Serotonergic sensory‐motor neurons mediate a behavioral response to hypoxia in pond snail embryos

Oxygen (O₂) is one of the most important environmental factors that affects both physiological processes and development of aerobic animals, yet little is known about the neural mechanism of O₂ sensing and adaptive responses to low O₂ (hypoxia) during development. In the pond snail, Helisoma trivolvis, the first embryonic neurons (ENC1s) to develop are a pair of serotonergic sensory‐motor cells that regulate a cilia‐driven rotational behavior. Here, we report that the ENC1‐ciliary cell circuit mediates an adaptive behavioral response to hypoxia. Exposure of egg masses to hypoxia elicited a dose‐dependent and reversible acceleration of embryonic rotation that mixed capsular fluid, thereby facilitating O₂ diffusion to the embryo. The O₂ partial pressures (Po₂) for threshold, half‐maximal, and maximal rotational response were 60, 28, and 13 mm Hg, respectively. During hypoxia, embryos relocated to the periphery of the egg masses where higher Po₂ levels occurred. Furthermore, intermittent hypoxia treatments induced a sensitization of the rotational response. In isolated ciliary cells, ciliary beating was unaffected by hypoxia, suggesting that in the embryo, O₂ sensing occurs upstream of the motile cilia. The rotational response of embryos to hypoxia was attenuated by application of the serotonin receptor antagonist, mianserin, correlated to the development of ENC1‐ciliary cell circuit, and abolished by laser‐ablation of ENC1s. Together, these data suggest that ENC1s are unique oxygen sensors that may provide a good single cell model for the examination of mechanistic, developmental, and evolutionary aspects of O₂ sensing. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 73–83, 2002     

Serotonergic sensory-motor neurons mediate a behavioral response to hypoxia in pond snail embryos

Oxygen (O(2)) is one of the most important environmental factors that affects both physiological processes and development of aerobic animals, yet little is known about the neural mechanism of O(2) sensing and adaptive responses to low O(2) (hypoxia) during development. In the pond snail, Helisoma trivolvis, the first embryonic neurons (ENC1s) to develop are a pair of serotonergic sensory-motor cells that regulate a cilia-driven rotational behavior. Here, we report that the ENC1-ciliary cell circuit mediates an adaptive behavioral response to hypoxia. Exposure of egg masses to hypoxia elicited a dose-dependent and reversible acceleration of embryonic rotation that mixed capsular fluid, thereby facilitating O(2) diffusion to the embryo. The O(2) partial pressures (Po(2)) for threshold, half-maximal, and maximal rotational response were 60, 28, and 13 mm Hg, respectively. During hypoxia, embryos relocated to the periphery of the egg masses where higher Po(2) levels occurred. Furthermore, intermittent hypoxia treatments induced a sensitization of the rotational response. In isolated ciliary cells, ciliary beating was unaffected by hypoxia, suggesting that in the embryo, O(2) sensing occurs upstream of the motile cilia. The rotational response of embryos to hypoxia was attenuated by application of the serotonin receptor antagonist, mianserin, correlated to the development of ENC1-ciliary cell circuit, and abolished by laser-ablation of ENC1s. Together, these data suggest that ENC1s are unique oxygen sensors that may provide a good single cell model for the examination of mechanistic, developmental, and evolutionary aspects of O(2) sensing.     

Early development of an identified serotonergic neuron in Helisoma trivolvis embryos: Serotonin expression,de-expression,and uptake

In early-stage embryos of Helisoma trivolvis, a bilateral pair of identified neurons (ENC1) express serotonin and project primary descending neurites that ramify in the pedal region of the embryo prior to the formation of central ganglia. Pharmacological studies suggest that serotonin released from ENC1 acts in an autoregulatory pathway to regulate its own neurite branching and in a paracrine or synaptic pathway to regulate the activity of pedal ciliary cells. In the present study, several key features of early ENC1 development were characterized as a necessary foundation for further experimental studies on the mechanisms underlying ENC1 development and its physiological role during embryogenesis. ENC1 morphology was determined by confocal microscopy of serotonin-immunostained embryos and by differential-interference contrast (DIC) microscopy of live embryos. The soma was located at an anteriolateral superficial position and contained several distinguishing features, including a large spherical nucleus with prominent central nucleolus, large granules in the apical cytoplasm, a broad apical dendrite ending in a sensory-like structure at the embryonic surface, and a ventral neurite. ENC1 first expressed serotonin immunoreactivity around stage E13, followed immediately by the appearance of an immunoreactive neurite (stage E14). Both the intensity of immunoreactivity and primary neurite length were consistently greater in the right ENC1 at early stages. Serotonin uptake, as indicated by 5,7-dihydroxytryptamine-induced fluorescence, first occurred between stages E18 and E25. At later stages of embryogenesis (after stage E65), serotonin immunoreactivity disappeared, whereas serotonin uptake and normal cell morphology were retained. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 361–376, 1998     

