Ultrastructure of neurons and synapses in the tentacle gastrodermis of the sea anemone Calliactis parasitica

Little is known about gastrodermal neurons and synapses in the tentacles of sea anemones. Using transmission electron microscopy of serial thin sections of Calliactis parasitica, we have identified both a sensory cell and a ganglion cell with granular vesicles originating from the Golgi complex and have identified four types of synapses in the tentacular gastrodermal nerve plexus. The sensory cell has a recessed apical cilium with a basal body and a perpendicularly oriented centriole, below which are several strands of striated rootlets surrounded by mitochondria. The ganglion cell lacks a cilium and resembles a bipolar neuron, with oppositely directed processes lying parallel to the basally located circular smooth muscle. Both one-way and two-way interneuronal synapses are present with 60- to 90-nm granular vesicles of various densities aligned at the paired electron-dense membranes and fine cross filaments in the intervening 13-nm cleft. Two types of neuroeffector synapses have been located. Dense granular vesicles are present at neuromuscular synapses, whereas less dense vesicles are present at neuroglandular synapses. Most of the synaptic vesicles range from 60 to 120 nm in diameter. Two types of nerve cells and a variety of synaptic loci provide morphological substrates for the spontaneous SS2 conduction pulses in the tentacular gastrodermis of C. parasitica. J Morphol 231:217–223, 1997. © 1997 Wiley-Liss, Inc.     

Two slow conduction systems co-ordinate shell-climbing behaviour in the sea anemone Calliactis parasitica.

1. Pulses in two slow conducting systems, the ectodermal SS 1 and the endodermal SS 2, were recorded during shell-climbing behaviour. The mean pulse interval of SS 1 pulses was 7-4 s and that of SS 2 pulses was 6-4 s. Activity in both systems may arise as a sensory response of tentacles to shell contact, but the SS 1 and SS 2 may not share the same receptors. 2. Electrical stimulation of the SS 1 and SS 2 together, at a frequency of 1 shock every 5 s, elicits shell-climbing behaviour in the absence of a shell. 3. Low-frequency nerve-net activity (about 1 pulse every 15 s) accompanies column bending during both normal and electrically elicited responses. This activity probably arises as a result of column bending and is not due to a sensory response to the shell.     

Control of mouth opening and pharynx protrusion during feeding in the sea anemone Calliactis parasitica.

1. Activity in all three known conducting systems (the nerve net, SS1, and SS2) may accompany feeding in Calliactis. The most marked response is an increase in pulse frequency in the SS2 (the endodermal slow conducting system) during mouth opening and pharynx protrusion. 2. Electrical stimulation of the SS2 at a frequency of one shock every 5 s elicits mouth opening and pharynx protrusion in the absence of food. 3. A rise in SS2 pulse frequency is also evoked by food extracts, some amino acids, and in particular by the tripeptide reduced glutathione, which produces a response at a concentration of 10(-5) M. 4. Although the SS2 is an endodermal system, the receptors involved in the response to food appear to be ectodermal. 5. The epithelium that lines the pharynx conducts SS1 pulses, but there is some evidence for polarization of conduction.     

Calitoxin, a neurotoxic peptide from the sea anemone Calliactis parasitica: amino acid sequence and electrophysiological properties

We have isolated a new toxin, calitoxin (CLX), from the sea anemone Calliactis parasitica whose amino acid sequence differs greatly from that of other sea anemone toxins. The polypeptide chain contains 46 amino acid residues, with a molecular mass of 4886 Da and an isoelectric point at pH 5.4. The amino acid sequence determined by Edman degradation of the reduced, S-carboxymethylated polypeptide chain and tryptic and chymotryptic peptides is Ile-Glu-Cys-Lys-Cys-Glu-Gly-Asp-Ala-Pro-Asp-Leu-Ser-His-Met-Thr-Gly-Thr- Val-Tyr - Phe-Ser-Cys-Lys-Gly-Gly-Asp-Gly-Ser-Trp-Ser-Lys-Cys-Asn-Thr-Tyr-Thr-Ala- Val-Ala - Asp-Cys-Cys-His-Glu-Ala. No cysteine residues were present in the peptide. Similarly to other sea anemone toxins, calitoxin interacts, in crustacean nerve muscle preparations, with axonal and not with muscle membranes, inducing a massive release of neurotransmitter that causes a strong muscle contraction. The low homology of CLX with RP II and ATX II toxins has implications regarding the role played by particular amino acid residues.     

