Regeneration of freshwater planarians Dugesia tigrina and Polycelis tenuis under the influence of methyl mercury compounds of natural origin

We studied the effects of methyl mercury compounds of natural origin on regeneration of the planarians Dugesia tigrina and Polycelis tenuis. Accumulation of methyl mercury in the planarian body leads to a delayed formation of photoreceptor organs in planarians of both species. After a significant traumatic load, the regeneration is suppressed and the death of some control and most experimental animals was observed. The intensity of joining additional cuts depends on the localization of body fragment with a cut and localization of a cut itself.     

Gtwnt-5 a member of the wnt family expressed in a subpopulation of the nervous system of the planarian Girardia tigrina

Wnt proteins are a family of highly conserved secreted glycoproteins that regulate cell-to-cell interactions during embryogenesis. They act as signaling molecules and take part in many crucial decisions throughout the development of organisms ranging from Hydra to human. We have isolated and characterized the expression of a member of the Wnt family, Gtwnt-5 gene in the planarian Girardia tigrina. Planarians are free-living members (Class Turbellaria) of the Phylum Platyhelminthes. They are best known for their high regenerative capabilities. These organisms have an apparently simple central nervous system (CNS) from a morphological perspective, with cephalic ganglia in the dorsal anterior region and two ventral main nerve cords along the body. However, a large number of planarian neural genes have recently been identified and therefore it is possible to define different molecular and functional domains in the planarian brain. The present study shows expression of Gtwnt-5 in a subpopulation of the whole CNS of intact organisms, being activated during regeneration. Gtwnt-5 reveals a differential spatial pattern: the expression is preferentially found in the most external region of the CNS. In addition, a kind of iterative pattern has been observed at the ganglia level, suggesting that the planarian brain might not be a continuous structure but compartmented or regionalized. Gtwnt-5 signal is also detected at the sensors of the worm: at the auricle level and all around the cephalic periphery. All these data provide us with a new neural marker for the planarian brain, and can be used to follow regeneration of the CNS.     

Myocyte differentiation and body wall muscle regeneration in the planarian Girardia tigrina

 Freshwater planarians (Platyhelminthes, Turbellaria) show a great degree of morphological plasticity, making them a useful model for studying cell differentiation and pattern restoration processes during regeneration. Using confocal microscopy and a monoclonal antibody specific for muscle cells (TMUS-13), we have monitored the restoration of the body wall musculature during head regeneration in whole-mount organisms. Our results show that until the 4th day of regeneration the blastema is occupied by very disorganized muscle fibers, that from this moment become progressively organized restoring the original muscle pattern. In addition to recognizing mature muscle cells, TMUS-13 also recognizes differentiating myocytes, allowing us to trace the origin of newly formed muscle cells. We report that myocytes are detected in the postblastema region as early as day 1 of regeneration. This is the first demonstration that, in addition to serving as a proliferative zone as previously described, overt differentiation begins in the postblastema, at least for muscle cells. We also show that the TMUS13 antigen is the myosin heavy-chain gene from planarians. Received: 15 April 1997 / Accepted: 7 July 1997     

GABA in the nervous system of the planarian Polycelis nigra

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in vertebrates and it has a similar inhibitory role in several invertebrate taxa. The transmitters serotonin, octopamine, catecholamines and histamine are present in flatworms while evidence for GABA is still lacking. Therefore, we have studied the occurrence of GABA-like immunoreactivity (IR) in the planarian nervous system. Specimens of Polycelis nigra were fixed in 4% 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide with 2% paraformaldehyde. The GABA-antiserum was raised in rabbits against GABA conjugated to keyhole limpet hemocyanin. Preabsorption with GABA-ovalbumin conjugate abolished all IR. The results were further confirmed with an monoclonal antibody and high pressure liquid chromatography (HPLC). In P. nigra GABA-like IR was seen as long, often varicose, sparsely distributed fibers in the ventral longitudinal nerve cords. IR was also located in a few cell somata in the brain and in the neuropil of the brain. The IR was restricted to the central nervous system and was absent in peripheral nerves and plexuses. The HPLC analysis supported the presence of GABA.Our results suggest that GABA is an interneuronal transmitter in P. nigra. The results also suggest a phylogenetically old origin of GABAergic neurotransmission.     

