Fumihiko Fukamauchi

Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers.

Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier. Here, we show the role in this process of TAG-1, a glycosyl-phosphatidyl-inositol–anchored cell adhesion molecule. In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems. In contrast, the localization of protein 4.1B, an axoplasmic partner of Caspr2, was only moderately altered. TAG-1, which is expressed in both neurons and glia, was able to associate in cis with Caspr2 and in trans with itself. Thus, a tripartite intercellular protein complex, comprised of these two proteins, appears critical for axo–glial contacts at juxtaparanodes. This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo–glial interactions.

Halothane prevents MK-801 neurotoxicity in the rat cingulate cortex.

Subcutaneous administration of the N-methyl-D-aspartic acid (NMDA) antagonist, MK-801, to adult rats causes a toxic vacuole reaction in neurons of the posterior cingulate cortex which is readily detected in histological sections 4 h following MK-801 administration. Certain drugs that facilitate neurotransmission at gamma-aminobutyric acidA (GABAA) receptors block this neurotoxic action of MK-801. The anesthetic actions of halothane (fluothane) are thought to be due, at least in part, to an interaction with GABAA receptors. In the present study, we investigated the effect of halothane on MK-801 neurotoxicity. When halothane was administered for either 1 or 2 h, then terminated immediately prior to MK-801 treatment, the vacuole reaction detected 4 h later was almost as severe as in controls not exposed to halothane. Administration of halothane for 1 h after MK-801 injection postponed but did not prevent a relatively full vacuole reaction. However, when rats were kept under halothane anesthesia continuously throughout the 4 h period following MK-801 administration, the vacuole reaction was completely prevented. We postulate that halothane blocks MK-801 neurotoxicity by a facilitative action at GABAA receptors. Because halothanes duration of action is fleeting compared to the very long duration of action of MK-801, the efficacy of halothane in blocking MK-801 neurotoxicity varies in direct proportion to the length of time following MK-801 treatment that the rat brain is exposed to halothane.

DIFFERENTIAL EXPRESSION OF c-fos mRNA IN RAT PREFRONTAL CORTEX, STRIATUM, N. ACCUMBENS AND LATERAL SEPTUM AFTER TYPICAL AND ATYPICAL ANTIPSYCHOTICS: AN IN SITU HYBRIDIZATION STUDY

The regional difference in the expression of c-fos mRNA induced by typical and atypical antipsychotics was determined in prefrontal cortex, striatum, N. accumbens and lateral septum in rats by in situ hybridization. Two typical antipsychotics, haloperidol (2 mg/kg) and fluphenazine (2 mg/kg), and three atypical antipsychotics, (-)sulpiride (100 mg/kg), clozapine (20 mg/kg) and OPC-14597 (40 mg/kg), were used. Brains were fixed with 4% paraformaldehyde 45 min after drug administration (i.p.). Brain sections of 30 microns-thickness were made in a cryostat and hybridized with 35S-labelled for c-fos oligonucleotide probe. These sections were apposed to X-ray films and the autoradiograms were semi-quantitatively analysed by computer-assisted densitometry. All antipsychotics used increased c-fos mRNA expression in N. accumbens shell, a region of the forebrain associated with limbic systems. On the other hand, two typical antipsychotics (haloperidol and fluphenazine) that cause a high incidence of acute motor side effects increased the expression of c-fos mRNA in the dorsolateral striatum, an extrapyramidal region primarily involved in motor control. Only clozapine induced c-fos mRNA in the medial prefrontal cortex and lateral septum. These results strongly suggest that the shell region of N. accumbens may be a common site of therapeutic action of antipsychotics.

Expression and agonist-induced down-regulation of mRNAs of m2- and m3-muscarinic acetylcholine receptors in cultured cerebellar granule cells.

Abstract: The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization. mRNA species for m2‐ and m3‐mAChRs but not ml‐ and m4‐mAChRs were detected in these cells. The expression of mRNAs of both m2‐ and m3‐mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 μM carbachol led to differential down‐regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 μM) and pirenzepine (10 μM), prevented carbachol‐induced m3‐mAChR mRNA down‐regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up‐regulation of m3‐mAChR mRNA. Thus, the mRNA species for both m2‐ and m3‐mAChR subtypes are differentially expressed in culture and down‐regulated by agonist stimulation. The loss of these mRNA species may play a role in the down‐regulation of mAChR binding sites that occurs after desensitization.

Developmental regulation of notochord-derived repulsion for dorsal root ganglion axons

During the initial stages of development, the notochord provides repulsive signals for dorsal root ganglion (DRG) axons via semaphorin 3A/neuropilin-1, axonin-1/SC2, and other unknown repulsive molecules. The notochord is known to produce aggrecan, one of the chondroitin sulfate proteoglycans (CSPGs). We report here that adding aggrecan to the culture medium cannot only induce DRG growth cone collapse, but also inhibit DRG axonal growth. Using cocultures composed of tissues derived from chick embryos or neuropilin-1-deficient mice treated with chondroitinase ABC, we show the direct evidence that CSPGs are involved in notochord-derived repulsion for DRG axons. At later developmental stages, CSPGs are involved in perinotochordal sheath-derived axon repulsion, but not in notochord core-derived repulsion. We further demonstrate that TAG-1/axonin-1/SC2 is not involved in mediating repulsive activities by CSPGs, but is required for notochord core-derived axon repulsion. Thus, notochord-derived multiple axon repulsions act in a spatiotemporal-specific manner to shape the initial trajectories of DRG axons.

