2-AG may be produced by CA1 pyramidal cells during Schaffer collateral stimulation , and the newly generated endocannabinoid may mediate depolarization-induced suppression of inhibition , if able to diffuse to the nearby GABAergic boutons, or a suppression of excitation . Thus MGL is exquisitely poised to terminate the actions of 2-AG at hippocampal synapses.In a landmark study published in 1990, Herkenham and co-workers took advantage of the newly developed cannabinoid agonist [3 H]CP-55,940, the same highly selective ligand that had helped identify cannabinoid receptors two years earlier , to investigate for the first time the distribution of cannabinoid binding sites in the brain . Their results showed that these sites strikingly coincide with the neural substrates for cannabinoid actions predicted from behavioral experiments and started a season of intense research on the CNS distribution of cannabinoid receptors. In the following pages, we will summarize the current status of this research, highlighting the correspondence between cannabinoid receptor distribution and behavioral effects of cannabimimetic agents. In the next sections, we focus on the cellular and subcellular localization of cannabinoid receptors and on the consequences of their physiological or pharmacological activation. Various radioactive ligands have been used to identify the sites of action of cannabimimetic drugs at the regional and cellular level . One surprising observation stemming from these binding experiments, and confirmed later with other neuroanatomical techniques,growing cannabis indoors is that cannabinoid receptors are much more densely expressed in the rat brain than are any other G protein-coupled receptors .
Indeed, in several brain regions cannabinoid receptors are present in densities that are comparable to those of GABA or glutamate receptor channels, which, owing to their relatively low ligand affinities, are highly concentrated at synapses to allow fast neurotrans mission to occur. This puzzling finding is still unexplained but can be conceptualized in the light of recent discoveries suggesting that the synaptic functions served by the endocannabinoid system may be much broader than previously suspected. These functions, which will be discussed in detail in section VC, appear to be primarily concerned with the short-range, activity-dependent regulation of synaptic strength and to extend to a diversity of CNS structures. The broad regulatory roles of the endocannabinoids also may be surmised from the diverse effects of cannabimimetic drugs on physiology and behavior. In both animals and humans, these agents elicit a wide, but very distinctive spectrum of biological responses , which are epitomized by a tetrad comprising rigid immobility , decreased motor activity, analgesia, and hypothermia. This tetrad assay, developed by Billy R. Martin and his collaborators , provides a convenient early screening to identify novel cannabimimetic drugs and highlights the role of the endocannabinoid system in motor behavior. Consistent with such a role, two brain regions that are intimately involved in movement regulation, the basal ganglia and the cerebellum, stand out among others for their very high densities of cannabinoid binding sites . On the other hand, the marked binding capacities observed in limbic areas of the cerebral cortex, especially the cingulate and frontal cortices, as well as the amygdala, concord with the potent analgesic and antihyperalgesic properties of cannabinoid agonists and with their impact on emotional reactivity . Although not as dense, significant cannabinoid binding is also found in other pain-processing areas of the CNS, including thePAG and the dorsal horn of the spinal cord.
An important property of cannabimimetic agents, which is not modeled by the tetrad assay, relates to the ability of these compounds to influence cognitive functions, including short term memory and attention . The high densities of cannabinoid binding sites in the hippocampus and other cortical structures provide a likely neural substrate for this property . Sheer density of CNS binding sites is not sufficient to precisely account for the spectrum of cannabinoid effects. Studies on the activation of G proteins by cannabinoid agonists in acutely dissected brain slices have revealed, indeed, the existence of an uneven coupling of cannabinoid receptors with G protein activation in different brain structures . For example, receptors in structures such as the hypothalamus and the thalamus, although relatively low in number, display very tight G protein coupling, suggesting that they may be more efficacious than receptors found elsewhere in the brain. The molecular basis for these regional variations is unclear at present, but they may help reconcile the comparatively low density of cannabinoid receptors found in the hypothalamus with the profound neuroendocrine effects of cannabinoid drugs .Similar distribution patterns have been found in other mammalian and non-mammalian species , implying that the endocannabinoid system may play conserved roles in vertebrate phylogeny .The mapping of brain cannabinoid binding sites by Herkenham et al. preceded by a few months the molecular identification of the first cannabinoid receptor, the G protein-coupled receptor that is now called CB1 . A related gene encoding a second cannabinoid sensitive G protein-coupled receptor, the CB2, was identified soon afterward . The CB1 receptor is distributed throughout the body but is predominantly found in neurons of the central and peripheral nervous systems. In contrast, the CB2 receptor is highly concentrated in immune cells and appears to be absent from CNS neurons . Genetic deletion studies have confirmed that CB1 receptors contribute in a major way to the behavioral effects of cannabimimetic drugs. Thus mutant mice lacking functional CB1 receptors do not exhibit the tetrad of behavioral responses evoked by cannabinoid agonists .
