Ciliary Channels Research Articles

On hydromagnetic two-phase gas-liquid flow in ciliary channel: An application of a metachronal rhythm

On hydromagnetic two-phase gas-liquid flow in ciliary channel: An application of a metachronal rhythm

Inhibition of a cyclic nucleotide-gated channel on neuronal cilia activates unfolded protein response in intestinal cells to promote longevity

Ciliary defects are linked to ciliopathies, but impairments in the sensory cilia of Caenorhabditis elegans neurons extend lifespan, a phenomenon with previously unclear mechanisms. Our study reveals that neuronal cilia defects trigger the unfolded protein response of the endoplasmic reticulum (UPR ER ) within intestinal cells, a process dependent on the insulin/insulin-like growth factor 1 (IGF-1) signaling transcription factor and the release of neuronal signaling molecules. While inhibiting UPR ER doesn’t alter the lifespan of wild-type worms, it normalizes the extended lifespan of ciliary mutants. Notably, deactivating the cyclic nucleotide-gated (CNG) channel TAX-4 on the ciliary membrane promotes lifespan extension through a UPR ER -dependent mechanism. Conversely, constitutive activation of TAX-4 attenuates intestinal UPR ER in ciliary mutants. Administering a CNG channel blocker to worm larvae activates intestinal UPR ER and increases adult longevity. These findings suggest that ciliary dysfunction in sensory neurons triggers intestinal UPR ER , contributing to lifespan extension and implying that transiently inhibiting ciliary channel activity may effectively prolong lifespan.

The TRPP2-dependent channel of renal primary cilia also requires TRPM3.

Primary cilia of renal epithelial cells express several members of the transient receptor potential (TRP) class of cation-conducting channel, including TRPC1, TRPM3, TRPM4, TRPP2, and TRPV4. Some cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by defects in TRPP2 (also called polycystin-2, PC2, or PKD2). A large-conductance, TRPP2-dependent channel in renal cilia has been well described, but it is not known whether this channel includes any other protein subunits. To study this question, we investigated the pharmacology of the TRPP2-dependent channel through electrical recordings from the cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin. The pharmacology was found to match that of TRPM3 channels. The ciliary TRPP2-dependent channel is known to be activated by depolarization and by increasing cytoplasmic Ca2+. This activation was greatly enhanced by external pregnenolone sulfate, an agonist of TRPM3 channels. Pregnenolone sulfate did not change the single-channel current-voltage relation. The channels were effectively blocked by isosakuranetin, a specific inhibitor of TRPM3 channels. Both pregnenolone sulfate and isosakuranetin were effective at concentrations as low as 1 μM. Knocking out TRPM3 by CRISPR/Cas9 genome editing eliminated the ciliary channel. Thus the channel is both TRPM3-dependent and TRPP2-dependent, suggesting that it may include both types of subunit. Knocking out TRPM3 did not change the level of TRPP2 protein in the cilia, so it is unlikely that the absence of functional ciliary channels results from a failure of trafficking.

Novel Insights into the Development and Function of Cilia Using the Advantages of the Paramecium Cell and Its Many Cilia.

Paramecium species, especially P. tetraurelia and caudatum, are model organisms for modern research into the form and function of cilia. In this review, we focus on the ciliary ion channels and other transmembrane proteins that control the beat frequency and wave form of the cilium by controlling the signaling within the cilium. We put these discussions in the context of the advantages that Paramecium brings to the understanding of ciliary motility: mutants for genetic dissections of swimming behavior, electrophysiology, structural analysis, abundant cilia for biochemistry and modern proteomics, genomics and molecular biology. We review the connection between behavior and physiology, which allows the cells to broadcast the function of their ciliary channels in real time. We build a case for the important insights and advantages that this model organism continues to bring to the study of cilia.

