Promotion of axonal maturation and prevention of memory loss in mice by extracts of Astragalus mongholicus.

BACKGROUND AND PURPOSE: Neurons with atrophic neurites may remain alive and therefore may have the potential to regenerate even when neuronal death has occurred in some parts of the brain. This study aimed to explore effects of drugs that can facilitate the regeneration of neurites and the reconstruction of synapses even in severely damaged neurons. EXPERIMENTAL APPROACH: We investigated the effects of extracts of Astragalus mongholicus on the cognitive defect in mice caused by injection with the amyloid peptide Abeta(25-35). We also examined the effect of the extract on the regeneration of neurites and the reconstruction of synapses in cultured neurons damaged by Abeta(25-35). KEY RESULTS: A. mongholicus extract (1 g kg(-1) day(-1) for 15 days, p.o.) reversed Abeta(25-35)-induced memory loss and prevented the loss of axons and synapses in the cerebral cortex and hippocampus in mice. Treatment with Abeta(25-35) (10 microM) induced axonal atrophy and synaptic loss in cultured rat cortical neurons. Subsequent treatment with A. mongholicus extract (100 microg/ml) resulted in significant axonal regeneration, reconstruction of neuronal synapses, and prevention of Abeta(25-35)-induced neuronal death. Similar extracts of A. membranaceus had no effect on axonal atrophy, synaptic loss, or neuronal death. The major known components of the extracts (astragalosides I, II, and IV) reduced neurodegeneration, but the activity of the extracts did not correlate with their content of these three astragalosides. CONCLUSION AND IMPLICATIONS: A. mongholicus is an important candidate for the treatment of memory disorders and the main active constituents may not be the known astragalosides.

Inhibition of itch-scratch response by fruits of Cnidium monnieri in mice.

We previously screened the anti-itching activities of 33 herbal medicines in substance P (SP)-induced itching model mice. One of the most potent antipruritogenic extracts, the methanol extract of fruits of Cnidium monnieri (Cnidii Fructus) was studied further. The chloroform-soluble fraction of the methanol extract markedly inhibited SP-induced scratching. Among 10 subfractions of the chloroform-soluble fraction, the CS-3 fraction had the most potent inhibitory effect on scratching. Each of 3 subfractions of CS-3 showed significant anti-scratching activities. However, inhibitory potencies were not different among the three and weaker than that of CS-3 itself at a same dose. These 3 subfractions of CS-3 mainly contained xanthotoxin, isopimpinellin, bergapten, imperatorin and osthol. Single administration of osthol did not inhibit SP-induced scratching, and imperatorin very weakly subsided scratching. These results suggest that the strong antipruritic action was focused on the CS-3 fraction of the C. monnieri methanol extract, and it might result from the combined effects of these coumarin derivatives, or by undetermined minor compounds.

Axonal transport of VR1 capsaicin receptor mRNA in primary afferents and its participation in inflammation-induced increase in capsaicin sensitivity.

Capsaicin receptors are expressed in primary sensory neurons and excited by heat and protons. We examined the inflammation-induced changes of the level of VR1 capsaicin receptor mRNA in sensory neurons and the sensitivity of primary afferents to capsaicin. Carrageenan treatment induced axonal transport of VR1 mRNA, but not that of preprotachykinin mRNA, from the dorsal root ganglia to central and peripheral axon terminals. The sensitivity of central terminals to capsaicin, which was estimated by measuring the capsaicin-evoked release of glutamate from the dorsal horn, was increased by peripheral inflammation, and such an increase was suppressed by inhibiting the RNA translation in the dorsal horn with cycloheximide and an intrathecal injection of VR1 antisense oligonucleotides. Thus, peripheral inflammation induces the axonal transport of VR1 mRNA, which may be involved in the hypersensitivity of primary afferents to capsaicin and the production of inflammatory hyperalgesia.

Dendrite extension by methanol extract of Ashwagandha (roots of Withania somnifera) in SK-N-SH cells.

Extension of dendrites and axons in neurons may compensate for and repair damaged neuronal circuits in the dementia brain. Our aim in the present study was to explore drugs activating neurite outgrowth and regenerating the neuronal network. We found that the methanol extract of Ashwagandha (roots of Withania somnifera; 5 microg/ml) significantly increased the percentage of cells with neurites in human neuroblastoma SK-N-SH cells. The effect of the extract was dose- and time-dependent mRNA levels of the dendritic markers MAP2 and PSD-95 by RT-PCR were found to be markedly increased by treatment with the extract, whereas those of the axonal marker Tau were not. Immunocytochemistry demonstrated the specific expression of MAP2 in neurites extended by the extract. These results suggest that the methanol extract of Ashwagandha promotes the formation of dendrites.

