Cholinergic and glutamatergic spontaneous and evoked excitatory postsynaptic currents in optic lobe neurons of cuttlefish, Sepia officinalis

Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from two different classes of neurons in the optic lobes of the cuttlefish brain and their synaptic activities analyzed and compared. The cell types were as follows: efferent centrifugal neurons, with cell bodies in the inner granule layer and axons projecting to the retina, and interneurons local to the medulla. For both neuronal groups, the sEPSCs reversal potentials were around 0 mV and there were no significant differences in their mean amplitude and rise times. However, the sEPSCs from the centrifugal neurons had a significantly higher frequency and faster decay time constant than those recorded from the medulla. Tetrodotoxin (TTX) reduced the mean frequency of the sEPSCs from both the medulla and centrifugal neurons by 69.66 +/- 4.05% and 57.80 +/- 3.87%, respectively, implying that more than half of these excitatory synaptic inputs were due to action potential-mediated release of neurotransmitter. Pharmacological examination revealed that the centrifugal neurons were driven by spontaneous synaptic inputs mediated by glutamatergic and cholinergic receptors, because co-application of the glutamate antagonist kynurenic acid (KYNA) and the nicotinic antagonist mecamylamine hydrochloride (MCM) resulted in complete blockade of these excitatory inputs. For the medulla neurons, the synaptic inputs were driven by glutamate and other transmitters yet to be identified. Evoked EPSCs (eEPSCs) were recorded from both types of neurons by stimulating the appropriate optic nerve bundles; in centrifugal neurons, the eEPSCs were blocked by co-application of KYNA and MCM, whereas in the medulla neurons, KYNA alone either totally or partially blocked the eEPSCs.     

Modulation of spontaneous and evoked EPSCs and IPSCs in optic lobe neurons of cuttlefish Sepia officinalis by the neuropeptide FMRF-amide

The effects of the neuropeptide FMRFa on spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs), as well as on evoked EPSCs and IPSCs, in two types of neurons within the central optic lobe of cuttlefish were examined using the whole-cell voltage-clamp technique. FMRFa (1-10 micro m) did not affect cell membrane resting potentials, but reversibly reduced both the frequency and amplitude of sEPSCs in neurons within the medulla region of the optic lobe while increasing the frequency and amplitude of their sIPSCs. For centrifugal neurons in the inner granule cell layer of the optic lobe, FMRFa (1-10 micro m) decreased both the frequency and amplitude of sEPSCs. In the presence of tetrodotoxin (0.5 micro m), neither the interevent interval, nor amplitude distributions of the miniature EPSCs or the miniature IPSCs, were affected by FMRFa, implying a presynaptic action of FMRFa on the optic lobe neurons. Bath application of the neuropeptide also abolished or reduced in amplitude the evoked EPSCs and increased the amplitude of evoked IPSCs in optic lobe neurons, showing that FMRFa induced similar effects on evoked as on spontaneous postsynaptic currents. These results demonstrate the complex range of modulatory effects FMRFa can have within central nervous system circuits.     

Microsatellite loci for the plate-and-pillar coral, Porities rus.

Little is known about species and population-level genetic relatedness for many reef building corals, despite their critical importance as the foundation of coral reef ecosystems. We report the isolation and characterization of seven polymorphic microsatellites from the plate-and-pillar coral, Porites rus. Across all loci, there was a maximum of 18 alleles for the 37 individuals tested. We also tested these primers on 12 other species of Porites. At least one locus amplified in most of the species. These markers should be useful in various genetic applications involving Porities spp.     

Microsatellite loci for the carpenter bee Xylocopa frontalis (Apidae, Xylocopini)

In the present study, a microsatellite-enriched genomic library was constructed and primers for 14 microsatellite loci were designed for Xylocopa frontalis. Twenty unrelated individuals were screened. All loci were polymorphic and the number of alleles per locus ranged from 6 to 17 (x?=?10.43). Observed (H_o) and expected (H_e) heterozygosities ranged from 0.350 to 0.950 and 0.674 to 0.898, respectively. All loci were in Hardy?CWeinberg equilibrium, except one. The microsatellite loci described in this study will contribute towards general biology studies of X. frontalis, intranidal genetic relationships and nest management for the pollination of passion fruit.     

