Decreased 14-3-3 expression correlates with age-related regional reductions in CNS dopamine and motor function in the pond snail, Lymnaea.

Ageing is associated in many organisms with a reduction in motor movements. We have previously shown that the rate of feeding movements of the pond snail, Lymnaea, decreased with age but the underlying cause is not fully understood. Here, we show that dopamine in the cerebro-buccal complex is an important signalling molecule regulating feeding frequency in Lymnaea and that ageing is associated with a decrease in CNS dopamine. A proteomic screen of young and old CNSs highlighted a group of proteins that regulate stress responses. One of the proteins identified was 14-3-3, which can enhance the synthesis of dopamine. We show that the Lymnaea 14-3-3 family exists as three distinct isoforms. The expression of the 29 kDa isoform (14-3-3Lym3) in the cerebro-buccal complex decreased with age and correlated with feeding rate. Using a 14-3-3 antagonist (R18) we were able to reduce the synthesis of L-DOPA and dopamine in ex vivo cerebro-buccal complexes. Together these data suggest that an age-related reduction in 14-3-3 can decrease CNS dopamine leading to a consequential reduction in feeding rate.

PACAP Modulates Acetylcholine-Elicited Contractions at Nicotinic Neuromuscular Contacts of the Land Snail.

In this study, we investigate the potentiating effect of PACAP27 on cholinergic neuromuscular transmission in the recently discovered flexor muscles of the land snail, Helix pomatia. Using immunohistochemistry, we show that PACAP and PAC1 receptors are present in nerve fibers innervating the flexor muscles but not in the muscle itself. We also observed that PACAP27 exerts both pre- and postsynaptic effects on the cholinergic synapse and performed tests using a broad spectrum of chemicals in order to explore the possible intracellular pathways through which PACAP mediates its stimulatory effect. Our pharmacological data demonstrate that PACAP27 presynaptically enhances the release of acetylcholine by activating the adenylate cyclase-cAMP-PKA pathway. Postsynaptically, PACAP27 was found to enhance muscle contractility by PKC-mediated signaling pathway resulting in an increased Ca(2+) release from intracellular stores. These findings suggest that regulation of Ca(2+) release may contribute to the stimulatory effect of PACAP. Our data are the first demonstration of the potentiating effect of PACAP27 at the molluscan excitatory neuromuscular contact.

The activity of isolated neurons and the modulatory state of an isolated nervous system represent a recent behavioural state.

Behavioural/motivational state is known to influence nearly all aspects of physiology and behaviour. The cellular basis of behavioural state control is only partially understood. Our investigation, performed on the pond snail Lymnaea stagnalis whose nervous system is useful for work on completely isolated neurons, provided several results related to this problem. First, we demonstrated that the behavioural state can produce long-term changes in individual neurons that persist even after neuron isolation from the nervous system. Specifically, we found that pedal serotonergic neurons that control locomotion show higher activity and lower membrane potential after being isolated from the nervous systems of hungry animals. Second, we showed that the modulatory state (the chemical neuroactive microenvironment of the central ganglia) changes in accordance with the nutritional state of an animal and produces predicted changes in single isolated locomotor neurons. Third, we report that observed hunger-induced effects can be explained by the increased synthesis of serotonin in pedal serotonergic neurons, which has an impact on the electrical activity of isolated serotonergic neurons and the intensity of extrasynaptic serotonin release from the pedal ganglia.

The activity of isolated snail neurons controlling locomotion is affected by glucose.

The involvement of serotonin in mediating hunger-related changes in behavioral state has been described in many invertebrates. However, the mechanisms by which hunger signals to serotonergic cells remain unknown. We tested the hypothesis that serotonergic neurons can directly sense the concentration of glucose, a metabolic indicator of nutritional state. In the snail Lymnaea stagnalis, we demonstrate that completely isolated pedal serotonergic neurons that control locomotion changed their biophysical characteristics in response to glucose application by lowering membrane potential and decreasing the firing rate. Additionally, the excitatory response of the isolated serotonergic neurons to the neuroactive microenvironment of the pedal ganglia was significantly lowered by glucose application. Because hunger has been reported to increase the activity of select neurons and their responses to the pedal ganglia microenvironment, these responses to glucose are in accordance with the hypothesis that direct glucose signaling is involved in the mediation of the hunger-related behavioral state.

Nicotinic acetylcholine receptors containing the α7-like subunit mediate contractions of muscles responsible for space positioning of the snail, Helix pomatia L. tentacle.

Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh) responses in muscles to determine the properties of the ACh receptor (AChR), the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium), methyllycaconitine, α-Bungarotoxin (αBgTx) and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na+ and Ca2+ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.

Novel peripheral motor neurons in the posterior tentacles of the snail responsible for local tentacle movements.