Of cilia, titin, and neurosteroids

Of cilia, titin, and neurosteroids

A method for measuring electrical signals in a primary cilium

BackgroundMost cells in the body possess a single primary cilium. These cilia are key transducers of sensory stimuli, and defects in cilia have been linked to several diseases. Evidence suggests that some transduction of sensory stimuli by the primary cilium depends on ion-conducting channels. However, the tiny size of the cilium has been a critical barrier to understanding its electrical properties. We report a novel method that allows sensitive, repeatable electrical recordings from primary cilia. Adherent cells were grown on small, spherical beads that could be easily moved within the recording chamber. In this configuration, an entire cilium could be pulled into a recording microelectrode.ResultsIn 47% of attempts, suction resulted in a seal with high input resistance. Single channels could be recorded while the cilium remained attached to the cell. When the pipette was raised into the air, the cell body was pulled off at the air-bath interface. The pipette retained the cilium and could then be immersed in various solutions that bathed the cytoplasmic face of the membrane. In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca2+ and transmembrane voltage.ConclusionsCiliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

Paramecium BBS genes are key to presence of channels in Cilia.

BackgroundChanges in genes coding for ciliary proteins contribute to complex human syndromes called ciliopathies, such as Bardet-Biedl Syndrome (BBS). We used the model organism Paramecium to focus on ciliary ion channels that affect the beat form and sensory function of motile cilia and evaluate the effects of perturbing BBS proteins on these channels.MethodsWe used immunoprecipitations and mass spectrometry to explore whether Paramecium proteins interact as in mammalian cells. We used RNA interference (RNAi) and swimming behavior assays to examine the effects of BBS depletion on ciliary ion channels that control ciliary beating. Combining RNA interference and epitope tagging, we examined the effects of BBS depletion of BBS 7, 8 and 9 on the location of three channels and a chemoreceptor in cilia.ResultsWe found 10 orthologs of 8 BBS genes in P. tetraurelia. BBS1, 2, 4, 5, 7, 8 and 9 co-immunoprecipitate. While RNAi reduction of BBS 7 and 9 gene products caused loss and shortening of cilia, RNAi for all BBS genes except BBS2 affected patterns of ciliary motility that are governed by ciliary ion channels. Swimming behavior assays pointed to loss of ciliary K+ channel function. Combining RNAi and epitope tagged ciliary proteins we demonstrated that a calcium activated K+ channel was no longer located in the cilia upon depletion of BBS 7, 8 or 9, consistent with the cells’ swimming behavior. The TRPP channel PKD2 was also lost from the cilia. In contrast, the ciliary voltage gated calcium channel was unaffected by BBS depletion, consistent with behavioral assays. The ciliary location of a chemoreceptor for folate was similarly unperturbed by the depletion of BBS 7, 8 or 9.ConclusionsThe co-immunoprecipitation of BBS 1,2,4,5,7,8, and 9 suggests a complex of BBS proteins. RNAi for BBS 7, 8 or 9 gene products causes the selective loss of K+ and PKD2 channels from the cilia while the critical voltage gated calcium channel and a peripheral receptor protein remain undisturbed. These channels govern ciliary beating and sensory function. Importantly, in P. tetraurelia we can combine studies of ciliopathy protein function with behavior and location and control of ciliary channels.

Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK(1) renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R-gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK(1) cells stained with anti-V2R antibodies and in LLC-PK(1) cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 microM AVP (trans) or forskolin (cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.

Spontaneous gating of olfactory cyclic-nucleotide-gated channels.

In vertebrates, cilia on the olfactory receptor neurons have a high density of cyclic-nucleotide-gated (CNG) channels. During transduction of odorous stimuli, cyclic AMP is formed. cAMP gates the CNG channels and this initiates the neuronal depolarization. Here it is shown that the ciliary CNG channels also open spontaneously. In the absence of odorants and second messengers, olfactory cilia have a small basal conductance to cations. Part of this conductance is similar to the cAMP-activated conductance in its sensitivity to channel inhibitors and divalent cations. The basal conductance may help to stabilize the neuronal membrane potential while limiting the sensitivity of odorant detection.

