This study aimed to explore the roles of microRNAs (miRNAs) in the post-transcriptional regulation of cocaine- and amphetamine-regulated transcript (CART) peptide in the bovine hypothalamus and to screen key regulatory miRNAs. Targetscan was used to predict the potential miRNAs binding to CART 3' untranslated regions (3'UTR). Bioinformatics analysis predicted 7 miRNA binding sites in the bovine CART 3'UTR, which were bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, bta-miR-758, bta-miR-877, and bta-miR-381, respectively. Reverse transcription-PCR (RT-PCR) was carried out to determine the endogenous expression of CART and target miRNAs in the bovine hypothalamus. All the 7 target miRNAs and CART were endogenously expressed in the bovine hypothalamus. The dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between CART 3'UTR and target miRNAs obtained from bioinformatics analysis. The dual-luciferase reporter gene assay confirmed that the 3'UTR of CART had a targeted binding relationship with the 7 target miRNAs. Cell experiments were conducted to examine the effects of target miRNAs on the messenger RNA (mRNA) and protein levels of exogenous CART and screen for key regulatory miRNAs. The results of cell experiments showed that the 7 miRNAs downregulated the mRNA level of CART, with bta-miR-491 demonstrating the strongest downregulating effect. Bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, and bta-miR-381 downregulated the protein level of CART, with bta-miR-381 exerting the strongest downregulating effect. Animal experiments were conducted to explore the effects of key regulatory miRNAs on the mRNA and protein levels of CART in the hypothalamus and the CART concentration in the serum. The results from animal experiments showed that miR-491 and miR-381 regulated the endogenous expression of CART in the hypothalamus and the concentration in the serum by binding to the CART 3'UTR. These results suggest that miR-491 and miR-381 are the main miRNAs regulating CART expression in the bovine hypothalamus, which can affect serum CART concentration by modulating endogenous CART expression.
Animals ; MicroRNAs/metabolism* ; Cattle ; Hypothalamus/metabolism* ; 3' Untranslated Regions/genetics* ; Nerve Tissue Proteins/metabolism* ; Gene Expression Regulation ; Binding Sites ; Base Sequence ; Computational Biology/methods* ; Cocaine- and Amphetamine-Regulated Transcript Protein

OBJECTIVES: To establish a rapid determination method with LC-MS/MS for cocaine and its metabolite benzoylecgonine in hair. METHODS: Deuterated internal standards （cocaine-D₃ and benzoylecgonine-D₈） were added to the decontaminated hair. After the extraction by ultrasonication with methanol, the compounds were separated by the Restek Allure PFP propyl column, and cocaine and benzoylecgonine were simultaneously analysed in multiple reaction monitoring mode. RESULTS: The cocaine and benzoylecgonine in hair showed a good linearity in the range of mass fraction between 0.02 and 10.00 ng/mg with the limits of detection of 0.01 ng/mg. CONCLUSIONS The developed method is simple and rapid with a good selectivity, which is suitable for the determination of cocaine and its metabolite benzoylecgonine in hair.
Chromatography, High Pressure Liquid ; Chromatography, Liquid/methods* ; Cocaine/metabolism* ; Hair/metabolism* ; Humans ; Reference Standards ; Reproducibility of Results ; Tandem Mass Spectrometry/methods*

The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli make addiction hard to cure by contributing to cocaine seeking and relapse. So it's of great importance to examine the neurobiological basis of addiction memory. Cocaine conditioned place preference (CPP) used in this study is a form of Pavlovian conditioning which can establish associations between drug and contextual factors. c-Fos and Zif268 are commonly used immediate early gene (IEG) makers to identify neurons that are activated after a stimulus or behavioral conditioning. This study was designed to reveal neuronal c-Fos, Zif268 expression pattern in 10 brain regions following cocaine context-associated reward memory retrieval in mice, combining animal behavioral study and immunofluorescence method. C57BL/6 mice were randomly divided into 3 groups: Saline retrieval, Cocaine retrieval, and No retrieval of cocaine groups. Cocaine retrieval and No retrieval of cocaine underwent CPP training (one side paired with cocaine, and the other side with saline) except that No retrieval of cocaine group didn't undergo CPP test. Saline retrieval group received saline injections (i.p) on both sides. The results showed that: Neuronal c-Fos, Zif268 protein expression levels in nucleus accumbens (NAc) core both were elevated in Cocaine retrieval group compared with those in Saline retrieval (Control) group during cocaine context-associated reward memory retrieval. Zif268 protein expression level in basolateral amygdala (BLA) was also elevated in Cocaine retrieval group compared with that in control mice. Elevation was not seen in other regions such as hippocampus, prefrontal cortex (PFC). Thus, NAc core and BLA were activated during cocaine context-associated reward memory retrieval. The results suggest that neurons that are activated in NAc core and BLA are crucial basis of cocaine context-associated reward memory.
Animals ; Basolateral Nuclear Complex ; cytology ; Cocaine ; pharmacology ; Conditioning (Psychology) ; Early Growth Response Protein 1 ; metabolism ; Hippocampus ; Memory ; Mice ; Mice, Inbred C57BL ; Neurons ; metabolism ; Nucleus Accumbens ; metabolism ; Prefrontal Cortex ; Proto-Oncogene Proteins c-fos ; metabolism ; Reward

