Kanako Hirata

Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals

Complexins are presynaptic proteins whose functional roles in synaptic transmission are still unclear. In cultured rat hippocampal neurons, complexins are distributed throughout the cell bodies, dendrites and axons, whereas synaptotagmin I and synaptobrevin/VAMP‐2, essential proteins for neurotransmitter release, accumulated in the synaptic‐releasing sites as early as 1 week in culture. With a maturation of synapses in vitro, complexins also accumulated in the synaptic release sites and co‐localized with synaptotagmin I and synaptobrevin/VAMP‐2 after 3–4 weeks in culture. Complexins I and II were expressed in more than 90 and 70% of the cultured neurons, respectively; however, they were largely distributed in different populations of synaptic terminals. In the developing rat brain, complexins were distributed in neuronal cell bodies in the early stage of postnatal development, but gradually accumulated in the synapse‐enriched regions with development. In mature presynaptic neurons of Aplysia buccal ganglia, injection of anticomplexin II antibody caused a stimulation of neurotransmitter release. Injection of recombinant complexin II and αSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant αSNAP, and vice versa. These results suggest that complexins are not essential but have some regulatory roles in neurotransmitter release from presynaptic terminals of mature neurons.

Cogeneration of neurons with a unique molecular phenotype in layers V and VI of widespread lateral neocortical areas in the rat

Monoclonal antibody PC3.1 detects a unique subpopulation of neurons located mainly in layer VI and, to a lesser extent, in layer V within the lateral neocortical areas in the rat. In an attempt to characterize these neurons, we determined the time of their generation in selected neocortical areas by a double-labeling experiment combining quantitative long-survival 3H-thymidine autoradiography and immunohistochemistry for the PC3.1 antigen. We found that the vast majority of PC3.1-positive neurons in both layers V and VI were generated concurrently at embryonic day 15 in all areas examined, demonstrating a strict correlation between the molecular identity of neurons and the time of their generation, irrespective of their final positions along the radial and tangential axes. In contrast, PC3.1- negative neurons, which should represent more diverse phenotypic identities, were generated during a more extended period of cortical development and tended to exhibit radial (inside-to-outside) and tangential (ventral-to-dorsal and rostral-to-caudal) neurogenetic gradients. Our findings indicate that laminar and tangential locations of cortical neurons are not established solely by a combination of mechanisms for the inside-out movement of newly generated neurons in each cortical area and for the broad tangential neurogenetic gradients. The results of this study suggest a distinct way of cortical development in which neurons with a common molecular phenotype are generated concurrently and migrate toward their eventual positions, which are not necessarily located in a single lamina. In addition, our results suggest some kind of tangential heterogeneity in the mechanism involved in neocortical histogenesis, supporting the concept of early regional specification within the neocortex.

Genetic interactions of pokkuri with seven in absentia, tramtrack and downstream components of the sevenless pathway in R7 photoreceptor induction in Drosophila melanogaster

The sevenless (sev) cascade plays an inductive role in formation of the R7 photoreceptor, whilst the pokkuri (pok) and tramtrack (ttk) gene products are known to repress R7 induction in developing ommatidia of Drosophila melanogaster. To elucidate how these positive and negative signalling mechanisms co-operate in the normal fate determination of R7, genetic interactions of mutations in the pok locus with ttk and downstream elements of sev including Gap1, raf1, rolled (r1) and seven in absentia (sina) were examined. The eye phenotype of a weak hypomorph, pok15, was enhanced dominantly by Gap1-mip, a recessive mutation in a gene encoding a down-regulator of Ras1, producing multiple R7 in ommatidia. Ras1 has been reported to activate r1-encoded mitrogen-activated protein (MAP) kinase via Raf1 that is associated physically with Rasl. Ommatidia of raf1c110 and rl2/rlEMS64 typically lacked R7 and a few outer photoreceptors. The pok1 mutation suppressed dominantly the raflc110 rl2/rlEMS64 eye phenotypes, allowing single R7 cells to develop in ommatidia. The raflc110 mutation improved adult viability of pok1 homozygotes. An in vitro experiment demonstrated that MAP kinase phosphorylates Pok protein. Ttk is a transcriptional repressor which binds to the regulatory sequence upstream of the fushi-tarazu (ftz), even skipped (eve) and engrailed (en) coding region. A reduced activity in ttk resulted in enhancement of the pok phenotype. ttk mutations produced extra R7 cells even in sina homozygotes whilst the pok mutation did not. This result indicates that Ttk represses R7 induction downstream of the sites where Pok and Sina function.

Colocalization of latexin with carboxypeptidase A in the infragranular neurons of cerebral cortex

Latexin is a carboxypeptidase A (CPA) inhibitor expressed in certain cortico-cortical projection neurons located in the infragranular layers of lateral cortical areas of adult rats and mice. To understand a physiological function of latexin in the cerebral cortex, we previously analyzed colocalization of latexin with protein phosphatase inhibitor-l and with Al-opioid receptor (K. Takiguchi-Hayashi & Y. Arimatsu, Neurosci. Res. Suppl. 21. S44, 1997). In the present study, we examined colocalization of latexin with CPA by immunohistochemistry. Restricted and similar distribution profiles were observed within the cerebral cortex for latexinand CPA-immunopositive neurons. In double immunofluorescence experiments in some cortical areas, it was shown that all of latexin-immunopositive neurons were also CPAimmunopositive. These observations suggest that latexin play a role together with CPA in the cortico-cortical circuit.

355 Presynaptic accumulation and differential localization of complexin isoforms in neuronal cells

Murase, Kazuyuki, Asai, Tatsuya, Ikeda, Hiroshi Presynaptic inhibition by mu opioids in fine afferent fibers is known to be important in the production of antinociception within the superficial spinal dorsal horn. However, little is know about the mechanism by which transmitter release is inhibited by mu opioids at the terminals. We applied a high-resolution (128x 128 pixels) optical imaging method to rat spinal cord slices, and presynaptic excitation was directly visualized. A mu-opioid receptor agonist, [d-AlaZ,N-Me-Phe4, GlyS-ol]-enkephalin, reversibly reduced the presynaptic excitation in lamina II which was evoked by dorsal root stimulation. Spatial analysis of the optical response revealed that the degree of inhibition at each pixel measuring 4.3-6.5 micrometer-squared area varied from 0 to 100% and that the variability occurred even among the neighbouring pixels. We propose that m opioids suppress transmitter release by blocking the spread of excitation along the axonal arbors and/or into the terminals.