Biomedical subjects

Leonid L Moroz

Publications and source records attributed to Leonid L Moroz.

15 recordsLinked to original sources

PubMed2005

Complete DNA sequence of the mitochondrial genome of the sea-slug, Aplysia californica: conservation of the gene order in Euthyneura.

We have sequenced and characterized the complete mitochondrial genome of the sea slug, Aplysia californica, an important model organism in experimental biology and a representative of Anaspidea (Opisthobranchia, Gastropoda). The mitochondrial genome of Aplysia is in the small end of the observed sizes of animal mitochondrial genomes (14,117 bp, NCBI Accession No. NC_005827). The Aplysia genome, like most other mitochondrial genomes, encodes genes for 2 ribosomal subunit RNAs (small and large rRNAs), 22 tRNAs, and 13 protein subunits (cytochrome c oxidase subunits 1-3, cytochrome b apoenzyme, ATP synthase subunits 6 and 8, and NADH dehydrogenase subunits 1-6 and 4L). The gene order is virtually identical between opisthobranchs and pulmonates, with the majority of differences arising from tRNA translocations. In contrast, the gene order from representatives of basal gastropods and other molluscan classes is significantly different from opisthobranchs and pulmonates. The Aplysia genome was compared to all other published molluscan mitochondrial genomes and phylogenetic analyses were carried out using a concatenated protein alignment. Phylogenetic analyses using maximum likelihood based analyses of the well aligned regions of the protein sequences support both monophyly of Euthyneura (a group including both the pulmonates and opisthobranchs) and Opisthobranchia (as a more derived group). The Aplysia mitochondrial genome sequenced here will serve as an important platform in both comparative and neurobiological studies using this model organism.

Animals↗

PubMed2005

Electroporation of neurons and growth cones in Aplysia californica.

Specific labeling of individual neurons and neuronal processes is virtually an everyday task for neuroscientists. Many traditional ways for delivery of intracellular dyes have limitations in terms of speed, efficiency and reproducibility. Electroporation is a fast, reliable and efficient method to deliver microscopic amounts of polar and charged molecules into neurons and their compartments such as individual neurites and growth cones. Here, we present a simple and highly effective procedure for intracellular labeling of individual Aplysia neurons both in intact ganglia and in cell culture. Pleural mechanoreceptor neurons have been used as illustrative examples to demonstrate applicability of direct and local labeling of the smallest individual neurites (< 2 microm) and single growth cones. Specifically, a 3-s train of 1.0 V hyperpolarizing pulses at 50 Hz effectively filled discrete neurites in contact with the tip of the micropipette with no dye transfer visible to other, non-contacted neurites. Application of this localized dye labeling technique to single neurites reveals a surprisingly complex morphology for patterns of axonal branching in culture. The protocol can be easily applied to a variety of models in neuroscience including accessible nervous systems of invertebrate animals.

Animals↗

PubMed2005

Two-color in situ hybridization in the CNS of Aplysia californica.

Aplysia californica is an attractive model organism for cellular and systems neuroscience. Currently, there is a growing body of sequence data from Aplysia that includes many interesting genes. To fully exploit this molecular data it must be integrated with the large body of physiological data that are already available for identified neurons in Aplysia networks. In situ hybridization is a powerful technique that enables this to be done. Expression patterns of selected mRNA transcripts can be mapped to individual cells in the central nervous system (CNS). Here, we describe a detailed non-radioactive in situ hybridization protocol optimized for whole-mount preparations of Aplysia ganglia. The indirect alkaline phosphatase-based chromogenic detection method we employ may be used with one or two colors in order to detect one or two different transcripts in the same preparation. The procedure is also compatible with intracellular dye labeling, making it possible to couple localization of transcripts with electrophysiological studies in positively identified neurons. Double labeling was done for transcripts encoding the neuropeptides FMRFamide and sensorin. The sensitive detection of mRNA and great preservation of CNS morphology makes this method a useful tool for analyzing expression patterns of neuron specific genes in Aplysia.

