Peripheral CB1 receptor blockade acts as a memory enhancer through a noradrenergic mechanism.

Peripheral inputs continuously shape brain function and can influence memory acquisition, but the underlying mechanisms have not been fully understood. Cannabinoid type-1 receptor (CB1R) is a well-recognized player in memory performance, and its systemic modulation significantly influences memory function. By assessing low arousal/non-emotional recognition memory in mice, we found a relevant role of peripheral CB1R in memory persistence. Indeed, the peripherally-restricted CB1R specific antagonist AM6545 showed significant mnemonic effects that were occluded in adrenalectomized mice, and after peripheral adrenergic blockade. AM6545 also transiently impaired contextual fear memory extinction. Vagus nerve chemogenetic inhibition reduced AM6545-induced mnemonic effect. Genetic CB1R deletion in dopamine ß-hydroxylase-expressing cells enhanced recognition memory persistence. These observations support a role of peripheral CB1R modulating adrenergic tone relevant for cognition. Furthermore, AM6545 acutely improved brain connectivity and enhanced extracellular hippocampal norepinephrine. In agreement, intra-hippocampal ß-adrenergic blockade prevented AM6545 mnemonic effects. Altogether, we disclose a novel CB1R-dependent peripheral mechanism with implications relevant for lengthening the duration of non-emotional memory.

Orally Active Peptide Vector Allows Using Cannabis to Fight Pain While Avoiding Side Effects.

The activation of cannabinoid CB1 receptors (CB1R) by Δ9-tetrahydrocannabinol (THC), the main component of Cannabis sativa, induces analgesia. CB1R activation, however, also causes cognitive impairment via the serotonin 5HT2A receptor (5HT2AR), a component of a CB1R-5HT2AR heteromer, posing a serious drawback for cannabinoid therapeutic use. We have shown that peptides reproducing CB1R transmembrane (TM) helices 5 and 6, fused to a cell-penetrating sequence (CPP), can alter the structure of the CB1R-5HT2AR heteromer and avert THC cognitive impairment while preserving analgesia. Here, we report the optimization of these prototypes into drug-like leads by (i) shortening the TM5, TM6, and CPP sequences, without losing the ability to disturb the CB1R-5HT2AR heteromer, and (ii) extensive sequence remodeling to achieve protease resistance and blood-brain barrier penetration. Our efforts have culminated in the identification of an ideal candidate for cannabis-based pain management, an orally active 16-residue peptide preserving THC-induced analgesia.

Peripheral and central CB1 cannabinoid receptors control stress-induced impairment of memory consolidation.

Stressful events can generate emotional memories linked to the traumatic incident, but they also can impair the formation of nonemotional memories. Although the impact of stress on emotional memories is well studied, much less is known about the influence of the emotional state on the formation of nonemotional memories. We used the novel object-recognition task as a model of nonemotional memory in mice to investigate the underlying mechanism of the deleterious effect of stress on memory consolidation. Systemic, hippocampal, and peripheral blockade of cannabinoid type-1 (CB1) receptors abolished the stress-induced memory impairment. Genetic deletion and rescue of CB1 receptors in specific cell types revealed that the CB1 receptor population specifically in dopamine ß-hydroxylase (DBH)-expressing cells is both necessary and sufficient for stress-induced impairment of memory consolidation, but CB1 receptors present in other neuronal populations are not involved. Strikingly, pharmacological manipulations in mice expressing CB1 receptors exclusively in DBH(+) cells revealed that both hippocampal and peripheral receptors mediate the impact of stress on memory consolidation. Thus, CB1 receptors on adrenergic and noradrenergic cells provide previously unrecognized cross-talk between central and peripheral mechanisms in the stress-dependent regulation of nonemotional memory consolidation, suggesting new potential avenues for the treatment of cognitive aspects on stress-related disorders.

Differential Pharmacological Regulation of Sensorimotor Gating Deficit in CB1 Knockout Mice and Associated Neurochemical and Histological Alterations.

The endocannabinoid system has been widely involved in the pathophysiology of sensorimotor gating deficits. This study aimed to evaluate the pharmacological modulation of the sensorimotor gating impairment induced by cannabinoid CB1 receptor (CB1r) deletion. For this purpose, the prepulse inhibition (PPI) paradigm was used to evaluate the effect of two antipsychotics drugs (risperidone and haloperidol) and a psychostimulant (methylphenidate) on the preattentional deficit presented by CB1KO mice. Furthermore, the effects of the CB1r antagonist AM251 on PPI were evaluated in WT mice. Real-time PCR and immunohistochemical studies were carried out to analyze dopamine transporter (DAT) and α-2C adrenergic receptor (ADRA2C) gene expressions and the distribution of parvalbumin (PV) and cholecystokinin-8 (CCK) immunoreactive (ir) cortical neurons, respectively. Neither risperidone nor haloperidol significantly modified the PPI of WT and CB1KO mice, whereas methylphenidate improved the preattentional deficit of CB1KO mice. In addition, treatment with AM251 (3 mg/kg; i.p.) significantly decreased the PPI of WT animals. The administration of methylphenidate increased DAT and ADRA2C gene expressions in CB1KO mice without producing any effect in WT animals. Immunohistochemical studies revealed that there were no significant changes in CCK immunolabeling between WT and CB1KO mice, whereas the radial distribution of PV-ir neurons was abnormal in CB1KO mice. These data further support the important role of CB1r in sensorimotor gating regulation and the therapeutic usefulness of methylphenidate for the treatment of psychiatric disorders with associated preattentional deficits.

Role of cannabinoid CB2 receptor in the reinforcing actions of ethanol.

This study examines the role of the cannabinoid CB2 receptor (CB2 r) on the vulnerability to ethanol consumption. The time-related and dose-response effects of ethanol on rectal temperature, handling-induced convulsions (HIC) and blood ethanol concentrations were evaluated in CB2 KO and wild-type (WT) mice. The reinforcing properties of ethanol were evaluated in conditioned place preference (CPP), preference and voluntary ethanol consumption and oral ethanol self-administration. Water-maintained behavior schedule was performed to evaluate the degree of motivation induced by a natural stimulus. Preference for non-alcohol tastants assay was performed to evaluate the differences in taste sensitivity. Tyrosine hydroxylase (TH) and µ-opioid receptor gene expressions were also measured in the ventral tegmental area and nucleus accumbens (NAcc), respectively. CB2 KO mice presented increased HIC score, ethanol-CPP, voluntary ethanol consumption and preference, acquisition of ethanol self-administration, and increased motivation to drink ethanol compared with WT mice. No differences were found between genotypes in the water-maintained behavior schedule or preference for non-alcohol tastants. Naïve CB2 KO mice presented increased µ-opioid receptor gene expression in NAcc. Acute ethanol administration (1-2 g/kg) increased TH and µ-opioid receptor gene expressions in CB2 KO mice, whereas the lower dose of ethanol decreased TH gene expression in WT mice. These results suggest that deletion of the CB2 r gene increased preference for and vulnerability to ethanol consumption, at least in part, by increased ethanol-induced sensitivity of the TH and µ-opioid receptor gene expressions in mesolimbic neurons. Future studies will determine the role of CB2 r as a target for the treatment of problems related with alcohol consumption.

Synaptic plasticity alterations associated with memory impairment induced by deletion of CB2 cannabinoid receptors.

In this study, the role of CB2r on aversive memory consolidation was further evaluated. Mice lacking CB2r (CB2KO) and their corresponding littermates (WT) were exposed to the step-down inhibitory avoidance test (SDIA). MAP2, NF200 and synaptophysin (SYN)-immunoreactive fibers were studied in the hippocampus (HIP) of both genotypes. The number of synapses, postsynaptic density thickness and the relation between the synaptic length across the synaptic cleft and the distance between the synaptic ends were evaluated in the HIP (dentate gyrus (DG) and CA1 fields) by electron microscopy. Brain-derived neurotrophic factor (BDNF), glucocorticoid receptor (NR3C1) gene expressions and mTOR/p70S6K signaling cascade were evaluated in the HIP and prefrontal cortex (PFC). Finally, the effects of acute administration of CB2r-agonist JWH133 or CB2r-antagonist AM630 on memory consolidation were evaluated in WT mice by using the SDIA. The lack of CB2r impaired aversive memory consolidation, reduced MAP2, NF200 and SYN-immunoreactive fibers and also reduced the number of synapses in DG of CB2KO mice. BDNF and NR3C1 gene expression were reduced in the HIP of CB2KO mice. An increase of p-p70S6K (T389 and S424) and p-AKT protein expression was observed in the HIP and PFC of CB2KO mice. Interestingly, administration of AM630 impaired aversive memory consolidation, whereas JWH133 enhanced it. Further functional and molecular assessments would have been helpful to further support our conclusions. These results revealed that CB2r are involved in memory consolidation, suggesting that this receptor could be a promising target for developing novel treatments for different cognitive impairment-related disorders.

Decreased cocaine motor sensitization and self-administration in mice overexpressing cannabinoid CB2 receptors.

The potential involvement of the cannabinoid CB2 receptors (CB2r) in the adaptive responses induced by cocaine was studied in transgenic mice overexpressing the CB2r (CB2xP) and in wild-type (WT) littermates. For this purpose, the acute and sensitized locomotor responses to cocaine, conditioned place preference, and cocaine intravenous self-administration were evaluated. In addition, we assessed whether CB2r were localized in neurons and/or astrocytes, and whether they colocalized with dopamine D1 and D2 receptors (D1Dr and D2Dr). Dopamine (DA) extracellular levels in the nucleus accumbens (NAcc), and gene expression of tyrosine hydroxylase (TH) and DA transporter (DAT) in the ventral tegmental area (VTA), and µ-opioid and cannabinoid CB1 receptors in the NAcc were also studied in both genotypes. CB2xP mice showed decreased motor response to acute administration of cocaine (10-20 mg/kg) and cocaine-induced motor sensitization compared with WT mice. CB2xP mice presented cocaine-induced conditioned place aversion and self-administered less cocaine than WT mice. CB2r were found in neurons and astrocytes and colocalized with D2Dr in the VTA and NAcc. No significant differences in extracellular DA levels in the NAcc were observed between genotypes after cocaine administration. Under baseline conditions, TH and DAT gene expression was higher and µ-opioid receptor gene expression was lower in CB2xP than in WT mice. However, both genotypes showed similar changes in TH and µ-opioid receptor gene expression after cocaine challenge independently of the pretreatment received. Importantly, the cocaine challenge decreased DAT gene expression to a lesser extent in cocaine-pretreated CB2xP than in cocaine-pretreated WT mice. These results revealed that CB2r are involved in cocaine motor responses and cocaine self-administration, suggesting that this receptor could represent a promising target to develop novel treatments for cocaine addiction.

Overexpression of CB2 cannabinoid receptors results in neuroprotection against behavioral and neurochemical alterations induced by intracaudate administration of 6-hydroxydopamine.

The role of CB2 cannabinoid receptors in the behavioral and neurochemical changes induced by intracaudate administration of 6-hydroxydopamine (6-OHDA) was evaluated. 6-OHDA (12 µg/4 µL) or its vehicle was injected in the caudate-putamen (CPu) of mice overexpressing the CB2 cannabinoid receptor (CB2xP) and wild type (WT) mice. Motor impairment, emotional behavior, and cognitive alterations were evaluated. Tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule 1 (Iba-1) were measured by immunocytochemistry in the CPu and/or substantia nigra (SN) of CB2xP mice and WT mice. Oxidative/nitrosative and neuroinflammatory parameters were also measured in the CPu and cortex of 6-OHDA-treated and sham-treated mice. 6-OHDA-treated CB2xP mice presented significantly less motor deterioration than 6-OHDA-treated WT mice. Immunocytochemical analysis of tyrosine hydroxylase in the SN and CPu revealed significantly fewer lesions in CB2xP mice than in WT mice. GFAP and Iba-1 immunostaining revealed less astrocyte and microglia recruitment to the treated area of the CPu in CB2xP mice. Malonyldialdehyde (MDA) concentrations were lower in the striatum and cerebral cortex of sham-treated CB2xP mice than in sham-treated WT mice. The administration of 6-OHDA increased MDA levels in both WT mice and CB2xP mice; it increased the oxidized (GSSG)/reduced (GSH) glutathione ratio in the striatum in WT mice alone compared with matched sham-treated controls. The results revealed that overexpression of CB2 cannabinoid receptors decreased the extent of motor impairment and dopaminergic neuronal loss, reduced the recruitment of astrocytes and microglia to the lesion, and decreased the level of various oxidative parameters. These results suggest that CB2 receptors offer neuroprotection against dopaminergic injury.

Comparison of the antinociceptive effects of ibuprofen arginate and ibuprofen in rat models of inflammatory and neuropathic pain.

AIMS: Ibuprofen arginate is a highly soluble salt formed by combining racemic ibuprofen with the amino acid l-arginine. This formulation is absorbed faster, and it is safe and effective in treating many forms of mild to moderate pain. We compared the analgesic effect of ibuprofen arginate and conventional ibuprofen in rat models of pain. MAIN METHODS: Mechanical and cold allodynia were assessed in the chronic constriction injury (CCI) model of neuropathic pain, and mechanical allodynia was also examined in capsaicin-injected rats (a model of central sensitization). Inflammatory hypersensitivity was assessed with the formalin test. Ibuprofen-l-arginine, ibuprofen, l-arginine or saline was administered orally on a daily basis after CCI or capsaicin injection, and the von Frey and cold plate tests were performed on days 1, 3 and 7 after CCI or capsaicin administration. In the formalin-induced inflammatory pain test, the drugs were administered 30 min before formalin injection. KEY FINDINGS: Ibuprofen only exerts an antinociceptive effect in the formalin model whereas ibuprofen-l-arginine exerts antinociceptive effects on both mechanical and cold allodynia induced by CCI, mechanical allodynia induced by capsaicin injection, and in phase 2 of the formalin test, exhibiting superior antinociceptive activity to ibuprofen in all these tests. l-Arginine only exerted antinociceptive effects on cold allodynia in CCI. SIGNIFICANCE: These results demonstrate that ibuprofen arginate has stronger antinociceptive effects than ibuprofen in all the models used, suggesting it might improve the therapeutic management of neuropathic and inflammatory pain.

Deletion of CB2 cannabinoid receptor induces schizophrenia-related behaviors in mice.

The possible role of the CB(2) receptor (CB(2)r) in psychiatric disorders has been considered. Several animal models use knockout (KO) mice that display schizophrenia-like behaviors and this study evaluated the role of CB(2)r in the regulation of such behaviors. Mice lacking the CB(2)r (CB(2)KO) were challenged in open field, light-dark box, elevated plus-maze, tail suspension, step down inhibitory avoidance, and pre-pulse inhibition tests (PPI). Furthermore, the effects of treatment with cocaine and risperidone were evaluated using the OF and the PPI test. Gene expression of dopamine D(2) (D(2)r), adrenergic-α(2C) (α(2C)r), serotonergic 5-HT(2A) and 5-HT(2C) receptors (5-HT(2A)r and 5-HT(2C)r) were studied by RT-PCR in brain regions related to schizophrenia. Deletion of CB(2)r decreased motor activity in the OF test, but enhanced response to acute cocaine and produced mood-related alterations, PPI deficit, and cognitive impairment. Chronic treatment with risperidone tended to impair PPI in WT mice, whereas it 'normalized' the PPI deficit in CB(2)KO mice. CB(2)KO mice presented increased D(2)r and α(2C)r gene expressions in the prefrontal cortex (PFC) and locus coeruleus (LC), decreased 5-HT(2C)r gene expression in the dorsal raphe (DR), and 5-HT(2A)r gene expression in the PFC. Chronic risperidone treatment in WT mice left α(2C)r gene expression unchanged, decreased D(2)r gene expression (15 µg/kg), and decreased 5-HT(2C)r and 5-HT(2A)r in PFC and DR. In CB(2)KO, the gene expression of D(2)r in the PFC, of α(2C)r in the LC, and of 5-HT(2C)r and 5-HT(2A)r in PFC was reduced; 5-HT(2C)r and 5-HT(2A)r gene expressions in DR were increased after treatment with risperidone. These results suggest that deletion of CB(2)r has a relation with schizophrenia-like behaviors. Pharmacological manipulation of CB(2)r may merit further study as a potential therapeutic target for the treatment of schizophrenia-related disorders.

The role of 5-HT1A receptors in research strategy for extensive pain treatment.

In the last few years, there has been a great increase in our understanding of pain mechanisms. Given the complexity of the mechanisms involved in pain modulation, it is surprising that the pharmacological control of pain through the application of relatively simple analgesics can be effective. Nevertheless, the application of single analgesics is not always effective in diverse painful conditions such as chronic pain syndromes. In these circumstances, we can take advantage of the complexity of pain regulation and try to identify new targets in these intricate processes. It is becoming clear that the combination of different mechanisms, which improves efficacy with reduced toxicity, is necessary for the reliable pharmacotherapy of pain, and is at the forefront in the search for better analgesics. Serotonin is involved at multiple levels in the regulation of nociception. In particular, the raphe nuclei may play a crucial role in integrating the nociceptive and affective information through descending projections to the spinal cord and ascending projections to the forebrain. In these nuclei, 5-HT1A receptors function as somatodendritic autoreceptors controlling the release of serotonin in terminal areas. Different studies have shown that, by preventing this inhibitory control of serotonin release, it is possible to enhance the analgesic effect of drugs that increase serotonin levels (i.e. antidepressants, opiates, and non-steroidal anti-inflammatory drugs) by facilitating descending, and also ascending, pathways involved in pain modulation. Therefore, 5-HT1A receptors may be used as a new target in the search for new pharmacological approaches in the augmentation of analgesia.

Influence of chronic treatment with olanzapine, clozapine and scopolamine on performance of a learned 8-arm radial maze task in rats.

Cognitive deficit is a significant symptom in schizophrenic patients. Use of atypical antipsychotics has been demonstrated to improve some cognitive functions in schizophrenics, as well as in patients with dementia. However, side effects like sedation and muscarinic antagonism induced by these drugs have detracted from this improvement. We are interested in determining the behavioural effect of acute and chronic treatments with olanzapine and clozapine, two atypical antipsychotics, in a paradigm of working memory, and the influence on behavioural response of possible motor effects during test performance. Unspecific muscarinic antagonist scopolamine has been used for comparison. Male Wistar rats were trained on the 8-arm radial maze up to an accuracy level in choice of 80%. Distance travelled in the maze was also measured during test performance. Acute olanzapine, clozapine and scopolamine caused significant impairment of correct performance. Rats treated with olanzapine and clozapine presented a decrease in motor activity level at the same time. After the test at acute dosage, rats were chronically treated for 14 days with olanzapine, clozapine or scopolamine and 24 h after the last dose were again tested in the 8-arm radial maze. Under this procedure, chronic treatment with olanzapine, clozapine and scopolamine did not impair correct task performance and did not modify distance travelled. We concluded that the sedative effect masked a possible effect on working memory after acute administration of olanzapine and clozapine, whereas chronic treatment with olanzapine, clozapine and scopolamine did not adversely affect working memory performance. In the case of scopolamine, it suggests that chronic muscarinic antagonism does not induce memory impairment and for atypical antipsychotics, it suggests that chronic treatment induced a tolerance to acute motor effects of these drugs.

Effect of the antidepressant nefazodone on the density of cells expressing mu-opioid receptors in discrete brain areas processing sensory and affective dimensions of pain.

RATIONALE: The principal use of antidepressants is in the treatment of depression and affective disorders. Antidepressants have also been used as an adjuvant to analgesics in pain treatment. However, in chronic treatment, their antinociceptive and antidepressive effects coexist simultaneously. Antidepressants can interact with the opioid system, which is also involved in regulating nociceptive processing and affective state. Chronic antidepressants could act by increasing mu-opioid receptor expression in many brain areas involved in the regulation of nociception and affective state. OBJECTIVES: The aim of this study was to evaluate the antinociceptive and antidepressant-like effects and the possible variations in mu-opioid receptor expression induced by a chronic nefazodone treatment in brain areas related to pain and affective state. METHODS: Wistar rats were chronically treated with nefazodone (10 and 25 mg/kg IP, twice a day, for 14 days). Twelve hours after the last day 14 dose of nefazodone, a tail-flick test was performed. After the administration of a daily dose of nefazodone, Porsolt's test was carried out 12 h after last dose. Two hours after completion of 14 days treatment, other animals were processed for mu-opioid receptor immunocytochemistry using polyclonal antisera raised in rabbits. Several brain regions were analyzed: the frontal and cingulate cortex, the dorsal raphe nucleus and the periaqueductal gray. RESULTS: Chronic nefazodone treatment induced a significant increase in tail-flick latency and a significant decrease in immobility time at total doses of 20 and 50 mg/kg per day ( P<0.05). In treated animals, the density of neural cells immunostained for mu-opioid receptor in the frontal and cingulate cortices, dorsal raphe nucleus and periaqueductal gray had increased after chronic nefazodone compared to controls. CONCLUSION: Therefore, chronic nefazodone induces antinociceptive and antidepressant-like effects in rats and increases mu-opioid receptor expression in brain areas related to pain and affective state. These results suggest that antidepressants could be effective on somatic and affective dimensions of pain and this action could be related to its influence on the opioid system.

The role of 5-HT1A/B autoreceptors in the antinociceptive effect of systemic administration of acetaminophen.

BACKGROUND: It has been proposed that serotonin participates in the central antinociceptive effect of acetaminophen. The serotonin activity in the brainstem is primarily under the control of 5-HT1A somatodendritic receptors, although some data also suggest the involvement of 5-HT1B receptors. In the presence of serotonin, the blockade of 5-HT(1A/B) receptors at the level of the raphe nuclei leads to an increase in serotonin release in terminal areas, thus improving serotonin functions. This study examines the involvement of 5-HT(1A/B) receptors in the antinociceptive effect of acetaminophen in mice. METHODS: The effects of acetaminophen (600 mg/kg intraperitoneal) followed by different doses of antagonists (WAY 100635 [0.2-0.8 mg/kg subcutaneous] and SB 216641 [0.2-0.8 mg/kg subcutaneous]) or agonists (8-OH-DPAT [0.25-1 mg/kg subcutaneous] and CP 93129 [0.125-0.5 mg/kg subcutaneous]) of 5-HT1A and 5-HT1B receptors, respectively, were determined in the hot-plate test in mice. RESULTS: Acetaminophen (300-800 mg/kg) showed a dose-dependent antinociceptive effect in the hot-plate test in mice. WAY 100635 (0.2-0.8 mg/kg; 5-HT1A antagonist) induced an increase in the antinociceptive effect of 600 mg/kg acetaminophen, but this increase was not dose related. Conversely, 8-OH-DPAT (0.25-1 mg/kg; 5-HT1A agonist) decreased the antinociceptive effect of acetaminophen. SB 216641 (0.2-0.8 mg/kg; 5-HT1B antagonist) induced a dose-related increase in the antinociceptive effect of acetaminophen, and CP 93129 (0.25 mg/kg; 5-HT1B agonist) significantly decreased the antinociceptive effect of acetaminophen. CONCLUSIONS: These results suggest that the combination of acetaminophen with compounds having 5-HT1A and 5-HT1B antagonist properties could be a new strategy to improve the analgesia of acetaminophen, thanks to its mild serotonergic properties.

Pindolol, a beta-adrenoceptor blocker/5-hydroxytryptamine(1A/1B) antagonist, enhances the analgesic effect of tramadol.

The ability of pindolol, a beta-adrenoceptor blocker/5-hydroxytryptamine(1A/1B) antagonist, to enhance the clinical antidepressant response to selective serotonin re-uptake inhibitors is generally attributed to a blocking of the feedback that inhibits the serotoninergic neuronal activity mediated by somatodendritic 5-hydroxytryptamine (5-HT)(1A) autoreceptors. The current study examined the ability of pindolol to enhance the analgesic effect of tramadol, an atypical centrally-acting analgesic agent with relatively weak opioid receptor affinity and which, like some antidepressants, is able to inhibit the re-uptake of 5-HT in the raphe nuclei. Racemic pindolol (2 mg/kg, s.c.), rendered analgesic a non-effective acute dose of tramadol (10-40 mg/kg, i.p.) in two nociceptive tests: a hot plate test in mice and a plantar test in rats. Moreover, (+/-)8-OH-DPAT (0.125-1 mg/kg, s.c.), a selective 5-HT(1A) agonist, reduces the analgesic effect of tramadol in the same tests. These results suggest an implication of the somatodendritic 5-HT(1A) receptors in the analgesic effect of tramadol and open a new adjuvant analgesic strategy for the use of this compound.