Lipidomic analysis of p-chlorophenylalanine-treated mice using continuous-flow two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: Although serotonin deficiency is involved with various physiological disorders such as Alzheimer's disease, Parkinson's disease, schizophrenia and depression, the serotonin-dependent pathomechanisms remain poorly understood, particularly from a lipidomics perspective. METHODS: This study therefore aimed to identify novel lipid biomarkers associated with serotonin deficiency by lipid profiling of p-chlorophenylalanine (pCPA)-treated, serotonin-deficient mice using continuous-flow normal-phase/reversed-phase two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry (NP/RP 2D LC/QTOFMS). Principal component analysis (PCA) was performed to distinguish significantly altered lipids between the pCPA-treated mice and control mice. RESULTS: Eighteen lipid biomarkers were associated with pCPA-induced serotonin deficiency. Specifically, lipid species of lysophosphatidylethanolamine (LPE), phosphatidylethanolamine (PE), sphingomyelin (SM), galactosylceramide (GalCer), glucotosylceramide (GluCer), lactosylceramide (LacCer) and triacylglycerol (TG) were down-regulated whereas glycerophosphocholine (PC) and phosphatidylinositol (PI) were up-regulated in the pCPA-treated mice compared with control mice. CONCLUSIONS: This work demonstrates the significant effects of serotonin deficiency on lipid metabolisms and will facilitate improved understanding of pathomechanisms in serotonin deficiency, particularly from a lipidomics perspective.

Anxiolytic and antidepressant like effects of natural food flavour (E)-methyl isoeugenol.

(E)-methyl isoeugenol (MIE) is a natural food flavour that constitutes 93.7% of an essential oil from Pimenta pseudocaryophyllus leaf. The leaf extracts of this species are used as a calming agent. As a ubiquitous food additive, the application of MIE for treating mood disorders appears to be globally attractive. Hence, we sought to evaluate general pharmacological activities, anticonvulsant, anxiolytic and antidepressant effects and the possible mechanisms of MIE actions. Administration of MIE was carried out prior to the exposure of a male Swiss mice to general behavioural tests, barbiturate sleep, PTZ-induced convulsion, light dark box (LDB), elevated plus maze (EPM), wire hanging, open field (OF) and forced swimming test (FST). The involvement of monoamine system was studied by mice pretreatment with WAY100635 (antagonist of 5-HT1A), α-methyl-p-tyrosine (AMPT; depletor of catecholamine) or p-chlorophenylalanine (PCPA; depletor of serotonin storage). There was no record of neurotoxic effect or animal's death during the course of general pharmacological tests. MIE at 250 and 500 mg kg(-1) potentiated the hypnotic effect of sodium pentobarbital. However, MIE did not protect against PTZ-induced convulsion. Except for MIE at 500 mg kg(-1), parameters evaluated in the LDB, EPM and OF demonstrated an anxiolytic like property of MIE. This effect was blocked by WAY100635 pretreatment. MIE at 500 mg kg(-1) elicited a reduction in locomotor activity of the mice in the OF. Anti-immobility effect of MIE 250 mg kg(-1) in the FST suggested an antidepressive like property. Unlike AMPT, pretreatment with PCPA reversed the antidepressant like effect of MIE. Our findings demonstrated anxiolytic and antidepressant like properties of (E)-methyl isoeugenol and suggested the participation of serotonergic pathways.

[Expression of cation-chloride cotransporters KCC2 and NKCC1 in brainstem of para- chlorophenylalanine-induced acute insomnia rats].

