Effect of 2-Amino-7-Phosphonoheptanoic Acid on Regional Brain Amino Acid Levels in Fed and Fasted Rodents

Abstract: 2-Amino-7-phosphonoheptanoic acid, an antagonist of excitation caused by dicarboxylic amino acids with a selective action on N -methyl-d-aspartate receptors, has been administered in an anticonvulsant dose (1 mmol/kg i.p.) to fed or fasted rats and mice. The drug impaired motor activity in fasted mice. Glucose and amino acids were determined in dissected regions of brain fixed by microwave irradiation. Glucose content was low in the brains of fasted rats and mice but was restored to normal (fed) concentration 45 min after the administration of 2-amino-7-phosphonoheptanoic acid in fasted mice. In fed animals, 2-amino-7-phosphonoheptanoic acid did not change brain aspartate concentration. In fasted animals, aspartate concentration was raised in most brain regions. In fasted rats and mice, 2-amino-7-phosphonoheptanoic acid significantly increased glutamine in rat cortex and mouse striatum, decreased glutamate content in rat striatum, and decreased aspartate concentration in all regions except mouse cortex and striatum. GABA levels were significantly decreased in rat striatum and hippocampus. These changes are consistent with an increased synaptic release of glutamate and aspartate following blockage of their post-synaptic action at selected sites.     

Implication of 5-HT2A receptors in the genetic mechanisms of the brain 5-HT system autoregulation

Brain serotonin (5-HT) system has been implicated in pathophysiology of anxiety, depression, drug addiction, and schizophrenia. 5-HT2A receptor is involved in the mechanisms of stress-induced psychopathology and impulsive behavior. Here, we investigated the role of 5-HT2A receptor in the autoregulation of the brain 5-HT system. The chronic treatment with agonist of 5-HT2A receptor DOI (1.0 mg/kg, i.p./14 days) produced considerable decrease of 5-HT2A receptor-mediated "head-twitches" in AKR/J mice indicating desensitization of 5-HT2A receptors. Chronic DOI treatment failed to alter 5-HT2A receptor gene expression in the midbrain, hippocampus and frontal cortex. At the same time, the increase in the expression of the gene encoding key enzyme of 5-HT synthesis, tryptophan hydroxylase 2 (TPH2), the increase in TPH2 activity and 5-HT levels and decreased expression of serotonin transporter (5-HTT) gene was found in the midbrain of DOI-treated mice. The results provide new evidence of receptor-gene cross-talk in the brain 5-HT system and the implication of 5-HT2A receptor in the autoregulation of the brain 5-HT system.     

Serotonin 5-HT2C receptor-mediated inhibition of the M-current in hypothalamic POMC neurons

Hypothalamic proopiomelanocortin (POMC) neurons are controlled by many central signals, including serotonin. Serotonin increases POMC activity and reduces feeding behavior via serotonion [5-hydroxytryptamine (5-HT)] receptors by modulating K(+) currents. A potential K(+) current is the M-current, a noninactivating, subthreshold outward K(+) current. Previously, we found that M-current activity was highly reduced in fasted vs. fed states in neuropeptide Y neurons. Because POMC neurons also respond to energy states, we hypothesized that fasting may alter the M-current and/or its modulation by serotonergic input to POMC neurons. Using visualized-patch recording in neurons from fed male enhanced green fluorescent protein-POMC transgenic mice, we established that POMC neurons expressed a robust M-current (102.1 ± 6.7 pA) that was antagonized by the selective KCNQ channel blocker XE-991 (40 μM). However, the XE-991-sensitive current in POMC neurons did not differ between fed and fasted states. To determine if serotonin suppresses the M-current via the 5-HT(2C) receptor, we examined the effects of the 5-HT(2A)/5-HT(2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on the M-current. Indeed, DOI attenuated the M-current by 34.5 ± 6.9% and 42.0 ± 5.3% in POMC neurons from fed and fasted male mice, respectively. In addition, the 5-HT(1B)/5-HT(2C) receptor agonist m-chlorophenylpiperazine attenuated the M-current by 42.4 ± 5.4% in POMC neurons from fed male mice. Moreover, the selective 5-HT(2C) receptor antagonist RS-102221 abrogated the actions of DOI in suppressing the M-current. Collectively, these data suggest that although M-current expression does not differ between fed and fasted states in POMC neurons, serotonin inhibits the M-current via activation of 5-HT(2C) receptors to increase POMC neuronal excitability and, subsequently, reduce food intake.     