Constitutive and permissive roles of nitric oxide activity in embryonic ciliary cells

Embryos of Helisoma trivolvis exhibit cilia-driven rotation within the egg capsule during development. In this study we examined whether nitric oxide (NO) is a physiological regulator of ciliary beating in cultured ciliary cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 1-1,000 microM) produced a dose-dependent increase in ciliary beat frequency (CBF). In contrast, the nitric oxide synthase (NOS) inhibitor 7-nitroindazole (10 and 100 microM) inhibited the basal CBF and blocked the stimulatory effects of serotonin (100 microM). NO production in response to serotonin was investigated with 4,5-diaminofluorescein diacetate imaging. Although SNAP (100 microM) produced a rise in NO levels in all cells, only 22% of cells responded to serotonin with a moderate increase. The cGMP analog 8-bromo-cGMP (8-Br-cGMP; 0.2 and 2 mM) increased CBF, and the soluble guanylate cyclase inhibitor LY-83583 (10 microM) blocked the cilioexcitatory effects of SNAP and serotonin. These data suggest that NO has a constitutive cilioexcitatory effect in Helisoma embryos and that the stimulatory effects of serotonin and NO work through a cGMP pathway. It appears that in Helisoma cilia, NO activity is necessary, but not sufficient, to fully mediate the cilioexcitatory action of serotonin.     

Pharmacological characterization of a serotonin receptor involved in an early embryonic behavior of Helisoma trivolvis

In contrast to the abundance of information on the many physiological and developmental actions of serotonin in molluscan nervous systems, comparatively little is known about the serotonin receptors involved in these responses. Embryos of the pulmonate gastropod, Helisoma trivolvis, display a cilia-driven rotational behavior that is regulated by endogenous serotonin. In the present study, two functional assays were used to determine some of the pharmacological properties of the receptors that mediate the cilio-excitatory action of serotonin. Timelaspe video microscopy was used to measure whole embryo rotation rat and cilia beat frequency in isolated cells. In dose-response experiments, serotonin was approximately 10 times more potent in stimulating cilia beat frequency over embryo rotation. In rotation experiments, 5-carboxyamidotryptamine and methysergide had effective agonist activity in dose ranges similar to that of serotonin (1 to 100 μM). In contrast, 8-hydroxydiproylaminotetralin HBr (8-OH-DPAT) displayed agonist activity of lower potency and effectiveness. Several compounds displayed antagonist activity in the 1 to 100 μM dose range, including mianserin, spiperone, ritanserin, 1-(1-naphthyl) piperazine, and Propranolol. α-Methylserotonin had mixed agonist–antagonist activity, and metoclopramide, MDL-72222, and ketanserin were inactive. Experiments on isolated cells suggested that the extremely effective antagonism displayed by mianserin in the embryo rotation assay was due to its specific activity at ciliary serotonin receptors. These results implicate the presence of a novel serotonin receptor on embryonic ciliated cells that is pharmacologically distinct from those previously characterized in vertebrate or invertebrate systems. 1994 John Wiley & Sons, Inc.     

Serotonergic cerebral cells control activity of cilia in the foregut of the pteropod mollusk <Emphasis Type="Italic">Clione limacina</Emphasis>

Bilaterally symmetrical pair of serotonergic cells, named C1 in Clione, has been described in the cerebral ganglia of all gastropod species. Here we describe a new role of C1 cells in gastropod mollusks: control of activity of ciliated epithelium in the foregut. Detailed morphological investigation of C1 neurons in the pteropod mollusk Clione limacina revealed that these cells among other destinations send their neurites into foregut where they produce intense arborization with large varicosities along the processes. Intracellular stimulation of a single C1 induced pronounced activation (often followed by inhibition) of cilia lining the foregut. This activation was substantially reduced by serotonin antagonist mianserin. Bath application of serotonin also induced transient increase in ciliary transport rate, followed by inhibition of ciliary activity up to its full cessation in some areas of isolated foregut. These data suggest that C1 in Clione may use serotonin to influence cilia in the foregut. Taking into account high homology of serotonergic cerebral cells across studied species we can speculate that these cells may be involved in the neural control of cilia in the foregut in other gastropod mollusks.     