As mentioned above, the tetrad only partially illustrates the complexity of cannabinoid actions and ostensibly excludes those involving cognitive systems. It is conceivable therefore that certain responses to cannabimimetic agents may be preserved in mutant CB1 /_x0005_ mice . This possibility is strongly supported by electrophysiological experiments, which show that CB1 /_x0005_ mice, although impaired in their CB1-mediated regulation of GABAergic transmission, retain an intact cannabinoid modulation of glutamate transmission . A parsimonious interpretation of these results, which is also consistent with current morphological data , is that glutamatergic axon terminals contain a cannabinoid-sensitive receptor that is molecularly distinct from CB1 . To understand the complex neurobiological effects of cannabinoid drugs and their endogenous counterparts, it is first necessary to precisely outline the neuronal cell types that express cannabinoid receptors. The molecular characterization of the CB1 receptor opened the way to in situ hybridization studies on the CNS distribution of this receptor’s mRNA . The subsequent development of specific antibodies allowed the comparison of mRNA and protein expression, and investigators could now delve in greater detail into the cellular and subcellular localizations of this receptor . In this section, we synthesize the rapidly growing body of data from several laboratories about CB1 cannabinoid receptor localization in particular cell types of given brain areas. Remarkably, these anatomical studies confirmed that,vertical grow rack likewise to the patterned distribution of cannabinoid binding sites in certain brain regions, expression of the CB1 receptor gene is restricted to specific cell types sub-serving distinct functional roles in certain neuronal networks, which may indeed account for the striking diversity of cannabinoid effects.Comprehensive in situ hybridization experiments have revealed three populations of brain cells that can be grouped according to their levels of CB1 mRNA . Cells with very high CB1 mRNA expression are found in many cortical regions, especially in the hippocampus, but also in the anterior olfactory nucleus, the neocortex, and the amygdala. Cells with moderate CB1 mRNA levels are characteristically present in the striatum and the cerebellum, whereas cells with very weak CB1 mRNA expression are widespread throughout the brain. Although this broad classification is generally accepted, more precise descriptions of CB1 mRNA distribution are still controversial. Discrepancies have been reported not only in the intensity of labeling among brain regions, but even in the presence or absence of CB1 mRNA in certain cell types . Subsequent immuno cytochemical studies have helped clarify some of these issues but have left others unsolved and, indeed, generated their own share of unexplained results. For example, recent reports of strong CB1 immuno staining in cerebellar Purkinje cells are in striking contrast to the lack of CB1 mRNA noted in these cells by many investigators . Surely, these problems will be appropriately addressed and resolved in due time . Meanwhile, here we will primarily discuss those results, which are unequivocally supported by a combination of multiple neuroanatomical and functional approaches.In situ hybridization and immunocytochemical studies consistently show that CB1 receptors are highly abundant in many forebrain areas, including the anterior olfactory nucleus, the hippocampal formation , the neocortex , and the basolateral as well as the cortical amygdaloid nuclei.
CB1-positive cells in these areas display a scattered distribution pattern, represent only a small percent of the total cell population, and belong to the heterogeneous population of GABAergic interneurons . In the forebrain, GABAergic interneurons can be divided into various classes based on the cell type selective expression of neurochemical markers, two prominent examples of which are the neuropeptide cholecystokinin and the calcium-binding protein parvalbumin . Double-labeling studies have revealed that only one subset of GABAergic interneurons contains CB1 receptors, those that also express and presumably release CCK. In contrast, other major interneuron types, such as those containing parvalbumin, lack CB1 receptors. This pattern of expression is common to most forebrain areas, having been found in the anterior cortical nucleus , the basolateral amygdala , the cortical amygdaloid nuclei , the hippo campal formation , and the neocortex . Moreover, an analogous pattern is also seen in the human hippo campal formation . This selective distribution implies that CB1 receptor-dependent effects of cannabinoids on many of the physiological processes related to these forebrain areas might involve the modulation of a particular sub-population of GABAergic interneurons and predicts that this interneuron population may be closely connected with the participation of the endocannabinoids in the short-range modulation of synaptic activity, which will be further discussed in section VC. Although the strong expression of CB1 receptors in GABAergic interneurons of the cortex is now well established, the presence of CB1 receptors in principal cells of the forebrain is still debated. Initial in situ hybridization studies reported a modest CB1 mRNA expression in principal neurons of the neocortex . Subsequent double-labeling experiments showed, however, that all CB1-expressing cells in this structure are also positive for the 65-kDa isoform of glutamic acid decarboxylase , the GABA-synthesizing enzyme that marks GABAergic cells . Moreover, although several investigators have reported low CB1 mRNA expression in principal neurons of the CA3 and CA1 sub-fields of the hippocampus , a more recent study suggested that CB1 labeling may be restricted to GABAergic interneurons . Even looking at the original figures claiming CB1 expression in pyramidal neurons , the density of labeling over the principal cells seems to be remarkably low compared with the interneuronal labeling . This very low expression pattern within the principal neurons of cortical networks is similar in most other forebrain areas and was found in the human brain as well . This disagreement could not be settled by immunocytochemical localization of the CB1 protein. Experiments with antibodies raised against the NH2 terminus of the CB1 receptor found labeling of principal neurons in many forebrain areas . However, these studies also report CB1 immunoreactivity in cell populations from other brain areas, which were found to benegative for CB1 mRNA in all in situ hybridization studies . Other investigators utilized different antibodies directed either against the NH2 or the COOH terminus of the CB1 protein, and unequivocally established antibody specificity with control tests on brains of mutant CB1 /_x0005_ mice . These carefully controlled studies found CB1 immunostaining only in GABAergic interneurons of the cortex . However, the fact that principal cells were not stained in these experiments does not rule out the possibility that a very low amount of CB1 protein, undetectable by the antibodies, may be present in principal cells. Moreover, targeting of the receptor to axon terminals could further decrease antibody access to the antigen and account for the lack of cell body staining. Indeed, several laboratories have reported that glutamatergic synaptic currents in neurons of the prefrontal cortex and hippocampus are inhibited by cannabinoid agonists via a presynaptic mechanism . Yet, the lack of CB1 immunore activity on axon terminals forming asymmetrical synapses strongly argues against the presence of CB1 receptors at these glutamatergic terminals .