Effects of Agomelatine on Oxidative Stress in the Brain of Mice After Chemically Induced Seizures

Agomelatine is a novel antidepressant drug with melatonin receptor agonist and 5-HT(2C) receptor antagonist properties. We analyzed whether agomelatine has antioxidant properties. Antioxidant activity of agomelatine (25, 50, or 75 mg/kg, i.p.) or melatonin (50 mg/kg) was investigated by measuring lipid peroxidation levels, nitrite content, and catalase activities in the prefrontal cortex, striatum, and hippocampus of Swiss mice pentylenetetrazole (PTZ) (85 mg/kg, i.p.), pilocarpine (400 mg/kg, i.p.), picrotoxin (PTX) (7 mg/kg, i.p.), or strychnine (75 mg/kg, i.p.) induced seizure models. In the pilocarpine-induced seizure model, all dosages of agomelatine or melatonin showed a significant decrease in TBARS levels and nitrite content in all brain areas when compared to controls. In the strychnine-induced seizure model, all dosages of agomelatine and melatonin decreased TBARS levels in all brain areas, and agomelatine at low doses (25 or 50 mg/kg) and melatonin decreased nitrite contents, but only agomelatine at 25 or 50 mg/kg showed a significant increase in catalase activity in three brain areas when compared to controls. Neither melatonin nor agomelatine at any dose have shown no antioxidant effects on parameters of oxidative stress produced by PTX- or PTZ-induced seizure models when compared to controls. Our results suggest that agomelatine has antioxidant activity as shown in strychnine- or pilocarpine-induced seizure models.     

Acute effects of caffeine injection on neutral amino acids and brain monoamine levels in rats

The injection of caffeine (100 mg/kg, i.p.) into male rats acutely increased brain levels of trytophan, serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). Blood levels of glucose, nonesterified fatty acids (NEFA) and insulin also increased, while those of the aromatic and branched-chain amino acids fell. Serum tryptophan levels either did not fall, or increased. Consequently, the serum ratio of trypthopahn to the sum of other large neutral amino acids (LNAA) increased. Less consistently noted were increases in serum free tryptophan levels. Brain tyrosine levels were not appreciably altered by caffeine, nor was the serum tyrosine ratio. In dose-response studies, 25 mg/kg of caffeine was the minimal effective dose needed to raise brain tryptophan, but only the 100 mg/kg dose elevated all three indoles in brain. In no experiments did caffeine, at any time or dose, alter brain levels of dopamine or norepinephrine. Caffeine thus probably raises brain tryptophan levels by causing insulin secretion, and thereby changing plasma amino acid levels to favor increased tryptophan uptake into brain. The rises in brain 5-HT and 5-HIAA may follow from the increase in brain tryptophan, although further data are required clearly to establish such a mechanism.     

Pulmonary responses to acute ozone exposure in fasted mice: effect of leptin administration.

Leptin is a satiety hormone that also has proinflammatory effects, including augmentation of ozone-induced pulmonary inflammation. The purpose of this study was to determine whether reductions in endogenous levels of leptin can attenuate pulmonary responses to ozone. To reduce serum leptin, we fasted mice overnight before ozone exposure. Fasting caused a marked reduction in serum leptin to approximately one-sixth the levels observed in fed mice, and continuous infusion of leptin via Alzet micro-osmotic pumps restored serum leptin to, but not above, fed levels. Ozone exposure (2 ppm for 3 h) caused a significant, approximately 40% increase in pulmonary resistance (P < 0.01) and increased airway responsiveness in fasted but not in fed mice. The increased effect of ozone on pulmonary mechanics and airway responsiveness in fasted mice was not observed when leptin was restored via continuous infusion. Ozone exposure caused pulmonary inflammation, as evident by increases in bronchoalveolar lavage cells, protein, and soluble tumor necrosis factor receptors. There was no effect of fasting status on ozone-induced changes in the bronchoalveolar lavage inflammatory profile, and leptin treatment did not alter these responses. Our results indicate that fasting augments ozone-induced changes in pulmonary mechanics and airway responsiveness in mice. These effects of fasting are the result of declines in serum leptin. The mechanistic basis for this protective effect of leptin in fasted mice remains to be determined but is not related to effects on ozone-induced inflammation.     

