Immunofluorescence staining for MGL using two different antibodies recognizing independent epitopes of the MGL protein resulted in a comparable distribution pattern, although the general density of staining was stronger for the antibody “MGL-mid” in human hippocampal sections . Notably, as with the DGL-α-immunostaining, the distribution pattern of MGL mirrored the laminar structure of the hippocampal formation and was found to be similar in mouse and human hippocampi . At higher magnification, the stratum oriens showed the strongest density of MGL-immunoperoxidase reactivity in the cornu ammonis , but profound staining was also observed in strata pyramidale and radiatum . This pattern of expression in the three sub-fields of the cornu ammonis was very similar. Immunoperoxidase labeling for MGL was also found in the hilus and in the stratum moleculare of the dentategyrus , with a somewhat stronger MGL immunoreactivity visible in the outer two-thirds of the dentate molecular layer . Interestingly, this latter intensity pattern was in contrast with the distribution of DGL-α, which was more abundant in the inner third of the dentate molecular layer . At even higher magnification, cell bodies of pyramidal cells and granule cells were only weakly or not at all MGL-positive. Moreover, apical dendrites of pyramidal and granule cells were also largely devoid of immunolabeling for MGL . On the other hand, the neuropil among these dendrites and throughout the dendritic layers contained a dense, punctate MGL-positive staining. These varicosities were small, distributed with different densities in distinct layers and were often arranged in an array-like manner ,vertical farm companies reminiscent of the DGL-α-immuno reactivity pattern at the light microscopic level .
To test the prediction that the comparable dotted immuno staining pattern for DGL-α and MGL is due to the similar sub-cellular compartmentalization of these two enzymes with opposing functions in the metabolism of 2-AG, we performed a high-resolution electron microscopic analysis of MGL-immunostaining in the human hippocampal formation. The same regions were selected for detailed investigations as for DGL-α, the stratum radiatum of the CA1 region and the inner third of stratum moleculare of the dentate gyrus . Importantly, both antibodies revealed an identical staining pattern at the ultrastructural level. In addition, no differences in MGL-immunostaining were observed between strata radiatum and moleculare. At asymmetric, presumably glutamatergic synapses, MGL immunoreactivity was restricted to presynaptic axon terminals, in contrast to the postsynaptic localization of DGL-α. These MGL-positive boutons terminated most often on dendritic spine heads, but occasionally dendritic shafts were also present among their postsynaptic targets. The DAB end product indicating the presence of the MGL protein was predominantly found in the central part of the axon terminals often close to synaptic vesicles and to active zone release sites , and could be consistently followed through consecutive ultrathin sections of the same terminals . Besides the immuno labeling in axon terminals, MGL-immuno reactivity also appeared in thin axonal segments that could be often identified as preterminal axons through serial sections. In contrast to axonal profiles, consistent MGL-immunoreactivity confirmed with both antibodies remained under detection thresholds at postsynaptic sides, dendritic shafts, cell bodies and in glial processes. Taken together, the abundance of MGL in axon terminals indicates that the majority of postsynaptically released 2-AG is inactivated presynaptically, close to its target, the CB1 cannabinoid receptor. Moreover, together with similar findings in the rodent hippocampus , these data also suggest that the entire molecular architecture of retrograde 2-AG signaling at excitatory synapses is evolutionarily conserved across species. Despite the compelling association of impaired endocannabinoid signaling with several neurological and psychiatric disorders , our knowledge regarding the molecular architecture of endocannabinoid system in the human brain is still limited.
