A similar presynaptic localization also has been suggested for CB1 receptors in dorsal root ganglion neurons, because resection of the dorsal root significantly decreases cannabinoid binding in the dorsal horn of the spinal cord . An important achievement in cannabinoid research was the development of specific antibodies recognizing CB1 receptors, which have become indispensable research tools . Antibodies raised against either the NH2 terminus or the COOH terminus of the CB1 protein provided crucial information about the precise localization of CB1 receptors at the regional, cellular, and sub-cellular levels. However, immunohistochemical studies require careful investigation and well-designed controls, since it is rare that an antibody is absolutely specific for the desired target protein. Thus reports claiming immunore activity for CB1 receptors in cells , which do not produce the mRNA of CB1, or immunolabeling of glial cells due to the antibody recognizing the antigen carrier protein should be viewed with caution . Essential in this regard was the generation of mutant CB1 /_x0005_ mice , which were instrumental to demonstrate antibody specificity and limit the confusion resulting from staining artifacts . Initial light microscopy studies revealed the existence of numerous CB1-immunoreactive fibers throughout the brain . Based on their distinctive morphological appearance, thin and rich in varicosities, these fibers were tentatively identified as axons. This identification received its first subcellular confirmation from work conducted in the rat hippocampus . The varicosities observed at the light microscopy level were found to correspond to axon terminals packed with synaptic vesicles and to be densely covered by CB1 receptors . Notably,cannabis grow racks when an antibody against the extracellular NH2 terminus of the CB1 receptor was used in combination with silver-impregnated gold particles, the particles were exclusively found at the outer surface of the axonal plasma membrane .

On the other hand, when the staining was carried out with a different antibody, specific for the COOH terminus, the gold particles only labeled the intracellular surface of the boutons . CB1-positive axons have a scattered pattern of distribution, which largely parallels that obtained with radioli-gand binding . An especially dense fiber mesh work is observed in the globus pallidus, the substantia nigra pars reticulata, and the entopeduncular nucleus, probably on axons deriving from the striatum. In many cortical areas, as well as in olfactory systems, CB1-immunoreactive axons are abundant and form pericellular baskets around CB1-negative cell bodies. Likewise, CB1-positive axons equipped with numerous boutons cover the somata of Purkinje cells in the cerebellum and shape the characteristic pinceaux structures around the axon initial segments . In addition, the stratum moleculare of the cerebellum also exhibits strong CB1 immunoreactivity, while leaving blank the dendritic tree of Purkinje cells . The cell origin of these fifibers can sometimes be inferred from the combination of cellular CB1 expression pattern and the distribution of CB1-positive axons. For example, in the cerebellum, the dense staining seen in the stratum moleculare likely results from axons of CB1-expressing granule cells, which constitute the so-called parallel fibers. In most cases, however, the cell origin and phenotype of CB1- carrying axons is still uncertain. Recent efforts have helped determine the precise subcellular distribution of CB1 receptors in the rodent somatosensory cortex, the hippocampus, and the amygdala, as well as in the human hippocampus . In these areas, CB1 receptors localize to specific types of axon terminals, and as a rule, boutons engaged in asymmetrical synapses do not carry CB1 receptors, whereas boutons engaged in symmetrical synapses do . This indicates that GABAergic, but not glutamatergic, axon terminals contain the receptors. GABAergic interneurons are extremely heterogeneous, however, and not all of them express CB1 receptors.

