This clear morphological finding is also supported by work with CB_x0005_ /_x0005_ mice, which suggests that an additional receptor, pharmacologically related, but molecularly distinct from the CB1, may mediate the cannabinoid modulation of glutamatergic transmission in the hippocampus . We will return to this hypothesis in section IVB1B.CB1 receptors are also present in several subcortical nuclei of the basal forebrain. Cells expressing moderate levels of this receptor are located mainly in the teniatecta, the lateral and medial septum, and the nuclei of the vertical and horizontal limbs of the diagonal band . Colocalization experiments show that CB1 receptors may be present in somatostatin-positive neurons of the lateral septum and in cholinergic cells in the medial septum and the nucleus basalis of Meynert .In keeping with the profound impact of cannabimimetic drugs on motor activity , in situ hybridization studies have invariably reported strong expression of CB1 mRNA in the striatum . Detailed analysis at the regional and cellular level uncovered a selective expression pattern in specific components of basal ganglia networks . In rodents, the highest density of CB1 mRNA is found in the dorsolateral portion of the striatum, where the transcript is primarily localized to GABAergic medium spiny cells, which constitute 90% of striatal neurons. In contrast, CB1 mRNA expression is rather low in two key output structures of the basal ganglia in the globus pallidus and in the substantia nigra. This is also true for the human basal ganglia, which lack however the dorsoventral gradient of mRNA expression seen in rodents . Although the globus pallidus and the substantia nigra pars reticulata contain little CB1 mRNA,cannabis plant growing cannabinoid binding is remarkably dense in these structures, implying that CB1 receptors may be mainly localized to the axons of striatonigral and striatopallidal GABAergic neurons .
Indeed, two colocalization studies have now established that CB1 mRNA is expressed by neurons that also contain high levels of the enzyme GAD65 and low levels of its higher molecular mass isoform, GAD67 . In a separate study, CB1 mRNA was found to be coexpressed with both preproenkephalin and prodynorphin , indicating that striatal projection neurons express CB1 receptors irrespectively of their specific target region . Interestingly, a small fraction of CB1-positive neurons contain neither preproenkephalin nor prodynorphin and express high levels of GAD67, which is typical of striatal interneurons. Thus, in addition to medium spiny projection cells, other neurons also may express CB1 mRNA. Because colocalization experiments revealed that CB1 is found neither in somatostatin-positive nor in cholinergic interneurons , the presumptive candidates are the remaining parvalbumin-containing cells . Indeed, Marsicano and Lutz demonstrated that 15% of the CB1-expressing neurons are positive for parvalbumin, providing direct evidence that striatal local-circuit neurons express CB1 receptors. It is important to reiterate that this expression pattern is opposite to the one found in cortical and amygdaloid structures, where parvalbumin-positive interneurons do not express CB1 receptors . While the above results are based on the presence of CB1 mRNA in striatal projection neurons and local-circuit cells, the cellular expression pattern has not been confirmed yet at the protein level, although the presence of the CB1 protein in striatal neurons has already been demonstrated by immunostaining .In situ hybridization studies have reported very low levels of CB1 mRNA expression in the thalamus . Subsequent work confirmed this finding both at the mRNA and at the protein level and extended it to the human brain . Neurons expressing moderate amounts of CB1 mRNA were observed in the habenula and the anterior dorsal part of thalamus, while CB1-immunoreactive cells were found in the reticular nucleus and zona incerta . Further studies are needed, however, to unambiguously identify these cells and solve remaining inconsistencies in the literature regarding their exact location in different nuclei. This need is further underscored by the finding that anterior and dorsal nuclei of the thalamus may express high levels of monoacylglycerol lipase, an intracellular serine hydrolase implicated in terminating the biological effects of the endocannabinoid, 2-AG .There is a coherent body of evidence indicating that the endocannabinoid system participates in the hypothalamic regulation of feeding and neuroendocrine function . Likewise, anatomical investigations agree in finding moderate levels of CB1 receptor expression in the ventromedial and anterior nuclei of the hypothalamus , while pharmacological experiments suggest that these receptors may be particularly well coupled to G proteins .
Importantly, a double-labeling study showed that CB1 receptors are colocalized with calretinin, a marker for glutamatergic neurons in select hypothalamic nuclei , but not with GAD65 . This suggests that glutamatergic, but not GABAergic, cells may express CB1 receptors in these nuclei. Other hypothalamic nuclei display very low levels of CB1 expression in a population of uniformly distributed cells. These nuclei include the medial and lateral preoptic nucleus, the magnocellular preoptic and hypothalamic nucleus, the premammilary nucleus and the lateral nucleus of the mammilary body, and the lateral hypothalamus . However, as elsewhere in the brain, there is still disagreement as to the precise identity and localization of hypothalamic CB1-expressing neurons, which will undoubtedly foster further scrutiny.The finding that noxious stimuli trigger anandamide release in the PAG, as assessed by in vivo microdialysis , implies that this midbrain structure may serve as a relay in the pain-processing circuit modulated by the endocannabinoids. Yet, a coherent description of the regional and cellular expression of CB1 receptors in the midbrain is still lacking. Although current data suggest that several midbrain nuclei may have very low to moderate expression of CB1 mRNA, they are in conflict regarding the exact identity of these nuclei . Immunostaining studies have shown that the superior colliculus contains CB1-positive neuronal cell bodies, but the identity of these cells was not determined . To be able to interpret the growing body of work on the analgesic and antihyperalgesic effects of cannabinoid agents, these morphological gaps need to be filled.Detailed morphological studies of the hindbrain are also rare. A notable exception is represented by the recent immunocy to chemical demonstration of CB1 receptors in the dorsal vagal complex of the ferret, which may be relevant to the autonomic and antiemetic effects of cannabinoid agonists . The exclusive presence of these receptors in local GABAergic interneurons, but not in preganglionic motor neurons , shows how this intriguing morphological leitmotif may recurrently be found at most levels of the neuraxis.CB1 receptor mRNA is highly abundant in the cerebellum . Owing to the well-determined circuitry of the cerebellar cortex, along with its laminar structure, the identification of neuronal elements expressing CB1 receptors in this region is relatively straightforward.
Strong expression levels are found in glutamatergic granule cells, but not in the GABAergic Purkinje cells . In the molecular layer, several large cells were also reported to express CB1,vertical grow rack which might belong to the basket and stellate cells . However, it is not known whether all cerebellar interneurons express CB1 or a subtype selectivity exists among them.One of the most important aspects of cannabinoids in terms of medicinal usefulness is their analgesic and antihyperalgesic effect at multiple stages of the pain-processing pathway, from high cognitive centers of the forebrain to the midbrain and down to peripheral tissues . The spinal cord, where cells expressing CB1 receptors have been extensively characterized, is obviously an integral component of this circuit. Most in situ hybridization and immuno staining studies agree that CB1 receptors are present in select neuronal populations of the spinal dorsal horn . In lamina II, GABAergic neurons expressing CB1 also contain nitric oxide synthase , a marker for a subset of spinal interneurons called islet cells . In addition, CB1- positive cells have also been found in lamina X, which surrounds the central canal of the spinal cord ; however, by using a different antibody, these cells could only be visualized after spinal transection.Primary sensory neurons in these ganglia are classified based on the selective expression of various neuropeptides [calcitonin gene-related peptide , substance P, somatostatin], or the responsiveness to neurotrophic factors [nerve growth factor , glial-derived growth factor , present in nociceptive neurons]. These cell-specific markers are rather heterogeneously colocalized with CB1 receptors. In a small population of dorsal root ganglion cells, CB1 receptors are present in CGRP and substance P-expressing neurons, but not in somatostatin-positive cells . This suggests that CB1 receptors may be expressed only by a subset of peptidergic nociceptive neurons, which represent 25% of all CB1- positive cells, whereas the remaining CB1-expressing cells may belong to other sub-populations of nociceptive or nonnociceptive neurons. Work in dorsal root ganglion cultures suggests that CB1 receptors colocalize with another nociceptor marker, the acid- and heat-sensitive vanilloid receptor 1 . Further triple immuno labeling experiments confirmed this observation and suggested that 25% of CB1-bearing neurons are nonnociceptive and that distinct types of nociceptive neurons express the receptor as well .
This highly heterogeneous distribution may contribute to explain the unprecedented analgesic effectiveness of cannabinoid agents, particularly in animal models of persistent pain of neuropathic origin .Based on the selective distribution of CB1 receptors in the CNS and their pervasive association with GABAergic interneurons, one would predict that the endocannabinoid system may play important and, possibly, unique roles in the local control of neuronal network activity. A growing body of functional evidence supports this prediction. For example, microdialysis experiments have found that anandamide is released in the striatum by activation of dopamine D2 receptor, where it may act as a short range mediator to counterbalance dopamine activity . Furthermore, an endocannabinoid substance, which remains unfortunately uncharacterized, has been recently identified as a key component in two related forms of trans-synaptic communication, known as depolarization induced suppression of inhibition and depolarization-induced suppression of excitation . In section V, we discuss how the endocannabinoid system may participate in these processes. But to do that, we first need to take a further step in the localization of CB1 receptors, down to the sub-cellular level. G protein-coupled receptors, such as the CB1, are embedded within the lipid bilayer of the plasma membrane. The membrane surface of a nerve cell can be subdivided into two functionally distinct spatial domains. The dendritic tree and cell body are equipped to receive synaptic contacts at specialized structures called active zones, where receptors for fast-acting neurotransmitters such as glutamate or GABA are concentrated. G protein coupled receptors are rarely associated with these structures; rather, a significant proportion of these receptors are found outside the synapse, within the so-called perisynaptic zone or even further away on the dendritic tree , where they can influence synaptic currents and neuronal excitability by triggering the formation of diffusible intracellular second messengers. Another group of G protein-coupled receptors is situated on axon terminals, where they are exquisitely poised to regulate the release of neurotransmitters, thereby controlling the final output of a neuron. Thus the question arises, in which neuronal surface domain are CB1 receptors localized? The most direct way to approach this question consists, when a receptor-specific antibody is available, in analyzing the sub-cellular distribution of the receptor by using electron microscopy. This approach can also provide a wealth of information on the structure and function of the synapse, such as the complement of neurotransmitters and additional membrane receptors present. Evidence from anatomical studies such as these, as well as functional experiments, indicates that CB1 receptors are predominantly found in axon terminal membranes, where they may be involved in the presynaptic regulation of neurotransmitter release.Indirect anatomical evidence for the localization of CB1 receptors on axon terminals was first provided by in situ hybridization and receptor binding experiments . These studies showed that, in the basal ganglia, CB1 receptor mRNA is almost exclusively localized to neurons within the striatum , whereas cannabinoid binding is strongest in the globus pallidus and the substantia nigra pars reticulata . This mismatch implies that CB1 receptors synthesized in the cell bodies of striatal projection neurons are transported to axon terminal fields in the pallidum and substantia nigra. In keeping with this hypothesis, ibotenic acid lesion of the rat striatum produces a marked loss of cannabinoid binding in these two regions .