Cannabinoid CB(1) receptor antagonist rimonabant attenuates reinstatement of ketamine conditioned place preference in rats

Recent evidence suggests that cannabinoid CB(1) receptors may represent effective targets for therapeutic agents used to treat cocaine and heroin relapse. However, the role of cannabinoid CB(1) receptors in the potential treatment for other drugs of abuse is still largely unknown. The present study was conducted to determine whether cannabinoid CB(1) receptors play a similar role in relapse to ketamine abuse. To establish a ketamine reinstatement model in the conditioned place preference paradigm, rats were trained to develop place preference conditioned by ketamine, which was subsequently extinguished through daily exposure to the test chambers in the absence of ketamine. On the day following the last extinction session, four groups of rats were injected with ketamine (1, 5, 10 and 15 mg/kg, i.p.) to reinstate previously extinguished conditioned place preference. To investigate the effects of rimonabant, a cannabinoid CB(1) receptor antagonist, on reinstatement of ketamine-induced place preference, different doses of rimonabant (0.1, 0.5 and 3 mg/kg, i.p) were injected 30 min prior to the ketamine (5 and 15 mg/kg, i.p.) priming injection in a separate group of rats. To determine whether rimonabant itself produces conditioned place preference or conditioned place aversion, rats were trained for conditioned place preference or place aversion with rimonabant (0, 0.1, 0.5, 3.0 mg/kg, i.p.). While ketamine priming injections reinstated extinguished place preference, rimonabant administration significantly attenuated the reinstatement of ketamine-induced place preference in a dose-dependent manner. Importantly, rimonabant itself did not produce conditioned place preference or place aversion. Since the reinstatement effects of ketamine administration were inhibited by rimonabant, these findings suggest that a cannabinoid CB(1) receptor antagonist may be useful in preventing relapse to ketamine abuse.     

Decreased basal endogenous opioid levels in diabetic rodents: effects on morphine and delta-9-tetrahydrocannabinoid-induced antinociception

We have previously demonstrated synergy between morphine and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) in the expression of antinociception in acute pain models and in arthritic models of chronic pain. Our data has been extended to include acute pain in both diabetic mice and rats. In diabetic mice, Δ⁹-THC p.o. was more active in the tail-flick test in the diabetic mouse than in the non-diabetic mouse. Morphine (s.c.) was less potent in diabetic than in non-diabetic mice [6.1 (5.1-7.2) versus 3.2 (2.4-4.1) mg/kg, respectively], an effect previously extensively documented in pre-clinical and clinical testing. In addition, the combination of Δ⁹-THC with morphine produced a greater-than-additive relief of acute pain in mice. In the rat, the induction of the diabetic state decreased the antinociceptive effect of morphine, an effect temporally related to a decreased release of specific endogenous opioids. Conversely, Δ⁹-THC retained the ability to release endogenous opioids in diabetic rats and maintained significant antinociception. Extrapolation of such studies to the clinical setting may indicate the potential for use of Δ⁹-THC-like drugs in the treatment of diabetic neuropathic pain, alone or in combination with very low doses of opioids.     

Discriminative stimulus effects of the cannabinoid CB<Subscript>1</Subscript> receptor antagonist rimonabant in rats

OBJECTIVE: To examine the discriminative stimulus effects of the cannabinoid CB(1) receptor (CB(1)R) antagonist/inverse agonist rimonabant (SR141716A) using a discriminated taste aversion (DTA) procedure. MATERIALS AND METHODS: Groups of rats were trained to discriminate between drug (5.6 or 3 mg/kg) and vehicle in DTA (t' = 20 min). The 30-min drinking opportunity after rimonabant pretreatment was followed by injection of lithium chloride (120 mg/kg) in the experimental (EXP) animals. When offered fluid after vehicle pretreatment, EXP animals subsequently were given intraperitoneal saline (NaCl, 10 ml/kg). Post-drinking treatment for controls (CONT) was NaCl irrespective of the pretreatment condition (rimonabant or vehicle). Tests examined other doses and drugs (t' = 20 min). RESULTS: The rimonabant analog AM251 (1 to 5.6 mg/kg) substituted for rimonabant. AM281 also appeared to substitute, but interpretation is complicated by unconditioned effects (drinking suppressed also in the CONT group). The CB(2)R antagonists SR144528 (18 and 30 mg/kg), AM630 (1 to 10 mg/kg), and the CB(1)R agonist methanandamide (mAEA, 3 and 10 mg/kg) did not substitute. There was a dose-related attenuation of the rimonabant-induced suppression of saccharin drinking when Delta9-tetrahydrocannabinol (Delta9-THC; 0.3 to 5.6 mg/kg), but not mAEA (1 to 10 mg/kg), was given together with rimonabant (3 mg/kg). Unconditioned effects occurred with the mAEA-rimonabant combination, not evident for combinations of rimonabant and Delta9-THC. mAEA (10 mg/kg) plus AM251 (5.6 mg/kg) resulted in strong unconditioned effects. CONCLUSION: Rimonabant induces a discriminative stimulus in DTA that continues to show potential for further examination of cannabinoid receptor antagonism.     

