Mechanoelectric transduction in nematocytes of a hydropolyp (Corynidae)

In sensitivity and ultrastructure of their cnidocil apparatus (CA), the nematocytes (stinging cells) of hydrozoans are analogous to hair cells of vertebrates and epidermal mechanoreceptors of insects. Intracellular recordings using current and voltage clamp in the capitate tentacles of the marine hydropolyp Stauridiosarsia producta (Corynidae) now revealed that depolarizing receptor potentials and receptor currents are generated in nematocytes (stenotele type) in response to mechanical stimulation of the CA. The responsive cells were identified by injection of Lucifer Yellow. For recording, the tentacles were isolated from the polyp and held by a suction capillary. Stimuli were applied by a glass probe moved electromagnetically or piezoelectrically.The mechanosensitivity of the nematocytes was found to be strictly limited to the CA. The characteristics of the mechanoelectric transduction were those typical of mechanoreceptor cells: phasic-tonic time course of an increase in membrane conductance; latency between stimulus and receptor response < 50 s; sigmoid relationship between receptor-response amplitude and stimulus amplitude; maximal increase in conductance of 15 nS; reversal potential between + 35 mV and — 10 mV; unspecific cation dependence and reversible blocking by streptomycin. The results suggest a direct mechanical control of unspecific cation channels such as has been found for mechanoreceptor cells.Suprathreshold receptor potentials elicit two forms of regenerative depolarization: non-inactivating, steplike potentials and action potentials. The latter can trigger discharge of the nematocyst.The discharge of nematocysts in the intact animal (without recording) in response to adequate stimuli was blocked by streptomycin and Na⁺ depletion in the same way as the receptor potential.Mechanoreceptor potentials are thus the beginning of a stimulus-induced electrical reaction cascade that ends in nematocyst discharge.     

Neural pathways and innervation of cnidocytes in tentacles of sea anemones

Our previously published studies are here reviewed detailing neuro-cnidocyte synapses, demonstrating putative neurotransmitter substances, and identifying complex neural pathways in sea anemones. Synapses were traced to their contacts on nematocytes and spirocytes by transmission electron microscopy of serial thin sections of tentacles. In five animals, cells containing microbasic p-mastigophores had synapses with clear vesicles, whereas cells containing basitrichous isorhizas had synapses with dense-cored vesicles, providing preliminary evidence for a selectivity of neurotransmitter types for different nematocysts. Either clear or dense-cored synaptic vesicles were also present at neuro-spirocyte contacts. Antho-RFamide immunoreactivity occurred in some anthozoan synaptic vesicles and immunogold labeling of serotonin was found at a neuro-spirocyte synapse. Neural pathways included direct innervation of spirocytes by sensory cells, sequential neuro-neuro-spirocyte and neuro-neuro-nematocyte synapses and reciprocal synapses involving axons of both sensory cells and ganglion cells. Such synaptic patterns resemble neuro-effector pathways found in higher animals and lay to rest the independent effector hypothesis for cnidocyte discharge in tentacles of sea anemones.     

Monoclonal antibodies against surface components of the mechano-and chemosensitive cnidocil complex of Hydra vulgaris

Summary Two monoclonal antibodies (Gc3.2 and Bd 2.2) against surface components of the cnidocil complex of Hydra vulgaris have been produced. In indirect immunofluorescence and in immunogold-labelling, the Gc 3.2-antibody stains the complete surface of all nematocytes, whereas other cellular surfaces are not labelled. The Bd 2.2-antibody, in contrast, produces only a small band of fluorescence on isolated cnidocils. This pattern of fluorescence and the corresponding immunogold-labelling indicate that the Bd 2.2-antibody exclusively binds to those intermembrane connectors that link the cnidocil and stereovillar cone in situ. In isolated and decnidociliated nematocytes, the tips of the stereovilli are also labelled by the Bd 2.2-antibody. Physiological experiments suggest that the Bd 2.2-antibody disturbs the reconstitution of intermembrane connectors during cnidocil regeneration. These data confirm the hypothesis that the intermembrane connectors are formed by two identical subunits located at the cnidociliar and stereovillar surfaces.     

synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses.     

