Remote memories are enhanced by COMT activity through dysregulation of the endocannabinoid system in the prefrontal cortex.

The prefrontal cortex (PFC) is a crucial hub for the flexible modulation of recent memories (executive functions) as well as for the stable organization of remote memories. Dopamine in the PFC is implicated in both these processes and genetic variants affecting its neurotransmission might control the unique balance between cognitive stability and flexibility present in each individual. Functional genetic variants in the catechol-O-methyltransferase (COMT) gene result in a different catabolism of dopamine in the PFC. However, despite the established role played by COMT genetic variation in executive functions, its impact on remote memory formation and recall is still poorly explored. Here we report that transgenic mice overexpressing the human COMT-Val gene (COMT-Val-tg) present exaggerated remote memories (>50 days) while having unaltered recent memories (<24 h). COMT selectively and reversibly modulated the recall of remote memories as silencing COMT Val overexpression starting from 30 days after the initial aversive conditioning normalized remote memories. COMT genetic overactivity produced a selective overdrive of the endocannabinoid system within the PFC, but not in the striatum and hippocampus, which was associated with enhanced remote memories. Indeed, acute pharmacological blockade of CB1 receptors was sufficient to rescue the altered remote memory recall in COMT-Val-tg mice and increased PFC dopamine levels. These results demonstrate that COMT genetic variations modulate the retrieval of remote memories through the dysregulation of the endocannabinoid system in the PFC.

Age-related changes in the diagnostic assessment of women with severe cervical lesions.

OBJECTIVES: To evaluate the effect of age on the diagnostic assessment of women with severe cervical intraepithelial neoplasia (CIN). METHODS: This retrospective observational study included 338 consecutive women with a diagnosis of CIN3 on cone specimen. Patients were divided into three groups according to age: < 35 years (Group A), 35-49 years (Group B), and ≥ 50 years (Group C). Clinical and colposcopic variables were compared, and human papillomavirus (HPV) genotype distribution was measured. RESULTS: The most common HPV genotype was HPV-16 (63.65%), followed by HPV-33 (7%), HPV-18 (6.2%), and HPV-31 (5.4%). The rate of the following high-grade lesion predictors was lower in Group C than in Groups A and B: HPV-16 infections (55.9% vs. 75% vs. 70.9%, respectively, p = 0.022); high-grade colposcopic impression (29.4% vs. 51.8% vs. 51.7%, respectively, p < 0.0001); and high-grade cytological changes (30.9% vs. 56.2% vs. 45.4%, respectively, p = 0.025). An endocervical lesion location was more frequent in Group C than in Groups A and B (55.6% vs. 6.8% vs. 11.8%, respectively, p < 0.0001). CONCLUSION: Women aged 50 years and older with CIN3 showed a significant reduction of high-grade lesion predictors along with physiological confounding cervical changes (transformation zone type 3 and endocervical lesion location). The diagnostic work-up of cervical lesions in older women should provide their potential consideration as a special population.

Neurobiological alterations at adult age triggered by adolescent exposure to cannabinoids.

Marijuana is consistently the most widely used illicit drug among teenagers and most users first experiment it in adolescence. Adolescence is the period between childhood and adulthood, encompassing not only reproductive maturation, but also cognitive, emotional and social maturation and is characterized by a brain in transition that differs anatomically and neurochemically from that of the adult. The endocannabinoid system plays an important role in this critical phase for cerebral development, therefore a strong stimulation by the psychoactive component of marijuana, delta-9-tetrahydrocanabinol, that acts through the cannabinoid system, might lead to subtle but lasting neurobiological changes that can affect adult brain functions and behaviour. The literature here summarized, exploiting animal models of cannabis consumption, points to the presence of subtle changes in the adult brain circuits after heavy cannabis consumption in adolescence. These alterations lead to impaired emotional and cognitive performance, enhanced vulnerability for the use of more harmful drugs of abuse, and may represent a risk factor for developing schizophrenia in adulthood. The few studies examining the neurobiological basis of the altered behaviours demonstrate the presence of stable alteration in the endocannabinoid system that can trigger subsequent alteration in synaptic protein and synaptic morphology, thus altering the responsiveness of selected brain areas to different internal and external stimuli. These pre-clinical observations are strengthened by literature in humans where longitudinal studies often support the experimental results. There is an urgent need of multidisciplinary approaches combining behaviour with neurochemical and genetic studies to build a scientific based opinion on the long-lasting consequences of cannabis use in adolescence.

Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex.

In the present study we explored with a multidisciplinary approach, the role of anandamide (AEA) in the modulation of anxiety behavior at the level of the prefrontal cortex (PFC). Low doses of the metabolically stable AEA analog, methanandamide, microinjected into the PFC, produced an anxiolytic-like response in rats, whereas higher doses induced anxiety-like behaviors. Pretreatment with the selective antagonist of CB1 or TRPV1 receptors (AM251 and capsazepine, respectively) suggested that the anxiolytic effect evoked by AEA might be due to the interaction with the CB1 cannabinoid receptor, whereas vanilloid receptors seem to be involved in AEA anxiogenic action. When AEA contents in the PFC were increased by microinjecting the selective inhibitor of fatty acid amide hydrolase (FAAH), URB597, we observed an anxiolytic response only at low doses of the compound and no effect or even an anxiogenic profile at higher doses. In line with this, a marked decrease of AEA levels in the PFC, achieved by lentivirus-mediated local overexpression of FAAH, produced an anxiogenic response. These findings support an anxiolytic role for physiological increases in AEA in the PFC, whereas more marked increases or decreases of this endocannabinoid might lead to an anxiogenic response due to TRPV1 stimulation or the lack of CB1 activation, respectively.

CB1 receptor stimulation in specific brain areas differently modulate anxiety-related behaviour.

There is a general consensus that the effects of cannabinoid agonists on anxiety seem to be biphasic, with low doses being anxiolytic and high doses ineffective or possibly anxiogenic. Besides the behavioural effects of cannabinoids on anxiety, very few papers have dealt with the neuroanatomical sites of these effects. We investigated the effect on rat anxiety behavior of local administration of THC in the prefrontal cortex, basolateral amygdala and ventral hippocampus, brain regions belonging to the emotional circuit and containing high levels of CB1 receptors. THC microinjected at low doses in the prefrontal cortex (10 microg) and ventral hippocampus (5 microg) induced in rats an anxiolytic-like response tested in the elevated plus-maze, whilst higher doses lost the anxiolytic effect and even seemed to switch into an anxiogenic profile. Low THC doses (1 microg) in the basolateral amygdala produced an anxiogenic-like response whereas higher doses were ineffective. All these effects were CB1-dependent and closely linked to modulation of CREB activation. Specifically, THC anxiolytic activity in the prefrontal cortex and ventral hippocampus was paralleled by an increase in CREB activation, whilst THC anxiogenic response in the basolateral amygdala was paralleled by a decrease in CREB activation. Our results suggest that while a mild activation of CB1 receptors in the prefrontal cortex and ventral hippocampus attenuates anxiety, a slight CB1 receptor stimulation in the amygdala results in an anxiogenic-like response. The molecular underpinnings of these effects involve a direct stimulation of CB1 receptors ending in pCREB modulation and/or a possible alteration in the fine tuning of local neuromodulator release.

Long lasting consequences of cannabis exposure in adolescence.

Despite the increasing use of cannabis among adolescents, there are little and often contradictory studies on the long-term neurobiological consequences of cannabis consumption in juveniles. Adolescence is a critical phase for cerebral development, where the endocannabinoid system plays an important role influencing the release and action of different neurotransmitters. Therefore, a strong stimulation by the psychoactive component of marijuana, delta-9-tetrahydrocanabinol (THC), might lead to subtle but lasting neurobiological changes that can affect adult brain functions and behaviour. The literature here summarized by use of experimental animal models, puts forward that heavy cannabis consumption in adolescence may induce subtle changes in the adult brain circuits ending in altered emotional and cognitive performance, enhanced vulnerability for the use of more harmful drugs of abuse in selected individuals, and may represent a risk factor for developing schizophrenia in adulthood. Therefore, the potential problems arising in relation to marijuana consumption in adolescence suggest that this developmental phase is a vulnerable period for persistent adverse effects of cannabinoids.

