Changes in cannabinoid receptor binding and mRNA levels in several brain regions of aged rats.

We have recently found that cannabinoid receptor binding and gene expression markedly decreased in extrapyramidal structures of aged rats. The present study was designed to analyze the possible existence of similar aging-induced changes in cannabinoid receptor binding and gene expression in brain regions other than extrapyramidal areas, but that also contain a significant population of cannabinoid receptors, such as the cerebellum, hippocampal structures, limbic and hypothalamic nuclei, the cerebral cortex and others. To this end, we analyzed cannabinoid receptor binding, using autoradiography, and cannabinoid receptor mRNA levels, using in situ hybridization, in slide-mounted brain sections obtained from young (3 month old) and aged (> 2 year old) rats. Results were as follows. In the cerebellum, aged rats exhibited a marked decrease in cannabinoid receptor binding in the molecular layer (-33.3%), although accompanied by no changes in mRNA levels in the granular layer. In the cerebral cortex, a small, although statistically significant, decrease in binding was found in the deep layer (VI) (-18.3%) of aged rats, whereas no changes were found in the superficial layer (I). As in the case of the cerebellum, mRNA levels did not change in the cerebral cortex layers (II-III and V-VI). The different regions of the Ammon's horn of the hippocampus exhibited similar cannabinoid receptor binding levels in aged and young rats. Interestingly, mRNA levels decreased in aged rats to a small, but statistically significant, extent (CA1: -26.1%; CA2: -21.6%; CA3: -14.4%). This was also seen in another hippocampal structure, the dentate gyrus (-14.6%), although in this region binding levels increased in aged rats (+28.4%). Two hypothalamic structures, the arcuate nucleus and the ventromedial hypothalamic nucleus, exhibited decreased cannabinoid receptor binding in aged rats (-31.1% and -30.3%, respectively), but this was not seen in the medial preoptic area. This was accompanied by no changes in mRNA levels in the ventromedial hypothalamic nucleus. In the limbic structures, aged rats exhibited similar binding levels to young rats. This was seen in the nucleus accumbens, septum nuclei and basolateral amygdaloid nucleus. However, mRNA levels slightly decreased in the basolateral amygdaloid nucleus (-13.4%), whereas they were not altered in the septum nuclei. Finally, other brain structures, such as the central gray substance and the brainstem, exhibited similar binding levels in aged and young rats. However, it is important to note that mRNA levels increased significantly (+211.2%) in the brainstem of aged rats, an area where the levels of binding and mRNA were very low in young rats. This marked increase may be related to an increase in the presence of glial elements in this region, as revealed by the increase in the immunoreactivity for glial fibrillary acidic protein observed in the brainstem of aged rats as compared to young animals. In summary, senescence was associated with changes in cannabinoid receptors in the cerebellum, the cerebral cortex, limbic and hypothalamic structures, the hippocampus and other brain regions. However, the changes observed (i) were not as marked and relevant as those early reported in extrapyramidal areas, and (ii) exhibited regional differences that might be attributed to the different roles played by these receptors in each region. Of particular relevance by their magnitude were the aging-induced decrease in binding found in the cerebellum and the hypothalamus, and the increase in mRNA levels observed in the brainstem. The latter might be related to an increase in the presence of glial cells which might contain cannabinoid receptor mRNA.

Pharmacological and biochemical interactions between opioids and cannabinoids.

Opioids and cannabinoids are among the most widely consumed drugs of abuse in humans. A number of studies have shown that both types of drugs share several pharmacological properties, including hypothermia, sedation, hypotension, inhibition of both intestinal motility and locomotor activity and, in particular, antinociception. Moreover, phenomena of cross-tolerance or mutual potentiation of some of these pharmacological effects have been reported. In recent years, these phenomena have supported the possible existence of functional links in the mechanisms of action of both types of drugs. The present review addresses the recent advances in the study of pharmacological interactions between opioids and cannabinoids, focusing on two aspects: antinociception and drug addiction. The potential biochemical mechanisms involved in these pharmacological interactions are also discussed together with possible therapeutic implications of opioid-cannabinoid interactions.

Sex- and age-related changes in catecholamine metabolism and release of rat adrenal gland.