Inhibitory effect of Byakko-ka-ninjin-to on itch in a mouse model of atopic dermatitis.

Byakko-ka-ninjin-to (BN) is composed of gypsum, the root of anemarrhena, ginseng, licorice and rice. The effect of BN on the inhibition of itch was studied using an NC mouse model of atopic dermatitis. BN (200 mg/kg, p.o.) significantly inhibited the scratching frequency in NC mice, and decreased the skin temperature by 1.97 degrees C. The cooling action on the skin by BN may be involved in the inhibitory mechanism of itch, at least in part, since cooling the skin is known to inhibit the itch sensation in humans. Although the myocyte-specific enhancer binding factor 2C (MEF2C) mRNA is known to be increased in the cerebral cortex correlated with the itch sensation and skin lesions in NC mice, BN did not affect the expression level of the MEF2C mRNA. This result suggests that the inhibitory effect of BN on itch does not relate to inhibition of MEF2C expression in the cerebral cortex. The present study indicates that BN has an inhibitory effect on itch, and may be a useful antipruritic drug for atopic dermatitis.

Inhibitory effects of methanol extracts of herbal medicines on substance P-induced itch-scratch response.

In a search for new anti-pruritic drugs we screened methanol extracts of 33 herbal medicines which have been used for cutaneous diseases for their antipruritic activity using substance P (SP) as a pruritogen in mice. When administered perorally 30 min before SP injection, methanol extracts of 6 of these herbal medicines, the root of Scrophularia ningpoensis Hemsl., the root of Patrinia villosa (Thunb.) Juss, the fruit of Forsythia suspenna Vahl, the rhizome of Cimicifuga dahurica (Turcz.) Maxim., the aerial part of Schizonepeta tenuifolia Briq. and the fruit of Cnidium monnieri (L.) Cuss, inhibited SP-induced itch-scratch response at a dose of 200 mg/kg with-out affecting locomotor activity. Dose dependence of these 6 extracts (50-500 mg/kg) was investigated and all of them inhibited SP-induced itch-scratch response, with extracts from Scrophularia ningpoensis, Schizonepeta tenuifolia and Cnidium monnieri showing particularly significant inhibition. The results suggest that these 6 methanol extracts have inhibitory activity against SP-induced itching.

Trigonelline-induced neurite outgrowth in human neuroblastoma SK-N-SH cells.

Extension of dendrites and axons in neurons may compensate and rescue damaged neuronal networks in the dementia brain. Our aim is to isolate and identify constituents of coffee beans exhibiting neurite outgrowth activity. Among the extracts of raw and roasted coffee beans, a methanol fraction of the ethanol extract (1 microg/ml) of raw beans increased significantly the percentage of cells with neurites in human neuroblastoma SK-N-SH cells. Among subfractions of this methanol fraction was a basic fraction (5 microg/ml) which exhibited significant neurite outgrowth activity. Finally, trigonelline in the basic fraction was identified to be active as far neurite extension was concerned. Treatment with trigonelline (30 microM) increased the percentage of cells with neurites at 3 and 6 d after treatment. In addition, the number of neurites reacting positively to phosphorylated neurofilament-H was increased by treatment with 30 microM trigonelline for 6 d, suggesting enhancement of axonal extension. These results show that trigonelline promotes functional neurite outgrowth.

The function and expression of sproutin, a novel neurite outgrowth factor.

TA20 cDNA was previously cloned as a neurite outgrowth factor from a hybridoma of mouse and rat cells, NG108-15. To clarify the detailed function and tissue distribution of this gene, homologous sequences of rat and mouse were identified. The cloned sequences had no homology with known genes, and was designated as sproutin. A predicted open reading frame of rat sproutin was transfected into human SK-N-SH cells. The over-expressed protein was distributed in cytoplasm and neurites, and caused an increase in the levels of microtuble associated proteins, but not that of phosphorylated neurofilament-H. The percentage of cells with neurites, the length of neurites and the number of neurites per cell were increased by sproutin transfection. Sproutin mRNA was brain specific. These results suggest that an increase in sproutin promotes dendritic extension.

