Lucı́a Garcı́a-Gil

Effects of chronic exposure to Δ9-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 Δ9-tetrahydrocannabinol (Δ9-THC), also occurs in most brain areas that contain these receptors. Results were as follows. The acute exposure to Δ9-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 Δ9-THC presumably due to an effect of the pre-bound drug. The specific binding measured in animals chronically (5 days) exposed to Δ9-THC decreased ranging from ≈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 CB1 receptors was unaltered after 5 days of a daily Δ9-THC administration. In addition, we have analyzed the levels of CB1 receptor mRNA in specific brain regions of animals chronically exposed to Δ9-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 Δ9-THC exposure in most of the brain regions studied, although this was not accompanied by parallel decreases in CB1 receptor mRNA levels. This might indicate that the primary action of Δ9-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.

Atypical Location of Cannabinoid Receptors in White Matter Areas during Rat Brain Development

Previous evidence suggests that the endogenous cannabinoid system could emerge and be operative early during brain development. In the present study, we have explored the distribution of specific binding for cannabinoid receptors in rat brain at gestational day 21 (GD21), postnatal days 5 (PND5) and 30 (PND30), and at adult age (>70 days after birth) by using autoradiography with [3H]CP‐55,940. Our results indicated that specific binding for cannabinoid receptors can be detected in the brain of rat fetuses at GD21 in the classic areas that contain these receptors in adulthood—in particular, in the cerebellum and the hippocampus and, to a lesser extent, in the basal ganglia, several limbic structures, and cerebral cortex. The density of cannabinoid receptors in all these structures increased progressively at all postnatal ages studied until reaching the classical adult values in 70‐day‐old animals. Interestingly, cannabinoid receptor binding can also be detected at GD21 in regions, in which they are scarcely distributed or not located in the adult brain and that have the particularity of all being enriched in neuronal fibers. Among these were the corpus callosum, anterior commissure, stria terminalis, fornix, white matter areas of brainstem, and others. This atypical location was quantitatively high at GD21, tended to wane at PND5, and practically disappeared at PND30 and in adulthood, with the only exception being the anterior commissure, which exhibited a moderate density for cannabinoid receptors. Moreover, the binding of [3H]CP‐55,940 to cannabinoid receptors in the white matter regions at GD21 seems to be functional and involves a GTP‐binding protein‐mediated mechanism. Thus, the activation of these receptors with an agonist such as WIN‐55,212‐2 increased the binding of [35S]‐guanylyl‐5′‐O‐(γ‐thio)‐triphosphate, measured by autoradiography, in the corpus callosum and white matter areas of brainstem of fetuses at GD21. This increase was reversed by coincubation of WIN‐55,212‐2 with SR141716, a cannabinoid receptor antagonist. As this antagonist is specific for the cerebral cannabinoid receptor subtype, called CB1, we can assert that the signal found for cannabinoid receptor binding in the fetal and early postnatal brain likely corresponds to this receptor subtype. Collectively, all these data suggest the existence of a transient period of the brain development in the rat, around the last days of the fetal period and the first days of postnatal life, in which CB1 receptors appear located in neuronal fiber‐enriched areas. During this period, CB1 receptors would be already functional acting through a GTP‐binding protein‐mediated mechanism. After this transient period, they progressively acquire the pattern of adult distribution. All this accounts for a specific role of the endogenous cannabinoid system in brain development. Synapse 26:317–323, 1997.

Maternal exposure to Δ9-tetrahydrocannabinol facilitates morphine self-administration behavior and changes regional binding to central μ opioid receptors in adult offspring female rats