Real-Time Confocal Imaging of Calcium Changes Upon Exocytosis in Paramecium

Exocytosis in the unicellular eukaryote Paramecium occurs rapidly upon stimulation with the biological secretagogue lysozyme (100 μM) in a Ca2+-dependent manner. Prior to stimulation each cell contains several thousand dense core secretory vesicles (trichocysts) docked beneath the cell membrane which can be induced to fuse with the plasma membrane either globally or locally. Fusion of the vesicle is followed by secretion and expansion of the trichocyst content from a 4 μm protein crystalline array to a 40 μm spear-like protein structure that are visible in the light microscope. The exocytosis microdomain of the cell membrane has been well characterized in earlier freeze fracture studies. The specific site of exocytosis was shown to consist of a rosette (fusion rosette) of 9 to 11 intramembrane particles surrounded by two outer rings of smaller sized particles. It is below these fusion rosettes that each of the secretory vesicles is docked and primed to release upon stimulation. In the absence of assembled fusion rosette normal release of trichocysts is blocked, as demonstrated with rosette defective mutants1. The fusion rosette has been postulated to function as calcium gates involved in exocytosis3. Paramecium has three known distinct Ca2+ channels: 1) the ciliary voltage-gated channel, 2) the hyper-polarization-activated channel, and 3) the anterior mechanosensory channel. Recently, we have described experiments indicating the presence of a fourth type of Ca2+ channel, a specific receptor operated Ca2+ conductance associated with lysozyme stimulated exocytosis. In this study we have visualized local (temporal and spatial) calcium fluxes associated with discrete exocytic events, using real time confocal microscopy (Nikon, RCM 8000) and calcium imaging with Indo-1 dual emission dye.

Morphological Changes in Rabbit Ciliary Epithelium and Blood–Aqueous Barriers after Intravitreal 10 −5MEndothelin-1

Morphological changes in the ciliary epithelium and blood–aqueous barriers were observed with a tracer, horseradish peroxidase, after 10μl of 10 −5 mendothelin-1 was injected intravitreally. A decrease of intraocular pressure and an increase of anterior chamber flare were found, and the structure of the ciliary epithelium changed drastically 6–7hr after the injection. Swelling of mitochondria appeared in both non-pigmented and pigmented epithelial cells of the ciliary body. Intercellular spaces and ciliary channels were markedly dilated in the iridial processes. Some non-pigmented epithelial cells in both iridial and ciliary processes appeared to be degenerated and necrotic. Horseradish peroxidase leaked into the posterior chamber via extremely dilated ciliary channels and necrotic cells. The dose of 10 −5 mendothelin-1 seems to be so large that a toxic action occurred. The mechanism of the effect of endothelin on intraocular pressure was also discussed.

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mM veratridine by immediate trichocyst exocytosis, provided [Ca2+]o (extracellular Ca2+ concentration) is between about 10(-4) to 10(-3) M as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec. Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca2+o or increasing [Ca2+]o to 10(-2) M inhibit exocytotic membrane fusion. Veratridine does not release Ca2+ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na+o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca2+]o) in the somatic, i.e., nonciliary cell membrane and that a Ca2+ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.

Gating properties of the cAMP-gated channel in toad olfactory receptor cells.