Adult rats were implanted with sleep-wake recording electrodes in our experiments. Polygraphic signs of undisturbed sleep-wake activities were recorded for 24 h before cocaine administration, cocaine withdrawal day 1 (acute), day 8 (subacute), and day 14 (subchronic). Western blot method was performed to examine the expression levels of adenosine receptor subtypes in hypothalamus and cerebellum. Non rapid eye movement (NREM) sleep was significantly increased during nighttime (P < 0.01) and daytime (P < 0.05) on withdrawal day 8. The increase of NREM sleep was significant during nighttime (P < 0.01) and slight during daytime on withdrawal day 14, whereas both daytime and nighttime rapid eye movement (REM) sleeps were reduced markedly (P < 0.01) on withdrawal day 8 and 14. In addition, A2A receptor level was significantly enhanced on cocaine withdrawal day 8 and day 14 (P < 0.05), whereas A1 receptor level reduced markedly on withdrawal day 14 (P < 0.05). However, compared with that in the control group, no significant changes existed among adenosine A1, A2A and A2B receptors in rat cerebellum on cocaine withdrawal day 1, day 8 and day 14. Our findings suggest that sleep disorder caused by subacute and subchronic cocaine abstinence may be associated with over-expression of adenosine A2A receptor in rat hypothalamus to some extent.
Animals ; Cocaine ; adverse effects ; Dyssomnias ; chemically induced ; Electroencephalography ; Hypothalamus ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptor, Adenosine A2A ; metabolism ; Substance Withdrawal Syndrome

OBJECTIVE: To examine the effects of cocaine on the activities of ATPase, LDH and SDH in cultured mouse splenocytes in vitro. METHODS: The ATPase, LDH and SDH activities in mouse splenocytes were detected at day 7 after continuous culturing the mouse cells exposed to cocaine hydrochloride in final concentration of 10, 20 and 100 microg/mL in vitro. RESULTS: The activities of ATPase, LDH and SDH in mouse splenocytes exposed to cocaine hydrochloride in final concentration of 10, 20 and 100 microg/mL were significantly decreased after continuous culturing for 7 days. CONCLUSION The present study demonstrated that cocaine could inhibit the activities of ATPase, LDH and SDH in cultured splenocytes in vitro.
Adenosine Triphosphatases/metabolism* ; Animals ; Cells, Cultured ; Cocaine/pharmacology* ; Dose-Response Relationship, Drug ; L-Lactate Dehydrogenase/metabolism* ; Male ; Mice ; Mice, Inbred Strains ; Spleen/enzymology* ; Succinate Dehydrogenase/metabolism*

A sensitive LC-MS/MS method to determine cocaine and its major metabolite benzoylecgonine in guinea pig' s hair has been established. About 20 mg of decontaminated hair sample was hydrolyzed with 0. 1 mol x L(-1) HCl at 50 degrees C overnight, in the presence of cocaine-d3 and benzoylecgonine-d8 used as internal standards, and then extracted with dichlormethane. The analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Positive electrospray ionization (ESI +) and multiple reactions monitoring (MRM) mode were used. The limit of detection (LOD) for cocaine and benzoylecgonine was 1 pg x mg(-1). The calibration curves of extracted standards were linear over the range from 5 pg x mg(-1) to 250 pg x mg(-1) (r2 > or = 0.9997). The method was validated and applied to the analysis of guinea pig's hair after a single dose administration of cocaine hydrochloride. Cocaine and benzoylecgonine were not only detected, but also quantified in guinea pigs hair.
Animals ; Chromatography, High Pressure Liquid ; Cocaine ; administration & dosage ; analogs & derivatives ; analysis ; metabolism ; Guinea Pigs ; Hair ; chemistry ; metabolism ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

OBJECTIVE: To investigate the reproductive system impairment induced by cocaine in adult male rats and the possible underlying mechanism. METHODS: Thirty adult male rats were randomly divided into experimental and control groups, with 15 rats in each group. Rats of the experimental group were injected cocaine hydrochloride (15 mg/kg body weight) subcutaneously daily for four weeks. The weight of body and testis, as well as the level of serum hormone of the rats were examined. In addition, the apoptosis rate of testicular tissue by TUNEL and the expression of Fas gene in testicular tissue were examined by immunohistochemistry. RESULTS: Compared with the control, the weight of testis in the cocaine exposed group decreased significantly (P<0.05), and the serum testosterone level decreased significantly (P<0.05). Moreover, both the apoptosis rate and the expression of Fas gene increased in the testicular tissue of rats in the cocaine exposed group in comparison to the control group (P<0.05). The apoptosis rate was significantly correlated with the expression of Fas gene (r=0.9012, P<0.05). CONCLUSION Cocaine may cause reproductive system injury in adult male rats, and Fas-mediated apoptosis may be one of the functional mechanisms involved in the reproductive system injuried by cocaine.
Adaptor Proteins, Signal Transducing/metabolism* ; Animals ; Apoptosis/drug effects* ; Cocaine/adverse effects* ; Forensic Toxicology ; Male ; Molecular Chaperones ; Nuclear Proteins/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatids/pathology* ; Substance-Related Disorders ; Testis/pathology* ; Testosterone/blood*

OBJECTIVETo investigate the protective effect of the roots of F. hirta against the cocaine-induced hepatotoxicity and it's active components.

