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The endocannabinoid system (ECS) plays an important role in neuroprotection, neuroplasticity, energy balance, modulation of stress, and inflammatory responses, acting as a critical link between the brain and the body's peripheral regions, while also offering promising potential for novel therapeutic strategies. Unfortunately, in humans, pharmacological inhibitors of different ECS enzymes have led to mixed results in both preclinical and clinical studies. As the ECS has been highly conserved throughout the eukaryotic lineage, the use of invertebrate model organisms like the pond snail Lymnaea stagnalis may provide a flexible tool to unravel unexplored functions of the ECS at the cellular, synaptic, and behavioral levels. In this study, starting from the available genome and transcriptome of L. stagnalis, we first identified putative transcripts of all ECS enzymes containing an open reading frame. Each predicted protein possessed a high degree of sequence conservation to known orthologues of other invertebrate and vertebrate organisms. Sequences were confirmed by qualitative PCR and sequencing. Then, we investigated the transcriptional effects induced by different stress conditions (i.e., bacterial LPS injection, predator scent, food deprivation, and acute heat shock) on the expression levels of the enzymes of the ECS in Lymnaea's central ring ganglia. Our results suggest that in Lymnaea as in rodents, the ECS is involved in mediating inflammatory and anxiety-like responses, promoting energy balance, and responding to acute stressors. To our knowledge, this study offers the most comprehensive analysis so far of the ECS in an invertebrate model organism., (© 2024 International Society for Neurochemistry.)

Dopa decarboxylase (DDC) protein is involved in the synthesis of dopamine and serotonin. Here, we show that in the silkworm Bombyx mori, a novel DDC splicing variant is selectively expressed in the brain and subesophageal ganglia. In Drosophila melanogaster, a neuron-specific isoform of DDC is known to be alternatively spliced in a similar manner.

In decapod crustaceans, molting is controlled by the pulsatile release of molt-inhibiting hormone (MIH) from neurosecretory cells in the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). A drop in MIH release triggers molting by activating the molting gland or Y-organ (YO). Post-transcriptional mechanisms ultimately control MIH levels in the hemolymph. Neurotransmitter-mediated electrical activity controls Ca 2+ -dependent vesicular release of MIH from the SG axon terminals, which may be modulated by nitric oxide (NO). In green shore crab, Carcinus maenas, nitric oxide synthase (NOS) protein and NO are present in the SG. Moreover, C. maenas are refractory to eyestalk ablation (ESA), suggesting other regions of the nervous system secrete sufficient amounts of MIH to prevent molting. By contrast, ESA induces molting in the blackback land crab, Gecarcinus lateralis. Double-label immunofluorescence microscopy and quantitative polymerase chain reaction were used to localize and quantify MIH and NOS proteins and transcripts, respectively, in the ESG, brain, and thoracic ganglion (TG) of C. maenas and G. lateralis. In ESG, MIH- and NOS-immunopositive cells were closely associated in the SG of both species; confocal microscopy showed that NOS was localized in cells adjacent to MIH-positive axon terminals. In brain, MIH-positive cells were located in a small number of cells in the olfactory lobe; no NOS immunofluorescence was detected. In TG, MIH and NOS were localized in cell clusters between the segmental nerves. In G. lateralis, Gl-MIH and Gl-crustacean hyperglycemic hormone (CHH) mRNA levels were ~10 5 -fold higher in ESG than in brain or TG of intermolt animals, indicating that the ESG is the primary source of these neuropeptides. Gl-NOS and Gl-elongation factor (EF2) mRNA levels were also higher in the ESG. Molt stage had little or no effect on CHH, NOS, NOS-interacting protein (NOS-IP), membrane Guanylyl Cyclase-II (GC-II), and NO-independent GC-III expression in the ESG of both species. By contrast, MIH and NO receptor GC-I beta subunit (GC-Iβ) transcripts were increased during premolt and postmolt stages in G. lateralis, but not in C. maenas. MIH immunopositive cells in the brain and TG may be a secondary source of MIH; the release of MIH from these sources may contribute to the difference between the two species in response to ESA. The MIH-immunopositive cells in the TG may be the source of an MIH-like factor that mediates molt inhibition by limb bud autotomy. The association of MIH- and NOS-labeled cells in the ESG and TG suggests that NO may modulate MIH release. A model is proposed in which NO-dependent activation of GC-I inhibits Ca 2+ -dependent fusion of MIH vesicles with the nerve terminal membrane; the resulting decrease in MIH activates the YO and the animal enters premolt., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Planorbid snails of the genus Biomphalaria are major intermediate hosts for the digenetic trematode parasite Schistosoma mansoni. Evidence suggests that levels of the neurotransmitter dopamine (DA) are reduced during the course of S. mansoni multiplication and transformation within the snail. This investigation used immunohistochemical methods to localize tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, in the nervous system of Biomphalaria. The two species examined, Biomphalaria glabrata and Biomphalaria alexandrina, are the major intermediate hosts for S. mansoni in sub-Saharan Africa, where more than 90% of global cases of human intestinal schistosomiasis occur. TH-like immunoreactive (THli) neurons were distributed throughout the central nervous system (CNS) and labeled fibers were present in all commissures, connectives, and nerves. Some asymmetries were observed, including a large distinctive neuron (LPeD1) in the pedal ganglion described previously in several pulmonates. The majority of TH-like immunoreactive neurons were detected in the peripheral nervous system (PNS), especially in lip and foot regions of the anterior integument. Independent observations supporting the dopaminergic phenotype of THli neurons included 1) block of LPeD1 synaptic signaling by the D2/3 antagonist sulpiride, and 2) the similar localization of aqueous aldehyde (FaGlu)-induced fluorescence. The distribution of THli neurons indicates that, as in other gastropods, dopamine functions as a sensory neurotransmitter and in the regulation of feeding and reproductive behaviors in Biomphalaria. It is hypothesized that infection could stimulate transmitter release from dopaminergic sensory neurons and that dopaminergic signaling could contribute to modifications of both host and parasite behavior., (© 2014 Wiley Periodicals, Inc.)

Using NADPH-diaphorase (NADPH-d) histochemistry, inducible nitric oxide synthase (iNOS)-immunohistochemistry and immunoblotting, we characterized the nitric oxide (NO)-producing neurons in the brain and thoracic ganglion of a shore crab subjected to a nociceptive chemical stimulus. Formalin injection into the cheliped evoked specific nociceptive behavior and neurochemical responses in the brain and thoracic ganglion of experimental animals. Within 5-10 min of injury, the NADPH-d activity increased mainly in the neuropils of the olfactory lobes and the lateral antenna I neuropil on the side of injury. Later, the noxious-induced expression of NADPH-d and iNOS was detected in neurons of the brain, as well as in segmental motoneurons and interneurons of the thoracic ganglion. Western blotting analysis showed that an iNOS antiserum recognized a band at 120 kDa, in agreement with the expected molecular mass of the protein. The increase in nitrergic activity induced by nociceptive stimulation suggests that the NO signaling system may modulate nociceptive behavior in crabs.