The role of the endogenous cannabinoid system in drug addiction.

This review aims to present the more recent knowledge on the role of the endocannabinoid system in drug addiction. For a long time, dopamine has been consistently associated with the reinforcing effects of most drugs of abuse but, recently, pharmacological evidence points to the possibility that pharmacological management of the endocannabinoid system might not only block the direct reinforcing effect of cannabis, opioids, nicotine and ethanol, but also prevent the relapse to various drugs of abuse including opioids, cocaine, nicotine, alcohol and amphetamine. Preclinical and clinical studies suggest that the manipulation of the endocannabinoid system through the CB(1) receptor antagonist SR-141716A (rimonabant) might constitute a new therapeutical strategy for treating addiction across different classes of abused drugs.

Chronic FAAH inhibition during nicotine abstinence alters habenular CB1 receptor activity and precipitates depressive-like behaviors.

The role of the endocannabinoid system in nicotine addiction is being increasingly acknowledged. Acute inhibition of anandamide (AEA) degradation efficiently reduces nicotine withdrawal-induced affective symptoms in rats and fatty acid amide hydrolase (FAAH), the degradation enzyme of AEA, has been proposed as a possible treatment against nicotine addiction. However, it is unclear whether chronic inhibition of AEA during nicotine abstinence will have beneficial or deleterious affective side-effects. Using a rat model of nicotine addiction, we found that, during abstinence, rats injected daily with a FAAH inhibitor (URB597) developed a depressive-like phenotype. Our results show that in the nicotine abstinent rats, URB597 induced low saccharin consumption, persistent immobility in the forced swim test and increased corticosterone levels in response to stress. In addition, URB597decreased CB1 receptor binding and activity in the habenula, a key structure in the control of nicotine-related emotional states. In contrast, non-treated abstinent rats showed increased CB1 receptor activity and behaviors comparable to controls. No FAAH inhibition-induced alterations were observed in animals that had a previous history of saline self-administration. Taken together, our results suggest that chronic FAAH inhibition prevents the homeostatic adaptations of habenular CB1 receptor function that are necessary for the recovery from nicotine dependence.

Chronic delta-9-tetrahydrocannabinol treatment increases cAMP levels and cAMP-dependent protein kinase activity in some rat brain regions.

When Delta(9)-tetrahydrocannabinol (Delta(9)-THC,15 mg/kg) was injected intraperitoneally twice a day for 6 days, tolerance to its analgesic effect appeared to be complete. Chronic exposure to Delta(9)-THC caused a significant reduction in CB1 receptor binding in all brain areas that contain this receptor. Cannabinoid receptor density was markedly reduced in the cerebellum (52%), hippocampus (40%) and globus pallidum (47%) compared to 30% in the cortex and striatum. Chronic exposure enhanced the cAMP pathway, as shown by the significant increase of cAMP levels and PKA activity in the areas with receptor down-regulation (cerebellum, striatum and cortex). We propose that the increase in cAMP cascade is part of the biochemical basis of cannabinoid tolerance.

Immune function alterations in mice tolerant to delta9-tetrahydrocannabinol: functional and biochemical parameters.

We studied the effect of acute (1 h) or chronic exposure (7 and 14 days) to delta9-tetrahydrocannabinol (delta9-THC) on immune parameters in male Swiss mice. One hour after a dose of 10 mg/kg s.c., the splenocyte proliferative response to ConA and NK activity were not inhibited, but there was a significant decrease in the production of IL-2. After 7 days of treatment, when mice were tolerant to delta9-THC-induced analgesia, these functional parameters were strongly inhibited and there was a persistent reduction in IL-2 and IFNgamma. With 14 days exposure to the drug, splenocyte proliferation was significantly reduced only with 5 microg/ml ConA, and NK activity was still significantly depressed (about 37%). IL-2 had returned to the control value, whereas IFNgamma was still 40% down. Flow cytometry analysis of spleen cell composition indicated no changes after the acute and 7 day treatments, but at 14 days there was a 20% decrease in the number of T lymphocytes, mirrored by a 26% increase of B lymphocytes. In conclusion, in vivo exposure to psychoactive doses of delta9-THC has profound effects on immune function. This implies some important questions in relation to the liberalization of marijuana and its therapeutic uses.

Mu opioid receptor signaling in morphine sensitization.

We used a previously reported model of morphine sensitization that elicited a complex behavioral syndrome involving stereotyped and non stereotyped activity. To identify the mechanism of these long-lasting processes, we checked the density of mu opioid receptors, receptor-G-protein coupling and the cyclic AMP (cAMP) cascade. In morphine-sensitized animals mu opioid receptor autoradiography revealed a significant increase in the caudate putamen (30% versus controls), nucleus accumbens shell (16%), prefrontal and frontal cortex (26%), medial thalamus (43%), hypothalamus (200%) and central gray (89%). Concerning morphine's activation of G proteins in the brain, investigated in the guanylyl 5'-[gamma-(35)S]thio]triphosphate ([(35)S]GTPgammaS) binding assay, a significant increase in net [(35)S]GTPgammaS binding was seen in the caudate putamen (39%) and hypothalamus (27%). In the caudate putamen this was due to an increase in the amount of activated G proteins, and in the hypothalamus to a greater affinity of G proteins for guanosine triphosphate (GTP). The main second messenger system linked to the opioid receptor is the cAMP pathway. In the striatum basal cAMP levels were significantly elevated in sensitized animals (70% versus controls) and [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) significantly inhibited forskolin-stimulated cAMP production in control (30%) but not in sensitized rats. In the hypothalamus no significant changes were observed in basal cAMP levels and DAMGO inhibition. These cellular events induced by morphine pre-exposure could underlie the neuroadaptive processes involved in morphine sensitization.

Chronic treatment with a synthetic cannabinoid CP-55,940 alters G-protein expression in the rat central nervous system.

Prolonged exposure of rats to the synthetic cannabinoid receptor ligand, CP-55,940 (0.4 mg/kg, i.p. for 11 days), induced tolerance to analgesia, to the reduction in spontaneous locomotor activity and the incidence of splayed hind limbs. One hour after the last injection on day 11, the rats were killed and in situ hybridization was used to investigate the effect of treatment on G-protein alpha-subunit expression throughout the brain. Chronic cannabinoid exposure markedly reduced G alpha(s), G alpha(i) and G alpha(o) mRNA levels. The message for the alpha(s)-subunit was decreased in all the brain areas containing the basal autoradiographic signal; the decrease ranging from 25% in the thalamus to 45% in the mesencephalon. Also the basal G alpha(i) expression was reduced in tolerant rats showing the greatest decrease in the forebrain (63%) in the cerebellum (58%) and in the mesencephalon (38%). The reduction in G alpha(o) expression (25%) was more localized, being present only in the rostral portion of the brain (cortex, striatum and olfactory area). The alterations in alpha-subunits gene expression were not followed by any change in the amount of proteins. Our results indicate that, besides the receptor modification, alteration to the G-protein expression could be a molecular event associated with the development of cannabinoid tolerance.

Effect of chronic exposure to naltrexone and opioid selective agonists on G protein mRNA levels in the rat nervous system.