Region-specific increase in glutamate release from dorsal horn of rats with adjuvant inflammation.

Glutamate is considered an important pain transmitter and responsible for inflammatory hyperalgesia, but quantitative and topographical changes in glutamate release in the dorsal horn during peripheral inflammation have not been characterized. To address this issue, image analysis with a confocal laser scanning microscope was performed for quantitatively mapping capsaicin-evoked glutamate release from the lumbar cord slice of rats following unilateral adjuvant inoculation to the hind-paw. Capsaicin induced glutamate release from laminae I, II and X in the spinal cord of the adjuvant-treated and untreated sides, without apparent release from laminae III-V. The concentration of released glutamate in laminae I, II and X was higher on the adjuvant-treated side than on the untreated side. The results suggest that adjuvant inflammation increases glutamate release from capsaicin-sensitive primary afferents in laminae I, II and X.

Existence of capsaicin-sensitive glutamatergic terminals in rat hypothalamus.

Capsaicin has been suggested to act not only on thin primary afferents but also on the hypothalamus, but the neurotransmitter(s) of central capsaicin-sensitive neurons are unknown. The present study was conducted to determine whether any central, especially hypothalamic, glutamatergic terminals were sensitive to capsaicin. Capsaicin evoked glutamate release from slices of hypothalamus and lumbar dorsal horn, but not cerebellum. Such capsaicin action was Ca2+ dependent and inhibited by the capsaicin antagonist capsazepine. Vanilloid receptor subtype 1 mRNA was widely distributed in the brain, with a marked level in the hypothalamus and cerebellum, but not in the spinal cord. The results suggest that there are glutamatergic terminals sensitive to capsaicin in the hypothalamus.

Intracisternal injection of opioids induces itch-associated response through mu-opioid receptors in mice.

We examined whether opioids, especially morphine, would centrally elicit scratching in mice and determined some characteristics of the scratch-inducing action of opioids. When intracisternally (i.c.) injected, morphine (0.1-3 nmol/mouse) produced a dose-dependent increase in scratching of the face, but not of the ears, head and body trunk. When injected intradermally into the rostral part of the back, morphine (at most potent i.c. dose of 3 nmol/mouse or higher) did not increase the scratching of the injected site. Facial scratching of the mouse induced by i.c. injection of morphine (0.3 nmol/mouse) was almost abolished by distraction and by naloxone (1 mg/kg, s.c.). [D-Ala2, N-Me-Phe4, Gly5-ol]Enkephalin (DAMGO) (0.03-2 nmol), but not [D-Pen2,5]enkephalin (DPDPE) and U-50,488, dose-dependently elicited facial scratching by i.c. injection. These results suggest that morphine and DAMGO increased facial scratching, probably mediated by central opioid mu-receptors in mice, and such scratching was due to a sensation, probably itching. The present animal model may be useful for analyzing opioid-mediated central itching.

Analysis of dissociated single neurons by simple and semi-quantitative RT-PCR (reverse transcription and polymerase chain reaction).

We have developed a simple and semi-quantitative method for mRNA determination in single cells using the reverse transcription and polymerase chain reaction (RT-PCR). The distinct features of this method are the highly efficient RNA harvest from whole dissociated cells and the ability to perform all RT procedures in one tube that allowed semi-quantitative determination of mRNA in dissociated cells. This method revealed that histamine H1-receptor mRNA was highly expressed in 5/28 small and 1/26 large dorsal root ganglion neurons of the mouse.

Increased expression of mRNA for myocyte-specific enhancer binding factor (MEF) 2C in the cerebral cortex of the itching mouse.

Since itch can be a subjective sensation, markedly affected by psychological conditions or sometimes originate in the central nervous system, the mechanisms of neuronal processing of itch signaling in the central nervous system should be studied. Therefore, we examined itch-related behaviors and nervous gene expressions of NC mice, which show severe dermatitis and atopy-like changes in inflammatory cells. Some NC mice spontaneously scratched their bodies and showed skin lesions, such as eczema, bleeding and alopecia from 2-6 months after birth. The mice with skin lesions scratched mainly the face, ears and rostral part of the body throughout the day, using their hind paws . The average scratching frequency was 126.7 +/- 36.8 (n = 4) and 5.3 +/- 4.7 (n = 3) per h in skin-lesioned and non-lesioned control mice, respectively. A differential display analysis of gene expressions in several regions in central and peripheral nervous systems was performed between these scratching and control groups. One of the genes that was expressed at a higher level in a scratching group than in the control group was myocyte-specific enhancer binding factor (MEF) 2C, in the cerebral cortex. The scratching was inhibited by intracerebroventricular injection of antisense oligodeoxynucleotide for MEF2C. These results raise the possibility that MEF2C may be involved in the sensation or perception of itch in the cerebral cortex.

