M. Cebeira

Cannabinoid tolerance and dependence: A review of studies in laboratory animals

Two are the issues on cannabis addiction that provoke more controversy from a research perspective. The first one is related to the development of tolerance phenomena and, in particular, of a dependence state after chronic cannabinoid consumption, with appearance of withdrawal signs when this is interrupted, that would be (or not) comparable to those observed for other drugs. A second controversial issue is related to the possibility that chronic cannabinoid consumption may increase the risk to consume other drugs of greater addictive power. Since the discovery in the 1990s of the endocannabinoid signaling system as the target for the action of plant-derived cannabinoids, many studies have addressed these two questions in laboratory animals and, although the results have resulted controversial in various aspects, the following conclusions seem evident: (i) prolonged exposure to plant-derived, synthetic or endogenous cannabinoid agonists in laboratory animals is currently associated with the development of tolerance for most of their pharmacological effects, (ii) tolerance is essentially due to adaptative phenomena consisting in pharmacodynamic events (down-regulation/desensitization of cannabinoid receptors), although some evidence exist on additional pharmacokinetic responses, (iii) the discontinuation of chronic cannabinoid treatment does not elicit abstinence responses spontaneously in most of the cases, presumably because the pharmacokinetic characteristics of cannabinoids, but these responses may be elicited after the blockade of cannabinoid CB1 receptors in cannabinoid-tolerant animals, (iv) these abstinent responses include mainly somatic signs and changes in various molecular processes affected during the abstinence to other drugs although the magnitude of these changes was currently lower in the case of cannabinoids, and (v) cannabinoid-tolerant animals do not appear to be more vulnerable to reinforcing properties of morphine, although the manipulation of the endocannabinoid signaling might serve to treat cannabis addiction and, in particular, the addiction to other drugs such as alcohol, nicotine or opioids. The present review article will address all these aspects trying to establish the bases for future research.

Cannabinoid receptors in rat brain areas: Sexual differences, fluctuations during estrous cycle and changes after gonadectomy and sex steroid replacement

Cannabinoid effects on brain dopaminergic activity vary as a function of gonadal status. In this work, we examined whether these variations might be due to sex steroid-dependent differences in brain cannabinoid receptors (CNr). Four experiments were done: (i) male versus females; (ii) females at each stage of the ovarian cycle; (iii) estradiol (E2) and/or progesterone (P)-replaced ovariectomized (OVX) females; and (iv) testosterone (T)-replaced orchidectomized males. The density of CNr in the medial basal hypothalamus fluctuated in females during the estrous cycle. The density was higher in diestrus and decreased in estrus. This parameter did not change after ovariectomy and E2 replacement. However, P increased the density of CNr when administered to OVX rats acutely treated with E2, but not administered alone or after chronic E2 treatment. In the striatum, the affinity of CNr was slightly higher in males than females, with no changes in density. Ovariectomy increased the affinity of CNr, which normalized only after administration of acute E2. Interestingly, the high affinity values observed in this area after P alone or combined with E2, corresponded to low densities as compared with intact females. In the limbic forebrain, the affinity for the cannabinoid ligand was also higher in males than females with no changes in density. Affinity was also higher in diestrus and lower in estrus, whereas density was unchanged. Ovariectomy decreased CNr density. A normal situation was found after administration of acute E2 or P alone, whereas chronic E2 markedly increased the density of CNr as compared with both intact and OVX females. Interestingly, this latter increase was prevented by coadministration of P. Orchidectomy did not affect CNr density, but administration of T produced a marked decrease. In the mesencephalon, the density and affinity of CNr was higher in males than females. Administration of P to OVX rats produced opposite effects, increasing the density when administered alone and decreasing it when administered to acute E2-replaced OVX rats. In summary, these results reveal the existence of subtle, sometimes more pronounced, sex dimorphisms, fluctuations along the ovarian cycle and changes after gonadectomy and sex steroid replacement in CNr density and affinity in certain brain areas. This supports the hypothesis of possible sex steroid-dependent differences in the sensitivity of certain neuronal processes to cannabinoid treatment.