The spontaneously active Br neuron from the brain-subesophageal ganglion complex of the garden snail Helix pomatia rhythmically generates regular bursts of action potentials with quiescent intervals accompanied by slow oscillations of membrane potential. We examined the involvement of the Na(+)/K(+) pump in modulating its bursting activity by applying a static magnetic field. Whole snail brains and Br neuron were exposed to the 10-mT static magnetic field for 15 min. Biochemical data showed that Na(+)/K(+)-ATPase activity increased almost twofold after exposure of snail brains to the static magnetic field. Similarly, (31)P NMR data revealed a trend of increasing ATP consumption and increase in intracellular pH mediated by the Na(+)/H(+) exchanger in snail brains exposed to the static magnetic field. Importantly, current clamp recordings from the Br neuron confirmed the increase in activity of the Na(+)/K(+) pump after exposure to the static magnetic field, as the magnitude of ouabain's effect measured on the membrane resting potential, action potential, and interspike interval duration was higher in neurons exposed to the magnetic field. Metabolic pathways through which the magnetic field influenced the Na(+)/K(+) pump could involve phosphorylation and dephosphorylation, as blocking these processes abolished the effect of the static magnetic field.

In our study we investigated the influence of dopamine (DA) on the caudal photoreceptor (CPR) in crayfish. Here we report the following: (a) the chromatographic determination of DA in the sixth abdominal ganglion (6th AG) shows a variation in the content during a 24-h cycle with the maximum value at dawn. (b) There are possibly dopaminergic neurons in the 6th AG with antityrosine hydroxylase antibodies. Immunopositive neurons (164) were located in the anterior and posterior regions of the 6th AG with the mean (± SE) diameter of their somata 23 ± 1 μm. In addition, there is immunopositive staining in axons, neuropilar fibers, and varicosities. (c) We also identified, using immunohistochemistry, 108 neurons in the sixth AG that contain dopamine D1-like receptors, with the mean (±SE) diameter of their somata 18 ± 1 μm. (d) We examined the exogenous action of DA on the electrical activity of the CPR in the isolated sixth AG by conventional extracellular-recording methods. This CPR displays spontaneous activity and phasic-tonic responses to light pulses. Topical application of dopamine to ganglia kept in the dark increased the spontaneous firing rate of the CPR, whereas the photoresponse of the CPR remained unchanged. The effect on the spontaneous activity is dose-dependent with an ED₅₀ of 33 μM, and is blocked by the dopamine D1-like antagonist SCH23390. These observations suggested that the DA is playing the role of a neurotransmitter or a neuromodulator of the CPR in the 6th AG in both species of crayfish, Procambarus clarkii and Cherax quadricarinatus., (Copyright © 2010 Wiley-Liss, Inc.)

Using immunocytochemistry combined with light and electron microscopy, the distribution and ultrastructure of tyrosine hydroxylase (TH)-immunoreactive neurons in the CNS of bivalve mollusc, Megangulus venulosus, have been studied under the influence of increased temperature and hypoxia. It has been established, that the stress causes changes in the amount of TH and in the structure of TH-immunopositive neurons in all ganglia. The most essential changes in CNS of M. venulosus were revealed after 60 min exposure to increased temperature and hypoxia; degenerative changes in large neurons, reduction of the synapses and reduction of TH-immunoreactivity in neurons and neuropil.

Neuronal nitric oxide (NO) levels are modulated through the control of catalytic activity of NO synthase (NOS). Although signals limiting excess NO synthesis are being extensively studied in the vertebrate nervous system, our knowledge is rather limited on the control of NOS in neurons of invertebrates. We have previously reported a transient inactivation of NOS in hibernating snails. In the present study, we aimed to understand the mechanism leading to blocked NO production during hypothermic periods of Helix pomatia. We have found that hypothermic challenge translocated NOS from the cytosol to the perinuclear endoplasmic reticulum, and that this cytosol to membrane trafficking was essential for inhibition of NO synthesis. Cold stress also downregulated NOS mRNA levels in snail neurons, although the amount of NOS protein remained unaffected in response to hypothermia. Our studies with cultured neurons and glia cells revealed that glia-neuron signaling may inhibit membrane binding and inactivation of NOS. We provide evidence that hypothermia keeps NO synthesis "hibernated" through subcellular redistribution of NOS.

Nitric oxide (NO) is a gas produced through the action of nitric oxide synthase that acts as a neurotransmitter in the central nervous system (CNS) of adult gastropod mollusks. There are no known reports of the presence of NOS-containing neurons and glial cells in young and adult Megalobulimus abbreviatus. Therefore, NADPH-d histochemistry was employed to map the nitrergic distribution in the CNS of young and adult snails in an attempt to identify any transient enzymatic activity in the developing CNS. Reaction was observed in neurons and fibers in all CNS ganglia of both age groups, but in the pedal and cerebral ganglia, positive neurons were more intense than in other ganglia, forming clusters symmetrically located in both paired ganglia. However, neuronal NADPH-d activity in the mesocerebrum and pleural ganglia decreased from young to adult animals. In both age groups, positive glial cells were located beneath the ganglionic capsule, forming a network and surrounding the neuronal somata. The trophospongium of large and giant neurons was only visualized in young animals. Our results indicate the presence of a nitrergic signaling system in young and adult M. abbreviatus, and the probable involvement of glial cells in NO production., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The MAPK/ERK pathway plays an important role in the regulation of gene expression during memory formation both in vertebrates and invertebrates. In the mollusk Helix lucorum, serotonin induces activation of MAPK/ERK in the central nervous system (CNS) upon food aversion learning. Such learning depends on a neuronal network in which specialized neurons play distinct roles so that they may exhibit different activation levels of the MAPK/ERK pathway. Here we performed a comparative analysis of MAPK/ERK activation in single neurons of the food-aversion network, focusing both on command neurons, which mediate withdrawal behavior and process information pertaining to the unconditioned stimulus, and on neurons of the procerebrum, the mollusk's olfactory center, which process information from the conditioned stimulus. By means of Western blots designed to detect micro amounts of proteins, we determined MAPK/ERK activation in these neurons and found that after food aversion learning phospho-ERK levels increased significantly in RPa(2/3) command neurons of the right parietal ganglia and in the procerebrum. Such an increase was prevented by injection of PD98095, an inhibitor of the ERK upstream kinase (MEK-1). In contrast, no activation of MAPK/ERK was detected in similar conditions in the corresponding neurons of the left parietal ganglia LPa(2/3). This asymmetry was verified after serotonin application to the CNS in order to mimic learning. Our results thus show that learning involves synchronous and asymmetric serotonin-dependent MAPK/ERK activation. Such an asymmetry may reflect lateralization of memory processes in the mollusk brain., (Copyright 2010 Elsevier Inc. All rights reserved.)

