Akiko Harada

Characterization of factors regulating lamina‐specific growth of thalamocortical axons

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer-specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina-specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol-linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers.

BDNF and NT‐3 promote thalamocortical axon growth with distinct substrate and temporal dependency

The role of neurotrophins in thalamic axon growth was studied by culturing embryonic rat thalamus on collagen‐coated substrate or fixed cortical slices in the presence of either brain‐derived neurotrophic factor (BDNF) or neurotrophin‐3 (NT‐3). Both BDNF and NT‐3 promoted axonal growth, but the axonal growth‐promoting activity depended on culture substrates. Axonal growth on collagen‐coated membrane was accelerated by BDNF, but not by NT‐3. In contrast, axonal outgrowth on fixed cortex was significantly enhanced by NT‐3, but not by BDNF. Semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of cultured thalamic cells demonstrated that culture substrates did not alter the expression of their receptors, trkB and trkC. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) staining further demonstrated that axonal growth promoted by neurotrophins was not due to reduction of cell death. Measurement of the developmental changes in BDNF and NT‐3 levels revealed that, in contrast to the rapid elevation of BDNF after the arrival of thalamocortical axons to their target layer, the regulation of NT‐3 protein accompanies the phase of their outgrowth in neocortex. These findings suggest that BDNF and NT‐3 promote thalamic axon growth in different manners in terms of substrate dependency and developmental stage.

Photosynthetic control of the plasma membrane H+-ATPase in Vallisneria leaves. I. Regulation of activity during light-induced membrane hyperpolarization

Abstract. In mesophyll cells of the aquatic angiosperm Vallisneria gigantea Graebner, red, blue, or blue plus far-red light induced a typical membrane hyperpolarization, whereas far-red light alone had little effect. Both N,N′-dicyclohexylcarbodiimide, a potent inhibitor of H+-ATPase, and carbonylcyanide m-chlorophenylhydrazone, an uncoupler, produced a considerable membrane depolarization in the dark-adapted cells and a complete suppression of the light-induced hyperpolarization. Although 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, did not affect the membrane potential in darkness, it completely inhibited the light-induced membrane hyperpolarization. In vivo illumination of the leaves with red light caused a substantial decrease in the Km for ATP, not only of the vanadate-sensitive ATP-hydrolyzing activity in leaf homogenate, but also of the ATP-dependent H+-transporting activity in plasma membrane (PM) vesicles isolated from the leaves by aqueous polymer two-phase partitioning methods. The effects of red light were negated by the presence of DCMU during illumination. In vivo illumination with far-red light had no effect on the Km for ATP of H+-transporting activity. These results strongly suggest that an electrogenic component in the membrane potential of the mesophyll cell is generated by the PM H+-ATPase, and that photosynthesis-dependent modulation of the enzymatic activity of the PM H+-ATPase is involved in the light-induced membrane hyperpolarization.

Photosynthetic control of the plasma membrane H+-ATPase in Vallisneria leaves. II. Presence of putative isogenes and a protein equipped with a C-terminal autoinhibitory domain

Abstract. In vitro treatment with trypsin of plasma membrane (PM) vesicles isolated from the leaves of Vallisneria gigantea Graebner, an aquatic monocot, produced a marked decrease in the Km for ATP and an increase in the Vmax of H+-transporting activity. Concomitantly, the removal of 8xa0kDa of the C-terminal domain from the 94-kDa PM H+-ATPase was confirmed by immunoblotting using different kinds of polyclonal antibody. Three partial clones of putative PM H+-ATPase genes (Vga1, 2, and 3) were isolated from leaves by reverse transcription polymerase chain reaction. Northern blotting analysis revealed that the expression level of Vga3 was high and that of the other two genes was much lower. The H+-transporting activity of PM vesicles was substantially suppressed in the presence of inorganic phosphate (Pi), which has been supposed to be a noncompetitive inhibitor of the PM H+-ATPase, coincident with an increase in the Km for ATP and a decrease in the Vmax. After treatment of the isolated PM vesicles with trypsin, the inhibitory effect of Pi was no longer evident. This result indicates that Pi inhibited the activity through the C-terminal autoinhibitory domain of the PM H+-ATPase. Furthermore, Pi increased the Km for ATP of the H+-transporting activity in the PM vesicles isolated from both dark-adapted and red-light-irradiated leaves. The results suggest that regulation of the Km for ATP through the operation of photosynthesis is independent of regulation through the cytoplasmic level of Pi.

Regulation of thalamic axon growth by neurotrophins

To reveal the role of neurotrophins in axonal growth and arborization in neocortical circuits, thalamic axon growth was studied on a fixed cortical slice. Rat thalamic blocks (E15) were juxtaposed to the lateral edge of cortical slices (P7) that had been fmed with paraforamaldehyde. Under these conditions the effect of intrinsic neurotrophic factors released from cortical cells could be largely excluded. After a few days in culture neurotrophins including NGF, BDNF, NT3 or NT4 (200 rig/ml) were added to the culture medium, and the cultures were further incubated for an additional several days. Then, axonal projection patterns were examined by implanting diI in thalamic explants. Thalamic axons extended longer in the deep layers (layers 5 and 6) than in the upper layers (layers 1 to 4) on fixed cortical slices. This elongation pattern was not changed by adding NGF, BDNF or NT4. However, axonal growth was increased in each layer in NT3-containing medium. ‘Ihis finding suggests that developing thalamic neurons extend axons that respond to neurotropbins through trkC receptors.

1008 Regulation of thalamic axon growth in the neocortex: Analysis of outgrowth on fixed cortical slices

Jun Aruga’, Osamu Minowa 2, Junko Kuno”, Hiroyuki Yaginuma3, Takeharu Naga?, Tetsuo Noda2, Katsuhiko Mikoshiba’ Zic genes were found as a gene family encoding zinc finger proteins expressed restrictedly in the adult mouse cerebellum. We recently showed that the genes are the vertebrate homologues of Drosophila odd-paired, which may play essential roles in parasegmental subdivision and in visceral mesoderm development. To clarify the role of Zic genes in the cerebellar development, we targeted Zicl gene, which is a major species in the adult CNS among the Zic family. Homozygous mutants showed remarkable ataxia during their postnatal life and most of them died within a month after birth. Morphologically, the cerebellum was hypoplastic and showed abnormal foliation pattern. The abnormality of cerebellar foliation could be detected as early as 17 days post coitum. These findings suggested that Zicl has an essential role in the vertebrate development. Further characterization on the family may reveal genetic interactions with other genes involved in cerebellar development.

1129 Axonal growth pattern of lateral geniculate nucleus neurons on fixed cortical slice

Vertebrate neural development is initiated during gastrulation by the inductive action of the Spemann’s organizer (presumptive dorsal mesoderm) on neighbouring ectoderm. We have previously shown that fibroblast growth factor (FGF) can mimic the action of the organizer to induce Xenopus ectoderm cells in culture to express position-specific neural markers along the anteroposterior axis and also that several Xenopus FGF receptor (XFGFR) genes are transcribed in gastrula ectoderm cells. To further elucidate whether these XFGFRs are involved in the neural induction in living organisms, detailed studies using the dominant-negative molecules for XFGFR were done by injecting m.RNAs of these molecules into early embryos. In the co-culture system with organizer cells/ectoderm cells, it is shown that blocking FGF-signalIing in ectoderm cells by overexpression of a dominant-negative form of XFGFR leads to disruption of the neural differentiation. Our findings indicate that FGF family member(s) are a promising candidate for the neural-inducing morphogen from the organizer.