Theanine, γ-glutamylethylamide,a unique amino acid in tea leaves,modulates neurotransmitter concentrations in the brain striatum interstitium in conscious rats

Theanine (γ-glutamylethylamide) is one of the major amino acid components in green tea and can pass through the blood-brain barrier. Recent studies suggest that theanine affects the mammalian central nervous system; however, the detailed mechanism remains unclear. In this study, we demonstrated the effect of theanine on neurotransmission in the brain striatum by in vivo brain microdialysis. Theanine injection into the rat brain striatum did not increase the concentration of excitatory neurotransmitters in the perfusate. On the other hand, theanine injection increased the concentration of glycine in the perfusate. Because it has been reported that theanine promotes dopamine release in the rat striatum, we investigated the glycine and dopamine concentrations in the perfusate. Co-injection of glycine receptor antagonist, strychnine, reduced theanine-induced changes in dopamine. Moreover, AMPA receptor antagonist, which regulates glycine and GABA release from glia cells, inhibited these effects of theanine and this result was in agreement with the known inhibitory effect of theanine at AMPA receptors.     

Effect of quantity and quality of dietary protein on choline acetyltransferase and nerve growth factor, and their mRNAs in the cerebral cortex and hippocampus of rats

The brain protein synthesis is sensitive to the dietary protein; however, the role of dietary protein on biomarkers including choline acetyltransferase and nerve growth factor (NGF) for the function of cholinergic neurons remains unknown in young rats. The purpose of this study was to determine whether the quantity and quality of dietary protein affects the concentration of NGF and activity of choline acetyltransferase, and their mRNA levels in the brains of young rats. Experiments were carried out on five groups of young rats (4 weeks) given the diets containing 0, 5, 20% casein, 20% gluten or 20% gelatin for 10 days. The activity of choline acetyltransferase in the cerebral cortex and hippocampus declined gradually with a decrease in quantity and quality of dietary protein. The concentration of NGF in the cerebral cortex and the mRNA levels of choline acetyltransferase in the cerebral cortex and hippocampus did not differ among groups. However, the concentration and mRNA level of NGF in the hippocampus was significantly lower in rats fed with lower quantity of protein or lower quality of protein. In the hippocampus, the mRNA levels of NGF significantly correlated with the NGF concentration when the quantity (r = 0.704, P < 0.01) and quality (r = 0.682, P < 0.01) of dietary protein was manipulated. It was further found that a significant positive correlation existed between the NGF concentration and the activity of choline acetyltransferase in the hippocampus (dietary protein quantity, r = 0.632, P < 0.05; dietary protein quality, r = 0.623, P < 0.05). These results suggest that the ingestion of lower quantity and quality of dietary protein are likely to control the mRNA level and concentration of NGF, and cause a decline in the activity of choline acetyltransferase in the brains of young rats.     

The effects of training and detraining on memory, neurotrophins and oxidative stress markers in rat brain

In the current investigation we tested how swimming training (T) (8 week, 5 times/week, 2 h/day), and detraining (DT) affects brain functions and oxidative stress markers in rat brain. The free radical concentration, measured by electron paramagnetic resonance, decreased in brain of T and DT rats compared to controls (C). The level of brain-derived neurotrophic factor (BDNF) increased as a result of training, but decreased below the control level after 6 weeks of detraining. In addition, the concentration of nerve growth factor (NGF) also declined with DT. The passive avoidance test was used to assess the memory of rats, and training-induced improvement was observed but the enhancement disappeared with detraining. When the content of mitochondrial electron transport complexes, as a potent free radical generator, was evaluated by the blue native gel method, no significant alterations were observed. The repair of nuclear and mitochondrial 8-oxodeoxyguanosine, as measured by the activity of OGG1, showed no significant difference. Therefore, the results suggest that regular exercise training improves memory, decreases the level of reactive oxygen species, and increase the production of BDNF and NGF. On the other hand, it appears that the beneficial effects of training are reversible in the brain, since detraining down-regulates the neurotrophin level, and memory. It is suggested that exercise training is more likely to beneficially effect the production of reactive oxygen species and the related oxidative damage.     