In this review a comparative analysis is performed of enzymological characteristics of cholinesterase (ChE) from optic ganglia of individuals of the Commander squid Berryteuthis magister caught in 8 zones of its habitation areal in the northern-western Pacific aquatorium, of ChE of the Pacific squid Todarodes pacificus as well as of the "standard" acetylcholinesterase from human erythrocytes and butyrylcholinesterase from horse blood serum. By the method of the substrate-inhibitor analysis there was shown heterogeneity of ChE preparations from the B. magister individuals from different habitation zones. Kinetic parameters of the enzymatic hydrolysis of 8 ester substrates are presented as well as the data on study of inhibitory specificity with use of 20 irreversible organophosphorus inhibitors, which show identity of ChE properties in the B. magister individuals from different habitation zones. Study of the process of the ChE reversible inhibition from the Commander squid individuals under action of 57 mono- and bisonium inhibitors has revealed differences in ChE properties of squid individuals from isolates in different zones of the habitation areal, which argues in favor of the existence of intraspecies groups of the Commander squid B. magister.

Nitric oxide (NO) is an evolutionarily conserved mediator of neural plasticity. Because NO is highly diffusible, signals from multiple sources might combine in space and time to affect the same target. Whether such cooperative effects occur will depend on the effective signaling range and on the distances of NO sources to one another and to their targets. These anatomical parameters have been quantified in only few systems. We analyzed the 3D architecture of NO synthase (NOS) expression in a sensory neuropil, the ventral association center (VAC) of the locust. High-resolution confocal microscopy revealed NOS immunoreactive fiber boutons in submicrometer proximity to both the axon terminals of sensory neurons and their postsynaptic target, interneuron A4I1. Pharmacological manipulation of NO signaling affected the response of A4I1 to individual wind-puff stimuli and the response decrement during repetitive stimulation. Mapping NOS immunoreactivity in defined volumes around dendrites of A4I1 revealed NOS-positive fiber boutons within 5 mum of nearly every surface point. The mean distances between neighboring NOS-boutons and between any point within the VAC and its nearest NOS-bouton were likewise about 5 mum. For an NO signal to convey the identity of its source, the effective signaling range would therefore have to be less than 5 mum, and shorter still when multiple boutons release NO simultaneously. The architecture is therefore well suited to support the cooperative generation of volume signals by interaction between the signals from multiple active boutons., ((c) 2010 Wiley-Liss, Inc.)

Comparative study of substrate specificity of monoamine oxidase (MAO) of optic ganglia of the Pacific squid Todarodes pacificus and the Commander squid Berryteuthis magister has been carried out. The enzyme of the Pacific squid, unlike that of the Commander squid, has been established to be able to deaminate not only tyramine, tryptamine, serotonin, benzylamine, and beta-phenylethylamine, but also histamine--substrate of diamine oxidase (DAO). In relation to all studied substrates, the MAO activity of optic ganglia of T. pacificus is several times higher as compared with B. magister. In the case of deamination of serotonin this difference was the greatest and amounted to 5 times. Semicarbazide, the classic DAO inhibitor, at a concentration of 10 mM did not inhibit catalytic activity of both studied enzymes. The substrate-inhibitory analysis with use of deprenyl and chlorogiline, specific inhibitors of different MAO forms, indicates homogeneity of the enzyme of the Pacific squid and heterogeneity of the Commander squid enzyme whose composition seems probably to contain at least two MAO forms. There are obtained quantitative differences in substrate specificity and reaction capability with respect to the inhibitors chlorgiline and deprenyl for MAO of optic ganglia of the studied squid species. These differences probably can be explained by significant differences in the evolutionary level of these biological species.

Summarized are results of the 40-year studies of the Russian biochemists on the comparative-enzymological characteristics of cholinesterase of optic ganglia of the Pacific squid Todarodes pacificus. The review includes the comparative evaluation of the cholinesterase activity of various hydrobiont tissues, the proof of enzymatic homogeneity of the tissue of the Pacific squid optic ganglia, data on substrate specificity with study of 18 ester substrates as well as detailed study of inhibitory specificity (61 irreversible inhibitors and 49 reversible onium inhibitors). Peculiarity of properties of this enzyme as compared with vertebrate and invertebrate cholinesterases is shown.

Expression, localization and regulation of different cAMP-dependent protein kinase A (PKA) subunits account for specificity in the intracellular cAMP/PKA signaling pathway. In Aplysia neurons, two classes of PKA (I and II) differing in their regulatory (R) subunits have been characterized. Type I is mostly soluble in the cell body, and type II enriched at the synaptic endings. Although both types are necessary for long-term changes in synaptic plasticity, their differences in cellular localization and expression suggest that they mediate distinct functions. By photoaffinity labeling studies, we previously observed a cAMP-binding 105 kDa band in extracts from Aplysia neurons as a putative third class of R subunit of PKA. Here, we have determined that the 105 kDa band is a high molecular weight complex (HMWC) containing alpha/beta-tubulin and PKA RI, but not RII. This hetero-complex is conserved in vertebrates since mouse brain extracts also contain it. The enrichment of the endogenous HMWC by subcellular fractionation and its synthesis in vitro indicate that it is mainly produced in the cytosol, and then transported to the synapses. The HMWC is functional as a cAMP-sensitive regulatory subunit of PKA since it binds catalytic subunit in the absence of cAMP. Furthermore, serotonin (5-HT) treatment, which produces long-term facilitation in neurons, induced its degradation. In mouse brain RI co-localized with tubulin in neuropils and in COS-7 cells discretely at the cell membrane. These observations suggest that the alpha/beta-tubulin anchoring type I PKA may have an important role in the formation of long-term synaptic plasticity.

The cAMP-dependent protein kinase (PKA) is known to play a critical role in both transcription-independent short-term or intermediate-term memory and transcription-dependent long-term memory (LTM). Although distinct phases of LTM already have been demonstrated in some systems, it is not known whether these phases require distinct temporal patterns of learning-induced PKA activation. This question was addressed in a robust form of associative LTM that emerges within a matter of hours after single-trial food-reward classical conditioning in the pond snail Lymnaea stagnalis. After establishing the molecular and functional identity of the PKA catalytic subunit in the Lymnaea nervous system, we used a combination of PKA activity measurement and inhibition techniques to investigate its role in LTM in intact animals. PKA activity in ganglia involved in single-trial learning showed a short latency but prolonged increase after classical conditioning. However, while increased PKA activity immediately after training (0-10 min) was essential for an early phase of LTM (6 h), the late phase of LTM (24 h) required a prolonged increase in PKA activity. These observations indicate mechanistically different roles for PKA in recent and more remote phases of LTM, which may underpin different cellular and molecular mechanisms required for these phases.

