A novel form of presynaptic CaMKII-dependent short-term potentiation between Lymnaea neurons.

Short-term plasticity is thought to form the basis for working memory, the cellular mechanisms of which are the least understood in the nervous system. In this study, using in vitro reconstructed synapses between the identified Lymnaea neuron visceral dorsal 4 (VD4) and left pedal dorsal 1 (LPeD1), we demonstrate a novel form of short-term potentiation (STP) which is 'use'- but not time-dependent, unlike most previously defined forms of short-term synaptic plasticity. Using a triple-cell configuration we demonstrate for the first time that a single presynaptic neuron can reliably potentiate both inhibitory and excitatory synapses. We further demonstrate that, unlike previously described forms of STP, the synaptic potentiation between Lymnaea neurons does not involve postsynaptic receptor sensitization or presynaptic residual calcium. Finally, we provide evidence that STP at the VD4-LPeD1 synapse requires presynaptic calcium/calmodulin dependent kinase II (CaMKII). Taken together, our study identifies a novel form of STP which may provide the basis for both short- and long-term potentiation, in the absence of any protein synthesis-dependent steps, and involve CaMKII activity exclusively in the presynaptic cell.

Integrative biology of an embryonic respiratory behaviour in pond snails: the 'embryo stir-bar hypothesis'.

Embryos of freshwater snails undergo direct development from single cell to juvenile inside egg masses that are deposited on vegetation and other substratum in pond, lake and stream habitats. Helisoma trivolvis, a member of the Planorbidae family of basommatophoran snails, has served as a model for studying the developmental and physiological roles for neurotransmitters during embryogenesis. Early studies revealed that H. trivolvis embryos from stage E15 to E30, the period between gastrulation and the trochophore-juvenile transition, display a cilia-driven behaviour consisting of slow basal rotation and transient periods of rapid rotation. The discovery of a bilateral pair of early serotonergic neurons, named ENC1, which project an apical process to the embryo surface and basal neurites to ciliated cells, prompted the hypothesis that each ENC1 is a dual-function sensory and motor neuron mediating a physiological embryonic response. This article reviews our past and present studies and addresses questions concerning this hypothesis, including the following. (1) What environmental signal regulates ENC1 activity and rotational behaviour? (2) Does ENC1 function as both a primary sensory and motor neuron underlying the rotational behaviour? (3) What are the sensory transduction mechanisms? (4) How does ENC1 regulate ciliary beating? (5) Do other basommatophoran species have similar neural-ciliary pathways and behavioural responses? (6) How is the behaviour manifest in the dynamic natural environment? In this review, we introduce the ;embryo stir-bar hypothesis', which proposes that embryonic rotation is a hypoxia-sensitive respiratory behaviour responsible for mixing the egg capsule fluid, thereby enhancing delivery of environmental oxygen to the embryo.

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.

Effect of serotonin on ciliary beating and intracellular calcium concentration in identified populations of embryonic ciliary cells.

Embryos of the pond snail Helisoma trivolvis express three known subtypes of ciliary cells on the surface of the embryo early in development: pedal, dorsolateral and scattered single ciliary cells (SSCCs). The pedal and dorsolateral ciliary cells are innervated by a pair of serotonergic sensory-motor neurons and are responsible for generating the earliest whole-animal behavior, rotation within the egg capsule. Previous cell culture studies on unidentified ciliary cells revealed that serotonin (5-hydroxytryptamine; 5-HT) produces a significant increase in the ciliary beat frequency (CBF) in a large proportion of ciliary cells. Both Ca2+ influx and a unique isoform of protein kinase C (PKC) were implicated in the signal transduction pathway underlying the cilio-excitatory response to 5-HT. The goal of the present study was to characterize the anatomical and physiological differences between the three known populations of superficial ciliary cells. The pedal and dorsolateral ciliary cells shared common structural characteristics, including flat morphology, dense cilia and lateral accessory ciliary rootlets. By contrast, the SSCCs had a cuboidal morphology, reduced number of cilia, increased ciliary length and absence of lateral accessory rootlets. In cultures containing unidentified ciliary cells, the calcium/calmodulin-dependent enzyme inhibitor calmidazolium (2 micromol l(-1)) blocked the stimulatory effect of 5-HT (100 micromol l(-1)) on CBF. In addition, 50% of unidentified cultured cells responded to 5-HT (100 micromol l(-1)) with an increase in [Ca2+]i. To facilitate the functional analyses of the individual populations, we developed a method to culture identified ciliary subtypes and characterized their ciliary and calcium responses to 5-HT. In cultures containing either pedal or dorsolateral ciliary cells, 5-HT (100 micromol l(-1)) produced a rapid increase in CBF and a slower increase in [Ca2+]i in all cells examined. By contrast, the CBF and [Ca2+]i of SSCCs were not affected by 100 micromol l(-1) 5-HT. Immunohistochemistry for two putative 5-HT receptors recently cloned from Helisoma revealed that pedal and dorsolateral ciliary cells consistently express the 5-HT(1Hel) protein. Intense 5-HT(7Hel) immunoreactivity was observed in only a subset of pedal and dorsolateral ciliary cells. Cells neighboring the SSCCs, but not the ciliary cells themselves, expressed 5-HT(1Hel) and 5-HT(7Hel) immunoreactivity. These data suggest that the pedal and dorsolateral ciliary cells, but not the SSCCs are a homogeneous physiological subtype that will be useful for elucidating the signal transduction mechanisms underlying 5-HT induced cilio-excitation.