Long-term biphasic effects of lithium treatment on phospholipase C-coupled m3-muscarinic acetylcholine receptors in cultured cerebellar granule cells

We have studied the long-term effects of lithium on neuronal morphology and the functional expression of phospholipase C-coupled m3-muscarinic acetylcholine receptors (mAChRs) in cerebellar granule cells. There was a biphasic dose-dependent effect on cell morphology following treatment with lithium for 7 days. At low concentrations (< or = 2 mM), this drug elicited an increase in the number and thickness of connecting nerve fibers, and the size of neuronal aggregates. At high concentrations (5-10 mM), lithium induced a severe deterioration of cell morphology, which ultimately resulted in neuronal death. Carbachol-induced phosphoinositide (PI) turnover was similarly affected by lithium treatment with a significant potentiation at concentrations up to 2 mM and a marked inhibition at doses higher than 5 mM due to lithium-induced neurotoxicity. The biphasic effect on mAChR-mediated PI hydrolysis was associated with corresponding changes in the maximal extent of carbachol-induced inositol phosphate accumulation, and was accompanied by similar changes in [3H]N-methyl-scopolamine binding to mAChRs and the levels of mRNAs for m3-mAChR and c-Fos. The up-regulation of m3-mAChR mRNA induced by low concentrations of lithium was associated with a down-regulation of m2-mAChR mRNA and no change in either total RNA or beta-actin mRNA. Lithiums effects on m2- and m3-mAChR mRNAs were time-dependent, requiring a pretreatment time of > or = 3 days. The biphasic effect was also demonstrated by the binding of [3H]ouabain to Na+, K(+)-ATPase, which was shown to be a convenient method for quantifying viable neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Paradoxical behavioral response to apomorphine in tenascin-gene knockout mouse

Tenascin is a large extracellular matrix glycoprotein which is highly expressed in the developing nervous system. To examine the role of tenascin in vivo, we have produced mice in which the tenascin-gene is inactivated. These animals did not easily habituate to unfamiliar circumstances and displayed hyperlocomotion. A dopamine receptor agonist, apomorphine, reduced this hyperlocomotion dose dependently, but this phenomenon was not due to the appearance of apomorphine-induced stereotypic behavior, suggesting that tenascin-gene mutant mice have a paradoxical behavioral response to apomorphine compared to wild-type mice.

Enhancement of mRNA expression of tissue-type plasminogen activator by L-threo-3, 4-dihydroxyphenylserine in association with ocular dominance plasticity

Tissue-type plasminogen activator (tPA) plays important roles in the regulation of synaptic plasticity in the hippocampus and cerebellum. We found that the expression of tPA mRNA in the visual cortex was increased significantly by the peripheral administration of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS; 100 mg/kg, i.p.), which we had previously shown to have a promotive effect on ocular dominance (OD) plasticity. When plasminogen activator inhibitor-1 (PAI-1; 100 muM in an osmotic minipump) was infused into the kitten visual cortex, OD plasticity was suppressed; i.e. a significantly large number of binocular cells was recorded in the PAI-1 infused cortex following monocular deprivation. These results, therefore, suggest that the PA system is involved in the promotive effect of L-threo-DOPS in OD plasticity.

Dopaminergic agents affected neuronal transmission of cholecystokinin in the rat brain

In order to study the interaction between cholecystokinin (CCK) and dopamine (DA), we prepared an anti-CCK-8 antibody with low cross reactivity, and observed effects of administered various dopaminergic agents on CCK-immunoreactivity (CCK-IR) in discrete brain regions of rats. CCK-8 IR (boiling water extraction) and CCK-33 IR (acetic acid extraction) were also measured in the same sample. A single administration of haloperidol decreased the CCK-8 IR in the corpus striatum and that of racemic sulpiride significantly decreased the CCK-8 IR in the frontal cortex and the limbic system. In contrast, a single administration of apomorphine or methamphetamine increased the CCK-8 IR in the same regions. These findings suggest that an acute response of the CCK system to administered dopaminergic agents may be due to a change in the rate of release of CCK-8, but not to a change in its synthesis in areas in which DA neurons originating in the midbrain innervate. After chronic administration of racemic sulpiride or methaphetamine, CCK-8 IR in various brain regions exhibited a tendency close to that of control.

Reduced mRNA expression of neuropeptide Y in the limbic system of tenascin gene disrupted mouse brain.

Tenascin-C (TN), an extracellular matrix glycoprotein which reveals both neurite outgrowth-promoting and growth-inhibiting effects, is generated in the central nervous system. A previous study reported that TN-gene null mutant mice display hyperlocomotion and do not easily habituate to unfamiliar environments. Additionally, these mice display poor appetite, abnormal circadian rhythm and low pregnancy rate. The present study demonstrated that neuropeptide Y (NPY) mRNA expression is reduced in the limbic area of the TN gene-deficient mouse brain as compared to wild-type mice. NPY has been shown to affect emotion, circadian rhythm and food intake, and the present results suggest that the some behavioural abnormalities exhibited by TN-mutant mice may be in part due to the low level of expression of NPY mRNA in the limbic system.