1. Inside-out membrane patches were excised from different parts of isolated olfactory receptor cells of the toad, and the gating properties of cAMP-gated channels were investigated under low concentrations of divalent cations. 2. At +50 mV, an outward current was observed when 1 mM cAMP was applied to the cytoplasmic side of membrane patches excised from cilia. cGMP had a similar effect. The dose-response relation for the cAMP-induced current could be fitted with the Hill equation with a coefficient of 1.5 for both cAMP and cGMP, and half-maximal concentration (K1/2) values of 19 microM (cAMP) and 16 microM (cGMP). 3. cAMP at low concentration (1 microM) induced step-like currents representing the opening of individual channels with a unitary conductance of about 30 pS. The I-V relation for the unitary events showed a weak outward rectification. 4. The relation between variance and mean current amplitude was well described with a parabola. Even at a saturating ligand concentration (1 mM cAMP) the current fluctuations did not disappear, indicating that fully liganded channels still switched between open and closed states. The maximum open probability was about 80% (+40 to +60 mV). 5. The current fluctuations at 1 mM cAMP were analysed with power spectral analysis. At +50 mV, frequency components lower than 10 Hz were well described with a Lorentzian function with a corner frequency of 1.7 +/- 0.2 Hz (+/- S.D.). In addition, higher flat frequency components were observed. At -50 mV the corner frequency became 0.6 +/- 0.1 Hz. 6. Membrane patches having a single cAMP-gated channel were obtained from the dendro-somatic membrane. These channels were very similar to the ciliary channels in electrophysiological characteristics. 7. The single cAMP-gated channel current showed flickering bursts that were interrupted with gaps. In saturating cAMP condition (1 mM), both burst- and gap-time histograms were fitted with single-exponential functions with time constants of 148 and 141 ms, respectively. 8. The channels were present at a high density of around 1750 microns-2 on the ciliary plasma membrane, as compared to 6 microns-2 on dendro-somatic membrane.

Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells.

Ultrastructural localization of Na+,K+-ATPase in rat ciliary epithelium was investigated quantitatively by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of Na+,K+-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha-subunit of Na+,K+-ATPase in the ciliary body. Gold particles were found mainly on the basolateral surfaces of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells with an approximately twofold higher labeling density in the PE cells. A few gold particles were also found on the apical and ciliary channel surfaces of the PE cells, whereas no significant binding was found on the apical surfaces of the NPE cells. The basolateral surfaces of PE and NPE cells are markedly infolded and are much greater in area than the apical surfaces. This means that Na+,K+-ATPase is almost exclusively located on the basolateral surfaces of both the NPE and PE cells. We suggest that the Na+,K+-ATPase of both the NPE and PE cells play an important role in the formation of aqueous humor.

Presence and distribution of Na+/K+-ATPase in the ciliary epithelium of the rabbit.

Regional differences in the localization of Na+/K+-ATPase in the ciliary epithelium of albino rabbits were studied histochemically using the method of Chayen et al. and ultra-histochemically using a cerium-based method. In addition, the incubation time necessary to achieve first signs of staining was investigated as an indication of Na+/K+-ATPase activity. In the entire pars plicata: prelenticular, postlenticular, as well as tips and valleys, staining was seen in the lateral infoldings of the non pigmented epithelium (NPE) after short incubation periods. Somewhat later, the apical cell membranes also stained. The ultrastructure of these cells, together with the staining pattern, point towards a functional significance of the NPE in active fluid secretion. The pigmented epithelium (PE) did not stain. In the iridial processes and in the area of the ciliary ridges staining first appeared in the apical cell membranes of the NPE, which form the typical ciliary channels. The basolateral infoldings of the NPE also stained, whilst the PE remained unstained. The difference in morphology and staining between pars plicata and iridial processes could indicate a difference in function, e.g. reabsorption of freshly secreted aqueous humour. In the pars plana, only the basolateral infoldings of the PE stained. A functional significance of this area in connection with the blood retina barrier is discussed.