The distribution of oxidizable mucosubstances and polysaccharides in the planarian Polycelis tenuis iijima

A chromic acid oxidation-silver technique was used to localize polysaccharide material in Polycelis tenuis at the electron microscope level. In the epithelium, staining was observed within apical vacuoles and on the free surfaces of the cells. A similar staining was observed in relation to the glycocalyx of the pharyngeal epithelia and that of the flame cells. Silver was deposited in the basement membrane. In the parenchyma, the major components giving a positive reaction were the cyanophil and mucous gland cells. Particularly strong silver staining (confirmed by X-ray microanalysis) was observed in the granules and Golgi apparatus of the cyanophil cells. IDPase activity was also found in relation to the Golgi apparatus and its secretory products. The overall distribution of mucopolysaccharide material was confirmed with the PAS and Alcian blue techniques. The fine structural localization of the Alcian blue was also determined using electron microscopy and X-ray microanalysis.     

Cell-, tissue-, and position-specific monoclonal antibodies against the planarian Dugesia (Girardia) tigrina

 To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation, and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual race of the freshwater planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining, we have found monoclonal antibodies against all tissues and cell types with the exception of neoblasts, the undifferentiated totipotent stem-cells in planarians. We have also detected position-specific antigens that label anterior, central, and posterior regions. Patterns of expression uncovered an unexpected heterogeneity among previously thought single cell types, as well as interesting cross-reactivities that deserve further study. Characterization of some of these monoclonal antibodies suggests they may be extremely useful as molecular markers for studying cell renewal and cell differentiation in the intact and regenerating organism, tracing the origin, lineage, and differentiation of blastema cells, and characterizing the stages and mechanisms of early pattern formation. Moreover, two position-specific monoclonals, the first ones isolated in planarians, will be instrumental in describing in molecular terms how the new pattern unfolds during regeneration and in devising the pattern formation model that best fits classical data on regeneration in planarians. Accepted: 16 September 1996     

Cell Death and Acid Phosphatase Activity in the Regenerating Planarian Polycelis tenuis Iijima

A combination of microscopical, cytochemical, and biochemical techniques have been employed to study the changes occurring during the first seven days of blastema formation and regeneration after decapitation in adult Polycelis tenuis worms. Fine structural data reveal evidence of cell fragmentation, selective cell deletion, and phagocytosis at and below the wound surface. Initially, (0–12 h regeneration) cell debris is phagocytosed by intact parenchymal and gastrodermal cells near the cut surface which is later sealed (24 h) by a stretching of marginal epidermal cells. Wound sealing is followed by a migration of newly differentiated rhabdite cells into the epithelium. Morphological evidence of a selective cell autolysis precedes evidence of an accumuluation of lipid and glycogen reserves in the parenchymal and gastrodermal cells and the later (48 h regeneration time) aggregation of undifferentiated mitotically active neoblasts beneath the wound.
Biochemical data reveal an early period of high acid phosphatase (p-nitrophenyl phosphatase and sodium-β-glycerophosphatase) activity 3–12 h after injury, followed by a further intense period of activity at 44–48 h after decapitation. The coincident cytochemical data show an increased level of acid phosphatase activity associated with cell lysis and death in the wound and blastema zone and also with the digestion of phagocytosed cell debris.     

Potassium channels in the Helix central nervous system: preliminary immunohistochemical studies. Short communication

Distribution of the potassium channel of Kv4.3 type was investigated in the central nervous system (CNS) of Helix pomatia by immunohistochemistry. Immunopositive neurons were found widely distributed in the CNS, present mostly in smaller groups in the different central ganglia but not in the visceral ganglion. Labeled fibers were characteristic for not only the neuropils of all ganglia but also the connective tissue sheath around the CNS and the aorta wall were richly innervated. Western blot analysis revealed a clear identity with the mammalian Kv4.3 subunit, suggesting an evolutionary conserved structure of this channel type. Our preliminary results provide a steady basis for further experiments aiming partly at the identification of other potassium channel types and partly the ultrastructural localization of Kv4.3.     