Animals↗

PubMed2005

Single cell glutamate analysis in Aplysia sensory neurons.

Glutamate is the major excitatory neurotransmitter in the central nervous system. However, techniques and assays available for the determination and detection of glutamate are limited. Here we have applied an effective glutamate assay toward the high-throughput analysis of single neurons. Initial physiological studies and recent immunohistochemical data strongly suggested that mechanosensory neurons could use L-glutamate as a co-transmitter (in addition to sensorin) in the sensory-motor synapse of Aplysia californica. We have evaluated the levels of glutamate in these cells and compared them to other non-sensory Aplysia neurons. Since this is the first report of this assay in single cellular analysis, a series of chemical and cellular controls were also done. Based on our results, we were able to determine the concentration levels inside single Aplysia sensory neurons to be 29 mM, with significant heterogeneity between individual cells. In comparison to the pleural mechanosensory neurons, non-sensory abdominal neurons contained approximately 3 mM glutamate. These elevated levels in the sensory neurons confirm the earlier findings [Dale N, Kandel ER. L-glutamate may be the fast excitatory transmitter of Aplysia sensory neurons. Proc Natl Acad Sci USA 1993;90:7163-7.], suggesting that glutamate plays a role as neurotransmitter in these cells.

Animals↗

PubMed2005

Calcium/calmodulin-dependent nitric oxide synthase activity in the CNS of Aplysia californica: biochemical characterization and link to cGMP pathways.

We characterized enzymatic activity of nitric oxide synthase (NOS) in the central nervous system of Aplysia californica, a popular experimental model in cellular and system neuroscience, and provided biochemical evidence for NO-cGMP signaling in molluscs. Aplysia NOS (ApNOS) activity, determined as citrulline formation, revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence and it was inhibited by 50% with 5mM of W7 hydrochloride (a potent Ca/CaM-dependent phosphodiesterase inhibitor). A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia. Specifically, the ApNOS was inhibited by 65-92% with 500 microM of L-NAME (a competitive NOS inhibitor) whereas d-NAME at the same concentration had no effect. S-Ethylisothiourea hydrobromide (5mM), a selective inhibitor of all NOS isoforms, suppressed ApNOS by 85%, l-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL, 5mM), an iNOS inhibitor, by 78% and L-thiocitrulline (5mM) (an inhibitor of nNOS and iNOS) by greater than 95%. Polyclonal antibodies raised against rat nNOS hybridized with a putative purified ApNOS (160 kDa protein) from partially purified central nervous system homogenates in Western blot studies. Consistent with other studies, the activity of soluble guanylyl cyclase was stimulated as a result of NO interaction with its heme prosthetic group. The basal levels of cGMP were estimated by radioimmunoassay to be 44.47 fmol/microg of protein. Incubation of Aplysia CNS with the NO donors DEA/NONOate (diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate - 1mM) or S-nitroso-N-acetylpenicillamine (1mM) and simultaneous phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (1mM) prior to the assay showed a 26-80 fold increase in basal cGMP levels. Addition of ODQ (1H-[1,2,4]oxadiazolo[4,3-a] quinoxaline-1-one - 1mM), a selective inhibitor of soluble guanylyl cyclase, completely abolished this effect. This confirms that NO may indeed function as a messenger in the molluscan CNS, and that cGMP acts as one of its effectors.

Animals↗

PubMed2005

Direct single cell determination of nitric oxide synthase related metabolites in identified nitrergic neurons.

