early in embryonic development, the pond snail Helisoma trivolvis exhibits a rotational behavior that is generated by beating of cilia in the dorsolateral and pedal bands. Although previous anatomical and pharmacological studies provided indirect evidence that a pair of serotonergic neurons, Embryonic Neurons C1 (ENC1s), is involved in regulating embryonic rotation, direct evidence linking ENC1 to ciliary function is still lacking. In the present study, we used laser microbeams to perturb ENC1 in vivo while monitoring ciliary activity in identified ciliary bands. A laser treatment protocol to specifically ablate ENC1 without damaging the surrounding cells was established. Unilateral laser treatment of ENC1 caused transient increases in the activity of the pedal and ipsidorsolateral cilia, lasting 30-50 min. In contrast, activity of cilia that were not anatomically associated with ENC1 was unaffected by laser treatment. Mianserin, an effective serotonin antagonist in Helisoma ciliated cells, decreased the overall CBF of pedal and dorsolateral cilia by reducing the occurrence of spontaneous CBF surges in these cilia. Finally, the cilioexcitatory action of ENC1 laser treatment was mimicked by serotonin and reduced in the presence of mianserin. These results suggest that laser treatment provokes a release of serotonin from ENC1, resulting in a prolonged elevation of activity in the target ciliary cells. We conclude that, in addition to their previously established role in regulating neurodevelopment, ENC1s also function as serotonergic motor neurons to regulate ciliary activity, and therefore the rotational behavior of early embryos.     

Innervation of dog ciliary ganglion

Summary There are species differences with regard to the composition of the ciliary ganglion. For instance, in rabbits and cats it consists solely of oculomotor nerves and has no sympathetic or sensory innervation. The purpose of this study is to clarify the participation of these nerves in the ciliary ganglion of the dog by histochemical methods. Cholinesterase (ChE) activity was studied by Karnovsky's method and catecholamine fluorescence by the glyoxylic acid method. Furthermore, the origins of the respective nerves were investigated by a serial preparation method, involving unilateral cervical sympathectomy and tracer dye injection in the ganglion. The results obtained were: (1) Ciliary ganglion cells showed intense ChE activity. Oculomotor nerve fibers leading to the ganglion showed moderate ChE activity, while the reaction in the short ciliary nerves was strong. (2) Aminergic nerves were present in the intercellular space of the ciliary ganglion, and bilateral or central innervation was suggested by the results of cervical sympathectomy. (3) Connection between the ciliary and trigeminal ganglia was proved by the dye tracer study. The results show that the ciliary ganglion in dogs is composed of oculomotor, trigeminal and sympathetic nerves.     

Innervation of dog ciliary ganglion

There are species differences with regard to the composition of the ciliary ganglion. For instance, in rabbits and cats it consists solely of oculomotor nerves and has no sympathetic or sensory innervation. The purpose of this study is to clarify the participation of these nerves in the ciliary ganglion of the dog by histochemical methods. Cholinesterase (ChE) activity was studied by Karnovsky's method and catecholamine fluorescence by the glyoxylic acid method. Furthermore, the origins of the respective nerves were investigated by a serial preparation method, involving unilateral cervical sympathectomy and tracer dye injection in the ganglion. The results obtained were: (1) Ciliary ganglion cells showed intense ChE activity. Oculomotor nerve fibers leading to the ganglion showed moderate ChE activity, while the reaction in the short ciliary nerves was strong. (2) Aminergic nerves were present in the intercellular space of the ciliary ganglion, and bilateral or central innervation was suggested by the results of cervical sympathectomy. (3) Connection between the ciliary and trigeminal ganglia was proved by the dye tracer study. The results show that the ciliary ganglion in dogs is composed of oculomotor, trigeminal and sympathetic nerves.     

