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Objective: To investigate whether a customised exercise programme influences pregnant women's psychological wellbeing and general health perception reflecting health-related quality of life (HRQoL) in late pregnancy.Design: A two-armed, two-centred randomised controlled trial.Setting: Trondheim and Stavanger University Hospitals, Norway.Population: A total of 855 healthy Caucasian pregnant women.Methods: The intervention group was offered a 12-week exercise programme between 20 and 36 weeks of pregnancy. One weekly group session was led by physiotherapists, in addition women were encouraged to follow a home exercise programme at least twice a week. The exercise programme followed standard recommendations and included both aerobic and strength training. The control group received regular antenatal care. Pretests and post-tests were performed at 18-22 and 32-36 weeks of pregnancy.Main Outcome Measures: In the original study primary outcome was gestational diabetes mellitus, but in this report the primary outcome is HRQoL. The questionnaire Psychological General Wellbeing Index (PGWBI) was used to assess psychological wellbeing and self-perceived general health before and after the intervention. PGWBI contains six subscales and it is also possible to summarise all items to a global score.Results: No association between antenatal exercise programme allocation and PGWBI (global score and subscales) was found. The study population was homogeneous and had high educational level.Conclusions: The results indicate that offering women an exercise programme during pregnancy does not seem to influence healthy pregnant women's psychological wellbeing and self-perceived general health. Further research is needed to investigate the effects of exercise in pregnancy on psychological wellbeing and self-perceived general health among women from different sociocultural subgroups.Tweetable Abstract: Exercise in pregnancy does not influence healthy pregnant women's health-related quality of life. [ABSTRACT FROM AUTHOR]

Introduction: Maternal nutritional and vitamin status during pregnancy may have long-term effects on offspring health and disease. The aim of this study was to examine the associations between maternal vitamin A and D status in pregnancy and offspring bone mineral content (BMC) at nine years of age., Methods: This is a post-hoc study of a randomized control trial including 855 pregnant women from two Norwegian cities; Trondheim and Stavanger. The women were randomized into an exercise intervention or standard antenatal care. Mother and child pairs for the present study were recruited from those still living in Trondheim after 8-10 years. Serum vitamin A (retinol) and vitamin D (25(OH)D) were measured in the 2 nd and 3 rd trimesters of pregnancy, and active vitamin D (1,25(OH) 2 D) in serum was measured in a subgroup. Spine BMC and trabecular bone score were measured in the children at nine years of age. Associations were analyzed with linear regression models., Results: A total of 119 mother and child pairs were included in the analyses. Vitamin A insufficiency (retinol < 1.05 µmol/L) and vitamin D deficiency (25(OH)D < 50 mmol/L) increased from ~7% to ~43% and from ~28% to ~33%, respectively, from the 2 nd to the 3 rd trimester. An increase in serum 1,25(OH) 2 D from the 2 nd to the 3 rd trimester was observed in the subgroup. There was a negative association between serum retinol in the 2 nd trimester and spine BMC in the boys, but not in the girls, when adjusted for maternal and child confounders. No other associations between maternal serum vitamin A or D and BMC in the children were found., Conclusion: We observed a high prevalence of vitamin A insufficiency and vitamin D deficiency during pregnancy. A negative association between mid-pregnancy vitamin A status and spine BMC was observed in boys, but not girls, while no associations were found between maternal vitamin D status and child BMC. The implications of optimal vitamin A and D status in pregnancy for offspring bone health, remains a subject for further investigations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Stunes, Mosti, Gustafsson, Børsting, Thorsby, Stafne and Syversen.)

Background: Research focusing on the association between serum vitamin D and oral health outcomes in children, such as dental caries and molar incisor hypomineralisation (MIH), shows inconsistent results. Previous studies have predominantly investigated dental caries and MIH as dichotomized outcomes, which limits the information on their distribution. In addition, the methods used for analysing serum vitamin D have varied. The present study aimed to investigate potential associations between serum vitamin D status measured by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) and the prevalence, as well as the number of teeth, affected by dental caries or MIH among 7-9-year-old Norwegian children., Methods: The study had a cross-sectional design and included 101 children aged 7-9 years. Serum 25-hydroxyvitamin D (25(OH)D) was measured and included as continuous (per 25 nmol/l) and categorised (insufficient (< 50 nmol/l) and sufficient (≥50 nmol/l)) exposure variables. Adjusted negative binomial hurdle models were used to investigate the potential associations between serum vitamin D and the oral health outcomes (dental caries and MIH) adjusted for sex, age, body mass index, season of blood draw, and mother's educational level., Results: Of the 101 children in the total sample, 27% had insufficient vitamin D levels (< 50 nmol/l). The descriptive analysis indicated that the children with insufficient vitamin D levels had a higher prevalence (33.3%) and a higher number of teeth affected by dental caries (mean (SD) = 0.7 (1.4)), compared to children with sufficient levels of vitamin D (21.6% and mean (SD) = 0.4 (0.8), respectively). The same holds for MIH, with a higher prevalence (38.5%) and a higher number of teeth affected (mean (SD) = 1.2 (2.3)), compared to children with sufficient levels of vitamin D (30.1% and mean (SD) = 0.8 (1.6), respectively). However, in the adjusted hurdle model analysis, neither the prevalence or number of teeth affected by caries or MIH showed statistically significant associations with having insufficient or lower vitamin D levels., Conclusions: Vitamin D status was not significantly associated with the prevalence and number of teeth affected by caries and MIH among the participating children. Large prospective studies with multiple serum vitamin D measurements and oral examinations throughout childhood are warranted to elucidate the relationship., (© 2024. The Author(s).)

Purpose: The study aimed to investigate associations between maternal vitamin D status during pregnancy and molar incisor hypomineralisation (MIH) and hypomineralised second primary molars (HSPM) among children., Methods: The study had a longitudinal design using prospectively collected data from 176 mother and child pairs. Mothers were initially recruited in a randomised controlled trial to assess a pregnancy exercise programme. Along with the 7-year follow-up, we invited the children to a dental examination. The exposure variable was maternal serum 25-hydroxyvitamin D in gestational weeks 18-22 and 32-36, categorised as insufficient (< 50 nmol/l) and sufficient (≥ 50 nmol/l). Negative binomial hurdle models were used to analyse potential associations between the exposure variables and MIH or HSPM. The models were adjusted for potential confounders., Results: Among the children (7-9 years old), 32% and 22% had at least one tooth with MIH or HSPM, respectively. A significant association was found between insufficient maternal vitamin D measured in gestational weeks 18-22 and the number of affected teeth among those with MIH at 7-9 years (adjusted RR = 1.82, 95% CI 1.13-2.93)., Conclusion: Considering any limitations of the present study, it has been shown that insufficient maternal serum vitamin D at mid-pregnancy was associated with a higher number of affected teeth among the offspring with MIH at 7-9 years of age. Further prospective studies are needed to investigate whether this finding is replicable and to clarify the role of maternal vitamin D status during pregnancy and MIH, as well as HSPM, in children., (© 2022. The Author(s).)

