Dopamine(DA)is a vital neurotransmitter,and accurate detection of its concentration is critical for both clinical diagnos-tics and neuroscience research.Due to its electrochemical activity,DA is commonly detected usin...Dopamine(DA)is a vital neurotransmitter,and accurate detection of its concentration is critical for both clinical diagnos-tics and neuroscience research.Due to its electrochemical activity,DA is commonly detected using electrochemical methods,which are favored for their simplicity,fast response time,and suitability for in vivo analysis.In this work,a highly sensitive DA electrochemical sensor was developed using an Au@MoS_(2)composite,created by modifying molybdenum disulfide(MoS_(2))nanosheets with gold nanoparticles through HAuCl_(4) reduction,and it was aimed at enhancing DA adsorption and improving detection performance.Scanning Electron Microscopy(SEM),transmission electron microscopy(TEM),Energy Dispersive Spectroscopy(EDS),X-ray photoelectron spectroscopy(XPS)and X-ray Diffraction(XRD)confirmed the suc-cessful synthesis of Au@MoS_(2)and the uniform distribution of gold nanoparticles across the MoS_(2)nanosheets.Then,the electrochemical characterization demonstrated that the Au@MoS_(2)/GCE exhibited distinct oxidation peaks in a 10μmol·L^(-1)DA solution,with significantly enhanced electrochemical activity compared to both unmodified GCE and pristine MoS_(2).Furthermore,differential pulse voltammetry(DPV)further revealed a strong linear relationship between DA concentration and the current response in the range of 800 nmol·L^(-1)to 10μmol·L^(-1),with a low detection limit(LOD)of 78.9 nmol·L^(-1)(S/N=3).Additionally,the sensor showed excellent selectivity against other interfering substances.Moreover,the laser-induced Au@MoS_(2)(LIAu@MoS_(2)),with its abundance of negatively charged surface defects,enabled the ultra-sensitive detection of the ultra-low concentrations of DA.In conclusion,the successfully fabricated Au@MoS_(2)based sensor offers advantages such as low cost,ease of operation,and scalability,making it a promising candidate for biosensing applications due to its enhanced DA detection capabilities.展开更多
OBJECTIVE Abnormal striatal dopaminergic and glutamatergic neurotransmis⁃sion is central to the pathophysiology of schizo⁃phrenia.In this study,we investigated the roles of M4 receptor interplay with D1 signaling in s...OBJECTIVE Abnormal striatal dopaminergic and glutamatergic neurotransmis⁃sion is central to the pathophysiology of schizo⁃phrenia.In this study,we investigated the roles of M4 receptor interplay with D1 signaling in stria⁃tal neurotransmission that affect glutamatergic transmission to control the etiology of neuropsy⁃chiatric disorders.METHODS To study dorsal striatum(DS)region-specific neuronal and behav⁃ioral responses modulated by M4 receptors,we used clustered regularly interspaced short palin⁃dromic repeats-associated protein 9 technology to generate mice lacking M4 in the dorsal stria⁃tum(DS-M4-KD).The M4 positive allosteric modu⁃lator,VU0467154,were used to study the phar⁃macologically profiles with M4 receptor stimula⁃tion in WT mice.Oxotremorine M(Oxo-M),a no subtype-selective muscarinic agonist,was used to show that mAchRs activation,in order to dissect the particular function of M4,in DS-M4-KD mice.Open filed test and forced swim test were used to assess the change of psychiatric-like behav⁃iors.Western blotting and immunohistochemistry were used to detect protein levels of phosphory⁃lation site of dopamine-and cAMP-regulated phosphoprotein of 32 ku(DARPP-32).Whole-cell patch-clamp recording was used to assess M4-mediated cholinergic inhibition of glutamater⁃gic synaptic input transmission.RESULTS West⁃ern blotting and immunohistochemistry assay showed VU0467154(5 mg·kg-1,ip)promoted phosphorylation of DARPP-32 at Thr75,and atten⁃uated D1-dependent phosphorylation of DARPP-32 at Thr34 within the mouse DS.Consistently,the Oxo-M(4μg,icv)also increased DARPP-32 phosphorylation at site Thr75 to reversed phos⁃phorylation at site Thr34 in WT mice,but not in DS-M4-KD mice.In parallel with altered DARPP-32 responses,VU0467154 or Oxo-M evoked a psychological stress response and reversed D1-induced hyperlocomotion in mice in open field test and force swim tests.However,Oxo-M sup⁃pression of D1-depengdeng behavioral respons⁃es was impaired in DS-M4-KD mice.Whole-cell patch recording showed that VU0467154 or Oxo-M mediated endogenous cholinergic inhibition of miniature excitatory postsynaptic currents through M4 receptors,which in turn suppressed D1-depen⁃dent glutamatergic synaptic transmission in the DS.CONCLUSION This study provides evidence for the role of M4 receptors in regulation of dopa⁃mine/DARPP-32 signaling and glutamate respons⁃es in the DS,and therefore modulation of psychi⁃atric behaviors associated with D1 signaling.This results indicate the mechanisms of treatments targeting M4 in psychiatric disorders.展开更多
OBJECTIVE Cognitive inflexibility plays a critical role in the compulsive drug taking,a central characteristic of drug addictions,yet its underlying neurochemical mechanisms are not well understood.The present study e...OBJECTIVE Cognitive inflexibility plays a critical role in the compulsive drug taking,a central characteristic of drug addictions,yet its underlying neurochemical mechanisms are not well understood.The present study examined the impact of morphine withdrawal on reversal learning.METHODS Reversal learning was tested in a four-choices digging task.Some brain tissues were harvested 2 h after the behavioral experiment for the further measurement.RESULTS We found that after long-term abstinence for a month from chronic morphine exposure,mice exhibited a profound reversal learning deficit.We further found that dopamine D2 receptor(D2R)system in the frontal-striatal circuit is significantly down-regulated,at both receptor and downstream signals levels.Subsequent pharmacological experiments demonstrated that aripiprazole,a D2R partial agonist,prevented the D2R downregulation and rescued the reversal learning deficit.CONCLUSION Together,our findings provide valuable insights into the causal relationship between D2R system in the frontal-striatal circuit and the cognitive inflexibility caused by abused drugs and offer a promising possibility of an effective therapeutic intervention for drug addictions.展开更多