Ciliary activity in the oviduct of cycling, pregnant, and muscarinic receptor knockout mice

The transport of the oocyte and the embryo in the oviduct is managed by ciliary beating and muscular contractions. Because nonneuronally produced acetylcholine influences ciliary beating in the trachea via the muscarinic receptors M2 and M3, we supposed that components of the cholinergic system may also modulate ciliary activity in the oviduct. To address this issue, we analyzed the expression profile of muscarinic receptors (CHRMs) in the murine oviduct by RT-PCR and assessed ciliary beat frequency (CBF) and cilia-driven particle transport speed (PTS) on the mucosal surface of opened oviductal segments in correlation with histomorphological investigations. RT-PCR of laser-assisted microdissected epithelium revealed expression of Chrm subtypes Chrm1 and Chrm3. In opened isthmic segments, particle transport was barely seen, correlating with a significantly lower number of ciliated cells compared to the ampulla. In the ampulla, basal PTS and CBF were high (71 μm/sec and 21 Hz, respectively) both in cycling and pregnant wild-type mice and in mice with targeted deletion of the Chrm genes Chrm1, Chrm3, Chrm4, and Chrm5. In contrast to the trachea, where basal ciliary activity was low and largely enhanced by muscarinic stimulation, muscarinic agonists and antagonists did not affect the high ampullar PTS. Our results imply that this high oviductal autonomous ciliary activity is independent from the intrinsic cholinergic system and serves to maintain optimal clearance of the tube throughout all stages of the estrous cycle and early pregnancy.     

Ovum transport in the rat oviductal ampulla in the absence of muscle contractility

Ovum transport in mammalian oviducts involves two main effectors: ciliary motility and muscle contractility. To study the relative contribution of cilia to ovum transport in the rat, we blocked smooth muscle activity with isoproterenol, a beta-adrenergic agonist, and measured transport rates of surrogate ova in situ. Transport rates before isoproterenol administration were 0.04 mm/s in the cephalic ampulla and 0.03 mm/s in the caudal ampulla; rates were unchanged after administration of isoproterenol. To determine if isoproterenol affected ciliary activity, we measured ciliary beat frequency with laser-scattering spectroscopy over the effective isoproterenol dosage. Isoproterenol did not cause a significant change in ciliary beat frequency. Our results show that in the rat oviductal ampulla, ciliary motion is capable of transporting ova in the absence of muscle contractility.     

The effect of hydroxyurea on ciliogenesis in ciliary epithelium of the mollusk Lymnaea stagnalis

Electron-microscopy study of the ciliary epithelium structure of the mollusk Lymnaea stagnalis was carried out under the action of hydroxyurea. By the method of radioautography, a high proliferative activity of the ciliary epithelium was established as the norm; a cluster distribution of cells, including the label, was noted. The presence of hydroxyurea in the mollusk organism was shown to inhibit proliferation. Scanning electron microscopy of the molluskan foot surface revealed clusters of nonciliated cells and of cells with short villi in control epithelial folds. Under hydroxyurea treatment for 24 h, such sites disappeared completely and ciliary epithelium looked uniform and was composed of cells with long cilia. By transmission electron microscopy, it was established that hydroxyurea did not affect the formation of the basal body and course of ciliogenesis. It has been suggested that hydroxyurea not only inhibits proliferative activity of epithelial cells, but also induces differentiation of unciliated into the ciliated cells.     

Comparison of mechanical agitation and calcium shock methods for preparation of a membrane fraction enriched in olfactory cilia

Calcium plays an important regulatory role in olfactory signal transduction. Many investigations into the regulation of the olfactory signaling pathway have been performed using fractions enriched in ciliary membranes from olfactory sensory neurons. The traditional method of preparing ciliary fractions uses high calcium concentrations, thought to dislodge cilia from the dendritic knobs of the olfactory neurons in the nasal epithelium. However, calcium, an important second messenger in the odorant signaling cascade, modulates the activity of many enzymatic reactions in this cascade. Pre-exposure of cilia to high calcium concentrations may modify these signaling events. Therefore, we sought to develop a method of isolating cilia-enriched membranes that avoids exposing the cilia to high calcium concentrations. Our method of isolation, referred to as the mechanical agitation method, involves mechanical disruption and sonication of the olfactory epithelium to dislodge the cilia. To evaluate this method of cilia preparation, basal adenylyl cyclase activity, as well as forskolin- and odorant-activated adenylyl cyclase, were analyzed. Specific activity of adenylyl cyclase and protein yield were compared for the mechanical agitation and the high calcium preparations. Immunoblots were analyzed for the presence of transduction components enriched in olfactory cilia: adenylyl cyclase type III (ACIII), heterotrimeric G-protein subunit Galphaolf and the 1 C2 isoform of phosphodiesterase (PDE 1 C2). Based on these analyses, the ciliary fraction prepared by the mechanical agitation method appears to be very similar to that prepared by the high calcium method, with a higher yield.     