The in situ hybridization technique was used to investigate the effect on G protein alpha subunit expression throughout the brain of rats chronically infused with naltrexone (70 micrograms/microliters, 1 microliter/h), DAGO (0.5 micrograms/microliter, 1 microliter/h), DADLE (11.4 micrograms/microliters, 1 microliter/h), DPDPE (3.4 micrograms/microliters, 1 microliter/h) and U-50,488H (4 micrograms/microliters, 1 microliter/h). Prolonged exposure to naltrexone did not modify G protein alpha subunit mRNA expression, whereas DADLE and U-50,488H, respectively, increased the levels of alpha s and alpha o mRNA in specific brain regions. In particular, a 15% increase in alpha s expression was only observed in the dorsomedial hypothalamic nucleus of rats undergoing chronic DADLE infusion: a 15% increase in alpha o levels was detected in the claustrum and endopiriform nucleus of rats chronically treated with U-50,488H. These are the first in vivo data to demonstrate that only chronic stimulation with an opioid agonist (morphine and/or DADLE and U-50,488H) is capable of modifying G protein alpha subunit mRNA. The regional selectivity of these modifications is discussed, together with the receptor specificity of the opioid effects.

Modulation of rat brain cannabinoid receptors after chronic morphine treatment.

Intraperitoneal injection of delta9-THC (7.5 mg/kg) in rats made tolerant to morphine by s.c. implantation of morphine pellets had a much greater analgesic effect than in placebo pellet plus delta9-THC treatment. To investigate whether this was due to some change in cannabinoid receptor levels and/or expression induced by chronic morphine, we designed this autoradiographic binding study coupled with in situ hybridization on sagittal sections of the treated rat brains. Binding showed a significant increase in CB1 receptor density (15%) specifically in the caudate-putamen, in parallel with a significant enhancement of CB1 mRNA in the same area (20%). We suggest that morphine chronic treatment leads to a functional modulation between the opioid and cannabinoid systems at least for analgesia in a specific area, in this case the striatum.

Chronic CP-55,940 alters cannabinoid receptor mRNA in the rat brain: an in situ hybridization study.

Using in situ hybridization we found that chronic treatment with CP-55,940 (0.4 mg kg-1, i.p. daily for 11 days), a synthetic cannabinoid receptor ligand, changed cannabinoid receptor mRNA levels in rat brain. CP-55,940 produced the expected tolerance: the decrease in locomotor activity (75%) caused by an acute dose was diminished to 25% after the 11 days of treatment. Thirty minutes after the last injection the animals were killed and in situ hybridization indicated that the levels of cannabinoid receptor mRNA in the caudate-putamen were reduced by 33%, with no alteration in the other brain areas. We suggest that the altered cannabinoid receptor expression is part of the adaptive changes underlying cannabinoid tolerance.

Endocannabinoids in the immune system and cancer.

The present review focuses on the role of the endogenous cannabinoid system in the modulation of immune response and control of cancer cell proliferation. The involvement of cannabinoid receptors, endogenous ligands and enzymes for their biosynthesis and degradation, as well as of cannabinoid receptor-independent events is discussed. The picture arising from the recent literature appears very complex, indicating that the effects elicited by the stimulation of the endocannabinoid system are strictly dependent on the specific compounds and cell types considered. Both the endocannabinoid anandamide and its congener palmitoylethanolamide, exert a negative action in the onset of a variety of parameters of the immune response. However, 2-arachidonoylglycerol appears to be the true endogenous ligand for peripheral cannabinoid receptors, although its action as an immunomodulatory molecule requires further characterization. Modulation of the endocannabinoid system interferes with cancer cell proliferation either by inhibiting mitogenic autocrine/paracrine loops or by directly inducing apoptosis; however, the proapoptotic effect of anandamide is not shared by other endocannabinoids and suggests the involvement of non-cannabinoid receptors, namely the VR1 class of vanilloid receptors. In conclusion, further investigations are needed to elucidate the function of endocannabinoids as immunosuppressant and antiproliferative/cytotoxic agents. The experimental evidence reviewed in this article argues in favor of the therapeutic potential of these compounds in immune disorders and cancer.

Influence of omega-conotoxin on morphine analgesia and withdrawal syndrome in rats.