Aim Baicalein is the major flavonoid obtained from the Scutellaria root. Our previous studies have dem- onstrated that baicalein has a clear positive effect on recovery in an experimental model of Parkinsonism. The pu...Aim Baicalein is the major flavonoid obtained from the Scutellaria root. Our previous studies have dem- onstrated that baicalein has a clear positive effect on recovery in an experimental model of Parkinsonism. The put- pose of this study was to investigate the role of baicalein in modulating dopamine （DA） metabolism in PC12 cells and to explore possible mechanisms of its actions. Methods The intracellular content and extracellular release of DA in both rotenone-treated and untreated PC12 cells were examined. Second, PC12 cells were first pretreated with baicalein （ 10 μmol · L^-1 ） for 10 rain, and then incubated with or without ionomycin （5 μmol · L^-1 ） for 10 rain to test whether short-term exposure to baicalein affected calcium-dependent or spontaneous DA release. Third, the intracellular and extracellular contents of DA and its related metabolites were examined. After treatment with baica- lein for 24 h, the The tyrosine hydroxylase （TH）, monoamine oxidase B （MAOB）, catechol-O-methyltransferase （COMT） and dopamine transporter （DAT） were detected by immunoblot analysis. Results The results showed that baicalein prevented rotenone-induced cytotoxicity and significantly increased the DA content in both rotenone- treated and untreated PC12 cells. Furthermore, it had no effect on ionomycin-induced or spontaneous DA release after short-term exposure but significantly increased DA content in a time- and dose-dependent manner after treat- ment for 6 h. Baicalein also significantly decreased the intracellular and extracellular homovanillic acid （HVA） content but increased the intracellular 3,4-dihydroxy phenylacetic acid （DOPAC） content. Finally, baicalein sig- nificantly decreased the expression of COMT and DAT, but it had no effect on the expression of TH and MAOB. Conclusion These data suggest that bacalein has the ability to increase DA content and modulate DA metabolism by inhibiting the expression of COMT and DAT. Our study provides evidence that baicalein may be a potential anti- PD drug that merits further study.展开更多
OBJECTIVE To explore the antipost-traumatic-stress-disorder(PTSD) effects and its probable mechanism of YQA14,a dopamine D3 receptor antagonist.METHODS Two PTSD animal models,the rat single prolonged stress(SPS) model...OBJECTIVE To explore the antipost-traumatic-stress-disorder(PTSD) effects and its probable mechanism of YQA14,a dopamine D3 receptor antagonist.METHODS Two PTSD animal models,the rat single prolonged stress(SPS) model and the mouse pre-shock model,were used in this experiment.In the SPS model,adult male Sprague-Dawley(SD) rats were randomly divided into control group,model group,positive group and YQA14 groups with different dosages.In the mouse pre-chock model,dopamine D3 receptor knockout(KO) and wild type(WT) mice were randomly divided into control group,model group,positive group and YQA14 group.After the establishment of animal models,the saline,sertraline(ig) and YQA14(ip)were administered to the animals in the control,model,positive control and test groups respectively.The open field test(OFT) was used to evaluate the locomotor activity while the contextual freezing(CF) measurement and elevated plus maze(EPM) test were used to evaluate the PTSD-like behaviors.RESULTS In the rat SPS model,neither SPS nor drug treatment affected the locomotor activity in rats.However,SPS rats showed significant PTSD-like behaviors with enhanced freezing time in CF(P<0.01) and decreased percentage of entries into open arms and time spent in open arms in EPM(P<0.05,P<0.01).Moreover,compared with the model group,the repeated administration of YQA14(3.125,6.25 and 12.5 mg·kg-1)significantly reduced the freezing time(P<0.01)and increased the percentages of entries into open arms and time spent in open arms(P<0.05).In the mouse pre-shock model,when both model groups showed significant higher freezing time compared with the respective control groups(P<0.05,P<0.01),YQA14 selectively alleviated the freezing time on WT mice(P<0.05) while had no effect on KO mice.In the EPM tests,the WT mice model group showed a significant reduction in the percentage of entries into open arms and time spent in open arms(P<0.05) while D3 R KO mice model group didn′ t show any reduction,compared with respective control groups.Furthermore,daily administration of YQA14 at 12.5 mg·kg-1 both significantly reduced the percentages of entries into and time spent in open arms(P<0.05) but not D3 R KO mice.None of the locomotor activity were significantly affected.CONCLUSION YQA14 could significantly alleviate the PTSD-like behaviors in rodents and the effects were mediated by the blockade of brain D3 receptors.展开更多
OBJECTIVE Dopamine receptors(DRs) are involved in the development and treatment of many neuropsychiatric disorders.Currently available dopaminergic drugs modulate both DRD2 and DRD3,leading to side effects and uncerta...OBJECTIVE Dopamine receptors(DRs) are involved in the development and treatment of many neuropsychiatric disorders.Currently available dopaminergic drugs modulate both DRD2 and DRD3,leading to side effects and uncertainty as to the roles each DR subtype plays physiologically.Our lab employed high throughput screening paradigms to discover highly selective modulators for the DRD3.METHODS The NIH Molecular Libraries Program 400,000 + small molecule library was screened using the Discove Rx Path Hunter?β-arrestin assay for compounds that activate the DRD3 without effects on the DRD2.Confirmation and counter-screens assessed selectivity and mechanisms of action.We identified 62 potential agonists,and chose the most promising to perform a structure-activity relationship(SAR) study to increase potency while maintaining selectivity.The lead compound identified through this process,ML417,was also characterized using bioluminescence resonance energy transfer(BRET)-based β-arrestin recruitment and G-protein activation assays as well as p-ERK assays.Potential neuroprotective properties of this compound were assessed using a SHSY5 Y neuronal cell model.RESULTS ML417 displays potent,DRD3-selective agonist activity in multiple functional assays.Binding and functional GPCR screens(>165 receptors) show ML417 has limited cross-reactivity with other GPCRs.ML417 also displays superior(compared to the reference compound pramipexole),dose-dependent protection against a decrease in neurite length induced by 10 μmol·L^(-1) of the neurotoxin,6-hydroxydopamine,in the SHSY5 Y cell model.CONCLUSION We have discovered and characterized ML417,a potent and highly selective DRD3 agonist.This compound will be useful as a research tool,and may prove useful as a therapeutic drug lead.展开更多
