Fernando Berrendero

The endogenous cannabinoid system and brain development

Cannabinoid receptors and their endogenous ligands constitute a novel modulatory system that is involved in specific brain functions, such as nociception, control of movement, memory and neuroendocrine regulation. Recently, it has also been suggested that this system is involved in brain development. Studies have used a variety of techniques to elucidate the effects of cannabinoids during development, as well as to characterize the presence of elements of the endogenous cannabinoid system (receptors and ligands) in the developing brain. Collectively, they suggest that endocannabinoids participate in brain development through the activation of second-messenger-coupled cannabinoid receptors.

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.

Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N‐arachidonoylethanolamine (anandamide) and 2‐arachidonoylglycerol (2‐AG), as well as other constituents of their lipid families, N‐acylethanolamines (NAEs) and 2‐monoacylglycerols (2‐MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46‐fold increase of cortical NAE concentrations (anandamide, 13‐fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2‐AG and other 2‐MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB1 receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up‐regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2‐AG, and CB1 receptor density.

The endogenous cannabinoid system and the basal ganglia: biochemical, pharmacological, and therapeutic aspects

New data strengthen the idea of a prominent role for endocannabinoids in the modulation of a wide variety of neurobiological functions. Among these, one of the most important is the control of movement. This finding is supported by 3 lines of evidence: (1) the demonstration of a powerful action, mostly inhibitory in nature, of synthetic and plant-derived cannabinoids and, more recently, of endocannabinoids on motor activity; (2) the presence of the cannabinoid CB(1) receptor subtype and the recent description of endocannabinoids in the basal ganglia and the cerebellum, the areas that control movement; and (3) the fact that CB(1) receptor binding was altered in the basal ganglia of humans affected by several neurological diseases and also of rodents with experimentally induced motor disorders. Based on this evidence, it has been suggested that new synthetic compounds that act at key steps of endocannabinoid activity (i.e., more-stable analogs of endocannabinoids, inhibitors of endocannabinoid reuptake or metabolism, antagonists of CB(1) receptors) might be of interest for their potential use as therapeutic agents in a variety of pathologies affecting extrapyramidal structures, such as Parkinsons and Huntingtons diseases. Currently, only a few data exist in the literature studying such relationships in humans, but an increasing number of journal articles are revealing the importance of this new neuromodulatory system and arguing in favour of the funding of more extensive research in this field. The present article will review the current knowledge of this neuromodulatory system, trying to establish the future lines for research on the therapeutic potential of the endocannabinoid system in motor disorders.

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.

Unilateral 6-hydroxydopamine lesions of nigrostriatal dopaminergic neurons increased CB1 receptor mRNA levels in the caudate-putamen.

It has been recently suggested that the effects of cannabinoids on motor behavior might be different in rats with lesions of nigrostriatal dopaminergic neurons than in controls. In the present study, we examined the possible alteration in the status of cannabinoid CB1 receptors in the basal ganglia of rats with unilateral lesions of those neurons caused by 6-hydroxydopamine. We used two different experimental groups depending on the duration of the period of recovery after the lesion, and comparisons were done between the lesioned and nonlesioned sides at the level of the basal ganglia. Both groups of lesioned rats exhibited a similar marked reduction in tyrosine hydroxylase (TH)-mRNA levels, measured by in situ hybridization, in the substantia nigra of the lesioned side. In the same way, lesioned rats exhibited the characteristic rotational behavior after a single injection of apomorphine and the intensity of this rotation was stable at the two times analyzed after the lesion. Also as expected, lesioned rats exhibited an increase in proenkephalin mRNA levels in the caudate-putamen, whereas mRNA levels of substance P decreased, although differences between the two times of recovery analyzed were observed in this case. We did not find any significant changes in CB1 receptor binding, measured by [3H]WIN-55,212,2 autoradiography, or in the activation of signal transduction mechanisms, measured by WIN-55,212,2-stimulated [35S]GTPgammaS binding autoradiography, between the lesioned and nonlesioned sides at the level of the lateral caudate-putamen, globus pallidus and substantia nigra in both groups of lesioned rats. However, we found a significant increase in levels of CB1 receptor-mRNA transcripts, measured by in situ hybridization, in the lesioned side in both the lateral and medial caudate-putamen. This occurred 7-10 weeks after the lesion, but the increase was markedly waned after 17-18 weeks. In summary, the unilateral 6-hydroxydopamine lesion of nigrostriatal dopaminergic neurons originated a marked increase in CB1 receptor-mRNA levels in cell bodies of striatal efferent neurons, although accompanied by no changes in CB1 receptor binding and activation of signal transduction mechanisms. This supports a critical role for dopamine in the control of CB1 receptor gene expression. However, the magnitude of the effect significantly waned as a function of the duration of the period after lesion.