Given the scarcity of data on diabetes prevalence and associated risk factors among women in rural Nepal, we aimed to examine this, using glycated hemoglobin (HbA1c) as a diagnostic tool. A cross-sectional survey addressing reproductive health and non-communicable diseases was conducted in 2012-2013 among non-pregnant, married women in Bolde, a rural district of Nepal. HbA1c ≥ 6.5% (48 mmol/mol) was used as diagnostic criterion for diabetes, a cut-off of 7.0% (53 mmol/mol) was used to increase the specificity. HbA1c was measured in 757 women (17-86 years). The prevalence of diabetes and prediabetes was 13.5% and 38.5%, respectively. When using 7.0% as a cut-off, the prevalence of diabetes was 5.8%. Aging, intake of instant noodles and milk and vegetarian food (ns) were associated with increased risk for diabetes. Waist circumference was higher among women with diabetes, although not significant. The women were uneducated (87.6%), and only 12% had heard about diabetes. In conclusion, we observed a higher prevalence of diabetes and prediabetes than anticipated among rural, Nepalese women. The increased risk was mainly attributed to dietary factors. In contrast to most previous studies in Nepal, we used HbA1c as diagnostic criterion.

Hypovitaminosis D is prevalent worldwide, and especially in South-Asia. According to the Institute of Medicine (IOM), 25(OH)D levels below 30 nmol/L are defined as vitamin D deficiency (VDD) and levels between 30−50 nmol/L as insufficiency (VDI). Besides its role in calcium homeostasis, it has been postulated that vitamin D is involved in metabolic syndrome. Given the scarcity of data on vitamin D status in Nepal, we aimed to examine the prevalence of VDD and VDI, as well as the determinants and association with metabolic parameters (lipids, HbA1c), in a cohort of women in rural Nepal. Altogether, 733 women 48.5 ± 11.7 years of age were included. VDD and VDI were observed in 6.3 and 42.4% of the participants, respectively, and the prevalence increased by age. Women reporting intake of milk and eggs > 2 times weekly had higher 25(OH)D levels than those reporting intake < 2 times weekly. Women with vitamin D levels < 50 nmol/L displayed higher levels of cholesterol, LDL-cholesterol, triglycerides, and HbA1c. Additionally, a regression analysis showed a significant association between hypovitaminosis D, dyslipidemia, and HbA1c elevation. In conclusion, VDI was prevalent and increased with age. Milk and egg intake > 2 times weekly seemed to decrease the risk of VDI. Moreover, hypovitaminosis D was associated with an adverse metabolic profile.

Objective: To assess the association between levels of vitamin D and urinary incontinence (UI) in pregnancy., Design: A cross-sectional study. Secondary analysis of a randomised controlled trial., Setting: Two university hospitals in Norway., Population: A total of 851 healthy, pregnant women >18 years in gestational weeks 18-22 with a singleton live fetus., Methods: Data on UI were collected from a questionnaire at inclusion and serum analysis of 25-hydroxy vitamin D (25(OH)D) was performed. Univariable and multivariable logistic regression analyses were applied to study associations between exposure and outcomes., Main Outcome Measures: Prevalence of self-reported UI, stress (SUI) and urge (UUI) or mixed UI., Results: In total, 230/851 (27%) of the participants were vitamin D insufficient (25(OH)D <50 nmol/l) and 42% reported to have any UI. Women with 25(OH)D <50 nmol/l were more likely to report any UI (P = 0.03) and SUI (P < 0.01) compared with women with 25(OH)D ≥50 nmol/l. In a univariable logistic regression analysis, serum levels of 25(OH)D <50 nmol/l was associated with increased risk of any UI (odds ratio [OR] 1.5 with 95% CI 1.0-2.1), SUI only (OR 1.7, 95% CI 1.2-2.4), but not mixed UI or UUI only (OR 0.8, 95% CI 0.5-1.5). In a multivariable logistic regression model, serum levels of 25(OH)D <50 nmol/l were associated with a higher risk of experiencing SUI only (OR 1.5, 95% CI 1.1-2.2)., Conclusions: Serum 25(OH)D <50 nmol/l was associated with increased risk of any UI, and SUI in particular., Tweetable Abstract: Low levels of vitamin D are associated with increased risk of urinary incontinence in pregnancy., (© 2020 The Authors. BJOG: An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd on behalf of Royal College of Obstetricians and Gynaecologists.)

Background: Vitamin D insufficiency is common in pregnant women worldwide. Regular prenatal exercise is considered beneficial for maternal and fetal health. There is a knowledge gap regarding the impact of prenatal exercise on maternal vitamin D levels. The objective of this study was to investigate whether a prenatal exercise program influenced serum levels of total, free and bioavailable 25-hydroxyvitamin D (25(OH)D) and related parameters. This is a post hoc analysis of a randomized controlled trial with gestational diabetes as the primary outcome., Methods: Healthy, pregnant women from two Norwegian cities (Trondheim and Stavanger) were randomly assigned to a 12-week moderate-intensity exercise program (Borg perceived rating scale 13-14) or standard prenatal care. The intervention group (n = 429) underwent exercise at least three times weekly; one supervised group training and two home based sessions. The controls (n = 426) received standard prenatal care, and exercising was not denied. Training diaries and group training was used to promote compliance and evaluate adherence. Serum levels of 25(OH)D, parathyroid hormone, calcium, phosphate, magnesium and vitamin D-binding protein were measured before (18-22 weeks' gestation) and after the intervention (32-36 weeks' gestation). Free and bioavailable 25(OH)D concentrations were calculated. Regression analysis of covariance (ANCOVA) was applied to assess the effect of the training regime on each substance with pre-intervention levels as covariates. In a second model, we also adjusted for study site and sampling month. Intention-to-treat principle was used., Results: A total of 724 women completed the study. No between-group difference in serum 25(OH)D and related parameters was identified by ANCOVA using baseline serum levels as covariates. The second model revealed a between-group difference in levels of 25(OH)D (1.9, 95% CI 0.0 to 3.8 nmol/L; p = 0.048), free 25(OH)D (0.55, 95% CI 0.10 to 0.99 pmol/L; p = 0.017) and bioavailable 25(OH)D (0.15 95% CI 0.01 to 0.29 nmol/L; p = 0.036). No serious adverse events related to regular exercise were seen., Conclusion: This study, a post hoc analysis, indicates that exercise may affect vitamin D status positively, and emphasizes that women with uncomplicated pregnancies should be encouraged to perform regular exercise., Trial Registration: ClinicalTrials.gov: NCT00476567 , registered May 22, 2007.