OBJECTIVE: To explore the possible roles of KCC2 and NKCC1 in the pathological mechanism of acute insomnia in rats. METHODS: A total of 18 Sprague-Dawley rats were randomly selected into model, interference and normal control groups.The expressions of KCC2 and NKCC1 in brainstem were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.The concentration of intracellular Cl(-) ([Cl(-)]i) in brainstem was detected by fluorescence probe MQAE with laser confocal microscopy. RESULTS: (1) Comparing with the control group, both KCC2 mRNA and protein expression were down-regulated in the model and interference groups (mRNA:0.196 ± 0.021 vs 0.939 ± 0.109, P < 0.05; 0.485 ± 0.026 vs 0.939 ± 0.109, P < 0.05; protein expression:0.363 ± 0.058 vs 0.967 ± 0.155, P < 0.05; 0.663 ± 0.106 vs 0.967 ± 0.155, P < 0.05).However they became up-regulated in the interference group versus the model group (mRNA: 0.485 ± 0.026 vs 0.196 ± 0.021, P < 0.05; protein expression:0.663 ± 0.106 vs 0.363 ± 0.058, P < 0.05). (2) Comparing with the control group, both NKCC1 mRNA and protein expression in the model group were slightly up-regulated.But statistical difference was insignificant (mRNA: 0.344 ± 0.026 vs 0.320 ± 0.019, P > 0.05; protein expression:0.244 ± 0.010 vs 0.230 ± 0.021, P > 0.05).There was down-regulation in the interference group versus the model and control groups (mRNA: 0.066 ± 0.031 vs 0.320 ± 0.019, P < 0.05; 0.066 ± 0.031 vs 0.344 ± 0.026, P < 0.05; protein expression:0.131 ± 0.012 vs 0.230 ± 0.021, P < 0.05; 0.131 ± 0.012 vs 0.244 ± 0.010, P < 0.05). (3) Comparing with the control group, [Cl(-)]i became up-regulated in the model group (0.0315 ± 0.0039 vs 0.0164 ± 0.0019, P < 0.05).It was down-regulated in the interference group versus the model group (0.0182 ± 0.0013 vs 0.0315 ± 0.0039, P < 0.05), but higher than control group without statistical difference (0.0182 ± 0.0013 vs 0.0164 ± 0.0019, P > 0.05). CONCLUSION: The down-regulation of KCC2 and rise of [Cl(-)]i in brainstem may participate in the pathological mechanism of acute insomnia in rats. And the mechanism of sedative-hypnotic diazepam may be operate through an up-regulation of KCC2, a down-regulation of NKCC1 and decreased [Cl(-)]i.

Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models.

Serotonin (5-HT) is a neurotransmitter that is involved in many behavioral functions, including the organization of defense, and its putative pathological correlate, anxiety and stress disorders. Recently, behavioral tests for anxiety have been proposed in zebrafish. Exposure to the novel tank test or to the light/dark test increased extracellular fluid 5-HT content in the brain; anxiety-like behavior correlated positively with 5-HT content in the novel tank test, while the correlation was negative in the light/dark test. Acute treatment with a low dose of fluoxetine was anxiolytic in the geotaxis test and anxiogenic in the scototaxis test, while treatment with a higher dose produced a hyperlocomotor effect in both tasks. Buspirone and WAY 100635 were anxiolytic in both tests, while SB 224289 was anxiolytic in the geotaxis and slightly anxiogenic in the scototaxis test. Serotonin depletion with pCPA was anxiogenic in the geotaxis and anxiolytic in scototaxis. These results underline the differential sensitivity of these tasks to assess serotonergic agents; alternatively, serotonin might regulate zebrafish behavior differently in the novel tank test and in the light/dark test.

[Effects of extracts from ziziphi spinosae semen and schisandrae chinensis fructus on amino acid neurotransmitter in rats with insomnia induced by PCPA].

OBJECTIVE: To observe the effects of extract from Ziziphus Spinosa Semen and Schisandrae Chinensis Fructus on the content of amino acid neurotransmitter in the hypothalamus of insomnia rats induced by P-Chlorophenylalanine (PCPA) and its mechanism. METHODS: The model of insomnia rats were established by PCPA intraperitoneal injection, after the modeling, all the therapeutic group were treated with corresponding drug for one week. The hypothalamus pathological changes of the rats were observed. The contents of GABA, Glu in the hypothalamus were detected by Elisa. The GABA, Glu protein expression were detected by immunohistochemical. GABA(A), R(alpha1) and GABA(A)R(gamma2) mRNA expressions were detected by RT-PCR. RESULTS: Compared with model group, the content of GABA in the hypothalamus of rats increased obviously in the alcohol-water group (P < 0.05 or P < 0.01), while the content of Glu decreased obviously (P < 0.05 or P < 0.01). CONCLUSION: The extract from Ziziphus Spinosae Semen and Schisandrae Chinensis Fructus has obviously Sedative-hypnotic effect. Its mechanism may be related to regulating the content of amino acid neurotransmitter in the hypothalamus of rats.