The effect of repeated tryptophan administration on body weight, food intake, brain lipid peroxidation and serotonin immunoreactivity in mice

Tryptophan as a circulating precursor of serotonin (5-HT) may suppress food intake and body weight. Tryptophan administration can enhance the generation of reactive oxygen species (ROS) by inducing oxidative pathway in vivo and in vitro. We have examined the effect of repeated tryptophan administration on food consumption, body weight, brain lipid peroxidation and 5-HT immunoreactivity. Tryptophan was given at the dose of 100 mg/kg/24 hr in 0.2 ml saline solution i.p. for 7 days to mice. Control mice received 0.9% NaCL solution at the same manner and volume. Body weights were recorded at the beginning and end of the experiments. Thiobarbituric acid reactive substance (TBARS), the last product of lipid peroxidation, was measured spectrophotometrically. Brain 5-HT levels were determined by the immunohistochemical method. Our findings indicate that the tryptophan suppresses food intake significantly in mice. Body weight decreased and brain TBARS levels increased significantly by repeated tryptophan treatment. Immunohistochemical detection showed that 5-HT levels increased by tryptophan administration. There is a link between increased 5-HT level and oxidative stress by tryptophan administration on brain tissue. Tryptophan at repeated doses should be exercised carefully in clinical practice.     

Aquaporin 4 regulates the effects of ovarian hormones on monoamine neurotransmission

Aquaporin 4 (AQP4) is the predominant water channels in the brain of mammals. Our previous study has reported that AQP4 knockout induced sex-specific alterations in neurotransmission, indicating that AQP4 might regulate the interaction between sex hormones and neurotransmission. In the present study, we found that AQP4 knockout decreased the concentrations of estrogen and progestogen. Further study showed that exogenous estrogen decreased DA and 5-HT in cortex, reduced DA and 5-HT in striatum, but increased 5-HT in hippocampus in AQP4+/+ male mice. However, in AQP4-/- male mice, exogenous estrogen almost did not alter the levels of neurotransmitters except for decreasing DA in cortex. In female mice, ovariectomy decreased DA in the striatum of AQP4+/+ mice, but did not alter the levels of DA in AQP4-/- mice. These findings reveal for the first time that AQP4 regulates not only water and ion homeostasis but also the functions of ovarian hormone and neurotransmitter.     

Chronic Stress Increases Serotonin and Noradrenaline in Rat Brain and Sensitizes Their Responses to a Further Acute Stress

The effects of 1 h/day restraint in plastic tubes for 24 days on the levels of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), tryptophan (TP), and noradrenaline (NA) in six regions of rat brain 20 h after the last restraint period were investigated. The levels of 5-HT, 5-HIAA, and NA but not TP increased in several regions. The effects of 1 h of immobilization on both control and chronically restrained rats were also studied. Immobilization per se did not alter brain 5-HT, 5-HIAA, and TP levels, but decreased NA in the pons plus medulla oblongata and hypothalamus. However, immobilization after chronic restraint decreased 5-HT, increased 5-HIAA, and decreased NA in most brain regions in comparison with values for the chronically restrained rats. We suggest that chronic restraint leads to compensatory increases of brain 5-HT and NA synthesis and sensitizes both monoaminergic systems to an additional acute stress. These changes may affect coping with stress demands.     