To ensure optimal calcium accrual in the fetal skeleton, a substantial rise occurs in 1,25-dihydroxyvitamin D (1,25(OH)2D), but is dependent on sufficient 25-hydroxyvitamin (25(OH)D). Large longitudinal studies addressing free 25(OH)D and 1,25(OH)2D during pregnancy are scarce. We aimed to assess levels of and relationship between 25(OH)D, 1,25(OH)2D, vitamin D-binding protein (DBP), parathyroid hormone (PTH), and free 25(OH)D during pregnancy; determinants of vitamin D status; and association between vitamin D indices or PTH and pregnancy outcomes (gestational diabetes mellitus and birthweight). Altogether 855 pregnant Norwegian Caucasian women from Trondheim and Stavanger (latitude 63°N and 58°N) were recruited; 94 were lost to follow-up. The study was originally a randomized controlled trial (2007-2009) with gestational diabetes as primary outcome. Data were collected in second and third trimester. In third trimester, 246 (34%) had vitamin D insufficiency and 52 (7%) deficiency (25(OH)D <50 and <30nmol/L, respectively). During wintertime in third trimester, 61 (47%) from Trondheim and 23 (51%) from Stavanger exhibited vitamin D insufficiency. PTH was elevated in 27 (3.7%). Estimate of change between trimesters was (95% CI): 25(OH)D -1.8 (-2.8 to -0.7) nmol/L, DBP 0.62 (0.57 to 0.66) μmol/L, calculated free 25(OH)D -1.7 (-2.0 to -1.4) pmol/L, PTH 0.81 (0.72 to 0.90) pmol/L, 1,25(OH)2D (sub-analysis) 31.4 (CI 24.7 to 38.2) pmol/L. A decrease in 1,25(OH)2D occurred in 45% of those with vitamin D deficiency, and they also exhibited lower levels than women with adequate vitamin D status. No association of vitamin D indices and PTH with pregnancy outcomes was observed. Women in Trondheim displayed lower 25(OH)D levels, despite minor latitudinal differences. Less than one-fifth adhered to the authorities' vitamin D recommendations. These findings demonstrate that hypovitaminosis D is prevalent among pregnant women living in northern latitudes, especially during the dark season, and there is an unmet need to ensure adequate vitamin D intake.

Spontaneous electrical activity is recorded in two species of marine cercariae, Cryptocotyle lingua and Himasthla elongata, with different types of swimming-by glass microelectrode recordings. Slow local field potentials (sLFPs) of low amplitude and fast high amplitude action potentials (APs) are found. The shape of the sLFPs is different in the species and correlates with the type of swimming. Fast high amplitude APs are recorded for the first time in cercariae. The limited number of APs included in the swimming pattern of larva suggests a key role for the spiking neurons in initiating the motility pattern in the cercaria and needs further research.

The neuromuscular system in cercariae of Moliniella anceps, Echinostoma revolutum, Cathaemasia hians, Psilochasmus oxyurus, Sphaeridiotrema globulus, Paramphistomum cervi and Diplodiscus subclavatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR sensory receptors were investigated. The general patterns of musculature, 5-HT- and FMRFamide-IR neuronal elements in the 12 species studied here and in paper I are similar to those observed in other cercariae and reflect the morphology of the groups. The musculature of the tail shows variations which are related to the different strategies of host finding. In the Echinostomatoidea and Paramphistomoidea, the striated musculature of the tail is well developed compared to that in the Xiphidiocercariae. Specialized muscle fibres were found in S. globulus, which are able to change the shape of the tail. Nine of the species studied have seven paired 5-HT-IR neurons in the body, and two species have eight. No correlation between the body size and the number of 5-HT-IR neurons was observed. However, the size of the neurons followed the body size. The number of 5-HT-IR neurons in the brain ganglia increased from the primitive to the advanced forms. The number of FMRFamide-IR transverse commissures in the body correlates with the size of the cercariae. Regardless of the differences in the second intermediate host, the distribution of α-tubulin-IR sensory receptors shows a high degree of conformity in all species except in P. cervi, which encysts on plants.

The neuromuscular system (NMS) in cercariae of Neoastiotrema trituri, Plagiorchis elegans, Omphalometra flexuosa, Skrjabinoeces similis and Prosthogonimus ovatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR sensory receptors were investigated, and they were found to be rather similar in all the cercariae studied. Four species have seven paired 5-HT-IR neurons in the body, and P. elegans has eight. N. trituri has three 5-HT-IR neurons in each brain ganglion, while the other species have four. A high degree of conformity in the structure of the NMS was observed, probably reflecting the close phylogenetic relationship and the similar strategy of host finding.

The neuromuscular system (NMS) in cercariae of Diplostomum pseudospathaceum, Cotylurus szidati, Australapatemon burti, Holostephanus volgensis, and Paracoenogonimus ovatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRF-amide-IR neuronal elements, and α-tubulin-IR in sensory receptors were investigated. The NMS in the five species studied were compared with each other and with three species of Schistosomatidae studied earlier (Bilharziella polonica, Trichobilharzia szidati, and Trichobilharzia franki). No major structural differences in the musculature, the 5-HT-IR or FMRF-IR neuronal elements were noticed between the cercariae. The minor variations observed in the musculature were related to the size and organization of the muscle fibers. The checked pattern formed by the transverse muscle fibers in the tail stems of D. pseudospathaceum, C. szidati, A. burti, H. volgensis, and P. ovatus was not observed in B. polonica, T. szidati, and T. franki. A trend in the differentiation of the longitudinal muscle fibers in the furca from evenly distributed fibers in H. volgensis and P. ovatus to many bundles in D. pseudospathaceum and two well-organized lateral bundles in C. szidati, A. burti, and Trichobilharzia spp. was observed. The transverse muscle fibers in the furca follow the same trend. The number of 5-HT-IR neurons in the cercarial bodies varied between 10 and 16. In cercariae of H. volgensis and P. ovatus, the central nervous system (CNS) was less centralized compared to the CNS in the other species studied, with only two 5-HT-IR marker neurons in each brain ganglion and the other neurons distributed evenly along the main cords. In the tails of H. volgensis and P. ovatus, many transverse 5-HT-IR comissures were found. In the tails of higher strigeidid cercariae, only a few crosslinks were observed. The number and distribution of sensory receptors on the bodies and tails of the cercarial species differed from each other. A trend in the differentiation of the sensory receptors in the tails was discerned. A process of grouping and decrease in number of ciliated receptors in the stem and in the furca from H. volgensis and P. ovatus to Schistosomatid cercariae took place.