Electrical properties and membrane currents in the ciliateDidinium

1. The electrical properties of the ciliateDidinium nasutum, aParamecium predator, have been investigated using techniques of constant current injection, voltage-clamp, exchange of solutions, and non-lethal deciliation. The electrical observations have been related to structural characteristics (Fig. 1). 2. The resting potential was primarily K-dependent. K concentrations beyond 1 mmol/1 depolarized the cell and reduced the input resistance. The resting conductance for Ca was low. Partial substitution of Ca by Mg changed the membrane potential very little and had no effect on the input resistance. Chloride did not influence the membrane properties. 3. The late V/I-relationship was characterized by pronounced outward and minimal inward rectification. It was very little modified by Ca concentrations ranging between 0.125 mmol/1 and 8 mmol/1. Raising the K concentration between 0.063 and 16 mmol/1 did not affect membrane rectification. 4. Membrane depolarization triggered a stimulus-graded action potential, which was Ca-dependent. The action potential rose with rates of up to 6 V/s; its overall duration was 100 to 500 ms. Repolarization occurred along a fast and a slow (shoulder) component and was followed by pronounced after-hyperpolarization. 5. Complete deciliation ofDidinium eliminated the regenerative depolarization of the membrane. The resting potential, input resistance and late current/voltage relationship were not modified by the removal of ciliary membranes. Under voltage-clamp deciliation removed the early inward current and the hump component of the late outward current. 6. Depolarizing voltage-clamp pulses of up to + 20 mV revealed a residual inward current following the early transient. The transient included two decay time constants (3.3 ms; 31.8 ms). An 0.8 nA persistent inward current was isolated using the difference current of ciliated and deciliated cells. A late outward current rose with depolarizations beyond 20 mV including an upward inflection (hump) about 50 ms after step onset. 7. A decreasing Ca/Ba mole-fraction increased the frequency and duration of spontaneous action potentials, and depressed the amplitudes of both, early inward and late outward current under voltage-clamp. In Ca-free Ba solution the unclamped membrane fired repetitively; the depolarizations included shoulders of 10 s or more. Inactivation of inward Ba currents was slow under depolarizing voltage-clamp; the late outward current was strongly depressed. In deciliated cells an early inward current was missing in Ca-free Ba solution, and the late I/V relation resembled that of deciliated cells in Ba-free Ca solution. 8. Tail currents following the late outward current of ciliated cells in Ca-solution and of deciliated cells in Ca- or Ba-solutions decayed with similar time constants. 9. In conclusion, the voltage-activated Ca conductance of the ciliary membrane ofDidinium compares well with data from ciliates so far studied. Unique is (i) a persistent inward current isolated using minor step depolarization or the difference current of ciliated and deciliated cells, (ii) a slow activating voltage-dependent K conductance, and (iii) a late, potentially Ca-dependent, outward current, which may be related to spatial separation of the cilia from the majority of the soma membrane. Ba interferes with the Ca channel which favours a previous two-binding site model. Ba can inhibit the K conductance from inside the cell after passing the ciliary Ca channel.

A new function of the non-pigmented epithelium of ciliary processes in the formation of aqueous humor

The invagination of the optic vesicle has, as one of its consequences, the apposition of the apices of the nonpigmented and pigmented epithelia of the ciliary processes of the eye. There are well developed intercellular ciliary "channels" formed at the apices of these cells. Embryologically the ciliary epithelia are homologous to the epithelia of the choroid plexus, a characteristically differentiated ependyma. These epithelial cells have a polarity. For example, the supranuclear region of the nonpigmented epithelia has Golgi apparatus. While stimulation of the epithelia of the choroid plexus by beta adrenergics or cholera toxin increases the rate of production of cerebrospinal fluid, similar stimulation of the adenylate complex within the ciliary epithelia decreases aqueous humor production. When the secretory function of the nonpigmented epithelial cell is activated, fluid is probably reabsorbed from the posterior chamber and secreted apically into the intercellular ciliary channels, in accord with cell polarity, thereby reducing net aqueous inflow. The fluid may enter the ciliary stroma thereafter and thus may become part of what may be called "uveal scleral" flow, or in this instance, uveal flow. The anatomy, physiology, and chemistry fit this hypothesis and form the basis for new investigations of a two-way transport across the ciliary epithelia.