Regulation of nerve growth factor mRNA levels in developing rat heart ventricle is not altered by sympathectomy

The survival of sympathetic and sensory neurons is known to be controlled by nerve growth factor (NGF) supplied by the targets of innervation, yet little is known about how target NGF synthesis is regulated. We have investigated the pattern of NGF mRNA expression in developing rat heart ventricle using a sensitive RNA blotting procedure. We find that the concentration of NGF mRNA increases steadily from Embryonic Day 17 to peak levels at 10-14 days postnatal and then declines about twofold and stabilizes at the level found in adults. The rise in NGF mRNA concentration correlates with the arrival and differentiation of sympathetic nerve terminals in the heart and the cessation of sympathetic cell death. To assess the role of innervating sympathetic neurons in regulating NGF mRNA expression, neonatal rats were sympathectomized by treatment with 6-hydroxydopamine and heart ventricles were assayed for NGF message. Although this treatment reduced ventricle norepinephrine content by 82%, no significant change in NGF mRNA concentration was observed. These results suggest that the developmental program of NGF mRNA production in the heart is not influenced by innervating sympathetic neurons.     

Regulation of nerve growth factor synthesis/secretion by catecholamine in cultured mouse astroglial cells

The nerve growth factor (NGF) synthesis/secretion by cultured mouse astroglial cells was modulated by catecholamine. In quiescent cells, epinephrine (EN) and dopamine (DA) markedly increased the NGF content in the conditioned medium (CM). Conversely, EN, DA, and norepinephrine (NE) decreased the NGF content in growing cells. Cholinergic agonists, metacholine and carbamylcholine, slightly increased the NGF content in quiescent cells, but showed no effects on growing cells. Other neurotransmitters tested had no effects on either growing or quiescent cells. These results suggest that catecholamine is one of the molecules responsible for regulation of NGF synthesis/secretion in the mouse brain.     

Intranasal Administration of Nerve Growth Factor Produces Antidepressant-Like Effects in Animals

Many works showed that nerve growth factor (NGF) injected into the brain of animal model emerges potential antidepressant effects. However, this route of administration significantly restricts the application of NGF clinically. Here, we reported that intranasal NGF could provide an alternative to intraventricular injection. The behavioral analysis showed that intranasal administration of NGF reduced the immobility time in forced swimming test (FST) and tail suspension test (TST) in mice. Likewise, intranasal NGF increased the sucrose intake and the locomotor activity in rats after unpredictable chronic mild stress (UCMS). Furthermore, intranasal NGF increased the levels of monoamine neurotransmitters (norepinephrine, dopamine) in the frontal cortex and hippocampus and affected the number of 5-bromodeoxyuridine (BrdU), c-fos and caspase-3 positive neurons in dentate gyrus of hippocampus in rats after UCMS. In summary, intranasal NGF had significant antidepressant effects on animal models of depression and this route of administration may provide a promising way to deliver NGF to brain in a therapeutic perspective.     

Facilitated Neurogenesis in the Developing Hippocampus After Intake of Theanine,an Amino Acid in Tea Leaves,and Object Recognition Memory

Theanine, γ-glutamylethylamide, is one of the major amino acid components in green tea. In this study, cognitive function and the related mechanism were examined in theanine-administered young rats. Newborn rats were fed theanine through dams, which were fed water containing 0.3% theanine, and then fed water containing 0.3% theanine after weaning. Theanine level in the brain was under the detectable limit 6 weeks after the start of theanine administration. Theanine administration did not influence locomotor activity in the open-field test. However, rearing behavior was significantly increased in theanine-administered rats, suggesting that exploratory activity is increased by theanine intake. Furthermore, object recognition memory was enhanced in theanine-administered rats. The increase in exploratory activity in the open-field test seems to be associated with the enhanced object recognition memory after theanine administration. On the other hand, long-term potentiation (LTP) induction at the perforant path-granule cell synapse was not changed by theanine administration. To check hippocampal neurogenesis, BrdU was injected into rats 3 weeks after the start of theanine administration, and brain-derived neurotropic factor (BDNF) level was significantly increased at this time. Theanine intake significantly increased the number of BrdU-, Ki67-, and DCX-labeled cells in the granule cell layer 6 weeks after the start of theanine administration. This study indicates that 0.3% theanine administration facilitates neurogenesis in the developing hippocampus followed by enhanced recognition memory. Theanine intake may be of benefit to the postnatal development of hippocampal function.     

