Mariluz Hernández

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.

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.

Cannabinoid–Dopamine Interaction in the Pathophysiology and Treatment of CNS Disorders

Endocannabinoids and their receptors, mainly the CB1 receptor type, function as a retrograde signaling system in many synapses within the CNS, particularly in GABAergic and glutamatergic synapses. They also play a modulatory function on dopamine (DA) transmission, although CB1 receptors do not appear to be located in dopaminergic terminals, at least in the major brain regions receiving dopaminergic innervation, e.g., the caudate‐putamen and the nucleus accumbens/prefrontal cortex. Therefore, the effects of cannabinoids on DA transmission and DA‐related behaviors are generally indirect and exerted through the modulation of GABA and glutamate inputs received by dopaminergic neurons. Recent evidence suggest, however, that certain eicosanoid‐derived cannabinoids may directly activate TRPV1 receptors, which have been found in some dopaminergic pathways, thus allowing a direct regulation of DA function. Through this direct mechanism or through indirect mechanisms involving GABA or glutamate neurons, cannabinoids may interact with DA transmission in the CNS and this has an important influence in various DA‐related neurobiological processes (e.g., control of movement, motivation/reward) and, particularly, on different pathologies affecting these processes like basal ganglia disorders, schizophrenia, and drug addiction. The present review will address the current literature supporting these cannabinoid‐DA interactions, with emphasis in aspects dealing with the neurochemical, physiological, and pharmacological/therapeutic bases of these interactions.

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.

Cannabinoids and gene expression during brain development

Cannabis is the most commonly used illicit drug in western societies, in particular among young people. It is consumed even by women during pregnancy and lactation, which result in a variety of disturbances in the development of their offspring, because, like other habit-forming drugs, cannabinoids, the psychoactive ingredients of marijuana, can cross the placental barrier and be secreted in the maternal milk. Through this way, cannabinoids affect the ontogeny of various neurotransmitter systems leading to changes in different behavioral patterns. Dopamine and endogenous opioids are among the neurotransmitters that result more affected by perinatal cannabinoid exposure, which, when animals mature, produce changes in motor activity, drug-seeking behavior, nociception and other processes. These disturbances are likely originated by the capability of cannabinoids to influence the expression of key genes for both neurotransmitters, in particular, the enzyme tyrosine hydroxylase and the opioid precursor proenkephalin. In addition, cannabinoids seem to be also able to influence the expression of genes encoding for neuron-glia cell adhesion molecules, which supports a potential influence of cannabinoids on the processes of cell proliferation, neuronal migration or axonal elongation in which these proteins are involved. In support of this possibility, CB1 receptors, which represent the major targets for the action of cannabinoids, are abundantly expressed in certain brain regions, such as the subventricular areas, which have been involved in these processes during brain development. Finally, cannabinoids might also be involved in the apoptotic death that occurs during brain development, possibly by influencing the expression of the Bcl-2/Bax system. Also in support of this option, CB1 receptors are transiently expressed during brain development in different group of neurons which do not contain these receptors in the adult brain. This paper will review all evidence relating cannabinoids to the expression of key genes for neural development, trying to establish the future research addressed to elucidate the mechanisms involved in the epigenetic action of cannabinoids during brain development.

Exposure to cannabinoids in the development of endogenous cannabinoid system

New data strengthen the idea of a prominent role for endocannabinoids in the modulation of a wide variety of neurobiological functions. Among these, two functions, control of movement and antinociception, have attracted the maximal interest because of the possibility that cannabinoids and related compounds might be used with a therapeutic purpose. However, the functions of endocannabinoids in the brain, and also in the periphery, are large and involve, not only the adulthood, but also the period of prenatal and postnatal development, when endocannabinoids have been reported to be significantly present and to play a role in processes of brain development as neuronal proliferation and migration, axonal elongation, synaptogenesis and/or myelinogenesis. The present review article will summarize the different studies carried out on this topic and will suggest futures lines of reseearch to clarify the role of endocannabinoids and their receptors in the development.

Colocalization of CB1 receptors with L1 and GAP-43 in forebrain white matter regions during fetal rat brain development: Evidence for a role of these receptors in axonal growth and guidance

There is recent evidence supporting the notion that the cannabinoid signaling system plays a modulatory role in the regulation of cell proliferation and migration, survival of neural progenitors, neuritic elongation and guidance, and synaptogenesis. This assumption is based on the fact that cannabinoid 1-type receptors (CB(1) receptors) and their ligands emerge early in brain development and are abundantly expressed in certain brain regions that play key roles in these processes. We have recently presented in vivo evidence showing that this modulatory action might be exerted through regulating the synthesis of the cell adhesion molecule L1 that is also a key element for those processes. To further explore this issue, we conducted here immunohistochemical studies aimed at determining the cellular substrates of CB(1) receptor-L1 interactions in the rat brain during late fetal development. In this period, we previously found that the activation of CB(1) receptors increased L1 synthesis in several forebrain white matter regions but not in gray matter areas. Using double labeling studies, we observed here colocalization of both proteins in fiber tracts including the corpus callosum, the adjacent subcortical white matter, the internal capsule and the anterior commissure. Experiments conducted with cultures of fetal rat cortical nerve cells revealed that L1 is present mainly in neurons but not in glial cells. This fact, together with the results obtained in the double labeling studies, would indicate that L1 and CB(1) receptors should possibly be present in axons elongating through these white matter tracts, or, alternatively, in migrating neurons. Further experiments confirmed the presence of CB(1) receptors in elongating axons, since these receptors colocalized with growth-associated protein 43 (GAP-43), a marker of growth cones, but not with synaptophysin, a marker of active synaptic terminals, in the same forebrain white matter regions. Lastly, using cultured fetal rat cortical neurons, we also observed that the activation of cannabinoid receptors increased the levels of the full-length L1 and altered those of some active proteolytic fragments of this protein whose generation has been associated with specific steps in the process of neuritic elongation in cultured neurons. In summary, we have demonstrated that the effects caused by cannabinoid agonists on L1 are facilitated by the colocalization of this cell adhesion molecule with CB(1) receptors in several forebrain white matter regions during fetal brain development. We have provided strong evidence that this phenomenon occurs in axons elongating through these white matter tracts, and we have explored in vitro how cannabinoid receptors influence L1 levels. Considering the role played by L1 in different events related to neural development, our observations support the occurrence of a physiological mechanism by which the cannabinoid system might regulate the process of axonal growth and guidance through regulating the synthesis and function of L1.