Behavioral and molecular characterization of cell-type specific populations governing fear learning and behavior is a promising avenue for the rational identification of potential therapeutics for fear-related disorde...Behavioral and molecular characterization of cell-type specific populations governing fear learning and behavior is a promising avenue for the rational identification of potential therapeutics for fear-related disorders.Identification of cell-type specific changes in neuronal translation following fear learning allows for targeted pharmacological intervention during fear extinction learning,mirroring possible treatment strategies in humans.Here we identify the central amygdala(Ce A)Drd2-expressing population as a fear-supporting population that is molecularly distinct from other,previously identified fear-supporting CeA populations.Sequencing of actively translating transcripts of Drd2 neurons identifies m RNAs that are differentially regulated following fear learning including Npy5r,Rxrg,Sst5r,Fgf3,Erb B4,Fkbp14,Dlk1,Ssh3 and Adora2a.Direct pharmacological manipulation of NPY5R,RXR,and ADORA2A confirms their importance in fear behavior and validates the present approach of identifying pharmacological targets for the modulation of emotional learning.展开更多
Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mic...Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mice were used to examine the lineage differentiation of SOX2-positive cells during the development of cerebral cortex.Primary NSCs/astrocytes culture,ChIP-seq and Western Blot were adopted to analyze and verify the expression of candidate genes.Pharmacological manipulation,neurosphere formation,photochemical ischemia,immunofluorescence staining and behavior tests were adopted to evaluate the effects of activating DRD2 signaling on astrocytic dedifferentiation.Results:Immunofluorescence staining demonstrated the NSC-astrocyte switch of SOX2-expression in the normal development of cerebral cortex.ChIP-seq revealed enrichment of DRD2 signaling by SOX2-bound enhancers in NSCs and SOX2-bound promoters in astrocytes.Western Blot and immunofluorescence staining verified the expression of DRD2 in NSCs and reactive astrocytes.Application of quinagolide hydrocholoride(QH),an agonist of DRD2,significantly promoted astrocytic dedifferentiation both in vitro and in vivo following ischemia.In addition,quinagolide hydrocholoride treatment improved locomotion recovery.Conclusion:Activating DRD2 signaling facilitates astrocytic dedifferentiation and may be used to treat ischemic stroke.展开更多
The motor cortex plays a key role in motor control as its output directly generates movement.However,it is known that the motor cortex also exhibits a high degree of plasticity that may be important in both health and...The motor cortex plays a key role in motor control as its output directly generates movement.However,it is known that the motor cortex also exhibits a high degree of plasticity that may be important in both health and disease.Here I provide evidence to support that dopaminergic innervation arising from the midbrain plays critical roles in the process of acquiring novel motor skills as well as recovery of motor functions after ischemic stroke.Based on extracel ular multi-unit recordings from conscious freely moving rats,we investigated the dynamics of neurons in the output layer 5b(L5b)of the primary motor cortex during the training of a forelimb reaching for food task.We found a subpopulation of task-recruited layer 5b neurons that not only became more movement-encoding during motor learning,but their activities were also more temporally aligned to motor execution.These phenomena were accompanied by the emergence of reproducibleneurodynamics of the L5b neuronal ensemble and traininginduced long-term synaptic plasticity of inputs to these neurons,but were highly disrupted by local denervation of dopaminergic inputs originating from the ventral tegmental area.In another series of experiments based on rodent models of focal ischemic stroke,administration of L-DOPA,the precursor of dopamine,significantly promoted motor function recovery.Morphological and biochemical examination of the perilesioned area provided evidence that neuroplasticity markers,dendritic arborisation and synaptic plasticity were increased by the treatment.Further examination of neuronal activity through in vivo recording revealed that motor cortical regions adjacent to the infarct had restored capability for synaptic plasticity.Finally,pharmacological blockade of either D1 or D2 receptors at the perilesioned region abolished the beneficial effects.Together,our findings highlight the importance of the plasticity of the motor cortex in health and disease that are mediated by the mesocortical dopaminergic pathway.展开更多
OBJECTIVE Dopamine(DA)plays important roles in Pavlovian conditioning by mediating reward,learning and motivation.While the conditioning stimulation(CS) is the most important inducement for reinstatement in addiction....OBJECTIVE Dopamine(DA)plays important roles in Pavlovian conditioning by mediating reward,learning and motivation.While the conditioning stimulation(CS) is the most important inducement for reinstatement in addiction.The present study investigated the specific role of the DA projections to nucleus accumbens(NAc) and medial prefrontal cortices(mPFC) from ventral tegmental area(VTA) in reinstatement induced by cue.METHODS(1)Optogenetic intracranial self-stimulation and reinstatement.DAT-Cre transgenic mice received an injection of adeno-associated viral vectors encoding channelrhodopsin2(ChR2) or control vector into the VTA resulting in the selective expression of these opsins in DA neurons.Then,we stimulated the VTA,NAc(core and shel) or mP FC [prelimbic cortex(PL) and infralimbic cortex(IL)] via an optical fiber.In the reinforcement test,the mice with ChR2 learned instrumental responses corresponding to the delivery of photostimulation into the VTA with multiple frequencies and during time;in the reinstatement phase,stimulation of the DA projections to NAc(core or shell) or mPFC(IL and PL) from VTA to induce reinstatement after 2 weeks of extinction of self-stimulation.(2)Reinstatement in cocaine self-administration.Virus encoding ChR2 or hM4 Di were injected into VTA of DAT-Cre transgenic mice.The mice with ChR2 and hM4 Di in DA neurons were trained to establish self-administration of cocaine.After 2 weeks of extinction,laser stimulation of the DA projections to NAc(core or shel) or mP FC(IL and PL) was conducted to induce reinstatement.After that,Clozapine was injected in NAc core to test the impacts of VTA-NAc core depression on the reinstatement induced by cue.