The transmission of impulses in the ectodermal slow conduction system of the sea anemone Calliactis parasitica (Couch).

1. The SS 1 fatigues in response to repetitive electrical stimulation. This fatigue is manifested by an increased conduction delay and a decreased SS 1 pulse amplitude. 2. Continued repetitive stimulation leads to the failure of the system. Recovery may take many seconds. Narrow strips of column fail more rapidly than wide strips. 3. The increased conduction delay is explained in terms of a decrease in the population of spiking cells. 4. A computer model is described and analysed. It suggests that conduction between electrically coupled ectoderm cells could be the basis for the SS1. The SS 1 may have properties not so far experimentally demonstrated; for example, under certain conditions it could behave as a local system.     

Delayed initiation of SS1 pulses in the sea anemone Calliactis parasitica: evidence for a fourth conducting system.

1. Single electrical shocks to the column sometimes elicit a series of 1-6 pulses in the SS1 (ectodermal slow system) but the first pulse does not appear until 5-28 s after stimulation. These pulses occur in addition to the early SS1 pulse which follows every shock and which has a conduction delay of less than 1 s. 2. The threshold of the delayed SS1 response is different from the thresholds of the three known conducting systems (through-conducting nerve net, SS1, and SS2). 3. In the case of stimulation of the column, the delayed SS1 pulses do not arise at the point of stimulation but probably originate in the tentacles or upper column. The pulse origin can shift during a single burst. 4. The pathway from the point of stimulation to the site of origin of delayed SS1 pulses is endodermal. We propose that this pathway represents a fourth conducting system (Delayed Initiation System--DIS). The DIS must connect, across the mesogloea, with the ectodermal SS1. The long pulse delay and repetitive firing may derive from pacemaker activity in the DIS. The DIS pacemakers closely resemble the pacemakers connected to the through-conducting nerve net. The DIS may be neuronal. 5. Delayed SS1 pulse bursts from unattached anemones showed an earlier onset, and more pulses/burst, than those from attached anemones. 6. Delayed SS1 pulses can also be evoked by electrical, and in some cases mechanical, stimulation of the pedal disc, tentacles, and pharynx, but there are regional differences in the number of pulses evoked, in their delay, and in their site of origin.     

Toxins of the sea anemone Epiactis prolifera

Fe³⁺-EDTA chelates react with the superoxide radical at physiological pH values (k = 1.3 × 10⁶M^?1 s^?1 at pH 7 but is lower at more alkaline pH values) but do not appear to catalyze O₂^? dismutation at a significant rate. Complexes of Fe³⁺ with desferrioxamine, bathophenanthroline, or diethylenetriaminepentaacetic acid react much more slowly, if at all. Fe²⁺ complexes of EDTA, ATP, and diethylenetriaminepentaacetic acid also react with O₂^? at alkaline pH values. The significance of these reactions in the mechanism of the “iron-catalyzed Haber-Weiss reaction” is discussed.     