Characterization and distribution of FMRFamide immunoreactivity in the rat central nervous system

FMRFamide immunoreactive material (irFMRFamide) was studied in rat brain and gastrointestinal tract. Highest irFMRFamide concentrations were found in tissues of the gastrointestinal tract and, in the brain, highest concentrations were found in the hippocampus, midbrain, brainstem and hypothalamus. High pressure liquid chromatographic characterization of irFMRFamide demonstrated that the immunoreactive material in brain, pancreas and duodenum was different from molluscan FMRFamide but it was also distinct from any known neuropeptide.     

The adenylate cyclase system of planaria Polycelis tenuis: activation by serotonin and guanine nucleotides

The particulate fraction prepared after homogenization of planaria Polycelis tenuis in a buffer containing 3 mM EDTA and 15 mM 2-mercaptoethanol possesses an adenylate cyclase activity which was enhanced two-fold by serotonin and 20-fold by the nucleotide analog guanosine 5'-(beta-gamma-imino)triphosphate, Gpp(NH)p; when present together, the two activators exhibited a marked synergistic effect. The effect of serotonin was dose dependent, with a KA of 2 micrometer and a Hill coefficient of 0.4. In the presence of 10 micrometer Gpp(NH)p, these values became 45 nM and 1.5, respectively. The effect of serotonin was due to an increase in the maximal velocity of the enzyme and was specifically inhibited by methiotepin. The effect of methiotepin was half-maximal at 0.2 micrometer in the absence of Gpp(NH)p and at 5.0 micrometer in its presence. Planaria thus appear to be the lowest organisms in which guanine nucleotides are active upon adenylate cyclase. As serotonin is normally present in planaria, it is postulated that a serotonin-dependent regulation of adenylate cyclase activity plays a physiological role in this species.     

Reorganization of the nervous system during regeneration in Dugesia tigrina]

The normal structure of the nervous system in Dugesia tigrina Girard and the total morphodynamics of the nervous system during regeneration have been studied by means of choline esterase assay. The nervous system reacts to local damages of the planarian body; accumulations of nervous elements form in the wound region. Following the transverse cut of a planarian, the regeneration of the nervous system is not reduced to the completion of lacking parts. In this case (as well as in that of asexual reproduction) the nervous system manifests a considerable morphological lability and undergoes morphological rearrangements accompanied by the appearance of additional, frequently unpaired, nerve trunks. The data obtained are to be taken into account in neurobiological studies on planarians.     

FMRFamide immunoreactive neurons in the central nervous system of the snail,Achatin a fulica

• 1.1. FMRFamide immunoreactive neurons were detected in the central nervous system of the snail, Achatina fulica.
• 2.2. FMRFamide immunoreactive neurons were found in all the ganglia comprising the central nervous system. In particular, the immunoreactivity was recognized in both the ordinary and giant neurons of the visceral and right parietal ganglia.
• 3.3. In the cerebral and pleural ganglia, FMRFamide immunoreactive neurons were found only in the ordinary neurons. The immunoreactivity was shown to have a tendency to form a group in the cerebral and pedal ganglia.

     

Thioredoxin and glutaredoxin system proteins—immunolocalization in the rat central nervous system

Background

The oxidoreductases of the thioredoxin (Trx) family of proteins play a major role in the cellular response to oxidative stress. Redox imbalance is a major feature of brain damage. For instance, neuronal damage and glial reaction induced by a hypoxic–ischemic episode is highly related to glutamate excitotoxicity, oxidative stress and mitochondrial dysfunction. Most animal models of hypoxia–ischemia in the central nervous system (CNS) use rats to study the mechanisms involved in neuronal cell death, however, no comprehensive study on the localization of the redox proteins in the rat CNS was available.

Methods

The aim of this work was to study the distribution of the following proteins of the thioredoxin and glutathione/glutaredoxin (Grx) systems in the rat CNS by immunohistochemistry: Trx1, Trx2, TrxR1, TrxR2, Txnip, Grx1, Grx2, Grx3, Grx5, and γ-GCS, peroxiredoxin 1 (Prx1), Prx2, Prx3, Prx4, Prx5, and Prx6. We have focused on areas most sensitive to a hypoxia–ischemic insult: Cerebellum, striatum, hippocampus, spinal cord, substantia nigra, cortex and retina.