The neuro-muscular system (NMS) in cercariae of the family Schistosomatidae from Belarus was studied with immunocytochemical methods and confocal scanning laser microscopy. The specimens of Bilharziella polonica were compared with Trichobilharzia szidati and Trichobilharzia franki. The patterns of F-actin in the musculature, 5-HT-immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR in sensory receptors and nerves were investigated. No indications of structural differences in the musculature, the 5-HT-IR, FMRF-IR neuronal elements and the general distribution of sensory receptors were noticed between cercariae of Trichobilharzia spp. The number of 5-HT-IR neurons in the cercarial bodies is 16. In cercaria B. polonica, the tail musculature is weaker than in Trichobilharzia spp. A detailed schematic picture of the NMS in the tail of Trichobilharzia spp. cercaria is given. The function of NMS elements in the tail is discussed.

The neuro-muscular system (NMS) of cercariae with different swimming patterns was studied with immunocytochemical methods and confocal scanning laser microscopy. Specimens of the continuously swimming Cercaria parvicaudata, Maritrema subdolum and Himasthla elongata were compared with specimens of the intermittently swimming Cryptocotyle lingua and the attached Podocotyle atomon. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRFamide-IR neuronal elements, α-tubulin-IR elements in the nervous and sensory systems and DAPI-stained nuclei were investigated. The general plan of the NMS was similar in all cercariae studied. No major structural differences in the patterns of muscle fibres were observed. However, in the tail of C. lingua, transverse muscle fibres connecting the bands of longitudinal muscles were found. No major structural differences in the 5-HT- or FMRFamide-IR nervous systems were observed. The number of 5-HT-IR neurones in the cercarial bodies varied between 12 and 14. The number and distribution of the α-tubulin-IR processes on the cercarial bodies and tails differed from each other. The relation between the number and structure of the α-tubulin-IR processes and the host finding strategy of the cercariae is discussed. A detailed schematic picture of the NMS in the tails of C. lingua and M. subdolum is presented.

Zic genes encode a conserved family of zinc finger proteins with essential functions in neural development and axial skeletal patterning in the vertebrate embryo. Zic proteins also function as Gli co-factors in Hedgehog signaling. Here, we report that Zic genes have a role in Myf5 regulation for epaxial somite myogenesis in the mouse embryo. In situ hybridization studies show that Zic1, 2, and 3 transcripts are expressed in Myf5-expressing epaxial myogenic progenitors in the dorsal medial dermomyotome of newly forming somites, and immunohistological studies show that Zic2 protein is co-localized with Myf5 and Pax3 in the dorsal medial lip of the dermomyotome, but is not expressed in the forming myotome. In functional reporter assays, Zic1 and Zic2, but not Zic3, potentiate the transactivation of Gli-dependent Myf5 epaxial somite-specific (ES) enhancer activity in 3T3 cells, and Zic1 activates endogenous Myf5 expression in 10T1/2 cells and in presomitic mesoderm explants. Zic2 also co-immunoprecipitates with Gli2, indicating that Zic2 forms complexes with Gli2 to promote Myf5 expression. Genetic studies show that, although Zic2 and Zic1 are activated normally in sonic hedgehog(-/-) mutant embryos, Myf5 expression in newly forming somites is deficient in both sonic hedgehog(-/-) and in Zic2(kd/kd) mutant mouse embryos, providing further evidence that these Zic genes are upstream regulators of Hedgehog-mediated Myf5 activation. Myf5 activation in newly forming somites is delayed in Zic2 mutant embryos until the time of Zic1 activation, and both Zic2 and Myf5 require noggin for their activation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study is the first detailed study of the organisation of the neuromuscular system of Cyathocephalus truncatus (Cestoda, Spathebothriidea). Five techniques have been used: (1) immunocytochemistry, (2) staining with TRITC-conjugated phalloidin, (3) NADPHdiaphorase histochemistry, (4) confocal scanning laser microscopy and (5) transmission electron microscopy. The patterns of nerves immunoreactive (IR) to antibodies towards serotonin (5-HT) and the invertebrate neuropeptide FMRFamide are described in relation to the musculature. The patterns of NADPHdiaphorase positive nerves and 5-HT-IR nerves are compared. The fine structure of the nervous system (NS) is described. The organisation of NS in the non-segmented, polyzoic C. truncatus differs clearly from that in the non-segmented, monozoic Caryophyllaeus laticeps and shows distinct similarities with the NS in pseudophyllidean cestodes. This supports the hypothesis that taxon Caryophyllidea and Spatheobothriidea form independent lineages within Eucestoda.

The effects of neuropeptide F (NPF; from Moniezia expansa) on the regeneration of Girardia tigrina were studied. The animals were decapitated and incubated in water (control) or NPF. The dynamics of the proliferation of the neoblasts in the developing tissue were studied during the course of regeneration by monitoring the mitotic index (MI). The effects of incubation in FMRFamide and GYIRFamide on the MI were also tested. The course of cephalic regeneration was followed with in vivo computer-assisted morphometry for up to 7 days. The development of the regenerating nervous system and the musculature was visualised by immunostaining with a primary antiserum to the C-terminal decapeptide of NPF (YFAIIGRPRFa) and tetramethylrhodamine-isothiocyanate-conjugated phalloidin, which stains F-actin in muscle filaments. The study showed that NPF had a stimulatory effect on the mitotic activity of the neoblasts. FMRFamide and GYIRFamide did not have this effect. NPF also stimulated the growth of the regenerating head and the growing nervous system and musculature. NPF is postulated to have a morphogenetic action in the regenerating animals.

Copulatory organs of eight species of the monophyletic taxon Childia were investigated in detail, using phalloidin fluorescence method and confocal microscopy. Childia species were shown to have one, two or several tubular stylets, conical to cylindrical in shape, composed of few to numerous needles. The musculature varied greatly, from the absence of seminal vesicle to extensively developed seminal vesicles with several additional types of specialized muscles. Ten copulatory organ characters were coded and mapped on the total evidence tree. The data obtained permitted to follow the evolution of the Childia stylet and to demonstrate that the structure of the stylet apparatus is largely consistent with the phylogeny of the group (CI=0.75). Possible function of different muscle specializations was discussed.