臭椿生物碱canthin-6-one对小鼠镇静催眠作用的研究

摘要 目的：研究臭椿生物碱canthin-6-one对小鼠的镇静催眠作用。方法：选昆明小鼠随机分为空白组、地西泮阳性药物组、canthin-6-one低、中、高给药组（5、10、20 mg?kg^-1），采用阈上剂量和阈下剂量戊巴比妥钠诱导小鼠睡眠，记录入睡小鼠比例、睡眠潜伏期和睡眠持续时间。此外，酶联免疫法测定小鼠脑组织中5-HT、NE、DA和GABA含量。结果：中、高剂量canthin-6-one可使小鼠的站立次数和自主活动次数均明显减少（P＜0.05），可使阈上剂量戊巴比妥钠诱导的小鼠的睡眠潜伏期缩短（P＜0.05）；各剂量组可使阈下剂量戊巴比妥钠诱导的小鼠入睡率提高（P＜0.05）。此外，中、高剂量canthin-6-one也可降低小鼠脑组织中5-HT和NE含量，增加GABA含量（P＜0.05），对DA的含量有影响，但无统计学意义（P＞0.05）。结论：Canthin-6-one有明显的协同戊巴比妥钠改善睡眠作用，其作用与相关神经递质（5-HT、NE、GABA）具有密切关系。     

Low-Dose Sarin Exposure Produces Long Term Changes in Brain Neurochemistry of Mice

Sarin is a toxic organophosphorus (OP) nerve agent that has been reported to cause long-term alterations in behavioral and neuropsychological processes. The present study was designed to investigate the effect of low dose sarin exposure on the monoamine neurotransmitter systems in various brain regions of mice. The rationale was to expand our knowledge about the noncholinergic neurochemical alterations associated with low dose exposure to this cholinesterase inhibitor. We analyzed the levels of monoamines and their metabolites in different brain areas after exposure of male C57BL/6 mice to a subclinical dose of sarin (0.4 LD50). Mice did not show any signs of cholinergic toxicity or pathological changes in brain tissue. At 1, 4 and 8 weeks post-sarin exposure brains were collected for neurochemical analysis. A significant decrease in the dopamine (DA) turnover, as measured by the metabolite to parent ratio, was observed in the frontal cerebral cortex (FC) at all time points tested. DA turnover was significantly increased in the amygdala at 4 weeks but not at 1 or 8 weeks after exposure. The caudate nucleus displayed a decrease in DA turnover at 1 week but no significant change was observed at 4 and 8 weeks suggesting a reversible effect. In addition to this, serotonin (5-HT) levels were transiently altered at various time points in all the brain regions studied (increase in FC, caudate nucleus and decrease in amygdala). Since there were no signs of cholinergic toxicity or cell death after sarin exposure, different non-cholinergic mechanisms may be involved in regulating these effects. Our results demonstrate that non-symptomatic dose of OP nerve agent sarin has potent long-term, region-specific effects on the monoaminergic neurotransmitter systems. Data also suggests differential effects of sarin on the various DA projections. These neurochemical alterations could be associated with long term behavioral and neuropsychological changes associated with low dose OP exposure.     

Additive effects of apomorphine and clonidine on serotonin neurons in the dorsal raphe

Effects of apomorphine (APO) and clonidine (CLON) on the mesostriatal and mesolimbic serotonergic systems were examined in the present study. Both drugs selectively elevated serotonin (5-HT) concentrations in the dorsal raphe and the striatum without significantly altering 5-HT measures in the median raphe and the hippocampus. Apomorphine also increased tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) levels in the dorsal raphe and 5-HIAA level in the striatum. Clonidine did not markedly alter tryptophan and 5-HIAA measures, while it decreased 5-HT turnover rate in both region, as indicated by the ratio of 5-HIAA/5-HT levels. Co-administration of APO and CLON, at doses of each drug exerted maximum effects on 5-HT alone, produced an additive effect on 5-HT in the dorsal raphe, while their effects on 5-HT and 5-HIAA in the striatum were counteracting each other. Effects of APO on 5-HT and 5-HIAA were attributed to the elevation of 5-HT precursor tryptophan, while effects of CLON on 5-HT and 5-HIAA were due to a decreased rate of 5-HT turnover. Therefore, the present results support the hypothesis that the additive effects of APO and CLON on dorsal raphe 5-HT are mediated through different receptors and neuropharmacological mechanisms.     