To examine the possible existence of changes in the adrenal catecholaminergic activity during aging, we analyzed the adrenal content of catecholamines (CA) and the activities of selected enzymes involved in their metabolism as well as the basal and the K+-stimulated release of these CA in incubated adrenal tissue of aged (greater than 22 months) and young (2 months) rats of both sexes. Adrenal contents of norepinephrine (NE) and epinephrine (E) of male rats were unaltered in aging, although aged males showed an increased activity of tyrosine hydroxylase (TH) and a decrease in phenylethanolamine-N-methyl transferase (PNMT) activity. In addition, the in vitro release of both CA as well as their content in the incubated adrenal tissue were higher in aged males than in young animals. However, the response of the adrenal of aged males to incubation with stimulatory concentrations of K+ was significantly lower than that observed in young males. Aged females showed increases in the adrenal content of E, although the activities of TH and PNMT were unaltered. As in aged males, the in vitro release of CA from incubated adrenal tissue was higher in aged females than in young rats, but the CA amounts measured in the incubated tissues were similar. Moreover, the response to stimulatory concentrations of K+ was lower in aged females than in young animals. In summary, these results clearly indicate that adrenal catecholaminergic activity is enhanced during aging, which could have important consequences for physiological functions regulated by the adrenal secretion. Also, some differences in the effects of aging could be observed between males and females.

The endocannabinoid system and Huntington's disease.

The research in Huntington's disease (HD) has been growing exponentially during the last decade, since the discovery of the genetic basis that leads to neurodegeneration. HD is one of several progressive neurodegenerative disorders, in which the underlying mutation is a CAG expansion encoding a polyglutamine tract in a specific protein, which in the case of HD, is called huntingtin. The first clinical symptoms of HD are generally psychiatric abnormalities, most commonly depression and mood disturbances. Involuntary choreiform movements and dementia develop over the next 15-20 years, and death generally results from complications derived from immobility. There is currently no cure, or even an effective therapy to offset the decline in mental and motor capabilities suffered by those affected by HD, but recent studies have started to examine the usefulness of different classes of new compounds. Among these, plant-derived, synthetic or endogenous cannabinoids have been proposed to have therapeutic value for the treatment of HD, since they act on cannabinoid CB(1) receptors located in the basal ganglia circuitry, that is affected by the striatal atrophy typical of HD. Recent studies have characterized the changes in these receptors, as well as their endogenous ligands, in the basal ganglia in a variety of animal models of HD. The results are indicative that the endocannabinoid system becomes hypofunctional in this disease, which could be related to the hyperkinesia typical of the earliest phases of this disease. In addition, it has been proposed that the loss of these receptors might be involved in the process of pathogenesis itself. This, together with the well-known protective properties of cannabinoid-related compounds, suggest that, in addition to a symptomatic usefulness, cannabinoids might also serve to delay or to arrest the development of this disease. The present article will review all recent data dealing with the biochemical, pharmacological and therapeutic bases that support a potential role of cannabinoids in the pathogenesis and/or therapeutic treatment of this motor disorder.

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.

Loss of cannabinoid receptor binding and messenger RNA levels and cannabinoid agonist-stimulated [35S]guanylyl-5'O-(thio)-triphosphate binding in the basal ganglia of aged rats.

Recent reports have demonstrated that cannabinoid receptor binding decreases in several neurodegenerative diseases related to extrapyramidal function. However, there is little evidence with regard to potential changes of these receptors during senescence. The present study was designed to determine the possible existence of ageing-induced changes in cannabinoid receptor binding and gene expression in extrapyramidal areas. To this end, we analysed cannabinoid receptor binding and basal and cannabinoid receptor agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate binding, by using autoradiography, and cannabinoid receptor messenger RNA levels, by using in situ hybridization, in slide-mounted brain sections obtained from young (three-month-old) and aged (>two-year-old) rats. Results were as follows. Aged rats exhibited a marked decrease in cannabinoid receptor binding in most of the basal ganglia, excepting the globus pallidus which had similar binding levels in both young and aged rats. The highest decreases were in the entopeduncular nucleus (-49.6%) and substantia nigra pars reticulata (-45.2%), whereas more moderate decreases were found in the lateral caudate putamen (-29%) and only a decremental trend in the medial caudate putamen (-13.1%). These decreases in cannabinoid receptor binding were paralleled by less marked increases in WIN 55212-2-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate binding in these structures in aged rats (% of agonist stimulation: 189% in the substantia nigra; 29.4% in the lateral caudate putamen) as compared to young rats (296% and 53.2%, respectively). Contrarily, the percentage of agonist stimulation was similar in the globus pallidus, an area where cannabinoid receptor binding did not change during ageing, of aged (205.5%) and young (215.5%) rats. In addition, aged rats also exhibited significant reductions in the cannabinoid receptor messenger RNA levels in the medial (-14.3%) and, in particular, in the lateral (-29.4%) caudate putamen, the area where the cell bodies of cannabinoid receptor-containing neurons, projecting to the substantia nigra, entopeduncular nucleus and globus pallidus, are located. In summary, the synthesis and binding levels of cannabinoid receptors markedly dropped in different structures of the extrapyramidal system of aged rats. Since these receptors, located in the basal ganglia, seem to play a role in motor control, this loss of cannabinoid receptors might be related to the motor impairment which progressively appears during senescence.

