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

Jezzini, Sami H.; Bodnárová, Michaela; Moroz, Leonid L.

doi:10.1016/j.jneumeth.2005.05.007

Cited by 35 publications

(37 citation statements)

References 35 publications

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“…The methods for probe preparation and in situ hybridization as well as Lucifer yellow double labeling have also been described previously (Jezzini et al, 2005). Briefly, in situ riboprobes were prepared using cloned full-length NOS cDNA from Aplysia californica (AcNOS-1; GenBank accession number AAK83069) (Sadreyev et al, 2000) (for details of the molecular data and the relationships between different NOS isoforms in mollusks, see Moroz et al, 2006;Moroz and Kohn, 2007).…”

Section: Methodsmentioning

confidence: 99%

Role of Nitric Oxide in Classical Conditioning of Siphon Withdrawal inAplysia

Antonov

Ha²,

Antonova³

et al. 2007

J. Neurosci.

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Nitric oxide (NO) is thought to be involved in several forms of learning in vivo and synaptic plasticity in vitro, but very little is known about the role of NO during physiological forms of plasticity that occur during learning. We addressed that question in a simplified preparation of the Aplysia siphon-withdrawal reflex. We first used in situ hybridization to show that the identified L29 facilitator neurons express NO synthase. Furthermore, exogenous NO produced facilitation of sensory-motor neuron EPSPs, and an inhibitor of NO synthase or an NO scavenger blocked behavioral conditioning. Application of the scavenger to the ganglion or injection into a sensory neuron blocked facilitation of the EPSP and changes in the sensory-neuron membrane properties during conditioning. Injection of the scavenger into the motor neuron reduced facilitation without affecting sensory neuron membrane properties, and injection of an inhibitor of NO synthase had no effect. Postsynaptic injection of an inhibitor of exocytosis had effects similar to injection of the scavenger. However, changes in the shape of the EPSP during conditioning were not consistent with postsynaptic AMPA-like receptor insertion but were mimicked by presynaptic spike broadening. These results suggest that NO makes an important contribution during conditioning and acts directly in both the sensory and motor neurons to affect different processes of facilitation at the synapses between them. In addition, they suggest that NO does not come from either the sensory or motor neurons but rather comes from another source, perhaps the L29 interneurons.

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Section: Methodsmentioning

confidence: 99%

Role of Nitric Oxide in Classical Conditioning of Siphon Withdrawal inAplysia

Antonov

Ha²,

Antonova³

et al. 2007

J. Neurosci.

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“…The DIG-labeled antisense probes were hybridized to NR1 mRNA in whole-mount CNS preparations, and the neurons containing the probe-target duplex were localized and visualized with alkaline phosphatase-conjugated anti-DIG antibody fragments (Boehriger Mannheim). The detailed in situ hybridization protocol has been described Walters et al, 2004;Jezzini et al, 2005).…”

Section: Cloning Of Full-length Cdna Encoding Nmda-like Receptorsmentioning

confidence: 99%

Molecular Characterization of NMDA-Like Receptors inAplysiaandLymnaea:Relevance to Memory Mechanisms

Kohn

Bobkova

et al. 2006

The Biological Bulletin

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Abstract. The N-methyl-D-aspartate (NMDA) receptor belongs to the group of ionotropic glutamate receptors and has been implicated in synaptic plasticity, memory acquisition, and learning in both vertebrates and invertebrates, including molluscs. However, the molecular identity of NMDA-type receptors in molluscs remains unknown. Here, we cloned two NMDA-type receptors from the sea slug Aplysia californica, AcNR1-1 and AcNR1-2, as well as their homologs from the freshwater pulmonate snail Lymnaea stagnalis, LsNR1-1 and LsNR1-2. The cloned receptors contain a signal peptide, two extracellular segments with predicted binding sites for glycine and glutamate, three recognized transmembrane regions, and a fourth hydrophobic domain that makes a hairpin turn to form a pore-like structure. Phylogenetic analysis suggests that both the AcNR1s and LsNR1s belong to the NR1 subgroup of ionotrophic glutamate receptors. Our in situ hybridization data indicate highly abundant, but predominantly neuron-specific expression of molluscan NR1-type receptors in all central ganglia, including identified motor neurons in the buccal and abdominal ganglia as well as groups of mechanosensory cells. AcNR1 transcripts were detected extrasynaptically in the neurites of metacerebral cells of Aplysia. The widespread distribution of AcNR1 and LsNR1 transcripts also implies diverse functions, including their involvement in the organization of feeding, locomotory, and defensive behaviors.

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“…VEs were dissected in Limulus saline (Warren and Pierce, 1982), fixed for 2 h on ice in 4% paraformaldehyde in phosphate-buffered saline (PBS: 11.9 mmol l −1 , pH 7.2, 0.5 mol l −1 NaCl) and processed for in situ hybridization as whole mounts (Jezzini et al, 2005). The probe was visualized with Fab fragments of an anti-digoxigenin-alkaline phosphatase antibody, followed by the NBT/BCIP reaction (Roche Applied Science).…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Opsins inLimuluseyes: Characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes

Battelle

Kempler

Saraf

et al. 2014

Journal of Experimental Biology

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The eyes of the horseshoe crab Limulus polyphemus have long been used for studies of basic mechanisms of vision, and the structure and physiology of Limulus photoreceptors have been examined in detail. Less is known about the opsins Limulus photoreceptors express. We previously characterized a UV opsin (LpUVOps1) that is expressed in all three types of Limulus eyes (lateral compound eyes, median ocelli and larval eyes) and three visible light-sensitive rhabdomeric opsins (LpOps1, -2 and -5) that are expressed in Limulus lateral compound and larval eyes. Physiological studies showed that visible light-sensitive photoreceptors are also present in median ocelli, but the visible light-sensitive opsins they express were unknown. In the current study we characterize three newly identified, visible lightsensitive rhabdomeric opsins (LpOps6, -7 and -8) that are expressed in median ocelli. We show that they are ocellar specific and that all three are co-expressed in photoreceptors distinct from those expressing LpUVOps1. Our current findings show that the pattern of opsin expression in Limulus eyes is much more complex than previously thought and extend our previous observations of opsin coexpression in visible light-sensitive Limulus photoreceptors. We also characterize a Limulus peropsin/RGR (LpPerOps1). We examine the phylogenetic relationship of LpPerOps1 with other peropsins and RGRs, demonstrate that LpPerOps1 transcripts are expressed in each of the three types of Limulus eyes and show that the encoded protein is expressed in membranes of cells closely associated with photoreceptors in each eye type. These finding suggest that peropsin was in the opsin repertoire of euchelicerates.

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

Cited by 35 publications

References 35 publications

Role of Nitric Oxide in Classical Conditioning of Siphon Withdrawal inAplysia

Role of Nitric Oxide in Classical Conditioning of Siphon Withdrawal inAplysia

Molecular Characterization of NMDA-Like Receptors inAplysiaandLymnaea:Relevance to Memory Mechanisms

Opsins inLimuluseyes: Characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes

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