Characterization and development of rotational behavior in Helisoma embryos: role of endogenous serotonin.

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.     

Characterization and development of rotational behavior in Helisoma embryos: Role of endogenous serotonin

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.     

Neuronal control of pedal sole cilia in the pond snail Lymnaea stagnalis appressa

5-HT (serotonin) is a ubiquitous neurotransmitter that produces ciliary beating in gastropods when applied topically, but ciliary beating caused by gastropod serotonergic neurons has been described in only three neuron pairs. We extend these results to the North American Lymnaea stagnalis appressa, which is a different species from the European Lymnaea stagnalis. We describe a non-serotonergic neuron pair, PeV1, which accelerates pedal sole mucociliary transport and a serotonergic neuron pair, PeD7, which slows mucociliary transport. We compare and discuss development and identified neurons in L. s. appressa and in L. stagnalis, which have homologs to L. s. appressa PeD7 and PeV1 neurons. In addition to PeD7 and PeV1 neurons, we test neurons immunoreactive to Tritonia pedal peptide antibodies with negative results for mucociliary transport. In characterizing PeD7 and PeV1 neurons, we find that PeV1 does not excite PeD7. In semi-intact preparations, a strong increase in PeD7 neuron activity occurs during tactile stimulation, but V1 neurons are inhibited during tactile stimulation. Following tactile stimulation, PeV1 neurons show strong activity. This suggests a distinct difference in function of the two neuron pairs, which both have their axons overlying pedal sole ciliary cells. Application of 5-HT to the pedal sole initiates mucociliary transport in 1.4–1.9 s with a time course similar to that seen when stimulating a PeV1 neuron. This result appears to be through a 5-HT_1A-like receptor on the pedal sole. We describe a possible external source of 5-HT on the pedal sole from 5-HT immunoreactive granules that are released with mucus.     

Roles of Ca2+ and protein kinase C in the excitatory response to serotonin in embryonic molluscan ciliary cells

We examined the roles of Ca2+ and protein kinase C (PKC) in the cilio-excitatory response to serotonin in pedal ciliary cells from Helisoma trivolvis embryos. Serotonin (5-hydroxytryptamine; 5-HT; 100 micromol/L) induced an increase in ciliary beat frequency (CBF) was abolished by microinjected BAPTA (50 mmol/L), but was only partially inhibited by the phospholipase C inhibitor U-73122 (10 micromol/L). The diacylglycerol analogs 1-oleoyl-2-acetyl-sn-glycerol (100 micromol/L) and 1,2-dioctanoyl-sn-glycerol (100 micromol/L) caused increases in [Ca2+]i that were smaller than those induced by serotonin. In the absence of extracellular Ca2+, 1,2-dioctanoyl-sn-glycerol (100 micromol/L) failed to elicit an increase in both CBF and [Ca2+]i. In contrast, the serotonin-induced increase in CBF persisted in the absence of extracellular Ca2+, although the increase in [Ca2+]i was abolished. PKC inhibitors bisindolylmaleimide (10 and 100 nmol/L) and calphostin C (10 nmol/L) partially inhibited the serotonin-induced increase in CBF, but didn't affect the serotonin-induced change in [Ca2+]i. These findings suggest that an intracellular store-dependent increase in [Ca2+]i mediates the cilio-excitatory response to serotonin. Furthermore, although PKC is able to cause an increase in [Ca2+]i through calcium influx, it contributes to the cilio-excitatory response to 5-HT through a different mechanism.     

Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis

The neurotransmitter serotonin has been shown to inhibit neurite outgrowth in specific identified neurons isolated from adult Helisoma. While in vivo experiments on Helisoma embryos have supported the hypothesis that endogenous serotonin regulates neurite outgrowth during embryonic development, direct effects of serotonin on embryonic neurons have not been measured. In the present study, cultures of dissociated embryonic neurons were used to test the direct actions of serotonin on developing embryonic neurons. Serotonin arrested neurite outgrowth in a significant percentage of elongating neurites in a dose-dependent manner. Furthermore, analysis of neurons with stable, nonelongating neurites revealed a novel response. Serotonin caused the reinitiation of neurite outgrowth in a significant percentage of nonelongating neurites. The arrestment of outgrowth and reinitiation of outgrowth occurred in similar percentages of elongating and nonelongating neurites, respectively. Parallel experiments on cultures of dissociated adult neurons were carried out to determine whether serotonin could also induce both inhibitory and stimulatory responses in adult cells. Serotonin arrested neurite outgrowth in a similar percentage of neurites to that observed in cultures of embryonic neurons. In contrast, serotonin did not reinitiate neurite outgrowth in a significant percentage of adult neurites. These data support the hypothesis that serotonin regulates neurite outgrowth in developing embryonic neurons. Furthermore, only some of these regulatory effects appear to be conserved from embryonic to adult neurons.     