NADPH-diaphorase (NADPH-d) is a histochemical marker for nitric oxide synthase (NOS) and is widely used to identify nitric oxide (NO) producing cells in the central nervous system (CNS) of both vertebrates and invertebrates. NADPH-d histochemistry was used to quantitatively characterize putative NO-producing neurons in the CNS of the Gray mussel Crenomytilus grayanus subjected to two kinds of stress, environmental pollution and hypoxia, the latter caused by the mollusk transportation in a small volume of water. Mussels were sampled from one relatively clean (reference) and four polluted sites in Amursky and Ussuriysky Bays (Peter the Great Bay, Sea of Japan) in August, 2003. The number of NADPH-d-positive neurons was estimated and enzyme activity was determined from the optical density of the formazan precipitate in the CNS ganglia at 0, 3, and 72 h after sampling. Just after sampling, NADPH-d-positive neurons were found in the cerebropleural, visceral, and pedal ganglia. The number and staining intensity of NADPH-d-positive neurons were significantly higher in the pedal ganglia than the other two ganglia. There were significant differences in the number of NADPH-d-positive neurons and enzyme activity between the mussels from the reference and heavily polluted stations. The proportion and staining intensity of NADPH-d-positive neurons were maximum in the pedal ganglia of the mussels from the heavily polluted station in Amursky Bay. Transportation of mussels in a limited volume of water for 3h resulted in a significant increase in the proportion and staining intensity of NADPH-d-positive neurons in all ganglia. In mollusks from all stations kept in aerated aquaria for 72 h, both the proportion and staining intensity of NADPH-d-positive neurons did not differ significantly from the initial level. However, the differences in the proportion and staining intensity of NADPH-d-positive neurons between the reference and heavily polluted stations were significant. The present results suggest that NO is involved in mollusk nerve cell adaptation to environmental changes.

Calmodulin and Abelson tyrosine kinase are key signaling molecules transducing guidance cues at the Drosophila embryonic midline. A reduction in the signaling strength of either pathway alone induces ectopic midline crossing errors in a few segments. When Calmodulin and Abelson signaling levels are simultaneously reduced, the frequency of ectopic crossovers is synergistically enhanced as all segments exhibit crossing errors. But as the level of signaling is further reduced, commissures begin to fuse and large gaps form in the longitudinal connectives. Quantitative analysis suggests that the level of Abelson activity is particularly important. Like Calmodulin, Abelson interacts with son-of-sevenless to increase ectopic crossovers suggesting all three contribute to midline repulsive signaling. Axons cross the midline in almost every segment if Frazzled is co-overexpressed with the Calmodulin inhibitor, but the crossovers induced by the Calmodulin inhibitor itself do not require endogenous Frazzled. Thus, Calmodulin and Abelson tyrosine kinase are key signaling molecules working synergistically to transduce both midline attractive and repulsive cues. While they may function downstream of specific receptors, the emergence of commissural and longitudinal connective defects point to a novel convergence of Calmodulin and Abelson signaling during the regulation of actin and myosin dynamics underlying a guidance decision.

One characteristic of organophosphate poisoning is the ability to increase excitability or induce epileptiform activity in nerve cells, but underlying mechanisms are not fully understood. We have previously reported that paraoxon, an organophosphate compound, at submicromolar concentrations effectively suppress Ca(2+) spikes and modulate the activity of snail neurons. This effect was unrelated to acetylcholinesterase (AChE) inhibition but was found to involve the direct or indirect modulation of ion channels [Vatanparast J, Janahmadi M, Asgari AR, Sepehri H, Haeri-Rohani A. Paraoxon suppresses Ca(2+) spike and afterhyperpolarization in snail neurons: relevance to the hyperexcitability induction. Brain Res 2006a;1083(1):110-7]. In the present study, the interaction of paraoxon with cAMP formation on the modulation of Ca(2+) spikes and neuronal excitability was examined. Forskolin, the activators of adenylate cyclase, suppressed afterhyperpolarization (AHP) and increased the activity of snail neurons without any significant effect on the Ca(2+) spike duration. Pretreatment with forskolin, although attenuated the suppressing effect of paraoxon on the duration of Ca(2+) spikes but also potentiated the paraoxon-induced hyperexcitability by enhancing the suppressive effects of paraoxon on AHP. Our findings support the possible involvement of cAMP formation in the paraoxon-induced AHP suppression and neuronal hyperexcitability, although activation of cAMP pathway may attenuates some effects of paraoxon.

Target-dependent increases in axon growth and varicosities accompany the formation of functional synapses between Aplysia sensory neurons and specific postsynaptic neurons (L7 and not L11). The enhanced growth is regulated in part by a target-dependent increase in the secretion of sensorin, the sensory neuron neuropeptide. We report here that protein kinase C (PKC) activity is required for synapse formation by sensory neurons with L7 and for the target-dependent increases in sensorin synthesis and secretion. Blocking PKC activity reversibly blocked synapse formation and axon growth of sensory neurons contacting L7, but did not affect axon growth of sensory neurons contacting L11 or axon growth of the postsynaptic targets. Blocking PKC activity also blocked the target-induced increase in sensorin synthesis and secretion. Sensorin then activates additional signaling pathways required for synapse maturation and synapse-associated growth. Exogenous anti-sensorin antibody blocked target-induced activation and translocation into sensory neuron nuclei of p42/44 mitogen-activated protein kinase (MAPK), attenuated synapse maturation, and curtailed growth of sensory neurons contacting L7, but not the growth of sensory neurons contacting L11. Inhibitors of MAPK or phosphoinositide 3-kinase also attenuated synapse maturation and curtailed growth and varicosity formation of sensory neurons contacting L7, but not growth of sensory neurons contacting L11. These results suggest that PKC activity regulated by specific cell-cell interactions initiates the formation of specific synapses and the subsequent synthesis and release of a neuropeptide to activate additional signaling pathways required for synapse maturation.

Western blot analysis was used to study the activation of MAP/ERK protein kinases responsible for controlling gene expression via phosphorylation of transcription factors CREB and ELK-1 in native common snails and animals with impaired abilities to form long-term types of conditioned aversive reflexes. Different periods of the formation of this reflex were found to be characterized by different levels of activation of MAP-ERK kinases. The extents of activation of MAP-ERK kinase cascade were different in ganglia (parietal-visceral, cerebral, and pedal) with different roles in the formation of this reflex. The dynamics of activation showed a wavelike nature, with peaks at 10 min and 4 h. Administration of the neurotoxin 5,7-DHT, which induces dysfunction of serotonin terminals and decreases the ability to acquire this type of learning, led to significant decreases in activation of the MAP-ERK kinase cascade at the early stages of learning, which is evidence for an important role for the serotoninergic system in inducing this cascade. Activation of the MAP/ERK kinase cascade 4 h after training was seen both in native and DHT-treated animals, which is probably evidence for activation of non-specific adaptive processes in response to the sensitizing unconditioned stimulus. Thus, the MAP/ERK kinase intracellular regulatory cascade, which plays an important role in the survival of neurons, the regeneration of neuron processes, and synaptic sprouting, also plays an important role in forming the serotonin-dependent food-aversive reflex in the common snail.

