K. Tomizawa

Enhanced synaptic transmission and reduced threshold for LTP induction in fyn‐transgenic mice

To elucidate the physiological role of Fyn, we analysed the properties of synaptic transmission and synaptic plasticity in hippocampal slices of mice overexpressing either wild‐type Fyn (w‐Fyn) or its constitutively active mutant (m‐Fyn). These fyn‐transgenes were driven by the calcium/calmodulin‐dependent protein kinase IIα promoter which turned on in the forebrain neurons including hippocampal pyramidal cells and in late neural development. In the hippocampal slices expressing m‐Fyn the paired‐pulse facilitation was reduced and the basal synaptic transmission was enhanced. A weak theta‐burst stimulation, which was subthreshold for the induction of long‐term potentiation (LTP) in control slices, elicited LTP in CA1 region of the slices expressing m‐Fyn. When a relatively strong stimulation was applied, the magnitude of LTP in m‐Fyn slices was similar to that in control slices. By contrast, the basal synaptic transmission and the threshold for the induction of LTP were not altered in the slices overexpressing wild‐type Fyn. To examine the effect of expression of m‐Fyn on GABAergic inhibitory system, we applied bicuculline, a GABAA receptor blocker, to the hippocampal slices. The ability of bicuculline to enhance excitatory postsynaptic potentials was attenuated in slices expressing m‐Fyn, suggesting that the overexpression of m‐Fyn reduced the GABAergic inhibition. The enhancement of synaptic transmission and the reduction of GABAergic inhibition may contribute to the enhanced seizure susceptibility in the mice expressing m‐Fyn. Thus, these results suggest that regulation of Fyn tyrosine kinase activity is important for both synaptic transmission and plasticity.

Calcineurin inhibitors, FK506 and cyclosporin A, suppress the NMDA receptor-mediated potentials and LTP, but not depotentiation in the rat hippocampus

The effects of FK506, a Ca2+/calmodulin-dependent phosphatase 2B (calcineurin) inhibitor, on the NMDA receptor-mediated potentials and synaptic plasticity were investigated in the CA1 region of the rat hippocampus. Bath application of FK506 (50 microM) produced a 45% inhibition on the NMDA receptor-mediated potentials. FK506 also inhibited the induction of long-term potentiation (LTP), but had no effect on the depotentiation in the CA1 hippocampus. Cyclosporin A (100 microM), another calcineurin inhibitor, mimicked the effects of FK506 on the NMDA responses and synaptic plasticity. These results suggest that FK506 inhibits the activity of NMDA receptors via the involvement of calcineurin. The differential effects of FK506 on LTP and depotentiation may attribute to the partial inhibition on the activity of NMDA receptors and the subsequent attenuation of intracellular Ca2+ increase.

Localization and developmental changes in the neuron-specific cyclin-dependent kinase 5 activator (p35nck5a) in the rat brain

Mammalian brains contain a cde2-like protein kinase which is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a brain-specific regulatory subunit with a molecular weight of 35,000. In this study, we examined the temporal and spatial expression patterns of p35nck5a in the developing rat brain. Northern blot analysis showed that p35nck5a messenger RNA expression was low in the brain of 12-day postcoitum rats, and increased to a much higher level from 18 days postcoitum to two weeks after birth, and then declined at three weeks after birth. These developmental changes in p35nck5a expression correlated with the changes in Cdk5-associated kinase activity during brain development. These data suggest that p35nck5a is the specific activator for Cdk5 in the brain. Immunohistochemical and in situ hybridization studies demonstrated the presence of p35nck5a protein in postmitotic neurons but not in glial cells at all stages of brain development, indicating that p35nck5a is a neuron-specific protein. In the adult brain, the protein was rich in cell bodies and dendrites, and only very low amounts were detected in axons. In fetal and neonatal brains, however, axonal pathways such as the corpus callosum and external capsule were also stained with anti-p35nck5a antibody. Our findings suggest that p35nck5a is neuron specific, and a specific activator for Cdk5, and the subcellular localization of the two is strictly regulated depending on brain development. Neuronal Cdc2-like kinase may play key roles in neuronal maturation, synaptic formation, and neuronal plasticity.

A monoclonal antibody stains radial glia in the adult zebrafish (Danio rerio) CNS

We have prepared a monoclonal antibody, denoted as C-4, which specifically recognizes astroglia with radial forms in the adult zebrafish brain. This report suggests that there are at least two different types of astroglia in the mature teleost brain, only one of which is recognized by C-4. Further, we have found that the C-4-binding astroglia are comprised of three morphologically distinct cellular types. Immunoblot analysis revealed that the antibody recognized only one protein band of approximately 30 kDa in the membrane fraction of the adult zebrafish brain. In the spinal cord, stained glial cells appeared to occur in the same location as ependymocytes. The processes and cell bodies of extra-ependymal cells, many adjacent to ependymocytes and a few near the pial surface, were also stained by the antibody. In the cerebellum, long processes stained by C-4 were found in the molecular layer, and these connected the cerebellum surface to the Purkinje-like cell layer. Long processes were also stained in the mesencephalon, but these were thicker than those in the spinal cord and they linked the two ventricles. The optic tectum, olfactory bulb and cranial nerves, including the optic nerves, were, however, completely devoid of the C-4 antigen. Double-immunofluorescence with antibodies against glial fibrillary acidic protein (GFAP) and C-4 demonstrated that C-4-positive cells were also GFAP-positive, although there was also a subset of GFAP-positive cells which were C-4-negative. The C-4 antibody is thus a useful tool for studying subtypes of GFAP-positive astroglia.

