Cannabinoid type 2 receptor activation inhibits MPP+-induced M1 differentiation of microglia through activating PI3K/Akt/Nrf2 signal pathway.

BACKGROUND: Growing evidence indicates that cannabinoid type 2 (CB2) receptor activation inhibits neuroinflammation in the pathogenesis of Parkinson's disease (PD). Nonetheless, the precise mechanisms of CB2 receptor-mediated neuroprotection have not been fully elucidated. The differentiation of microglia from the M1 to M2 phenotype plays a vital role in neuroinflammation. METHODS: In the present study, we investigated the effect of CB2 receptor activation on the M1/M2 phenotypic transformation of microglia treated with 1-methyl-4-phenylpyridinium (MPP+). The M1 phenotype microglia markers, including inducible nitric oxide (iNOS), interleukin 6 (IL-6), and CD86, and the M2 phenotype microglia markers, including arginase-1 (Arg-1), IL-10, and CD206, were detected by western blots and flow cytometry. The levels of phosphoinositide-3-kinase (PI3K)/Akt and nuclear factor erythroid 2-related factor 2 (Nrf2) were determined by Western blots. Subsequent addition of Nrf2 inhibitors initially revealed the specific mechanism by which CB2 receptors affect phenotypic changes in microglia. RESULTS: Our results showed that pretreatment with JWH133 significantly inhibited the MPP+-induced up-regulation of M1 phenotype microglia markers. Meanwhile, JWH133 increased the levels of M2 phenotype microglia markers. JWH133-mediated effects were blocked by co-treatment with AM630. Mechanism studies found that MPP+ treatment downregulated PI3K, Akt phosphorylated proteins, and nuclear Nrf2 protein. JWH133 pretreatment promoted PI3K/Akt activation and facilitated nuclear translocation of Nrf2, which was reversed by the PI3K inhibitor. Further studies showed that Nrf2 inhibitors inverted the effect of JWH133 on microglia polarization. CONCLUSION: The results indicate that CB2 receptor activation promotes MPP+-induced microglia transformation from M1 to M2 phenotype through PI3K/Akt/Nrf2 signaling pathway.

Roles of the Cannabinoid System in the Basal Ganglia in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disease usually caused by neuroinflammation, oxidative stress and other etiologies. Recent studies have found that the cannabinoid system present in the basal ganglia has a strong influence on the progression of PD. Altering the cannabinoid receptor activation status by modulating endogenous cannabinoid (eCB) levels can exert an anti-movement disorder effect. Therefore, the development of drugs that modulate the endocannabinoid system may be a novel strategy for the treatment of PD. However, eCB regulation is complex, with diverse cannabinoid receptor functions and the presence of dopaminergic, glutamatergic, and γ-aminobutyric signals interacting with cannabinoid signaling in the basal ganglia region. Therefore, the study of eCB is challenging. Here, we have described the function of the cannabinoid system in the basal ganglia and its association with PD in three parts (eCBs, cannabinoid receptors, and factors regulating the cannabinoid metabolism) and summarized the mechanisms of action related to the cannabinoid analogs currently aimed at treating PD. The shortcomings identified from previous studies and the directions that should be explored in the future will provide insights into new approaches and ideas for the future development of cannabinoid-based drugs and the treatment of PD.

Attribute nonattendance in COVID-19 vaccine choice: A discrete choice experiment based on Chinese public preference.