Nitric oxide synthase (NOS) in the brain of the cephalopod Sepia officinalis

Nitric oxide synthase-like protein (NOS) is shown to be present in specific regions of the central nervous system (CNS) of the cephalopod mollusc Sepia officinalis (cuttlefish). NOS activity, which is Ca(2+)/calmodulin-dependent, was determined by measuring the conversion of L-[(14)C]arginine in L-[(14)C]citrulline. The partially purified NOS from brain and optic lobes exhibited on SDS-PAGE a band at 150 kDa that was immunolabelled by antibodies raised against the synthetic peptide corresponding to the amino acids 1,414-1,429 of the C-terminus of rat nNOS. This same antibody was then used for immunohistochemical staining of serial sections of the cuttlefish CNS to reveal localized specific staining of cell bodies and fibers in several lobes of the brain. Staining was found in many lower motor centers, including cells and fibers of the inferior and superior buccal lobes (feeding centers); in some higher motor centers (anterior basal and peduncle lobes); in learning centers (vertical, subvertical, and superior frontal lobes); and in the visual system [retina and deep retina (optic lobe)]. Immunopositivity was also found in the olfactory lobe and organ and in the sucker epithelium. These findings suggest that nitric oxide (NO) may be involved as a signaling molecule in feeding, motor, learning, visual, and olfactory systems in the cuttlefish brain. The presence of NOS in the cephalopod "cerebellum" and learning centers is discussed in the context of the vertebrate CNS.     

Microsatellite loci characterised in a megapode, the Australian brush-turkey Alectura lathami

We isolated 137 unique Australian brush-turkey (Alectura lathami) microsatellite sequences. Twenty-five loci when characterised in 24 individuals displayed 2–17 alleles per locus. Four loci were found to be Z-linked based on a complete lack of heterozygotes in females and sequence similarity to the chicken Z chromosome. Due to the small genetic distance among species within this family, a proportion of these loci are expected to be of utility for genetic studies of other megapodes, many of which are endangered.     

Microsatellite loci for Symbiodinium goreaui and other Clade C Symbiodinium

The genus Symbiodinium comprises a diverse group of dinoflagellates known for their obligate relationship with reef–building corals. Members of the sub-genus ‘clade C’ are abundant, geographically wide-spread, as well as genetically and ecologically diverse. Coral colonies harboring clade C are often the most exposed to physical stressors. The genotypic diversity, dispersal and genetic connectivity exhibited by these Symbiodinium are the subjects of an increasing number of population genetic studies utilizing microsatellites. Here we describe 18 new microsatellite loci and test their utility across four common clade C types. We obtained multi-locus genotypes with individual level resolution in each of these types. Our results indicate that multi-locus genotypes can be obtained for many members of clade C using a subset of these markers. Data from these markers may assess population dynamics during coral bleaching and recovery as well as resolve boundaries to genetic recombination among these closely related, yet ecologically distinct, lineages.     

Microsatellite loci development for rare Colorado Sclerocactus (Cactaceae)

The genus Sclerocactus (Cactaceae) consists of 15 species, which have a confused taxonomic history due to morphological similarities and distribution overlap. Habitat loss and hybridization are of concern and have established cause for genetic investigations to further understand the genus and develop conservation strategies. Thirteen variable microsatellite loci were identified using S. glaucus and S. parviflorus and were tested in three additional species: S. wetlandicus, S. brevispinus and S. cloverii. The mean number of alleles per locus ranged from 2 to 11, with an average of 6.6. The observed and expected mean heterozygosity ranged from 0.00 to 0.90 and 0.26 to 0.90, respectively. These loci will aid in determining levels of hybridization, diversity and taxonomy of Sclerocactus.     

Distribution of neurokinin A-like and serotonin immunoreactivities within the vertical lobe complex in Sepia officinalis

Immunohistochemistry, using antibodies raised against mammalian neurokinin A (NKA) and serotonin (5-HT), was applied in double-staining experiments to map these molecules within the vertical lobe complex (inferior frontal, superior frontal, post-frontal, vertical, subvertical and precommissural lobes). NKA-like and 5-HT immunoreactivities were detected in all the lobes of the vertical lobe complex but were never colocalized in cell bodies or fibres. Except for the cell layers of the superior frontal lobe, both types of labelled cell bodies were observed in all the lobes. Both types of immunoreactive fibres were detected in all the neuropils and interestingly revealed clear subdivisions within some lobes, e.g., 5-HT-IR fibres were more abundant in the peripheral part of the vertical lobe whereas NKA-IR ones were widely observed in both the peripheral and central parts. In cephalopods, the vertical lobe complex is involved in learning and memory; thus, our results strongly suggest that one or more NKA-like and 5-HT molecules may function as neurochemical messengers in these cognitive processes.     