Role of nerve growth factor-tyrosine kinase receptor A signaling in paclitaxel-induced peripheral neuropathy in rats

The mechanisms underlying paclitaxel-induced peripheral neuropathy remain unknown. Nerve growth factor (NGF) is a representative neurotrophic factor that maintains neuronal function, promotes survival, and mediates neuropathic pain. We investigated expression levels of NGF and its receptors in the dorsal root ganglia (DRG) and spinal dorsal horn (DH) following paclitaxel treatment. Intraperitoneal (I.P.) administration of paclitaxel induced significant mechanical hypersensitivity and cold allodynia in rats, significantly increased the expression of NGF and its receptor tyrosine kinase receptor A (trkA) in the DRG, and increased NGF expression in the DH. In contrast, paclitaxel treatment did not alter the mRNA levels of NGF or its receptors in the DRG, DH, sciatic nerve, or hindpaw skin. Moreover, expression of NEDD4-2, a negative regulator of trkA, was significantly increased in the DRG of paclitaxel-treated rats. Intrathecal (I.T.) administration of the tyrosine kinase receptor inhibitor k252a significantly alleviated mechanical hypersensitivity in paclitaxel-treated rats. Our results suggest that NGF–trkA signaling is involved in mechanical allodynia in paclitaxel-induced neuropathy.     

Time-dependent changes of amino acids in the serum, liver, brain and urine of rats administered with theanine.

Time-dependent changes of theanine (gamma-glutamylethylamide) and other amino acids in various tissues of rats were investigated during the 24 hrs after theanine administration. When theanine (4 g/kg of body weight) was intragastrically administered to rats, the concentrations of theanine in the serum, liver and brain were significantly increased 1 hr after its administration, and thereafter gradually decreased, but reached the maximum level in the brain after 5 hrs. Theanine in these tissues had completely disappeared 24 hrs after its administration. In contrast, the administration of theanine resulted in the concentrations of theanine, urea, ethylamine and glutamic acid in the urine being significantly enhanced. These results suggest that theanine might be degraded via glutamic acid.     

Eukaryotic translation initiation factor 4E (eIF4E) expression in the brain tissue is induced by infusion of nerve growth factor into the mouse cisterna magnum: an in vivo study

In many cell types translation can be regulated by an expression of the translation initiation factor. Eukaryotic translation initiation factor eIF4E, which binds to the 5′ cap structure of mRNA, plays an important role in translation regulation and it has been suggested that it is implicated in increased protein synthesis promoted by growth factors. In this study the effects of nerve growth factor (NGF) infusion into the cerebrospinal fluid (CSF) on eIF4E expression and phosphorylation in mouse brain tissue have been investigated. We investigated NGF as it is one of the most important growth factors and it is an important factor in cerebral cortical development, stimulating neuronal precursor proliferation. eIF4E level is also increased in response to infusion of NGF into the CSF. The present study shows that eIF4E is phosphorylated in the brain tissues treated with NGF. It is concluded that NGF regulates protein synthesis in the nervous tissue by enhancing expression and phosphorylation of eIF4E.     