OBJECTIVES: The global coronavirus disease 2019 (COVID-19) pandemic has not been well controlled, and vaccination could be an effective way to prevent this pandemic. By accommodating attribute nonattendance (ANA) in a discrete choice experiment (DCE), this paper aimed to examine Chinese public preferences and willingness to pay (WTP) for COVID-19 vaccine attributes, especially the influence of ANA on the estimated results. METHODS: A DCE was designed with four attributes: effectiveness, protection period, adverse reactions and price. A random parameter logit model with an error component (RPL-EC) was used to analyse the heterogeneity of respondents' preferences for COVID-19 vaccine attributes. Two equality constraint latent class (ECLC) models were used to consider the influence of ANA on the estimated results in which the ECLC-homogeneity model considered only ANA and the ECLC-heterogeneity model considered both ANA and preference heterogeneity. RESULTS: Data from 1,576 samples were included in the analyses. Effectiveness had the highest relative importance, followed by adverse reactions and protection period, which were determined by the attributes and levels presented in this study. The ECLC-heterogeneity model improved the goodness of fit of the model and obtained a lower probability of ANA. In the ECLC-heterogeneity model, only a small number of respondents (29.09%) considered all attributes, and price was the most easily ignored attribute (64.23%). Compared with the RPL-EC model, the ECLC-homogeneity model obtained lower WTPs for COVID-19 vaccine attributes, and the ECLC-heterogeneity model obtained mixed WTP results. In the ECLC-heterogeneity model, preference group 1 obtained higher WTPs, and preference groups 2 and 3 obtained lower WTPs. CONCLUSIONS: The RPL-EC, ECLC-homogeneity and ECLC-heterogeneity models obtained inconsistent WTPs for COVID-19 vaccine attributes. The study found that the results of the ECLC-heterogeneity model considering both ANA and preference heterogeneity may be more plausible because ANA and low preference may be confused in the ECLC-homogeneity model and the RPL-EC model. The results showed that the probability of ANA was still high in the ECLC-heterogeneity model, although it was lower than that in the ECLC-homogeneity model. Therefore, in future research on DCE (such as the field of vaccines), ANA should be considered as an essential issue. PUBLIC CONTRIBUTION: Chinese adults from 31 provinces in mainland China participated in the study. All participants completed the COVID-19 vaccine choice questions generated through the DCE design.

Scope Issue in Contingent Valuation Studies of the COVID-19 Vaccine: The Case of China.

BACKGROUND: Assessing the public's willingness to pay (WTP) for the coronavirus disease 2019 (COVID-19) vaccine by the contingent valuation (CV) method can provide a relevant basis for government pricing. However, the scope issue of the CV method can seriously affect the validity and reliability of the estimation results. AIM: To examine whether there are scope issues in respondents' WTP for the COVID-19 vaccine and to further verify the validity and reliability of the CV estimate results. METHOD: In this study, nine different CV double-bounded dichotomous choices (DBDC) hypothetical COVID-19 vaccine scenarios were designed using an orthogonal experimental design based on the vaccine's attributes. A total of 2450 samples from 31 provinces in Mainland China were collected to independently estimate the public's WTP in these nine scenarios with logistic, normal, log-logistic and log-normal parameter models. Based on this estimation, several external scope tests were designed to verify the validity and reliability of the CV estimate results. RESULTS: In the 20 pairs of COVID-19 vaccine scenarios, 6 pairs of scenarios were classified as negative scope issues, therefore not passing the external scope test. Of the remaining 14 pairs of scenarios, only four pairs of scenarios completely passed the external scope test, and one pair of scenarios partially passed the external scope test. Significant negative scope and scope insensitivity issues were revealed. CONCLUSION: In the context of a dynamic pandemic environment, the findings of this study reveal that the CV method may face difficulty in effectively estimating respondents' WTP for the COVID-19 vaccine. We suggest that future studies be cautious in applying the CV method to estimate the public's WTP for the COVID-19 vaccine.

Levo-tetrahydropalmatine inhibits α4ß2 nicotinic receptor response to nicotine in cultured SH-EP1 cells.

Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4ß2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 µM) induced inward currents, co-application of l-THP (3 µM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 µM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4ß2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4ß2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42ß23-nAChR) and cytisine-activated, low-sensitive one (α43ß22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4ß2-nAChR function, which may underlie its inhibition on nicotine addiction.

Protective effects of berberine against MPTP-induced dopaminergic neuron injury through promoting autophagy in mice.

Berberine, an isoquinoline alkaloid isolated from Coptis chinensis, has been widely studied for its efficacy in the treatment of neurodegenerative diseases. However, the detailed mechanisms are unknown. In this study, the effects of berberine on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice model of Parkinson's disease were investigated. We showed that treatment with berberine significantly ameliorates the degeneration of dopaminergic neurons in substantia nigra compacta (SNc) and improves motor impairment in MPTP-treated mice. Berberine also significantly decreased the level of α-synuclein and enhanced the microtubule-associated protein light chain 3 (LC3-II)-associated autophagy in the SN of MPTP-treated mice. Furthermore, adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) was activated by berberine. Berberine's actions were abolished by pre-treatment with 3-methyladenine (an autophagy inhibitor) or compound c (an AMPK inhibitor) in the MPP+-treated SH-SY5Y cells. These results suggested that the protective effects of berberine on the toxicity of MPTP could be attributed to berberine-enhanced autophagy via the AMPK dependent pathway.