Effects of pre- and perinatal exposure to hashish extracts on the ontogeny of brain dopaminergic neurons

The changes induced by maternal exposure to cannabinoids in the maturation of nigrostriatal, tuberoinfundibular and mesolimbic dopaminergic activities of rat offspring 15-40 days old were studied. In the striatum, tyrosine hydroxylase activity was constantly decreased during cannabinoid exposure in males. This decrease was correlative to increased number of D1 and D2 dopaminergic receptors. Both effects were also observed after the drug withdrawal caused by weaning on day 24. In females, the most consistent effect appeared on day 20, when decreased dopamine content and number of D1 receptors were observed. Both effects disappeared after drug withdrawal, but the reduction in the number of D1 receptors was again observed 40 days after birth. In the limbic area, cannabinoid exposure caused a decrease in the number of D1 receptors in 15-day-old females, along with decreases in the content of dopamine and its metabolite, L-3,4-dihydroxyphenylacetic acid. Changes in receptors disappeared on subsequent days, but increases in L-3,4-dihydroxyphenylacetic acid content and in its ratio with dopamine (L-3,4-dihydroxyphenylacetic acid/dopamine) were observed on day 20 followed by a decrease in the neurotransmitter content on day 30. In males, tyrosine hydroxylase activity increased on day 30, followed by an increase in L-3,4-dihydroxyphenylacetic acid content and L-3,4-dihydroxyphenylacetic acid/dopamine ratio on day 40. In the hypothalamus, the cannabinoid effects were always manifested after the cessation of drug exposure. Thus, a rise in L-3,4-dihydroxyphenylacetic acid/dopamine ratio was observed in 30-day-old females, and it was followed by a decrease on day 40, accompanied by a decrease in the anterior pituitary content of dopamine. Rise in prolactin release was not significant. In males, tyrosine hydroxylase activity was increased 30 days after birth, while L-3,4-dihydroxyphenylacetic acid content decreased. On day 40, L-3,4-dihydroxyphenylacetic acid content increased, paired to a rise in L-3,4-dihydroxyphenylacetic acid/dopamine ratio and anterior pituitary content of dopamine and to a decrease in the prolactin release. Perinatal exposure to cannabinoids altered the normal development of nigrostriatal, mesolimbic and tuberoinfundibular dopaminergic neurons, as reflected by changes in several indices of their activity. These changes were different regarding the sex and brain areas. Cannabinoid effects were more marked and constant in the striatum of males, while alterations in limbic neurons were mostly transient and those in hypothalamic neurons occurred after drug withdrawal. A long-term impact of these early changes on the neurological processes of adulthood is plausible.

The endogenous cannabinoid receptor ligand, anandamide, inhibits the motor behavior: role of nigrostriatal dopaminergic neurons.

The present study has been designed to test whether the recently described endogenous ligand for the cannabinoid receptor, arachidonylethanolamide, termed anandamide, can mimic the effects produced by exogenous cannabinoids on motor behavior and to test possible neurochemical substrates for this potential effect. To this end, adult male rats were submitted to an acute i.p. injection of anandamide, delta 9-tetrahydrocannabinol (THC) or vehicle. Animals were behaviorally tested ten minutes after injection of the drug and, then, sacrificed and their brains used for dopaminergic analyses. Ambulation was not significantly affected by the treatment with either THC or anandamide, but a very pronounced increase was observed in the time spent in inactivity in rats treated with either THC or anandamide. This was accompanied by a marked decrease in the frequency of spontaneous non-ambulatory activities, such as grooming and rearing, although only the administration of THC decreased shaking behavior. The anandamide-induced decrease in grooming was dose-dependent, but the decrease in rearing was higher with the dose of 3 mg/kg than with the dose of 10 mg/kg. The administration of anandamide also caused a dose-dependent decrease in the activity of tyrosine hydroxylase and in the ratio between the number of D1 and D2 receptors in the striatum. Moreover, the administration of 3 mg/kg of anandamide significantly decreased the contents of dopamine and L-3,4-dihydroxyphenylacetic acid in the striatum although lesser and higher doses were less effective. THC only tended to decrease these parameters. No changes were seen in dopaminergic activity in the limbic forebrain after either cannabimimetics. In summary, anandamide, as well as THC, decreases motor behavior. This effect was paralleled by reduction in the activity of nigrostriatal dopaminergic neurons. However, subtle differences in the behavioral and neurochemical effects between anandamide and THC could be observed.

Changes in rat brain cannabinoid binding sites after acute or chronic exposure to their endogenous agonist, anandamide, or to δ9-tetrahydrocannabinol