Microsatellite loci for genetic analysis of the arctic gadids Boreogadus saida and Arctogadus glacialis

We report sets of 19 and 16 microsatellite loci for the examination of the population genetics of Boreogadus saida and Arctogadus glacialis, respectively. Six of these loci were developed from a collection of 9,497 expressed sequences from B. saida while the remaining loci were found in the literature and optimized for use in B. saida and A. glacialis. The numbers of alleles observed for each locus ranged from 3 to 33 in B. saida and 1–22 in A. glacialis. Observed heterozygosities ranged from 0.02 to 0.93 in B. saida and 0.17–1.0 in A. glacialis. Species specific differences were observed for the loci providing new tools for the identification of these two morphologically similar arctic gadids. The loci presented here can be used to distinguish between the two species and fill fundamental biological knowledge gaps, thus promoting conservation of these important fishes.     

Evidence of early nervous differentiation and early catecholaminergic sensory system during Sepia officinalis embryogenesis

Within Mollusca, cephalopods exhibit a particularly complex nervous system. The adult brain is formed from the fusion of several “typical” molluscan ganglia but it remains poorly understood how these ganglia emerge, migrate, and differentiate during embryogenesis. We studied the development of both central and peripheral nervous system by antibodies raised against α‐tubulin and tyrosine hydroxylase (TH) in Sepia officinalis embryos to visualize neurites and catecholamine‐containing neurons, respectively. In early embryos, when organs start delineating, some ganglia already exhibited a significant fiber network. TH‐like immunoreactivity was detected in these fibers and in some primary sensory neurons in the embryo periphery. These data attest to the occurrence of an early embryonic sensory nervous system, likely effective, transient in part, and in relation to the perception of external cues. Concerning the peripheral nervous network, the stellate ganglia emerged as a plexus of numerous converging axons from TH‐like immunoreactive sensory cells, first at the mantle edge, and then in the whole mantle surface. Later, TH‐immunopositive motor fibers, originating from the stellate ganglia, penetrated the circular muscles of the mantle. These patterns reveal the setup of a mantle midline with likely attractive and repulsive properties. Our findings seem to challenge the widespread, still accepted, view of a late differentiation of cephalopod ganglia, and provides significant data for further investigations about axonal guidance during cephalopod development. J. Comp. Neurol. 517:539–549, 2009. © 2009 Wiley‐Liss, Inc.     

Motor and behavioral responses obtained by stimulation with chronic electrodes of the optic lobe of Sepia officinalis.

A new technique using a stimulating chronically-implanted electrode has allowed us to study the motor responses induced by electrical stimulation of the optic lobe in a freely swimming Sepia. Electrical stimulation of the cortex of the optic lobe produces no motor response; this is in agreement with the results of preceding authors. The stimulation of the neuropil of the optic lobe by monopolar electrode produces many different motor responses, in support of Boycott's results obtained by the same type of excitation in acute experiments. However, the field of stimulation of these electrodes could not always have been the same and it is possible that we were sometimes stimulating nervous structures close to the optic lobe. Stimulation by a bipolar electrode, however, which does not have this advantage, induces only two very different motor responses: an ipsilateral rotation and an 'alarm reaction", so called because of its similarities to the 'attentive immobilization" of higher vertebrates. These two reactions are very complex and their different components are linked together as in a behavioural response from an intact animal. These reactions present very different characteristics of excitability. They are obtained from many areas in the neuropil of the optic lobe, within which there does not seem to be any preferential localization. These results emphasize the importance of the optic lobe in motor control.     