Cannabinoid CB2 receptors are expressed in glutamate neurons in the red nucleus and functionally modulate motor behavior in mice.

Cannabinoids produce a number of central nervous system effects via the CB2 receptor (CB2R), including analgesia, antianxiety, anti-reward, hypoactivity and attenuation of opioid-induced respiratory depression. However, the cellular distributions of the CB2Rs in the brain remain unclear. We have reported that CB2Rs are expressed in midbrain dopamine (DA) neurons and functionally regulate DA-mediated behavior(s). Unexpectedly, high densities of CB2-like signaling were also found in a neighboring motor structure - the red nucleus (RN) of the midbrain. In the present study, we systematically explored CB2R expression and function in the RN. Immunohistochemistry and in situ hybridization assays showed high densities of CB2R-immunostaining and mRNA signal in RN magnocellular glutamate neurons in wildtype and CB1-knockout, but not CB2-knockout, mice. Ex vivo electrophysiological recordings in midbrain slices demonstrated that CB2R activation by JWH133 dose-dependently inhibited firing rates of RN magnocellular neurons in wildtype, but not CB2-knockout, mice, while having no effect on RN GABA neurons in transgenic GAD67-GFP reporter mice, suggesting CB2-mediated effects on glutamatergic neurons. In addition, microinjection of JWH133 into the RN produced robust ipsilateral rotations in wildtype, but not CB2-knockout mice, which was blocked by pretreatment with either a CB2 or DA D1 or D2 receptor antagonist, suggesting a DA-dependent effect. Finally, fluorescent tract tracing revealed glutamatergic projections from the RN to multiple brain areas including the ventral tegmental area, thalamus, and cerebellum. These findings suggest that CB2Rs in RN glutamate neurons functionally modulate motor activity, and therefore, constitute a new target in cannabis-based medication development for motor disorders.

Therapeutic potential of ATP-sensitive potassium channels in Parkinson's disease.

In the past decade, there was an increasing interest in the therapeutic potential targeting ATP-sensitive potassium (KATP) channels for an effective treatment of Parkinson's disease (PD). KATP channels are widely expressed in the central nervous system and were reported to mediate the degeneration and death of nigral dopamine neurons in the pathogenesis of PD. This review aims to address the pivotal roles of KATP channels played in the mechanisms underlying PD pathogenesis, and provide possible directions for further research from different perspectives, such as the vulnerability of dopamine neurons in the substantia nigra, neurotransmitter releasing, iron metabolism in the brain, α-synuclein secretion and mitochondrial dysfunction, which are off critical importance in the investigation of KATP channels-targeted precise therapeutic interventions for PD.

Myricetin reduces cytotoxicity by suppressing hepcidin expression in MES23.5 cells.

Multiple studies implicate iron accumulation in the substantia nigra in the degeneration of dopaminergic neurons in Parkinson's disease. Indeed, slowing of iron accumulation in cells has been identified as the key point for delaying and treating Parkinson's disease. Myricetin reportedly plays an important role in anti-oxidation, anti-apoptosis, anti-inflammation, and iron chelation. However, the mechanism underlying its neuroprotection remains unclear. In the present study, MES23.5 cells were treated with 1 × 10-6 M myricetin for 1 hour, followed by co-treatment with 400 nM rotenone for 24 hours to establish an in vitro cell model of Parkinson's disease. Our results revealed that myricetin alleviated rotenone-induced decreases in cell viability, suppressed the production of intracellular reactive oxygen species, and restored mitochondrial transmembrane potential. In addition, myricetin significantly suppressed rotenone-induced hepcidin gene transcription and partly relieved rotenone-induced inhibition of ferroportin 1 mRNA and protein levels. Furthermore, myricetin inhibited rotenone-induced phosphorylation of STAT3 and SMAD1 in MES23.5 cells. These findings suggest that myricetin protected rotenone-treated MES23.5 cells by potently inhibiting hepcidin expression to prevent iron accumulation, and this effect was mediated by alteration of STAT3 and SMAD1 signaling pathways.

