J.A. Ramos

Analysis of cannabinoid receptor binding and mRNA expression and endogenous cannabinoid contents in the developing rat brain during late gestation and early postnatal period

Cannabinoid CB1 receptors emerge early in the rat brain during prenatal development, supporting their potential participation in events related to neural development. In the present investigation, we completed earlier studies, analyzing CB1 receptor binding and mRNA expression by using autoradiography and in situ hybridization, respectively, in the brain of rat fetuses at gestational day (GD) 21 and of newborns at postnatal days (PND) 1 and 5, in comparison with the adult brain. These analyses were paralleled by quantitation of levels of anandamide and its precursor, N‐arachidonoyl‐phosphatidylethanolamine (NAPE), and of 2‐arachidonoyl‐glycerol (2‐AG), carried out by using gas chromatography / mass spectrometry of the tri‐methyl‐sylyl‐ether derivatives. As expected, CB1 receptor binding was detected at GD21 in a variety of brain structures. In most of them, such as the hippocampus, cerebral cortex, cerebellum, basal ganglia, and limbic nuclei, there were no marked differences in the density of CB1 receptors in animals at GD21 as compared to early newborns (PND1 and 5), although it markedly increased in these regions in adulthood. However, with the exception of the cerebellum and, in part, the caudate‐putamen, the pattern observed for binding in these regions was clearly different from that observed for mRNA expression of the CB1 receptor, which currently exhibited the highest levels at PND1 and the lowest in the adult brain. This was also seen in the basolateral amygdaloid nucleus, ventromedial hypothalamic nucleus, medial habenula, and other structures. In the caudate‐putamen and, particularly, in the cerebellum, mRNA expression was higher in the adult brain as compared with other ages. As previously reported, specific binding for CB1 receptors was also detected at GD21 in white matter areas, such as the corpus callosum, anterior commissure, fornix, fimbria, stria medullaris, stria terminalis, and fasciculus retroflexum. With the exception of the anterior commissure and the fimbria, specific binding progressively decreased at PND1 and PND5 until disappearing in the adult brain. In the fimbria, the highest values of binding were seen at PND1, but binding also completely disappeared in the adult brain, whereas in the anterior commissure, specific binding at PND1 and PND5 was lesser than that observed at GD21 and, particularly, in adulthood. CB1 receptor mRNA expression was not detected in these white matter areas, thus dismissing the possible presence of these receptors in glial cells rather than in neuronal axons. However, mRNA expression was detected in the brainstem, an area also rich in white matter, and it mostly correlated with receptor binding, exhibiting a progressive decrease from GD21 up to adulthood. CB1 receptor mRNA expression was also detected at GD21 in atypical areas where binding was not detected. These areas are proliferative regions, such as the subventricular zones of the neocortex, striatum, and nucleus accumbens. This atypical location only persisted at PND1 and PND5 in the striatal subventricular zone, but disappeared in the adult brain. We also found measurable levels of different endogenous cannabinoids in the developing brain. High levels of 2‐AG, comparable to those found in the adult brain, were measured at GD21, whereas significantly lower levels were measured for anandamide and NAPE at this fetal age compared with the levels found in the adult brain. Levels of anandamide and NAPE increased during the early postnatal period until reaching the maximum in the adult brain. By contrast, 2‐AG levels peaked at PND1, with values approximately twofold higher than those found at the other ages. In summary, all these data demonstrate that the endogenous cannabinoid system, constituted by endogenous ligands and receptor signaling pathways, is present in the developing brain, which suggests a possible specific role of this system in key processes of neural development. Synapse 33:181–191, 1999.