A simplified method to generate serotonergic neurons from mouse embryonic stem and induced pluripotent stem cells

We have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancer-driven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders.     

Expression patterns of HNK-1 carbohydrate and serotonin in sea urchin, amphioxus, and lamprey, with reference to the possible evolutionary origin of the neural crest

We examined deuterostome invertebrates, the sea urchin and amphioxus, and an extant primitive vertebrate, the lamprey, for the presence of structures expressing the HNK-1 carbohydrate and serotonin. In sea urchin embryos and larvae, HNK-1 positive cells were localized in the ciliary bands and in their precursor ectoderm. Serotonergic cells were exclusively observed in the apical organs. In juvenile amphioxus, primary sensory neurons in the dorsal nerve cords were HNK-1 immunoreactive. The juvenile amphioxus nerve cords contained anti-serotonin immunoreactive nerve fibers that seem to be the Rohde axons extending from amphioxus interneurons, the Rohde cells. In lamprey embryos, migrating neural crest cells and primary sensory neurons, including Rohon-Beard cells, expressed the HNK-1 carbohydrate. Lamprey larvae (ammocoetes) contained cell aggregates expressing both the HNK-1 carbohydrate and serotonin in the pronephros and in the regions adjacent to the gut epithelium. Some of these cell aggregates were present in the anti-serotonin positive visceral motor nerve net. Motor neurons and Müller fibers were serotonergic in ammocoetes. Comparison of the expression patterns of HNK-1 carbohydrate among sea urchins, amphioxus and lampreys seem to suggest the possible evolutionary origin of the neural crest, that is, ciliary bands in dipleurula-type ancestors evolved into primary sensory neurons in chordate ancestors, as inferred from Garstang's auricularia hypothesis, and the neural crest originated from the primary sensory neurons.     

Long-term regulation of serotonergic activity in the rat brain via activation of protein kinase A.

Serotonergic neurons located at the base of the mammalian brain innervate practically every region of the brain and the spinal cord. These neurons exhibit spontaneous electrical discharges in a rhythmical way. Their firing frequency is modulated by serotonin autoreceptors which also regulate intracellular cAMP levels. We have investigated how elevated levels of cAMP alter the development and the functional properties of serotonergic neurons in culture. To study the influence of cAMP on the expression of genes underlying serotonergic activity, a quantitative RT-PCR approach using internal standards was developed. Cultures of embryonic rat brain serotonergic neurons were continuously treated with cAMP analogues. Increased cAMP levels had three effects. First, the neuronal morphology was changed towards that typical for mature serotonergic neurons. Second, the expression of tryptophan hydroxylase, the rate-limiting enzyme in serotonin production, was increased in dibutyryl-cAMP treated cultures. Third, the expression of the inhibitory autoreceptor (5-HT1A) was down-regulated. These results suggest the existence of a mechanism by which the neurons react to synaptic input regulating intracellular cAMP levels. Increased cAMP concentrations affect the development and cause a prolonged activation of serotonergic transmission. Since 5-HT1A receptors inhibit cAMP formation, their down-regulation argues against a negative feedback control in this system, consistent with observations in vivo.     