A brain constituent, the N-amide derivative of arachidonic acid, termed anandamide, has been recently proposed as a possible endogenous ligand for the cannabinoid receptor. The present study has been designed to examine whether the acute or chronic exposure to anandamide affected the binding of cannabinoid receptors in specific brain areas as occurred with the exogenous cannabinoid agonist, delta 9-tetrahydrocannabinol (THC). To this end, we measured the maximum binding capacity (Bmax) and the affinity (Kd) of cannabinoid receptors, by using [3H]CP-55,940 binding assays, in membranes obtained from several brain areas of male rats acutely or chronically treated with anandamide or THC. Results were as follows. The acute administration of either anandamide or THC increased the Bmax of cannabinoid receptors in the cerebellum and, particularly, in the hippocampus. This effect was also observed after 5 days of a daily exposure to either anandamide or THC. However, whereas the increase in the Bmax after the acute treatment seems to be caused by changes in the receptor affinity (high Kd), the increase after the chronic exposure may be attributed to an increase in the density of receptors. On the contrary, the [3H]CP-55,940 binding to cannabinoid receptors in the striatum, the limbic forebrain, the mesencephalon, and the medial basal hypothalamus was not altered after the acute exposure to anandamide or THC. However, the chronic exposure to THC significantly decreased the Bmax of these receptors in the striatum and nonsignificantly in the mesencephalon. This effect was not elicited after the chronic exposure to anandamide and was not accompanied by changes in the Kd.(ABSTRACT TRUNCATED AT 250 WORDS)

Motor disturbances induced by an acute dose of Δ9-tetrahydrocannabinol: Possible involvement of nigrostriatal dopaminergic alterations

Exposure to cannabinoids has been reported to affect several neurotransmitter systems and their related behaviors. The present study has been designed to further explore the effects of cannabinoids on motor behavior and test the involvement of nigrostriatal dopaminergic neurotransmission and other neurotransmitters as possible neurochemical targets for these cannabinoid effects. Male rats treated with an oral dose of delta 9-tetrahydrocannabinol (THC), the main psychoactive ingredient of cannabinoid derivatives, or vehicle were used 1 h after treatment for analyses of spontaneous motor and stereotypic activities together with neurochemical analyses of the nigrostriatal dopaminergic activity. Treatments and analyses were performed in the dark phase of photoperiod because it corresponds to the maximum behavioral expression in the rat. Neurochemical analyses were measurements of presynaptic activity--dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents, tyrosine hydroxylase (TH) activity, and in vitro DA release--and postsynaptic sensitivity--number and affinity of D1 and D2 receptors--in the striatum. In addition, measurements of 5-hydroxytryptamine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) contents were also performed to evaluate serotoninergic activity in the striatum. An oral dose of THC produced a loss of spontaneous motor activity, measured in both actimeter and open-field test, and a decrease in the frequency of several stereotypic behaviors, such as rearing and self-grooming. This decrease was correlated to a low number of D1-dopaminergic receptors in the striatum, although neither DA and DOPAC contents nor TH activity and D2 receptors were altered.(ABSTRACT TRUNCATED AT 250 WORDS)

An acute dose of δ9-tetrahydrocannabinol affects behavioral and neurochemical indices of mesolimbic dopaminergic activity

Cannabinoid consumption has been reported to affect several neurotransmitter systems and their related behaviors. The present study has been designed to examine cannabinoid effects on certain behaviors, which have been currently located in the limbic forebrain, in parallel to their effects on mesolimbic dopaminergic neurons. To this end, male rats treated with an oral dose of delta 9-tetrahydrocannabinol (THC) or vehicle were used 1 h after treatment for two different behavioral tests or neurochemical analyses of mesolimbic dopaminergic activity. Treatments, behavioral tests and sacrifice were performed in the dark phase of photoperiod because it corresponds to the maximum behavioral expression in the rat. Behavioral tests were a dark-light emergence test, which allows measurements of emotional reactivity, and a socio-sexual approach behavior test, which allows measurements of sexual motivation and also of spontaneous and stereotypic activities. Neurochemical analyses consisted of measurements of dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents, tyrosine hydroxylase activity, in vitro DA release and number and affinity of D1 receptors in the limbic forebrain. Results were as follows. THC exposure markedly altered the pattern executed by the animals in both tests. Concretely, THC-exposed animals exhibited a low number of visits to an incentive female in addition to high time spent in the vicinity of an incentive male, both observed in the socio-sexual approach behavior test, and an increased emergence latency to go out of a dark compartment in the dark-light emergence test. However, the fact that THC also decreased spontaneous activity and the frequency of rearing and self-grooming behaviors, in addition to the observations of either low total number of visits to both incentive sexual areas or high escape latency to go out of a light compartment, when the animal is placed in this compartment, also suggest the possible existence of an accompanying motor deficit. These behavioral effects were accompanied by increases in DA and DOPAC contents and in D1 receptor density in the limbic forebrain and to a slight decrease in the pattern of K(+)-evoked DA release in vitro from perifused limbic fragments, with no changes in the remaining neurochemical parameters. Collectively, these results allow us to conclude that acute THC markedly altered the behavioral pattern executed by the animals in a socio-sexual approach behavior test and in a dark-light emergence test, presumably indicating loss of sexual motivation and increased emotionality, although also accompanied by motor deficiencies.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute effects of δ-9-tetrahydrocannabinol on dopaminergic activity in several rat brain areas

