Michiko Ohtomi

Distribution of FMRFamide-like immunoreactivity in the brain and neurohypophysis of the lamprey, Lampetra japonica

SummaryDistribution of molluscan cardio-excitatory tetrapeptide Phe—Met—Arg—Phe—NH2 (FMRFamide) was determined by means of immunohistochemistry in the brain and neurohypophysis of the lamprey, Lampetra japonica. Many FMRFamide-like immunoreactive neurons were found in the periventricular nuclear region and in a region near the mammillary recess. Neurons situated in the former region were larger. The immunoreactive cell groups were shown to be located at sites differing from those of the AF-positive cell groups. The fibers of immunoreactive neurons extended in all directions within the brain and towards the spinal cord, some reaching the third ventricle and capillaries. Thus, FMRFamide-like immunoreactive peptides appear to function as neurotransmitters or neuromodulators and possibly also as neurohormones. FMRFamide-like immunoreactive material was rarely observed in the posterior neurohypophysis (neural lobe), but was noted to be present to a limited extent in the caudal part of the anterior neurohypophysis (median eminence). It would thus follow that FMRFamide-like immunoreactive neurons may not necessarily be related to the hypothalamo-neural lobe system, but may possibly be associated with the hypothalamoadenohypophysial system. The pineal body showed no FMRFamide-like immunoreactivity.

Identification of addicsin/GTRAP3-18 as a chronic morphine-augmented gene in amygdala.

Using subtractive cloning, we identified a 1.4 kb mRNA that was ubiquitously expressed in various tissues; this mRNA was highly up-regulated in amygdala nuclei in mice when morphine was repeatedly administered but not when an opiate-receptor antagonist was co-administered. The mRNA encodes a 23 kDa protein, designated ‘addicsin’. This contains two putative PKC-phosphorylation motifs and several hydrophobic regions, and was recovered in a soluble protein fraction of brain lysate. Its primary structure showed 98% identity with that of rat glutamate-transporter-associated protein 3-18 (GTRAP3-18), a putative modulator of neural glutamate-transporter EAAC1. Up-regulation of addicsin expression by morphine may affect glutamate uptake in the amygdala, causing mice to develop morphine tolerance and dependence.

Selective complement C1s deficiency caused by homozygous four-base deletion in the C1s gene.

The complement system plays an important role in defense mechanisms by promoting the adherence of microorganisms to phagocytic cells and lysis of foreign organisms. Deficiencies of the first complement components, C1r/C1s, often cause systemic lupus erythema-tosus-like syndromes and severe pyogenic infections. Up to now no genetic analysis of the C1r/C1s deficiencies has been carried out. In the present work, we report the first genetic analysis of selective C1s deficiency, the patient having a normal amount of C1r. C1s RNA with a normal size was detected in patient’s subcutaneous fibroblasts (YKF) by RNA blot analysis and RT-PCR. The amount of C1s RNA was approximately one-tenth of the RNA from the human chondrosarcoma cell line, HCS2/8. In contrast, the levels of C1r and β-actin RNA of YKF were similar to that of HCS2/8. Sequence analysis of C1s cDNA revealed a deletion at nucleotides 1087–1090 (TTTG), creating a stop codon (TGA) at position 94 downstream of the mutation site. Direct sequencing of the gene between the primers designed on intron 9 and exon 10 indicated the presence of the deletion on exon 10 of the gene. Quantitative Southern blot hybridization suggested the mutation was homozygous. The 4-bp deletion on exon 10 was also found in the patient’s heterozygous mother who had normal hemolytic activity.

Toxicity of coelomic fluid of the earthworm Eisenia foetida to vertebrates but not invertebrates: probable role of sphingomyelin

The coelomic fluid (CF) of the earthworm Eisenia foetida exhibits a wide variety of biological activities. We found that the CF was not toxic to 42 species, belonging to seven invertebrate phyla, almost all in aquatic adults and larvae exposed to CF. Eleven teleostean species tested died in 0.2-1% CF mostly between 10 and 120 min and the effects were dose-dependent. Tadpoles of the toad Bufo japonicus formosus died in 0.4-2% CF between 80 and 225 min depending upon size, with larger tadpoles surviving longer. Before dying, all experimental tadpoles developed curled and shrunken tails. The Okinawa tree lizard, soft-shelled turtle, Japanese quail, mouse and rat all died after i.v. injection of CF (above 20 microl/kg). Thus, CF was not toxic to invertebrates, but toxic to vertebrates. After heating, CF lost its toxicity to fish, tadpoles and mice. Both CF and lysenin incubated with sphingomyelin-liposomes (SM-liposomes) were no longer toxic, suggesting the involvement of SM in the toxicity. Lysenin, which is a constituent of CF and known to bind specifically to sphingomyelin, exhibited toxicity similar to that of CF. Thus, lysenin in CF is probably responsible for the toxic effects of CF by binding to SM in vertebrate tissues. The bodies of invertebrates might contain little or no SM, while those of vertebrates do contain SM. The coelomic fluid of the earthworm Pheretima communissima has no toxicity to mouse.