Effects of cannabinoids on adrenaline release from adrenal medullary cells.

1. The objective of the present study was to analyse the peripheral effects of cannabinoids on adrenaline release from adrenal chromaffin cells. 2. In pithed rabbits with electrically stimulated sympathetic outflow, intravenous injection of the cannabinoid receptor agonists WIN55212-2 and CP55940 (5, 50 and 500 microg x kg(-1)) markedly lowered the plasma adrenaline concentration. The effect of WIN55212-2 was attenuated by the selective CB1 cannabinoid receptor antagonist SR141716A (500 microg x kg(-1)). WIN55212-3 (same doses as WIN55212-2), the enantiomer of WIN55212-2 lacking affinity for cannabinoid receptors, had no effect on the plasma adrenaline concentration. 3. In rabbit isolated adrenal glands, the release of adrenaline elicited by electrical stimulation was measured by fast cyclic voltammetry. Electrically-evoked adrenaline release was inhibited by WIN55212-2 (0.3, 1, 3 and 10 microM) and this effect was antagonized by SR141716A (1 microM). The non-cholinergic component of adrenaline release observed after blockade of nicotinic (by hexamethonium 100 microM) and muscarinic (by atropine 0.5 microM) acetylcholine receptors was not depressed by WIN55212-2. WIN55212-3 (10 microM) had no effect on adrenaline release. 4. No detectable specific CB1 receptor binding and mRNA expression were found in rabbit adrenal glands with autoradiography and in situ hybridization. 5. The results show that cannabinoids inhibit adrenaline secretion in rabbit isolated adrenal glands; the likely mechanism is a presynaptic CB1 receptor-mediated inhibition of acetylcholine release from preganglionic sympathetic neurons. The inhibition of adrenaline secretion in adrenal glands most probably accounts for the decrease in the plasma adrenaline concentration observed after cannabinoid administration in pithed rabbits.

Possible role of dopamine and noradrenaline in the regulation of prolactin secretion from an ectopic anterior pituitary gland in female rats.

It was recently reported that anterior pituitary tissue transplanted to an ectopic site contains measurable amounts of dopamine and noradrenaline. To examine the possibility of local catecholaminergic control of prolactin secretion from ectopic pituitaries, pituitary grafted and sham-operated female rats were submitted to several pharmacological treatments modifying catecholamine synthesis. Administration of a single dose of alpha-methyl-p-tyrosine (alpha-MPT) significantly reduced dopamine content in the graft, while noradrenaline content was not modified. Similar changes in the contents of dopamine and noradrenaline after alpha-MPT administration were observed in the hypothalamus and in the in-situ pituitary in both grafted and sham-operated rats. Plasma concentrations of prolactin were increased in both grafted and sham-operated rats after administration of alpha-MPT. A single injection of L-3,4-dihydroxyphenylalanine (L-DOPA) increased dopamine content in the ectopic pituitary gland without altering the noradrenaline content, and produced similar effects in the hypothalamus and in-situ pituitary of grafted and control rats. Plasma prolactin concentrations were decreased by L-DOPA in both pituitary grafted and control rats. Administration of DL-treo-dihydroxyphenylserine (DOPS) increased noradrenaline content in the ectopic pituitary and reduced plasma prolactin concentrations in pituitary grafted rats. In contrast, injection of DOPS to control rats increased both hypothalamic noradrenaline content and plasma prolactin concentrations. These results suggest that dopamine and noradrenaline present in the ectopic pituitary tissue have a role in mediating prolactin release from pituitary transplants.

Effects of anandamide on hepatic fatty acid metabolism.