Bisphenol-A suppresses neurite extension due to inhibition of phosphorylation of mitogen-activated protein kinase in PC12 cells

An endocrine disrupter, bisphenol-A is widely used in the production of plastics and coatings. Recently, it was reported that bisphenol-A affected neurotransmitters in the mammalian brain. On the basis of these reports, it was considered that bisphenol-A affected neuronal differentiation. In this study, the morphological changes in nerve growth factor (NFG)-induced differentiation caused by bisphenol-A were confirmed using a PC12 cell system. When a low concentration of bisphenol-A was added to medium containing NGF, it inhibited neurite extension. In addition, to clarify whether bisphenol-A affects the early and late stages of the NGF-signaling pathway in cell differentiation, changes of phosphorylation of MAP kinases and cAMP-response element binding protein (CREB) in PC12 cells treated with and without BPA in medium containing NGF were investigated using western blot analysis. As results, bisphenol-A significantly inhibited phosphorylation of CREB and ERK1/2 MAPK.     

Transforming growth factor-beta 1 in the rat brain: increase after injury and inhibition of astrocyte proliferation

Transforming growth factor-beta 1 (TGF-beta 1) has been shown to up-regulate the synthesis of nerve growth factor (NGF) in cultured rat astrocytes and in neonatal brain in vivo (Lindholm, D., B. Hengerer, F. Zafra, and H. Thoenen. 1990. NeuroReport. 1:9-12). Here we show that mRNA encoding TGF-beta 1 increased in rat cerebral cortex after a penetrating brain injury. The level of NGF mRNA is also transiently increased after the brain trauma, whereas that of brain-derived neurotrophic factor remained unchanged. In situ hybridization experiments showed a strong expression of TGF-beta 1 4 d after the lesion in cells within and in the vicinity of the wound. Staining of adjacent sections with OX-42 antibodies, specific for macrophages and microglia/brain macrophages, revealed a similar pattern of positive cells, suggesting that invading macrophages, and perhaps reactive microglia, are the source of TGF-beta 1 in injured brain. Both astrocytes and microglia express TGF-beta 1 in culture, and TGF-beta 1 mRNA levels in astrocytes are increased by various growth factors, including FGF, EGF, and TGF-beta itself. TGF-beta 1 is a strong inhibitor of astrocyte proliferation and suppresses the mitotic effects of FGF and EGF on astrocytes. The present results indicate that TGF-beta 1 expressed in the lesioned brain plays a role in nerve regeneration by stimulating NGF production and by controlling the extent of astrocyte proliferation and scar formation.     

Administration of Theanine,a Unique Amino Acid in Tea Leaves,Changed Feeding-Relating Components in Serum and Feeding Behavior in Rats

We identified an effect of γ-glutamylethylamide (theanine) on feeding in a rat study. Oral theanine suppressed the food intake of rats. The serum glucose level did not differ from the control, but the insulin concentration was reduced and the corticosterone concentration was increased by theanine. We suggest that the effect of theanine on feeding involved hormones.     

Effects of Panax ginseng on the nerve growth factor expression in testosterone induced benign prostatic hyperplasia

The prostatic hyperplasia in benign prostatic hyperplasia (BPH) leads to obstructive micturition symptoms. Previous studies showed that pontine micturition center (PMC), ventrolateral periaqueductal gray (vlPAG), and medial preopticnucleus (MPA) regions in the brain have been known to regulate the urinary bladder function. The present study shows the influences of Panax ginseng on nerve growth factor (NGF) expressions in PMC, vlPAG, and MPA regions in the brain. Wistar rats were used for the present study. The rats split into four groups; 4 groups (n = 6) in control group, BPH-induced group, BPH-induced and P. ginseng-treated group, and BPH-induced and finasteride-treated group. BPH in rats was induced by testosterone and the animals were evaluated for NGF expression in PMC, vlPAG, and MPA regions in the brain. The NGF expression was identified using immunohistochemistry (IHC). The NGF expression by IHC showed spots with dark brown color. In our results, NGF expressions in PMC, vlPAG, and MPA regions in the brainstem of the BPH-induced group showed increase than the control animal. These increased NGF expressions in three regions were decreased using treatment with P. ginseng (200 mg/kg). These results suggest that P. ginseng has therapeutic effects on the symptoms of BPH and is associated with the regulation of NGF expression in the brain. In conclusion, the administration of P. ginseng helps nerve growth factor activation.     

