Beneficial effects of cannabinoids (CB) in a murine model of allergen-induced airway inflammation: role of CB1/CB2 receptors

The endocannabinoid system (ECS) consists of two cannabinoid (CB) receptors, namely CB₁ and CB₂ receptor, and their endogenous (endocannabinoids) and exogenous (cannabinoids, e.g. delta-9-tetrahydrocannabinol (THC)) ligands which bind to these receptors. Based on studies suggesting a role of THC and the ECS in inflammation, the objective of this study was to examine their involvement in type I hypersensitivity using a murine model of allergic airway inflammation. THC treatment of C57BL/6 wildtype mice dramatically reduced airway inflammation as determined by significantly reduced total cell counts in bronchoalveolar lavage (BAL). These effects were greatest when mice were treated during both, the sensitization and the challenge phase. Furthermore, systemic immune responses were significantly suppressed in mice which received THC during sensitization phase. To investigate a role of CB_1/2 receptors in this setting, we used pharmacological blockade of CB₁ and/or CB₂ receptors by the selective antagonists and moreover CB₁/CB₂ receptor double-knockout mice (CB₁^−/−/CB₂^−/−) and found neither significant changes in the cell patterns in BAL nor in immunoglobulin levels as compared to wildtype mice. Our results indicate that the activation of the ECS by applying the agonist THC is involved in the development of type I allergies. However, CB₁/CB₂ receptor-independent signalling seems likely in the observed results.     

d-Aspartate activates mGlu receptors coupled to polyphosphoinositide hydrolysis in neonate rat brain slices

The d-amino acid, d-aspartate, is abundant in the developing brain, yet its function is unknown. Addition of d-aspartate to hippocampal or cortical slices prepared from 8- to 9-day-old rats stimulated polyphosphoinositide (PI) hydrolysis to a slightly greater extent than l-glutamate. The action of d-aspartate was concentration-dependent with an apparent EC₅₀ value of 1 mM and a maximal stimulation of 6- and 20-fold in cortical and hippocampal slices, respectively. Stimulation of PI hydrolysis by d-aspartate was largely reduced by pharmacological blockade of mGlu5 metabotropic glutamate receptors with 2-methyl-6-(phenylethynyl)pyridine. These findings suggest that d-aspartate behaves as an endogenous agonist of mGlu5 receptors during early postnatal life.     

A metabotropic glutamate 2/3 receptor antagonist, MGS0039, increases extracellular dopamine levels in the nucleus accumbens shell

(1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (MGS0039), a potent and selective metabotropic glutamate 2/3 (mGlu 2/3) receptor antagonist, exhibits antidepressant-like activities in some animal models. In the present study, we examined the effect of MGS0039 on extracellular dopamine levels in the rat nucleus accumbens (NAc) shell using in vivo microdialysis evaluation because accumbal dopamine has been implicated in depression. Local application of MGS0039 into the NAc shell at 10 microM significantly increased extracellular dopamine levels in the NAc shell in freely moving rats. In contrast, local application of 10 microM of LY354740, an mGlu 2/3 receptor agonist, significantly decreased extracellular dopamine levels in the same brain region. These findings suggest that dopamine release in the NAc shell is regulated by mGlu 2/3 receptors, and that the effect on dopamine levels in the NAc shell may partially explain the antidepressant-like properties of mGlu 2/3 receptor antagonists.     

CB2 receptor and amyloid pathology in frontal cortex of Alzheimer's disease patients

The cannabinoid system seems to play an important role in various neurodegenerative diseases including Alzheimer's disease (AD). The relationship of cannabinoid receptors (CB₁R and CB₂R) to cognitive function and neuropathological markers in AD remains unclear. In the present study, postmortem cortical brain tissues (Brodmann area 10) from a cohort of neuropathologically confirmed AD patients and age-matched controls were used to measure CB₁R and CB₂R by immunoblotting. Correlational analyses were performed for the neurochemical and cognitive data. CB₁R expression was significantly decreased in AD. Levels of CB₁R correlated with hypophagia, but not with any AD molecular marker or cognitive status (Mini Mental State Examination score). The level of CB₂R was significantly higher (40%) in AD. Increases in the expression of the glial marker glial fibrillar acidic protein were also found. CB₂R expression did not correlate with cognitive status. Interestingly, expression levels of CB₂R correlated with two relevant AD molecular markers, Aβ₄₂ levels and senile plaque score. These results may constitute the basis of CB₂R-based therapies and/or diagnostic approaches.     