The host-parasite relationship, Tenebrio molitor-Hymenolepis diminuta, was analyzed. The learning behavior of infected and uninfected (control) beetles in a T-maze was compared. The infected beetles moved much slower in the T-maze than the controls. The infected beetles reached the same level of learning as the controls. However, they needed more trials than the controls. The effect of the infection was already distinct after the first week and even higher after the second week. This indicates that the initial phase of infection caused stress in the beetles. Longer infection did not worsen their ability to learn. Thus, the parasites clearly changed the behavior of their intermediate host and probably made them more susceptible to their final host, the rat.

The spatial relationship between the musculature and the NADPH-diaphorase (NADPH-d) activity, 5-HT and FMRFamide immunoreactivities in redia, cercaria and adult Echinoparyphium aconiatum was studied using scanning electron microscopy (SEM), NADPH-d histochemistry, immunocytochemistry, and confocal scanning laser microscopy (CSLM). TRITC-conjugated phalloidin was used to stain the musculature. Staining for NADPH-d was observed in the central (CNS) and peripheral nervous system (PNS) of all three stages. NADPH-d positive nerves occurred very close to muscle fibres. 5-HT-immunoreactive (5-HT-IR) nerve cells and fibres occurred in the CNS and PNS and close to muscle fibres. FMRFamide-IR nerve fibres were observed in the CNS and PNS of adult worms. This is the first time, the presence of the NADPH-d has been demonstrated in the larval as well as the adult stages of a fluke.

The formation of cGMP in homogenates of the adult rat-tapeworm Hymenolepis diminuta was followed with a radiometric assay during 3 h after stimulation with the nitric oxide donor sodium nitroprusside (SNP) and in the presence of isobutylmethylxanthine (IBMX). The level of cGMP was stable in worms incubated with IBMX during the first hour. After 3 h of incubation, the level of cGMP had declined by 27%. Addition of SNP stimulated the formation of cGMP during the first hour of incubation. After 3 h of incubation, a two-fold decline in cGMP formation was observed. The rate of nitric oxide (NO) release by the worm was determined by a spectrophotometric assay for the accumulation of nitrites and nitrates, the stable degradation products of NO, using the Griess reaction. The results are discussed from the perspective of the current concept on the role of the nitrergic mechanisms in the flatworm nervous system.

The pattern of muscles in the actively swimming predatory rotifer Asplanchnopus multiceps is revealed by staining with tetramethyl-rhodamine isothiocyanate (TRITC)-labelled phalloidin and confocal scanning laser microscopy (CSLM). The major components of the musculature are: prominent semicircular muscles of the corona; paired lateral, dorsal and ventral retractors in the trunk; a network of six seemingly complete circular muscles and anastomosing longitudinal muscles in the trunk; two short foot retractors, originating from a transverse muscle in the lower third of the trunk. The sphincter of the corona marks the boundary between the head and the trunk. The muscular patterns in rotifers with different lifestyles differ clearly, therefore, the muscular patterns seem to be determined by the mode of locomotion and feeding behaviour.

The Nemertodermatida are a small group of microscopic marine worms. Recent molecular studies have demonstrated that they are likely to be the earliest extant bilaterian animals. What was the nervous system (NS) of a bilaterian ancestor like? In order to answer that question, the NS of Nemertoderma westbladi was investigated by means of indirect immunofluorescence technique and confocal scanning laser microscopy. The antibodies to a flatworm neuropeptide GYIRFamide were used in combination with anti-serotonin antibodies and phalloidin-TRITC staining. The immunostaining revealed an entirely basiepidermal NS. A ring lying outside the body wall musculature at the level of the statocyst forms the only centralisation, the "brain". No stomatogastric NS has been observed. The GYIRFamide immunoreactive part of the "brain" is formed of loosely packed nerve fibres with multiple small neurones and a few large ones. The peptidergic and aminergic patterns of the NS do not correspond to each other: the former is more developed on the ventral side, the latter is more pronounced on the dorsal side. A pair of GYIRFamide immunoreactive nerve cords innervates the ventral side of the animal, the mouth and the male genital opening. The nemertodermatids studied to-date display no common NS pattern. Possible synapomorphies of the Acoelomorpha are discussed. The study demonstrates that the nemertodermatid NS possesses a number of plesiomorphic features and appears more primitive than the NS in other worms, except the Xenoturbellida. The bilaterian ancestor supposedly possessed only a basiepidermal nerve net and had no centralised brain-like structures and no stomatogastric NS.

We studied the pattern of cGMP immunostaining (IS) after stimulation with a nitric oxide donor in the presence of an inhibitor of phosphodiesterase in adult Hymenolepis diminuta. cGMP-IS was detected in the peripheral nervous system, especially in nerve fibres close to the body muscle fibres. cGMP-IS also occurred in terminals beneath the basal lamina of the tegument and between the muscle fibres of the suckers. The pattern of cGMP-IS was compared to that of 5-HT-IS and GYIRFamide-IS. TRITC-conjugated phalloidin was used to stain the musculature.

The activity of neuronal nitric oxide synthase (nNOS) in homogenates of adult Fasciola hepatica was measured by the direct radiometric assay of the production of L-[3H]citrulline. This is the first radiometric study of the activity of nNOS in a fluke. The effect of arginase was tested. In the presence of L-valine, which is an inhibitor of arginase, the formation of L-[3H]citrulline decreased from 12% to 38%, depending on the time of incubation. This means that the arginase activity in the worm is high, and has to be taken into consideration when measuring the activity of nNOS. When co-factors, such as H4B, and NADPH, were omitted the formation of L-[3H]citrulline decreased significantly (29%). The effects of several nNOS inhibitors were tested. N(omega)-nitro-L-arginine (L-NAME), aminoguanidine and S-methyl-L-thiocitrulline added at a concentration of 1 mM inhibited the L-[3H]citrulline formation by 28%, 15% and 14%, respectively. Chelation of Ca2+ with 1 mM EGTA resulted in a 40% decrease in the formation of L-[3H]citrulline. These results indicate the presence of nNOS activity in homogenates of F. hepatica.

In order to advance our knowledge of the nitrergic nervous system in flatworms, the patterns of the NADPH-diaphorase (NADPH-d) reaction and cGMP immunoreactivity, after stimulation with a nitric oxide donor in the presence of an inhibitor of phosphodiesterase, were investigated in cercaria of Diplostomum chromatophorum. This is the first time the presence of NADPH-d activity has been detected in a larval fluke, and the first time the presence of cGMP immunoreactivity has been detected in a flatworm. The NADPH-d reaction occurs in the ventral sucker, the hind body and the tail. cGMP immunoreactivity was detected in the muscle cells of the body and in two pairs of sensory cells at the anterior end of the body and in the middle of the furca. The sensory cells also showed 5-HT immunoreactivity. The spatial relationship between the cGMP and the 5-HT immunoreactivities was studied. By staining with TRITC-labelled phalloidin, the pattern of the muscle fibres was revealed.