A Computational Model of Dynein Activation Patterns that Can Explain Nodal Cilia Rotation

Normal left-right patterning in vertebrates depends on the rotational movement of nodal cilia. In order to produce this ciliary motion, the activity of axonemal dyneins must be tightly regulated in a temporal and spatial manner; the specific activation pattern of the dynein motors in the nodal cilia has not been reported. Contemporary imaging techniques cannot directly assess dynein activity in a living cilium. In this study, we establish a three-dimensional model to mimic the ciliary ultrastructure and assume that the activation of dynein proteins is related to the interdoublet distance. By employing finite-element analysis and grid deformation techniques, we simulate the mechanical function of dyneins by pairs of point loads, investigate the time-variant interdoublet distance, and simulate the dynein-triggered ciliary motion. The computational results indicate that, to produce the rotational movement of nodal cilia, the dynein activity is transferred clockwise (looking from the tip) between the nine doublet microtubules, and along each microtubule, the dynein activation should occur faster at the basal region and slower when it is close to the ciliary tip. Moreover, the time cost by all the dyneins along one microtubule to be activated can be used to deduce the dynein activation pattern; it implies that, as an alternative method, measuring this time can indirectly reveal the dynein activity. The proposed protein-structure model can simulate the ciliary motion triggered by various dynein activation patterns explicitly and may contribute to furthering the studies on axonemal dynein activity.     

Ciliary function of the frog oro-pharyngeal epithelium

Summary The palate epithelium of the frog was examined by scanning electron microscopy, light microscopy and high speed cine micrography. The cilia remain stationary for much of the time in the end-of-effective stroke position. Each beat cycle begins with a forwardly-directed recovery stroke lasting about 60 ms, followed by an effective stroke towards the oesophagus lasting about 12 ms. Activity can often be correlated with the presence of mucus, which is carried as strands on the tips of the ciliary effective strokes whilst the recovery strokes move beneath the mucus. Coordination of ciliary activity was very variable; local antiplectic metachrony of the recovery strokes could almost always be seen, and on very active epithelia effective strokes were associated with approximately diaplectic waves (either to left or right), but any particular pattern of coordinated activity was transient and quickly transformed to another pattern. Beating and coordination of these short cilia were compared with those of cilia propelling water.     

The mechanism of membrane response to chilling. Effect of temperature on phospholipid deacylation and reacylation reactions in the cell surface membrane

The ciliary membrane of Tetrahymena pyriformis is physically and metabolically remote from the main centers of lipid metabolism. Nevertheless, it possesses an independent capacity to modify its phospholipid molecular species composition rapidly under stress. The role of ciliary phospholipid deacylating and reacylating enzymes in this phenomenon has been evaluated. Isolated cilia showed substantial phospholipase A (combined A1 and A2), acyl-CoA synthetase and acyltransferase activities. Activities of all the three enzymes of cilia from 39 degrees C-grown cells were greatly reduced when the cilia were incubated at 15 degrees C. In contrast, the phospholipase A and acyltransferase activities in cilia from 15 degrees C-grown cells were surprisingly high at 15 degrees C and twice as high at 37 degrees C as were the equivalent activities in preparations from 39 degrees C-grown cells. While the in vivo substrate specificity of phospholipase A could not be meaningfully assessed, the acyltransferases exhibited a temperature-dependent substrate specificity in vivo. Growth temperature also affected the positional distribution of fatty acids incorporated into ciliary phospholipids in vivo. The ability of acyltransferases to utilize added [14C] acyl-CoA could be markedly stimulated, and their lipid class specificity could be significantly altered in vitro by supplementing the incubation mixture with exogenous lysophospholipid acceptors. These findings suggest that the rate-limiting factor in acyl chain turnover is not the activity of acyltransferases per se but rather the availability of suitable substrates and acceptors. Therefore, we postulate that temperature alters the rate and specificity of ciliary membrane phospholipid retailoring primarily by controlling the in situ phospholipase A activity.     