Peripheral synapses at identifiable mechanosensory neurons in the spider Cupiennius salei : synapsin-like immunoreactivity

Indirect immunocytochemical tests were used at the light- and electron-microscopic levels to investigate peripheral chemical synapses in identified sensory neurons of two types of cuticular mechanosensors in the spider Cupiennius salei Keys.: (1) in the lyriform slit-sense organ VS-3 (comprising 7–8 cuticular slits, each innervated by 2 bipolar sensory neurons) and (2) in tactile hair sensilla (each supplied with 3 bipolar sensory cells). All these neurons are mechanosensitive. Application of a monoclonal antibody against Drosophila synapsin revealed clear punctate immunofluorescence in whole-mount preparations of both mechanoreceptor types. The size and overall distribution of immunoreactive puncta suggested that these were labeled presynaptic sites. Immunofluorescent puncta were 0.5–6.8 μm long and located 0.5–6.6 μm apart from each other. They were concentrated at the initial axon segments of the sensory neurons, while the somata and the dendritic regions showed fewer puncta. Western blot analysis with the same synapsin antibody against samples of spider sensory hypodermis and against samples from the central nervous system revealed a characteristic doublet band at 72 kDa and 75 kDa, corresponding to the apparent molecular mass of synapsin in Drosophila and in mammals. Conventional transmissionelectron-microscopic staining demonstrated that numerous chemical synapses (with at least 2 vesicle types) were present at these mechanosensory neurons and their surrounding glial sheath. The distribution of these synapses corresponded to our immunofluorescence results.Ultrastructural examination of anti-synapsin-stained neurons confirmed that reaction product was associated with synaptic vesicles. We assume that the peripheral synaptic contacts originate from efferents that could exert a complex modulatory influence on mechanosensory activity. Received: 20 April 1998 / Accepted: 18 August 1998     

Different nematocytes have different synapses in the sea anemone Aiptasia pallida (Cnidaria,Anthozoa)

Sea anemones feed by discharging nematocysts into their prey, but the pathway for control of nematocyst discharge is unknown. The purpose of this study was to investigate the ultrastructural evidence of neuro-nematocyte synapses and to determine the types of synaptic vesicles present at different kinds of nematocyst-containing cells. The tip and middle of tentacles from small specimens of Aiptasia pallida were prepared for electron microscopy and serial micrographs were examined. We found clear vesicles in synapses on mastigophore-containing nematocytes and dense-cored vesicles in synapses on basitrich-containing nematocytes and on one cnidoblast with a developing nematocyst. In addition, we found reciprocal neuro-neuronal and sequential neuro-neuro-nematocyte synapses in which dense-cored vesicles were present. It was concluded that : (1) neuro-nematocyte synapses are present in sea anemones, (2) different kinds of synaptic vesicles are present at cells containing different types of nematocysts, (3) synapses are present on cnidoblasts before the developing nematocyst can be identified and these synapses may have a trophic influence on nematocyst differentiation, and (4) both reciprocal and sequential synapses are present at the nematocyte, suggesting a complex pathway for neural control of nematocyst discharge. J. Morphol. 238:53–62, 1998. © 1998 Wiley-Liss, Inc.     

Cytoskeleton-membrane interactions in the cnidocil complex of hydrozoan nematocytes

