Ryota Matsuo

Identification and Cataloging of Genes Induced by Long‐Lasting Long‐Term Potentiation in Awake Rats

Abstract: Maintenance of long‐term potentiation (LTP) requires de novo gene expression. Here we report the direct isolation, using PCR‐differential display, of genes whose expression level was altered after induction of long‐lasting LTP in the hippocampus of freely moving awake rats. Differential display using 480 primer combinations revealed 17 cDNA bands that showed a reproducible change in expression level. These cDNAs represented at least 10 different genes (termed RM1‐10), all of which showed up‐regulation at 75 min after LTP induction and a return to basal expression levels within 24 h. Three of these genes were known only from expressed sequence tags (RM1‐3), two were known genes whose up‐regulation by LTP has not been described (GADD153/CHOP and ler5), and five were known genes whose up‐regulation by LTP has already been reported (MAPK phosphatase, NGFI‐A/zif268, vesl‐1S/homer‐1a, Ag2, and krox‐20). We characterized the expression profiles of genes in the two former categories with respect to NMDA receptor dependency, tissue specificity, and developmental regulation using northern blotting and semiquantitative RT‐PCR. The up‐regulation of all five of these genes was NMDA receptor‐dependent and correlated with the persistence of LTP, suggesting that these genes may play functional roles in prolonged LTP maintenance.

Regulated expression of an actin-associated protein, synaptopodin, during long-term potentiation

We report NMDA receptor‐dependent expression of synaptopodin mRNA in the dentate granule cells of the hippocampus following induction of long‐term potentiation (LTP) in vivo. Synaptopodin did not belong to immediate‐early genes, as de novo protein synthesis was required for the induction of synaptopodin gene transcription. An increased level of synaptopodin mRNA was observed at 75 min and 3.5 h after the onset of LTP. Importantly, there was correlation between the induction of mRNA expression and the persistence of LTP. Synaptopodin immunoreactivity was elevated specifically in synaptic layers, middle and outer molecular layers of dentate gyrus where LTP was induced. As synaptopodin is an actin‐associated protein present in spine neck and implicated in the modulation of cell morphology, our results suggest that synaptopodin, by regulating the dynamics of the actin cytoskeleton, contributes to the morphological change in spine shape considered to be important for the maintenance of synaptic plasticity.

A novel nitric oxide synthase expressed specifically in the olfactory center

Nitric oxide (NO) plays important roles in the olfactory center of various animals. In the terrestrial slug, NO is indispensable for field potential oscillation in the higher olfactory center, the procerebrum (PC), and also for odor learning. Here we identify a novel NO synthase (NOS) gene, limNOS2, in the terrestrial slug. The mRNA (approximately 10kb) of limNOS2 encodes a protein consisting of 1616 amino acids, including a PDZ domain. The protein has 70.0% sequence identity with the previously identified limNOS1 gene. In contrast to limNOS1, however, limNOS2 is expressed specifically in the PC. Moreover, most of the cells in the PC contain limNOS2 mRNA, indicating that the nonbursting neurons, the major constituent of the PC, have this mRNA. The expression pattern of limNOS2 conforms well to the pattern of NOS enzymatic histochemical staining. Our present findings indicate that limNOS2 is responsible for most of the NO generation in the PC.

Delayed-onset amnesia caused by protein synthesis inhibition in odor-taste associative memory of the terrestrial slug Limax valentianus

The terrestrial slug Limax avoids the odor of innately preferred food after it is presented with the aversive taste of quinidine. This type of associative memory persists for several weeks. We investigated effects of protein synthesis inhibitors on the retention of this odor-taste associative memory. Anisomycin or cycloheximide applied to the slug 30 min prior to conditioning impaired the memory retention as late as two or more days after the conditioning, while the retention of a short-term memory was normal for up to 24 h. In contrast, the inhibition of protein synthesis by these inhibitors decays within several hours. In Limax, the onset of amnesia is unusually delayed when protein synthesis is suppressed at the time of conditioning.