(3) Photometry of VTA DA neurons in reinstatement.DAT-Cre transgenic mice were received an injection of AAV-DIO-Gcamp6 m into VTA.After cocaine self-administration and extinction,mice with Gcamp6 m were challenged by cue(paired with cocaine previously) and the photometry of VTA DA neurons was conducted during the reinstatement.RESULTS(1)The enhanced self-stimulation behavior was positive correlation with the stimulation of DA neurons in VTA according to the increasing frequency of stimulation and extent stimulation duration time.Furthermore,DA receptor antagonists significantly depressed the frequency curve.(2) Only stimulation of the projections to the NAc core from the VTA significantly induced reinstatement after extinction of self-stimulation,neither shell nor mPFC(PL or IL).(3) Depression of VTA-NAc core projection significantly inhibited the reinstatement induced by cue.(4) DA neurons in VTA were activated when the cue appeared during the period of reinstatement test.CONCLUSION Mesocorticolimbic DA system directly modulate the reinforcement dependant on DA receptor.The activity of DA neurons in VTA is necessary for cue induced relapse.Importantly,projections to NAc core from VTA perform the unique effects in reinstatement.展开更多
OBJECTIVE To investigate the motility,dopamine,serotonin and pro-BDNF level of Centella asiatica(CA)comparable to the combination of levodopa and benzerazid HCL on zebrafish model Parkinson′s.METHODS Rotenone 5μg...OBJECTIVE To investigate the motility,dopamine,serotonin and pro-BDNF level of Centella asiatica(CA)comparable to the combination of levodopa and benzerazid HCL on zebrafish model Parkinson′s.METHODS Rotenone 5μg·L-1 induced to adult zebrafish(8months)for 28 dto made zebrafish model Parkinson′s.CA concentration were used 5 and 10μg·mL-1.The combination of levodopa 100 g and 25 mg benzerazid(L-DOPA agonist)was given at 1 μg·mL-1 at the same time to rotenone,and CA as well.Motility assessment conducted for every 7dstarted on day 0until day 28.Five fish were subjected in the 2L tank(25cm×16.5cm×12cm).Three vertical lines were drawn on the tank at equal distances,dividing the tank into four zones(the length of each zone was 6.25cm).Locomotor activity was measured for 5min by counting the number of lines that adult zebrafish crossed.Fish were sacrificed by decapitating on ice water.Dopamine level measured from whole brain by ELISA,serotonin and pro-BDNF by immunoreactivity at substantia nigra.RESULTS The locomotor activity of rotenone treated fish were significantly decrease starts at 7 dcompared to control group.Interestingly on 10μg·mL-1 group there are increasing motility start at day 7 and slightly decrease until day 28,but on L-DOPA group increasing motility at day 7 followed by decreasing motility significantly(P<0.05)until day 28.Dopamine level of rotenone group decreased compared to control group,CA 10μg·mL-1 and L-DOPA group increased significantly to rotenone group which CAμg·mL-1 higher than L-DOPA group.Serotonin and pro-BDNF level had the same profile.Its significantly increased on rotenone group compared to control group.Serotonin on group with CA administration significantly decreased compared to rotenone group,while rotenone plus CA 10μg·mL-1 had no significant differet to L-DOPA group(P>0.05).Pro-BDNF on rotenone with CA 5μg·mL-1 had no significant difference,while compare to rotenone with CA 10-1 μg·mLgroup and L-DOPA group were significantly different(P<0.05).CONCLUSION Rotenone produces reliable Parkinson′s zebrafish model by decreasing motility and dopamine level.CA was more stable,increasing motility than L-DOPA administration.CA and L-DOPA increased dopamine level,but decreasing serotonin and pro-BDNF.展开更多
We have reported that the central mechanism of acupuncture-induced PRL secretion in non-lactating rats are related to antagonizing hypothalamic dopamine activity; noradrenaline system played little significant role in...We have reported that the central mechanism of acupuncture-induced PRL secretion in non-lactating rats are related to antagonizing hypothalamic dopamine activity; noradrenaline system played little significant role in the acupuncture effect; Υ-aminobutyric-acid system perhaps participated in this effect.This paper further provided evidence that central serotonin and EOP play a stimulatory role in the acupuncture induced secretion of prolactin; acupuncture may antagonize inhibitory effect of H<sub>2</sub> histamine receptor activation on prolactin secretion; the possible role of H<sub>1</sub>-receptor needs further investigation.展开更多
OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 depende...OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.展开更多
OBJECTIVE Wide spread abuse of synthetic cathinones found in bath salts preparations has resulted in regulation of some cathinones internationally.Chemists skirt these laws by altering the chemical structures of first...OBJECTIVE Wide spread abuse of synthetic cathinones found in bath salts preparations has resulted in regulation of some cathinones internationally.Chemists skirt these laws by altering the chemical structures of first-generation cathinones(ie,MDPV,methylone,and mephedrone),resulting in second-generation cathinones(eg,α-PVP,α-PPP,MDPPP,and MDPBP).Although MDPV is a more effective reinforcer than cocaine,little is known about the reinforcing effectiveness of secondgeneration cathinones.To test the hypothesis that synthetic cathinones with higher selectivity for DAT relative to SERT are more effective reinforcers.METHODS Monoamine transporter inhibition was determined using synaptosomes prepared from rat brains.The relative reinforcing effectiveness of intravenously self-administered MDPV,MDPBP,MDPPP,α-PVP,α-PPP,and cocaine were directly compared through evaluations of (1)dose-response curves under a progressive ratio(PR)schedule of reinforcement and (2)demand curves obtained for each drug in male Sprague-Dawley rats.RESULTS Rank order selectivity for DAT/SERT wasα-PVP>MDPV>α-PPP≈MDPBP>MDPPP>cocaine.Comparisons of the maximum number of infusions obtained under a PR schedule of reinforcement(α-PVP>MDPV>α-PPP>MDPBP≈MDPPP>cocaine)and the essential value obtained for each drug in demand analyses(α-PVP>MDPV>α-PPP≈MDPBP≈MDPPP>cocaine)suggest relative reinforcing effectiveness is related to DAT/SERT selectivity.CONCLUSION These data provide evidence that DAT/SERT selectivity accounts for select synthetic cathinones functioning as more effective reinforcers than cocaine and may predict the abuse-related effects of novel synthetic cathinones in humans.展开更多
基金supported by the Young Talent Innovation Team Support Project from Zhengzhou University(No.32213280)the scientific research program of innovation platform in State Tobacco Monopoly Administrationthe State Key Program of National Natural Science Foundation of China(Grant No.32130083).
文摘Dopamine(DA)is a vital neurotransmitter,and accurate detection of its concentration is critical for both clinical diagnos-tics and neuroscience research.Due to its electrochemical activity,DA is commonly detected using electrochemical methods,which are favored for their simplicity,fast response time,and suitability for in vivo analysis.In this work,a highly sensitive DA electrochemical sensor was developed using an Au@MoS_(2)composite,created by modifying molybdenum disulfide(MoS_(2))nanosheets with gold nanoparticles through HAuCl_(4) reduction,and it was aimed at enhancing DA adsorption and improving detection performance.Scanning Electron Microscopy(SEM),transmission electron microscopy(TEM),Energy Dispersive Spectroscopy(EDS),X-ray photoelectron spectroscopy(XPS)and X-ray Diffraction(XRD)confirmed the suc-cessful synthesis of Au@MoS_(2)and the uniform distribution of gold nanoparticles across the MoS_(2)nanosheets.Then,the electrochemical characterization demonstrated that the Au@MoS_(2)/GCE exhibited distinct oxidation peaks in a 10μmol·L^(-1)DA solution,with significantly enhanced electrochemical activity compared to both unmodified GCE and pristine MoS_(2).Furthermore,differential pulse voltammetry(DPV)further revealed a strong linear relationship between DA concentration and the current response in the range of 800 nmol·L^(-1)to 10μmol·L^(-1),with a low detection limit(LOD)of 78.9 nmol·L^(-1)(S/N=3).Additionally,the sensor showed excellent selectivity against other interfering substances.Moreover,the laser-induced Au@MoS_(2)(LIAu@MoS_(2)),with its abundance of negatively charged surface defects,enabled the ultra-sensitive detection of the ultra-low concentrations of DA.In conclusion,the successfully fabricated Au@MoS_(2)based sensor offers advantages such as low cost,ease of operation,and scalability,making it a promising candidate for biosensing applications due to its enhanced DA detection capabilities.
文摘OBJECTIVE Abnormal striatal dopaminergic and glutamatergic neurotransmis⁃sion is central to the pathophysiology of schizo⁃phrenia.In this study,we investigated the roles of M4 receptor interplay with D1 signaling in stria⁃tal neurotransmission that affect glutamatergic transmission to control the etiology of neuropsy⁃chiatric disorders.METHODS To study dorsal striatum(DS)region-specific neuronal and behav⁃ioral responses modulated by M4 receptors,we used clustered regularly interspaced short palin⁃dromic repeats-associated protein 9 technology to generate mice lacking M4 in the dorsal stria⁃tum(DS-M4-KD).The M4 positive allosteric modu⁃lator,VU0467154,were used to study the phar⁃macologically profiles with M4 receptor stimula⁃tion in WT mice.Oxotremorine M(Oxo-M),a no subtype-selective muscarinic agonist,was used to show that mAchRs activation,in order to dissect the particular function of M4,in DS-M4-KD mice.Open filed test and forced swim test were used to assess the change of psychiatric-like behav⁃iors.Western blotting and immunohistochemistry were used to detect protein levels of phosphory⁃lation site of dopamine-and cAMP-regulated phosphoprotein of 32 ku(DARPP-32).Whole-cell patch-clamp recording was used to assess M4-mediated cholinergic inhibition of glutamater⁃gic synaptic input transmission.RESULTS West⁃ern blotting and immunohistochemistry assay showed VU0467154(5 mg·kg-1,ip)promoted phosphorylation of DARPP-32 at Thr75,and atten⁃uated D1-dependent phosphorylation of DARPP-32 at Thr34 within the mouse DS.Consistently,the Oxo-M(4μg,icv)also increased DARPP-32 phosphorylation at site Thr75 to reversed phos⁃phorylation at site Thr34 in WT mice,but not in DS-M4-KD mice.In parallel with altered DARPP-32 responses,VU0467154 or Oxo-M evoked a psychological stress response and reversed D1-induced hyperlocomotion in mice in open field test and force swim tests.However,Oxo-M sup⁃pression of D1-depengdeng behavioral respons⁃es was impaired in DS-M4-KD mice.Whole-cell patch recording showed that VU0467154 or Oxo-M mediated endogenous cholinergic inhibition of miniature excitatory postsynaptic currents through M4 receptors,which in turn suppressed D1-depen⁃dent glutamatergic synaptic transmission in the DS.CONCLUSION This study provides evidence for the role of M4 receptors in regulation of dopa⁃mine/DARPP-32 signaling and glutamate respons⁃es in the DS,and therefore modulation of psychi⁃atric behaviors associated with D1 signaling.This results indicate the mechanisms of treatments targeting M4 in psychiatric disorders.
文摘OBJECTIVE Cognitive inflexibility plays a critical role in the compulsive drug taking,a central characteristic of drug addictions,yet its underlying neurochemical mechanisms are not well understood.The present study examined the impact of morphine withdrawal on reversal learning.METHODS Reversal learning was tested in a four-choices digging task.Some brain tissues were harvested 2 h after the behavioral experiment for the further measurement.RESULTS We found that after long-term abstinence for a month from chronic morphine exposure,mice exhibited a profound reversal learning deficit.We further found that dopamine D2 receptor(D2R)system in the frontal-striatal circuit is significantly down-regulated,at both receptor and downstream signals levels.Subsequent pharmacological experiments demonstrated that aripiprazole,a D2R partial agonist,prevented the D2R downregulation and rescued the reversal learning deficit.CONCLUSION Together,our findings provide valuable insights into the causal relationship between D2R system in the frontal-striatal circuit and the cognitive inflexibility caused by abused drugs and offer a promising possibility of an effective therapeutic intervention for drug addictions.