Effects of ghrelin on the electrical activities of substantia nigra dopaminergic neurons treated with MPP.

Ghrelin, a 28 amino acid brain-gut peptide, has attracted increasing attention for its neuroprotective effect in Parkinson's disease (PD). In view of the pivotal role of excitability of dopaminergic neurons in substantia nigra pars compacta (SNc) in the function of nigrostriatal system, it is of great significance to elucidate the regulation of electrical activity of dopaminergic neurons by ghrelin, especially in PD pathogenesis. In this study, we tackled this issue by probing the effects of ghrelin on the electrophysiological properties of dopaminergic neurons in acute application of Methyl-4-phenylpyridinium (MPP+), a potent parkinsonizing agent in primates and rodents, with whole cell patch clamp technique. We first observed that MPP+ (10, 20 and 50 µM) inhibited the spontaneous firing activity of dopaminergic neurons with dose-dependent and time-dependent properties. MPP+ also hyperpolarized the membrane potential, inhibited the evoked firing activity and reduced the amplitude of the inward rectification characteristic (Sag) in dopaminergic neurons. Importantly, ghrelin (100 nM) could improve the above effects of MPP+ on the electrical activities of dopaminergic neurons. The potential mechanism of this phenomenon may be that ghrelin upregulated hyperpolarization-activated cyclic nucleotide-gated channel current (Ih) to antagonize the inhibition of MPP+ on Ih, thereby improving the electrical activities of dopaminergic neurons.

Activation of cannabinoid receptor 2 protects rat hippocampal neurons against Aß-induced neuronal toxicity.

Alzheimer's disease (AD) is a dementing, neurodegenerative disorder characterized by increased accumulation of beta-amyloid peptides (Aß), degeneration of hippocampal neurons and the gradual development of learning and memory deficits. Therapeutically, there are still no ideal medicines available and this represents an urgent need for the development of new strategies to treat AD. Emerging lines of evidence suggest that modulation of the cannabinoid system exhibits neuroprotective effects in various neurological diseases, including AD. However, a consensus is yet to emerge as to the impact of hippocampal cannabinoid receptor 2 (CB2R) in protection of hippocampal neurons against Aß induced neuronal toxicity. Here, we report that chronic treatment of primary hippocampal neuronal cultures with 100 nM Aß1-42 oligomers for 7 days results in neurotoxicity, which includes increases in lactate dehydrogenase (LDH) levels, suggesting an Aß1-42 -induced neuron apoptosis. Further, chronic Aß1-42 reduces the ratio of phosphorylated Akt (pAkt)/Akt, in turn decreases neuronal Bcl-2/Bax ratio, and leads to an increase of caspase-3, which likely underlines the signal pathway of chronic Aß1-42-induced neuron apoptosis. Interestingly, pre-treatments of CB2R agonist (JWH133, 10 µM) with Aß1-42 prevents Aß1-42-induced the decrease of pAkt/Akt ratio, the decrease of Bcl-2/Bax ratio, and the increase of caspase-3, and protects hippocampal neurons against Aß1-42-induced apoptosis. All neuroprotective effects of JWH133 are abolished by a selective CB2R antagonist, AM630. Taken together, the activation of hippocampal CB2Rs protects neurons against Aß1-42 toxicity, and the CB2R-mediated enhancement of the pAkt signaling is likely involved in the protection of hippocampal neurons against Aß1-42-induced neuronal toxicity.

JWH133 inhibits MPP+-induced inflammatory response and iron influx in astrocytes.