In vivo temporal property of GABAergic neural transmission in collateral feed-forward inhibition system of hippocampal–prefrontal pathway

Anatomical evidence suggests that rat CA1 hippocampal afferents collaterally innervate excitatory projecting pyramidal neurons and inhibitory interneurons, creating a disynaptic, feed-forward inhibition microcircuit in the medial prefrontal cortex (mPFC). We investigated the temporal relationship between the frequency of paired synaptic transmission and gamma-aminobutyric acid (GABA)ergic receptor-mediated modulation of the microcircuit in vivo under urethane anesthesia. Local perfusions of a GABAa antagonist (-)-bicuculline into the mPFC via microdialysis resulted in a statistically significant disinhibitory effect on intrinsic GABA action, increasing the first and second mPFC responses following hippocampal paired stimulation at interstimulus intervals of 100-200 ms, but not those at 25-50 ms. This (-)-bicuculline-induced disinhibition was compensated by the GABAa agonist muscimol, which itself did not attenuate the intrinsic oscillation of the local field potentials. The perfusion of a sub-minimal concentration of GABAb agonist (R)-baclofen slightly enhanced the synaptic transmission, regardless of the interstimulus interval. In addition to the tonic control by spontaneous fast-spiking GABAergic neurons, it is clear the sequential transmission of the hippocampal-mPFC pathway can phasically drive the collateral feed-forward inhibition system through activation of a GABAa receptor, bringing an active signal filter to the various types of impulse trains that enter the mPFC from the hippocampus in vivo.

Regulation of neurite extension by expression of LECT2 and neurotrophins based on findings in LECT2-knockout mice.

Leukocyte cell-derived chemotaxin 2 (LECT2) was first isolated as a chemotactic factor from phytohemagglutinin-activated human T-cell leukemia SKW-3 cells. LECT2 is expressed in various tissues, including in the brain, stomach and liver, but the functions of LECT2 in the brain remains unclear. To elucidate these functions, we investigated the influence of a deficiency of LECT2 on the morphology of cultured hippocampal neurons during neuronal development, and examined the expression of neurotrophins (NGF, BDNF, and NT-3) and their receptors (TrkA, TrkB, TrkC, and p75NTR) in these neurons. The extension of axons and dendrites in neurons from LECT2-knockout (LECT2-KO) mice was shorter than that in neurons from wild-type mice during culture and significantly less than that in wild-type mice after 4 days in culture. Moreover, neurons from LECT2-KO mice showed different expression of NGF, BDNF and NT-3 during culture compared to wild-type mice. Our results show that LECT2 regulates the extension of axons and dendrites and the expressions of NGF, BDNF and NT-3 during neuronal development.

Synaptic plasticity dynamics in the hippocampal-prefrontal pathway in vivo.

We examined the effect of long-term potentiation (LTP) on paired-pulse responses across varied stimulus intensities and interstimulus intervals (ISIs), at ascending synapses from the intermediate and ventral hippocampus to the medial prefrontal cortex in urethane-anesthetized rats. LTP significantly shifted the median effective stimulus towards lower intensities in the intermediate route, and increased at 25-ms ISI the paired-pulse response, which was inversely proportional to the stimulus intensity. In the ventral route, the paired-pulse response varied with ISI rather than intensity, and increased at 50-ms and 100-ms ISI after LTP. The intermediate synaptic plasticity significantly exhibited total amplifier dynamics with wide ranges of frequency at lower intensity and intensity at 100-ms ISI in contrast to the ventral one.

The Catalytic Effect of Crown Ethers on Benzoin Condensation

Aromatic aldehydes were treated with potassium cyanide in the presence of a crown ether in water or in aprotic solvents to give benzoins in high yields, although similar reactions in the absence of the crown ether proceeded sluggishly in general. The catalysis is believed to be due to the ability of the crown ether to bring anions into the organic phase.

Regulation of katanin-P60 levels by LECT2 adjusts microtubular morphology

Leukocyte cell-derived chemotaxin 2 (LECT2) facilitates neuritic extension in cultured hippocampal neurons during initial development. However, the functions of LECT2 in neuritic extension are poorly understood. To elucidate these functions, we examined microtubular morphology and levels of katanin-P60, a microtubule-severing enzyme, in cultured hippocampal neurons from wild-type mice and LECT2 knockout (KO) mice. Microtubules in LECT2-KO mice exhibited fragmentation and were shorter than those of wild-type mice. Furthermore, the expression of katanin-P60 in LECT2-KO mice was significantly elevated when compared with wild-type mice at 1 day in vitro (DIV1) and 4. Our results suggest that LECT2 regulates neuritic extension through microtubular morphallaxis through the control of katanin-P60 levels.