Clarifying CB2 receptor-dependent and independent effects of THC on human lung epithelial cells

Marijuana smoking is associated with a number of abnormal findings in the lungs of habitual smokers. Previous studies revealed that Δ⁹-tetrahydrocannabinol (THC) caused mitochondrial injury in primary lung epithelial cells and in the cell line, A549 [Sarafian, T. A., Kouyoumjian, S., Khoshaghideh, F., Tashkin, D. P., and Roth, M. D. (2003). Delta 9-tetrahydrocannabinol disrupts mitochondrial function and cell energetics. Am J Physiol Lung Cell Mol Physiol 284, L298-306; Sarafian, T., Habib, N., Mao, J. T., Tsu, I. H., Yamamoto, M. L., Hsu, E., Tashkin, D. P., and Roth, M. D. (2005). Gene expression changes in human small airway epithelial cells exposed to Delta9-tetrahydrocannabinol. Toxicol Lett 158, 95-107]. The role of cannabinoid receptors in this injury was unclear, as was the potential impact on cell function. In order to investigate these questions, A549 cells were engineered to over-express the type 2 cannabinoid receptor (CB2R) using a self-inactivating lentiviral vector. This transduction resulted in a 60-fold increase in CB2R mRNA relative to cells transduced with a control vector. Transduced cell lines were used to study the effects of THC on chemotactic activity and mitochondrial function. Chemotaxis in response to a 10% serum gradient was suppressed in a concentration-dependent manner by exposure to THC. CB2R-transduced cells exhibited less intrinsic chemotactic activity (p < 0.05) and were 80- to 100-fold more sensitive to the inhibitory effects of THC. Studies using SR144528, a selective CB2R antagonist, verified that these effects were mediated by the CB2R. Marijuana smoke extract, but not smoke extracts from tobacco or placebo marijuana cigarettes, reproduced these effects (p < 0.05). THC decreased ATP level and mitochondrial membrane potential (Ψ_m) in both control and CB2R-transduced cells. However, these decreases did not play a significant role in chemotaxis inhibition since cyclosporine A, which protected against ATP loss, did not increase cell migration. Moreover, CB2R-transduced cells displayed higher Ψ_m than did control cells. Since both Ψ_m and chemotaxis are regulated by intracellular signaling, we investigated the effects of THC on the activation of multiple signaling pathways. Serum exposure activated several signaling events of which phosphorylation of IκB-α and JNK was regulated in a CB2R- and THC-dependent manner. We conclude that airway epithelial cells are sensitive to both CB2R-dependent and independent effects mediated by THC.     

Additive subthreshold dose effects of cannabinoid CB(1) receptor antagonist and selective serotonin reuptake inhibitor in antidepressant behavioral tests

The main clinically used antidepressant drugs are selective monoamine reuptake inhibitors, including selective serotonin reuptake inhibitors (citalopram, sertraline), selective dopamine reuptake inhibitor (nomifensine) and selective noradrenaline reuptake inhibitor (reboxetine), but they have various side effects. Because cannabinoid CB(1) receptor antagonists (SR141716A, AM251) enhance monoamine release, they might be beneficial in the therapy of affective disorders. We hypothesized that the use of monoamine reuptake inhibitors in combination with cannabinoid CB(1) receptor antagonists would allow a lower dose of monoamine reuptake inhibitors to be used in the therapy of depression, thereby reducing or eliminating the side effects. To test this hypothesis, we examined the combination of SR141716A or AM251 with citalopram, sertraline, nomifensine or reboxetine at subthreshold doses to see whether these combinations would show an additive effect in the forced swimming test and the tail suspension test with mice. Subthreshold doses of cannabinoid CB(1) receptor antagonist and selective serotonin reuptake inhibitors, which separately had no effect on the immobility of mice in the tests, showed a clear effect when the drugs were administered at 40 and 30 min, respectively, before the tests, without any change of motor activity. Therefore, the use of subthreshold doses of these agents in combination might be useful to enhance mainly serotonergic neurotransmission, and to reduce or eliminate the side effects of citalopram and sertraline.     