Abstract In this work, we examined the acute effects of two doses of δ-9-tetrahydrocannabinol (THC) on several pre- and postsynaptic biochemical measures of dopaminergic activity in the striatum, limbic forebrain, and hypothalamic-anterior pituitary area of adult male rats. The exposure to a low dose of THC (0.5 mg/kg bw) decreased the number of striatal D 2 dopaminergic binding sites, but did not affect their affinity. Treatment with a higher dose of THC was ineffective. In addition, both doses decreased the number of D 1 dopaminergic binding sites in the limbic forebrain without changing their affinity. We did not find any changes in the dopamine (DA) or L -3,4-dihydroxyphenylacetic acid (DOPAC) content, or in the DOPAC/DA ratio, in either the striatum or limbic forebrain. THC treatment produced a dose-related decline in plasma prolactin (PRL) levels. Furthermore, both the basal and DA-inhibited in vitro release were PRL were reduced in animals exposed to THC in a dose-dependent manner. This inhibitory effect of THC on PRL release was accompanied by a decreased DOPAC/DA ratio in the medial basal hypothalamus that, in turn, may be a result of the fall in PRL levels rather than a direct action of the drug. These data show that acute exposure to THC can alter brain dopaminergic neurotransmission. Our results suggest that the reduction of PRL release following THC exposure, both in vivo and in vitro, might be elicited by a direct action of THC on the pituitary.

Comparisons between brain dopaminergic neurons of juvenile and aged rats: Sex-related differences

It is known that several aspects of dopaminergic neurotransmission deteriorate with advanced age. In the present report, we have studied the possible existence of sexual differences in these aging-induced changes. Thus, we measured several pre- and postsynaptic biochemical parameters, indicative of the activity of dopaminergic neurons, in striatum, limbic forebrain and hypothalamic-anterior pituitary area of aged (24-26 months) and young (2 months) rats of both sexes. Tyrosine hydroxylase (TH) activity, as well as the number of D2-dopaminergic receptors, decreased in the striatum of aged rats, especially in the males in which the decrease in the number of receptors was associated with an increase in their affinity. In addition, the ratio between dopamine (DA) and its intraneuronal metabolite, L-3,4-dihydroxyphenyl-acetic acid (DOPAC), which can be used as an index of neurotransmitter turnover, was increased in aged females in parallel with a decreased DA content. In the limbic forebrain, TH activity was also decreased during aging, but only in males, whereas the DOPAC/DA ratio was increased in females, although in parallel with an increased DOPAC production. Finally, in the hypothalamic-anterior pituitary area, aging only affected the females, in which increased plasma prolactin levels were observed. This effect might be the result of a low responsiveness of pituitary lactotrophs to DA released from hypothalamic neurons, in spite of high prolactin levels producing a constant, although ineffective, stimulation of the activity of these neurons, as reflected by the high DOPAC content and DOPAC/DA ratio observed in the medial basal hypothalamus. In summary, these data allow us to suggest that the activity of brain dopaminergic neurons is modified with aging and there are significant differences as a function of sex and brain area.

Changes in brain dopaminergic indices induced by perinatal exposure to cannabinoids in rats

Perinatal exposure to cannabinoid derivatives has been shown to produce effects on brain development. In this study, we evaluated the changes induced by maternal exposure to hashish crude extract (HCE) during gestation and lactation in several biochemical indices of dopamine activity in the striatum and the limbic forebrain of offspring. Studies were performed either during the HCE exposure or after drug withdrawal. Perinatal exposure to HCE reduced the number of striatal D1 binding sites in females and increased the L-3,4-dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratio, whereas an increase in the number of striatal D2 binding sites, with a reduction in their affinity, and a decrease in the activity of tyrosine hydroxylase (TH) were observed in males. The DOPAC/DA ratio was also increased in the limbic forebrain in HCE-exposed females, but there were no changes in binding site parameters. Most of these effects disappeared after cessation of cannabinoid treatment, but the decrease in striatal TH activity in males was maintained during drug withdrawal. Interestingly, the affinity of D2 receptors in the striatum of females, the number of striatal D1 receptors in males, and the limbic TH activity in males increased after the cessation of drug treatment. These results allow us to conclude that: (1) the effects of perinatal exposure to HCE were different depending on the sex and the specific brain area studied; and (2) most of the effects disappeared after cessation of cannabinoid treatment, although some new changes then appeared.