Indeed, only a sub-population of GABAergic interneurons, those that utilize CCK as a peptide cotransmitter, was found to be CB1 positive , whereas those marked by parvalbumin were not . Because CCK- and parvalbumin-positive interneurons have distinct roles in the regulation of cortical activity, it is likely that endocannabinoid substances also have specific functions in the modulation of cortical network properties. This notion is strongly supported by the retrograde messenger role of endocannabinoids in DSI, which is clearly restricted to select inhibitory synapses within the hippocampus .Outside the cortex, detailed information on the subcellular distribution of CB1 receptors is only available for the peripheral nervous system, where CB1 receptors also appear to be concentrated at nerve endings. In the rat and guinea pig lung, sparse nerve fibers bearing CB1 receptors are found among bronchial smooth muscle cells . Although such fibers rarely form true synapses, immunogold labeling reveals that CB1 receptors are located close to vesicle accumulations, where they may act to modulate neurotransmitter release. Importantly, neuropeptide Y, a neurochemical marker for noradrenergic sympathetic nerve fibers , was found to colocalize with CB1 in these axon terminals . Accordingly, cannabinoids potently inhibit norepinephrine release in peripheral tissues and organs through a presynaptic mechanism .Although anatomical studies may reveal the precise localization site of a particular receptor type, they may only provide predictions about its functional importance. In the last decade, two major approaches, electrophysiological recordings and neurochemical release studies, contributed fundamentally to our understanding of the physiological role of endocannabinoids and the consequences of cannabinoid receptor activation. Most of these studies point to the same conclusion as anatomical studies, i.e., CB1 receptors presynaptically regulate the release of certain types of neurotransmitters from axon terminals. The major goal of these studies is to establish which of the numerous types of neurotransmitters are influenced by cannabinoids at certain brain areas.

Not surprisingly, the release of nearly all major neurotransmitter types was shown to be affected by cannabinoid agents. Similarly to CB1-specific antibodies in anatomical experiments, the development of pharmacological probes, such as selective CB1 receptor agonists and antagonists, was indispensable to advance the field . However, as is the case with immunohistochemical experiments, the establishment of the role of CB1 receptors in many of the described processes requires careful evaluation. Recent studies using CB1 /_x0005_ mice provided evidence that conventional cannabinoid receptor ligands, as well as the endocannabinoids, are not exclusively selective for CB1 receptors . In the following sections, we survey the various lines of pharmacological evidence for the existence of presynaptic cannabinoid receptors on many different types of axons in several brain areas and aim to evaluate in the light of anatomical data whether CB1 or another molecular target may underlie certain effects of cannabinoids.In the hippocampus,cannabis grow system electrophysiological and neurotransmitter release experiments concord in indicating that cannabimimetic agents modulate GABA release via a presynaptic mechanism. Whole cell patch-clamp experiments show that cannabinoid agonists decrease amplitude and frequency of GABAA receptor-mediated inhibitory postsynaptic currents elicited by action potentials . These effects are mediated by CB1 receptors, because they are blocked by the CB1 antagonist SR141716A and are completely absent in CB_x0005_ /_x0005_ mice . The presynaptic action of cannabinoids was suggested by the lack of effect on the amplitude of miniature IPSCs , as well as by a reduction in vesicle release probability . These data are in striking agreement with the anatomical studies showing the presynaptic localization of CB1 receptors on GABAergic axon terminals. In the basolateral amygdala, which has a morphological architecture in many respects similar to the hippocampus, cannabinoid agonists produce comparable responses. The compounds inhibit synaptic GABAA-mediated currents in principal neurons of this region, but cause no such effect in the central nucleus, which does not contain CB1 receptors . The significance of these findings was also recently confirmed in vivo in the prefrontal cortex . In accordance with the exclusive expression of CB1 by GABAergic neurons in the neocortex , the cannabinoid receptor agonist WIN 55,212–2 reduced cortical GABA levels, which was prevented by the cannabinoid receptor antagonist SR 141716A . Moreover, neurochemical release experiments extended the validity of this finding from the rat to the human hippocampus . Taken together, these results indicate that GABAergic axon terminals are one of the major targets of cannabinoids in cortical networks, where they reduce the release of GABA in a CB1 receptor-mediated manner.Results from a variety of cortical tissue preparations are consistent in indicating that cannabinoid agonists can reduce excitatorysynaptic neurotransmission.