Expression of mRNA for a neuronal differentiation factor, TA2O, in developing rat brains.

In our previous study, a novel factor, TA2O, was isolated from NGlO8-l5 cells. The TA2O mRNA was increased by stimulation which also induced neuronal differentiation. Neuronal cells overexpressed with TA2O extended long neurites and stopped cell growth (Tohda et al., 1995, Neurosci. Res., 23: 21-27). We investigated the expression pattern of TA2O mRNA in developing rat brains to predict physiological roles of TA2O. TA2O mRNA began to increase between embryonic days 13 and 16. TA2O mRNA was observed mainly in neocortical, hippocampal and precerebellar neuroepithelium on embryonic day 16. Although the level of TA2O mRNA in the cerebral cortex was higher before birth than after birth, the level in cerebellar Purkinje cells increased gradually even after birth. The high expression level of TA2O mRNA in the hippocampus was maintained before and after birth. Thus, TA2O was expressed highly in brain regions in which neurons were changing morphologically and qualitatively, suggesting that TA2O may be involved in neuronal formation in vivo.

Visualization of glutamate release from rat spinal cord with a confocal laser scanning microscope.

Visualization of the release of an excitatory neurotransmitter, glutamate (Glu), from a slice preparation of the brain and spinal cord may be of great advantage in studying the release of Glu from a small population of neurons. When capsaicin (10 mu M) was applied to a slice of the rat spinal cord immersed in a medium containing glutamate dehydrogenase (GDH), an oxidized form of nicotinamide adenine dinucleotide (NAD+), and tetrodotoxin, we observed an apparent increase of fluorescence in superficial laminae and lamina X using a confocal laser scanning microscope. Such an increase was not observed in the absence of either NAD+ or GDH, was inhibited by removal of extracellular Ca2+, and was terminated by capsazepine (100 mu M). In contrast to capsaicin, Glu release evoked by high K+ was observed in all laminae throughout the grey matter. The present results suggest that this system enables us to see the site of the release of Glu as an image and that capsaicin releases this amino acid mainly in superficial laminae and lamina X in the spinal cord.

A novel factor, TA20, involved in neuronal differentiation: cDNA cloning and expression.

Combined treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) and dibutyryl cyclic AMP (diBu-cAMP) induced significantly longer neurites than treatment with each alone in NG108-15 cells. We performed differential screening to identify genes expressed only by treatment with TPA plus diBu-cAMP but not by that with diBu-cAMP for 72 h alone in NG108-15 cells, and isolated a novel gene, TA20. Over-expression of the gene in NG108-15 and neuroblastoma N18TG-2 cells caused intense neurite elongation and suppressed cell growth. TA20 did not cause, however, any morphological changes in glioma C6Bu-1 cells. These results suggest that TA20 is a novel neuronal differentiation factor.

Possible involvement of botulinum ADP-ribosyltransferase sensitive low molecular G-protein on 5-hydroxytryptamine (5-HT)-induced inositol phosphates formation in 5-HT2c cDNA transfected cells.

To clarify the involvement of botulinum ADP-ribosyltransferase sensitive low molecular G-proteins in 5-hydroxytryptamine (5-HT)-induced stimulation of phosphatidylinositol turnover, we examined the effects of 5-HT on inositol phosphates formation in COS 7 cells transfected with 5-HT2c receptor cDNA, but did not in non-transfected or vector-transfected cells. A typical 5-HT2c receptor antagonist mianserin (0.3-3 microM) inhibited the 5-HT-induced inositol phosphates formation. Treatment with botulinum toxin D preparation (20 micrograms/ml, 8 h) that contained botulinum C3 ADP-ribosyltransferase, blocked the 5-HT-induced inositol phosphate formation, although botulinum toxin A preparation that did not contain the enzyme did not have an influence. These results support our previous findings suggesting that low molecular weight G-proteins ADP-ribosylated by botulinum ADP-ribosyltransferase are involved in phospholipase C activity.