Summary Each cnidocil complex of the hydrozoans Tubularia larynx and Hydra vulgaris consists of 9 or 7–10 large stereovilli (=stereocilia), respectively, and a modified cilium, the cnidocil. The cnidocils comprise the regular 9 microtubule doublets, up to 30 additional microtubules, as well as a central filament body. Adjacent stereovilli are linked together by intermembrane connectors forming the stereovillar cone. The distal tips of the stereovilli surround the cnidocil in a closed tubular arrangement measuring up to 0.7 m in length. Within this contact region the cnidocil is linked to the stereovillar tube by another set of intermembrane connectors, which seem to hold the cnidocil in a central position within the stereovillar cone. Stereovillar membrane and actin core are linked by 16-nm long cross bridges, which display a periodicity of 16 nm and emerge from the actin core. Within the cnidocils periodically arranged membrane-cytoskeleton bridges are uniformly restricted to the contact region. Here, 24-nm long cross bridges, which are spaced by a regular distance of 20 nm, interconnect the A-tubules of the microtubule doublets and the membrane. The cnidociliary membrane is differentiated into distinct domains as revealed by freeze-fracturing. Within the contact region of the nematocytes of Tubularia larynx, intramembrane particles are arranged in 9 rows of 700 nm length and 50 nm width, separated by particlefree areas. Intramembrane particles are irregularly distributed distal to the contact region. Considering recent physiological results we presume that the latter represent chemoreceptor units, while mechanical stimuli are transmitted via the intermembrane connectors and the microtubule-membrane bridges to mechanosensitive channels within the domain of the cnidociliary membrane in the contact region.     

The ciliated sensory cell of Stauridiosarsia producta (Cnidaria,Hydrozoa) — a nematocyst-free nematocyte?

Summary A mechanosensitive ciliated cell type of the polyp Stauridiosarsia producta (Hydrozoa) was investigated by means of electron microscopy. These cells bear at their apical cell surface a modified cilium, a set of seven stereovilli, a so-called pseudovillar system and a large vacuole. Cilium and stereovilli are interconnected like the cnidocil apparatus of hydrozoan nematocytes which is responsible for mechanoelectric transduction. The vacuole is enclosed by and linked to the pseudovillar system by a microtubular basket. Considering its structural organization and physiological activities the ciliated sensory cell closely resembles a nematocyte that has lost its ability ot produce a nematocyst.     

Secondary sensory cells in the gravity receptor system of the statocyst of Octopus vulgaris

Summary The presence of secondary sensory cells in the Octopus gravity receptor system has been demonstrated. In serial thin sections of the receptor cells (hair cells) no axons were found leaving the cells. Instead, synapses were observed with synaptic vesicles lying inside the receptor cells. Both data clearly indicate that the receptor hair cells represent secondary sensory cells. In addition, efferent contacts to the receptor cells could be confirmed.This work was supported in part by grant Wo 160/5 of the Deutsche Forschungsgemeinschaft to Prof. Dr. H.G. WolffThe experimental work was done in part at the Zoological Station in Naples and at the Sechenov Institute of Evolutionary Physiology and Biochemistry of the USSR Academy of Sciences (Laboratory of Prof. Dr. Ya.A. Vinnikov), Leningrad, USSR. The authors thank Prof. Vinnikov and Dr. Tsirulis for stimulating discussions     

Glutamatergic and GABAnergic control in the tentacle effector systems of Hydra vulgaris

In addition to their role in orchestrating body and tentacle contractions, hydra’s nerves control the behavior of nematocysts; precisely how is still a work in progress. There are strong indications that the classical neurotransmitters, glutamate and GABA (γ-amino-butyric acid), play an essential role in effecting stenotele and desmoneme discharge. In experiments on isolated tentacles of Hydra vulgaris, in which cnidocils were mechanically deflected with a piezo-electrically-driven glass micropipette, stenoteles and desmonemes respond to differences in applied force in a dose-dependent manner. GABA, working through its metabotropic receptor, appears to be involved with the recruitment of desmonemes. Desmonemes in distant battery cells or in another part of a given battery cell were discharged by stimulating a desmoneme cnidocil in the presence of bath-applied GABA or its metabotropic agonist, baclofen. The effect was blocked by phaclofen, its metabotropic antagonist. Neither GABA nor baclofen affected stenotele discharge. GABA_A agonists had no effect on nematocyst discharge. Glutamate caused a significant increase in number of stenoteles responding to direct mechanical stimuli, but did not effect desmoneme discharge. The effect was mimicked by NMDA (n-methyl-d-aspartate) together with kainate, or by NMDA plus AMPA (amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid), but not with any ionotropic agonist alone. The effect was blocked by D-AP 5 (d- (−)–2-amino–5-phosphopentanoic acid), a specific NMDA antagonist, or CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), a specific kainate/AMPA antagonist. A glutamatergic mechanism working through ionotropic glutamate receptors appears to lower the firing threshold of stenoteles, perhaps␣by permitting the entry of Ca²⁺ into the cell through the early evolved NMDA/kainite/AMPA mechanism.     