Obesity-resistant S5B rats showed greater cocaine conditioned place preference than the obesity-prone OM rats

Background

Dopamine (DA) and the DA D2 receptor (D2R) are involved in the rewarding and conditioned responses to food and drug rewards. Osborne-Mendel (OM) rats are genetically prone and S5B/P rats are genetically resistant to obesity when fed a high-fat diet. We hypothesized that the differential sensitivity of these two rat strains to natural rewards may also be reflected in sensitivity to drugs of abuse. Therefore, we tested whether OM and S5B/P rats showed a differential preference to cocaine using conditioned place preference (CPP). To also evaluate whether there is specific involvement of the D2R in this differential conditioning sensitivity, we then tested whether the D2R agonist bromocriptine (BC) would differentially affect the effects of cocaine in the two strains.

Methods

OM and S5B/P rats were conditioned with cocaine (5 or 10 mg/kg) in one chamber and saline in another for 8 days. Rats were then tested for cocaine preference. The effects of BC (0.5, 1, 5, 10, 20 mg/kg) on cocaine preference were then assessed in subsequent test sessions.

Results

OM rats did not show a significant preference for the cocaine-paired chamber on test day. Only the S5B/P rats showed cocaine CPP. Later treatment with only the highest dose of BC resulted in reduced cocaine CPP in S5B/P rats when treated with 5 mg/kg cocaine and in OM rats treated with 10 mg/kg cocaine.

Conclusion

Our results indicated that obesity-resistant S5B rats showed greater cocaine CPP than the obesity-prone OM rats. These findings do not support a theory of common vulnerability for reinforcer preferences (food and cocaine). However, they show that BC reduced cocaine conditioning effects supporting at least a partial regulatory role of D2R in conditioned responses to drugs.     

Dose-dependent cocaine place conditioning and D1 dopamine antagonist effects in male Japanese quail

The dopamine D1 receptor subtype has been implicated in drug reward processes in mammals. Two experiments investigated whether dose-dependent differences in cocaine conditioned place preference (CPP) would be obtained in an avian species and whether these cocaine effects were mediated by the dopamine D1 antagonist R(+/-)-SCH23390. In Experiment 1, male birds were given intraperitoneal injections of 1, 3, 10, or 30 mg/kg of cocaine hydrochloride, paired with a chamber that contained distinct visual cues. On alternate days, they received saline paired with a chamber containing different visual cues. A CPP test was given after four pairings of cocaine with the distinct chamber. In Experiment 2, 0.0025, 0.025, or 0.25 mg/kg of SCH 23390 or saline was administered 15 min prior to cocaine (3 mg/kg) and placement into the least preferred chamber. CPP was observed at 1, 3, and 10 mg/kg doses of cocaine but not at 30 mg/kg or saline. All doses of SCH 23390 attenuated cocaine-induced CPP. The findings suggest that cocaine administration results in a dose-dependent CPP, and that similar with mammals, it may be mediated by D1 receptors in an avian species. Thus, the avian species may be a beneficial comparative model for drug studies, especially those involving visual cue mechanisms of drug reward.     

A novel near-infrared fluorescence imaging probe that preferentially binds to cannabinoid receptors CB2R over CB1R

The type 2 cannabinoid receptors (CB₂R) have gained much attention recently due to their important regulatory role in a host of pathophysiological processes. However, the exact biological function of CB₂R and how this function might change depending on disease progression remains unclear and could be better studied with highly sensitive and selective imaging tools for identifying the receptors. Here we report the first near infrared fluorescence imaging probe (NIR760-XLP6) that binds preferentially to CB₂R over the type 1 cannabinoid receptors (CB₁R). The selectivity of the probe was demonstrated by fluorescence microscopy using DBT-CB₂ and DBT-CB₁ cells. Furthermore, in mouse tumor models, NIR760-XLP6 showed significantly higher uptake in DBT-CB₂ than that in DBT-CB₁ tumors. These findings indicate that NIR760-XLP6 is a promising imaging tool for the study of CB₂R regulation.     