Incubation of rat hepatocytes with anandamide (arachidonoylethanolamide) inhibited acetyl-CoA carboxylase activity and fatty acid synthesis de novo without affecting fatty acid synthase. This was concomitant to a decrease in the intracellular levels of malonyl-CoA. Likewise, anandamide depressed both cholesterol synthesis de novo and the incorporation of exogenous palmitate into triacylglycerols and phospholipids. On the other hand, anandamide stimulated in parallel both carnitine palmitoyltransferase I activity and ketogenesis from palmitate, though ketogenesis from octanoate was unaffected. The effects of anandamide on hepatic fatty acid synthesis and oxidation were: (a) mimicked by arachidonic acid, a product of anandamide breakdown by anandamide amidase; (b) prevented by phenylmethylsulfonyl fluoride, an inhibitor of anandamide amidase; and (c) not affected by bisindolylmaleimide, a specific inhibitor of protein kinase C. Furthermore, delta 9-tetrahydrocannabinol had no effect on any of the parameters determined, ruling out the possibility that the effects of anandamide on hepatic fatty acid metabolism are mediated by the peripheral cannabinoid receptor. The results thus indicate that anandamide might function as a carrier of arachidonic acid in the modulation of hepatic fatty metabolism.

Possible role of the cytochrome P450-linked monooxygenase system in preventing delta 9-tetrahydrocannabinol-induced stimulation of tuberoinfundibular dopaminergic activity in female rats.

The administration of delta 9-tetrahydrocannabinol (THC) or related cannabinoids markedly affected neurobehavioral and neuroendocrine indices in male rodents but usually failed to affect those indices in females. We examined whether inhibition of the cytochrome P450-linked monooxygenase system in female rats is able to elicit the effects of THC on one of the most characteristic targets of cannabinoid action, tuberoinfundibular dopaminergic neurons, whose activity is known to increase after cannabinoid exposure in males. It was found that the administration of THC to ovariectomized rats acutely replaced with estradiol (to discard problems derived from differences in the estrogenic status) did not affect either dopamine and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents and tyrosine hydroxylase activity in the medial basal hypothalamus or the density of D2-dopaminergic receptors in the anterior pituitary. However, the administration of THC to estrogen-replaced ovariectomized rats that had been pretreated with two separately administered inhibitors of cytochrome P450, piperonyl butoxide or metyrapone, significantly increased DOPAC content in the medial basal hypothalamus, with no changes in the other parameters. Collectively, these results indicate that the metabolism of THC to inactive compounds might play a protective role in females, counteracting the effects of this cannabinoid on tuberoinfundibular dopaminergic activity because pharmacological inhibition of cytochrome P450-linked monooxygenase system elicited a significant stimulation of these neurons by THC.

Time course of the effects of different cannabimimetics on prolactin and gonadotrophin secretion: evidence for the presence of CB1 receptors in hypothalamic structures and their involvement in the effects of cannabimimetics.

Several reports have demonstrated that (-)-delta9-tetrahydrocannabinol (delta9-THC) and arachidonylethanolamide [anandamide (AEA)] were able to inhibit prolactin (PRL) secretion from the anterior pituitary gland in male rodents, whereas ovarian phase-dependent effects were seen in females. However, in most of these studies, the analysis of PRL levels was performed at times longer than 30 min after cannabinoid administration. In the present study, we examined the time course of the effects of three different cannabimimetics, delta9-THC, AEA, and AM356 (R-methanandamide), a more stable analog of AEA, on PRL and gonadotrophin secretion in male Wistar rats. In addition, we characterized the presence of cannabinoid receptors in hypothalamic structures related to neuroendocrine control and studied their potential involvement in the effects of cannabimimetics. We found that the three compounds decreased plasma luteinizing hormone (LH) levels, although only the effects of delta9-THC were statistically significant. The inhibitory effect was already apparent at 40 min after administration, but only in the case of delta9-THC did it persist up to 180 min after administration. No significant changes were seen in plasma follicle-stimulating hormone (FSH) levels after the administration of any of the three different cannabimimetics at any of the four times analyzed. Both AEA and AM356 produced a significant decrease in plasma PRL levels, which appeared at 20 min after administration and persisted up to 60 min, waning after this time. Interestingly, the time course of the effect of delta9-THC resembled that of AEA and AM356 only during the later part of the response, because delta9-THC produced a marked increase in plasma PRL levels at 20 min, no changes at 40 min and a decrease from 60 min up to 180 min. In additional experiments, we tried to elucidate which of these two phases observed after delta9-THC administration was mediated by the activation of cannabinoid receptors. These receptors are present in hypothalamic structures related to neuroendocrine control, with the highest densities in the arcuate nucleus (dorsal area) and the medial preoptic area, and the lowest in the lateral hypothalamic area, although none of these regions exhibited high densities for this receptor as compared with classical regions containing cannabinoid receptors, such as the basal ganglia. The activation of these receptors by delta9-THC seems to be involved in the inhibitory phase of the effect of this cannabinoid on PRL release, but not in the early stimulation; when these receptors were blocked with a specific antagonist, SR141716, the stimulation by delta9-THC was still observed, but the late inhibition was abolished. In summary, AEA and AM356 markedly decreased PRL release and slightly decreased LH secretion, with no changes on FSH release. delta9-THC also produced a marked inhibition of LH secretion, but its effects on PRL were biphasic with an early stimulation not mediated by the activation of cannabinoid receptors, followed by a late and cannabinoid receptor-mediated inhibition. Their site of action may well be the hypothalamic structures related to neuroendocrine control, which contain a small, but probably very active, population of cannabinoid receptors.