Hair Cell Interactions in the Statocyst of Hermissenda

Hair cells in the statocyst of Hermissenda crassicornis respond to mechanical stimulation with a short latency (<2 ms) depolarizing generator potential that is followed by hyperpolarization and inhibition of spike activity. Mechanically evoked hyperpolarization and spike inhibition were abolished by cutting the static nerve, repetitive mechanical stimulation, tetrodotoxin (TTX), and Co⁺⁺. Since none of these procedures markedly altered the generator potential it was concluded that the hyperpolarization is an inhibitory synaptic potential and not a component of the mechanotransduction process. Intracellular recordings from pairs of hair cells in the same statocyst and in statocysts on opposite sides of the brain revealed that hair cells are connected by chemical and/or electrical synapses. All chemical interactions were inhibitory. Hyperpolarization and spike inhibition result from inhibitory interactions between hair cells in the same and in opposite statocysts.     

The cnidarian cnidocyte,a hightech cellular weaponry

The members of the phylum Cnidaria (corals, sea anemones, medusae) are all equipped with stinging cells (cnidocytes, nematocytes), which serve mainly in prey capture and defense. The secretory product of these cells is a most complicated extrusome consisting of a cyst containing a tubule and a liquid matrix. Mechanical stimulation of the cell's cnidocil apparatus by a prey or an offender leads via bioelectrical signal transduction to the explosive discharge of the cnidocyst. In stenoteles of Hydra this process, during which the tubule is everted out of the cyst, takes less than 3 msec. The forces involved are partly due to spring-like tensions stored in the collagenous structural compartment, and partly to an osmotically generated intracapsular pressure, which can amount to 150 bar (1.5 × 10⁷ Pa). The osmotic machinery depends on the presence in the cyst's matrix of inorganic cations (either K⁺, Mg²⁺ or Ca²⁺) and rare polyanions (poly-γ-L-glutamates), which, so far, have not been reported from recently evolved eukaryotes. The discharging cyst acts like a self-reloading syringe, injecting poison and other components into the target. Since the cnidocytes are incapable of regenerating their exocytosed cysts, they have to be replaced by new cells derived by differentiation from pluripotent stem cells (interstitial cells).     

Fine structure of the nematocytes of Polypodium hydriforme Ussov (Cnidaria)

The fine structure of the stinging cells (nematocytes) and stinging capsules (nematocysts) is described for Polypodium hydriforme. a freshwater coclenterate with a prominent endoparasitie stage in its life cycle. All the nematocysts belong to the type of lesser glutinants (atrichous isorhiza) and fall into three size classes. The internal structure of the capsules is similar in the three classes. A novel type of organization of the cnidocil apparatus of the nematocysts is described. The cnidocil lacks a root fibre and its kinctosome sits directly on the operculum of the nematocyst, so that the entire cnidocil apparatus has a radial rather than bilateral symmetry. It is compared with that of other types of nematocytes and its similarity with the mechanoreccptors of the coelentcratcs is noted. The possible place of the Polypodium nematocytes in the evolution of the collar receptors of the Metazoa is discussed.     