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.

Coordinated development of identified serotonergic neurons and their target ciliary cells in Helisoma trivolvis embryos.

Embryonic neuron C1s (ENC1s) are bilateral serotonergic neurons that function as cilioexcitatory motor neurons in embryonic development of the pond snail, Helisoma trivolvis. Recent experiments demonstrated that these neurons stimulate cilia-driven embryo rotation in response to hypoxia. In the present study, a comprehensive anatomic analysis of these cells and their target ciliary structures was done to address the following questions: (1) Does ENC1 have a morphology consistent with an oxygen-sensitive sensory cell; (2) Is the development of ENC1's neurite outgrowth pathway coordinated with the development of its target effectors, the pedal and dorsolateral ciliary bands; and (3) What is the anatomic basis of ENC1-ciliary communication? By using an array of microscopic techniques on live and serotonin-immunostained embryos, we found that each ENC1 possessed an apical dendrite that was capped with an integral dendritic knob penetrating the embryo surface. The dendritic knobs contained both microvilli and nonmotile cilia that suggested a sensory transduction role. Each ENC1 also possessed a descending projection, whose development was characterized by the rapid formation of the primary neurite pathway between stages E13 and E15, with the primary neurite of the right ENC1 developing in advance of its contralateral homologue. Secondary neurite branches formed between stages E15 and E30 in a spatiotemporal pattern that closely matched the development of the dorsolateral and pedal bands of cilia. Both dorsolateral and pedal ciliated cells formed basal processes that contacted ENC1 neurites. Finally, gap junction profiles were observed at neurite-neurite, neurite-ciliary cell, and ciliary cell-ciliary cell apposition sites, whereas putative chemical synaptic profiles were observed at neurite-neurite and neurite-ciliary cell apposition sites.

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.

Causes and consequences of pathogenic processes in evolution: implications from experimental epilepsy in animals.

Examples from experimental epilepsy in animals are used to illustrate the view that a crucial role of the transfer of mechanisms from compensatory into pathogenic (e.g. lethal ones in the course of a disease), is played by the power of pathologic stimuli. In the genesis of epilepsy it is suggested that a critical increase of endogenous factors may underlie the conversion of the absence form of epilepsy into a generalized self-supporting form. The ability to precipitate endogenous self-augmenting mechanisms of diseases may have increased in the course of evolution. The lethal result of a serious pathogenic process leads to the suggestion that organisms cope with the disease by dying. This prevents spreading of the putative infectious disease within the population. This mechanism of disease aggravation could play a role in the survival of the species and in further evolutionary progress. This may explain why certain species may have survived in evolution and supports the theory of synthetic evolution.

The role of TNF-alpha in amygdala kindled rats.

In the present study, the interaction between epileptogenesis and the immune system were studied in a kindling model. First, the effects of a single administration of TNF-alpha (5.0 microg/kg, i.p.) on seizure and EEG activity were investigated in amygdala-kindled rats. TNF-alpha treated rats showed more prolonged epileptiformic discharges than control rats. TNF-alpha also induced a decrease in the power of delta band and an increase in theta and alpha activity. In addition, a marked increase in the power of beta and gamma band was observed. The EEG changes were most numerous in the frontal cortex and amygdala. All effects were registered 24 h after TNF-alpha administration. Finally, electrical stimulation enhanced the level of TNF-alpha in blood serum from 1.9 +/- 1.5 to 12.7 +/- 3.8 pg/ml and in brain tissue 56.8 +/- 6.0 to 109.2 +/- 6.0 pg/mg, as was determined via the ELISA method. It can be concluded that there is a mutual facilitative interaction of both epileptogenic and cytokine-derived mechanisms on this type of seizure. The changes in the power spectrum of the EEG after TNF-alpha might contribute to intensify thalamic-derived facilitation of epileptic discharge in cortical structures.