Endocannabinoid-mediated synaptically evoked suppression of GABAergic transmission in the cerebellar cortex

Presynaptic CB₁ cannabinoid receptors are frequently targets of endogenous cannabinoids (endocannabinoids) released from postsynaptic neurons. It is known that the glutamatergic afferent input to a neuron can trigger endocannabinoid production and that the released endocannabinoid can suppress the glutamatergic input. We tested the hypothesis that activation of the glutamatergic input to a neuron leads to an endocannabinoid-mediated suppression of the GABAergic afferent input to the same neuron. Spontaneous postsynaptic currents (sPSCs) were recorded with patch-clamp techniques in Purkinje cells in mouse cerebellar brain slices. Activation of the climbing fiber-mediated glutamatergic input to Purkinje cells led to a suppression of the sPSCs by 34±3%. This suppression was mostly due to suppression of GABAergic spontaneous inhibitory postsynaptic current (sIPSCs), because 93% of the sPSCs recorded in Purkinje cells were GABAergic sIPSCs. Blockade of ionotropic, but not metabotropic glutamate receptors, prevented the suppression. The climbing fiber activation led to an increase in calcium concentration in the Purkinje cells, and this increase was necessary for the suppression of sPSCs, because the suppression did not occur when the calcium increase was prevented by BAPTA. No sPSC suppression was observed in the presence of the CB₁ antagonist rimonabant or the diacylglycerol lipase inhibitor orlistat. In a further series of experiments GABAergic sIPSCs were recorded: these sIPSCs were also suppressed after climbing fiber activation, and the suppression was sensitive to the CB₁ antagonist SLV319. Finally, the GABAergic synaptic transmission between molecular layer interneurons and Purkinje cells was directly studied on simultaneously patch-clamped neuron pairs. Climbing fiber activation led to suppression of the interneuron → Purkinje cell synaptic transmission. The results point to a novel form of endocannabinoid-mediated heterosynaptic plasticity. The endocannabinoid production in a neuron is triggered by its glutamatergic synaptic input and is dependent on an increase in intracellular calcium concentration. The produced endocannabinoid, in turn, suppresses the GABAergic synaptic input to the neuron by activating CB₁ cannabinoid receptors.     

Comparison of Changes in Glutamate Levels in the Nucleus Accumbens of the Rat Brain during Food Consumption in Conditions of Blockade of Dopamine D1 and D2 Receptors

The effects of blockade of D₁ and D₂ dopamine receptors in the nucleus accumbens on changes in glutamate levels in the intercellular space of this structure during food consumption were studied in Sprague–Dawley rats by intracerebral microdialysis combined with HPLC. These experiments showed that food consumption was accompanied by decreases in glutamate levels in the intercellular spaces of the nucleus accumbens. Blockade of D₁ dopamine receptors with SCH-23390 (0.01 mM) produced no changes in the dynamics of glutamate release during food consumption. Food consumption in conditions of blockade of D₂ dopamine receptors with raclopride (0.01 mM) induced increases in glutamate levels. These data suggest that glutamate levels during food consumption are controlled by the dopaminergic system of the nucleus accumbens, mediated by D₂ but not D₁ dopamine receptors.     

Reinforcing and locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice

Both ionotropic and metabotropic glutamate receptors (mGluRs) are involved in the behavioral effects of pyschostimulants; however, the specific contributions of individual mGluR subtypes remain unknown. Here we show that mice lacking the mGluR5 gene do not self-administer cocaine, and show no increased locomotor activity following cocaine treatment, despite showing cocaine-induced increases in nucleus accumbens (NAcc) dopamine (DA) levels similar to wild-type (WT) mice. These results demonstrate a significant contribution of mGlu5 receptors to the behavioral effects of cocaine, and suggest that they may be involved in cocaine addiction.     

Mesoaccumbens dopamine signaling alteration underlies behavioral transition from tolerance to sensitization to morphine rewarding properties during morphine withdrawal

Although the firing activity of dopamine (DA) neurons in the ventral tegmental area (VTA) and the behavioral response to morphine rewarding properties alter as opiate withdrawal, little is known about the dynamic changes in DA signal pathway from the VTA to the nucleus accumbens (NAc) during prolonged withdrawal, and whether the changes are indicative of vulnerability to relapse of drug abuse. Here we report that morphine spontaneously withdrawn (SW) rats are incapable of responding to small dose of morphine-induced conditioned place preference (CPP) from 24h-SW to 30d-SW, but recover response at 45d-SW. Interestingly, mesoaccumbens DA signaling, including the firing of DA neurons in the VTA, contents of DA and its metabolic ratio, and the membrane level of dopamine D₁ receptor in the NAc elicited by morphine challenge, display a similar pattern of time-dependent changes during morphine withdrawal. Moreover, blockade of D₁ receptor abolishes this behavioral transition. In addition, a strong correlation was found between % change in CPP score and membrane D₁ receptor level induced by morphine challenge. These results indicate a time-dependent behavioral switch from tolerance to sensitization during the prolonged withdrawal, which could offer a window for therapeutic intervention via manipulations of D₁ receptors.     