[The isolation of novel intracellular factors by differential screening methods].

Synaptic plasticity, a physiological basis of learning and memory, is mainly classified into two categories: 1) relatively short-term changes in electrical activities and 2) more long-lasting morphological changes in synapses. Studies on neuronal differentiation have provided detailed clarification of many of the morphological changes in synapses. Although it has been demonstrated that neuronal differentiation is induced by a variety of stimuli, the mechanism of neuronal differentiation has never been sequentially understood. Since there must be unknown factors relevant to these complicated processes, it is important to find and identify the novel intracellular factors that are able to induce the differentiation of neurons. Differential screening is useful cloning method to identify molecules without any information about their structures. Genes expressed in a distinct pattern among two or more groups, eg. different drug-treated cells, tissues and so on, can be isolated. To identify novel neuronal differentiation factors, we differentially screened approximately 500,000 primary clones from the cDNA library of NG108-15 cells treated with TPA and diBu-cAMP for 72 hr. Using two single strand cDNA probes, which were reverse-transcribed from poly(A)+ RNA, TA-20 was isolated from cells treated with TPA and diBu-cAMP (probe TA) or from cells treated with diBu-cAMP alone (probe A) for 72 hr. Clones that hybridized preferentially to the probe TA were further investigated by Southern and Northern blots. Thus the identified clone TA20 is a novel gene and plays functional roles as a neuronal differentiation factor.

Distinct induction of c-fos mRNA in NG108-15 cells transfected with muscarinic m1 and m3 receptors.

The differences of intracellular signalling mechanisms between muscarinic acetylcholine m1 and m3 receptors, which are coupled with polyphosphoinositide turnover, were examined by using m1- and m3-transfected NG108-15 cells. The c-fos mRNA was induced by 1 mM acetylcholine peak at 60 min in both m1 and m3 cells. The c-fos induction in m1 cells was inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), but was not inhibited by prolonged treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA), suggesting that intracellular Ca2+ and calmodulin are involved in the induction. The c-fos induction in m3 cells was inhibited by BAPTA-AM and prolonged treatment with TPA, but was not influenced by W-7, suggesting that protein kinase C is mainly involved in m3-induced c-fos expression. Acetylcholine induced an increase in inositol phosphates and a transient increase in the intracellular concentration of Ca2+ in both m1 and m3 cells. Sustained stimulation of acetylcholine strongly increased the inositol monophosphate content in m3 cells, but that of inositol trisphosphate and inositol diphosphate in m1 cells. These results suggest that the difference between m1- and m3-induced c-fos mRNA induction mechanisms is due to the difference in respective properties in polyphosphoinositide turnover.

Increase in neurite formation and acetylcholine release by transfection of growth-associated protein-43 cDNA into NG108-15 cells.

We previously reported that growth-associated protein-43 (GAP-43) could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in accumulation of GAP-43. In the present study, to clarify the functional significance of GAP-43 for neurite maintenance and acetylcholine (ACh) release, we prepared NG-G11 cells by transfection of GAP-43 cDNA into NG108-15 cells. NG-G11 cells expressed GAP-43 mRNA at levels approximately twice that in nontransfected or vector-transfected cells under control conditions and after treatment with dibutyryl cyclic AMP (diBu-cAMP) or 12-O-tetradecanoylphorbol 13-acetate (TPA) plus diBu-cAMP. Neurite outgrowth after addition of diBu-cAMP was greater in NG-G11 than in control cells. In NG-G11 cells, neurites elongated by treatment with diBu-cAMP for 72 h were maintained after removal of the drug. Treatment with TPA plus diBu-cAMP for 24 h induced neurite outgrowth in NG-G11 cells, although control cells required 72 h. Depolarization by 50 mM KCl induced ACh release in both NG-G11 and control cells treated with diBu-cAMP or TPA/diBu-cAMP. Although removal of the drugs following diBu-cAMP treatment reversed ACh release to nontreated levels in control cells, a high-K(+)-induced level of ACh release remained in NG-G11 cells after removal of diBu-cAMP. ACh release induced by TPA plus diBu-cAMP for 24 h was further enhanced after removal of the drugs in NG-G11 cells, but it was not seen in control cells. These results suggest that levels of GAP-43 mRNA are correlated with neurite maintenance and the level of ACh release. Thus, GAP-43 may be involved in neuronal differentiation in NG108-15 cells.