Effects of physical exercise on liver ATP levels in fasted and phosphate-injected rats

The purpose of the present study was to investigate the effects of exercise (30 min, 23 m/min, 0% grade) on the hepatic levels of ATP in fasted adrenodemedullated rats, with an intraperitoneal injection of sodium phosphate (Na (2) PO (4 ), 0.91 mM) or saline (NaCl). Sodium phosphate was injected to determine if the postulated decrease in liver ATP during exercise may be changed by providing an excess of phosphate. At the end of exercise, a piece of liver was rapidly freeze clamped and used for the enzymatic determination of ATP levels. Liver ATP, in saline-injected rats, was significantly (P < 0.05) decreased by fasting, compared to fed rats (𝒳 +/- SE: 3. 21 +/- 0.2 vs 2.86+/- 0.2 micromol/g). Exercise in fasted rats decreased even more the ATP response in liver (2.58 +/- 0.14 micromol/g). Injection of Na (2) PO (4) did not significantly (P > 0. 05) alter the pattern of ATP response following these 3 conditions (3.35 +/- 0.14 vs 3.0 +/-0.12 vs 2.57 +/- 0.1 micromol/g), ATP levels being significantly (P <0.05) decreased by the fast and the exercise in the fasted state. Fasting and exercise resulted in a significant (P < 0.05) decrease in liver glycogen and plasma glucose concentrations and an increase in free fatty acid levels in both NaCl- and Na (2 )PO (4) -injected groups. In both injection conditions, beta-hydroxybutyrate and peripheral insulin concentrations were respectively, increased and decreased (P < 0.05) by fasting, while norepinephrine and portal glucagon were decreased (P > 0.05) following exercise. The main effect of the injection of Na ( 2) PO (4) was a stimulation (P < 0.05) of peripheral glucagon response following exercise. It is concluded that exercise results in a decrease in liver ATP levels even in fasted rats and that this decrease is not corrected by Na (2 )PO( 4) administration. The decreased liver ATP levels might be involved in the metabolic adaptations to exercise.     

Effects of Repeated Low-dose Soman Exposure on Monoamine Levels in Different Brain Structures in Mice

In order to better understand the effects of repeated low-dose exposure to organophosphorus (OPs) on physiological and behavioural functions, we analysed the levels of endogenous monoamines (serotonin and dopamine) in different brain areas after repeated exposure of mice to sublethal dose of soman. Animals were injected once a day for 3 days with 0.12 LD50 of soman (47 μg/kg, i.p.). They did not show either severe signs of cholinergic toxicity or pathological changes in brain tissue. 24 h after the last injection of soman, inhibition of cholinesterase was similar in plasma and brain (32% and 37% of inhibition respectively). Afterwards, recovery of cholinesterase activity was faster in the plasma than in the brain. Dopamine levels were not significantly modified. On the other hand, we observed a significant modification of the serotoninergic system. An increase of the 5-HIAA/5-HT ratio was maintained for 2 and 4 weeks after exposure in the hippocampus and the striatum respectively. This study provides the first evidence of a modification of the 5-HT turnover in the hippocampus and the striatum after repeated low-dose intoxication with a nerve agent. Further experiments are necessary to evaluate the relationship between these modifications and the unexpected neuropsychological disorders usually reported after chronic exposure of organophosphorus.     

Serum increases CYP1A1 induction by 3-methylcholanthrene

Mice with a targeted null mutation of the serotonin 5-HT(2C) receptor gene exhibit hyperphagia that leads to a late-onset obesity. Here we show that oxygen consumption was decreased in fed and fasted obese mutants. No phenotypic differences were observed in uncoupling protein-1 (UCP-1) mRNA levels in brown adipose tissues and UCP-3 mRNA in skeletal muscle. UCP-2 mRNA levels were significantly increased in white adipose tissue (4-fold) and skeletal muscle (47%) in older obese mutant mice, whereas UCP-2 mRNA in liver are significantly increased in both young lean (54% increase) and older obese (52% increase) mutant mice. In contrast, 5-HT(2C) receptor mutants displayed age-dependent decreases in beta 3-adrenergic receptor (beta 3-AR) mRNA levels in white adipose tissue, however, no such changes were observed in brown adipose tissue. These results indicate that a mutation of 5-HT(2C) receptor gene leads to a secondary decrease in beta 3-AR gene expression that is related to enhanced adiposity.     