Three flexor muscles of the posterior tentacles of the snail Helix pomatia have recently been described. Here, we identify their local motor neurons by following the retrograde transport of neurobiotin injected into these muscles. The mostly unipolar motor neurons (15-35 µm) are confined to the tentacle digits and send motor axons to the M2 and M3 muscles. Electron microscopy revealed small dark neurons (5-7 µm diameter) and light neurons with 12-18 (T1 type) and 18-30 µm diameters (T2 type) in the digits. The diameters of the neurobiotin-labeled neurons corresponded to the T1 type light neurons. The neuronal processes of T1 type motor neurons arborize extensively in the neuropil area of the digits and receive synaptic inputs from local neuronal elements involved in peripheral olfactory information processing. These findings support the existence of a peripheral stimulus-response pathway, consisting of olfactory stimulus-local motor neuron-motor response components, to generate local lateral movements of the tentacle tip ("quiver"). In addition, physiological results showed that each flexor muscle receives distinct central motor commands via different peritentacular nerves and common central motor commands via tentacle digits, respectively. The distal axonal segments of the common pathway can receive inputs from local interneurons in the digits modulating the motor axon activity peripherally without soma excitation. These elements constitute a local microcircuit consisting of olfactory stimulus-distal segments of central motor axons-motor response components, to induce patterned contraction movements of the tentacle. The two local microcircuits described above provide a comprehensive neuroanatomical basis of tentacle movements without the involvement of the CNS.

Pharmacological analysis of response latency in the hot plate test following whole-body static magnetic field-exposure in the snail Helix pomatia.

PURPOSE: To study the effect of single, 30-min long, whole-body, homogeneous static magnetic field (SMF)-exposure of magnetic induction 147 ± 3 mT on the response latency of the snail Helix pomatia. MATERIALS AND METHODS: The response was investigated using the hot plate test. RESULTS: The effect caused by exposure to SMF was compared to sham-exposure and resulted in significant differences (up to 47.1%, p < 0.001). The response latency depended on the day-night cycle; response latency was higher by 51.2% (p < 0.001) during the night. This trend also held for SMF-exposure (28.6%, p < 0.001). Serotonin alone increased response latency (55.7%, p < 0.001), whereas serotonin antagonist tryptamine decreased it (- 97.8%, p < 0.001). Using naloxone, response latency decreased (- 52.5%, p < 0.001); however both SMF-exposure and serotonin in combination with naloxone rose it back to above the control level (116.9%, p < 0.001 or 150.2%, p < 0.001, respectively). CONCLUSIONS: This study provides evidence that SMF-exposure mediates peripheral thermal nociceptive threshold by affecting the serotonerg as well as the opioiderg system.

Neuronal background of positioning of the posterior tentacles in the snail Helix pomatia.

The location of cerebral neurons innervating the three recently described flexor muscles involved in the orientation of the posterior tentacles was investigated by applying parallel retrograde Co- and Ni-lysine tracing via the olfactory and the peritentacular nerves. Their innervation patterns in the flexor muscles were studied by applying anterograde neurobiotin tracings via these nerves. The labeled neurons are clustered in eight groups in the cerebral ganglion. They send both common and distinct innervation pathways to the flexor and the tegumental muscles and to the tentacular retractor muscle. The common pathway reaches the muscles via the olfactory nerve, whereas the distinct pathways innervate via the internal and external peritentacular nerves. The three anchoring points of the three flexor muscles at the base of the tentacle outline the directions of three force vectors generated by the contraction of the muscles and enable the protracted tentacle to bend around a basal pivot. In the light of earlier physiological and the present anatomical findings, we suggest that the common innervation pathway to the muscles is required for tentacle withdrawal and the retractor mechanism, whereas the distinct pathways primarily serve the bending of the protracted posterior tentacles during foraging.

Contraceptive efficacy of a combined oral contraceptive containing ethinyloestradiol 20 µg/drospirenone 3mg administered in a 24/4 regimen: a pooled analysis of four open-label studies.

OBJECTIVE: To determine the contraceptive efficacy of a low-dose combined oral contraceptive containing ethinyloestradiol (EE) 20 µg/drospirenone (drsp) 3mg administered for 24 days followed by a four-day hormone-free interval (24/4 regimen), by pooling data from four clinical trials. STUDY DESIGN: Data on the occurrence of pregnancy during treatment in four open-label trials that enrolled healthy female volunteers aged 17-36 years, who received EE 20 µg/drsp 3mg in a 24/4 regimen for 7 or 13 cycles, were pooled. RESULTS: Sixteen pregnancies occurred among 2386 women during 729,537 days (26,055 cycles) of treatment exposure, resulting in a Pearl Index (PI) of 0.80 (upper two-sided 95% confidence interval (CI) limit of 1.30). Of these pregnancies, seven were defined as method failures, resulting in an adjusted PI of 0.41 (upper two-sided 95% CI limit of 0.85), based on 616,607 days (22,022 cycles) of treatment. The Kaplan-Meier estimate for the one-year cumulative probability of contraceptive protection was 99.21%. CONCLUSIONS: The EE 20 µg/drsp 3mg combined oral contraceptive administered in a 24/4 regimen has acceptable contraceptive efficacy.