Downregulation of rat brain cannabinoid binding sites after chronic Δ9-tetrahydrocannabinol treatment

Specific cannabinoid receptors have been recently described in extrapyramidal and limbic areas and presumably might mediate the effects of marijuana exposure on behavioral processes related to those areas. In this work, we examined whether cannabinoid receptors exhibit downregulation as a consequence of the chronic exposure to delta 9-tetrahydrocannabinol (THC), which might explain certain tolerance phenomena observed in relation to motor and limbic effects of marijuana. To this end, we first characterized the binding of cannabinoid receptors, by using [3H]CP-55,940 binding assays, in the striatum, limbic forebrain, and ventral mesencephalon of male rats, and, second, we measured the density and affinity of those receptors in these brain areas after 7 days of a daily treatment with THC. Development of a tolerance phenomenon was behaviorally tested by using an open-field technique. Results were as follows. The three areas studies presented specific and saturable binding for the cannabinoid ligand, as revealed by their corresponding association and dissociation curves, displacement by THC, saturation curves, and Scatchard plots. A chronic treatment with THC produced the expected tolerance phenomenon: The decrease caused by an acute dose in spontaneous locomotor (49.4%) and exploratory (59.7%) activities and, mainly, the increase in the time spent by the rat in inactivity (181.7%) were diminished after 7 days of daily treatment (39.4, 40.4, and 31.7%, respectively). This tolerance was accompanied by significant decreases in the density of cannabinoid receptors in the striatum and limbic forebrain, the areas where nerve terminals for nigrostriatal and mesolimbic dopaminergic systems, respectively, which play an important role in those processes, are located.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Enhancement of Anandamide Formation in the Limbic Forebrain and Reduction of Endocannabinoid Contents in the Striatum of Δ9-Tetrahydrocannabinol-Tolerant Rats

Abstract: Recent studies have shown that the pharmacological tolerance observed after prolonged exposure to synthetic or plant‐derived cannabinoids in adult rats is accompanied by down‐regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid‐tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope dilution gas chromatography/mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N‐arachidonoylphosphatidylethanolamine (NArPE), and 2‐arachidonoylglycerol (2‐AG) in several brain regions of adult male rats treated daily with Δ9‐tetrahydrocannabinol (Δ9‐THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN‐55,212‐2‐stimulated guanylyl‐5′‐O‐(γ‐[35S]thio)‐triphosphate ([35S]GTPγS) binding to test the development of the well known down‐regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN‐55,212‐2‐stimulated [35S]GTPγS binding decreased in most of the brain areas of Δ9‐THC‐tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of Δ9‐THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents observed in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2‐AG were unchanged in brain regions from Δ9‐THC‐tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.

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.

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.

Time-course of the cannabinoid receptor down-regulation in the adult rat brain caused by repeated exposure to ?9-tetrahydrocannabinol