In the present study, we provide evidence that the enzymatic machinery responsible for the metabolism of the endocannabinoid 2-AG is also present in the human brain; its distribution follows the topographic layout of excitatory, glutamatergic pathways in the human hippocampal formation; and finally, its enzymes are restricted to complementary subcellular compartments at excitatory synapses. DGL-α, the key serine hydrolase in the biosynthesis of 2-AG is found postsynaptically. In contrast, MGL, the primaryserine hydrolase responsible for hydrolyzing 2-AG is localized presynaptically. Together with the presynaptic position of CB1 cannabinoid receptors on glutamatergic axon terminals in the human hippocampus , these data suggest that the molecular architecture of 2-AG signaling underlies 2-AG’s postulated function as a retrograde synaptic messenger. Moreover, these findings also indicate that retrograde 2-AG signaling is an evolutionarily conserved feature of hippocampal excitatory synapses and its similar organization in rodents and humans may help to offer plausible strategies for human medical research based on experimental findings obtained in rodents. An important implication of the present findings is the central role of DGL-α and 2-AG in the regulation of excitatory synaptic communication in the human hippocampus. Immunostaining for DGL-α at the light microscopic level resulted in an abundant punctate staining throughout the neuropil, which delineated the layered structure of the human hippocampal formation. On the other hand, characteristic profiles, like cell bodies and major dendritic trunks were weakly or not at all labeled. The granular pattern and its uneven, layered distribution suggest that DGL-α has a compartmentalized distribution at the subcellular level. The intense staining and its overlap with glutamatergic afferent pathways indicate that this compartment may be the glutamatergic synapse. Indeed, further electron microscopic examination revealed that DGL-α is exclusively found in postsynaptic spine heads receiving asymmetric, presumably excitatory glutamatergic synapses. This characteristic postsynaptic position was found both in stratum radiatum of the CA1 sub-field and in stratum moleculare of the dentate gyrus. On the other hand, dendritic shafts from which these DGL-α-containing spines protrude, axon terminals and glial profiles were not consistently labeled suggesting that even if these subcellular domains hold low, at present undetectable, levels of the DGL-α enzyme, the majority of 2-AG biosynthesis occurs postsynaptically at glutamatergic synapses in the human hippocampal formation.
This peculiar subcellular position of DGL-α highlights its key function in the initiation of synaptic endocannabinoid signaling, whose human occurrence has been postulated based on numerous animal studies, but has never been demonstrated in human nervous tissue before. Using electron microscopy, a series of recent neuroanatomical findings reported a very similar postsynaptically compartmentalized distribution of DGL-α in several brain areas in rodents, for example in the prefrontal cortex , in the hippocampus , in the striatum , in the ventral tegmental area , in the cerebellum , in the auditory brainstem and even in the dorsal horn of the spinal cord . Thus, we propose that the matching postsynaptic localization of DGL-α in the human hippocampus and in many rodent brain areas indicates that DGL-α is an evolutionarily conserved component of excitatory synapses and thereby its synaptic functions established in animal experiments can be extrapolated to the human brain as well. What may be the synaptic function, which necessitates principal neurons to target DGL-α so precisely into dendritic spine heads? DGL-α synthesizes 2-AG from diacylglycerol, the common second messenger produced upon Gq/11-coupled receptor activation and phospholipase C-β activity . The most abundant Gq/11-coupled receptor and PLC-β-type enzyme in hippocampal dendritic spine heads is the metabotropic glutamate receptor type 5 and PLC-β1, respectively , and indeed, activation of mGluR5 leads to endocannabinoid-mediated retrograde synaptic suppression ,vertical farming racks and the elevation of 2-AG levels through PLC-β1 and DGL-α activity . Because 2-AG inhibits glutamate release from excitatory nerve terminals in hippocampal neurons , this negative feed-back pathway operating as a “synaptic circuit-breaker” may have a pivotal functional significance in controlling network excitability during neuronal insults leading to excitotoxicity . Although the medical importance of such a protective messenger system is obvious, it is not yet clear if the same molecular machinery is functional at excitatory synapses of human neurons as well. The postsynaptic accumulation of DGL-α at excitatory synapses in human hippocampal samples described in the present study along with evidence that mGluR5 is also present post synaptically at excitatory synapses both in the human hippocampus and in primate cortical areas underlies this notion, though similar data are not yet available for PLC-βs in humans. Interestingly, an independent structural support derives from the striking similarity in the density of DGL-α and mGluR5- immunostaining in relation to given hippocampal layers. For example, in the dentate gyrus, higher concentration of DGL-α was found in the inner third of the molecular layer than in the outer two-thirds of stratum moleculare both in rodents and in humans , underlying the observation that excitatory inputs of granule cells received from mossy cells may be more tightly controlled by endocannabinoids than afferents from the entorhinal cortex . Similarly to DGL-α distribution, the density of mGluR5-immunostaining is more pronounced in the inner molecular layer both in rodents and in humans . Finally, among glutamatergic terminal types, the concentration of CB1 receptors is also highest in those arborizing in the inner molecular layer .