D-24851, a novel synthetic microtubule inhibitor, exerts curative antitumoral activity in vivo, shows efficacy toward multidrug-resistant tumor cells, and lacks neurotoxicity.

N-(pyridin-4-yl)-[1-(4-chlorbenzyl)-indol-3-yl]-glyoxyl-amid (D-24851) is a novel synthetic compound that was identified in a cell-based screening assay to discover cytotoxic drugs. D-24851 destabilizes microtubules and blocks cell cycle transition specifically at G2-M phase. The binding site of D-24851 does not overlap with the tubulin binding sites of known microtubule-destabilizing agents like vincristine or colchicine. In vitro, D-24851 has potent cytotoxic activity toward a panel of established human tumor cell lines including SKOV3 ovarian cancer, U87 glioblastoma, and ASPC-1 pancreatic cancer cells. In vivo, oral D-24851 treatment induced complete tumor regressions (cures) in rats bearing Yoshida AH13 sarcomas. Of importance is that the administration of curative doses of D-24851 to the animals revealed no systemic toxicity in terms of body weight loss and neurotoxicity in contrast to the administration of paclitaxel or vincristine. Interestingly, multidrug-resistant cell lines generated by vincristine-driven selection or transfection with the Mr 170,000 P-glycoprotein encoding cDNA were rendered resistant toward paclitaxel, vincristine, or doxorubicin but not towards D-24851 when compared with the parental cells. Because of its synthetic nature, its oral applicability, its potent in vitro and in vivo antitumoral activity, its efficacy against multidrug-resistant tumors, and the lack of neurotoxicity, D-24851 may have significant potential for the treatment of various malignancies.

Effects of delta-sleep-inducing peptide on NMDA-induced convulsive activity in rats.

Acute experiments on rats showed that the ED100 of NMDA for induction of clonic convulsions was 0.53 microgram, while the ED100 of NMDA for inducing tonic extension of the forelimbs was 5.02 micrograms/animal. Determination of these parameters after administration of delta-sleep-inducing peptide (100 micrograms/kg, i.p.) revealed 2.3- and 4.46-fold increases. These results provide evidence for a neuroprotective role of delta-sleep-inducing peptide in relation to excitatory amino acid receptor agonists.

Delta-sleep-inducing peptide and its analogs and the serotoninergic system in the development of anticonvulsive influences.

Experiments on rats were carried out to study the effects of administration of delta-sleep-inducing peptide (DSIP) and its analogs (9-14) into the reticular part of the substantia nigra and ventral hippocampus on picrotoxin- and kainate-induced epileptic activity. Additionally, the uptake of [3H]tryptophan by brain structures was studied. Intranigral and intrahippocampal microinjections of peptide and its analogs were found to have anticonvulsant effects against both picrotoxin- and kainate-induced epileptic activity. Studies of the effects of DSIP and its structural analogs on the uptake of tryptophan by brain structures showed that peptides predominantly increased uptake of this amino acid. It is suggested that brain structures which modulate tryptophan uptake are largely responsible for the anticonvulsant actions of DSIP and its analogs. The results obtained here provide evidence that the serotoninergic system is not of key importance in mediating the anticonvulsant effects of DSIP and its analogs.

Effects of delta-sleep-inducing peptide in cerebral ischemia in rats.

Experimental studies were carried out to investigate the neuroprotective effects of delta sleep-inducing peptide in animals with cerebral ischemia induced by bilateral compression of both carotid arteries, and to compare the efficacy of this peptide with that of MK-801. These studies led to the conclusion that the peptide had pronounced anti-ischemic effects, which were evident within 24 h and consisted of reductions in the severity of postural abnormalities in rats with bilateral cerebral ischemia, along with a reduction in lethality. Comparison of the efficacies of peptide and MK-801 showed the peptide to have the greater neuroprotective effect. These results are regarded as providing an experimental basis for using the peptide as a therapeutic agent in patients with stroke.

[Changes in the blastogenic lymphocyte transformation during kindling induced by picrotoxin in rats]. / Izmeneniia blastnoi transformatsii limfotsitov pri kindlinge, vyzvannom primeneniem pikrotoksina u krys.