Opiates and cannabinoids are among the most widely consumed habit-forming drugs in humans. Several studies have demonstrated the existence of interactions between both kind of drugs in a variety of effects and experimental models. The present study has been focused to determine whether perinatal delta9-tetrahydrocannabinol (Delta9-THC) exposure affects the susceptibility to reinforcing effects of morphine in adulthood and whether these potential changes were accompanied by variations in mu opioid receptor binding in brain regions related to drug reinforcement. Adult female rats born from mothers that were daily treated with delta9-THC during gestation and lactation periods, exhibited a statistically significant increase in the rate of acquisition of intravenous morphine self-administration behavior when compared with females born from vehicle-exposed mothers, an effect that did not exist in delta9-THC-exposed male offspring. This increase was significantly greater on the last day of acquisition period. There were not significant differences when the subjects were lever pressing for food. In parallel, we have also examined the density of mu opioid receptors in the brain of adult male and female offspring that were exposed to Delta9-THC during the perinatal period. Collectively, perinatal exposure to delta9-THC produced changes in mu opioid receptor binding that differed regionally and that were mostly different as a function of sex. Thus, delta9-THC-exposed males exhibited a lower density for these receptors than their respective oil-exposed controls in the caudate-putamen area as well as in the amygdala (posteromedial cortical nucleus). On the contrary, delta9-THC-exposed females exhibited higher density of these receptors than their respective oil-exposed controls in the prefrontal cortex, the hippocampus (CA3 area), the amygdala (posteromedial cortical nucleus), the ventral tegmental area and the periaqueductal grey matter, whereas the binding was lower than control females only in the lateral amygdala. These results support the notion that perinatal delta9-THC exposure alters the susceptibility to morphine reinforcing effects in adult female offspring, in parallel with changes in mu opioid receptor binding in several brain regions.

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.

Perinatal Δ9-tetrahydrocannabinol exposure reduces proenkephalin gene expression in the caudate-putamen of adult female rats

Abstract Perinatal Δ 9 -tetrahydrocannabinol ( Δ 9 -THC) exposure in rats affects several behavioral responses, such as opiate self-administration behavior or pain sensitivity, that can be directly related to changes in opioidergic neurotransmission. In addition, we have recently reported that the administration of naloxone to animals perinatally exposed to Δ 9 -THC produced withdrawal responses, that resemble those observed in opiate-dependent rats. The purpose of the present study was to examine the basal opioid activity in the brain of adult male and female rats that had been perinatally exposed to Δ 9 -THC. To this aim, proenkephalin mRNA levels were measured, by using in situ hybridization histochemistry, in the caudate-putamen, nucleus accumbens, central amygdala and prefrontal cingulate cortex. The results showed a marked reduction in proenkephalin mRNA levels in the caudate-putamen of Δ 9 -THC-exposed females as compared to oil-exposed females, whereas no changes were observed between Δ 9 -THC- and oil-exposed males. There were no differences in proenkephalin mRNA levels in the nucleus accumbens, central amygdala and prefrontal cingulate cortex between males and females perinatally exposed to Δ 9 -THC and their respective controls, although a certain trend to decrease was observed in Δ 9 -THC-exposed females. In summary, perinatal exposure to Δ 9 -THC exposure decreased proenkephalin gene expression in the caudate-putamen of adult rats, although this effect exhibited a marked sexual dimorphism since it was only seen in females. This result is in agreement with a previous observation from our laboratory that females, but not males, that had been perinatally exposed to Δ 9 -THC, self-administered more morphine in adulthood. This suggests that low levels of proenkephalin mRNA may be used as a predictor of greater vulnerability to opiates.

Cannabinoid receptor and WIN-55,212-2-stimulated [35S]GTPγS binding and cannabinoid receptor mRNA levels in the basal ganglia and the cerebellum of adult male rats chronically exposed to Δ9-tetrahydrocannabinol

AbstractThe 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 Δ9-tetrahydrocannabinol-(Δ9-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-(γ-thio)-triphosphate ([35S]-GTPγS) autoradiography;3.Cannabinoid receptor mRNA expression, quantitated by in situ hybridization. Results were as follows. As expected, the exposure to Δ9-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 Δ9-THC administration. However, in the substantia nigra, the magnitude of WIN-55,212-2-stimulated [35S]-GTPγS binding was lesser in Δ9-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]-GTPγS binding levels in Δ9-THC-tolerant rats and controls. Finally, animals chronically exposed to Δ9-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 Δ9-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 Δ9-THC-tolerant rats. The chronic exposure to Δ9-THC also resulted in decreased cannabinoid receptor binding in the cerebellum, although without affecting mRNA expression.