The use of well-characterized antibodies raised to neuronal signal substances and their application through immunocytochemistry and confocal scanning laser microscopy has revolutionized studies of the flatworm nervous system (NS). Data about flatworm neuropeptides and the spatial relationship between neuropeptides and other neuronal signal substances and muscle fibers are presented. Neuropeptides form a large part of the flatworm NS. Neuropeptides are especially important as myoexcitatory transmitters or modulators, controlling the musculature of the attachment organs, the stomatogastric and the reproductive systems.

In order to broaden the information about the organisation of the nervous system in the Plathelminthes, an immunocytochemical (ICC) study of the free-living flatworm Castrella truncata (Dalyellioida) has been performed. This is the first time a representative of the taxon Rhabdocoela has been studied with the ICC technique. Antibodies to 5-HT and FMRF-amide and confocal scanning laser microscope were used. 5-HT and FMRF-amide immunoreactivity was observed in the neuropile of the brain, in the cerebral neurones, in the three pairs of longitudinal nerve cords and the adjoining neurones, in the pharyngeal nerve ring and in twelve neurones associated with the pharynx. 5-HT and FMRF-amide immunoreactivity occurs in separate sets of neurones. Only FMRF-amide immunoreactivity was observed in a peculiar ellipse-shaped structure in the brain, and in the genital plexus. The type of orthogon is discussed. Staining of the muscular system with TRITC conjugated phalloidin revealed muscle patterns that have not been described previously.

Sonic hedgehog (Shh) is a secreted signaling molecule for tissue patterning and stem cell specification in vertebrate embryos. Shh mediates both long-range and short-range signaling responses in embryonic tissues through the activation and repression of target genes by its Gli transcription factor effectors. Despite the well-established functions of Shh signaling in development and human disease, developmental target genes of Gli regulation are virtually unknown. In this study, we investigate the role of Shh signaling in the control of Myf5, a skeletal muscle regulatory gene for specification of muscle stem cells in vertebrate embryos. In previous genetic studies, we showed that Shh is required for Myf5 expression in the specification of dorsal somite, epaxial muscle progenitors. However, these studies did not distinguish whether Myf5 is a direct target of Gli regulation through long-range Shh signaling, or alternatively, whether Myf5 regulation is a secondary response to Shh signaling. To address this question, we have used transgenic analysis with lacZ reporter genes to characterize an Myf5 transcription enhancer that controls the activation of Myf5 expression in the somite epaxial muscle progenitors in mouse embryos. This Myf5 epaxial somite (ES) enhancer is Shh-dependent, as shown by its complete inactivity in somites of homozygous Shh mutant embryos, and by its reduced activity in heterozygous Shh mutant embryos. Furthermore, Shh and downstream Shh signal transducers specifically induce ES enhancer/luciferase reporters in Shh-responsive 3T3 cells. A Gli-binding site located within the ES enhancer is required for enhancer activation by Shh signaling in transfected 3T3 cells and in epaxial somite progenitors in transgenic embryos. These findings establish that Myf5 is a direct target of long-range Shh signaling through positive regulation by Gli transcription factors, providing evidence that Shh signaling has a direct inductive function in cell lineage specification.

Embryological and genetic studies of mouse, bird, zebrafish, and frog embryos are providing new insights into the regulatory functions of the myogenic regulatory factors, MyoD, Myf5, Myogenin, and MRF4, and the transcriptional and signaling mechanisms that control their expression during the specification and differentiation of muscle progenitors. Myf5 and MyoD genes have genetically redundant, but developmentally distinct regulatory functions in the specification and the differentiation of somite and head muscle progenitor lineages. Myogenin and MRF4 have later functions in muscle differentiation, and Pax and Hox genes coordinate the migration and specification of somite progenitors at sites of hypaxial and limb muscle formation in the embryo body. Transcription enhancers that control Myf5 and MyoD activation in muscle progenitors and maintain their expression during muscle differentiation have been identified by transgenic analysis. In epaxial, hypaxial, limb, and head muscle progenitors, Myf5 is controlled by lineage-specific transcription enhancers, providing evidence that multiple mechanisms control progenitor specification at different sites of myogenesis in the embryo. Developmental signaling ligands and their signal transduction effectors function both interactively and independently to control Myf5 and MyoD activation in muscle progenitor lineages, likely through direct regulation of their transcription enhancers. Future investigations of the signaling and transcriptional mechanisms that control Myf5 and MyoD in the muscle progenitor lineages of different vertebrate embryos can be expected to provide a detailed understanding of the developmental and evolutionary mechanisms for anatomical muscles formation in vertebrates. This knowledge will be a foundation for development of stem cell therapies to repair diseased and damaged muscles.

The developmental signaling functions of cell surface heparan sulfate proteoglycans (HSPGs) are dependent on their sulfation states. Here, we report the identification of QSulf1, the avian ortholog of an evolutionarily conserved protein family related to heparan-specific N-acetyl glucosamine sulfatases. QSulf1 expression is induced by Sonic hedgehog in myogenic somite progenitors in quail embryos and is required for the activation of MyoD, a Wnt-induced regulator of muscle specification. QSulf1 is localized on the cell surface and regulates heparan-dependent Wnt signaling in C2C12 myogenic progenitor cells through a mechanism that requires its catalytic activity, providing evidence that QSulf1 regulates Wnt signaling through desulfation of cell surface HSPGs.

This is the first detailed description of the nitrergic nervous system in a fluke. In this study, the authors analysed the distribution of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in neuronal and nonneuronal tissues of the adult fluke Fasciola hepatica and compared this with the distribution of the musculature using tetramethylrhodamine isothiocyanate-phalloidin. To assess the correlation between the number of muscle cells in different parts of the fluke and the NADPH-d-stained cells, the nuclei were stained with Hoechst 333 42, which is specific for chromatin. The spatial relation between the NADPH-d-positive nerves and the 5-hydroxytryptamine (serotonin; 5-HT)-immunoreactive (-IR) and GYIRFamide-IR nervous elements was also examined. The methods complement each other. NADPH-d-positive staining occurs in both in neuronal tissue and nonneuronal tissue. Large, NADPH-d-stained neurones were localised in the nervous system. The oral and ventral suckers are innervated with many large NADPH-d-stained neurones. In addition, the NADPH-d staining reaction follows closely the muscle fibres in both the suckers, in the body, and in the ducts of the reproductive organs. The presence of NADPH-d activity along muscle fibres in F. hepatica and in other flatworms supports a possible myoinhibitory role for nitric oxide. Neuronal nitric oxide synthase in flatworms may form a novel drug target, which would facilitate the development of a novel anthelminthic., (Copyright 2001 Wiley-Liss, Inc.)