The biochemical characterization of individual nitrergic (NO releasing) neurons is a non-trivial task both in vertebrate and invertebrate preparations. In spite of numerous efforts, there are limited data related to intracellular concentrations of essential metabolites involved in NO synthesis and degradation. This situation creates controversies in both identification of nitrergic neurons and the selection of reliable reporters of NOS activity in heterogeneous cell populations. We take advantage of identified neurons from the pulmonate mollusc Lymnaea stagnalis to perform direct single cell microanalysis of intracellular concentrations of the major nitric oxide synthase (NOS) related metabolites such as arginine, citrulline, argininosuccinate, NO(2)(-),and NO(3)(-). Capillary electrophoresis protocols have been developed to quantitate levels of these metabolites in single identified neurons from the buccal, cerebral, and pedal ganglia using laser-induced fluorescence and conductivity detection. The limits of detection (LODs) for arginine (Arg) and citrulline (Cit) are 84 amol (11nM) and 110 amol (15 nM), respectively, and LODs for NO(2)(-)and NO(3)(-) are <200 amol (<10nM) each. We report that intracellular concentrations of NOS related metabolites are in the millimolar range and less than 1% of a single cell is required for microchemical analysis. From four cell types tested, only the esophageal motoneuron B2 contains active NOS, and they also contain surprisingly high nitrite levels (up to 5mM) compared to other neurons tested (peptidergic B4, dopaminergic RPeD1, and serotonergic CGC). These B2 neurons also exhibit an Arg/Cit ratio susceptible to the selective NOS inhibitor l-iminoethyl-N-ornithine whereas others neurons do not even though they all may contain NOS transcripts. On the contrary, we found that absolute concentrations of other NOS related metabolites including nitrates are not reliable markers of NOS activity and demonstrate the need for multiple assays for NOS activity.

Animals↗

PubMed2004

Monitoring real-time release of ATP from the molluscan central nervous system.

The further understanding of neuronal function is imperative for the prevention and treatment of neurofunctional disorders. To aid in this realization, novel methods for monitoring neuronal cell function must be developed and characterized. In this study, we report the application of real-time imaging of luciferase-catalyzed ATP chemiluminescence for the investigation of ATP release from whole central nervous systems of the freshwater snail Lymnaea stagnalis. Release of ATP from Lymnaea ganglia varied among the different ganglia as well as within individual ganglia. Furthermore, the magnitude of ATP release varied following the stimulation of neurons with common neurotransmitters.

Adenosine Triphosphate↗

PubMed2004

Identification and distribution of a two-pore domain potassium channel in the CNS of Aplysia californica.

A cDNA encoding a potassium channel of the two-pore domain family (K2p) of leak channels was cloned from the CNS of the marine opisthobranch Aplysia californica. This is the first sequence of the K2p family identified in molluscs and has been named AcK2p1. The deduced amino acid sequence is homologous to channels of the mammalian two-pore domain halothane inhibited (THIK) subfamily, bearing 46% identity to THIK-1 (KCNK 13) and 48% to THIK-2 (KCNK12). We used in-situ hybridization to analyze the distribution of this class of channels in the CNS. AcK2p1 is specifically expressed in many central neurons of all major ganglia including the largest identified neurons MCC, R2 and LP1. The highest expression of AcK2p1 was detected in an asymmetrical and distinct cluster of up to 30 cells located at the dorsal-medial region of the right pleural ganglion. The neuron-specific distribution seen in the molluscan CNS is consistent with data from mammals that indicate THIK is only expressed in restricted neuronal populations, suggesting its involvement in both the maintenance of neuronal phenotype and in the specific functional role of these neurons in their respective networks.

Amino Acid Sequence↗

PubMed2004

Somatotopic organization and functional properties of mechanosensory neurons expressing sensorin-A mRNA in Aplysia californica.