The Drosophila genes ninaB and ninaD, encoding a beta-carotene oxygenase and a type B scavenger receptor respectively, are essential for the biosynthesis of the 3-hydroxyretinal chromophore of rhodopsin. We analyzed transgenic reporter strains and performed in situ hybridization to show that both ninaB and ninaD are expressed in the adult brain but not retinal tissues. Developmental RT-PCR and tissue expression studies showed that ninaB is only expressed in the adult brain, while ninaD is expressed in the adult brain, the adult body, and many larval tissues. The data support a model in which NinaD is required for uptake and storage of dietary carotenoids throughout the larval and adult stages of development. Beta-carotene is transported to the adult brain, where cellular uptake by NinaD allows cleavage by the NinaB enzyme to produce retinal. Retinal is then transported to the retina for rhodopsin biogenesis.

The biochemical characterization of nitric oxide synthase (NOS) and its distribution in the central nervous system (CNS) were studied in the heteropteran bug Triatoma infestans. NOS-like immunoreactivity was found in the brain, subesophageal ganglion, and thoracic ganglia by using immunocytochemistry. In the protocerebrum, NOS-immunoreactive (IR) somata were detected in the anterior, lateral, and posterior soma rinds. In the optic lobe, numerous immunostained somata were observed at the level of the first optic chiasma, around the lobula, and in the proximal optic lobe. In the deutocerebrum, NOS-IR perikarya were mainly observed in the lateral soma rind, surrounding the sensory glomeruli, and a few cell bodies were seen in association with the antennal mechanosensory and motor neuropil. No immunostaining could be detected in the antennal nerve. The subesophageal and prothoracic ganglia contained scattered immunostained cell bodies. NOS-IR somata were present in all the neuromeres of the posterior ganglion. Western blotting showed that a universal NOS antiserum recognized a band at 134 kDa, in agreement with the expected molecular weight of the protein. Analysis of the kinetics of nitric oxide production revealed a fully active enzyme in tissue samples of the CNS of T. infestans.

Nitric oxide (NO) is a diffusible signaling molecule with evolutionarily conserved roles in neural plasticity. Prominent expression of NO synthase (NOS) in the primary olfactory centers of mammals and insects lead to the notion of a special role for NO in olfaction. In insects, however, NOS is also strongly expressed in non-olfactory chemo-tactile centers of the thoracic nerve cord. The functional significance of this apparent association with various sensory centers is unclear, as is the extent to which it occurs in other arthropods. We therefore investigated the expression of NOS in the pereopod ganglia of crayfish (Pacifastacus lenisculus and Procambarus clarkii). Conventional NADPH diaphorase (NADPHd) staining after formaldehyde fixation gave poor anatomic detail, whereas fixation in methanol/formalin (MF-NADPHd) resulted in Golgi-like staining, which was supported by immunohistochemistry using NOS antibodies that recognize a 135-kDa protein in crayfish. MF-NADPHd revealed an exceedingly dense innervation of the chemo-tactile centers. As in insects, this innervation was provided by a system of prominent intersegmental neurons. Superimposed on a putatively conserved architecture, however, were pronounced segmental differences. Strong expression occurred only in the anterior three pereopod ganglia, correlating with the presence of claws on pereopods one to three. These clawed pereopods, in addition to their role in locomotion, are crucially involved in feeding, where they serve both sensory and motor functions. Our findings indicate that strong expression of NOS is not a universal feature of primary sensory centers but instead may subserve a specific requirement for sensory plasticity that arises only in particular behavioral contexts., (2007 Wiley-Liss, Inc.)

Analysis of mechanism of reversible inhibition of human erythrocyte acetylcholinesterase (AChE), of horse serum cholinesterase (ChE), and ChE of optical ganglia tissue of individuals of the Commander squid Berryleuthis magister from various habitat zones was studied under effect thionphosphonates (P=S), derivatives of piperidine, morpholine, perhydroazepine as well as several heterocyclic model compounds. Data of comparative inhibitory specificity have allowed us to suggest that thionphosphonates are sorbed in the area of cholinesterase esterase center at the expense of phosphoryl part of the inhibitor molecule, rather than of its heterocyclic grouping. An advantage in the antienzyme efficiency of thionphosphonates (P=S) over phosphonates (P=O) is revealed. The ion strength effect is used for analysis of contribution of the hydrophobichydrophilic interaction in the enzyme-inhibitor system.

The circadian clock modulates the induction of long-term sensitization (LTS) in Aplysia such that long-term memory formation is significantly suppressed when animals are trained at night. We investigated whether the circadian clock modulated core molecular processes necessary for memory formation in vivo by analyzing circadian regulation of basal and LTS-induced levels of phosphorylated mitogen-activated protein kinase (P-MAPK) and Aplysia CCAAT/enhancer binding protein (ApC/EBP). No basal circadian regulation occurred for P-MAPK or total MAPK in pleural ganglia. In contrast, the circadian clock regulated basal levels of ApC/EBP protein with peak levels at night, antiphase to the rhythm in LTS. Importantly, LTS training during the (subjective) day produced greater increases in P-MAPK and ApC/EBP than training at night. Thus, circadian modulation of LTS occurs, at least in part, by suppressing changes in key proteins at night. Rescue of long-term memory formation at night required both facilitation of MAPK and transcription in conjunction with LTS training, confirming that the circadian clock at night actively suppresses MAPK activation and transcription involved in memory formation. The circadian clock appears to modulate LTS at multiple levels. 5-HT levels are increased more when animals receive LTS training during the (subjective) day compared with the night, suggesting circadian modulation of 5-HT release. Circadian modulation also occurred downstream of 5-HT release because animals treated with 5-HT to induce LTS exhibited significantly greater LTS when treated during the (subjective) day compared with the night. Together, our studies suggest that the circadian clock modulates LTS at multiple steps and locations during the formation of long-term memory.

A sonication method for the homogeneous extraction of acetylcholinesterase (AChE) from the synganglia of adult ticks is described. The method provides for the extraction of sufficient AChE for multiple assays of enzyme activity in the presence of specific organophosphate (OP) inhibitors for the rapid diagnosis of OP-insensitivity and assignment of homozygous susceptible (SS), heterozygous resistant (RS), and homozygous resistant (RR) genotypes to individual ticks. A single synganglion from adult ticks of either gender and various stages of feeding can successfully be used for AChE extraction. The study presents the results of diagnostic screening of four Boophilus microplus strains for OP-insensitivity. The extraction method and these findings should find utility in support of researchers involved in the mitigation of acaricide resistance in tick populations worldwide, and in particular, the Cattle Fever Tick Surveillance and Quarantine Program maintained by USDA-APHIS/Veterinary Services along the south Texas border that prevents reentry of Boophilus spp. into the United States from endemic populations in Mexico.

Background: The ability of animals to make morphine has been in question for the last 30 years. Studies have demonstrated that animals do contain morphine precursors and metabolites, as well as the ability to use some morphine precursors to make morphine., Material/methods: The present study uses excised ganglia from the marine invertebrate Mytilus edulis as well as whole animals. Morphine and dopamine levels were determined by high performance liquid chromatography coupled to electrochemical detection and radioimmunoassay. Tissues and whole animals were also exposed to morphine precursors and exposed to the CYP2D6 inhibitor quinidine and the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT). Additionally, via RT-PCR, a cDNA fragment of the CYP2D6 enzyme in the ganglia of M. edulis was identified., Results: Pedal ganglia incubated with either tyramine or tyrosine, or whole animals receiving injections, exhibited a statistically significant concentration- and time-dependent increase in their endogenous morphine and dopamine levels (2.51 +/- 0.76 ng/g for tyrosine and 2.39 +/- 0.64 ng/g for tyramine compared to approximately 1.0 ng/g morphine wet weight). Incubation with quinidine and/or AMPT diminished ganglionic morphine and dopamine synthesis at various steps in the synthesis process. We also demonstrated that CYP2D6 mediates the tyramine to dopamine step in this process, as did tyrosine hydroxylase in the step from tyrosine to L-DOPA. Furthermore, via RT-PCR, we identified a cDNA fragment of the CYP2D6 enzyme in the ganglia, which exhibits 94% sequence identity with its human counterpart. Evidence that tyrosine and tyramine were, in part, being converted to dopamine then morphine, and that this process can be inhibited by altering either or both CYP2D6 or tyrosine hydroxylase, is also provided., Conclusions: It appears that animals have the ability to make morphine. This process also appears to be dynamic in that the inhibition of one pathway allows the other to continue with morphine synthesis. Moreover, dopamine and morphine synthesis were coupled.

The role of auditory circuitry is to decipher relevant information from acoustic signals. Acoustic parameters used by different insect species vary widely. All these auditory systems, however, share a common transducer: tympanal organs as well as the Drosophila flagellar ears use chordotonal organs as the auditory mechanoreceptors. We here describe the central neural projections of the Drosophila Johnston's organ (JO). These neurons, which represent the antennal auditory organ, terminate in the antennomechanosensory center. To ensure correct identification of these terminals we made use of a beta-galactosidase-expressing transgene that labels JO neurons specifically. Analysis of these projection pathways shows that parallel JO fibers display extensive contacts, including putative gap junctions. We find that the synaptic boutons show both chemical synaptic structures as well as putative gap junctions, indicating mixed synapses, and belong largely to the divergent type, with multiple small postsynaptic processes. The ultrastructure of JO fibers and synapses may indicate an ability to process temporally discretized acoustic information., ((c) 2005 Wiley-Liss, Inc.)

Although an important role for the mitogen-activated protein kinase (MAPK) has been established for memory consolidation in a variety of learning paradigms, it is not known if this pathway is also involved in appetitive classical conditioning. We address this question by using a single-trial food-reward conditioning paradigm in the freshwater snail Lymnaea stagnalis. This learning paradigm induces protein synthesis-dependent long-term memory formation. Inhibition of MAPK phosphorylation blocked long-term memory consolidation without affecting the sensory and motor abilities of the snails. Thirty minutes after conditioning, levels of MAPK phosphorylation were increased in extracts from the buccal and cerebral ganglia. These ganglia are involved in the generation, modulation, and plasticity of the feeding behavior. We also detected an increase in levels of MAPK phosphorylation in the peripheral tissue around the mouth of the snails where chemoreceptors are located. Although an increase in MAPK phosphorylation was shown to be essential for food-reward conditioning, it was also detected in snails that were exposed to the conditioned stimulus (CS) or the unconditioned stimulus (US) alone, suggesting that phosphorylation of MAPK is necessary but not sufficient for learning to occur.

In a number of neuronal models of learning, signaling by the neurotransmitter nitric oxide (NO), synthesized by the enzyme neuronal NO synthase (nNOS), is essential for the formation of long-term memory (LTM). Using the molluscan model system Lymnaea, we investigate here whether LTM formation is associated with specific changes in the activity of members of the NOS gene family: Lym-nNOS1, Lym-nNOS2, and the antisense RNA-producing pseudogene (anti-NOS). We show that expression of the Lym-nNOS1 gene is transiently upregulated in cerebral ganglia after conditioning. The activation of the gene is precisely timed and occurs at the end of a critical period during which NO is required for memory consolidation. Moreover, we demonstrate that this induction of the Lym-nNOS1 gene is targeted to an identified modulatory neuron called the cerebral giant cell (CGC). This neuron gates the conditioned feeding response and is an essential part of the neural network involved in LTM formation. We also show that the expression of the anti-NOS gene, which functions as a negative regulator of nNOS expression, is downregulated in the CGC by training at 4 h after conditioning, during the critical period of NO requirement. This appears to be the first report of the timed and targeted differential regulation of the activity of a group of related genes involved in the production of a neurotransmitter that is necessary for learning, measured in an identified neuron of known function. We also provide the first example of the behavioral regulation of a pseudogene.

We have isolated and characterized the cDNAs for nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) from the terrestrial slug Limax marginatus, and examined the presence and distribution of their mRNAs in the central nervous system using histological techniques and a reverse transcription-polymerase chain reaction method. Our results showed that both bursting and nonbursting neurons in the procerebral lobes contain the mRNAs for both NOS and sGC. We further found that the oscillation frequency of electrical activity in the procerebral lobes increases with increasing intracellular concentrations of cyclic GMP (cGMP). Taken together with previous data on the NO-induced cGMP-like immunoreactivity and on the anatomical distribution of neurites and the localization of synapses of bursting and nonbursting neurons, our present results suggest that NO-induced changes in cGMP concentration modulate the oscillation frequency in the procerebral lobes by acting on the olfactory input pathways, but possibly not on the output pathways, in slugs. ., ((c) 2004 Wiley Periodicals, Inc)

Recent studies of long-term synaptic plasticity and long-term memory have demonstrated that the same functional endpoint, such as long-term potentiation, can be induced through distinct signaling pathways engaged by different patterns of stimulation. A critical question raised by these studies is whether different induction pathways either converge onto a common molecular mechanism or engage different molecular cascades for the maintenance of long-term plasticity. We directly examined this issue in the context of memory for sensitization in the marine mollusk Aplysia. In this system, training with a single tail shock normally induces short-term memory (<30 min) for sensitization of tail-elicited siphon withdrawal, whereas repeated spaced shocks induce both intermediate-term memory (ITM) (>90 min) and long-term memory (>24 hr). We now show that a single tail shock can also induce ITM that is expressed selectively at the trained site (site-specific ITM). Although phenotypically similar to the form of ITM induced by repeated trials, the mechanisms by which site-specific ITM is induced and maintained are distinct. Unlike repeated-trial ITM, site-specific ITM requires neither protein synthesis nor PKA activity for induction or maintenance. Rather, the induction of site-specific ITM requires calpain-dependent proteolysis of activated PKC, yielding a persistently active PKC catalytic fragment (PKM) that also serves to maintain the memory in the intermediateterm temporal domain. Thus, two unique forms of ITM that have different induction requirements also use distinct molecular mechanisms for their maintenance.