A pharmacological evidence of positive association between mouse intermale aggression and brain serotonin metabolism.

The neurotransmitter serotonin (5-HT) is involved in the regulation of mouse intermale aggression. Previously, it was shown that intensity of mouse intermale aggression was positively associated with activity of the key enzyme of 5-HT synthesis - tryptophan hydroxylase 2 (TPH2) in mouse brain. The aim of the present study was to investigate the effect of pharmacological activation or inhibition of 5-HT synthesis in the brain on intermale aggression in two mouse strains differing in the TPH2 activity: C57BL/6J (B6, high TPH2 activity, high aggressiveness) and CC57BR/Mv (BR, low TPH2 activity, low aggressiveness). Administration of 5-HT precursor L-tryptophan (300 mg/kg, i.p.) to BR mice significantly increased the 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels in the midbrain as well as the number of attacks and their duration in the resident-intruder test. And vice versa, administration of TPH2 inhibitor p-chlorophenylalanine (pCPA) (300 mg/kg, i.p., for 3 consecutive days) to B6 mice dramatically reduced the 5-HT and 5-HIAA contents in brain structures and attenuated the frequency and the duration of aggressive attacks. At the same time, L-tryptophan or pCPA did not influence the percentage of aggressive mice and the attack latency reflecting the threshold of aggressive reaction. This result indicated that the intensity of intermale aggression, but not the threshold of aggressive reaction is positively dependent on 5-HT metabolism in mouse brain.

Evaluation of object-based attention in mice.

The deficits of attention result in significant impairment in daily life, and pharmacological intervention to improve attention is the most effective treatment in clinics. However, methods which are suitable for the large scale preclinical screening of attention-improving compounds or drugs are few in the field. In this study, we have developed object-based attention task as a simple and wherever-practical method that suitable for quick drug screening in mice. Treatment with p-chlorophenylalanine (pCPA) (200mg/kg/day, i.p.) for three consecutive days reduced the prefrontal cortical content of serotonin and dopamine, and increased turn-over of dopamine while decreasing turn-over of norepinephrine in the prefrontal cortex on day 7. Auditory attention and working memory, but not long-term object memory after a long (10 min) object (two objects)-exposure period, were impaired on day 7 after the same treatment paradigm with pCPA. Novel object recognition ability immediately (<10s) after a short (3 min) object (on two objects)-exposure period was not impaired after pCPA treatment. However, novel object recognition ability immediately (<10s) after a short (3 min), but not long (6 min), object (five objects)-exposure period was impaired after pCPA treatment. For the verification, the current task, the object-based attention task, was confirmed in an attention deficit model induced by acute phencyclidine (1mg/kg, i.p.) treatment in mice. It was implied that the object-based attention task would assist the behavioral screening process of pharmacological studies on attention-improving drugs.

[Behavior of female and male mice submitted to action of p-chlorophenylalanine in prenatal ontogenesis].

Effect of serotonin (5-HT) deficit produced by administration ofp-chlorophenylalanine at a dose of 400 mg/kg to pregnant female mice on the day 8 of gestation and on the subsequent behavior of their offspring (hybrids F1 (C57BL/CBA)) was studied. The 5-HT deficit in prenatal ontogenesis leads to the following changes of behavior: 1) females and males of the experimental group show a higher level of the explorative activity in the "open field" than control animals; 2) in females of the experimental group at the age of 90 days, unlike control females and males of experimental and control groups, the explorative activity is extinguished at the threefold testing in the "open field"; 3) females of the experimental group have a decreased level of anxiety in tests "elevated plus-maze" and the "dark-light chamber". Males of the experimental group, on the contrary, have an elevated level of anxiety. The obtained data show that the 5-HT deficit at the prenatal period affects various aspects of behavior. The degree of the changes produced by the prenatal 5-HT deficit can have different manifestation depending on sex of the animals.

Behavioral alteration in the adult rats prenatally exposed to para-chlorophenylalanine.