These actions are probably exerted at a presynaptic locus, for three reasons: 1) cannabinoid agonists increase paired-pulse facilitation, 2) they do not change postsynaptic responses to glutamate or kainate applications, and 3) they cause a characteristic increase in response failures and coefficient of variation of excitatory postsynaptic currents . The ability of the CB1 antagonist SR141716A to prevent these inhibitory responses suggested early on that CB1 receptors might be involved. Nevertheless, the fact that careful anatomical analyses negated this hypothesis sent the field up an apparent cul-de-sac: how could cannabinoid agonists inhibit glutamate release if CB1 receptors are only weakly, if at all, expressed by glutamatergic neurons and are absent from glutamatergic terminals ? The use of CB1 /_x0005_ mice offered a solution to this conundrum. Cannabimimetic agents reduce glutamatergic EPSCs in CB1 /_x0005_ mice to the same degree as they do in wild-type ones, although they no longer affect GABAergic IPSCs . The most economical hypothesis compatible with this result is that glutamatergic axon terminals contain a novel cannabinoid-sensitive site, which is blocked by SR141716A, but is molecularly distinct from the cloned CB1 receptor. Further pharmacological characterization revealed that the new cannabinoid-sensitive receptor has an order of magnitude lower affinity for WIN55,212–2 compared with CB1 , as the EC50 for the suppression of EPSCs was 2.01 M, whereas for IPSCs 0.24 M . In addition, cannabinoid effects on EPSCs could be antagonized by the vanilloid antagonist capsazepine, and mimicked by the agonist capsaicin, whereas vanilloid compounds were without effect on GABAergic IPSCs . These data clearly indicate that cannabinoid receptors controlling IPSCs versus EPSCs are pharmacologically distinct. The latter type is unlikely to be the vanilloid receptor VR1, since WIN55,212–2 does not bind to VR1 on sensory nerves . Moreover, VR1 forms a nonselective cation channel , whereas cannabinoid effects on glutamatergic EPSCs are mediated via a pertussis toxin-sensitive G protein-coupled process , which is in accordance with the ability of WIN 55,212–2 to stimulate [ 35S]GTP S binding in several brain regions of CB1 knockout mice . It is reasonable therefore to conclude that a cannabinoid-sensitive receptor other than CB1 or VR1 is located on glutamatergic, but not on GABAergic, axons in the hippocampus and possibly other brain areas . C) CANNABINOID EFFECTS ON ACETYLCHOLINE RELEASE IN CORTICAL AREAS. The cannabinoid receptor agonist WIN 55,212–2 decreases acetylcholine release from electrically stimulated rat hippocampal slices . This effect is mimicked by other synthetic cannabinoid agonists, as well as by the endocannabinoid anandamide, and is prevented by the CB1 antagonists SR141716A and AM281.The role of CB1 receptors in these responses, suggested by the effects of CB1 antagonists, is further supported by anatomical and genetic data. CB1 receptors are expressed by neurons in the medial septum and ventral diagonal band, where cholinergic innervation of the hippocampus originates . In the monkey forebrain, septal CB1-immunoreactive cells, along with other CB1- positive neurons in the nucleus basalis of Meynert , express choline acetyltransferase , the synthetic enzyme for acetylcholine . Furthermore, the cannabinoid modulation of acetylcholine release was reduced in “knockdown” experiments with antisense oligonucleotides and abolished in the hippocampus and the neocortex of CB1 knock-out mice . Although unequivocal anatomical demonstration of CB1 receptors on cholinergic axon terminals is still needed, physiological evidence also supports their existence. In hippo campal slices perfused with a Ca2-free, K-rich medium containing the Na channel blocker tetrodotoxin, cannabinoid agonists attenuate Ca2 -evoked acetylcholine release, probably by inhibition of voltage-gated Ca2 channels . Importantly, a parallel result was obtained in cortical and hippo campal synaptosomes, again implying a presynaptic site of action . What is the functional significance of these in vitro findings? Cholinergic innervation of cortical brain regions is thought to play an important role in cognitive processes, many of which are strongly impaired by cannabinoid treatment . An appealing causal link between these observations is strengthened by the finding that cannabinoid agonists reduce acetylcholine levels in rat cortical and hippocampal microdialysates, when administered at relatively high doses . However, recent experiments uncovered that lower doses of these drugs cause an opposite effect, elevating acetylcholine level in the prefrontal cortex and the hippocampus .