The activation of cannabinoid receptors during early postnatal development reduces the expression of cell adhesion molecule L1 in the rat brain.

Cannabinoid CB(1) receptors and their ligands emerge early in brain development and are abundantly expressed in certain brain regions that play key roles in processes related to cell proliferation and migration, neuritic elongation and guidance, and synaptogenesis. This would support the notion that the cannabinoid system might play a modulatory role in the regulation of these processes. We have recently presented preliminary in vivo evidence showing that this modulatory action might be exerted, among others, through regulating the levels of several key elements in these processes, such as the L1 protein. This was observed in various white matter areas of the rat forebrain. Because these preliminary in vivo experiments focused only in fetal ages, we concentrated now in the period of early postnatal development. To this end, we analyzed the effects of the cannabinoid agonist Delta(9)-tetrahydrocannabinol (Delta(9)-THC) daily administered since the 5th day of gestation on mRNA levels for L1 in different brain structures of rat neonates at different postnatal ages (PND1, PND5 and PND12). Our results revealed that Delta(9)-THC exposure affected the levels of L1 transcripts in specific brain structures only in PND1, these effects disappearing during further days. Thus, we found reduced L1-mRNA levels in grey matter regions, such as the cerebral cortex, septum nuclei, striatum, dentate gyrus and CA3 subfield of the Ammon horn. White matter areas and subventricular zones were, however, more resistant to Delta(9)-THC exposure at this postnatal age in contrast with the previous data obtained in the fetal brain. Importantly, the effects were influenced by gender of animals, since the reductions were always more marked in females than males, also in contrast with the data reported for the fetal brain. In summary, the cannabinoid system seems to modulate the levels of L1 in several brain structures during specific periods of development [late gestation (previous data) and very early postnatal days (present data)], which correlates with the periods in which we had previously found an atypical distribution of CB(1) receptors in the developing brain. However, the magnitude of the effects of cannabinoids on L1 was influenced by two factors: gender and age of development. Considering the role played by L1 in different events related to neural development, our observations might support the occurrence of a physiological mechanism by which the cannabinoid system might regulate processes such as cell proliferation and migration, neuritic elongation and guidance, and synaptogenesis.

Possible direct effect of prolactin on catecholamine synthesis and release in rat adrenal medulla: in vitro studies

It has been recently shown that chronic increase in circulating prolactin (PRL) levels can affect the catecholamine (CA) synthesis and release in the adrenal medulla of female and male rats. However, it is not established if this effect is directly exerted on the adrenomedullary cells. To elucidate this question, we have studied the possible capacity of PRL to modify the in vitro synthesis and release of CA in isolated adrenal medullas of female rats. The study has been performed in animals with hyperprolactinemia induced by pituitary grafts (GRAFT) or with low PRL levels induced by ovariectomy (OVX), and in their respective sham-operated controls (SHAM),in order to establish if the previous plasma levels of this hormone could modify the effects observed in vitro. Basal release of adrenaline (A), noradrenaline (NA) and total CA observed during the first h of tissue incubation was similar in the three groups of animals. However, OVX animals exhibited a decreased release of both CA in the second h of incubation. This low CA release was partially reversed after the exposure of the incubated adrenal medullas of these animals to a medium containing PRL, whereas this hormone was ineffective to modify the CA release in SHAM and GRAFT animals. Moreover, PRL caused a significant decrease in the A production by the adrenal medullas of SHAM animals, since the amount of A released plus the amount of A stored after incubation in presence of PRL was significantly decreased. Finally, the exposure of the adrenomedullary cells from GRAFT rats to PRL was followed by an increase in the contents of both NA and A in the tissues. Since the total amount of CA produced was not altered, we suppose that these increases reflected alterations induced by PRL in the CA storage ability but not in the synthesis of these amines. Hence, PRL seems to be able to alter the activity of the adrenal medulla by a direct action on this gland. However, the nature of its action seems to be different depending on the previous plasma levels of this hormone.

Cannabinoid signaling system: does it play a function in cell proliferation and migration, neuritic elongation and guidance and synaptogenesis during brain ontogenesis?

The cannabinoid signaling system is located during brain development in a position concordant with playing a modulatory function in the regulation of neuronal and glial cell proliferation and migration, survival of neural progenitors, axonal elongation and synaptogenesis, and differentiation of oligodendrocytes and formation of myelin. This assumption is based on the fact that CB1 receptors and their ligands emerge early in brain development and are transiently expressed in certain brain regions that play key roles in these processes. We have recently proposed that this modulatory action might be exerted through regulating L1 and other cell adhesion molecules, that are also key elements for those processes. The present commentary will address these two questions trying to summarize all the available evidence and to suggest the future directions for research.