Effects of cannabinoids on prolactin and gonadotrophin secretion: involvement of changes in hypothalamic gamma-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.

Effects of perinatal exposure to delta 9-tetrahydrocannabinol on the fetal and early postnatal development of tyrosine hydroxylase-containing neurons in rat brain.

The exposure of pregnant rats to delta 9-tetrahydrocannabinol (delta 9-THC), the main psychoactive constituent of Cannabis sativa, during the perinatal period affects the gene expression and the activity of tyrosine hydroxylase (TH) in the brains of their offspring at peripubertal and adult ages. In the present work we explored whether these effects also appear during fetal and early neonatal periods, when TH expression plays an important role in neural development. To this end, the mRNA amounts for TH and the amounts and activity of this enzyme, in addition to catecholamine (CA) contents, were analyzed in the brain of fetuses at different gestational days (GD) and of newborns at two postnatal ages, which had been daily exposed to delta 9-THC or vehicle from d 5 of gestation. Results were as follows. The exposure to delta 9-THC markedly affected the expression of the TH gene in the brain of fetuses at GD 14. Thus, the amounts of its mRNA at this age were higher in delta 9-THC-exposed fetuses than in controls. This corresponded with a marked rise in the amounts of TH protein and in the activity of this enzyme at this age. Normalization was found in these parameters at GD16. However, a marked sexual dimorphism in the response of TH gene to cannabinoid exposure appeared from GD18 and was particularly evident at GD21, when TH-mRNA amounts increased in developing female brains, but decreased in developing male brains exposed to delta 9-THC, effects that were mostly prolonged to early postnatal ages. However, these changes did not correspond always with parallel changes in the amounts and activity of TH and in CA contents, as occurred in GD14, suggesting that delta 9-THC would not be affecting the basal capability to synthesize CAs in TH-containing neurons, but would affect the responsiveness of TH gene. We found only a marked increase in the production of L-3,4-dihydroxyphenylacetic acid, the main intraneuronal dopamine metabolite, in female newborns exposed to delta 9-THC. Collectively, our results support the belief that the perinatal exposure to delta 9-THC affects the expression of the TH gene and, sometimes, the activity of this enzyme in brain catecholaminergic neurons in certain critical periods of fetal and early neonatal brain development. These results support the notion that cannabinoids are able to affect the gene expression of specific key proteins for catecholaminergic development, and that these alterations might be the origin of important long-term neurobehavioral effects caused by perinatal cannabinoid exposure at peripubertal and adult ages.

delta 9-Tetrahydrocannabinol increases activity of tyrosine hydroxylase in cultured fetal mesencephalic neurons.