BACKGROUND: We investigated the anti- inflammatory effect of type II cannabinoid receptor (CB2 receptor) activation and their relationship to iron influx on 1-methyl-4-phenylpyridinium (MPP+) treated astrocytes. METHODS AND RESULTS: By western blots, real-time PCR and ELISA, the expressions of CB2 receptor, divalent metal transporter-1 (DMT1), cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), interleukin-1ß (IL-1ß) and tumor necrosis factor- α (TNF-α) were measured. Iron influx into astrocytes was determined by the quenching of calcein fluorescence. We found that pre-treatment with JWH133, a selective CB2 receptor agonist, significantly suppressed the MPP+-induced up-regulation of COX-2, iNOS, IL- 1ß and TNF-α in astrocytes. In addition, JWH133 significantly inhibited the MPP+-induced up- regulation of DMT1. Further studies indicated that JWH133 suppressed the MPP+-accelerated iron influx into astrocytes. These effects were blocked by co-treatment with AM630, the CB2 receptor antagonist. CONCLUSIONS: These results suggest that activation of CB2 receptor inhibit MPP +-induced inflammatory response and iron influx in astrocytes.

L-type Calcium Channels are Involved in Iron-induced Neurotoxicity in Primary Cultured Ventral Mesencephalon Neurons of Rats.

In the present study, we investigated the mechanisms underlying the mediation of iron transport by L-type Ca2+ channels (LTCCs) in primary cultured ventral mesencephalon (VM) neurons from rats. We found that co-treatment with 100 µmol/L FeSO4 and MPP+ (1-methyl-4-phenylpyridinium) significantly increased the production of intracellular reactive oxygen species, decreased the mitochondrial transmembrane potential and increased the caspase-3 activation compared to MPP+ treatment alone. Co-treatment with 500 µmol/L CaCl2 further aggravated the FeSO4-induced neurotoxicity in MPP+-treated VM neurons. Co-treatment with 10 µmol/L isradipine, an LTCC blocker, alleviated the neurotoxicity induced by co-application of FeSO4 and FeSO4/CaCl2. Further studies indicated that MPP+ treatment accelerated the iron influx into VM neurons. In addition, FeSO4 treatment significantly increased the intracellular Ca2+ concentration. These effects were blocked by isradipine. These results suggest that elevated extracellular Ca2+ aggravates iron-induced neurotoxicity. LTCCs mediate iron transport in dopaminergic neurons and this, in turn, results in elevated intracellular Ca2+ and further aggravates iron-induced neurotoxicity.

Cocaine potently blocks neuronal α3ß4 nicotinic acetylcholine receptors in SH-SY5Y cells.

Cocaine is one of the most abused illicit drugs worldwide. It is well known that the dopamine (DA) transporter is its major target; but cocaine also acts on other targets including nicotinic acetylcholine receptors (nAChRs). In this study, we investigated the effects of cocaine on a special subtype of neuronal nAChR, α3ß4-nAChR expressed in native SH-SY5Y cells. α3ß4-nAChR-mediated currents were recorded using whole-cell recordings. Drugs were applied using a computer-controlled U-tube drug perfusion system. We showed that bath application of nicotine induced inward currents in a concentration-dependent manner with an EC50 value of 20 µM. Pre-treatment with cocaine concentration-dependently inhibited nicotine-induced current with an IC50 of 1.5 µM. Kinetic analysis showed that cocaine accelerated α3ß4-nAChR desensitization, which caused a reduction of the amplitude of nicotine-induced currents. Co-application of nicotine and cocaine (1.5 µM) depressed the maximum response on the nicotine concentration-response curve without changing the EC50 value, suggesting a non-competitive mechanism. The cocaine-induced inhibition of nicotine response exhibited both voltage- and use-dependence, suggesting an open-channel blocking mechanism. Furthermore, intracellular application of GDP-ßS (via recording electrode) did not affect cocaine-induced inhibition, suggesting that cocaine did not alter receptor internalization. Moreover, intracellular application of cocaine (30 µM) failed to alter the nicotine response. Finally, cocaine (1.5 µM) was unable to inhibit the nicotine-induced inward current in heterologous expressed α6/α3ß2ß3-nAChRs and α4ß2-nAChRs expressed in human SH-EP1 cells. Collectively, our results suggest that cocaine is a potent blocker for native α3ß4-nAChRs expressed in SH-SY5Y cells.

Berberine alleviates rotenone-induced cytotoxicity by antioxidation and activation of PI3K/Akt signaling pathway in SH-SY5Y cells.