Recent studies have demonstrated that the pharmacological tolerance observed after prolonged exposure to plant or synthetic cannabinoids in adult individuals seems to have a pharmacodynamic rather than pharmacokinetic basis, because down‐regulation of cannabinoid receptors was assessed in the brain of cannabinoid‐tolerant rats. In the present study, we have examined the time‐course of cannabinoid receptor down‐regulation by analyzing cannabinoid receptor binding, using autoradiography, and mRNA expression, using in situ hybridization, in several brain structures of male adult rats daily exposed to Δ9‐tetrahydrocannabinol (Δ9‐THC) for 1, 3, 7, or 14 days. With only the exception of a few number of areas, most of the brain regions exhibited a progressive decrease in cannabinoid receptor binding. Two facts deserve to be mentioned. First, the pattern of this down‐regulation process presented significant regional differences in terms of onset of the decrease and magnitude reached. Second, the loss of cannabinoid receptor binding was usually accompanied by no changes in its mRNA expression. Thus, some structures, such as most of the subfields of the Ammons horn and the dentate gyrus in the hippocampus, exhibited a rapid (it appeared after the first injection) and marked (it reached approximately 30% of decrease after 14 days) reduction of cannabinoid receptor binding as a consequence of the daily Δ9‐THC administration. However, no changes occurred in mRNA levels. Decreased binding was also found in most of the basal ganglia, but the onset of this reduction was slow in the lateral caudate‐putamen and the substantia nigra (it needed at least three days of daily Δ9‐THC administration), and, in particular, in the globus pallidus (more than 3 days). The magnitude of the decrease in binding was also more moderate, with maximal reductions always less than 28%. No changes were seen in the entopeduncular nucleus and only a trend in the medial caudate‐putamen. However, the decrease in binding in some basal ganglia was, in this case, accompanied by a decrease in mRNA levels in the lateral caudate‐putamen, but this appeared after 7 days of daily Δ9‐THC administration and, hence, after the onset of binding decrease. In the limbic structures, cannabinoid receptor binding decreased in the septum nuclei (it needed at least 3 days of daily Δ9‐THC administration), tended to diminish in the nucleus accumbens and was unaltered in the basolateral amygdaloid nucleus, with no changes in mRNA levels in these last two regions. Binding also decreased in the superficial and deep layers of the cerebral cortex, but only accompanied by trends in mRNA expression. The decrease in binding was initiated promptly in the deep layer (after the first injection) and it reached more than 30% of reduction after 14 days of daily Δ9‐THC administration, whereas, in the superficial layer, it needed more than 3 days of daily Δ9‐THC administration and reached less than 30% of reduction. Finally, no changes in binding and mRNA levels were found in the ventromedial hypothalamic nucleus. In summary, the daily administration of Δ9‐THC resulted in a progressive decrease in cannabinoid receptor binding in most of the brain areas studied, and it was a fact that always occurred before the changes in mRNA expression in those areas where these existed. The onset of the decrease in binding exhibited regional differences with areas, such as most of the hippocampal structures and the deep layer of the cerebral cortex, where the decrease occurred after the first administration. Other structures, however, needed at least 3 days or more to initiate receptor binding decrease. Two structures, the entopeduncular nucleus and the ventromedial hypothalamic nucleus, were unresponsive to chronic Δ9‐THC administration, whereas others, the medial caudate‐putamen and the basolateral amygdaloid nucleus, only exhibited trends. Synapse 30:298–308, 1998.

Loss of mRNA levels, binding and activation of GTP-binding proteins for cannabinoid CB1 receptors in the basal ganglia of a transgenic model of Huntington's disease.

Data obtained from the basal ganglia of postmortem Huntingtons disease (HD) brains have revealed that the level of cannabinoid CB1 receptors in striatal efferent neurons decreases in parallel to the dysfunction and subsequent degeneration of these neurons. These findings, and others from rat models of HD generated by lesions with mitochondrial toxins, suggest that the loss of CB1 receptors may be involved in the pathogenesis of the disease. To explore further the changes in the endocannabinoid system, as well as the potential of endocannabinoid-related compounds, we examined the status of CB1 receptors in the HD94 transgenic mouse model of HD. These mice express huntingtin exon 1 with a polyglutamine tract of 94 repeats in a tissue-specific and conditional manner using the tet regulatable system. They develop many features of HD, such as striatal atrophy, intraneuronal aggregates and progressive dystonia. In these animals, we analyzed mRNA levels for the CB1 receptor, in addition to the number of specific binding sites and the activation of GTP-binding proteins by CB1 receptor agonists. mRNA transcripts of the CB1 receptor were significantly decreased in the caudate-putamen of HD transgenic mice compared to age-matched littermate controls. The decrease concurred with a marked reduction in receptor density in both the caudate-putamen and its projection areas such as the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata. Furthermore, the efficacy of CB1 receptor activation was reduced in the globus pallidus, as determined by agonist-induced [35S]GTPgammaS binding, and tended towards a decrease in the substantia nigra. None of these changes was seen in the cerebral cortex and hippocampus, despite high levels of expression of the mutant protein in these regions. The decrease in CB1 receptor levels was accompanied by a decrease in the proenkephalin-mRNA levels but not in substance P-mRNA levels. Taken together, these results suggest that the loss of CB1 receptor might be preferential to the enkephalinergic CB1 receptor-containing striatopallidal neurons, and further implicate the CB1 receptor to the subsequent HD symptomatology and neuropathology.