Glutamatergic neurotransmission in the procerebrum (Olfactory Center) of a terrestrial mollusk

The terrestrial slug Limax has the ability to learn odor associations. This ability depends on the function of the procerebrum, the secondary olfactory center in the brain. Among the various neurotransmitters that are thought to be involved in the function of the procerebrum, glutamate is one of the most important molecules. However, the existence and function of glutamate in this system have been proposed solely on the basis of a few lines of indirect evidence from pharmacological experiments. In the present study, we demonstrated the existence and release of glutamate as a neurotransmitter in the procerebrum of Limax, by using three different techniques: 1) immunohistochemistry of glutamate, 2) in situ hybridization to mRNA of the vesicular glutamate transporter, and 3) real‐time imaging of glutamate release within the procerebrum using the glutamate optical sensor EOS2. The release of glutamate within the cell mass layer of the procerebrum was synchronized with oscillation of the local field potential and had the same physiological properties as this oscillation; both were blocked by a serotonin antagonist and were propagated in an apical to basal direction in the procerebrum. Our observations suggest strongly that the oscillation of the local field potential is driven by the glutamate released by bursting neurons in the procerebrum.

FMRFamide regulates oscillatory activity of the olfactory center in the slug

In the olfactory center of terrestrial animals, changes in the oscillatory frequency of the local field potential (LFP) are thought to be involved in olfaction‐based behavior and olfactory memory. The terrestrial slug Limax has a highly developed olfactory center, the procerebrum, in which the LFP spontaneously oscillates. Although changes in the oscillatory frequency are thought to correspond to the preference for specific odors, our knowledge about the mechanism of this frequency regulation is limited. To clarify the mechanism of the bidirectional frequency changes in the procerebrum, we focused on the neuropeptide Phe‐Met‐Arg‐Phe‐NH2 (FMRFamide), which is known to have neuromodulatory functions in invertebrate nervous systems. Application of FMRFamide decreased the oscillatory frequency via G‐protein‐mediated cascades. Immunohistochemistry showed that FMRFamide‐like‐immunoreactive neuronal cell bodies are located in the cell mass layer of the procerebrum, projecting their neurites to the neuropile layers. The procerebrum was shown to also receive innervation from other regions of the cerebral ganglion. Furthermore, according to their morphological and projection characteristics, FMRFamide‐containing neurons belong to the subpopulations of both bursting and nonbursting neurons in the procerebrum. The mRNA splice variant encoding multiple copies of canonical FMRFamide was specifically expressed in the procerebrum. Taking into account previous results showing that serotonin increases the oscillatory frequency, our results indicate that FMRFamide and serotonin both regulate the LFP frequency but in exactly the opposite direction in the olfactory center of the terrestrial slug.

Genomic structure of nitric oxide synthase in the terrestrial slug is highly conserved

Nitric oxide (NO) is a key molecule in olfactory information processing across animal species. To gain insight into the genetic basis of NO generation, we investigated the genomic structure of nitric oxide synthase (NOS) in the terrestrial slug Limax because slugs use olfaction as their primary food detection system. The full length cDNA of limNOS encodes a protein consisting of 1632 amino acids that has a PSD-95/discs-large/ZO-1 (PDZ) domain in its N-terminus and 6 other cofactor-binding domains. The limNOS gene consists of 33 exons and spans at least 107 kb of the genome. Almost all the exon-intron boundaries are conserved in Limax and human nNOS and the organization of the Limax genome is more similar to that of humans than to Drosophila, indicating that there was an accelerated evolution of the Drosophila genome during evolution. These results imply that there was a highly conservative selective pressure imposed on NOS gene structure during the evolution of mollusks and vertebrates.