In the present work, effects of maternal administration of para-chlorophenylalanine (PCPA), a serotonin synthesis inhibitor, on behavior of adult offspring were studied. Pregnant rats were injected intraperitoneally with PCPA (200/100/100/50 mg/kg) either on the gestational days (GD) 8-11 or 14-17, or with vehicle at the same days. Behavioral parameters, in an open field, the Porsolt forced swim test and the Morris water maze test were evaluated at the age of 3-3.5 months in the male and female offspring. The prenatal PCPA increased activity in an open field in the offspring treated on either GD 8-11 or 14-17. The highest levels of the activity were revealed in the male and female offspring treated on GD 14-17. Besides, the PCPA treatment on GD 8-11 or 14-17 facilitated the intersession habituation of activity to repeated exposures to an open field in the male offspring. Both male and female offspring treated on GD 14-17 showed an increased immobility in the Porsolt forced swim test and a significant learning impairment in the Morris water maze. Thus, it has been shown that administration of PCPA to pregnant rats might cause significant changes in the adult offspring behavior. These results provide further evidence that unfavorable influence may have more adverse effects on the behavioral development of rats when exposed during the final trimester of pregnancy than during the second trimester.

Behavioral effects of pubertal anabolic androgenic steroid exposure in male rats with low serotonin.

The goal of this study was to assess the interactive effects of chronic anabolic androgenic steroid (AAS) exposure and brain serotonin (5-hydroxytryptamine, 5-HT) depletion on behavior of pubertal male rats. Serotonin was depleted beginning on postnatal day 26 with parachlorophenylalanine (PCPA 100 mg/kg, every other day); controls received saline. At puberty (P40), half the PCPA-treated rats and half the saline-treated rats began treatment with testosterone (T, 5 mg/kg, 5 days/week). Behavioral measures included locomotion, irritability, copulation, partner preference, and aggression. Animals were tested for aggression in their home cage, both with and without physical provocation (mild tail pinch). Brain levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were determined using HPLC. PCPA significantly and substantially depleted 5-HT and 5-HIAA in all brain regions examined. Chronic T treatment significantly decreased 5-HT and 5-HIAA in certain brain areas, but to a much lesser extent than PCPA. Chronic exposure to PCPA alone significantly decreased locomotor activity and increased irritability but had no effect on sexual behavior, partner preference, or aggression. T alone had no effect on locomotion, irritability, or sexual behavior but increased partner preference and aggression. The most striking effect of combining T+PCPA was a significant increase in attack frequency as well as a significant decrease in the latency to attack, particularly following physical provocation. Based on these data, it can be speculated that pubertal AAS users with low central 5-HT may be especially prone to exhibit aggressive behavior.

Experimental phenylketonuria: metabolic studies in rat liver.

The "in vivo" effects of L-phenylalanine on the gluconeogenic pathway in the liver of fasted rats with experimentally induced phenylketonuria-like characteristics have been investigated. Significant increases of the fructose 6-phosphate, glucose 6-phosphate and glucose concentrations were observed. The study of the effect of L-phenylalanine on the cytoplasmic and mitochondrial redox state and energy charge showed an increase in the mitochondrial NAD+/NADH ratio while the energy charge was virtually unchanged. The effects of phenylalanine and its metabolic derivatives (phenylacetate, phenylethylamine, phenyl-lactate, o-hydroxyphenylacetate and phenylpyruvate) on the activity of lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in rat liver have been also investigated. Phenylpyruvate inhibited the lactate dehydrogenase activity with a Ki of 5.3 mM. Phenylpyruvate also inhibited both the mitochondrial (Ki = 4 mM) and cytoplasmic (Ki = 5 mM) malate dehydrogenase activities. Phenylpyruvate, phenylacetate and o-hydroxyphenylacetate inhibited the 3-hydroxybutyrate dehydrogenase activity with Ki values of 0.7, 6.0 and 9.5 mM respectively.

[Exogenous psychosis in a patient with carcinoid syndrome following treatment with p-chlorophenylalanine (PCPA)]. / Exogene Psychose eines Patienten mit Karzinoidsyndrom nach Behandlung mit p-Chlorophenylalanin (PCPA)

A case is reported of a patient with carcinoid syndrome who developed a exogenous psychosis while under treatment with the serotonin-inhibitor p-chlorophenylalanine (PCPA). Partial symptoms similar to delirium and schizophrenia were exhibited. The attached literature survey focuses on the psychological side effects of PCPA treatment. A discussion follows concerning noteworthy phenomenological similarities between the case reported and certain forms of amphetamine psychosis. Common biochemical mechanisms are hypothetically stated.