Administration of theanine, a unique amino acid in tea leaves, changed feeding-relating components in serum and feeding behavior in rats

We identified an effect of gamma-glutamylethylamide (theanine) on feeding in a rat study. Oral theanine suppressed the food intake of rats. The serum glucose level did not differ from the control, but the insulin concentration was reduced and the corticosterone concentration was increased by theanine. We suggest that the effect of theanine on feeding involved hormones.     

Association of overactive bladder and stress urinary incontinence in rats with pudendal nerve ligation injury

Approximately one-third of patients with stress urinary incontinence (SUI) also suffer from urgency incontinence, which is one of the major symptoms of overactive bladder (OAB) syndrome. Pudendal nerve injury has been recognized as a possible cause for both SUI and OAB. Therefore, we investigated the effects of pudendal nerve ligation (PNL) on bladder function and urinary continence in female Sprague-Dawley rats. Conscious cystometry with or without capsaicin pretreatment (125 mg/kg sc), leak point pressures (LPPs), contractile responses of bladder muscle strips to carbachol or phenylephrine, and levels of nerve growth factor (NGF) protein and mRNA in the bladder were compared in sham and PNL rats 4 wk after the injury. Urinary frequency detected by a reduction in intercontraction intervals and voided volume was observed in PNL rats compared with sham rats, but it was not seen in PNL rats with capsaicin pretreatment that desensitizes C-fiber-afferent pathways. LPPs in PNL rats were significantly decreased compared with sham rats. The contractile responses of detrusor muscle strips to phenylephrine, but not to carbachol, were significantly increased in PNL rats. The levels of NGF protein and mRNA in the bladder of PNL rats were significantly increased compared with sham rats. These results suggest that pudendal nerve neuropathy induced by PNL may be one of the potential risk factors for OAB, as well as SUI. Somato-visceral cross sensitization between somatic (pudendal) and visceral (bladder) sensory pathways that increases NGF expression and alpha(1)-adrenoceptor-mediated contractility in the bladder may be involved in this pathophysiological mechanism.     

Effect of estradiol and soy phytoestrogens on choline acetyltransferase and nerve growth factor mRNAs in the frontal cortex and hippocampus of female rats.

We report here the effects of oral micronized estradiol and soy phytoestrogens on uterine weight, choline acetyltransferase (ChAT) and nerve growth factor (NGF) mRNAs in the frontal cortex and hippocampus of ovariectomized young and retired breeder rats. Within each age category, 15 bilaterally ovariectomized rats were randomized equally into three groups: control (OVX), estradiol (E2), and soy phytoestrogens (SBE). The OVX rats were fed a casein/lactalbumin-based control diet; the E2 rats were fed with the control diet with added estradiol; and the SBE rats were fed with the control diet with added soy phytoestrogens. After 8 weeks of treatment, blood, uteri, frontal cortex, and hippocampus were collected at necropsy. Results showed that the uterine weights and serum estradiol concentrations were significantly higher in the E2 group compared with those in the OVX and SBE groups. In the hippocampus of young rats, E2 treatment resulted in significantly higher NGF mRNA levels than no treatment (OVX), and NGF mRNA levels in the SBE group were intermediate between the E2 and OVX groups. ChAT mRNA levels were significantly higher in the frontal cortex of E2 and SBE-treated retired breeder rats compared to OVX retired breeder rats. There were no differences among treatment groups for ChAT mRNA levels in the frontal cortex of young rats and in the hippocampus of both young and retired breeder rats. Our data suggest that soy phytoestrogens may function as estrogen agonists in regulating ChAT and NGF mRNAs in the brain of female rats.     

Structure and expression of the chicken beta nerve growth factor gene.

The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult.