María Ángeles Villanúa

Acute administration of the CB1 cannabinoid receptor antagonist SR 141716A induces anxiety-like responses in the rat

ANIMAL models have revealed that psychoactive cannabinoids induce both anxiolytic and anxiety-like reactions which are dose- and context-dependent. In the present study we examined the acute actions of the CB1 cannabinoid receptor antagonist SR 141716A in both the defensive withdrawal test and the elevated plus-maze in rats. Acute administration of SR 141716A (0.1, 1 and 3 mg kg−1) induced defensive responses in both anxiety tests, at a dose of 3 mg kg−1. This dose had no effect on horizontal locomotor activity and did not activate the hypothalamus–pituitary–adrenal axis, although several cannabinoid withdrawal-like behavioural symptoms were observed. These results demonstrate that blockade of the endogenous cannabinoid tone might induce anxiety-like responses in rats.

CB1 cannabinoid receptor antagonist-induced opiate withdrawal in morphine-dependent rats

RECENT reports have provided evidence of a link between the endogenous brain cannabinoid system and the endogenous central opioid systems. Here we report that the selective CB1 receptor antagonist SR 141716A induced behavioral and endocrine alterations associated with opiate withdrawal in morphine-dependent animals in a dose-dependent manner and that naloxone induced an opiate withdrawal syndrome in animals made cannabinoid-dependent by repeated administration of the potent cannabinoid agonist HU-210. Additionally CB1 and μ-opioid receptor mRNAs were co-localized in brain areas relevant for opiate withdrawal such as the nucleus accumbens, septum, dorsal striatum, the central amygdaloid nucleus and the habenular complex. These results suggest that CB1 cannabinoid receptors may play a role in the neuroadaptive processes associated with opiate dependence, and they lend further support for the hypothesis of a potential role of cannabinoid receptors in the neurobiological changes that culminate in drug addiction.

Extrapyramidal and neuroendocrine effects of AM404, an inhibitor of the carrier-mediated transport of anandamide.

A selective inhibitor of the carrier-mediated transport of endogenous cannabinoids, N-(4-hydroxyphenyl)-arachidonylethanolamide (AM404), has been recently synthesized and proposed as a useful tool for studying the physiological effects of endogenous cannabinoids and as a potential therapeutic agent in a variety of diseases. In the present study, we have examined the effects of this compound in two important brain processes in which a role for anandamide and other endogenous cannabinoids has been claimed: neuroendocrine regulation and extrapyramidal motor activity. A single and well-characterized dose of AM404, which presumably resulted in a significant elevation of the levels of endogenous cannabinoids, produced a marked decrease in plasma prolactin (PRL) levels, with no changes in luteinizing hormone (LH) levels. This decrease in PRL levels was accompanied by an increase in the activity of tyrosine hydroxylase (TH) in the medial basal hypothalamus. Both decreased PRL secretion and increased hypothalamic TH activity have been reported to occur after the administration of anandamide. Administration of AM404 also produced a marked motor inhibition in the open-field test, as also reported for anandamide, with a decrease in ambulatory and exploratory activities and an increase in the time spent in inactivity. This was accompanied by a decrease in the activity of TH in the substantia nigra, an effect also previously observed for anandamide.

Characterization of the acute endocrine actions of (−)-11-hydroxy-Δ8-tetrahydrocannabinol-dimethylheptyl (HU-210), a potent synthetic cannabinoid in rats

In the present study we have characterized the effects of the acute administration of the synthetic cannabinoid (-)-11-hydroxy-delta8-tetrahydrocannabinol-dimethylheptyl (HU-210, 4, 20 and 100 microg/kg), on the secretion of prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, adrenocorticotropic hormone and corticosterone in adult male rats. HU-210 administration resulted in a dose-dependent inhibition of plasma growth hormone, follicle-stimulating hormone and luteinizing hormone 60 min after the acute intraperitoneal injection, starting at 20 microg/kg. Plasma adrenocorticotropic hormone and corticosterone levels revealed a dose-dependent activation of the pituitary-adrenal axis after acute exposure to HU-210. Plasma prolactin levels reflected a biphasic action of HU-210: the 4 microg/kg dose resulted in high prolactin levels and the 20 and 100 microg/kg doses induced a decrease in the levels of this hormone. The time course of the endocrine effects of HU-210 was examined using the 20 microg/kg dose and was found to parallel the onset of the immobility and hypothermic effects of this cannabinoid. HU-210 (20 microg/kg) was also found to block the hormonal surges of luteinizing hormone, follicle-stimulating hormone and prolactin occurring during the afternoon of the proestrus phase in adult female rats. This dose induced activation of tubero-infundibular dopaminergic neurons, as reflected by the decrease in hypothalamic contents of dopamine in both males and females in the afternoon of the proestrus phase. The actions of HU-210 during early postnatal development revealed a delayed maturation of the endocrine response to HU-210, with respect to the behavioral effects. The findings of the present study reveal that HU-210 induces a set of endocrine alterations closely related to those described for natural cannabinoids such as delta9-tetrahydrocannabinol but at doses 50-200 times lower than those required for delta9-tetrahydrocannabinol.

Effects of cannabinoids on prolactin and gonadotrophin secretion : Involvement of changes in hypothalamic γ-aminobutyric acid (GABA) inputs

CB1 cannabinoid receptors are located in hypothalamic nuclei and their activation alters several hypothalamic neurotransmitters resulting in, among other things, decreased prolactin (PRL) and luteinizing hormone (LH) secretion from the anterior pituitary gland. In the present study, we addressed two related objectives to further explore this complex regulation. First, we examined whether changes in gamma-aminobutyric acid (GABA) and/or dopamine (DA) inputs in the medial basal hypothalamus might occur in parallel to the effects resulting from the activation of CB1 receptors on PRL and gonadotrophin secretion in male rats. Thus, the acute administration of (-)-delta9-tetrahydrocannnabinol (delta9-THC) produced, as expected, a marked decrease in plasma PRL and LH levels, with no changes in follicle-stimulating hormone (FSH) levels. This was paralleled by an increase in the contents of GABA, but not of DA, in the medial basal hypothalamus and, to a lesser extent, in the anterior pituitary gland. The co-administration of delta9-THC and SR141716, a specific antagonist for CB1 receptors, attenuated both PRL and LH decrease and GABA increase, thus asserting the involvement of the activation of CB1 receptors in these effects. As a second objective, we tested whether the prolonged activation of these receptors might induce tolerance with regard to the decrease in PRL and LH release, and whether this potential tolerance might be related to changes in CB1-receptor binding and/or mRNA expression. The chronic administration of R-methanandamide (AM356), a more stable analog of anandamide, the putative endogenous cannabinoid ligand, produced a marked decrease in plasma PRL and LH levels, with no changes in FSH. The decreases were of similar magnitude to those caused by a single injection of this cannabimimetic ligand, thus suggesting the absence of tolerance. In parallel, the analysis of CB1-receptor binding and mRNA expression in several hypothalamic structures proved that the acute or chronic administration of AM356 did not affect either the binding or the synthesis of these receptors. In summary, the activation of CB1 receptors in hypothalamic nuclei produced the expected decrease in PRL and LH secretion, an effect which might be related to an increase in GABAergic activity in the hypothalamus-anterior pituitary axis. The prolonged activation of these receptors for five days did not elicit tolerance in terms of an attenuation in the magnitude of the decrease in PRL and LH, and, accordingly, did not alter CB1-receptor binding and mRNA levels in the hypothalamic nuclei examined.

Maternal Exposure to Low Doses of Δ9-Tetrahydrocannabinol Facilitates Morphine-Induced Place Conditioning in Adult Male Offspring

The possible existence of an increased susceptibility to the reinforcing properties of morphine was analyzed in male and female rats born from mothers exposed to delta9-tetrahydrocannabinol (THC, 1, 5, or 20 mg/kg) during gestation and lactation. Maternal exposure to low doses of THC (1 and 5 mg/kg), relevant for human consumption, resulted in an increased response to the reinforcing effects of a moderate dose of morphine (350 microg/kg), as measured in the place-preference conditioning paradigm (CPP) in the adult male offspring. These animals also displayed an enhanced exploratory behavior in the defensive withdrawal test. However, only females born from mothers exposed to THC 1 mg/kg exhibited a small increment in the place conditioning induced by morphine. The possible implication of the hypothalamo-pituitary-adrenal axis (HPA) was analyzed by monitoring plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone in basal and moderate-stress conditions (after the end of the CPP test). Female offspring perinatally exposed to THC (1 or 5 mg/kg) displayed high basal levels of corticosterone and a blunted adrenal response to the HPA-activating effects of the CPP test. However, male offspring born from mothers exposed to THC (1 or 5 mg/kg) displayed the opposite pattern: normal to low basal levels of corticosterone, and a sharp adrenal response to the CPP challenge. The present study reveals that maternal exposure to low doses of THC results in an increased sensitivity to the reinforcing effects of morphine in the adult male offspring, and in sexually dimorphic behavioral and endocrine alterations in the adaptative responses to stressors such as novelty or place-preference testing. These results support the growing evidence of the importance of monitoring the long-term consequences of maternal consumption of cannabis derivatives.

Fenofibrate, a PPARα agonist, decreases atrogenes and myostatin expression and improves arthritis-induced skeletal muscle atrophy

Arthritis is a chronic inflammatory illness that induces cachexia, which has a direct impact on morbidity and mortality. Fenofibrate, a selective PPARα activator prescribed to treat human dyslipidemia, has been reported to decrease inflammation in rheumatoid arthritis patients. The aim of this study was to elucidate whether fenofibrate is able to ameliorate skeletal muscle wasting in adjuvant-induced arthritis, an experimental model of rheumatoid arthritis. On day 4 after adjuvant injection, control and arthritic rats were treated with 300 mg/kg fenofibrate until day 15, when all rats were euthanized. Fenofibrate decreased external signs of arthritis and liver TNFα and blocked arthritis-induced decreased in PPARα expression in the gastrocnemius muscle. Arthritis decreased gastrocnemius weight, which results from a decrease in cross-section area and myofiber size, whereas fenofibrate administration to arthritic rats attenuated the decrease in both gastrocnemius weight and fast myofiber size. Fenofibrate treatment prevented arthritis-induced increase in atrogin-1 and MuRF1 expression in the gastrocnemius. Neither arthritis nor fenofibrate administration modify Akt-FoxO3 signaling. Myostatin expression was not modified by arthritis, but fenofibrate decreased myostatin expression in the gastrocnemius of arthritic rats. Arthritis increased muscle expression of MyoD, PCNA, and myogenin in the rats treated with vehicle but not in those treated with fenofibrate. The results indicate that, in experimental arthritis, fenofibrate decreases skeletal muscle atrophy through inhibition of the ubiquitin-proteasome system and myostatin.

Differential Effects of Chronic Treatment with Either Dopamine D1 or D2 Receptor Agonists on the Acute Neuroendocrine Actions of the Highly Potent Synthetic Cannabinoid HU-210 in Male Rats

Acute exposure to delta 9-tetrahydrocannabinol (THC), the main psychoactive constituent of marijuana, produces a well-characterized set of neuroendocrine effects. The recent description of both brain cannabinoid receptors (CB-1) and anandamide, their proposed endogenous ligand, has renewed the interest in cannabinoid actions in the brain. However, the neurobiological mechanisms underlying the neuroendocrine effects of natural cannabinoids are not yet fully understood because of several mechanisms involved in their actions. In this work we have studied the role of hypothalamic dopaminergic receptors in the mediation of the acute neuroendocrine effects of (-)-delta 8-tetrahydrocannabinol-dimethyl-heptyl (HU-210, 20 micrograms/kg), a highly potent agonist of CB-1. The use of this low dose of HU-210 precludes the multiple unspecific effects which appear with an equipotent dose of THC. Rats were exposed during 21 days to either the dopamine (DA) D2 receptor agonist quinpirole (1 mg/kg, daily), the DA D1 receptor agonist SKF 38393 (8 mg/kg, twice a day) or vehicle (twice a day). Twenty-four hours after the last injection, a single dose of HU-210 (20 micrograms/kg) was administered intraperitoneally, and the animals were sacrificed 90 min later. Acute exposure to HU-210 produced both a decrease in plasma prolactin and a rise of plasma corticosterone levels. HU-210 treatment also resulted in both an increase in the L-3,4-dihydroxyphenylacetic acid/DA ratio and a decrease in noradrenaline contents, measured in the medial basal hypothalamus. These neuroendocrine actions were prevented by chronic exposure to quinpirole, but not after chronic SKF 38393 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal Exposure to the Synthetic Cannabinoid HU-210: Effects on the Endocrine and Immune Systems of the Adult Male Offspring

Natural and synthetic cannabinoid receptor agonists have been described to exert profound effects on both the neuroendocrine integration and the functional responses of the immune system. In the present study, Wistar rats were exposed to the highly potent cannabinoid agonist HU-210 (1, 5 and 25 μg/kg) during gestation and lactation and the ensuing effects on several endocrine and immune parameters of the adult male offspring were analyzed. Perinatal exposure to HU-210 partially affected the distribution of lymphocyte subpopulations in the spleen and peripheral blood. The major changes observed occur after maternal exposure to the 25 μg/kg dose of HU-210. There was a reduction in the T-helper subpopulation in the spleen and a dose-related decrease in the rate of Thelper/Tcytotoxic in peripheral blood lymphocytes. Concanavalin-A and lipopolysaccharide-induced proliferation were normal in all the groups tested. In the same animals, perinatal exposure to HU-210 did not affect basal levels of growth hormone, IGF-1, prolactin, or follicle-stimulating hormone. Basal values of luteinizing hormone were elevated in animals given the 1 μg/kg dose of HU-210. Corticosterone levels were reduced in the animals exposed to the higher dose of HU-210 during gestation and lactation. These animals exhibited a decreased responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis to the stimulation with a single injection of HU-210 (20 μg/kg, i.v.) at adult ages, which may reflect the onset of long-lasting tolerance to the HPA-activating properties of cannabinoids. The opposite pattern of response was found in the animals given the 1 μg/kg dose, in which a sensitization of the corticosterone response to acute HU-210 was observed. The present work reveals that maternal exposure to cannabinoids results in minor changes in the development of the immune system, but may induce long-lasting alterations in the functional status of the HPA axis.

Adipose tissue loss in adjuvant arthritis is associated with a decrease in lipogenesis, but not with an increase in lipolysis

Adjuvant-induced arthritis is a model of rheumatoid arthritis that induces cachexia. In other cachectic situations, there is an increase in lipolysis resulting in a loss of adipose tissue mass. The aim of this work was to analyse the effect of chronic arthritis, induced by adjuvant injection, on white adipose tissue (WAT). For this purpose, rats were killed 10 days after adjuvant injection, when the first external symptoms appeared, on days 15 and 22 when the external signs of the illness reach their severest level. As arthritis decreases food intake, a pair-fed group was also included. Serum concentrations of insulin, leptin, adiponectin, glycerol and nitrites, as well as gene expression of leptin, adiponectin, hormone-sensitive lipase (HSL), fatty acid synthase (FAS), tumour necrosis factor alpha and zinc-alpha(2)-glycoprotein (ZAG) were determined. Arthritis decreased food intake between days 5 and 16, but not during the last 5 days of the experiment. There was a marked decrease in relative adipose tissue weight and in serum leptin and adiponectin as well as in their gene expression in WAT in arthritic rats. Arthritis decreased the gene expression of FAS in the WAT. However, none of these effects was found in pair-fed rats. Arthritis did not increase lipolysis, since arthritic rats have lower serum concentrations of glycerol, HSL mRNA in WAT, as well as liver ZAG mRNA than the pair-fed or control rats. These data suggest that in chronic arthritis the decrease in white adipose mass is secondary to a reduced adipose lipogenesis, and this effect is not mainly due to the decrease in food intake.