THE ANATOMY AND FINE STRUCTURE OF THE EYE IN FISH. VI CILIARY TYPE TISSUE IN NINE SPECIES OF TELEOSTS

The eyes of teleost fishes do not have ciliary bodies. Therefore there is no ciliary epithelium per se, the tissue normally assumed to secrete aqueous humor. When examined at the electron microscope level a layer of nonpigmented cells on the back of the fish iris shows many similarities to the ciliary epithelium of mammals. The tissue of fish iris has strategically located zonulae occludents similar to those forming the blood-aqueous barrier in mammals. There is a marked lateral interdigitation of cells as seen in mammalian ciliary tissue and as seen in the specific salt absorbing cells found in the gills of brackish water adapted crabs. The teleost tissue also has numerous intercellular spaces (ciliary channels?) distributed in the same fashion as in mammalian ciliary epithelium. Although there is no morphological evidence for the secretion of aqueous humor, there is indirect evidence that the nonpigmented cells absorb salt to produce the hypotonic aqueous humor that is unique to teleosts.

Experimental brucei trypanosomiasis: Immunosuppression of the blood-aqueous barrier

Experimental Trypanosoma brucei infection induces intense protein leakage into the anterior chamber of cats' and dogs' eyes, with a cyclitis-like symptomatology, preceded by hypersensitivity of conjunctiva and respiratory mucosae, together with lacrimation, and facial oedema. The pherogram of the aqueous humor is the replica of the serum pherogram at the end-stage of the disease, and is identical to that of secondary aqueous. Absence of any significant changes in serum proteins and immunoglobulins, and the poor cellular reaction at tissue level point towards immune unresponsiveness subsequent to experimental protozoal infection. Light microscopy reveals, at the level of the ciliary processes two kinds of cystic formation, the walls of which are formed by ciliary epithelium. One type contains amorphous material next to trypanosomes, macrophages and plasmocyte-like cells; the second type contains also fibrillar structures possibly collagen. Ultramicroscopy suggests these cystic formations to be enlarged ciliary channels. The fenestrated capillary wall showed no changes, either after withdrawal of aqueous, or after toxic parasite-action; the presence of large protein molecules and trypanosomes in the anterior chamber is due to damage to the ciliary epithelium.

Ultrastructural and cytochemical studies on the sensory organelles and nervous system ofDipetalonema viteae(Nematoda: Filarioidea)

The sensory organelles and some components of the nervous system of the adult filarial wormDipetalonema viteaehave been studied by means of electron microscopy. The sensory organelles contain modified cilia innervated by nerve axons. The cilia lie within cuticle-lined channels. The amphids each contain nine such cilia, but the phasmids and the papillae contain only one cilium. The amphidial and phasmidial cilia are exposed to the external environment and it is suggested that I they function as chemoreceptors. The cilia within the cephalic and caudal papillae are modified so as to produce a greater area of sensitivity. The ciliary channels in the cephalic papillae do not open to the exterior and these papillae therefore probably function as mechanoreceptors. The caudal papillae of the male worm are structurally similar to the cephalic papillae, except that the ciliary channels are open to the external environment. It is suggested that these papillae function both as mechanoreceptors and chemoreceptors. The terminal pair of caudal papillae differ from all the other papillae. The ciliary channels of all the sensory organelles are surrounded by supporting cells, but multivesicular cells are only associated K with the amphids, phasmids and cephalic papillae. The nerve-ring encircles the muscular region of the oesophagus; it is composed entirely of nerve axons. The nerve axons contain two kinds of vesicle. The nervering is surrounded by an ensheathing cell. The major nerve bundles are carried by the hypodermal cords, but they are not embedded in the hypodermal cytoplasm. The nerve axons at the surface of the cord form synapses with the innervation processes of the somatic muscle cells. Electron cytochemical techniques have been used to investigate the distribution of cholinesterases in the structures described. Acetylcholinesterase has been identified in the amphids, phasmids, cephalic papillae and nerve-ring; it is suggested that acetylcholine functions as a chemical transmitter at these sites. Pseudocholinesterase has been identified in the amphids, phasmids and cephalic papillae, but not the nerve-ring; it is present in considerably smaller quantities than acetylcholinesterase.