PCB disruption of the hypothalamus-pituitary-interrenal axis involves brain glucocorticoid receptor downregulation in anadromous Arctic charr

We examined whether brain glucocorticoid receptor (GR) modulation by polychlorinated biphenyls (PCBs) was involved in the abnormal cortisol response to stress seen in anadromous Arctic charr (Salvelinus alpinus). Fish treated with Aroclor 1254 (0, 1, 10, and 100 mg/kg body mass) were maintained for 5 mo without feeding in the winter to mimic their seasonal fasting cycle, whereas a fed group with 0 and 100 mg/kg Aroclor was maintained for comparison. Fasting elevated plasma cortisol levels and brain GR content but depressed heat shock protein 90 (hsp90) and interrenal cortisol production capacity. Exposure of fasted fish to Aroclor 1254 resulted in a dose-dependent increase in brain total PCB content. This accumulation in fish with high PCB dose was threefold higher in fasted fish compared with fed fish. PCBs depressed plasma cortisol levels but did not affect in vitro interrenal cortisol production capacity in fasted charr. At high PCB dose, the brain GR content was significantly lower in the fasted fish and this corresponded with a lower brain hsp70 and hsp90 content. The elevation of plasma cortisol levels and upregulation of brain GR content may be an important adaptation to extended fasting in anadromous Arctic charr, and this response was disrupted by PCBs. Taken together, the hypothalamus-pituitary-interrenal axis is a target for PCB impact during winter emaciation in anadromous Arctic charr.     

Effects of clofibrate on plasma tryptophan, growth hormone, and prolactin and on brain tryptophan and serotonin in prepubertal rats

The effects of clofibrate administration (200 mg/kg, po) on somatic growth, plasma levels of lipids, tryptophan, growth hormone (GH), and prolactin (PRL), as well as on brain concentrations of tryptophan and 5-hydroxytryptamine (5-HT) were studied in prepubertal male rats. The drug did not significantly alter ponderal growth, but an appreciable reduction of tail length was observed in rats treated for 30 days. Triglyceride concentrations in plasma showed a 43% diminution after 30 days of treatment, whereas free fatty acid (FFA) levels were not modified. Clofibrate administration for 7, 15, or 30 days caused a fall in total tryptophan and a significant increase of the free fraction in plasma with no change in brain tryptophan levels. Brain 5-HT was generally unaffected but a marked elevation of this parameter was noted in rats treated for 15 days. Plasma GH and PRL concentrations remained unaltered. It may be concluded from these findings that the slight reduction of somatic growth, the diminution of triglycerides, and the increase of free tryptophan in plasma, induced by chronic clofibrate treatment, are not associated with variations in brain tryptophan and 5-HT levels or with modifications of plasma GH and PRL titers.     

Involvement of 5-HT<Subscript>2A</Subscript> receptors in genetic mechanisms of autoregulation of brain 5-HT system

The brain serotonin (5-HT) system has been implicated in the pathophysiology of anxiety, depression, drug addiction, and schizophrenia. 5-HT_2A receptors are involved in the mechanisms of stressinduced psychopathology and impulsive behavior. In this work, we investigated the role of 5-HT_2A receptors in the autoregulation of the brain 5-HT system. Chronic treatment with DOI, a 5-HT_2A receptor agonist (1.0 mg/kg, i.p./14 days), produced a considerable decrease in the number of 5-HT_2A receptor-mediated head twitches in AKR/J mice, indicating the desensitization of 5-HT_2A receptors. Chronic DOI treatment did not affect the expression of the 5-HT_2A receptor gene in the midbrain, hippocampus and frontal cortex. At the same time, an increase in the expression of the gene encoding a key enzyme of 5-HT synthesis, tryptophan hydroxylase-2 (TPH-2), accompanied with an increase in TPH-2 activity and 5-HT levels, and decreased expression of the serotonin transporter (5-HTT) gene were observed in the midbrain of DOI-treated mice. These results provide new evidence of receptor-gene cross-talk in the brain 5-HT system and implication 5-HT_2A receptors in the autoregulation of the brain 5-HT system.