Molecular Evidence for Convergence and Parallelism in Evolution of Complex Brains of Cephalopod Molluscs: Insights from Visual Systems.

Coleoid cephalopods show remarkable evolutionary convergence with vertebrates in their neural organization, including (1) eyes and visual system with optic lobes, (2) specialized parts of the brain controlling learning and memory, such as vertical lobes, and (3) unique vasculature supporting such complexity of the central nervous system. We performed deep sequencing of eye transcriptomes of pygmy squids (Idiosepius paradoxus) and chambered nautiluses (Nautilus pompilius) to decipher the molecular basis of convergent evolution in cephalopods. RNA-seq was complemented by in situ hybridization to localize the expression of selected genes. We found three types of genomic innovations in the evolution of complex brains: (1) recruitment of novel genes into morphogenetic pathways, (2) recombination of various coding and regulatory regions of different genes, often called "evolutionary tinkering" or "co-option", and (3) duplication and divergence of genes. Massive recruitment of novel genes occurred in the evolution of the "camera" eye from nautilus' "pinhole" eye. We also showed that the type-2 co-option of transcription factors played important roles in the evolution of the lens and visual neurons. In summary, the cephalopod convergent morphological evolution of the camera eyes was driven by a mosaic of all types of gene recruitments. In addition, our analysis revealed unexpected variations of squids' opsins, retinochromes, and arrestins, providing more detailed information, valuable for further research on intra-ocular and extra-ocular photoreception of the cephalopods.

Phylogenomics meets neuroscience: how many times might complex brains have evolved?

The origin of complex centralized brains is one of the major evolutionary transitions in the history of animals. Monophyly (i.e. presence of a centralized nervous system in urbilateria) vs polyphyly (i.e. multiple origins by parallel centralization of nervous systems within several lineages) are two historically conflicting scenarios to explain such transitions. However, recent phylogenomic and cladistic analysis suggests that complex brains may have independently evolved at least 9 times within different animal lineages. Indeed, even within the phylum Mollusca cephalization might have occurred at least 5 times. Emerging molecular data further suggest that at the genomic level such transitions might have been achieved by changes in expression of just a few transcriptional factors - not surprising since such events might happen multiple times over 700 million years of animal evolution. Both cladistic and genomic analyses also imply that neurons themselves evolved more than once. Ancestral polarized secretory cells were likely involved in coordination of ciliated locomotion in early animals, and these cells can be considered as evolutionary precursors of neurons within different lineages. Under this scenario, the origins of neurons can be linked to adaptations to stress/injury factors in the form of integrated regeneration-type cellular response with secretory signaling peptides as early neurotransmitters. To further reconstruct the parallel evolution of nervous systems genomic approaches are essential to probe enigmatic neurons of basal metazoans, selected lophotrochozoans (e.g. phoronids, brachiopods) and deuterostomes.

5-HT and 5-HT-SO4, but not tryptophan or 5-HIAA levels in single feeding neurons track animal hunger state.

Serotonin (5-HT) is an intrinsic modulator of neural network excitation states in gastropod molluscs. 5-HT and related indole metabolites were measured in single, well-characterized serotonergic neurons of the feeding motor network of the predatory sea-slug Pleurobranchaea californica. Indole amounts were compared between paired hungry and satiated animals. Levels of 5-HT and its metabolite 5-HT-SO4 in the metacerebral giant neurons were observed in amounts approximately four-fold and two-fold, respectively, below unfed partners 24 h after a satiating meal. Intracellular levels of 5-hydroxyindole acetic acid and of free tryptophan did not differ significantly with hunger state. These data demonstrate that neurotransmitter levels and their metabolites can vary in goal-directed neural networks in a manner that follows internal state.

Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut.

The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH approximately 3 units*mm(-1)) in which an extremely alkaline (pH approximately 11) anterior midgut lies between near-neutral posterior midgut and gastric cecum (pH 7-8). A plasma membrane H(+) V-ATPase energizes this alkalinization but the ion carriers involved are unknown. Capillary zone electrophoresis of body samples with outlet conductivity detection showed a specific transepithelial distribution of chloride and bicarbonate/carbonate ions, with high concentrations of both anions in the midgut tissue: 68.3 +/- 5.64 and 50.8 +/- 4.21 mM, respectively. Chloride was higher in the hemolymph, 57.6 +/- 7.84, than in the lumen, 3.51 +/- 2.58, whereas bicarbonate was higher in the lumen, 58.1 +/- 7.34, than the hemolymph, 3.96 +/- 2.89. Time-lapse video assays of pH profiles in vivo revealed that ingestion of the carbonic anhydrase inhibitor acetazolamide and the ion exchange inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), at 10(-4) M eliminates lumen alkalinization. Basal application of these inhibitors in situ also reduced gradients recorded with self-referencing pH-sensitive microelectrodes near the basal membrane by approximately 65% and 85% respectively. Self-referencing chloride-selective microelectrodes revealed a specific spatial profile of transepithelial chloride transport with an efflux maximum in anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes. These data suggest that an H(+) V-ATPase-energized anion exchange occurs across the apical membrane of the epithelial cells and implicate an electrophoretic Cl(-)/HCO(3)(-) exchanger and carbonic anhydrase as crucial components of the steady-state alkalinization in anterior midgut of mosquito larvae.

Distribution of nitric oxide synthase immunoreactivity in the nervous system and peripheral tissues of Schistosoma mansoni.

The distribution of nitric oxide synthase (NOS) immunoreactivity and putative NOS activity in adult Schistosoma mansoni was analysed using 3 different types of NOS antibodies and NADPH-diaphorase histochemistry. Although potential involvement of the gaseous radical nitric oxide (NO) in host response to infection by schistosomes has been suggested, there is little or no information available regarding the role, or even the presence, of the NO pathway in schistosomes themselves. Here, we demonstrate that antibodies against neuronal NOS (nNOS) and inducible NOS (iNOS) isoforms stain adult worms with distinctive patterns; anti-endothelial NOS (eNOS) shows no selective labelling. nNOS-like immunoreactivity is found in the main nerve cords and the peripheral nervous system. Putative sensory neurons with apical neuronal processes leading to the tegument of male worms are also immunoreactive for nNOS. Anti-iNOS labels a variety of predominantly non-neuronal tissues, showing intense labelling at or near the surface of the worm and in components of the gastrointestinal tract. The distribution of NADPH-diaphorase reactivity (a histochemical marker of NOS), is generally similar to the pattern of NOS immunoreactivity, including labelling of neuronal-like cells as well as developing eggs. These results suggest that an NOS-like enzyme is present in S. mansoni, and indicate potential roles for the different NOS isoforms in neuronal signalling, reproduction and development.

In situ analysis of pH gradients in mosquito larvae using non-invasive, self-referencing, pH-sensitive microelectrodes.

The alkaline environment, pH approximately 11, in the anterior midgut lumen of mosquito larvae is essential for normal nutrition and development. The mechanism of alkalization is, however, unknown. Although evidence from immunohistochemistry, electron microscopy and electrophysiology suggests that a V-ATPase is present in the basal membranes of the epithelial cells, its physiological role in the alkalization process has not been demonstrated. To investigate a possible role of the V-ATPase in lumen alkalization, pH gradients emanating from the hemolymph side of the midgut in semi-intact mosquito larvae were measured using non-invasive, self-referencing, ion-selective microelectrodes (SERIS). Large H+ concentration gradients, with highest concentrations close to the basal membrane (outward [H+] gradients), were found in the anterior midgut, whereas much smaller gradients, with concentrations lowest close to this membrane (inward [H+] gradients), were found in the gastric caeca and posterior midgut. Similar region-specific pH gradients, with consistent anterior-to-posterior profiles, were observed in individuals of two Aedes species, Aedes aegypti from semi-tropical Florida and Aedes canadensis from north-temperate Massachusetts. The gradients remained in a steady state for up to 6 h, the maximum duration of the recordings. Bafilomycin A1 (10(-5), 10(-7 )mol x l(-1)) on the hemolymph side greatly diminished the [H+] gradients in the anterior midgut but had no effect on the gradients in the gastric caecum and posterior midgut. These physiological data are consistent with the previous findings noted above. Together, they support the hypothesis that a basal, electrogenic H+ V-ATPase energizes luminal alkalization in the anterior midgut of larval mosquitoes.

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia.

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.

Distribution of NADPH-diaphorase reactivity and effects of nitric oxide on feeding and locomotory circuitry in the pteropod mollusc, Clione limacina.

The action of nitric oxide (NO) and the distribution of putative nitric oxide synthase-containing cells in the pelagic pteropod mollusc Clione limacina were studied using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and conventional microelectrode techniques in the isolated central nervous system and in semi-intact preparations. The majority of NADPH-d-reactive neuronal somata were restricted to the cerebral ganglia. The labeled cells were small in diameter (20-30 microm) and were located in the medial areas of the ganglia. A pair of symmetrical neurons was found in the peripheral "olfactory organ." NADPH-d-reactive non-neuronal cells were detected in the periphery and were mainly associated with secretorylike cells and organs of the renopericardial system. The NO donor, diethylamine NO complex sodium salt (10-100 microM), activated neurons from both feeding and locomotory circuits. The cGMP analog, 8-Br-cGMP, mimicked the effects of NO on neurons. We suggest that NO is an endogenous neuromodulator involved in the control of some aspects of feeding and locomotor behavior of Clione.

Giant identified NO-releasing neurons and comparative histochemistry of putative nitrergic systems in gastropod molluscs.

Gastropod molluscs provide attractive model systems for behavioral and cellular analyses of the action of nitric oxide (NO), specifically due to the presence of many relatively giant identified nitrergic neurons in their CNS. This paper reviews the data relating to the presence and distribution of NO as well as its synthetic enzyme NO synthase (NOS) in the CNS and peripheral tissues in ecologically and systematically different genera representing main groups of gastropod molluscs. Several species (Lymnaea, Pleurobranchaea, and Aplysia) have been analyzed in greater detail with respect to immunohistochemical, biochemical, biophysical, and physiological studies to further clarify the functional role of NO in these animals.

Cost-benefit analysis potential in feeding behavior of a predatory snail by integration of hunger, taste, and pain.

Hunger/satiation state interacts with appetitive and noxious stimuli to determine feeding and avoidance responses. In the predatory marine snail Pleurobranchaea californica, food chemostimuli induced proboscis extension and biting at concentration thresholds that varied directly with satiation state. However, food stimuli also tended to elicit avoidance behavior (withdrawal and avoidance turns) at concentration thresholds that were relatively low and fixed. When the feeding threshold for active feeding (proboscis extension with biting) was exceeded, ongoing avoidance and locomotion were interrupted and suppressed. Noxious chemostimuli usually stimulated avoidance, but, in animals with lower feeding thresholds for food stimuli, they often elicited feeding behavior. Thus, sensory pathways mediating appetitive and noxious stimuli may have dual access to neural networks of feeding and avoidance behavior, but their final effects are regulated by satiation state. These observations suggest that a simple cost-benefit computation regulates behavioral switching in the animal's foraging behavior, where food stimuli above or below the incentive level for feeding tend to induce feeding or avoidance, respectively. This decision mechanism can weigh the animal's need for nutrients against the potential risk from other predators and the cost of relative energy outlay in an attack on prey. Stimulation of orienting and attack by low-level noxious stimuli in the hungriest animals may reflect risk-taking that can enhance prey capture success. A simple, hedonically structured neural network model captures this computation.

Nitric oxide selectively enhances cAMP levels and electrical coupling between identified RPaD2/VD1 neurons in the CNS of Lymnaea stagnalis (L.).

The isolated CNS of the freshwater mollusc Lymnaea stagnalis was used as a model to study the role of cAMP in NO-mediated mechanisms. The NO donor, DEA/NO (10(-5)-10(-3) M) increased cAMP concentrations in the cerebral, pedal, pleural, parietal and visceral ganglia. In contrast, in the buccal ganglia the same doses of DEA/NO decreased the level of cAMP production. The NOS inhibitor, L-NNA (10(-4) M) increased cAMP concentrations in all areas of the CNS. L-arginine (1 mM), a metabolic precursor of NO, mimicked the action of the NO-donor. The coefficient of electrical coupling between two viscero-parietal peptidergic neurons (VD1/RPaD2) was enhanced by both DEA/NO (10(-4) M) and 8-Br-cAMP (10(-4) M) whereas 8-Br-cGMP (2x10(-4) M) reduced the coupling. We suggest that cAMP-dependent mechanisms are involved in neuronal NO signaling in this simpler nervous system.

Nitric oxide synthase imunolabeling in the molluscan CNS and peripheral tissues.

