Miquel Martin

Involvement of CB1 cannabinoid receptors in emotional behaviour.

Abstract.Rationale: Endogenous and exogenous cannabinoids acting through the CB1 cannabinoid receptors are implicated in the control of a variety of behavioural and neuroendocrine functions, including emotional responses, and learning and memory processes. Recently, knockout mice deficient in the CB1 cannabinoid receptor have been generated, and these animals result in an excellent tool to evaluate the neurophysiology of the endogenous cannabinoid system. Objectives: To establish the role of the CB1 cannabinoid receptor in several emotional-related behavioural responses, including aggressiveness, anxiety, depression and learning models, using CB1 knockout mice. Methods: We evaluated the spontaneous responses of CB1 knockout mice and wild-type controls under different behavioural paradigms, including the light/dark box, the chronic unpredictable mild stress, the resident–intruder test and the active avoidance paradigm. Results: Our findings showed that CB1 knockout mice presented an increase in the aggressive response measured in the resident–intruder test and an anxiogenic-like response in the light/dark box. Furthermore, a higher sensitivity to exhibit depressive-like responses in the chronic unpredictable mild stress procedure was observed in CB1 knockout mice, suggesting an increased susceptibility to develop an anhedonic state in these animals. Finally, CB1 knockout mice showed a significant increase in the conditioned responses produced in the active avoidance model, suggesting an improvement of learning and memory processes. Conclusions: Taken together these findings demonstrate that endogenous cannabinoids through the activation of CB1 receptors are implicated in the control of emotional behaviour and participate in the physiological processes of learning and memory.

Cocaine, but not morphine, induces conditioned place preference and sensitization to locomotor responses in CB1 knockout mice

The involvement of cannabinoid CB1 receptors in morphine and cocaine motivational effects was investigated using CB1 knockout mice. For this purpose, we evaluated the rewarding effects in the place conditioning paradigm and the sensitization to the locomotor responses induced by these drugs. The hyperlocomotion induced by acute morphine administration (15 mg/kg, s.c.) was preserved, but the sensitization to this locomotor response induced by chronic morphine treatment was abolished in CB1 mutant mice. Morphine (5 mg/kg, s.c.) induced conditioned place preference in wild‐type mice but failed to produce any response in knockout mice, indicating the inability of morphine to induce rewarding effects in the absence of CB1 cannabinoid receptors. When the aversive effects of morphine withdrawal were investigated using the place aversion paradigm, no differences between genotypes were observed. Acute cocaine (10 mg/kg, i.p.) induced hyperlocomotor responses in wild‐type and knockout mice and a chronic cocaine treatment produced a similar sensitization to this response in both genotypes. In the conditioning place preference paradigm, cocaine (20 mg/kg, i.p.) produced rewarding responses in both wild‐type and knockout mice. These results demonstrate that CB1 receptors are essential for adaptive responses produced by chronic morphine but not by chronic cocaine treatment.

Crucial Role of CB2 Cannabinoid Receptor in the Regulation of Central Immune Responses during Neuropathic Pain

Neuropathic pain is a clinical manifestation of nerve injury difficult to treat even with potent analgesic compounds. Here, we used different lines of genetically modified mice to clarify the role played by CB2 cannabinoid receptors in the regulation of the central immune responses leading to the development of neuropathic pain. CB2 knock-out mice and wild-type littermates were exposed to sciatic nerve injury, and both genotypes developed a similar hyperalgesia and allodynia in the ipsilateral paw. Most strikingly, knock-outs also developed a contralateral mirror image pain, associated with an enhanced microglial and astrocytic expression in the contralateral spinal horn. In agreement, hyperalgesia, allodynia, and microglial and astrocytic activation induced by sciatic nerve injury were attenuated in transgenic mice overexpressing CB2 receptors. These results demonstrate the crucial role of CB2 cannabinoid receptor in modulating glial activation in response to nerve injury. The enhanced manifestations of neuropathic pain were replicated in irradiated wild-type mice reconstituted with bone marrow cells from CB2 knock-outs, thus demonstrating the implication of the CB2 receptor expressed in hematopoietic cells in the development of neuropathic pain at the spinal cord.

Interferon-γ Is a Critical Modulator of CB2 Cannabinoid Receptor Signaling during Neuropathic Pain

Nerve injuries often lead to neuropathic pain syndrome. The mechanisms contributing to this syndrome involve local inflammatory responses, activation of glia cells, and changes in the plasticity of neuronal nociceptive pathways. Cannabinoid CB2 receptors contribute to the local containment of neuropathic pain by modulating glial activation in response to nerve injury. Thus, neuropathic pain spreads in mice lacking CB2 receptors beyond the site of nerve injury. To further investigate the mechanisms leading to the enhanced manifestation of neuropathic pain, we have established expression profiles of spinal cord tissues from wild-type and CB2-deficient mice after nerve injury. An enhanced interferon-γ (IFN-γ) response was revealed in the absence of CB2 signaling. Immunofluorescence stainings demonstrated an IFN-γ production by astrocytes and neurons ispilateral to the nerve injury in wild-type animals. In contrast, CB2-deficient mice showed neuronal and astrocytic IFN-γ immunoreactivity also in the contralateral region, thus matching the pattern of nociceptive hypersensitivity in these animals. Experiments in BV-2 microglia cells revealed that transcriptional changes induced by IFN-γ in two key elements for neuropathic pain development, iNOS (inducible nitric oxide synthase) and CCR2, are modulated by CB2 receptor signaling. The most direct support for a functional involvement of IFN-γ as a mediator of CB2 signaling was obtained with a double knock-out mouse strain deficient in CB2 receptors and IFN-γ. These animals no longer show the enhanced manifestations of neuropathic pain observed in CB2 knock-outs. These data clearly demonstrate that the CB2 receptor-mediated control of neuropathic pain is IFN-γ dependent.

Acute antinociceptive responses in single and combinatorial opioid receptor knockout mice: distinct mu, delta and kappa tones.

We have examined responses of mice lacking mu, delta and kappa opioid receptor (MOR, DOR and KOR, respectively) genes, as well as combinatorial mutants, in several pain models. This is the first truly comparative study of all three opioid receptor‐deficient mice, with genotypes and gender analysis using mice on the hybrid 50% 129/SV : 50% C57BL/6 genetic background. In the tail‐immersion test, only KOR−/− females showed decreased withdrawal latencies. This modification was also found in MOR/KOR and MOR/DOR/KOR, but not MOR/DOR mutants. The hotplate test revealed increased nociceptive sensitivity for MOR−/−, a phenotype which was also observed in double mutants involving the MOR deletion, and in the triple mutants. The tail‐pressure test showed increased response for both MOR−/− and DOR−/− mutants, a modification which was enhanced in the triple‐mutant mice. In the formalin test, MOR−/− and DOR−/− mice showed increased responses in the early and late phases, respectively, while the triple mutant tended to show enhanced nociception in both phases. Finally, the enhanced response of KOR−/− mice in the writhing test, which we have demonstrated previously, was confirmed in double MOR/KOR‐ and triple‐mutant mice. Together, the data support the existence of an antinociceptive opioid tone. Each receptor presents a distinct pattern of activities, with mu receptors influencing responses to mechanical, chemical and thermal nociception at a supraspinal level, kappa receptors involved in spinally mediated thermal nociception and chemical visceral pain, and delta receptors modulating mechanical nociception and inflammatory pain. Phenotypes of mutant mice were subtle, suggesting a low endogenous opioid tone in the regulation of physiological pain.

CB1 knockout mice display impaired functionality of 5‐HT1A and 5‐HT2A/C receptors

Interaction between brain endocannabinoid (EC) and serotonin (5‐HT) systems was investigated by examining 5‐HT‐dependent behavioral and biochemical responses in CB1 receptor knockout mice. CB1 knockout animals exhibited a significant reduction in the induction of head twitches and paw tremor by the 5‐HT2A/C receptor selective agonist (±) DOI, as well as a reduced hypothermic response following administration of the 5‐HT1A receptor agonist (±)‐8‐OH‐DPAT. Additionally, exposure to the tail suspension test induced enhanced despair responses in CB1 knockout mice. However, the tricyclic antidepressant imipramine and the 5‐HT selective reuptake inhibitor fluoxetine induced similar decreases in the time of immobility in the tail suspension test in CB1 receptor knockout and wild‐type mice. No differences were found between both genotypes with regard to 5‐HT2A receptor and 5‐HT1A receptors levels, measured by autoradiography in different brain areas. However, a significant decrease in the ability of both, the 5‐HT1A receptor agonist (±)‐8‐OH‐DPAT and the 5‐HT2A/C receptor agonist (−)DOI, to stimulate [35S]GTPγS binding was detected in the hippocampal CA1 area and fronto‐parietal cortex of CB1 receptor knockout mice, respectively. This study provides evidence that CB1 receptors are involved in the regulation of serotonergic responses mediated by 5‐HT2A/C and 5‐HT1A receptors, and suggests that a reduced coupling of 5‐HT1A and 5‐HT2A receptors to G proteins might be involved in these effects.

Development and expression of neuropathic pain in CB1 knockout mice.