Ciliary Neuronotrophic Factor Stimulates Choline Acetyltransferase Activity in Cultured Chicken Retina Neurons

It has been demonstrated that cultured cholinergic retinal neurons from 8-day-old chicken embryos respond to a polypeptide factor present in retinal cell-conditioned medium (RCM) and in retinal extracts. Compared with control cultures, the activity of acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; ChAT) is enhanced more than twofold in neuronal retinal cultures grown for 7 days in the presence of RCM. The present study demonstrates that both ciliary neuronotrophic factor (CNTF), which is characterized by its trophic activity on parasympathetic ciliary neurons, and RCM exhibit identical stimulatory effects on ChAT activity in retinal monolayer cultures. Similarly, RCM supports the in vitro survival of ciliary neurons to the same extent as CNTF. The active species in RCM has a molecular weight (20,900 +/- 1,000) identical to that of CNTF, as determined by preparative sodium dodecyl sulfate gel electrophoresis. The results indicate that cholinergic retinal neurons represent a central neuronal target for CNTF or a closely related protein.     

Swimming Speed of Larval Snail Does Not Correlate with Size and Ciliary Beat Frequency

Many marine invertebrates have planktonic larvae with cilia used for both propulsion and capturing of food particles. Hence, changes in ciliary activity have implications for larval nutrition and ability to navigate the water column, which in turn affect survival and dispersal. Using high-speed high-resolution microvideography, we examined the relationship between swimming speed, velar arrangements, and ciliary beat frequency of freely swimming veliger larvae of the gastropod Crepidula fornicata over the course of larval development. Average swimming speed was greatest 6 days post hatching, suggesting a reduction in swimming speed towards settlement. At a given age, veliger larvae have highly variable speeds (0.8–4 body lengths s⁻¹) that are independent of shell size. Contrary to the hypothesis that an increase in ciliary beat frequency increases work done, and therefore speed, there was no significant correlation between swimming speed and ciliary beat frequency. Instead, there are significant correlations between swimming speed and visible area of the velar lobe, and distance between centroids of velum and larval shell. These observations suggest an alternative hypothesis that, instead of modifying ciliary beat frequency, larval C. fornicata modify swimming through adjustment of velum extension or orientation. The ability to adjust velum position could influence particle capture efficiency and fluid disturbance and help promote survival in the plankton.     

Immunohistochemical and ultrastructural studies on ciliary sense organs of arrow worms (Chaetognatha)

We examined epibenthic and pelagic species of Chaetognatha (Spadellidae and Sagittidae) using immunohistofluorescence and confocal laser scanning microscopy to detect tubulin and cell nuclei in whole-mount preparations and scanning and transmission electron microscopy to visualize the ultrastructural organisation of their ciliary sense organs. All chaetognaths bear three types of ciliary sense organs distributed throughout the body: (1) transversally oriented ciliary fence organs, (2) longitudinally (parallel to the anterior-posterior axis) oriented ciliary tuft organs, and (3) a ciliary loop, the corona ciliata. This study targets the ciliary fence as well as the ciliary tuft organs. Two types of primary receptor cells constitute the ciliary fence and ciliary tuft organs. The first type of receptor cells forms a single cell line along the midline axis of the organs, whereas the second type of receptor cells forms multiple lines of cells at either side of type 1 cells. Each receptor cell extends a single, non-locomotory cilium from its narrow apex collared by slender, non-reinforced microvilli; however, both types of sensory cells considerably differ on ultrastructural level. Type 1 sensory cells have thicker cilia than those protruded by the type 2 sensory cells which are characterized by rootlets consisting of an elongated, amorphous distal as well as a cross-striated proximal portion. These results likely reveal that both types of sensory cells have distinct functions.     