Pre-treatment with high doses of cocaine decreases the reinforcing effects of cocaine in the conditioned place preference paradigm

The aim of the present study was to determine if pre-exposure to high doses of cocaine can subsequently alter the rewarding effects of this drug. Adult male mice received a pretreatment of physiological saline, or 12.5 or 25 mg/kg of cocaine (one injection a day for five days). After an interval of six days without injections, the rewarding effects of low doses of cocaine (0.5, 1 or 1.5 mg/kg) were evaluated in the conditioned place preference (CPP) paradigm. Doses of 1 and 1.5 mg/kg induced a clear CPP in animals pre-treated with saline but were ineffective in those pre-treated with 25 mg/kg of cocaine. Only the dose of 1.5 mg/kg induced CPP in mice pre-treated with 12.5 mg/kg of cocaine. Our results, which reveal a decrease in the conditioned rewarding effects of threshold doses of cocaine, demonstrate that exposure to high doses of this drug can alter the reward system.     

CRIP1a switches cannabinoid receptor agonist/antagonist-mediated protection from glutamate excitotoxicity

A shared pathology among many neurological and neurodegenerative disorders is neuronal loss. Cannabinoids have been shown to be neuroprotective in multiple systems. However, both agonists and antagonists of the CB₁ cannabinoid receptor are neuroprotective, but the mechanisms responsible for these actions remain unclear. Recently a CB₁ receptor interacting protein, CRIP1a, was identified and found to alter CB₁ activity. Here we show that in an assay of glutamate neurotoxicity in primary neuronal cortical cultures CRIP1a disrupts agonist-induced neuroprotection and confers antagonist-induced neuroprotection.     

CaMKII: a biochemical bridge linking accumbens dopamine and glutamate systems in cocaine seeking

Increases in dopamine and glutamate transmission in the nucleus accumbens independently promote the reinstatement of cocaine seeking, an animal model of relapse. Here we have tested whether cocaine reinstatement in rats depends on interactions between accumbal dopamine and glutamate systems that are mediated by Ca(2+)/calmodulin-mediated kinase II (CaMKII). We show that stimulation of D1-like dopamine receptors in the nucleus accumbens shell reinstates cocaine seeking by activating L-type Ca(2+) channels and CaMKII. Cocaine reinstatement is associated with D1-like dopamine receptor-dependent increases in accumbens shell CaMKII phosphorylated on Thr286 and glutamate receptor 1 (GluR1) phosphorylated on Ser831 (a known CaMKII phosphorylation site), in addition to increases in cell-surface expression of GluR1-containing AMPA receptors in the shell. Consistent with these findings, cocaine reinstatement is attenuated by intra-shell administration of AAV10-GluR1-C99, a vector that impairs the transport of GluR1-containing AMPA receptors. Thus, CaMKII may be an essential link between accumbens shell dopamine and glutamate systems involved in the neuronal plasticity underlying cocaine craving and relapse.     

Inhibition of the cannabinoid 2 receptor in CNS-injury induced immunodeficiency syndrome

Central nervous system (CNS) injury is classified as an independent factor, increasing patients’ susceptibility to infections. The concept of infection susceptibility and impaired immune function is referred to as CNS-injury induced immunodeficiency syndrome (CIDS). The endocannabinoid system, an important homeostatic system that can modulate immune function, contributes to the consequences of an acute CNS injury. The actions of the endocannabinoid system are mediated via cannabinoid receptors, cannabinoid 1 (CB₁R) and cannabinoid 2 (CB₂R), the latter of which are highly expressed by immune cells and upregulated as a result of infectious and non-infectious stressors. While the role of the CB₂R in CNS immunity is primarily anti-inflammatory, focusing on the inhibition of the CB₂R pathways may be of benefit for therapeutic targeting of the immunosuppression in CIDS. We hypothesize that inhibition of the CB₂R will result in a decrease in the immunosuppression seen in CIDS, providing the patient protection against common infections such as pneumonia and urinary tract infections. However, due to the high variability of the patients’ immune status during and after an acute CNS injury, identifying the most effective therapeutic window and CB₂R antagonist dosage for effective immunostimulation is pivotal.     