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present and biochemically active in the central nervous system of the pond snail Lymnaea stagnalis.

PACAP is a highly conserved adenylate cyclase (AC) activating polypeptide, which, along with its receptors (PAC1-R, VPAC1, and VPAC2), is expressed in both vertebrate and invertebrate nervous systems. In vertebrates, PACAP has been shown to be involved in associative learning, but it is not known if it plays a similar role in invertebrates. To prepare the way for a detailed investigation into the possible role of PACAP and its receptors in a suitable invertebrate model of learning and memory, here, we undertook a study of their expression and biochemical role in the central nervous system of the pond snail Lymnaea stagnalis. Lymnaea is one of the best established invertebrate model systems to study the molecular mechanisms of learning and memory, including the role of cyclic AMP-activated signaling mechanisms, which crucially depend on the learning-induced activation of AC. However, there was no information available on the expression of PACAP and its receptors in sensory structures and central ganglia of the Lymnaea nervous system known to be involved in associative learning or whether or not PACAP can actually activate AC in these ganglia. Here, using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and immunohistochemistry, we established the presence of PACAP-like peptides in the cerebral ganglia and the lip region of Lymnaea. The MALDI-TOF data indicated an identity with mammalian PACAP-27 and the presence of a squid-like PACAP-38 highly homologous to vertebrate PACAP-38. We also showed that PACAP, VIP, and maxadilan stimulated the synthesis of cAMP in Lymnaea cerebral ganglion homogenates and that this effect was blocked by the appropriate general and selective PACAP receptor antagonists.

Efficacy and safety of a low-dose combined oral contraceptive containing drospirenone 3 mg and ethinylestradiol 20 mcg in a 24/4-day regimen.

OBJECTIVE: The study was conducted to assess the efficacy of a low-dose combined oral contraceptive (COC) containing drospirenone (drsp) 3 mg/ethinylestradiol (EE) 20 mcg administered for 24 days of active treatment followed by a 4-day hormone-free interval (24/4 regimen). STUDY DESIGN: In this open-label uncontrolled study conducted in 50 European centers, healthy females aged 18-35 years with a body mass index of less than 30 kg/m(2) received drsp 3 mg/EE 20 mcg 24/4 over 13 cycles. The primary efficacy variable was the number of unintended pregnancies. RESULTS: Five pregnancies occurred among 1101 women over 13,248 treatment cycles, resulting in a Pearl Index (PI) of 0.49 with an upper two-sided 95% CI limit of 1.14. Of these pregnancies, three were attributed to noncompliance with tablet use resulting in an adjusted PI for 'perfect use' of 0.22 (upper limit of two-sided 95% CI: 0.80) based on 11,755 cycles. CONCLUSION: Drospirenone 3 mg/EE 20 mcg 24/4 is a highly effective COC in nonobese women.

PACAP has anti-apoptotic effect in the salivary gland of an invertebrate species, Helix pomatia.

Pituitary adenylate cyclase activating polypeptide (PACAP) shows a remarkable sequence similarity among species and several studies provide evidence that the functions of PACAP have also been conserved among vertebrate species. Relatively little is known about its presence and functions in invertebrates. The aim of the present study was to investigate whether the well-known anti-apoptotic effect of PACAP can also be demonstrated in invertebrates. This effect was studied in the salivary gland of a molluscan species, Helix pomatia. In this work, we first showed the presence of PACAP-like immunoreactivity in the Helix salivary gland by means of immunohistochemistry. Radioimmunoassay measurements showed that PACAP38-like immunoreactivity dominated in the salivary gland of both active and inactive snails and its concentration was higher in active than in inactive animals in contrast to PACAP27-like immunoreactivity, which did not show activity-dependent changes. PACAP induced a significant elevation of cAMP level in salivary gland extracts. Application of apoptosis-inducing agents, dopamine and colchicine, led to a marked increase in the number of terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells in the salivary gland, which was significantly attenuated by PACAP treatment. In a similar manner, the number of caspase-positive cells was reduced after co-application of dopamine and PACAP. Taken together, the data indicate that PACAP activates cAMP in a molluscan species and we show, for the first time, that PACAP is anti-apoptotic in the invertebrate Helix pomatia.

Behavioural and neural deficits induced by rotenone in the pond snail Lymnaea stagnalis. A possible model for Parkinson's disease in an invertebrate.