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.

Role of endocannabinoids in brain development

In addition to those functions that have been extensively addressed in this special issue, such as nociception, motor activity, neuroendocrine regulation, immune function and others, the endogenous cannabinoid system seems to play also a role in neural development. This view is based on a three-fold evidence. A first evidence emerges from neurotoxicological studies that showed that synthetic and plant-derived cannabinoids, when administered to pregnant rats, produced a variety of changes in the maturation of several neurotransmitters and their associated-behaviors in their pups, changes that were evident at different stages of brain development. A second evidence comes from studies that demonstrated the early appearance of elements of the endogenous cannabinoid system (receptors and ligands) during the brain development. The atypical location of these elements during fetal and early postnatal periods favours the notion that this system may play a role in specific molecular events related to neural development. Finally, a third evidence derives from studies using cultures of fetal glial or neuronal cells. Cannabinoid receptors are present in some of these cultured cells and their activation produced a set of cellular effects consistent with a role of this system in the process of neural development. All this likely supports that endocannabinoids, early synthesized in nervous cells, play a role in events related to development, by acting through the activation of second messenger-coupled cannabinoid receptors.

Time-dependent differences of repeated administration with Δ9-tetrahydrocannabinol in proenkephalin and cannabinoid receptor gene expression and G-protein activation by μ-opioid and CB1-cannabinoid receptors in the caudate–putamen

The purpose of the present study was to examine the time-related effects of repeated administration of Delta9-tetrahydrocannabinol during 1, 3, 7 and 14 days on cannabinoid and mu-opioid receptor agonist-stimulated [35S]GTPgammaS binding, and CB1 cannabinoid receptor and proenkephalin gene expression in the caudate-putamen. Repeated administration with Delta9-tetrahydrocannabinol produced a time-related reduction in cannabinoid receptor synthesis and activation of signal transduction mechanisms in the caudate-putamen. Indeed, WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased 24% on day 1 and then progressively decreased finding a 42% decrease on day 14. Similarly, CB1 cannabinoid receptor mRNA levels decreased (22%) on day 3, reaching 50% reduction on day 7. In contrast, a pronounced increase is detected in DAMGO-stimulated [35S]GTPgammaS binding and proenkephalin mRNA levels in the caudate-putamen. The highest degree of increase was reached on day 7 of the treatment (35% of proenkephalin mRNA levels and 62% of DAMGO-stimulated [35S]GTPgammaS binding) and then values slightly decreased on day 14. Taken together, the results of the present study indicate that, in the caudate-putamen, repeated administration with Delta9-tetrahydrocannabinol produces a time-related increase in proenkephalin gene expression and mu-opioid receptor activation of G-proteins, and a time-related decrease in CB1 cannabinoid receptor gene expression and reduction in CB1 cannabinoid receptor activation of G-proteins. These results also suggest a possible interaction between the cannabinoid and opioid systems in the caudate-putamen which may be potentially relevant in the understanding of the alterations of motor behavior that occur after prolonged exposure to cannabinoids.