文摘Aim Baicalein is the major flavonoid obtained from the Scutellaria root. Our previous studies have dem- onstrated that baicalein has a clear positive effect on recovery in an experimental model of Parkinsonism. The put- pose of this study was to investigate the role of baicalein in modulating dopamine （DA） metabolism in PC12 cells and to explore possible mechanisms of its actions. Methods The intracellular content and extracellular release of DA in both rotenone-treated and untreated PC12 cells were examined. Second, PC12 cells were first pretreated with baicalein （ 10 μmol · L^-1 ） for 10 rain, and then incubated with or without ionomycin （5 μmol · L^-1 ） for 10 rain to test whether short-term exposure to baicalein affected calcium-dependent or spontaneous DA release. Third, the intracellular and extracellular contents of DA and its related metabolites were examined. After treatment with baica- lein for 24 h, the The tyrosine hydroxylase （TH）, monoamine oxidase B （MAOB）, catechol-O-methyltransferase （COMT） and dopamine transporter （DAT） were detected by immunoblot analysis. Results The results showed that baicalein prevented rotenone-induced cytotoxicity and significantly increased the DA content in both rotenone- treated and untreated PC12 cells. Furthermore, it had no effect on ionomycin-induced or spontaneous DA release after short-term exposure but significantly increased DA content in a time- and dose-dependent manner after treat- ment for 6 h. Baicalein also significantly decreased the intracellular and extracellular homovanillic acid （HVA） content but increased the intracellular 3,4-dihydroxy phenylacetic acid （DOPAC） content. Finally, baicalein sig- nificantly decreased the expression of COMT and DAT, but it had no effect on the expression of TH and MAOB. Conclusion These data suggest that bacalein has the ability to increase DA content and modulate DA metabolism by inhibiting the expression of COMT and DAT. Our study provides evidence that baicalein may be a potential anti- PD drug that merits further study.
基金National Key Research and DevelopmentProgram of China (2016YFC0800907)MedicalInnovation Program (16CXZ033)+2 种基金National KeyBasic Research Program (2015CB553504)National Natural Science Foundation of China(8157340581373385).
文摘OBJECTIVE To explore the antipost-traumatic-stress-disorder(PTSD) effects and its probable mechanism of YQA14,a dopamine D3 receptor antagonist.METHODS Two PTSD animal models,the rat single prolonged stress(SPS) model and the mouse pre-shock model,were used in this experiment.In the SPS model,adult male Sprague-Dawley(SD) rats were randomly divided into control group,model group,positive group and YQA14 groups with different dosages.In the mouse pre-chock model,dopamine D3 receptor knockout(KO) and wild type(WT) mice were randomly divided into control group,model group,positive group and YQA14 group.After the establishment of animal models,the saline,sertraline(ig) and YQA14(ip)were administered to the animals in the control,model,positive control and test groups respectively.The open field test(OFT) was used to evaluate the locomotor activity while the contextual freezing(CF) measurement and elevated plus maze(EPM) test were used to evaluate the PTSD-like behaviors.RESULTS In the rat SPS model,neither SPS nor drug treatment affected the locomotor activity in rats.However,SPS rats showed significant PTSD-like behaviors with enhanced freezing time in CF(P<0.01) and decreased percentage of entries into open arms and time spent in open arms in EPM(P<0.05,P<0.01).Moreover,compared with the model group,the repeated administration of YQA14(3.125,6.25 and 12.5 mg·kg-1)significantly reduced the freezing time(P<0.01)and increased the percentages of entries into open arms and time spent in open arms(P<0.05).In the mouse pre-shock model,when both model groups showed significant higher freezing time compared with the respective control groups(P<0.05,P<0.01),YQA14 selectively alleviated the freezing time on WT mice(P<0.05) while had no effect on KO mice.In the EPM tests,the WT mice model group showed a significant reduction in the percentage of entries into open arms and time spent in open arms(P<0.05) while D3 R KO mice model group didn′ t show any reduction,compared with respective control groups.Furthermore,daily administration of YQA14 at 12.5 mg·kg-1 both significantly reduced the percentages of entries into and time spent in open arms(P<0.05) but not D3 R KO mice.None of the locomotor activity were significantly affected.CONCLUSION YQA14 could significantly alleviate the PTSD-like behaviors in rodents and the effects were mediated by the blockade of brain D3 receptors.
基金supported by National Institute of Neurological Disorders and Stroke Intramural Research Program
文摘OBJECTIVE Dopamine receptors(DRs) are involved in the development and treatment of many neuropsychiatric disorders.Currently available dopaminergic drugs modulate both DRD2 and DRD3,leading to side effects and uncertainty as to the roles each DR subtype plays physiologically.Our lab employed high throughput screening paradigms to discover highly selective modulators for the DRD3.METHODS The NIH Molecular Libraries Program 400,000 + small molecule library was screened using the Discove Rx Path Hunter?β-arrestin assay for compounds that activate the DRD3 without effects on the DRD2.Confirmation and counter-screens assessed selectivity and mechanisms of action.We identified 62 potential agonists,and chose the most promising to perform a structure-activity relationship(SAR) study to increase potency while maintaining selectivity.The lead compound identified through this process,ML417,was also characterized using bioluminescence resonance energy transfer(BRET)-based β-arrestin recruitment and G-protein activation assays as well as p-ERK assays.Potential neuroprotective properties of this compound were assessed using a SHSY5 Y neuronal cell model.RESULTS ML417 displays potent,DRD3-selective agonist activity in multiple functional assays.Binding and functional GPCR screens(>165 receptors) show ML417 has limited cross-reactivity with other GPCRs.ML417 also displays superior(compared to the reference compound pramipexole),dose-dependent protection against a decrease in neurite length induced by 10 μmol·L^(-1) of the neurotoxin,6-hydroxydopamine,in the SHSY5 Y cell model.CONCLUSION We have discovered and characterized ML417,a potent and highly selective DRD3 agonist.This compound will be useful as a research tool,and may prove useful as a therapeutic drug lead.