The marginal sphincter of the sea anemoneCalliactis parasitica

Journal of Comparative Physiology A -     

Ammonium metabolism in the symbiotic sea anemone Anemonia viridis

The temperate sea anemone Anemonia viridis (Forskål) forms an endosymbiotic association with dinoflagellate algae commonly referred to as zooxanthellae. It is now well established that under appropriate environmental conditions, these associations can be autotrophic for carbon. Under such conditions, many of these symbioses, including A. viridis, not only retain excretory ammonium, but can take up ammonium added to the surrounding seawater. The flux from inorganic to organic nitrogen will be via the free amino acid pools and in A. viridis these were found to be markedly different between zooxanthellae and host with glycine and taurine dominant in the latter. When anemones were maintained with 20 M ammonium, the concentration of free amino groups increased in the zooxanthellae but appeared not to change in the host. There was no evidence that the ratio of glutamine – glutamate in zooxanthellae changed when anemones were maintained with 20 M ammonium for 47 days. These ratios imply that zooxanthellae from this temperate symbiosis may not be nitrogen-limited. GDH was detected in both zooxanthellae and host where it was most active with the coenzyme NADPH. In addition, GDH showed activity when glutamine replaced ammonium as the substrate, indicating that the host may have alternative means to assimilate ammonium. Zooxanthellae were shown to possess GOGAT activity in the presence of a ferredoxin analogue. This suggests that in vivo zooxanthellae could assimilate ammonium via the activity of GS linked with ferredoxin-dependent GOGAT. Given evidence from other studies of rapid ammonium assimilation and essential amino acid synthesis in symbiotic host tissue, it appears that the capacity of cnidarians to metabolise nitrogen may at present be underestimated.     

Spontaneous phrenic nerve activity in the exteriorized fetal lamb.

Phrenic nerve activity and tracheal pressure changes were recorded in four exteriorized fetal lambs (120-135 days gestation) from lightly anesthetized ewes to study possible mechanisms involved in the establishment of rhythmical breathing patterns. Two types of spontaneous neural activity were found. The first consisted of high-frequency multiunit bursts (mean duration 820 ms; range 450-2,500 ms) that preceded a gasp. Individual units within these bursts reached peak discharge frequencies as high as 40 impulses/s. The second type of neural activity consisted of single-unit, low-frequency (1-14 impulses/s), irregular background discharges lasting up to several seconds without changes in tracheal pressure. Occasionally, higher frequency bursts of single-unit activity were detected that were also unassociated with tracheal pressure changes. The data indicate that the neural correlate of a fetal gasp includes high-frequency synchronized bursting activity in the phrenic nerve. In addition, background phrenic activity can be detected in the exteriorized fetal lamb that reflects central nervous activity in the absence of tracheal pressure changes.     

Biological activity of sea anemone proteins: II. Cytolysis and cell line toxicity

Potent cytolytic activity was exhibited by proteins extracted from three sea anemones viz. Heteractis magnifica, Stichodactyla haddoni and Paracodylactis sinensis by affecting the red blood corpuscles (RBC) and the mouse fibroblast cell line (L929) and leukemia cell line (P388). Crude toxin of all the three anemone species induced spontaneous hemolysis of chicken, goat and human erythrocytes. The crude toxin of H. magnifica (0.98 mg/ml) elicited hemolysis at levels of 4096, 512 and 4096 HU (hemolytic unit) in chicken, goat and human erythrocytes respectively. Subsequently, the crude toxin of S. haddoni (0.82 mg/ml) exhibited a hemolytic activity of 256, 128 and 512 HU and that of P. sinensis (0.60 mg/ml) had a hemolytic activity of 128, 4096 and 512 HU. Most of the partially purified proteins of these anemones also exhibited the activity against the three different erythrocytes. The viability of L929 and P388 was adversely affected on adding the crude toxins. The symptoms of toxicity shown by the cells were rounding, lysis and detachment from the substratum. These effects were the least in S. haddoni, as compared to those the crude toxins of the other two species. Inhibition of growth of L929 exhibited by the toxin of the three species ranged between 61.08 and 93.38%. Similarly, inhibition of the growth of P388 ranged between 51.32 and 86.16%. The present investigation reveal the cytotoxic nature of anemone toxins.