Δ9-tetrahydrocannabinol increases activity of tyrosine hydroxylase in cultured fetal mesencephalic neurons

The exposure of pregnant rats to Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive constituent ofCannabis 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 Δ9-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 Δ9-THC. In addition, TH activity was also approx twofold higher in cells obtained from fetuses exposed daily to Δ9-THC from d 5 of gestation than in cells obtained from control fetuses, when both were exposed to basal media. This effect of Δ9-THC on TH activity seems to be produced via the activation of cannabinoid receptors, in particular the CB1 subtype, which would presumably be located in these cells. This is because the exposure to medium containing both Δ9-THC and SR141716A, a specific antagonist for CB1 receptors, abolished the effect observed with Δ9-THC alone. SR141716A alone was without effect on TH activity. Collectively, our results support the notion that Δ9-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.

Perinatal Δ9-Tetrahydrocannabinol Exposure Did Not Alter Dopamine Transporter and Tyrosine Hydroxylase mRNA Levels in Midbrain Dopaminergic Neurons of Adult Male and Female Rats

Abstract GARCIA-GIL, L., J. A. RAMOS, T. RUBINO, D. PAROLARO AND J. J. FERNANDEZ-RUIZ. Perinatal Δ9-tetrahydrocannabinol exposure did not alter dopamine transporter and tyrosine hydroxylase mRNA levels in midbrain dopaminergic neurons of adult male and female rats. NEUROTOXICOL TERATOL 20(5) 549–553, 1998.—We have recently demonstrated that the magnitude of L -3,4-dihydroxyphenylacetic acid (DOPAC) lowering effect caused by amphetamine in midbrain dopaminergic neurons of adult rats was lesser in animals that had been perinatally exposed to Δ9-tetrahydrocannabinol (Δ9-THC) than controls. In the present study, we have examined whether this loss in the responsiveness to amphetamine might be due to changes at the level of dopamine transporter (DAT), the main molecular site for the action of amphetamine, following the perinatal exposure to Δ9-THC. To this end, we have analyzed DAT mRNA levels, by using in situ hybridization, in the substantia nigra and ventral tegmental area, the areas where cell bodies of DAT-containing midbrain neurons are located, of adult male and female rats that had been perinatally exposed to Δ9-THC. In addition, we also analyzed mRNA levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis. Results were as follows. Both adult male and female rats that had been perinatally exposed to Δ9-THC exhibited similar mRNA levels to controls for both DAT and TH in the substantia nigra as well as in the ventral tegmental area. This observation makes it difficult to support the idea that the differences found in adulthood after pharmacological challenges were caused by irreversible changes at the level of gene expression for these two key proteins.

Cannabinoid receptor and WIN-55,212-2-stimulated [35S]GTPγS 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 Ammons 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 Ammons 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 Ammons 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.

Cannabinoid receptor binding and mRNA levels in several brain regions of adult male and female rats perinatally exposed to Δ9-tetrahydrocannabinol

The present study was designed to elucidate whether perinatal delta9-tetrahydrocannabinol (delta9-THC) exposure results in changes in cannabinoid receptor binding and mRNA levels in adulthood. Most of the brain areas studied, including the basal ganglia, the cerebellum, the limbic structures, and most of the hippocampal regions exhibited no changes in cannabinoid receptor binding and mRNA levels in adulthood as a consequence of the perinatal delta9-THC exposure. However, some subtle changes could be appreciated in specific regions, although their physiological relevance seems uncertain. For example, delta9-THC-exposed males exhibited a small decrease in binding in the superficial layer of the cerebral cortex, an effect that was not seen in delta9-THC-exposed females and in mRNA levels for both males and females. In the CA2 layer of the Ammons horn, there was an increase in mRNA levels of delta9-THC-exposed animals, although this was statistically significant only in males. However, the more marked and probably relevant changes were seen in the arcuate nucleus, where delta9-THC-exposed males exhibited an increase in binding, whereas this tended to decrease in delta9-THC-exposed females. In an additional experiment, we analyzed the motor response of these animals to a challenge with SR141716, a specific antagonist for cannabinoid receptors. The delta9-THC-exposed animals tended to show a higher response to SR141716 challenge, with changes apparently more marked in delta9-THC-exposed females, although they did not reach statistical significance. In summary, perinatal cannabinoid exposure does not appear to significantly alter cannabinoid receptor binding and mRNA expression in the brain of adult rats, as well as the motor response caused by the blockade of these receptors with a specific antagonist. There were some changes in the status of cannabinoid receptors but they were very small and, hence, of debatable physiological relevance. The most significant of these effects was the increase in binding observed in the arcuate nucleus of delta9-THC-exposed males.