Berberine, an isoquinoline alkaloid isolated from traditional Chinese medicine, has been widely studied for its efficacy in the treatment of neurodegenerative diseases. However, berberine-mediated neuroprotection in the pathogenesis of Parkinson's disease is still uncertain. In this study, the effects of berberine on rotenone-induced neurotoxicity in SH-SY5Y cells were investigated. The results showed that berberine treatment significantly alleviated rotenone-induced decrease in the cell viability in SH-SY5Y cells. Further studies demonstrated that berberine suppressed the production of intracellular reactive oxygen species, restored the mitochondrial transmembrane potential, increased Bcl-2/Bax ratio, and decreased caspase-3 activation that induced by rotenone. Furthermore, berberine also restored the phosphorylation of Akt, which was downregulated by rotenone in SH-SY5Y cells. These results suggest that berberine protects rotenone-treated SH-SY5Y cells by antioxidation and activation of PI3K/Akt signaling pathway.

Mechanisms of cannabinoid CB2 receptor-mediated reduction of dopamine neuronal excitability in mouse ventral tegmental area.

BACKGROUND: We have recently reported that activation of cannabinoid type 2 receptors (CB2Rs) reduces dopamine (DA) neuron excitability in mouse ventral tegmental area (VTA). Here, we elucidate the underlying mechanisms. METHODS: Patch-clamp recordings were performed in mouse VTA slices and dissociated single VTA DA neurons. FINDINGS: Using cell-attached recording in VTA slices, bath-application of CB2R agonists (JWH133 or five other CB2R agonists) significantly reduced VTA DA neuron action potential (AP) firing rate. Under the patch-clamp whole-cell recording model, JWH133 (10 µM) mildly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not miniature inhibitory postsynaptic currents (mIPSCs). JWH133 also did not alter evoked EPSCs or IPSCs. In freshly dissociated VTA DA neurons, JWH133 reduced AP firing rate, delayed AP initiation and enhanced AP after-hyperpolarization. In voltage-clamp recordings, JWH133 (1 µM) enhanced M-type K+ currents and this effect was absent in CB2-/- mice and abolished by co-administration of a selective CB2R antagonist (10 µM, AM630). CB2R-mediated inhibition in VTA DA neuron firing can be mimicked by M-current opener (10 µM retigabine) and blocked by M-current blocker (30 µM XE991). In addition, enhancement of neuronal cAMP by forskolin (10 µM) reduced M-current and increased DA neuron firing rate. Finally, pharmacological block of synaptic transmission by NBQX (10 µM), D-APV (50 µM) and picrotoxin (100 µM) in VTA slices failed to prevent CB2R-mediated inhibition, while intracellular infusion of guanosine 5'-O-2-thiodiphosphate (600 µM, GDP-ß-S) through recording electrode to block postsynaptic G-protein function prevented JWH133-induced reduction in AP firing. INTERPRETATION: Our results suggest that CB2Rs modulate VTA DA neuron excitability mainly through an intrinsic mechanism, including a CB2R-mediated reduction of intracellular cAMP, and in turn enhancement of M-type K+ currents. FUND: This research was supported by the Barrow Neuroscience Foundation, the BNI-BMS Seed Fund, and CNSF (81771437).

Cocaine Directly Inhibits α6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons.

Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits α6N/α3Cß2ß3-nAChR (α6*-nAChRs) function. Human α6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic α6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure α6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human α6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 µM. Interestingly, in the presence of 30 µM cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine's EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both α6N/α3Cß2ß3-nAChRs and α6M211L/α3ICß2ß3-nCAhRs similarly, suggesting that cocaine may not act on the α3 transmembrane domain of chimeric α6N/α3Cß2ß3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes α6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed α6*-nAChRs. These findings suggest that α6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence.

An unexpected improvement in spatial learning and memory ability in alpha-synuclein A53T transgenic mice.