Novel pharmacological targets based on receptor heteromers

Studies performed in the last 10 years have provided solid evidence indicating that G-protein-coupled receptors are expressed on the plasma membrane as homo and heterodimers. The first consequence of this fact is that homo and heterodimers are the true targets of natural (hormones, neurotransmitters) and synthetic drugs. Furthermore a given receptor in a heteromer may display a different functional and/or pharmacological profile than the same receptor characterized as monomer or as homodimer. Recent evidence indicates that receptor heteromers are sensors that lead to a fine-tuning in neurotransmission or hormone regulation; mainly this is achieved by a modification of the signaling pathways activated via a given receptor when it is forming a given heteromer. Quite often antagonists display variable affinities when a given receptor is expressed with different heteromeric partners. This fact should be taken into account in the development of new drugs. Finally it should be pointed out that radioligand binding data has to be analyzed by a model that considers receptors as dimers and not as monomers. This model provides a novel approach to characterize drugs interacting with the orthosteric center (agonists/antagonists) or with allosteric centers (allosteric regulators).     

Adolescent cannabinoid exposure attenuates adult female sexual motivation but does not alter adulthood CB1R expression or estrous cyclicity

Adolescence is a developmental period characterized by neuronal remodeling and the maturation of adult emotionality, reproductive behavior and social behavior. We examined whether chronic cannabinoid exposure in adolescent rats alters female sexual motivation, estrous cyclicity, sucrose preference, and CB₁R expression in adulthood. Female rats were administered with the synthetic cannabinoid agonist, CP-55,940 (0.4 mg/kg, intraperitoneal), daily during adolescent development (PND 35-45). In a subset of subjects, socio-sexual motivation was investigated in adulthood (PND 75-86) using a runway apparatus. Estrous cyclicity was tracked in adulthood via vaginal cytology and a single-mount test. A two-bottle sucrose preference test was also conducted to determine whether predicted changes in socio-sexual motivation might be linked to alterations in hedonic processing. CB₁R expression was examined in two separate subsets of subjects, one sacrificed following drug treatment (PND 46) and one before behavioral testing (PND 74). Drug treatment significantly decreased adult preference for a male conspecific (sexual motivation), as assessed by both Run Time and Proximity Time, but did not affect estrous cyclicity or sucrose preference. CP-55,940 treatment also induced immediate, but transient, decreases in CB₁R expression in the ventromedial nucleus of the hypothalamus and amygdala. Drug treatment did not affect CB₁R expression in the nucleus accumbens (core or shell) or globus pallidus at either time point. We suggest that the endocannabinoid system may play a role in the maturation of neuroendocrine axes and adult female reproductive behavior, and that chronic exposure to cannabinoids during adolescence disrupts these neurodevelopmental processes.     

Role of GLT-1 transporter activation in prevention of cannabinoid tolerance by the β-lactam antibiotic, ceftriaxone, in mice

Recently, it has been indicated that beta lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Furthermore, these antibiotics have been shown to prevent the development of tolerance and dependence to opioids. Since cannabinoid tolerance is known to be similar to opioids, our purpose was to examine the effect of ceftriaxone on the development of tolerance to WIN 55,212-2, a cannabinoid agonist. The tail flick test, a rectal thermometer, and the ring test were used for evaluating the degree of tolerance to the analgesic, hypothermic, and cataleptic effects of WIN 55,212-2, respectively. Within one week, animals became completely tolerant to analgesic, hypothermic and cataleptic effects of WIN 55,212-2 (6 mg/kg). Ceftriaxone, with its higher doses (100-200 mg/kg), attenuated the development of tolerance to the analgesic and hypothermic effects of WIN 55,212-2, but had no effect on its cataleptic action. Dihydrokainic acid (10 mg/kg), a GLT-1 transporter inhibitor, prevented this effect of ceftriaxone. Our results suggest that repeated treatment with ceftriaxone prevents the development of tolerance to the analgesic and hypothermic effects of cannabinoids, and GLT-1 activation appears to play a key role in this preventive effect of beta-lactam antibiotics.     