Casein kinase II and calcineurin modulate TRPP function and ciliary localization

Cilia serve as sensory devices in a diversity of organisms and their defects contribute to many human diseases. In primary cilia of kidney cells, the transient receptor potential polycystin (TRPP) channels polycystin-1 (PC-1) and polycystin-2 (PC-2) act as a mechanosensitive channel, with defects resulting in autosomal dominant polycystic kidney disease. In sensory cilia of Caenorhabditis elegans male-specific neurons, the TRPPs LOV-1 and PKD-2 are required for mating behavior. The mechanisms regulating TRPP ciliary localization and function are largely unknown. We identified the regulatory subunit of the serine-threonine casein kinase II (CK2) as a binding partner of LOV-1 and human PC-1. CK2 and the calcineurin phosphatase TAX-6 modulate male mating behavior and PKD-2 ciliary localization. The phospho-defective mutant PKD-2(S534A) localizes to cilia, whereas a phospho-mimetic PKD-2(S534D) mutant is largely absent from cilia. Calcineurin is required for PKD-2 ciliary localization, but is not essential for ciliary gene expression, ciliogenesis, or localization of cilium structural components. This unanticipated function of calcineurin may be important for regulating ciliary protein localization. A dynamic phosphorylation-dephosphorylation cycle may represent a mechanism for modulating TRPP activity, cellular sensation, and ciliary protein localization.     

Association of SPARC (osteonectin, BM-40) with extracellular and intracellular components of the ciliated surface ectoderm of Xenopus embryos

SPARC (Secreted Protein, Acidic, Rich in Cysteine) was detected by immunohistochemistry in the sensorial layer of the bilayered embryonic epidermis of Xenopus laevis during neurulation, when a subset of the sensorial cells are selected to differentiate into ciliated cell precursors. After the ciliated cells had intercalated into the outer layer and had undergone ciliogenesis, intense SPARC immunostaining was associated with the cilia and remained associated with the cilia throughout their persistence on the epidermis. Circumferential SPARC immunostaining was also detected at the interface between surface epithelial cells. Animal cap explants indicated that the embryonic activation of SPARC expression in the dorsal ectoderm does not require signaling from factors secreted by the underlying mesoderm. Immunoelectron microscopy revealed that SPARC is intimately associated with the 9 + 2 microtubule arrays of cilia. Our data indicate that SPARC plays a role in the development and function of the surface ciliated epidermis of Xenopus embryos. We propose that the counter-adhesive activity of SPARC facilitates the intercalation of ciliary cell precursors to the surface epithelial layer, where its Ca(2+)-binding abilities promote cell-cell adhesion. Based on its association with ciliary microtubule arrays, we also propose that intracellular SPARC may play a role in regulating ciliary beat frequency and polarity.     

Localized cytosolic alkalization and its functional impact in ciliary cells

Using confocal microscopy we demonstrate that ciliary cells from airway epithelium maintain two qualitatively distinct cytosolic regions in terms of pH regulation. While the bulk of the cytosol is stringently buffered and is virtually insensitive to changes in extracellular pH (pHo), the values of cytosolic pH in the vicinity of the ciliary membrane is largely determined by pHo. Variation of pHo from 6.2 up to 8.5 failed to affect ciliary beat frequency (CBF). Application of NH(4)Cl induced profound localized alkalization near cilia, which did not depress ciliary activity, but resulted in strong and prolonged enhancement of CBF. Calmodulin and protein kinase A (PKA) functionality was essential for the alkalization-induced CBF enhancement. We suggest that the ability of airway epithelium to sustain unusually strong but localized cytosolic alkalization near cilia facilitates CBF enhancement through altering the binding constants of Ca2+ to calmodulin and promotion of Ca2+-calmodulin complex formation. The NH4Cl-induced elevations in cytosolic pH and Ca2+ concentration act synergistically to activate calmodulin-dependent processes, cAMP pathway, and, thereby, stimulate CBF.     

Ciliary and nervous structures in juvenile females of the annelid Dinophilus gyrociliatus (O. Schmidt, 1848) (Annelida: Polychaeta)

Ciliary and nerve structures were described in juvenile female Dinophilus gyrociliatus (O. Schmidt, 1848) after immunochemical staining with tubulin, serotonin, and FMRFamide antibodies. Anti-tubulin antibodies revealed the following external structures: two head and seven body ciliary bands, a ventral ciliary band, and head ciliary fields. Gut cilia and five pairs of protonephridia were detected inside the body. The nervous system consists of an oval headed neuropile with anterior and posterior nerves extending from it, seven longitudinal nerve cords, commissures, and circular nerves. Anti-serotonin antibodies revealed the head neuropile, neurons at the base of the ventral ciliary band, an oesophageal ring, and seven longitudinal ventral cords. Anti-FMRFamide antibodies revealed approximately ten neurons in the cerebral ganglion, five longitudinal cords, and the oesophageal and caudal-nerve rings. The presented data suggest the simplification of the nervous system structure in D. gyrociliatus, which probably reflects pedomorphosis.