Development- and activity-dependent regulation of SNAP-25 phosphorylation in rat brain

Synaptosomal-associated protein of 25kDa (SNAP-25), a member of the SNARE proteins essential for neurotransmitter release, is phosphorylated at Ser(187) in PC12 cells and in the rat brain in a protein kinase C-dependent manner. It remains unclear how the phosphorylation of SNAP-25 is regulated during development and by neuronal activity. We studied the mode of SNAP-25 phosphorylation at Ser(187) in the rat brain using an anti-phosphorylated SNAP-25 antibody. Both the expression and phosphorylation of SNAP-25 increased remarkably during the early postnatal period, but their onsets were quite different. SNAP-25 expression was detected as early as embryonic Day 18, whereas the phosphorylation of SNAP-25 could not be detected until postnatal Day 4. A delay in the onset of phosphorylation was also observed in cultured rat hippocampal neurons. The phosphorylation of SNAP-25 was regulated in a neuronal activity-dependent manner and, in the rat hippocampus, decreased by introducing seizures with kainic acid. These results clearly indicated that the phosphorylation of SNAP-25 at Ser(187) is regulated in development- and neuronal activity-dependent manners, and is likely to play important roles in higher brain functions.

Effects of tentacle amputation and regeneration on the morphology and activity of the olfactory center of the terrestrial slug Limax valentianus

SUMMARY The tentacles of pulmonates regenerate spontaneously following amputation. The regenerated tentacle is equipped with all the elements necessary for normal olfactory functioning, and the slugs can behave as well as they did before the tentacle amputation. However, it is not known what changes occur to the olfactory center procerebrum in the brain at the morphological and physiological levels. Here, we investigated the innervation of tentacular nerves into the procerebrum by examining the size of the terminal mass (input layer from tentacular nerves) of the procerebrum and also by staining afferent nerves immunohistochemically at 15, 58 and 75 days following unilateral amputation of the superior and inferior tentacles. The size of the terminal mass was significantly decreased, and the Phe-Met-Arg-Phe-NH2ergic (FMRFamidergic) afferent nerves disappeared by 15 days following the tentacle amputation. However, the size of the terminal mass had recovered substantially by 58 days, as the tentacle regenerated. The FMRFamidergic innervation into the cerebral ganglion was also restored by this time. An extended recovery (75 days), however, did not result in any further increase in the size of the terminal mass. We also recorded the local field potential (LFP) oscillation in the procerebrum. We found that the oscillatory frequency of the LFP had decreased at 15 days following the tentacle amputation but had recovered at 58 and 75 days. These results suggest that the amputation and regrowth of the tentacle are accompanied by the respective degeneration and re-innervation of olfactory nerves, and these changes in the innervation status affect the basal state of LFP oscillation.

DNA Endoreplication in the Brain Neurons during Body Growth of an Adult Slug

Endoreplication is DNA synthesis without cell division. Giant neurons observed in the brains of mollusks are thought to be generated as a result of DNA endoreplication. It has been hypothesized that neuronal size becomes larger in parallel with an increase in body size and that DNA endoreplication is involved in this process to meet the increasing demand for macromolecules in neurons. There is, however, no experimental evidence for this hypothesis to date. In the present study, we investigated the following quantitatively: (1) the size of the brain and each ganglion, (2) the size of identified neurons, (3) the total number of neurons undergoing DNA endoreplication, (4) the total number of the neurons containing a cardioexcitatory peptide, and (5) the gene expression level per neuron, using terrestrial slugs whose body growth was regulated through the amount of food supplied in the laboratory. The body growth was accompanied by increases in the sizes of both neurons and ganglia and triggered more frequent DNA endoreplication events in each ganglion of the growth-promoted slugs, without increasing the total number of neurons. Increase in the neuronal size also involved the increase in the amount of transcripts expressed in a single neuron. This is the first quantitative evidence showing that the DNA endoreplication, neuronal size, and gene expression are increased concomitantly with body growth in adult mollusks.