Prolonged cannabinoid exposure alters GABA(A) receptor mediated synaptic function in cultured hippocampal neurons

Developing cannabinoid-based medication along with marijuana's recreational use makes it important to investigate molecular adaptations the endocannabinoid system undergoes following prolonged use and withdrawal. Repeated cannabinoid administration results in development of tolerance and produces withdrawal symptoms that may include seizures. Here we employed electrophysiological and immunochemical techniques to investigate the effects of prolonged CB1 receptor agonist exposure on cultured hippocampal neurons. Approximately 60% of CB1 receptors colocalize to GABAergic terminals in hippocampal cultures. Prolonged treatment with the cannabinamimetic WIN 55,212-2 (+ WIN, 1 μM, 24 h) caused profound CB1 receptor downregulation accompanied by neuronal hyperexcitability. Furthermore, prolonged + WIN treatment resulted in increased GABA release as indicated by increased mIPSC frequency, a diminished GABAergic inhibition as indicated by reduction in mIPSC amplitude and a reduction in GABA_A channel number. Additionally, surface staining for the GABA_A β_2/3 receptor subunits was decreased, while no changes in staining for the presynaptic vesicular GABA transporter were observed, indicating that GABAergic terminals remained intact. These findings demonstrate that agonist-induced downregulation of the CB1 receptor in hippocampal cultures results in neuronal hyperexcitability that may be attributed, in part, to alterations in both presynaptic GABA release mechanisms and postsynaptic GABA_A receptor function demonstrating a novel role for cannabinoid-dependent presynaptic control of neuronal transmission.     

Chronic treatment and withdrawal of the cannabinoid agonist WIN 55,212-2 modulate the sensitivity of presynaptic receptors involved in the regulation of monoamine syntheses in rat brain

Brain monoamines are involved in many neurochemical and behavioral effects of cannabinoids, but little is known on the regulation of noradrenaline, dopamine, and serotonin (5-HT) synthesis in cannabinoid addiction. This study investigated in rat brain the chronic effects of the potent cannabinoid agonist WIN 55,212-2 and of rimonabant-precipitated withdrawal, as well as the sensitivity of synthesis-modulating inhibitory receptors, on the accumulation of l-3,4-dihydroxyphenylalanine (DOPA) and 5-HTP after decarboxylase inhibition. Acute WIN (8 mg/kg; 1 h) increased DOPA synthesis in cortex (52%), hippocampus (51%), and cerebellum (56%) and decreased DOPA accumulation in striatum (31%). Acute WIN also decreased the synthesis of 5-HTP in all brain regions (40–53%). Chronic WIN (2–8 mg/kg; 5 days) and/or antagonist-precipitated withdrawal induced tolerance to the acute effects of WIN on the accumulation of DOPA (cortex and striatum) and 5-HTP (all brain regions). The inhibitory effect of clonidine (α₂-agonist; 1 mg/kg) on the accumulation of DOPA (15–41%) and 5-HTP (22–41%) was markedly decreased or abolished after chronic WIN and precipitated withdrawal, mainly in noradrenergic and serotonergic brain regions, which indicated desensitization of α₂-autoreceptors and α₂-heteroreceptors regulating the synthesis of noradrenaline and 5-HT. In WIN-dependent rats (chronic and withdrawal states), the effect of a low dose of (±)-8-hydroxy-2-(di-n-propylamino)-tetralin (5-HT_1A agonist; 0.1 mg/kg) on the accumulation of precursor amino acids was markedly potentiated in cerebellum and striatum, indicating the induction of supersensitivity of 5-HT_1A-autoreceptors and 5-HT_1A-heteroreceptors that regulate the synthesis of 5-HT, noradrenaline, and dopamine in these brain regions. These chronic adaptations in presynaptic receptor function could play a relevant role in cannabinoid addiction. A preliminary report of this work was presented at the XII Congress of the Spanish Society of Neuroscience, Valencia, Spain, September 5–9, 2007 (Esteban et al. 2007).