A previous study reported that a peptide, sensorin-A, is expressed exclusively in mechanosensory neurons having somata in central ganglia of Aplysia. The present study utilized in situ hybridization, staining by nerve back-fill and soma injection, and electrophysiological methods to characterize the locations, numbers, and functions of sensorin-A-expressing neurons and to define the relationships between soma locations and the locations of peripheral axons and receptive fields. Approximately 1,000 cells express sensorin-A mRNA in young adult animals (10-30 g) and 1,200 cells in larger adults (100-300 g). All of the labeled somata are in the CNS, primarily in the abdominal LE, rLE, RE and RF, pleural VC, cerebral J and K, and buccal S clusters. Expression also occurs in a few sparsely distributed cells in most ganglia. Together, receptive fields of all these mechanosensory clusters cover the entire body surface. Each VC cluster forms a somatotopic map of the ipsilateral body, a "sensory aplunculus." Cells in the pleural and cerebral clusters have partially overlapping sensory fields and synaptic targets. Buccal S cells have receptive fields on the buccal mass and lips and display notable differences in electrophysiological properties from other sensorin-A-expressing neurons. Neurons in all of the clusters have relatively high mechanosensory thresholds, responding preferentially to threatening or noxious stimuli. Synaptic outputs to target cells having defensive functions support a nociceptive role, as does peripheral sensitization following noxious stimulation, although additional functions are likely in some clusters. Interesting questions arise from observations that mRNA for sensorin-A is present not only in the somata but also in synaptic regions, connectives, and peripheral fibers.

Action Potentials↗

PubMed2004

Nitric oxide regulates swimming in the jellyfish Aglantha digitale.

The cnidarian nervous system is considered by many to represent neuronal organization in its earliest and simplest form. Here we demonstrate, for the first time in the Cnidaria, the neuronal localization of nitric oxide synthase (NOS) in the hydromedusa Aglantha digitale (Trachylina). Expression of specific, fixative-resistant NADPH-diaphorase (NADPH-d) activity, characteristic of NOS, was observed in neurites running in the outer nerve ring at the base of the animal and in putative sensory cells in the ectoderm covering its tentacles. At both sites, diphenyleneiodonium (10(-4) M) abolished staining. Capillary electrophoresis confirmed that the NO breakdown products NO2- and NO3- were present at high levels in the tentacles, but were not detectable in NADPH-d-negative areas. The NADPH-d-reactive neurons in the tentacles send processes to regions adjacent to the inner nerve ring where swimming pacemaker cells are located. Free-moving animals and semi-intact preparations were used to test whether NO is involved in regulating the swimming program. NO (30-50 nM) and its precursor L-arginine (1 mM) stimulated swimming, and the effect was mimicked by 8-Br-cGMP (50-100 microM). The NO scavenger PTIO (10-100 microM) and a competitive inhibitor of NOS, L-nitroarginine methyl ester (L-NAME, 200 microM), significantly decreased the swimming frequency in free-moving animals, while its less-active stereoisomer D-nitroarginine methyl ester (D-NAME, 200 microM) had no such effect. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 5-20 microM), a selective inhibitor of soluble guanylyl cyclase, suppressed spontaneous swimming and prevented NO-induced activation of the swimming program. We suggest that an NO/cGMP signaling pathway modulates the rhythmic swimming associated with feeding in Aglantha, possibly by means of putative nitrergic sensory neurons in its tentacles.

Animals↗

PubMed2004

Simple cDNA normalization using kamchatka crab duplex-specific nuclease.

We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.

Animals↗

PubMed2004

RFa-related peptides are algogenic: evidence in vitro and in vivo.

RFamide (RFa)-related peptides modulate pain processing in the mammalian CNS. The effects of these peptides are generally considered as 'anti-opioid'. They also decrease the rate of desensitization of acid-sensing ionic channels (ASICs), putative nociceptors in dorsal root ganglia neurons [C. Askwith et al. (2000) Neuron, 26, 133-141]. We have tested the role of mollusc-derived peptide, FMRFa (Phe-Met-Arg-Phe-amide) and its synthetic analogues in peripheral nociception. Here we demonstrate that RFa-related peptides powerfully excite the majority of C-fibres in the skin-nerve preparation of rat: 76% of 55 tested fibres with the conduction velocity below 2 m/s responded with long-lasting discharges to the application of peptides (20 microm). When injected subcutaneously in vivo (mice), they initiate nociceptive behaviour. We confirm the data on humans [S. Ugawa et al. (2002) J. Clin. Invest., 110, 1185-1190]: the activation of C-fibres by acid is inhibited by channel blocker of ASICs, amiloride. However, there is no correlation in the sensitivity of C-fibres to RFa peptides, protons and amiloride: 74% of tested RFa-sensitive C-fibres were insensitive to protons and in 67% of cases the response to peptides was insensitive to amiloride. Thus, powerful excitatory/algogenic action of RFa-related peptides cannot be interpreted solely in terms of their interaction with ASICs. The peptides do not activate any conductance in the somatic membrane of dorsal root ganglion neurons of rats and probably affect still unidentified molecular target(s) responsible for nociceptive signalling.