NOS immunoreactivity was assayed in CNS and peripheral tissues of the sea slugs Pleurobranchaea californica, Tritonia diomedea and Aplysia californica using different antisera against mammalian nitric oxide synthase in Western blots. Polyclonal anti-nNOS labeled at 250, 185, 170, 155, 100, 75, and 65 kD in extracts of Pleurobranchaea CNS, salivary gland and esophagus but not of gills or muscle. The labeling pattern for Tritonia in bands at 250, 200, 120/110, 100, 69, 65, and 60 kD differed somewhat. Anti-nNOS labeling in Aplysia was markedly different, with bands labeled only at 69 and 60 kD in CNS extracts, and at 200, 190, 69 and 60 kD in salivary and esophagus extracts. The wide variation in NOS immunoreactivity is consistent with species differences in tissue localization and biochemical properties of molluscan NOS isoforms.

Single-cell analyses of nitrergic neurons in simple nervous systems.

Understanding the role of the gaseous messenger nitric oxide (NO) in the nervous system is complicated by the heterogeneity of its nerve cells; analyses carried out at the single cell level are therefore important, if not critical. Some invertebrate preparations, most especially those from the gastropod molluscs, provide large, hardy and identified neurons that are useful both for the development of analytical methodologies and for cellular analyses of NO metabolism and its actions. Recent modifications of capillary electrophoresis (CE) allow the use of a small fraction of an individual neuron to perform direct, quantitative and simultaneous assays of the major metabolites of the NO-citrulline cycle and associated biochemical pathways. These chemical species include the products of NO oxidation (NO2-/NO3-), l-arginine, l-citrulline, l-ornithine, l-argininosuccinate, as well as selected NO synthase inhibitors and cofactors such as NADPH, biopterin, FMN and FAD. Diverse cotransmitters can also be identified in the same nitrergic neuron. The sensitivity of CE methods is in the femtomole to attomole range, depending on the species analysed and on the specific detector used. CE analysis can be combined with prior in vivo electrophysiological and pharmacological manipulations and measurements to yield multiple physiological and biochemical values from single cells. The methodologies and instrumentation developed and tested using the convenient molluscan cell model can be adapted to the smaller and more delicate neurons of other invertebrates and chordates.

Capillary electrophoresis analysis of nitric oxide synthase related metabolites in single identified neurons.

Intracellular concentrations of L-citrulline (Cit) and its metabolites are related to nitric oxide synthase (NOS) activity, an enzyme producing the intercellular messenger NO in animal tissues including the nervous system. A capillary electrophoresis system using laser-induced fluorescence detection is described, and methods are developed to monitor the levels of L-arginine (Arg), Cit, and related molecules in identified neurons of the marine slugs, Pleurobranchaea californica and Aplysia californica. The limits of detection for Arg, Cit, L-arginino-succinate, L-ornithine, and L-arginine phosphate range from 50 amol to 17 fmol (5 nM to 17 microM in the neurons under study); these detection limits are significantly lower than actual intracellular levels of the metabolites, allowing the direct assay of single cells. The levels of NOS metabolites in individual neurons varied form 6 (Arg) and 4 mM (Cit) in putative NOS-containing neurons down to < 1 microM (undetectable) levels in many putative NOS-negative cells. The Arg/Cit ratio is independent of cell volume, correlates with NADPH-diaphorase staining, and appears to be a characteristic parameter for the presence of NOS activity in identified neurons.

Serotonin immunoreactivity in the central nervous system of the marine molluscs Pleurobranchaea californica and Tritonia diomedea.

The central nervous systems of the marine molluscs Pleurobranchaea californica (Opisthobranchia: Notaspidea) and Tritonia diomedea (Opisthobranchia: Nudibranchia) were examined for serotonin-immunoreactive (5-HT-IR) neurons and processes. Bilaterally paired clusters of 5-HT-IR neuron somata were distributed similarly in ganglia of the two species. In the cerebropleural ganglion complex, these were the metacerebral giant neurons (both species), a dorsal anterior cluster (Pleurobranchaea only), a dorsal medial cluster including identified neurons of the escape swimming network (both species), and a dorsal lateral cluster in the cerebropleural ganglion (Pleurobranchaea only). A ventral anterior cluster (both species) adjoined the metacerebral giant somata at the anterior ganglion edge. Pedal ganglia had the greatest number of 5-HT-IR somata, the majority located near the roots of the pedal commissure in both species. Most 5-HT-IR neurons were on the dorsal surface of the pedal ganglia in Pleurobranchaea and were ventral in Tritonia. Neither the buccal ganglion of both species nor the visceral ganglion of Pleurobranchaea had 5-HT-IR somata. Afew asymmetrical 5-HT-IR somata were found in cerebropleural and pedal ganglia in both species, always on the left side. The clustering of 5-HT-IR neurons, their diverse axon pathways, and the known physiologic properties of their identified members are consistent with a loosely organized arousal system of serotonergic neurons whose components can be generally or differentially active in expression of diverse behaviors.