Contributions of mGlu1 and mGlu5 receptors to interactions with N-methyl-D-aspartate receptor-mediated responses and nociceptive sensory responses of rat thalamic neurons

The nociceptive responses of rat ventrobasal thalamus neurons can be reduced by N-methyl-D-aspartate antagonists and by selective metabotropic glutamate receptor mGlu1 antagonists. The recent development of the mGlu5-selective antagonist 6-methyl-2-(phenylethynyl)-pyridine now allows the direct probing of the possible involvement of mGlu5 receptors in thalamic nociceptive responses. Extracellular recordings were made from single neurons in the ventrobasal thalamus and immediately overlying dorsal thalamic nuclei of adult urethane-anaesthetized rats using multi-barrel electrodes. Responses of neurons to iontophoretic applications of the mGlu5-selective agonist (R,S)-2-chloro-5-hydroxyphenylglycine were selectively reduced during continuous iontophoretic applications of 6-methyl-2-(phenylethynyl)-pyridine. Similar applications of 6-methyl-2-(phenylethynyl)-pyridine reduced neuronal responses to noxious thermal stimuli to 53+/-9.5% of control responses. Co- application by iontophoresis of N-methyl-D-aspartate and metabotropic glutamate receptor agonists resulted in a mutual potentiation of excitatory responses. This effect could be reduced by either 6-methyl-2-(phenylethynyl)-pyridine or the mGlu1 antagonist LY367385.These results, taken together with previous data, suggest that acute thalamic nociceptive responses are mediated by a combination of mGlu1, mGlu5 and N-methyl-D-aspartate receptor activation, and that co-activation of these receptors produces a synergistic excitatory effect. Thus blockade of any of these receptor types would have a profound effect on the overall nociceptive response.     

Rac1信号通路在小鼠可卡因情景线索记忆过程中的作用

目的:探讨Rac1信号通路在小鼠可卡因情景线索记忆过程中的作用。方法:将成年雄性C57BL/6J小鼠随机分为对照组(control)、可卡因配对的条件性位置偏爱组(CPP)和Rac1抑制剂NSC23766处理组(NSC),利用CPP实验检测可卡因是否可诱导小鼠产生CPP,利用立体定位技术注射AAV-GFP病毒于小鼠海马CA1区并做免疫荧光染色观察海马CA1区神经元的形态;利用Western Blot及GST-pull down方式检测蛋白表达及活性变化;采用Rac1抑制剂NSC23766阻断信号通路探讨其对小鼠可卡因诱导的CPP的影响。结果:可卡因可以诱导小鼠形成CPP,并导致Rac1 GTPase活性增强及其下游信号分子cofilin磷酸化活性升高,并最终导致海马CA1区锥体神经元发生神经可塑性变化。结论:Rac1信号通路通过影响小鼠海马CA1区锥体神经元发生结构可塑性,从而参与调控可卡因情景线索记忆。     

The role of NMDA receptor binding sites in ethanol place conditioning

Little is known about the specific role of glutamate, in particular its actions at N-methyl-D-aspartate (NMDA) receptors, in ethanol reward. Pretreatment with channel blockers MK-801 and ketamine, NMDA NR2B receptor subunit antagonists ifenprodil and CP-101,606, and the glycine(B) partial agonist (+)-HA-966 did not alter acquisition of ethanol-induced conditioned place preference (CPP) in mice. However, pretreatment with the competitive antagonist CGP-37849 attenuated acquisition of ethanol-induced CPP. Follow-up experiments indicated that CGP-37849 also blocked acquisition of ethanol-induced and lithium chloride-induced conditioned place aversion but did not produce rewarding or aversive effects on its own. These results suggest that the NMDA receptor glutamate binding site is important for ethanol place conditioning. Moreover, these results suggest CGP-37849 modulates ethanol place conditioning by impairing the ability to learn these tasks.     

Conditioned increases in behavioral activity and accumbens dopamine levels produced by intravenous cocaine

In vivo microdialysis, behavioral activity assessments, and a conditioned place preference (CPP) test were used to investigate dopaminergic correlates of cocaine-conditioned behaviors. Over 12 days, rats were given either intravenous cocaine (4.2 mg/kg) or saline (6 cocaine and 6 saline infusions) daily in distinctively different environments. The following day, rats were tested in the cocaine- and saline-paired environments; 48 hr later, CPP was determined. The cocaine-associated environment elicited greater nucleus accumbens dopamine (NAcc DA) levels, hyperactivity, and place preference, though the emergence of DA increases was not in synchrony with peak behavioral activation. Although conditioned behavioral effects after repeated cocaine are well documented, direct evidence of increased NAcc DA in response to a cocaine-paired environment has not been previously reported. Discrepancies with previous work are attributed to a number of methodological differences.