Animals↗

PubMed2002

Ascorbic acid assays of individual neurons and neuronal tissues using capillary electrophoresis with laser-induced fluorescence detection.

Ascorbic acid is an important cellular metabolite involved in many biochemical pathways. A method to quantitate ascorbic acid and dehydroascorbic acid in individual neurons and neuronal tissues is described with detection limits of 320 pM (430 zmol). The method uses microvial sampling, derivatization with 4,5-dimethyl-1,2-phenylenediamine, capillary electrophoresis separation, and laser-induced fluorescence detection and quantifies the ascorbic acid and dehydroascorbic acid levels with less than a 15-min total analysis time including sample preparation and derivatization. Ascorbic acid and dehydroascorbic acid levels are measured using functionally characterized and identified neurons of Aplysia californica, Pleurobranchaea californica, and Lymnaea stagnalis -three well-recognized models in cellular and system neuroscience. Multiple assays of a particular identified neuron (e.g., metacerebral cells from Aplysia) show a high level of reproducibility, while endogenous intracellular concentrations of ascorbate are neuron-specific. Ascorbic acid concentrations in the neurons studied range from 0.19 to 6.2 mM for Aplysia and 0.12 to 0.22 mM for Lymnaea. In contrast, concentrations of ascorbic acid observed in heterogeneous tissues such as ganglia (with connective tissues, glia, blood vessels, neuropile, and areas with intercellular spaces), 4-190 microM, are significantly lower than the single-cell values.

Animals↗

PubMed2002

Interfering with nitric oxide measurements. 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid.

4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N(2)O(3) from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry.

Animals↗

PubMed2002

High-resolution microanalysis of nitrite and nitrate in neuronal tissues by capillary electrophoresis with conductivity detection.

Nitrites and nitrates are widely used reporters of endogenous activity of nitric oxide synthases (NOS), an important group of enzymes producing the gaseous signal molecule nitric oxide (NO). However, due to the great chemical heterogeneity of neuronal tissues, standard analytical protocols for evaluation of neuronal nitrite/nitrate concentrations are inefficient. We optimized a high-performance capillary zone electrophoresis (CZE) technique to analyze nitrite/nitrate concentrations in submicroliter samples from mammalian neuronal tissues. The measurements were made using a PrinCE 476 computerized capillary electrophoresis system with a Crystal 1000 contact conductivity detector. Isotachophoretic stacking injection of 1000- to 10000-fold diluted samples, which had been pretreated with a custom-designed solid-phase microextraction (SPME) cartridge, was employed to assay micromolar and nanomolar nitrite and nitrate levels in the presence of the high millimolar chloride concentrations characteristic of many biological samples. In the presented technique, a 10-microl volume of diluted ganglionic sample was used for chloride removal and sample cleanup. The method yields high analytical performance, including good reproducibility, resolution, and accuracy. The limits of detection relative to undiluted sample matrix were 8.9 nM (0.41 ppb) and 3.54 nM (0.22 ppb) for nitrite and nitrate, respectively. In addition, this technique resolves other anions that are present in neuronal tissues at sub-nanomolar concentrations and can be broadly applied for high-throughput anionic profiling. In rat dorsal root ganglia, endogenous levels of nitrate (231+/-29 microM; n=6) and nitrite (24-96 microM) were found. These concentrations exceeded those previously found in neuronal tissue homogenates using different techniques.

Electrophoresis, Capillary↗