Single neuron analysis by capillary electrophoresis with fluorescence spectroscopy.

A technique to identify and quantitate simultaneously more than 30 compounds in individual neurons is described. The method uses nanoliter volume sampling, capillary electrophoresis separation, and wavelength-resolved native fluorescence detection. Limits of detection (LODs) range from the low attomole to the femtomole range, with 5-hydroxytryptamine (or serotonin [5-HT]) LODs being approximately 20 attomoles. Although the cellular sample matrix is chemically complex, the combination of electrophoretic migration time and fluorescence spectral information allows positive identification of aromatic monoamines, aromatic amino acids and peptides containing them, flavins, adenosine- and guanosine-nucleotide analogs, and other fluorescent compounds. Individual identified neurons from Aplysia californica and Pleurobranchaea californica are used to demonstrate the applicability and figures of merit of this technique.

Non-enzymatic production of nitric oxide (NO) from NO synthase inhibitors.

The gaseous signal molecule, nitric oxide (NO*), is generated enzymatically by NO synthase (NOS) from L-arginine. Overproduction of NO contributes to cell and tissue damage as sequelae of infection and stroke. Strategies to suppress NO synthesis rely heavily on guanidino-substituted L-arginine analogs (L-NAME, L-NA, L-NMMA, L-NIO) as competitive inhibitors of NOS, which are often used in high doses to compete with millimolar concentrations of intracellular arginine. We show that these analogs are also a source for non-enzymatically produced NO. Enzyme-independent NO release occurs in the presence of NADPH, glutathione, L-cysteine, dithiothreitol and ascorbate. This non-enzymatic synthesis of NO can produce potentially toxic, micromolar concentrations of NO and can oppose the effects of NOS inhibition. NO production driven by NOS inhibitors was demonstrated ex vivo in the central nervous and peripheral tissues of gastropod molluscs Aplysia and Pleurobranchaea using electron paramagnetic resonance and spin-trapping techniques. These results have important implications for therapeutic regulation of NO homeostasis.

Nitrite and nitrate levels in individual molluscan neurons: single-cell capillary electrophoresis analysis.

Cell and tissue concentrations of NO2- and NO3- are important indicators of nitric oxide synthase activity and crucial in the regulation of many metabolic functions, as well as in nonenzymatic nitric oxide release. We adapted the capillary electrophoresis technique to quantify NO2- and NO3- levels in single identified buccal neurons and ganglia in the opisthobranch mollusc Pleurobranchaea californica, a model system for the study of the chemistry of neuron function. Neurons were injected into a 75-microm separation capillary and the NO2- and NO3- were separated electrophoretically from other anions and detected by direct ultraviolet absorbance. The limits of detection for NO2- and NO3- were <200 fmol (<4 microM in the neurons under study). The NO2- and NO3- levels in individual neurons varied from 2 mM (NO2-) and 12 mM (NO3-) in neurons histochemically positive for NADPH-diaphorase activity down to undetectable levels in many NADPH-diaphorase-negative cells. These results affirm the correspondence of histochemical NADPH-diaphorase activity and nitric oxide synthase in molluscan neurons. NO2- was not detected in whole ganglion homogenates or in hemolymph, whereas hemolymph NO3- averaged 1.8 +/- 0.2 x 10(-3) M. Hemolymph NO3- in Pleurobranchaea was appreciably higher than values measured for the freshwater pulmonate Lymnaea stagnalis (3.2 +/- 0.2 x 10(-5) M) and for another opisthobranch, Aplysia californica (3.6 +/- 0.7 x 10(-4) M). Capillary electrophoresis methods provide utility and convenience for monitoring NO2-/NO3- levels in single cells and small amounts of tissue.