Nucleotide-metabolizing enzymes play an important role in the regulation of nucleotide levels. In the present report, we demonstrated an enzyme activity with different kinetic properties in membrane preparations of the nervous ganglia and digestive gland from Helix aspersa. ATPase and ADPase activities were dependent on Ca2+ and Mg2+ with pH optima approximately 7.2 and between 6.0 and 8.0 in digestive gland and nervous ganglia, respectively. The enzyme activities present in membrane preparations of these tissues preferentially hydrolyzed triphosphate nucleotides. In nervous ganglia, the enzyme was insensitive to the classical ATPases inhibitors. In contrast, in digestive gland, N-ethylmaleimide (NEM) produced 45% inhibition of Ca(2+)-ATP hydrolysis. Sodium azide, at 100 microM and 20 mM, inhibited Mg(2+)-ATP hydrolysis by 36% and 55% in digestive gland, respectively. The presence of nucleotide-metabolizing enzymes in these tissues may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in these species.

In the present study, we investigated the underlaying mechanism of nitric oxide (NO) and cGMP on the decline of a Ca2+-activated potassium (KCa) current in U-cells of the right parietal ganglion of the pulmonate snail, Helix pomatia. Using a two-electrode voltage-clamp technique, we activated a KCa-current either by opening of endogenous voltage-gated Ca2+-channels during depolarizing voltage steps or by ionophoretic injection of Ca2+ via a third electrode containing 100 mM Ca2+. KCa-current amplitude in U-cells was sensitive to Ba2+, TEA, iberiotoxin, kaliotoxin and charybdotoxin (ChTX), but not to 4-aminopyridine (4-AP) (up to 30 mM) and apamin (up to 300 nM). Thus, the biophysical and pharmacological profile of the KCa-current in U-cells shares similarities with the large-conductance KCa channel (BKCa). The NO-donor sodium nitroprusside (SNP) or S-nitro-N-acetylpenicillamine (SNAP) as well as NO-gas decreased the KCa-current amplitude and decreased the rate of KCa-current activation elicited by Ca2+-injection. Decline of the current amplitude and decrease of activation of KCa-current were qualitatively mimicked by the membrane-permeable cGMP analogue dibutyryl-cGMP (db-cGMP). NO-induced decrease of KCa-current was blocked by methylene blue (50 microM), an inhibitor of the guanylyl-cyclase, and by erytho-9-(2-hydroxyl-3-nonyl) adenine (EHNA) (100 microM), an inhibitor of the cGMP-stimulated phosphodiesterase 2 (PDE2). These experiments suggest that the NO-mediated decrease of KCa-current in U-cells results from synthesis of cGMP by activation of a guanylyl-cyclase and subsequent activation of PDE2.

The anatomical and functional relationship between neurons expressing nitric oxide (NO) synthase and molluscan cardioexcitatory (FMRFamide)-like neuropeptides was studied in the central ganglia of Helix lucorum (Pulmonata, Gastropoda), applying NADPHdiaphorase (NADPHd) histochemistry to visualize NO synthase and immunocytochemistry to demonstrate FMRFamide (FMRFa) at the light microscopic level. The NO production of the ganglia was detected by the colorimetric Griess determination of nitrite, a breakdown product of NO. Effects of the NO synthase substrate amino acid L-arginine, the NO synthase inhibitor Nomega-nitro-L-arginine (NOARG), synthetic FMRFa and the FMRFa sensitive ion channel blocker amiloride hydrochloride on nitrite production were also tested. NADPHd reaction labeled nerve cells and fibers in the procerebra, mesocerebra and metacerebra within the cerebral ganglia, and cell clusters in the postcerebral ganglia. FMRFa immunolabeling could be observed within subpopulations of NADPHd positive cells and in pericellular varicose fibers surrounding NADPHd stained neurons. Nitrite production of the ganglia was stimulated by L-arginine (10- 20 mM) but was decreased by NOARG (1-2 mM). Synthetic FMRFa (0.830-3.340 mM) increased the nitrite production in a dose dependent manner, but was ineffective in the presence of NOARG. Amiloride hydrochloride (7.890 mM) reduced the FMRFa evoked nitrite production in all ganglia. This is the first description of an anatomical relationship between putative NO producing and FMRFa containing cells, suggesting a possible regulatory role of FMRFa in the NO mediated signaling in an invertebrate nervous system., (Copyright 2004 S. Karger AG, Basel)

The presence and localization of NADPH-diaphorase (NADPH-d) in the radial nerve cords of Patiria pectonifera was shown by electron histochemistry. NADPH-d-positive structures were found in ectoneural and hyponeural regions of the radial nerve cord. Ultrastructural localization of NADPH-d was detected in neurons, sensory cells, supporting cells, and in the nerve plexus. The highest enzymatic activity in ectoneural region of the radial nerve cord is due, presumably, to the involvement of NADPH in sensory signal processing.

Sex differentiation and gametogenesis represent critical steps in the reproductive process and are subject to hormonal control by serotonin, dopamine and steroids such as estradiol-17beta and testosterone. The purpose of this study sought to examine the endocrine-disrupting activity that a primary-treated municipal effluent might have on the metabolism of biogenic amine levels. First, serotonin receptors transfected in Chinese hamster ovary (CHO) cells were used to screen for the presence of serotonin receptor agonist or antagonist. Second, one group of Elliptio complanata mussels were exposed to single compounds likely to be found in municipal wastewaters and another group was exposed in situ to the municipal effluent plume for 90 days in experimental cages. Results showed that solid phase C-8 extracts of surface water downstream a municipal effluent could activate the transport of serotonin by receptors at a distance of at least 5 km from its outfall thereby indicating the presence of serotonin mimics in the effluent dispersion plume. Levels of serotonin and monoamine oxidase (MAO) activity in nerve ganglia of mussels exposed for 90 days to the municipal effluent were, respectively, reduced and increased at a distance 10-km downstream. Injections of estradiol-17beta and nonylphenol in mussels decreased the levels of serotonin and dopamine, but increased MAO activity in the gonad and nerve ganglia. Exposure to estrogenic chemicals present in municipal effluents may therefore alter the normal metabolism of serotonin and dopamine, both of which are involved in sexual differentiation in bivalves and fish. Chemicals acting through E2 receptor-mediated pathways and serotonin receptors are likely to cause the observed effects.