The exposure of pregnant rats to delta 9-tetrahydrocannabinol (delta 9-THC), the main psychoactive constituent of Cannabis sativa, during gestation and lactation, affects the gene expression and the activity of tyrosine hydroxylase (TH) in the brain of their offspring, measured at fetal and early postnatal ages, when the expression of this enzyme plays an important role in neural development. In the present article, we have examined whether delta 9-THC is able to affect TH activity in cultured mesencephalic neurons obtained from fetuses at gestational d 14. Thus, TH activity increased approximately twofold in cells obtained from naive fetuses when exposed for 24 h to medium containing delta 9-THC. In addition, TH activity was also approx twofold higher in cells obtained from fetuses exposed daily to delta 9-THC from d 5 of gestation than in cells obtained from control fetuses, when both were exposed to basal media. This effect of delta 9-THC on TH activity seems to be produced via the activation to cannabinoid receptors, in particular the CB1 subtype, which would presumably be located in these cells. This is because the exposure to medium containing both delta 9-THC and SR141716A, a specific antagonist for CB1 receptors, abolished the effect observed with delta 9-THC alone. SR141716A alone was without effect on TH activity. Collectively, our results support the notion that delta 9-THC increased TH activity in cultured mesencephalic neurons, as previously observed in vivo, and that this effect was produced by activation of CB1 receptors, which seem to be operative at these early ages. All this points to a role for the endogenous cannabimimetic system in brain development.

Cannabinoid receptor and WIN-55,212-2-stimulated [35S]GTP gamma S binding and cannabinoid receptor mRNA levels in the basal ganglia and the cerebellum of adult male rats chronically exposed to delta 9-tetrahydrocannabinol.

The inhibition of motor behavior in rodents caused by the exposure to plant or synthetic cannabinoids has been reported to develop tolerance after repeated exposure. This tolerance seems to have a pharmacodynamic basis, since downregulation of cannabinoid receptors in motor areas, basal ganglia and cerebellum, has been demonstrated in cannabinoid-tolerant rats. The present study was designed to further explore this previous evidence by analyzing simultaneously in several motor areas of delta 9-tetrahydrocannabinol- (delta 9-THC)-tolerant rats: 1. Cannabinoid receptor binding, by using [3H]WIN-55,212-2 autoradiography; 2. Cannabinoid receptor activation of signal transduction mechanisms, by using WIN-55,212-2-stimulated [35S]-guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]-GTP gamma S) autoradiography; 3. Cannabinoid receptor mRNA expression, quantitated by in situ hybridization. Results were as follows. As expected, the exposure to delta 9-THC for 5 d resulted in a decrease of cannabinoid receptor binding in the molecular layer of the cerebellum, medial, and lateral caudate-putamen and, in particular, entopeduncular nucleus. We also found decreased cannabinoid receptor binding in the superficial and deep layers of the cerebral cortex, two regions used as a reference to test the specificity of changes observed in motor areas. There were only two brain regions, the globus pallidus and the substantia nigra, where the specific binding for cannabinoid receptors was unaltered after 5 d of a daily delta 9-THC administration. However, in the substantia nigra, the magnitude of WIN-55,212-2-stimulated [35S]-GTP gamma S binding was lesser in delta 9-THC-tolerant rats than controls, thus suggesting a possible specific change at the level of receptor coupling to GTP-binding proteins. This was not seen neither in the globus pallidus nor in the lateral caudate-putamen, where agonist stimulation produced similar [35S]-GTP gamma S binding levels in delta 9-THC-tolerant rats and controls. Finally, animals chronically exposed to delta 9-THC also exhibited a decrease in the levels of cannabinoid receptor mRNA in the medial and lateral caudate-putamen, but there were no changes in the cerebellum (granular layer) and cerebral cortex. In summary, the chronic exposure to delta 9-THC resulted in a decrease in cannabinoid receptor binding and mRNA levels in the caudate-putamen, where cell bodies of cannabinoid receptor-containing neurons in the basal ganglia are located. However, this decrease particularly affected the receptor binding levels in those neurons projecting to the entopeduncular nucleus, but not in those projecting to the globus pallidus and substantia nigra, although, in this last region, a specific decrease in the efficiency of receptor activation of signal transduction mechanisms was seen in delta 9-THC-tolerant rats. The chronic exposure to delta 9-THC also resulted in decreased cannabinoid receptor binding in the cerebellum, although without affecting mRNA expression.

Chronic administration of cannabinoids regulates proenkephalin mRNA levels in selected regions of the rat brain.