The nervous system of young and adult Amphilina foliacea was studied with immunocytochemical, electron microscopical and spectrofluorometrical methods. The general neuroanatomy is described in detail. New data on the structure and development of the brain were obtained. The 5-HT and GYIRFamide-immunoreactivities occur in separate sets of neurones. The innervation of the reproductive organs is described. The fine structure of 2 types of neurones in the CNS, a sensory neurone, a 'glial' cell type, the neuropile and the synapses are described. The level of 5-HT varies between 0.074 and 0.461 microg/g wet weight. This is the first detailed study of the nervous system of A. foliacea. Earlier data on the structure of the nervous system in A. foliacea published in Russian are introduced into the discussion. The study provides data that can be used when considering the phylogenetic position of Amphilinidea.

The taxa Nemertodermatida and Acoela have traditionally been considered closely related and classified as sister groups within the Acoelomorpha Ehlers 1984 (Platyhelminthes). Recent molecular investigations have questioned their respective position. In this study, the 5-HT and FMRFamide immunoreactivity (IR) in the nervous system of two nemertodermatids, Nemertoderma westbladi and Meara stichopi, is described. The 5-HT immunoreactive pattern differs in the two nemertodermatids studied. In M. stichopi, two loose longitudinal bundles of 5-HT-immunoreactive fibres and an basi-epidermal nerve net were observed. In N. westbladi the 5-HT-IR shows a ring-shaped commissural structure, different from the commissural brain of acoels. In both nemertodermatids, FMRFamide immunoreactive nerve fibres followed the 5-HT-immunoreactive fibres. It is demonstrated that the Nemertodermatida have neither a 'commissural brain' structure similar to that of the Acoela, nor a 'true', ganglionic brain and orthogon, typical for other Platyhelminthes. The question of the plesiomorphic or apomorphic nature of the nervous system in Nemertodermatida cannot yet be answered. The neuroanatomy of the studied worms provides no synapomorphy supporting the taxon Acoelomorpha.

The ultrastructure of two types of secretory glands in the scolex of preadults of Proteocephalus longicollis is described for the first time in the present report. The gland cells contain extensive cisternae of granular endoplasmic reticulum and Golgi complexes, which participate in the production of secretory globules. Type I scolex glands produce electron-dense globules of various size. The secretory globules enter the secretory canal, openings of which were not observed in the preadults. The secretory product of type I was found at the inner sucker surface and in the tegument of the sucker edges. In addition, electron-dense globules in adult worms are secreted via an eccrine mechanism. Type II scolex glands are characterized by secretory globules of lower electron density and occur mainly in preadults. The electron-lucent, membrane-bound secretory globules are transported via microtubule-lined ducts opening to the exterior at the tegumental surface. Secretory globules of type II are released by an eccrine process.

The ultrastructure of five types of presumed sensory receptors in the scolex of preadults of Proteocephalus longicollis is described. Two types of nonciliate sensory receptors are situated on the inner surface of the lateral sucker. They differ from each other in the shape, presence, or absence of a large rootlet, electron-dense collars, desmosomes, microtubules, and/or vesicles. In addition, three types of ciliate sensory receptors are found along the edges of the lateral suckers. They can be differentiated by the length of the cilium, by the number of electron-dense collars (one or two), and by types of vesicles. Four types of vesicles were found inside the ciliate sensory receptors. One type of ciliate sensory receptor occurring in preadults differs markedly from any of the sensory receptors previously described in adult P. longicollis.

The free radical nitric oxide (NO) is a neuronal messenger which is synthesized from L-arginine and O2 by nitric oxide synthase (NOS). In the synthesis NO and L-citrulline are produced in a stoichiometric 1:1 relation. The activity of NOS was analysed in homogenates of the rat tapeworm Hymenolepis diminuta by measuring the formation of L-[3H]citrulline after incubation with L-[3H]arginine. The nature of NOS in H. diminuta was determined by studying the effect of 3 types of NOS inhibitors: (1) L-NAME, (2) EGTA, (3) 7-nitro-indazole. All inhibitors caused a significant but not complete reduction in the formation of L-[3H]citrulline. The results are discussed against the background of nerve cells and fibres positive for NADPH-diaphorase staining in H. diminuta.

In order to test the role of nitric oxide in flatworms, Mesocestoides vogae tetrathyridia were incubated together with L-arginine, which is the substrate for nitric oxide synthesis, or with NG-nitro-L-arginine, which is an irreversible inhibitor of nitric oxide synthase. Normally, tetrathyridia attach to each other with the aid of their suckers, forming clusters. The rate of cluster formation was followed during the incubations. L-Arginine stimulated, and NG-nitro-L-arginine clearly inhibited, the cluster formation. This is the first time that an effect of nitric oxide has been observed in a flatworm. In addition, the pattern of the NADPH-diaphorase histochemical reaction in the nervous system and the pattern of F-actin filaments in the musculature stained with TRITC-labelled phalloidin were studied. NADPH-d staining occurred in the brain and the main nerve cords but also followed the muscle fibres stained with phalloidin. The pattern of the NADPH-d reaction was compared with that of 5-HT immunoreactivity. The implications of the results are discussed in relation to the background of data on neuronal signal substances in M. vogae.

A new category of signalling molecules--transmitter gases--has appeared. Nitric oxide (NO) is generated by nitric oxide synthase (NOS) and diffuses as a short-lived transcellular messenger through the plasma membrane. NADPH-diaphorase (NADPH-d) is a marker enzyme for NO-producing neurons. In this study the pattern of NADPH-d stained neurons in Diphyllobothrium dendriticum is described and compared to the pattern of aminergic and peptidergic neuronal elements and to that of the musculature stained with TRITC-labelled phalloidin. NADPH-d staining was observed in neurons in the bilobed brain and along the 2 main nerve cords and in nerve fibres close to the body musculature and the musculature of the reproductive ducts, the walls of the testicular follicles and in sensory endings in the tegument. The NADPH-d staining reaction and the 5-HT or FMRFamide immunoreactivities occur in separate sets of neurons.

The ultrastructure of six type of putative sense receptors including three nonciliate (Types I-III) and three ciliate (Types IV-VI) types found in the tegument of the scolex of adult Proteocephalus exiguus has been described for the first time. Type I has a simple tegumental sensory ending, containing one electron-dense collar, circular septate desmosome, electron-lucent vesicles and microtubules. Type II is represented by a receptor with a large rootlet. Type III is a receptor with a very long cross-striated rootlet, with two electron-dense collars and septate junction. The ciliated receptors differ in the length of the cilium, in the number of electron-dense collars and in the shape of sensory bulb. A comparison of structural features of receptors in different systematic group within Platyhelminthes is made.