The effect of omega-conotoxin on opiate analgesia and withdrawal syndrome was investigated in rats. omega-Conotoxin given i.c.v. and i.p. caused weak analgesia in the tail-flick test. When the toxin (20 ng/rat) was given i.c.v. immediately before morphine (1.5 micrograms/rat i.c.v.) the resultant analgesic effect was additive. In contrast, the analgesia elicited by morphine (3 micrograms/rat i.c.v.) was greatly reduced after 24-h pretreatment with the toxin (20 ng/rat i.c.v.). The systemic administration of the toxin (10 micrograms/kg i.p.) did not affect morphine analgesia whether omega-conotoxin was coadministered with morphine (2.5 mg/kg i.p.) or was given 24 h before the opiate (5 mg/kg i.p.). omega-Conotoxin i.c.v. injected in morphine-dependent rats 15 min before naloxone challenge significantly attenuated the abstinence syndrome. On the contrary systemic administration of omega-conotoxin failed to suppress the morphine withdrawal syndrome. The present results suggest that omega-conotoxin affects both acute and chronic effects of morphine.

Cholera toxin antagonizes morphine-induced catalepsy through a cyclic AMP-independent mechanism.

We studied the effect of intracerebroventricular pretreatment with pertussis toxin and cholera toxin on morphine catalepsy in rats. Pertussis toxin (1 micrograms/rat, two, three and six days before) did not affect catalepsy evoked by central morphine. Cholera toxin (1 micrograms/rat) did not affect morphine catalepsy after 24 h and 48 h, but significantly reduced it (about 60%) after three and five days. Ten days later the morphine response had totally recovered. This effect was selective, since morphine analgesia was not modified. The reduction of catalepsy appeared unrelated to the ability of cholera toxin to raise cAMP levels, as demonstrated by the different time course of changes in striatal cholera toxin-stimulated adenylate cyclase activity. The effect required an intact cholera toxin molecule and did not occur with a similar dose of cholera toxin-B subunit. These findings demonstrate that catalepsy is an opioid effect not linked to pertussis toxin-sensitive G proteins and suggest that the Gs protein might be involved.

In situ hybridization reveals specific increases in G alpha s and G alpha o mRNA in discrete brain regions of morphine-tolerant rats.

In situ hybridization histochemistry has been used to detect the basal distribution of mRNA encoding the alpha subunit of Gs, Go and Gi2 proteins throughout the rat brain. Based on these data we investigated the effect of chronic morphine on the content of these G protein alpha subunits mRNA. We observed an increase in the expression of alpha s and alpha o messages of chronically morphine-treated animals, while no changes were seen in alpha i2 mRNA. Specifically a 30% increase in expression for alpha s was seen only in the paraventricular nucleus of hypothalamus and a 20% elevation for alpha o was detected in the claustrum and endopiriform nucleus. Immunoblotting analysis was used to correlate the changes in alpha s and alpha o messages with equivalent changes in protein levels. Chronic morphine significantly increased alpha s amounts in the hypothalamus (70%), and produced a minor elevation (30%) in G alpha o levels in the olfactory area. Our results indicate that in discrete brain regions altered G protein expression is part of the adaptive changes underlying opiate tolerance.

Intestinal effect of morphine 6-glucuronide: in vivo and in vitro characterization.

Morphine 6-glucuronide, a major metabolite of morphine with potent analgesic actions, is a potent inhibitor of intestinal motility when administered to rats by the intracerebroventricular (i.c.v.) route. Morphine 6-glucuronide was 62-fold more active than morphine in inhibiting gastrointestinal transit, whereas it was only 25-fold more potent in abolishing intestinal migrating myoelectric complexes. Pretreatment with naloxone (5 micrograms/rat i.c.v.) completely prevented the disappearance of migrating myoelectric complexes induced by the morphine metabolite. In contrast, in the guinea pig ileum bioassay, morphine 6-glucuronide and morphine inhibited the electrically evoked contractions of the tissue with similar potency, although in the guinea pig ileum binding assay the metabolite showed 4-fold lower affinity for the opiate receptor. The low naloxone Ke values against morphine 6-glucuronide or morphine indicated that the action of both drugs in guinea pig ileum was mediated by mu-opioid receptors.