This study was designed to examine the interactions between the cannabinoid and enkephalinergic systems in the rat brain. To this aim, we have examined the effects of subchronic (5 days) administration (10 mg.kg-1.day-1; i.p.) of delta 9 -tetrahydrocannabinol (THC) or R-methanandamide (AM356) and chronic (18 days) administration with the synthetic cannabinoid receptor agonist CP-55,940 (1 mg.kg-1.day-1; i.p) on proenkephalin (PENK) mRNA levels in several brain regions of the rat. Twenty micrometer brain sections from striatum, nucleus accumbens, paraventricular nucleus, ventromedial nucleus, periaqueductal grey matter and mammillary nucleus were hybridized with an oligonucleotide probe complementary to PENK using in situ hybridization technique. Subchronic administration of THC or AM356 increased PENK mRNA levels in the ventromedial nucleus of the hypothalamus, (82%) and (39%), in the periaqueductal grey matter, (97%) and (49%), and mammillary nucleus, (43%) and (9%), respectively. In contrast, both drugs were without effect in the striatum and nucleus accumbens. On the other hand, chronic administration of CP-55,940 increased PENK mRNA levels in the striatum (44%), nucleus accumbens (25%), paraventricular (31%) and ventromedial nuclei of the hypothalamus (41%). These results revealed that chronic cannabinoid administration increases opioid gene expression in the rat central nervous system and suggest an interaction between the cannabinoid and enkephalinergic systems that may be part of a molecular integrative response to behavioral and neurochemical alterations that occur in cannabinoid drug abuse.

Effects of chronic exposure to delta9-tetrahydrocannabinol on cannabinoid receptor binding and mRNA levels in several rat brain regions.

Previous data showed the development of tolerance to a variety of pharmacological effects of plant and synthetic cannabinoids when administered chronically. This tolerance phenomenon has been related both to enhancement of cannabinoid metabolism and, in particular, to down-regulation of brain CB1 cannabinoid receptors, although this has been only demonstrated in extrapyramidal areas. In the present study, we have tested, by using autoradiographic analysis of CB1 receptor binding combined with analysis of CB1 receptor mRNA levels in specific brain regions by Northern blot, whether the reduction in binding levels of CB1 receptors observed in extrapyramidal areas after a chronic exposure to delta9-tetrahydrocannabinol (delta9-THC), also occurs in most brain areas that contain these receptors. Results were as follows. The acute exposure to delta9-THC usually resulted in no changes in the specific binding of CB1 receptors in all brain areas studied, discarding a possible interference in binding kinetic of the pre-bound administered drug. The only exceptions were the substantia nigra pars reticulata and the cerebral cortex, which exhibited decreased specific binding after the acute treatment with delta9-THC presumably due to an effect of the pre-bound drug. The specific binding measured in animals chronically (5 days) exposed to delta9-THC decreased ranging from approximately 20 up to 60% of the specific binding measured in control animals in all brain areas. Areas studied included cerebellum (molecular layer), hippocampus (CA1, CA2, CA3, CA4 and dentate gyrus), basal ganglia (medial and lateral caudate-putamen and substantia nigra pars reticulata), limbic nuclei (nucleus accumbens, septum nucleus and basolateral amygdaloid nucleus), superficial (CxI) and deep (CxVI) layers of the cerebral cortex and others. There were only two brain regions, the globus pallidus and the entopeduncular nucleus, where the specific binding for CB receptors was unaltered after 5 days of a daily delta9-THC administration. In addition, we have analyzed the levels of CB1 receptor mRNA in specific brain regions of animals chronically exposed to delta9-THC, in order to correlate them with changes in CB1 receptor binding. Thus, we observed a significant increase in CB1 receptor mRNA levels, but only in the striatum, with no changes in the hippocampus and cerebellum. In summary, CB1 receptor binding decreases after chronic delta9-THC exposure in most of the brain regions studied, although this was not accompanied by parallel decreases in CB receptor mRNA levels. This might indicate that the primary action of delta9-THC would be on the receptor protein itself rather than on the expression of CB1 receptor gene. In this context, the increase observed in mRNA amounts for this receptor in the striatum should be interpreted as a presumably compensatory effect to the reduction in binding levels observed in striatal outflow nuclei.

Immunocytochemical demonstration of CB1 cannabinoid receptors in the anterior lobe of the pituitary gland.

