To reveal the site of synthesis of the endocannabinoid 2-AG in the human hippocampus by determining the localization of its predominant synthesizing enzyme DGL-α , we first sought to identify an antibody with unequivocal specificity for this transmembrane serine hydrolase. Therefore, DGL-α- immunostaining was performed and compared in hippocampal sections derived from wildtype or DGL-α knockout mice. Using an affinity-purified antibody raised against a large intracellular loop on the C-terminus of DGL-α , immunoperoxidase reaction revealed at low magnification that the general dense distribution of DGL-α-immunostaining followed the topographic arrangement of glutamatergic pathways in the wild-type hippocampus. In contrast, the immunoreactive material was almost fully absent in the DGL-α knockout hippocampus confirming the specificity of the “DGL-α INT” antibody. At higher magnification, the differences in staining intensity between the somatic and dendritic layers were even more pronounced. While nuclei and cell bodies in the principal cell layers were largely devoid of DGL-α-immunoreactivity, an intense punctate staining pattern was observed throughout the neuropil in those layers, which contain a high density of excitatory synapses in the hippocampus. This was in accordance with the observations we have reported earlier using this antibody in the hippocampus and in other regions. On the other hand, this punctate labeling was largely missing in DGL-α knockout hippocampi. Therefore, in the next set of experiments, we incubated hippo campal sections derived from human subjects together with hippo campal sections derived from wild-type C57BL/6 mice using the “DGL-α INT” antibody. At low magnification, immunofluorescence staining for DGL-α was unevenly distributed throughout the human hippo campal formation.
This pattern followed the laminar organization of the hippocampus and was found to be largely similar in mice. At higher magnification,drying rack cannabis the highest density of DGL-α- immunoperoxidase reactivity was observed in the strata oriens and radiatum of the cornu ammonis subfields, and in the inner molecular layer of the dentate gyrus , whereas somewhat weaker, but still significant density of DGL-α-immunoreactivity was found in the strata pyramidale and lacunosum-moleculare of the cornu ammonis and in the outer two-third of the stratum moleculare. Somata of pyramidal cells and dentate gyrus granule cells contained only very low amount of DGL-α-immunolabeling. At even higher magnification, the punctate staining pattern also showed striking similarities with the pattern observed in wild-type mice. This widespread granular pattern of DGL-α-immunoreactivity was visible throughout the hippo campal formation, but its distribution varied with regard to given subcellular profiles. For example, in the stratum radiatum of the CA1 sub-field, DGL-α-positive granules were distributed along the main trunk of the apical dendrites of pyramidal cells, whereas the trunk itself was devoid of immunostaining. Similarly, apical and possibly oblique dendrites of granule cells also appeared to be outlined on their surface by dense DGL-α-immunolabeling.To reveal the precise subcellular position of DGL-α in principal cells of the human hippocampus, we first tested the specificity of the “DGL-α INT” antibody at the ultrastructural level. Hippocampal sections from mice with different genotypes were processed together within the same incubation wells to ensure identical treatment throughout the imunostaining procedure. Further highresolution electron microscopic analysis in samples taken from the stratum radiatum of the CA1 sub-field of wild-type hippocampus revealed that DGL-α-immunoreactivity was predominantly concentrated in dendritic spine heads receiving asymmetric, putative excitatory synapses, in accordance with previous findings. Altogether, at least ~24% of dendritic spine heads were unequivocally positive for DGL-α immunoreactivity in our wild-type random samples ; this ratio should be treated as a conservative estimation restricted by epitope accessibility.
In contrast, under identical staining condition, only two out of 201 spine heads contained weak immunoperoxidase reaction end product in sections taken from the DGL-α knockout mouse , indicating the low level of background in this immunostaining experiment. To determine whether in the human hippocampus the same subcellular domain, namely the postsynaptic spine head, corresponds to the punctate staining pattern observed at the light microscopic level, hippocampal sections from human subjects with DGL-α- immunostaining were also processed for further electron microscopic analysis. Two regions were selected for detailed investigations, the stratum radiatum of the CA1 region and the inner third of the stratum moleculare of the dentate gyrus. In both regions, the DAB end product of the immunoperoxidase staining procedure, representing the subcellular position of DGL-α, was concentrated in dendritic spine heads protruding from DGL-α-immunonegative dendritic shafts. Because the majority of hippocampal GABAergic interneurons, including for example basket cells are aspiny, therefore the widespread occurrence of DGL-α in this characteristic subcellular compartment also reveals that principal cells express this enzyme in the human hippocampus. Notably, the DAB precipitate was consistently present within the spine heads through consecutive ultrathin sections. In contrast to this high concentration of DGL-α in dendritic spines, intensity of DGL-α-immunoreactivity did not reach the detection threshold in other subcellular profiles like excitatory and inhibitory axon terminals, or glial processes in the human hippocampus. Taken together, these data ultimately confirm previous findings that DGL-α accumulates postsynaptically in dendritic spines of principal cells in the mouse hippocampus and suggest that this 2-AG-synthesizing enzyme has a conserved function in the regulation of retrograde endocannabinoid signaling based on its entirely similar postsynaptic localization at excitatory synapses in the mouse and human hippocampus.If the enzyme responsible for 2-AG biogenesis is postsynaptically located , whereas its receptor is presynaptically positioned , then the next important question is where the 2-AG signal is terminated at excitatory synapses in the human hippocampus. Because MGL knockout mice have not yet become available to use as specificity controls, we employed two independent antibodies recognizing different epitopes of the MGL protein to characterize the regional distribution and subcellular localization of 2-AG’s principal hydrolyzing enzyme, MGL in the human hippocampal formation.
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,commercial greenhouse supplies 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.Immunoperoxidase labeling for MGL was also found in the hilus and in the stratum moleculare of the dentate gyrus , with a somewhat stronger MGLimmunoreactivity 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 , reminiscent of the DGL-α-immunoreactivity pattern at the light microscopic level.To test the prediction that the comparable dotted immunostaining pattern for DGL-α and MGL is due to the similar subcellular 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, MGLimmuno reactivity 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 immunolabeling in axon terminals, MGL-immunoreactivity 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.