Whether this intensity difference reflects the higher density of excitatory synapses in the inner molecular layer both in rodents , and in humans or it is due to synapse-specific variations in the regulation of synaptic 2-AG signaling needs to be established in further experiments. Nevertheless, the weaker density of MGL-immunostaining in the inner molecular layer indicating reduced capacity for 2-AG inactivation by MGL at mossy cell synapses gives some indirect support for the latter possibility and emphasizes that the termination of 2-AG signaling may be specifically regulated in the human hippocampus as well.We provide anatomical evidence that MGL, the main degrading enzyme of 2-AG has a widespread distribution in the human hippocampal formation. The nature of MGL immunoreactivity was comparable to DGL-α-immunostaining at the light microscopic level, with the profuse punctate labeling covering the neuropil and outlining the laminar structure of the hippocampus. Electron microscopic analysis uncovered that this compartmentalized staining pattern is due to the accumulation of immunolabeling at excitatory synapses. However, in striking contrast to the postsynaptically localized DGL-α enzyme, MGL was present presynaptically in glutamatergic axon terminals. This anatomical observation is in agreement with previous findings obtained in the rodent hippocampus , and it is also supported by recent physiological experiments demonstrating that MGL limits the duration of synaptic depression at hippocampal excitatory synapses . In addition, although further immunohistochemical studies using antibodies with higher sensitivity may reveal that MGL is not fully restricted to glutamatergic synapses, the present findings indicate that the highest concentration of MGL protein is likely located in excitatory boutons in the human hippocampus.It is estimated that approximately 85% of the brain’s 2-AG hydrolysis activity is accounted for this serine hydrolase . Its widespread distribution in excitatory axon terminals in the human hippocampus suggests that MGL may also play a similarly important role in 2- AG hydrolysis in the human brain. Together with our previous findings demonstrating the ubiquitous presence of CB1 cannabinoid receptors on the same excitatory terminals , these data collectively corroborate that MGL is the key enzyme terminating synaptic 2-AG signaling after activation of presynaptic CB1 receptors in the human hippocampal formation. Given that acute in vivo administration of JLZ184, the most potent selective inhibitor of MGL currently available, replicates nearly all of the characteristic behavioral effects of Δ9 – tetrahydrocannabinol by protecting endogenously released 2-AG from degradation , it is conceivable to hypothesize that JLZ184 may have a similar effect on the human brain based on the similar neuroanatomical localization of MGL in rodents and humans. Therefore, although MGL inhibitors hold great therapeutic potential in several medical applications , their predicted psychoactive side effects based on their cannabimimetic properties in animals , should be taken into consideration when pondering the use of these compounds in humans. Women who smoke marijuana have several good reasons to quit if they plan to get pregnant: maternal marijuana use has been associated with lower infant weight at birth1, as well as subtle cognitive deficits in childhood and adolescence. Studies in animals support these findings. The active constituent of marijuana, ∆9-tetrahydro cannabinol , binds to most nerve cells and some immune cells. Prenatal exposure of rats and mice to chemicals that engage these receptors reduces birth weight and impairs exploratory behavior and memory in offspring. Adding new grounds for concern, experiments in mice now unveil how cannabinoid signaling intricately regulates embryo implantation, the sequence of events leading to the adhesion of the embryo to the uterine wall. The new study, by Dey and colleagues, appears in a recent issue of the Proceedings of the National Academy of Sciences.