METHODCocaine hydrochloride was subcutaneously injected to make male ICR mice liver wounded. Male ICR mice were randomly ig administered with the F. hirta decoction. The dose groups are 100, 200, 300 g x kg(-1) herb materials per body weight. Cocaine hydrochloride was subcutaneously injected into the mice after the administration. The serum ALT, AST activity and the activity of CAT in liver homogenate were assayed, and liver change of pathomorphism was evaluated to prove the effect of the F. hirta decoction on cocaine-induced hepatotoxicity. And the activity of psoralean which was separated from the F. hirta decoction by bioassay-guided fractionation, was proofed in the same method.

RESULTWe find that the F. hirta decoction shows a distinct effect on reducing serum transferase. The serum transferase and the content CAT in liver homogenate were dose-related reduced, and the histopathological examination found a significantly change of the liver tissues. And the psoralean, qua the mainly component, shows the same effect.

CONCLUSIONF. hirta has the protective effect against the cocaine-induced hepatotoxicity. Psoralean is the basis.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Catalase ; metabolism ; Chemical and Drug Induced Liver Injury ; Cocaine ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Ficus ; chemistry ; Ficusin ; isolation & purification ; pharmacology ; Liver ; drug effects ; enzymology ; pathology ; Liver Diseases ; blood ; prevention & control ; Male ; Mice ; Mice, Inbred ICR ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Random Allocation

BACKGROUNDCocaine addiction may involve complex neuroadaptations, including many changes of genes expression. Dopamine D3 receptors play an important role in cocaine addiction; however, its role in cocaine induced gene expression change is poorly understood. To identify the changes in gene expression induced by repeated cocaine exposure through D3 dopamine receptors, we compared the expression of four molecules: Janus kinase 2 (Jak2), g-aminobutanoic acid receptor subunit alpha 1 (GABAAalpha1), glutamate receptor AMPA3 alpha 3 (GluR 3) and stromal cell derived factor 1 (SDF1). These four have been implicated in mediating the actions of cocaine in the nucleus accumbens (NAc) and caudoputamen (CPu) in mice after acute and repeated cocaine exposure.

METHODSFor the acute and repeated injections, the mice were divided into four groups: 30 mg/kg cocaine, nafadotride 0.5 mg/kg + cocaine 30 mg/kg, nafadotride 0.5 mg/kg, and saline as the basal group. The expression of Jak2, GABAAalpha1, GluR 3 and SDF1 were assayed by Western blot, quantitative real-time RT-PCR and immunohistochemistry.

RESULTSTwenty-four hours after seven consecutive days of repeated cocaine exposure, the expression of GABAAalpha1 decreased in cocaine group compared with basal line and further decreased in the cocaine + nafadotride group and remained at basal level in the nafadotride group. Similarly, the Jak2 expression decreased in cocaine group compared with base line. However, the levels of Jak2 increased in cocaine + nafadotride group compared with cocaine group, while remained at basal level in nafadotride group.

CONCLUSIONSGABAAalpha1 and Jak2 may be involved in chronic cocaine induced neuroadaptations. D3 dopamine receptors play an important role in the expression of these genes.

Animals ; Brain ; drug effects ; metabolism ; Cocaine ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Immunohistochemistry ; Janus Kinase 2 ; analysis ; genetics ; Male ; Mice ; Receptors, Dopamine D3 ; physiology ; Receptors, GABA-A ; analysis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

AIMTo investigate the effects and the possible mechanism of cocaine on the neurons of lateral habenular nucleus (LHb).

METHODSWe observed the effects on c-Fos protein expression in lateral habenular nucleus and medial habenular nucleus after injecting cocaine into a belly cavity and spontaneous and evoked discharge of pain-correlative unit through iontophoresis of cocaine into LHb. The delayed rectifier K+ current was recorded in the acute isolated LHb neuron in whole-cell mode.

RESULTS(1) The c-Fos protein expression was increased by cocaine treatment in LHb, but little effect in MHb. (2) Iontophoresis of cocaine into LHb increased the discharges of pain excitation unit and enhanced excitation response to noxious stimulation, but it decreased the discharges of pain inhibition unit and its responses to noxious stimulation in LHb. Cocaine inhibited the delayed rectifier K+ current.

CONCLUSIONCocaine can excite the LHb and increase its sensitivity. The probable mechanism is that cocaine inhibits the delayed rectifier K+ channels.

Animals ; Cocaine ; pharmacology ; Habenula ; drug effects ; metabolism ; physiology ; Proto-Oncogene Proteins c-fos ; metabolism ; Rats ; Rats, Wistar