trans-Resveratrol and grape disease resistance. A dynamical study by high-resolution laser-based techniques

Two modern laser-based techniques were synchronously applied to study the dynamics of the trans-resveratrol activity in Botrytis cinerea-infected grapes. Direct analysis of trans-resveratrol in both infected and noninfected grapes (Vitis vinifera, Aledo variety) was performed by using an analytical technique incorporating laser desorption coupled with laser resonant ionization and time-of-flight mass spectrometry. On the other hand, one of the most sensitive on-line methods for trace gas detection, laser photoacoustic spectroscopy, was used to investigate the involvement of the plant hormone ethylene (C(2)H(4)) in the B. cinerea grapes interaction and its temporal relationship with the trans-resveratrol content upon infection. The trans-resveratrol content and the ethylene released by noninfected grapes showed an opposite behavior. In this case, a high trans-resveratrol content corresponds to a low ethylene emission. For the B. cinerea-infected grapes, ethylene emission rises up after 48 h when the analogous content of trans-resveratrol started to decrease irreversibly. Moreover, the activity of trans-resveratrol as natural pesticide has been investigated by exogenous application on grapes. A short submerge (5 s) of the grapes in 1.6 x 10(-4) M solution of trans-resveratrol delays the increase of C(2)H(4) emission with about 48 h and produces a decrease of the C(2)H(4) concentration and its emission rate. The treatment has positive effects on fruit conservation during storage; it doubled the normal shelf-life of grapes at room temperature, maintaining their post-harvest quality within 10 d.     

The kynurenine metabolic pathway in the eye: studies on 3-hydroxykynurenine, a putative cataractogenic compound.

The rabbit lens has an elevated content of 3-hydroxykynurenine (30HKYN) in spite of a very low activity of the enzymes leading to its synthesis. The iris/ciliary body, on the contrary, has very high activity of 30HKYN synthesizing enzymes but a content of 30HKYN lower than that of the lens. These observations suggest that 30HKYN is formed in the iris/ ciliary body, released into the aqueous humor and then taken up into the lens where it may be used for the synthesis of UV filtering products. An excessive accumulation of 30HKYN in the lens has been associated with cataract formation. We found that available selective inhibitors of kynurenine hydroxylase reduced 30HKYN synthesis in both the lens and the iris/ciliary body.     

Effect of in vitro cultivation andMycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures

Summary Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains ofMycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels. This study was supported in part by Grant AI 12559 from the National Institutes of Health. The supply of cholera toxin from Dr. R. A. Finkelstein is most appreciated as are the assistance and advice of J. A. Engelhardt and Y. D. B. Stahl.     

Precise spatiotemporal control of optogenetic activation using an acousto-optic device

Light activation and inactivation of neurons by optogenetic techniques has emerged as an important tool for studying neural circuit function. To achieve a high resolution, new methods are being developed to selectively manipulate the activity of individual neurons. Here, we report that the combination of an acousto-optic device (AOD) and single-photon laser was used to achieve rapid and precise spatiotemporal control of light stimulation at multiple points in a neural circuit with millisecond time resolution. The performance of this system in activating ChIEF expressed on HEK 293 cells as well as cultured neurons was first evaluated, and the laser stimulation patterns were optimized. Next, the spatiotemporally selective manipulation of multiple neurons was achieved in a precise manner. Finally, we demonstrated the versatility of this high-resolution method in dissecting neural circuits both in the mouse cortical slice and the Drosophila brain in vivo. Taken together, our results show that the combination of AOD-assisted laser stimulation and optogenetic tools provides a flexible solution for manipulating neuronal activity at high efficiency and with high temporal precision.     