Nitric oxide synthase expression in the central nervous system of Sepia officinalis: an in situ hybridization study

We recently reported the molecular cloning of nitric oxide synthase (NOS) mRNA from Sepia officinalis (SoNOS) using a strategy that involves hybridization of degenerate PCR primers to highly conserved NOS regions, combined with a RACE procedure. Here, in situ hybridization study has been performed on serial sections of the cuttlefish central nervous system to reveal localized specific staining of cell bodies in several lobes of the brain. Staining was found in many lower motor centres, including cells of the inferior and superior buccal lobes (feeding centres); in some higher motor centres (anterior basal and peduncle lobes); in learning centres (vertical, subvertical and superior frontal lobes); and in the visual system [medulla and deep retina (optic lobe)]. Positive staining was also found in the olfactory lobe. NOS-expressing cells have been detected also in the interbasal lobe. Double labelling experiments, performed on consecutive sections, showed that neurons containing NOS immunoreactivity were also positive in in situ hybridization staining. All these data support the presence of NOS in several systems in the cuttlefish brain.     

Microsatellite loci for testing temporal changes in the population genetics of the Florida mouse (Podomys floridanus)

The Florida mouse (Podomys floridanus) is endemic to Florida and is considered vulnerable by the IUCN due to extensive habitat loss. Here, we describe a panel of 76 microsatellite DNA markers for population genetic studies of this species. Fourteen of these loci were examined to estimate genetic diversity for 39 specimens collected in Florida in 1957 and 2006. DNA extractions from the 1957 specimens were carried out from snips of skin taken from museum voucher skins, whereas the 2006 samples were extracted from fresh liver tissue. The use of museum skins for reconstructing the genetics of historical populations is becoming a critical part of understanding past, present, and future genetic trends for threatened species. Mean expected heterozygosity for the two populations was 0.73 and mean observed heterozygosity was 0.75. The number of alleles per locus ranged from 2 to 15. All microsatellites were polymorphic, and exhibited variability across time.     

Microsatellite loci for the blue swimming crab (Callinectes bellicosus) (Crustacea: Portunidae) from the Gulf of California, Mexico

Twelve microsatellite markers (six di-nucleotides, four tri-nucleotides and two tetra-nucleotides) were isolated and characterized for Callinectes bellicosus, a commercial crustacean species from the Gulf of California, Mexico. One locus was monomorphic and 11 loci were polymorphic in 32 individual samples from a single location. Overall polymorphic loci, the number of alleles per locus ranged from 2 to 24 (average 10.0), the observed heterozygosity ranged from 0.094 to 0.969 (average 0.603), and the expected heterozygosity varied from 0.089 to 0.935 (average 0.597). One locus deviated significantly from Hardy–Weinberg equilibrium due to an excess of heterozygotes, while another locus showed evidence for the presence of a null allele. No evidence of linkage disequilibrium was found among pair of loci. These markers will be helpful to estimate the level of genetic connectivity over a small spatial and temporal scale in order to identify stocks for the management of this small-scale fishery in the Gulf of California.     

Microsatellite loci isolated from the lowland tapir (Tapirus terrestris), one of the largest Neotropical mammal

We describe ten microsatellite loci from the lowland tapir (Tapirus terrestris), one of the last members of the Neotropical megafauna and considered a vulnerable species. A total of 74 alleles were detected, ranging from four to thirteen alleles per locus. The expected heterozygosity ranged from 0.34 to 0.88, with a mean of 0.635. All but one locus conformed to the Hardy–Weinberg expectations. These microsatellite loci can be used in population genetic studies of the genus Tapirus, in which all species are threatened globally.     

Microsatellite loci for Symbiodinium A3 (S. fitti) a common algal symbiont among Caribbean Acropora (stony corals) and Indo-Pacific giant clams (Tridacna)

We developed ten polymorphic microsatellite loci for S. fitti (type A3), and tested their utility on a Caribbean population associated with the coral Acropora palmata and an Indo-Pacific population harbored by the giant clam Tridacna maxima. Our analyses identified from 3 to 8 alleles for each haploid locus. Diversity (R) indices were 0.48 for the Indo-Pacific and 0.81 for the Caribbean. Greater than 95% of the samples possessed a single, symbiont, multilocus genotype (MLG). Among their many uses for the study of coral-algal symbioses, population genetic markers provide insight on the potential for symbiont dispersal, can be used to assess symbiont population stability/longevity in hospite, and partition symbiont diversity into reproductively isolated operational taxonomic units (i.e. species).     

Characterization of 22 microsatellite loci for conservation genetic studies of an endemic anemonefish,Amphiprion latezonatus

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