文摘Behavioral and molecular characterization of cell-type specific populations governing fear learning and behavior is a promising avenue for the rational identification of potential therapeutics for fear-related disorders.Identification of cell-type specific changes in neuronal translation following fear learning allows for targeted pharmacological intervention during fear extinction learning,mirroring possible treatment strategies in humans.Here we identify the central amygdala(Ce A)Drd2-expressing population as a fear-supporting population that is molecularly distinct from other,previously identified fear-supporting CeA populations.Sequencing of actively translating transcripts of Drd2 neurons identifies m RNAs that are differentially regulated following fear learning including Npy5r,Rxrg,Sst5r,Fgf3,Erb B4,Fkbp14,Dlk1,Ssh3 and Adora2a.Direct pharmacological manipulation of NPY5R,RXR,and ADORA2A confirms their importance in fear behavior and validates the present approach of identifying pharmacological targets for the modulation of emotional learning.
文摘Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mice were used to examine the lineage differentiation of SOX2-positive cells during the development of cerebral cortex.Primary NSCs/astrocytes culture,ChIP-seq and Western Blot were adopted to analyze and verify the expression of candidate genes.Pharmacological manipulation,neurosphere formation,photochemical ischemia,immunofluorescence staining and behavior tests were adopted to evaluate the effects of activating DRD2 signaling on astrocytic dedifferentiation.Results:Immunofluorescence staining demonstrated the NSC-astrocyte switch of SOX2-expression in the normal development of cerebral cortex.ChIP-seq revealed enrichment of DRD2 signaling by SOX2-bound enhancers in NSCs and SOX2-bound promoters in astrocytes.Western Blot and immunofluorescence staining verified the expression of DRD2 in NSCs and reactive astrocytes.Application of quinagolide hydrocholoride(QH),an agonist of DRD2,significantly promoted astrocytic dedifferentiation both in vitro and in vivo following ischemia.In addition,quinagolide hydrocholoride treatment improved locomotion recovery.Conclusion:Activating DRD2 signaling facilitates astrocytic dedifferentiation and may be used to treat ischemic stroke.
基金The project supported by NSFC grant(SZRIA0314025)SRFDP-HKRGC grant(2900703)+1 种基金HKGRF grant(2140903)HMRF grant(02130976)
文摘The motor cortex plays a key role in motor control as its output directly generates movement.However,it is known that the motor cortex also exhibits a high degree of plasticity that may be important in both health and disease.Here I provide evidence to support that dopaminergic innervation arising from the midbrain plays critical roles in the process of acquiring novel motor skills as well as recovery of motor functions after ischemic stroke.Based on extracel ular multi-unit recordings from conscious freely moving rats,we investigated the dynamics of neurons in the output layer 5b(L5b)of the primary motor cortex during the training of a forelimb reaching for food task.We found a subpopulation of task-recruited layer 5b neurons that not only became more movement-encoding during motor learning,but their activities were also more temporally aligned to motor execution.These phenomena were accompanied by the emergence of reproducibleneurodynamics of the L5b neuronal ensemble and traininginduced long-term synaptic plasticity of inputs to these neurons,but were highly disrupted by local denervation of dopaminergic inputs originating from the ventral tegmental area.In another series of experiments based on rodent models of focal ischemic stroke,administration of L-DOPA,the precursor of dopamine,significantly promoted motor function recovery.Morphological and biochemical examination of the perilesioned area provided evidence that neuroplasticity markers,dendritic arborisation and synaptic plasticity were increased by the treatment.Further examination of neuronal activity through in vivo recording revealed that motor cortical regions adjacent to the infarct had restored capability for synaptic plasticity.Finally,pharmacological blockade of either D1 or D2 receptors at the perilesioned region abolished the beneficial effects.Together,our findings highlight the importance of the plasticity of the motor cortex in health and disease that are mediated by the mesocortical dopaminergic pathway.
基金National Key Research and Development (2016YFC0800907)Project 973 (2015CB553504)+1 种基金National Natural Science Foundation of China(81573405)Beijing Nova Program(xx2014A014).
文摘OBJECTIVE Dopamine(DA)plays important roles in Pavlovian conditioning by mediating reward,learning and motivation.While the conditioning stimulation(CS) is the most important inducement for reinstatement in addiction.The present study investigated the specific role of the DA projections to nucleus accumbens(NAc) and medial prefrontal cortices(mPFC) from ventral tegmental area(VTA) in reinstatement induced by cue.METHODS(1)Optogenetic intracranial self-stimulation and reinstatement.DAT-Cre transgenic mice received an injection of adeno-associated viral vectors encoding channelrhodopsin2(ChR2) or control vector into the VTA resulting in the selective expression of these opsins in DA neurons.Then,we stimulated the VTA,NAc(core and shel) or mP FC [prelimbic cortex(PL) and infralimbic cortex(IL)] via an optical fiber.In the reinforcement test,the mice with ChR2 learned instrumental responses corresponding to the delivery of photostimulation into the VTA with multiple frequencies and during time;in the reinstatement phase,stimulation of the DA projections to NAc(core or shell) or mPFC(IL and PL) from VTA to induce reinstatement after 2 weeks of extinction of self-stimulation.(2)Reinstatement in cocaine self-administration.Virus encoding ChR2 or hM4 Di were injected into VTA of DAT-Cre transgenic mice.The mice with ChR2 and hM4 Di in DA neurons were trained to establish self-administration of cocaine.After 2 weeks of extinction,laser stimulation of the DA projections to NAc(core or shel) or mP FC(IL and PL) was conducted to induce reinstatement.After that,Clozapine was injected in NAc core to test the impacts of VTA-NAc core depression on the reinstatement induced by cue.(3) Photometry of VTA DA neurons in reinstatement.DAT-Cre transgenic mice were received an injection of AAV-DIO-Gcamp6 m into VTA.After cocaine self-administration and extinction,mice with Gcamp6 m were challenged by cue(paired with cocaine previously) and the photometry of VTA DA neurons was conducted during the reinstatement.RESULTS(1)The enhanced self-stimulation behavior was positive correlation with the stimulation of DA neurons in VTA according to the increasing frequency of stimulation and extent stimulation duration time.Furthermore,DA receptor antagonists significantly depressed the frequency curve.(2) Only stimulation of the projections to the NAc core from the VTA significantly induced reinstatement after extinction of self-stimulation,neither shell nor mPFC(PL or IL).(3) Depression of VTA-NAc core projection significantly inhibited the reinstatement induced by cue.(4) DA neurons in VTA were activated when the cue appeared during the period of reinstatement test.CONCLUSION Mesocorticolimbic DA system directly modulate the reinforcement dependant on DA receptor.The activity of DA neurons in VTA is necessary for cue induced relapse.Importantly,projections to NAc core from VTA perform the unique effects in reinstatement.