Toxic effects of mechlorethamine on mammalian respiratory mucociliary epithelium in primary culture

Mechlorethamine (HN2) is an alkylating agent usually used in cancer chemotherapy. Nevertheless, HN2 is extremely toxic and its use is accompanied by severe side-effects that may cause lung complications. Many studies report the morphological and biochemical modifications induced by sulfur mustard (SM) but no report has been published concerning the toxic effects of HN2 on the ultrastructural and functional activity of surface respiratory epithelial cells. This study was performed on rabbit tracheal epithelium (RTE) cells in primary culture. The functional activity of the culture was evaluated by measuring the ciliary beating frequency (CBF) of the ciliated cells using a videomicroscopic method, and the culture growth was determined by an image analysis system. The morphological aspects of the cells were analyzed by light, scanning electron, and transmission electron microscopy. An important inhibition of cell growth was observed associated with a detachment of the outgrowth cells. Morphological changes were expressed by vacuolization, increases in the intercellular spaces, and by disorganization of the cytoskeleton associated with a specific attack of the ciliated cells that show ciliary blebbing. The sudden CBF inhibition is more likely due to the detachment and the death of the ciliated cells than to a specific ciliotoxic effect of HN2. All these observations demonstrated the high sensitivity of respiratory epithelial cells to HN2 and showed that HN2-induced injuries were irreversible, and time- and dose-dependent.Abbreviations CBF ciliary beating frequency - HN2 nitrogen mustard, or mechlorethamine - RTE rabbit tracheal epithelium - SEM scanning electron microscopy - SM sulfur mustard - TEM transmission electron microscopy     

Synchronization between beating cilia.

A novel quantitative parameter is proposed to define and measure the degree of synchronization between two small ciliary areas. These areas can be close to or far from one another. The Pearson correlation factor is used to define the degree of synchronization by a single number. This approach is based on a computerized, dual photoelectric method which simulataneously measures the scattered light from two small areas on the ciliary epithelium or its tissue culture. The measurements were performed on tissue culture from frog's palate epithelium. It was found that: (a) the degree of synchronization decreases, as a function of distance; (b) the correlation is fairly high even at relatively large separations, when measured on the same patch; (c) on a given patch the synchronization factor is independent of the direction of the metachronal wave; (d) close disconnected ciliary cells exhibit fairly high correlation; (e) disconnected randomly choosen ciliary cells at relatively large separation distances exhibit relatively low correlation, smaller by a factor of 2 than the correlation factor at the same distances when measured along the metachronal wave; (f) the average frequencies' ratio and the metachronal wavelength can be used as first-order indicators of ciliary synchronization; (g) there is a spread of metachronal wavelengths even over a single well-organized patch.     

High glucose-6-phosphatase activity in non-pigmented epithelial cells of rabbit ciliary body.

For study of the origin of glucose in the aqueous humor, glucose-6-phosphatase (G6Pase) and hexokinase activities, and glycogen, were cytochemically examined in the ciliary body (CB) of rabbit. G6Pase activity was also assayed biochemically. The staining reaction for G6Pase activity was strong in the non-pigmented epithelium (NPE) in the pars plana and tips of ciliary processes in the region containing large ciliary pockets within the pars plicata. NPE cells contained abundant reaction product for G6Pase activity in the endoplasmic reticulum (ER) and nuclear envelope. However, NPE in other regions of the CB and pigmented epithelium (PE) of CB, and other areas surrounding the anterior and (PE) of CB, and other areas surrounding the anterior and posterior chambers, showed weak or no G6Pase staining reaction. Biochemical G6Pase activity in the whole ciliary body was relatively high. Both NPE and PE in the pars plana and the tips showed strong staining reaction for hexokinase activity but no staining for glycogen. Furthermore, NPE cells in the tips bore large aggregates of smooth ER and many Golgi apparati. These suggest that the high G6Pase activity in NPE cells in the pars plana and the tips is related to glucose release into the aqueous humor.