Parkinson's disease is a neurodegenerative disorder, related to the loss of dopamine (DA)-containing neurons in the substantia nigra. In experimental animals, both vertebrates and invertebrates, rotenone, a commercially available organic pesticide, induces symptoms of Parkinson's disease. We found that that rotenone is toxic to the pond snail Lymnaea stagnalis (4-day LC50 0.8 microM). Rotenone, at concentrations from 0.1 to 5 microM, caused progressive and irreversible behavioural deficits in both acute and chronic exposure. Chronic exposure to 0.5 microM rotenone led to a progressive decrease in spontaneous locomotion and in feeding, reaching almost 100% inhibition of both behaviours by the 7th day of rotenone treatment. In the central nervous system preparation made on the 7th day of treatment the postsynaptic potentials evoked by the identified dopaminergic RPeD1 neuron disappeared whereas the synaptic inputs received by the RPeD1 from a peptidergic neuron (VD4) were still functional. Immunostaining revealed that the tyrosine hydroxylase immunoreactivity decreased below the detectable level in both the RPeD1 cell body and its axonal processes. Finally, HPLC assay showed a significant (25%) decrease in DA level in the CNS by the 7th day of rotenone treatment. We conclude that, as in vertebrates, rotenone disrupts feeding and locomotion of the model mollusc Lymnaea stagnalis. One possible target of rotenone is the dopaminergic neurons in the CNS. We therefore suggest that Lymnaea stagnalis is a suitable invertebrate model for the study of Parkinson's disease, allowing direct analysis of the response of dopaminergic systems to rotenone at behavioural, cellular and neuronal levels.

Neuroanatomical, immunocytochemical, and physiological studies of the pharyngeal retractor muscle and its putative regulatory neurons playing a role in withdrawal and feeding in the snail, Helix pomatia.

We describe the neurons regulating two separate functions of the pharyngeal retractor muscle (PRM), namely sustained contraction during body withdrawal and rhythmic phasic contractions during feeding, in the snail, Helix pomatia. The distribution of central neurons innervating the PRM is organized into two main units; one in the buccal-cerebral ganglion complex, the other in the subesophageal ganglion complex. Serotonin- (5-HT-), FMRFamide- (FMRFa-), and tyrosine-hydroxylase-immunostained neurons are present among the PRM neurons that densely innervate the PRM. 5HT both decreases and increases the amplitude of the electrically evoked contraction between concentrations of 0.1 microM and 1 microM. Dopamine (DA) only decreases the amplitude of contraction at a 1-microM threshold concentration. In contrast, FMRFa increases the amplitude of the contraction and slightly elevates the tone of the PRM but requires a higher threshold (10 microM). Assay by high-performance liquid chromatography of 5HT and DA in the PRM has shown that the 5HT level decreases during locomotion but increases during feeding, whereas the DA level increases during locomotion but slightly decreases during feeding. Thus, different segments of the PRM are innervated by neurons from different loci within the central nervous system. The segments of the PRM distal to the pharynx are innervated from loci of the subesophageal ganglion complex suggesting that they mediate withdrawal. The proximal segment of the PRM is innervated from cerebral and buccal loci indicating that these neurons mediate the feeding rhythm produced by buccal and cerebral feeding central pattern generators to induce rhythmic phasic contractions in the PRM during feeding.

Crustacean cardioactive peptide (CCAP)-related molluscan peptides (M-CCAPs) are potential extrinsic modulators of the buccal feeding network in the pond snail Lymnaea stagnalis.

We combine electrophysiological and immunocytochemical analyses in the snail Lymnaea stagnalis of M-CCAP1 and M-CCAP2, two molluscan peptides with structure similar to crustacean cardioactive peptide CCAP, originally isolated from the snail Helix pomatia. Both M-CCAP peptides (M-CCAP1 and M-CCAP2, 1 microM) had an excitatory effect, depolarizing all the identified neurons of the buccal feeding network (including motoneurons: B1, B2, B4 and modulatory interneurons SO, OC: 62 neurons in 33 preparations). Additionally, in 67% of preparations, rhythmic activity (fictive feeding) was recorded with a mean rate of 7 cycles/min. No significant difference in the proportion of preparations showing fictive feeding or mean feeding rate was found between M-CCAP1 and M-CCAP2. The extrinsic feeding modulator, the serotonergic CGC neuron, responds by increase of the spontaneous activity after M-CCAP application (9 of 18 preparations). Crustacean CCAP (1 microM) evokes a slight membrane depolarization in 3 out of 8 preparations but never evokes fictive feeding. Immunostaining revealed no cell bodies in the buccal ganglia, but a dense network of CCAP immunopositive fibers arborizing in the buccal neuropil. Many of these fibers originate from a symmetrical pair of CCAP-immunoreactive cerebro-buccal interneurons, which are the most likely candidates for extrinsic modulatory interneurons in the buccal feeding network. Our data are the first results suggesting that M-CCAP-peptides exist as effective modulators in mollusc.