An immunosuppressant, FK506, protects against neuronal dysfunction and death but has no effect on electrographic and behavioral activities induced by systemic kainate.

Kainate is a potent agonist of an excitatory amino acid receptor subtype in the central nervous system, and causes neuronal death in several regions of the brain. Neurons are preferentially killed in the hippocampus, especially in the CA1 region, by systemic administration of kainate. It is speculated that functional alterations occur in the neurons preceding death. We examined the effect of FK506 on kainate-induced neuronal death and functional alterations in the rat hippocampal CA1 region. FK506 had no effect on electrographic and behavioral seizure activities induced by kainate; however, it prevented neuronal death measured seven days after administration. Although neither death nor morphological alterations of neurons were observed in the CA1 region 24 h after administration, the neurons exhibited decreased excitatory postsynaptic potentials and enhanced long-term potentiation. This functional alteration was not detected in the rats administered FK506 prior to kainate. Taken together, these observations indicate that functional alteration precedes neuronal death in rats systemically administered kainate and that FK506 prevents both. It is suggested that FK506 exerts its neuroprotective effect not by attenuating electrographic and behavioral seizure activities, but by protecting neurons from kainate-induced functional disorders.

Effects of vasopressin and involvement of receptor subtypes in the rat central amygdaloid nucleus in vitro.

Effects of arginine-vasopressin (AVP) on neurons in the central amygdaloid nucleus (ACe) were investigated with rat brain slice preparations using extracellular recording methods. Of 160 ACe neurons tested, 70 cells (44%) were excited and 9 cells (6%) were inhibited by bath application of AVP at 3 x 10(-7) M. The excitatory effects of AVP were dose-dependent and the threshold concentration was approximately 10(-10) to 10(-9) M. The excitatory effects of AVP persisted under blockade of synaptic transmission by perfusing with Ca2+-free and high-Mg2+ medium, whereas the inhibitory effects were abolished by synaptic blockade. AVP-induced effects were mimicked by a V1-receptor agonist and completely blocked by a selective V1-antagonist. V2-agonist produced no effects on ACe neurons and V2-antagonist had no effect on AVP-induced excitation. These results showed that the excitatory effect of AVP on ACe neurons was produced by a direct action through the V1-receptors, whereas the inhibitory response of ACe neurons to AVP seemed to be produced by an indirect action. The results of this study suggest that AVP is involved in the amygdala-relevant functions as a neurotransmitter or a neuromodulator.

Ex vivo culture of isolated zebrafish whole brain

We have succeeded in culturing whole zebrafish brains ex vivo for 1 week. While isolated cells and tissue slices have previously been employed for neurobiological studies, these techniques are limited, because while local networks may be preserved, their original context in the whole brain is lost. Culture of the whole brain would facilitate the study of cells and systems within an intact brain infrastructure. Our culture method entailed isolating the whole brain and placing it on a sterile and porous membrane, after which it was maintained with a conditioned medium in a six-well plate in a CO2 incubator at 28.5 degrees C. Whole brains cultured by this simple method were relatively unaltered in terms of their morphology, cytoarchitecture, immunohistochemistry and ability to transport horse radish peroxidase (HRP). This method of cultivation may be very useful for neurobiological research.

A novel monoclonal antibody recognizes a previously unknown subdivision of the habenulo-interpeduncular system in zebrafish

The habenulo-interpeduncular system is an evolutionarily conserved structure found in the brain of almost all vertebrates. We prepared a monoclonal antibody (6G11) which very specifically recognizes only a part of this system. 6G11 is a monoclonal antibody prepared from a neuronal membrane protein in adult zebrafish brain. In western blot analysis of the adult zebrafish brain, the antibody recognized a 95 kDa protein, and the class of the antibody was determined to be IgM. The 6G11 antigen was not detected in zebrafish muscle, intestine, testis or ovary. A group of neurons stained by the 6G11 antibody was located in the caudomedial part of the zebrafish habenula. The 6G11-immunopositive neurons extended their axons into the fasciculus retroflexus (FR). One group of immunopositive neurons projected toward the interpeduncular nucleus (IPN), especially to the intermediate and the central subnucleus (type 1 neuron). The other group projected to the ventral midline at the level of the raphe nucleus; these axons passed ipsilaterally beside the IPN and converged in the ventral midline under the raphe nucleus (type 2 neuron). Both type 1 and type 2 fibers are relatively minor components of the FR. Little has previously been known about this topological pattern in any species. The 6G11 monoclonal antibody could be a useful tool for expanding knowledge of the habenulo-interpeduncular system.

Monoclonal antibody stains oligodendrocytes and Schwann cells in zebrafish (Danio rerio)

We prepared a monoclonal antibody that recognizes oligodendrocytes and Schwann cells in zebrafish. On immunoblots, the antibody mainly recognized three protein bands of 34 kDa in a membrane fraction from adult zebrafish brain. Medaka fish (Oryzias latipes) also possessed the same protein bands in a membrane fraction. The antibody did not stain neurons, but stained cells in fiber tracts and cranial and spinal nerves. In order to determine the nature of these cells, the staining pattern of the monoclonal antibody was compared with that of a myelin basic protein antiserum. Both antibodies stained oligodendrocytes and Schwann cells in fixed sections from the adult zebrafish. Both antigens were also co-localized in cultured glial cells. Taken together, these results indicate that the new monoclonal antibody recognizes myelinating glial cells in zebrafish and will be useful for the analysis of piscine glia.