In order to solve the problem of whether the Acoela have a brain of a common flatworm type, an immunocytochemical study was performed of the pattern of FMRF-amide in the nervous system of four species of Acoela, Anaperus biaculeatus, Childia groenlandica, Antinoposthia beklemischevi and Mecynostomum sp. In all species a FMRF-amide positive bilobed brain-like structure lacking neuropile was observed. This brain is composed of large multipolar nerve cells with short processes. The FMRF-amide immunoreactivity appears as spots in the cytoplasm. Short lateral branches originating from the periphery of the brain were observed in Anaperus biaculeatus. No immunoreactive nerve cords were detected. The results indicate that the brain-like structure of Acoela is not homologous with the brains of other Plathelminthes and implies an independent evolution of Acoela.

The free radical nitric oxide (NO), which is synthesized by nitric oxide synthase (NOS), has recently been discovered to function as a neuronal messenger. The presence of NOS was detected in the nervous system of adult Hymenolepis diminuta with NADPH-diaphorase (NADPH-d) histochemistry. The NADPH-d histochemical reaction is regarded as a selective marker for NOS in neuronal tissue. NADPH-d staining was observed in nerve fibres in the main and minor nerve cords and the transverse ring commissures, and in cell bodies in the brain commissure, along the main nerve cords, in the suckers and the rostellar sac. NADPH-d staining was also observed in the wall of the internal seminal vesicle and the genital atrium. The pattern of NADPH-d staining was compared with that of the 5-HT immunoreactive nervous elements. The NADPH-d staining reaction and the 5-HT immunoreactivity occur in separate sets of neurons. This is the first time the NADPH-d reaction has been demonstrated in the nervous system of a flatworm, indicating that NOS is present and that NO can be produced at this level of evolution.

Neuropeptides and serotonin in the cestode, Proteocephalus exiguus: an immunocytochemical study. International Journal for Parasitology 25: 673-682. Neuropeptide F (NPF), RFamide and serotonin (5-HT) immunoreactivities have been detected in the nervous system of P. exiguus procercoids and adults, using an indirect immunocytochemical technique in conjunction with confocal scanning laser microscopy. The peptidergic nervous system of the procercoid is well developed, with two brain ganglia, three pairs of longitudinal nerve cords, transverse ring commissures and nerves in the suckers, all showing NPF-immunostaining. Strong NPF- and RF-immunostaining was observed in the CNS and PNS of the adult worm. The distribution patterns of the two neuropeptides were similar. Immunoreactivity for 5-HT was found only in the CNS.

The relationship between serotonin (5-HT) and motility in tetrathyridia of Mesocestoides vogae (syn. M. corti) was studied with the aid of reserpine. Reserpine decreases the content of 5-HT as measured spectrofluorometrically and immunocytochemically and, furthermore, inhibits the motility, thus indicating a connection between the two. The results support the hypothesis about 5-HT being an excitatory neurotransmitter of motor activity in M. vogue. New neuroanatomical details were revealed by immunocytochemical staining.

The organization of the nervous system of Procerodes littoralis (Tricladida, Maricola, Procerodidae) was studied by immunocytochemistry, using antibodies to authentic flatworm neuropeptide F (NPF) (Moniezia expansa). Compared to earlier investigations of the neuroanatomy of tricladid flatworms, the pattern of NPF immunoreactivity in Procerodes littoralis reveals differences in the following respects: 1. Shape and structure of the brain. 2. Number and composition of longitudinal nerve cords. 3. Shape of branches of, and transverse connections between, main ventral nerve cords. 4. Composition of the pharyngeal nervous system. The rich innervation by NPF immunoreactive (IR) fibres and cells of the subepithelial muscle layer, the pharynx musculature and the musculature of the male copulatory apparatus indicates a neurotransmitter or neuromodulatory influence on muscular activity.

The flatworms occupy a position at the base of the metazoan phylogenetic tree; they have a bilateral symmetric nervous system and an archaic brain. The following aspects, brought into focus by the use of new methods, will be dealt with in the present paper. 1. The high degree of diversity on all levels of the flatworm nervous system (NS). 2. The concept of main nerve cords is defined and the use of this concept in avoiding confusions in the terminology of nerve cords is stressed. 3. The archaic nature of the stomatogastric NS is reviewed. 4. The new data about neuronal celltypes implying advanced features at this low phylogenetic level. 5. The ultrastructural studies of neuronal cells indicating (A) that a common secretory cell type containing dense-core vesicles is archaic and a likely progenitor cell type for conventional neurons of advanced flatworms and (B) that an independent evolution of synaptic structures and glial cells has occurred inside the flatworm taxon. 6. The multitude of neuroactive substances demonstrated by light microscopic histofluorescence, immunocytochemistry, liquid chromatography, and HPLC. The cholinergic, aminergic, and peptidergic substances often occur in different neuronal compartments.

The nervous system (NS) of Dugesia tigrina has been studied by immunocytochemical double-staining, using the authentic flatworm neuropeptide, neuropeptide F (NPF), and serotonin (5-HT) on cryosections. This technique provides a precise morphological (descriptive) account of the NS. The results show that the central nervous system is shaped like a horseshoe. The brain is composed of two lateral lobes connected by three commissures, one antero-dorsal in front of the cerebral eyes and two, more ventral, behind the eyes. The pair of main nerve cords extend from the lateral lobes of the brain to the tail end of the worm. Cross sections reveal a very close contact between lateral branches from the main cords and the submuscular plexus. Thin cord-like lateral nerves are formed by longitudinal plexal fibres. No dorsal cords were observed. The patterns of immunoreactivity to NPF and 5-HT differ from each other in several respects. In the walls of gut diverticula only NPF immunoreactive (IR) cells and fibres were observed. Only NPF-immunoreactive cells occur in the parenchyma along dorso-ventral nerve fibres connecting the dorsal and ventral parts of the submuscular plexus. The number of 5-HT-immunoreactive cells associated with the main nerve cords (MCs) is greater than that of the NPF-immunoreactive cells, and the spongy structure of the MCs is more apparent following immunostaining for 5-HT. Thin 5-HT-immunoreactive fibres were observed in the subepithelial plexus, penetrating the basal lamina and innervating a rhabdite-free ventro-lateral sensory area along the body periphery. The correspondence between MCs in the lower flatworms (Catenulida and Macrostomida) and the Seriata (Tricladida and Proseriata) confirms the status of the MCs in flatworms as the most important and stable neuronal characteristic, and constitutes support for the hypothesized common origin of the MCs in flatworms.