Both exogenous and endogenous cannabinoids can influence hormone secretion from the anterior pituitary gland. A large body of information proves that the primary target of these effects is the neuroendocrine hypothalamus. However, recent studies using cannabinoid (CB) receptor autoradiography, messenger RNA in-situ hybridization and in-vitro analysis, indicate direct effects of cannabinoids at the level of the anterior pituitary gland itself. In the present paper, the immunocytochemical distribution of CB in the adult rat anterior pituitary was studied using specific polyclonal antibodies against CB1 (central) and CB2 (peripheral) receptors. Due to its resolution, this method allowed identification of individual anterior pituitary cells possessing cannabinoid receptors. The specific hormone immunoreactive cells with receptor-like immunoreactivity were compared on adjacent sections. CB1-like immunoreactivity (CB1ir) was found in the lactotroph cells as well as in luteinizing hormone (LH) secreting gonadotrophs. The CB1ir positive material present in the cytoplasm of these cells was less homogeneous than the hormone immunoreactive material, and it was also seen at the periphery of the cells, presumably on the cell membrane. No CB1ir was found in growth hormone (GH) secreting cells and it was hardly seen in the corticotrophs. No CB1ir was detected in the posterior pituitary. CB2ir was not observed in any part of the pituitary gland. The results support the view that the site of action of cannabinoids on neuroendocrine regulatory mechanisms may be both at pituitary and hypothalamic levels. We suggest that at least the direct effect of cannabinoids on the regulation of LH and prolactin secretion is mediated via CB1 cannabinoid receptors in the anterior pituitary.

Cannabinoid receptor and WIN-55,212-2-stimulated [35S]GTPgammaS binding and cannabinoid receptor mRNA levels in several brain structures of adult male rats chronically exposed to R-methanandamide.

We and others have recently demonstrated that the pharmacological tolerance observed after prolonged exposure to plant and synthetic cannabinoids in adult individuals seems to have a pharmacodynamic basis, based on the observed down-regulation of cannabinoid receptors in the brain of cannabinoid-tolerant rats. However, we were unable to elicit a similar receptor down-regulation after a chronic exposure to anandamide, the first discovered endogenous cannabinoid, possibly because of its rapid metabolic breakdown in arachidonic acid and ethanolamine. The present study was designed to progress in these previous studies, by using R-methanandamide, a more stable analog, instead anandamide. In addition, we examined not only cannabinoid receptor binding, but also WIN-55,212-2-stimulated [35S]-GTPgammaS binding, by autoradiography, and cannabinoid receptor mRNA levels, by in situ hybridization. Results were as follows. The daily administration of R-methanandamide for a period of five days produced decreases in cannabinoid receptor binding in the lateral caudate-putamen, cerebellum, entopeduncular nucleus and substantia nigra. The remaining areas, the medial caudate-putamen, globus pallidus, cerebral cortex (layers I and VI), hippocampus (dentate gyrus and Ammon's horn) and several limbic structures (nucleus accumbens, septum nuclei and basolateral amygdaloid nucleus), exhibited no changes in cannabinoid receptor binding. Similarly, the levels of cannabinoid receptor mRNA expression decreased in the lateral and medial caudate-putamen and in the CA1 and CA2 subfields of the Ammon's horn in the hippocampus after the chronic exposure to R-methanandamide, whereas the remaining areas showed no changes. WIN-55,212-2-stimulated [35S]-GTPgammaS binding did not change in the lateral caudate-putamen, cerebral cortex (layer I), septum nuclei and hippocampal structures (dentate gyrus and Ammon's horn) of animals chronically exposed to R-methanandamide, whereas a certain trend to decrease could be observed in the substantia nigra and deep layer (VI) of the cerebral cortex in these animals. In summary, as reported for other cannabinoid receptor agonists, the prolonged exposure of rats to R-methanandamide, a more stable analog of anandamide, was able to produce cannabinoid receptor-related changes in contrast with the absence of changes observed early with the metabolically labile anandamide. The observed changes exhibited an evident regional pattern with areas, such as basal ganglia, cerebellum and hippocampus, responding to chronic R-methanandamide treatment while regions, such as the cerebral cortex and limbic nuclei, not responding.

Presence of cannabinoid binding sites in the brain from early postnatal ages.

The present study demonstrates the presence of cannabinoid receptors in the brain from early postnatal ages. Specific and saturable binding was observed in the forebrain and remaining brain from early postnatal ages (2 and 5 days after birth). Female neonate forebrain exhibited a higher receptor density at 2 days after birth than males, but this trend was inverted at 5 days. From postnatal day 10, the receptors could be measured in more defined brain areas, i.e. the striatum, limbic forebrain and ventral mesencephalon. The ontogeny of the receptors in these three areas was relatively similar, exhibiting a progressive increase which maximised on days 30 or 40 and then subsequently decreased to adult values. Subtle sexual dimorphism was found in the striatum and ventral mesencephalon but not the limbic forebrain.