Picrotoxin-induced kindling was shown to suppress the blastogenic response to bacterial lipopolysaccharide and phytogemagglutinin in male Wistar rats. The delta-sleep-inducing peptide as well as carbamazepine prevented the epileptogenic effects of picrotoxin. Carbamazepine was also effective against decreasing of phytogemagglutinin-induced blastogenic response.

[The delta sleep-inducing peptide, its analogs and the serotoninergic system in the development of anticonvulsant action]. / Del'ta-son indutsiruiushchii peptid, ego analogi i serotoninergicheskaia sistema v razvitii protivosudorozhnogo deistviia.

A seizure-protecting effect of the delta-sleep-inducing peptide (DSIP) and its analogues was revealed. An intensive sorption of H3 tryptophan occurred under the effect of the DSIP and its analogues. The data obtained suggests that the serotoninergic system plays no important part in the seizure-protecting effect.

[Effects of delta-sleep-inducing peptide during ischemic injury of the rat brain]. / Effekty del'ta-son-indutsiruiushchego peptida v usloviiakh ishemicheskogo povrezhdeniia golovnogo mozga u krys.

An anti-ischemic effect of the delta-sleep-inducing peptide (DSIP) was found in rats. The DSIP effect was more obvious than that of the MK-801. The data obtained is discussed considering a possible use of the DSIP for brain stroke prophylaxis.

[The effect of the delta sleep-inducing peptide on NMDA-induced seizure activity in rats]. / Vliianie del'ta-son indutsiruiushchego peptida na sudorozhnuiu aktivnost', vyzvannuiu NMDA u krys.

The ED100 of the NMDA inducing clonic seizures (0.53 mcg) or tonic extension of forelimbs (5.02 mcg) increased their efficiency 2.3-fold and 4.46-fold, resp., due to the delta-sleep-inducing peptide administration in rats. The data obtained suggests a neuroprotective effect of the peptide on agonists of excitatory amino acid receptors action.

Effect of intranigral dosage with delta-sleep-inducing peptide and its analogs on movement and convulsive activity in rats.

Studies were carried out in rats on the effects of the administration of delta-sleep-inducing peptide (DSIP) and its analogs (1-4) into the reticular part of the substantia nigra on movement and convulsive activity. Intranigral microinjection of DSIP, and of DSIP-1 and DSIP-4, reduced horizontal and vertical movement activity as well as excursions to the center of the open field. DSIP, DSIP-2, and DSIP-3 had anticonvulsant effects, consisting of increases in the latent periods of the first convulsion and clonicotonic convulsions induced by picrotoxin, and reductions in the mean intensity of convulsions. It is suggested that changes in the structure of DSIP are accompanied by alterations in the strength of the effects of this peptide on horizontal and convulsive activity after dosage into the reticular part of the substantia nigra. The results indicating that these peptides have protective activity in experimental convulsive syndrome suggest that a relationship exists between DSIP-induced reductions in movement activity and the anticonvulsive efficacy of DSIP analogs when administered intranigrally, this being one of the components of the nigrodependent mechanisms of inhibition of convulsions.

[A rotational syndrome induced by administration of the delta sleep-inducing peptide into the reticular portion of the rat substantia nigra]. / Rotatsionnyi sindrom, vyzyvaemyi vvedeniem del'ta-son-indutsiruiushchego peptida v retikuliarnuiu chast' chernogo veshchestva krys.

Unilateral intranigral administration of a delta-sleep-induced peptide (DSIP) evoked a contralateral rotation. Naloxon prevented development of the effect whereas haloperidol administration enhanced the DSIP cataleptogenic effect.

Chemical kindling: implications for antiepileptic drugs - sensitive and resistant epilepsy models.

The efficacy of phenobarbital (PB) and phenytoin (PHT) was evaluated against the convulsions in chemically (picrotoxin, PTX) kindled rats. Two protocols were used: assessment of seizures immediately after the completion of the kindling procedure and after the 2-week postkindling PTX-free period, as compared with acute PTX seizures. Kindled convulsions were more sensitive than acute PTX seizures to the antiepileptic action of PB and PHT. On the other hand, the "postkindling state" was characterized by the enhancement of the severity of the convulsions in comparison with both kindled and acute PTX seizures and dramatic increase in the resistance to the action of antiepileptic drugs (AEDs). It is concluded that the two paradigms--kindling proper and "postkindling"--could be applied as models for AED-sensitive and AED-resistant animal epilepsy models correspondingly.