We measured the effects in vitro of pure and commercial pesticides on Ca(2+)-activated ATPase and cholinesterase (ChE) activities in the nervous system of the slug Phyllocaulis soleiformis. The pesticides used in this study included carbamate and organophosphates, which acts as reversible and irreversible anticholinesterases, respectively. Both enzymes were insensitive to pure carbofuran (1 mM), glyphosate (1 mM) and malathion (120 microM). However, the carbamate carbofuran, in the commercial formulation Furandan 350S, inhibited ATPase and ChE activities. The organophosphate glyphosate used in the commercial preparation of Gliz 480CS inhibited ATPase activity and increased cholinesterase activity. These effects are likely due to the action of adjuvant substances of the chemical formulation. The commercial formulation (Malatol 500CE) did not alter enzymes activities. Our results suggest that cholinesterase present in the slug nervous tissue has a different behavior to those identified in vertebrate nervous tissue, since it was insensitive to pure compounds, known as anticholinesterases in vertebrates. Considering the insensitivity of the Ca(2+)-activated ATPase, we suggested that the purinergic neurotransmission and other roles of ATP might not be affected by the pure pesticides tested.

Daily patterns of melatonin and 5-methoxytryptophol (5-ML) concentrations and of aryl alkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities have been measured in the cerebroid ganglions, visceral ganglions, and ocular tentacles of the gastropod mollusc Helix aspersa maxima. Melatonin concentrations are very low in all the studied structures, except a small peak at the end of the night in the cerebroid ganglions. 5-ML, which is quite undetectable in the cerebroid and visceral ganglions, shows clear daily variations in the ocular tentacles with low values in the middle of the light period and high values during the night. These results are opposite to what is known on daily variations of 5-ML in vertebrates. AA-NAT activity was not detected, while the presence of an HIOMT-like activity supports the hypothesis that 5-ML is synthesized in the ocular tentacles. The temporal relationships existing between the 5-ML rhythm in the ocular tentacles and the hemolymph suggest that 5-ML could be released in the general circulation. These preliminary results suggest that 5-ML could be an informative molecule involved in adaptative processes in the snail and they reinforce the hypothesis that the different 5-methoxyindoles could be implicated in the integration of environmental information.

Fixation-resistant NADPH-diaphorase (NADPHd) activity is used widely as a marker for nitric oxide synthase (NOS). In frozen sections, NADPHd histochemistry yields high anatomic definition. In whole-mounts, however, poor penetration of the reagents, background staining, and tissue opacity severely limit its application. Here we report a combination of new methods that significantly improves whole-mount NADPHd staining. We demonstrate these methods in the thoracic ganglia of a large insect, the locust Schistocerca gregaria, in which NADPHd has been analyzed previously using both whole-mounts and serial section reconstructions. The penetration of the staining reagents was markedly improved after fixation in methanol/formalin compared to phosphate-buffered formaldehyde. Methanol/formalin also reduced nonspecific NADPHd and enhanced the selective staining. Penetration was further enhanced by incubation regimens that exploit the temperature- or pH-dependence of NADPHd. In combination with methanol/formalin fixation, this permitted staining to develop evenly throughout these comparatively large invertebrate ganglia. These improvements were complemented by a new clearing technique that preserves the NADPHd staining, gives excellent transparency, and avoids distortion of specimen morphology. The new methods revealed the three-dimensional architecture of NADPHd expression in locust ganglia in unprecedented detail and may similarly improve whole-mount detection of NADPHd in other invertebrate and vertebrate preparations.

Nitric oxide (NO), produced by nitric oxide synthase (NOS) in brain tissue, is essential for a variety of kinds of learning in vertebrates. In invertebrates, there are clear examples of an association between NO signalling and olfaction, feeding behaviour and learning. The role of NO as a neurotransmitter in the manipulative behaviour of Sepia officinalis was tested. Manipulative behaviour requires extensive chemotactile sensory processing, fine motor control and probably motor learning processes. NADPH-diaphorase activity (a reliable histochemical marker for nitric oxide synthase) was found in sensory epithelia and in the axial nerve cord of the arms. NOS inhibitor injections (L-NAME) produced an increase in the latency of prey paralysis. By placing mechanical constraints on the base of the fifth periopods of the crab, we prevented the cuttlefish from injecting cephalotoxin and, thus, forced it to change injection sites. We showed that L-NAME pretreatment did not affect the flexibility of the manipulative behaviour. The implications of the involvement of NO in the acquisition of chemo-tactile information and in the programming of the motor skills of the manipulative behaviour is discussed.

Effect of single and binary treatments of plant-derived molluscicides on different enzymes--acetylcholinesterase (AChE), lactic dehydrogenase (LDH) and acid/alkaline phosphatase (ACP/ALP)--in the nervous tissue of the harmful terrestrial snail Achatina fulica were studied. Sublethal in vivo 24-h exposure to 40% and 80% LC(50) of Azadirachta indica oil, Cedrus deodara oil, Allium sativum bulb powder, Nerium indicum bark powder and binary combinations of A. sativum (AS) + C. deodara (CD) and CD + A. indica (AI) oils significantly altered the activity of these enzymes in the nervous tissue of Achatina fulica. The binary treatment of AS + CD was more effective against AChE, LDH, and ALP than the single ones. However, binary treatment of AI + CD was more effective against ALP., (Copyright 2003 John Wiley & Sons, Ltd.)

Receptor protein tyrosine phosphatases (RPTPs) are thought to play important functions in pathfinding and target recognition by growing neuronal processes. The leech RPTPs HmLAR1 and HmLAR2 are expressed selectively by central neurons, Comb cells, and peripheral muscle tissues in the Hirudo medicinalis embryo. To explore the functions of HmLARs, we have sought to determine their physiological substrates. We report here the cloning and embryonic expression of Lena, the leech homolog of Enabled, a cytosolic protein implicated in actin-based cell motility. Lena is expressed in embryonic central neurons and in the Comb cell. We present experimental evidences indicating that Lena associates selectively with the intracellular domain of HmLAR1 and HmLAR2. Additionally, RNA interference (RNAi) of HmLAR1 in intact leech embryos leads to the hyperphosphorylation of Lena. We propose, therefore, that Lena is an in vivo substrate of HmLAR1 in neurons and perhaps of HmLAR2 in the Comb cells.