基金The project supported by Directorate General of Higher Education,Ministry of Education and Research Technology of Indonesia,Through Fundamental Research grant 2014
文摘OBJECTIVE To investigate the motility,dopamine,serotonin and pro-BDNF level of Centella asiatica(CA)comparable to the combination of levodopa and benzerazid HCL on zebrafish model Parkinson′s.METHODS Rotenone 5μg·L-1 induced to adult zebrafish(8months)for 28 dto made zebrafish model Parkinson′s.CA concentration were used 5 and 10μg·mL-1.The combination of levodopa 100 g and 25 mg benzerazid(L-DOPA agonist)was given at 1 μg·mL-1 at the same time to rotenone,and CA as well.Motility assessment conducted for every 7dstarted on day 0until day 28.Five fish were subjected in the 2L tank(25cm×16.5cm×12cm).Three vertical lines were drawn on the tank at equal distances,dividing the tank into four zones(the length of each zone was 6.25cm).Locomotor activity was measured for 5min by counting the number of lines that adult zebrafish crossed.Fish were sacrificed by decapitating on ice water.Dopamine level measured from whole brain by ELISA,serotonin and pro-BDNF by immunoreactivity at substantia nigra.RESULTS The locomotor activity of rotenone treated fish were significantly decrease starts at 7 dcompared to control group.Interestingly on 10μg·mL-1 group there are increasing motility start at day 7 and slightly decrease until day 28,but on L-DOPA group increasing motility at day 7 followed by decreasing motility significantly(P<0.05)until day 28.Dopamine level of rotenone group decreased compared to control group,CA 10μg·mL-1 and L-DOPA group increased significantly to rotenone group which CAμg·mL-1 higher than L-DOPA group.Serotonin and pro-BDNF level had the same profile.Its significantly increased on rotenone group compared to control group.Serotonin on group with CA administration significantly decreased compared to rotenone group,while rotenone plus CA 10μg·mL-1 had no significant differet to L-DOPA group(P>0.05).Pro-BDNF on rotenone with CA 5μg·mL-1 had no significant difference,while compare to rotenone with CA 10-1 μg·mLgroup and L-DOPA group were significantly different(P<0.05).CONCLUSION Rotenone produces reliable Parkinson′s zebrafish model by decreasing motility and dopamine level.CA was more stable,increasing motility than L-DOPA administration.CA and L-DOPA increased dopamine level,but decreasing serotonin and pro-BDNF.
基金Project supported by the National Natural Science Fund
文摘We have reported that the central mechanism of acupuncture-induced PRL secretion in non-lactating rats are related to antagonizing hypothalamic dopamine activity; noradrenaline system played little significant role in the acupuncture effect; Υ-aminobutyric-acid system perhaps participated in this effect.This paper further provided evidence that central serotonin and EOP play a stimulatory role in the acupuncture induced secretion of prolactin; acupuncture may antagonize inhibitory effect of H<sub>2</sub> histamine receptor activation on prolactin secretion; the possible role of H<sub>1</sub>-receptor needs further investigation.
文摘OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.
基金supported by NIH and NIDA(R01DA039146T32DA031115)NIH Intramural Research Programs
文摘OBJECTIVE Wide spread abuse of synthetic cathinones found in bath salts preparations has resulted in regulation of some cathinones internationally.Chemists skirt these laws by altering the chemical structures of first-generation cathinones(ie,MDPV,methylone,and mephedrone),resulting in second-generation cathinones(eg,α-PVP,α-PPP,MDPPP,and MDPBP).Although MDPV is a more effective reinforcer than cocaine,little is known about the reinforcing effectiveness of secondgeneration cathinones.To test the hypothesis that synthetic cathinones with higher selectivity for DAT relative to SERT are more effective reinforcers.METHODS Monoamine transporter inhibition was determined using synaptosomes prepared from rat brains.The relative reinforcing effectiveness of intravenously self-administered MDPV,MDPBP,MDPPP,α-PVP,α-PPP,and cocaine were directly compared through evaluations of (1)dose-response curves under a progressive ratio(PR)schedule of reinforcement and (2)demand curves obtained for each drug in male Sprague-Dawley rats.RESULTS Rank order selectivity for DAT/SERT wasα-PVP>MDPV>α-PPP≈MDPBP>MDPPP>cocaine.Comparisons of the maximum number of infusions obtained under a PR schedule of reinforcement(α-PVP>MDPV>α-PPP>MDPBP≈MDPPP>cocaine)and the essential value obtained for each drug in demand analyses(α-PVP>MDPV>α-PPP≈MDPBP≈MDPPP>cocaine)suggest relative reinforcing effectiveness is related to DAT/SERT selectivity.CONCLUSION These data provide evidence that DAT/SERT selectivity accounts for select synthetic cathinones functioning as more effective reinforcers than cocaine and may predict the abuse-related effects of novel synthetic cathinones in humans.