Results and conclusions

Previous studies implied that these redox proteins may be distributed in most cell types and regions of the CNS. Here, we have observed several remarkable differences in both abundance and regional distribution that point to a complex interplay and crosstalk between the proteins of this family.

General significance

We think that these data might be helpful to reveal new insights into the role of thiol redox pathways in the pathogenesis of hypoxia–ischemia insults and other disorders of the CNS.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

The Morphogenetic Effects of Exogenous Sex Steroid Hormones in the Planarian Girardia tigrina (Turbellaria,Tricladida)

Biophysics - Abstract—Morphogenetic effects of two exogenous sex steroid hormones, testosterone and estradiol, during planarian regeneration have been studied. Anterior regenerants, which...     

Some aspects of the physiological role of ion channels in the nervous system

Recent analyses of the genomes of several animal species, including man, have revealed that a large number of ion channels are present in the nervous system. Our understanding of the physiological role of these channels in the nervous system has followed the evolution of biophysical techniques during the last century. The observation and the quantification of the electrical events associated with the operation of the ionic channels has been, and still is, one of the best tools to analyse the various aspects of their contribution to nerve function. For this reason, we have chosen to use electrophysiological recordings to illustrate some of the main functions of these channels. The properties and the roles of Na+ and K+ channels in neuronal resting and action potentials are illustrated in the case of the giant axons of the squid and the cockroach. The nature and role of the calcium currents in the bursting behaviour of the neurons are illustrated for Aplysia giant neurons. The relationship between presynaptic calcium currents and synaptic transmission is shown for the squid giant synapse. The involvement of calcium channels in survival and neurite outgrowth of cultured neurons is exemplified using embryonic cockroach brain neurons. This same neuronal preparation is used to illustrate ion channel noise and single-channel events associated with the binding of agonists to nicotinic receptors. Some features of the synaptic activity in the central nervous system are shown, with examples from the cercal nerve giant-axon preparation of the cockroach. The interplay of different ion conductances involved in the oscillatory behaviour of the Xenopus spinal motoneurons is illustrated and discussed. The last part of this review deals with ionic homeostasis in the brain and the function of glial cells, with examples from Necturus and squids.     

Planarians in pharmacology: parthenolide is a specific behavioral antagonist of cocaine in the planarian Girardia tigrina

Planarians are traditional animal models in developmental and regeneration biology. Recently, these organisms are arising as vertebrate-relevant animal models in neuropharmacology. Using an adaptation of published behavioral protocols, we have described the alleviation of cocaine-induced planarian seizure-like movements (pSLM) by a naturally-occurring sesquiterpene lactone, parthenolide. Interestingly, parthenolide does not prevent the expression of pSLM induced by amphetamines; in vertebrates, amphetamines interact with the same protein target as cocaine. Parthenolide is also unable to prevent pSLM elicited by the cholinergic com-pounds nicotine and cytisine or by the glutamatergic agents L- or D- glutamic acid or NMDA. Thus, we conclude that parthenolide is a specific anti-cocaine agent in this experimental organism.     

Allopatric and sympatric populations of Polycelis nigra in competition with P. tenuis (Turbellaria; Tricladida)

Two allopatric populations of Polycelis nigra (MuUller), one from lowland productive Llyn Maelog on Anglesey and the other from upland unproductive Llyn Dinas in Snowdonia, were each subjected to competition in the laboratory from the same Polycelis tenuis Ijima population from productive Llyn Llygeirian, also on Anglesey, where it coexists with P. nigra. The triclads were fed weekly on crushed Asellus (Isopoda) and their biomasses calculated monthly over five months. Results suggest that sympatric P. nigra are more successful competitors against P. tenuis than allopatric P. nigra. The data also suggest that the inferior competitive ability of allopatric P. nigra may be a consequence of both exploitative and interference competition. Coexistence of P. nigra with P. tenuis appears to increase P. nigra's tolerance to environmental stress; the current experiments and observations during several decades of research have indicated poorer survival and growth in allopatric than sympatric populations.