Growing evidence suggests, as Parkinson's disease (PD) progresses, that its non-motor symptoms appear prior to or in parallel with its motor deficits. Alpha-synuclein A53T transgenic mouse (A53T) is an essential tool to investigate the onsets and the extents of PD non-motor symptoms. Our aim is to investigate spatial learning and memory ability in A53T mice. In our rotarod tests, no motor coordination impairments were detected in mice of 3, 6, 9, and 12 months old. We then investigated their spatial learning and memory ability through Morris water maze in 3- and 9-month-old mice. No significant difference in escape latency was detected among the A53T mice and the control mice. However, an unexpected improvement in spatial learning and memory ability was observed in the probe session among the A53T mice. Reversal learning by Morris water maze also indicated that 3- and 9-month-old A53T mice exhibited a better cognitive flexibility compared to their littermate controls. Further studies by western blots showed that alpha-synuclein expressions in hippocampus of the A53T mice were noticeably up-regulated. The immunofluorescence staining of 5-bromo-2-deoxyuridine (Brdu) and doublecortin (DCX) demonstrated that neither the Brdu-positive neurons nor the Brdu/DCX positive neurons in hippocampus were significantly altered between the two groups. These results suggest that our A53T mice exhibit improved spatial learning and memory ability prior to their motor coordination deficits. These results are not induced by neurogenesis in the hippocampus.

The 2-aminoethoxydiphenyl borate analog, DPB161 blocks store-operated Ca2+ entry in acutely dissociated rat submandibular cells.

Cellular Ca2+ signals play a critical role in cell physiology and pathology. In most non-excitable cells, store-operated Ca2+ entry (SOCE) is an important mechanism by which intracellular Ca2+ signaling is regulated. However, few drugs can selectively modulate SOCE. 2-Aminoethoxydiphenyl borate (2APB) and its analogs (DPB162 and DPB163) have been reported to inhibit SOCE. Here, we examined the effects of another 2-APB analog, DPB161 on SOCE in acutely-isolated rat submandibular cells. Both patch-clamp recordings and Ca2+ imaging showed that upon removal of extracellular Ca2+ ([Ca2+]o=0), rat submandibular cells were unable to maintain ACh-induced Ca2+ oscillations, but restoration of [Ca2+]o to refill Ca2+ stores enable recovery of these Ca2+ oscillations. However, addition of 50 µM DPB161 with [Ca2+]o to extracellular solution prevented the refilling of Ca2+ store. Fura-2 Ca2+ imaging showed that DPB161 inhibited SOCE in a concentration-dependent manner. After depleting Ca2+ stores by thapsigargin treatment, bath perfusion of 1 mM Ca2+ induced [Ca2+]i elevation in a manner that was prevented by DPB161. Collectively, these results show that the 2-APB analog DPB161 blocks SOCE in rat submandibular cells, suggesting that this compound can be developed as a pharmacological tool for the study of SOCE function and as a new therapeutic agent for treating SOCE-associated disorders.

Isradipine attenuates MPTP-induced dopamine neuron degeneration by inhibiting up-regulation of L-type calcium channels and iron accumulation in the substantia nigra of mice.

The aim of this study is to investigate the effects of L-type calcium channels (LTCCs) on MPTP-induced dopamine (DA) neuron degeneration and iron accumulation in the substantia nigra (SN) of mice. By real-time PCR and western blots, we first quatified expressions of L-type Cav1.2 and Cav1.3 calcium channel α1 subunits in the SN of experimental mice treated with MPTP. We found that the expressions of Cav1.2 and Cav1.3 calcium channel α1 subunits markedly increased after MPTP treatment for 2 and 3 weeks. Secondly, we observed the effects of isradipine, a LTCC antagonist, on MPTP-induced DA neuron degeneration and iron accumulation in the SN. Our results showed that isradipine treatment prevented against MPTP-induced Cav1.2 and Cav1.3 calcium channel α1 subunits up-regulation in the SN. We also found that isradipine prevented against MPTP-induced DA neuron depletion in the SN and partly restored the DA content in the striatum. Moreover, we found that isradipine inhibited the increase of iron positive cells in the SN of the MPTP-treated mice. Finally, we investigated the effects of isradipine on cellular iron accumulation in the dopaminergic MES23.5 cell line. Our studies showed that MPP+ treatment accelerated iron influx in the MES23.5 cells. Treatment with Bayk8644 further aggravated iron accumulation. Treatment with isradipine prevented against MPP+-induced iron influx in the MES23.5 cells. These results suggest that up-regulation of LTCCs may be responsible for the DA neuron degeneration in the MPTP-treated mice, The LTCCs may directly contribute to iron influx into DA neurons, and isradipine may suppress cellular iron accumulation and prevents neurodegeneration.