Secondary hair cells and afferent neurones of the squid statocyst receive both inhibitory and excitatory efferent inputs

Summary Intracellular recordings were obtained from the hair cells and afferent neurones of the angular acceleration receptor system of the statocyst of the squid,Alloteuthis subulata. Electrical stimulation of the efferent fibres in the crista nerve (minor) evoked responses in all of the secondary hair cells recorded from (n=211). 48% of the secondary air cells responded with a small depolarization, 15% with a hyperpolarization, and 37% with a depolarization followed by a hyperpolarization. The depolarizations and hyperpolarizations had mean stimulus to response delays of 6.7 ms and 24 ms, and reversal potentials of about –1 mV and –64 mV, respectively. Both types of potential increased in amplitude, up to a point, when the stimulus shock was increased and facilitation and/or summation effects could be obtained by applying multiple shocks. These data, together with the fact that both responses could be blocked by bath application of cobalt or cadmium, indicate that the secondary hair cells receive both inhibitory and excitatory efferent inputs and that these are probably mediated via chemical synapses. No efferent responses were seen in the primary hair cells but both depolarizing and hyperpolarizing efferent responses were obtained from the afferent neurones.     

Ultrastructural Evidence for Neuromuscular Systems in Coelenterates

Cellular interrelationships and synaptic connections in tentaclesof several species of coelenterates were examined by means ofelectron microscopy to determine if neuromuscular pathways werepresent. The presence of sensory cells, ganglion cells, epitheliomuscularcells, interneuronal synapses, and neuromuscular junctions suggeststhat neuromuscular pathways are present in coelenterates. Nakedaxons without sheath cells form several synapses en passantwith the same and with different epitheliomuscular cells aswell as with nematocytes and other neurons. Interneuronal synapsesand neuromuscular and neuronematocyte junctions have clear ordense-cored vesicles (700–1500 Å in diameter) associatedwith a dense cytoplasmic coat on the presynaptic membrane, acleft (100–300 Å in width) with intracleft filaments,and a subsynaptic membrane with a dense cytoplasmic coat. Atscyphozoan neuromuscular junctions there is a subsurface cisternaof endoplasmic reticulum, which is separated from the epitheliomuscularcell membrane by a narrow cytoplasmic gap (100–300 Åin width) . Neuromuscular junctions in coelenterates resembleen passant axonal junctions with smooth muscle in higher animals. Morphological evidence is presented for a simple reflex involvinga two-cell (sensory or ganglion-epitheliomuscular cell) or three-cell(sensory-ganglion-epitheliomuscular cell) pathway that may resultin the coordinated contraction of the longitudinal muscle intentacles of coelenterates.     

Arrangement of accessory cells and nematocytes bearing mastigophores in the tentacles of the cubozoan Chironex fleckeri

Nematocytes containing microbasic mastigophores are intimately associated with accessory cells in the epidermis of Chironex fleckeri. Large microbasic mastigophores may be surrounded by seven to nine such cells. Each accessory cell possesses an apical portion containing secretory droplets and a basal portion which carries a radially oriented fibre linking the cell to the underlying mesogloea. The fibre is capable of projecting and retracting the accessory cell. Junctional complexes occur between accessory cells and the apical regions of neighbouring mastigophores. Each nematocyte bearing a mastigophore contains a triggering apparatus consisting of a cnidocil surrounded by microvilli. This apparatus protrudes from an invagination in the apical region of the nematocyte and is exposed when the mastigophore is in the fire-ready position. A basket of filaments which make contact with microvilli surrounds the apical end of the nematocyst like a collar. The basket is linked via fibrous bundles which envelop the mastigophore to radially oriented fibres basally. These fibres are capable of projecting and retracting the mastigophore and its associated triggering apparatus. Up to nine such fibres were observed to be associated with a single large microbasic mastigophore. Microtubules averaging 25 nm in diameter and linked via cross bridges to electrondense material were detected in the radial fibres of both nematocytes and accessory cells. Retraction of the accessory cells and projection of nematocytes result in mastigophores being brought to the firing line and in the exposure of the cnidocil apparatus.     

Ultrastructural localization of monoamines in the epidermis of Lumbricus terrestris (L.)