Neuropathic pain is a clinical manifestation characterized by the presence of spontaneous pain, allodynia and hyperalgesia. Here, we have evaluated the involvement of CB1 cannabinoid receptors in the development and expression of neuropathic pain. For this purpose, partial ligation of the sciatic nerve was performed in CB1 cannabinoid receptor knockout mice and their wild-type littermates. The development of mechanical and thermal allodynia, and thermal hyperalgesia was evaluated by using the von Frey filaments, cold-plate and plantar tests, respectively. Pre-surgical tactile and thermal withdrawal thresholds were similar in both genotypes. In wild-type mice, sciatic nerve injury led to a neuropathic pain syndrome characterized by a marked and long-lasting reduction of the paw withdrawal thresholds to mechanical and thermal stimuli. These manifestations developed similarly in mice lacking CB1 cannabinoid receptors. We have also investigated the consequences of gabapentin administration in these animals. Gabapentin (50 mg/kg/day, i.p.) induced a similar suppression of mechanical and thermal allodynia in both wild-type and CB1 knockout mice. Mild differences between genotypes were observed concerning the effect of gabapentin in the expression of thermal hyperalgesia. Taken together, our results indicate that CB1 cannabinoid receptors are not critically implicated in the development of neuropathic pain nor in the anti-allodynic and anti-hyperalgesic effects of gabapentin in this model.

Neurofilament proteins and cAMP pathway in brains of μ-, δ- or κ-opioid receptor gene knock-out mice: effects of chronic morphine administration

Abstract Opiate addiction is associated with abnormalities of neurofilament (NF) proteins and upregulation of cAMP signaling in the brain, which may modulate neuronal plasticity. This study investigated, using gene-targeted mice lacking μ -, δ - or κ -opioid receptors, the role of these receptors in modulating the basal activity and the chronic effects of morphine on both intracellular targets. In WT mice, chronic treatment (5 days) with morphine (20–100 mg/kg) resulted in decreases in the immunodensity of neurofilament (NF)-L in the cerebral cortex (14–23%). In contrast, chronic morphine did not decrease NF-L in cortices of μ -, δ -, and κ -KO mice, suggesting the involvement of the three types of opioid receptors in this effect of morphine. Also, the marked increase in phosphorylated NF-H induced by chronic morphine in WT mice (two-fold) was abolished in μ -KO mice. In cortex and/or striatum of μ -, δ - and κ -KO mice, the basal immunodensities of G α i1/2 proteins, the catalytic isoform (C α ) of protein kinase A (PKA) and the total content of cAMP response element-binding protein (CREB, the nuclear target of PKA) were not different from those of WT mice. In contrast, phosphorylated CREB (the active form of this transcription factor) was reduced in cortex and/or striatum (23–26%) of μ - and δ -KO mice, but not in κ -KO animals. These results suggest that the endogenous opioid tone acting on μ -/ δ -receptors tonically stimulate CREB activation in the brain. In cortex and/or striatum of WT mice, chronic morphine did not induce upregulation of the main components of the cAMP signaling pathway. In contrast, chronic morphine treatment in μ -KO mice, but not in δ - or κ -KO, resulted in a paradoxical upregulation of G α i1/2 (12–19%), PKA (19–21%,) and phosphorylated CREB (21–73%), but not total CREB, in cortex and/or striatum. The induction of heterologous receptor adaptations in μ -KO mice may explain this paradoxical effect of morphine.

Operant behavior to obtain palatable food modifies neuronal plasticity in the brain reward circuit

Palatability enhances food intake by hedonic mechanisms that prevail over caloric necessities. Different studies have demonstrated the role of endogenous cannabinoids in the mesocorticolimbic system in controlling food hedonic value and consumption. We hypothesize that the endogenous cannabinoid system could also be involved in the development of food-induced behavioral alterations, such as food-seeking and binge-eating, by a mechanism that requires neuroplastic changes in the brain reward pathway. For this purpose, we evaluated the role of the CB1 cannabinoid receptor (CB1-R) in the behavioral and neuroplastic changes induced by operant training for standard, highly caloric or highly palatable isocaloric food using different genetics, viral and pharmacological approaches. Neuroplasticity was evaluated by measuring changes in dendritic spine density in neurons previously labeled with the dye DiI. Only operant training to obtain highly palatable isocaloric food induced neuroplastic changes in neurons of the nucleus accumbens shell and prefrontal cortex that were associated to changes in food-seeking behavior. These behavioral and neuroplastic modifications induced by highly palatable isocaloric food were dependent on the activity of the CB1-R. Neuroplastic changes induced by highly palatable isocaloric food are similar to those produced by some drugs of abuse and may be crucial in the alteration of food-seeking behavior leading to overweight and obesity.

Central and peripheral consequences of the chronic blockade of CB1 cannabinoid receptor with rimonabant or taranabant.

J. Neurochem. (2010) 112, 1338–1351.