Summary Receptor cells in the epithelium and the basiepithelial nerve net of the prostomium of Lumbricus terrestris were investigated with electron microscope with special regard to the presence of monoamines. The receptor cells are found in groups of about 40 intermingled with supportive cells. After pretreatment with -methyl-noradrenaline and fixation with potassium permanganate a few receptor cells in each group and some nerve fibres in the basiepithelial nerve net contain small granular vesicles (about400 Å) characteristic for monoaminergic neurons. The distribution and relative number of these receptor cells and nerve fibres coincide well with previous reports on fluorescent receptor cells and varicose fibres. That the monoamine-storing small granular vesicles not are visualized until pretreatment with -methyl-noradrenaline is in accordance with recent microspectrofluorometric analysis, which shows that dopamine is the only primary monoamine present in the epithelium.In the epithelium there are occasional receptor cells and nerve fibres containing large vesicles (1000–1800 Å) which resemble the neurosecretory vesicles in the central nervous system. Photoreceptor cells having an intracellular cavity with microvilli and cilia have infrequently been observed at the base of the epithelium.No synapses on the mucous cells have been noticed. Nor have any synaptic specializations been observed in the basiepithelial nerve net. The morphological conditions necessary for the existence of possible axo-axonal synapses are briefly discussed.This work was supported by grants from the Helge Ax: son Johnson Foundation and the Magn. Bergvall Foundation.     

Soil acidity factors and nodulation ofTrifolium repens

Summary Effects of factors associated with soil acidity (low pH, low calcium, high aluminium and high manganese) on theTrifolium repens-Rhizobium trifolii symbiosis were investigted under laboratory conditions using an axenic solution-culture technique. 200 μM manganese increased root elongation in the range pH 4.3–5.5, but had no effect on root hair formation, the number of Rhizobium in the rhizosphere, or nodule formation. Root elongation and root hair formation were unaffected at pH 4.3 when 500 or 1000μM calcium was supplied, whereas multiplication of Rhizobium in the rhizosphere and nodulation were inhibited at pH 4.3 and 4.7.50–1000μM calcium had no effect either on the multiplication of Rhizobium in the range pH 4.3–5.5, or on nodule formation in the absence of aluminium. 50 μM aluminium inhibited, root elongation and root hair formation at pH 4.3 and 4.7; the effect on root elongation was reduced by increasing the calcium concentration from 50 to 1000μM. 50μM aluminium also inhibited Rhizobium multiplication in the rhizosphere and reduced nodule formation at pH 5.5 (at which aluminium precipitated out of solution), but root elongation and root hair formation were unaffected. These, effects of aluminium at pH 5.5 may explain the poor response to inoculation by white clover in acid mineral soils after liming.     

Characterization of glycinergic synapses in vertebrate retinas

Summary Glycine is one of the essential neurotransmitters modulating visual signals in retina. Glycine activates Cl^- permeable receptors that conduct either inhibitory or excitatory actions, depending on the Cl⁻ electrical–chemical gradient (E _Cl) positive or negative to the resting potential in the cells. Interestingly, both glycine-induced inhibitory and excitatory responses are present in adult retinas, and the effects are confined in the inner and outer retinal neurons. Glycine inhibits glutamate synapses in the inner plexiform layer (IPL), resulting in shaping light responses in ganglion cells. In contrast, glycine excites horizontal cells and On-bipolar dendrites in the outer plexiform layer (OPL). The function of glycinergic synapse in the outer retina represents the effect of network feedback from a group of centrifugal neurons, glycinergic interplexiform cells. Moreover, immunocytochemical studies identify glycine receptor subunits (α1, α2, α3 and β) in retinas, forming picrotoxin-sensitive α-homomeric and picrotoxin-insensitive α/β-heteromeric receptors. Glycine receptors are modulated by intracellular Ca²⁺ and protein kinas C and A pathways. Extracellular Zn²⁺ regulates glycine receptors in a concentration-dependent manner, nanomolar Zn²⁺ enhancing glycine responses, and micromolar Zn²⁺ suppressing glycine responses in retinal neurons. These studies describe the function and mechanism of glycinergic synapses in retinas.