Further high-resolution immunostaining and electron microscopic analysis in the human hippocampal formation and neocortex have narrowed down the presence of CB1 receptors to GABAergic boutons and also to glutamatergic axon terminals . Together, these findings contribute to the hypothesis that 2-AG may be a synaptic messenger in the human nervous system. However, despite their potential therapeutic significance and their prominent mRNA expression levels in the human hippocampus , the precise location of two key enzymes, DGL-α and MGL, known to regulate 2-AG signaling at chemical synapses in rodents have not yet been investigated in detail in the human brain. The aim of our study was therefore to uncover the precise molecular organization of the 2-AG signaling pathway at excitatory synapses in the human hippocampus by using novel antibodies with confirmed target specificity for DGL-α and MGL, as well as light and high-resolution electron microscopy. Control hippocampi were kindly provided by the Lenhossék Human Brain Program, Semmelweis University, Budapest. Control subjects died suddenly from causes not directly involving any brain disease, and none of them had a history of any neurological disorders. The control subjects were processed for autopsy in the Department of Forensic Medicine of the Semmelweis University Medical School, Budapest, and the brains were removed 2–5 h after death. Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. Tissue was obtained and used in a manner compliant with the Declaration of Helsinki. All procedures were approved by the Regional and Institutional Committee of Science and Research Ethics of Scientific Council of Health . After postmortem removal, the hippocampal tissue was immediately dissected into 3- to 4- mm-thick blocks, and immersed in a fixative containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.2% picric acid in phosphate buffer . The blocks were first rinsed for 6 h at room temperature in the fixative, which was replaced every hour with a fresh solution.
The blocks were then post fixed overnight in the same fixative solution,vertical farming startup but without glutaraldehyde. In the case of one control brain, both the internal carotid and vertebral arteries were cannulated 4 h after death, and the brain was perfused with physiological saline followed by a fixative solution containing 4% paraformaldehyde and 0.2% picric acid in PB . The hippocampus was removed after perfusion, and was cut into 3- to 4-mm-thick blocks, and was post fixed in the same fixative solution overnight. Subsequently, 60-µm-thick coronal sections were prepared from the blocks with a Leica VTS-1000 Vibratome for immunohistochemistry.Immunostaining of the slices was performed as described previously . Briefly, after washing in PB , sections were cryoprotected in 10% sucrose and in 30% sucrose in PB for 15 min and overnight, respectively, then freeze thawed four times in an aluminium-foil boat over liquid nitrogen to enhance penetration of the antibodies without destroying the ultrastructure. Residual sucrose was washed from the tissue in PB and then endogenous peroxidase activity was blocked for 10 min by treatment with 1% H2O2 dissolved in PB. Subsequently, all washing steps and antibody dilutions were carried out in Tris-buffered saline . After extensive washing in TBS , sections were first blocked with 5% normal goat serum for 45 min, washed in PB for 15 min, and then incubated with the primary antibody of interest for 48 h. The following polyclonal, affinity-purified primary antibodies were used in the present study: rabbit anti-DGL-α ; rabbit anti-MGL ; and rabbit anti-MGL . The specificity of the DGL-α-INT antibody was confirmed by the lack of immunostaining in hippocampal sections derived from DGL-α knockout mice . The specificity of the two MGL antibodies was supported by immunostaining in HEK293 cells transiently expressing a V5 epitope-tagged MGL; by the lack of immunostaining in neurons pre incubated with 5 µg/ml of the corresponding immunizing protein; and by the identical staining pattern in hippocampal sections at the electron microscopic level with the two antibodies raised against independent epitopes of the MGL protein .
After primary antibody incubation, human and mouse hippocampal sections were washed extensively in TBS , then treated first with biotinylated goat anti-rabbit IgG for 2 h, washed again three times in TBS, and then incubated with avidin-biotinylated horseradish peroxidase complex for 1.5 h. This step was followed again by washing in TBS and in Tris buffer , and finally the immunoperoxidase reaction was developed using 3,3- diaminobenzidine as chromogen and 0.01% H2O2 dissolved in TB. After the development of immunostaining, sections were washed in PB, treated with 1% OsO4 in 0.1 M PB for 20 min, dehydrated in ascending series of ethanol and acetonitrile, and embedded in Durcupan . During dehydration, sections were also treated with 1% uranyl acetate in 70% ethanol for 20 min. After overnight incubation in Durcupan, the sections were mounted onto glass slides and coverslips were sealed by polymerization of Durcupan at 56 °C for 48 h. Light microscopic analysis of immunostaining was carried out with a Nikon Eclipse 80i upright microscope , and light micrographs were taken with a DS-Fi1 digital camera . For electron microscopic investigations, selected immunoreactive profiles and regions were photographed and re-embedded for further ultrathin sectioning. Series of consecutive ultrathin sections were collected on Formvar-coated single-slot grids and counter stained with lead citrate for 2 min.For immunofluorescence double staining, after freeze-thawing and intense washing, the sections were first blocked with 5% normal donkey serum for 45 min, and then incubated with mouse anti-NeuN and either with rabbit anti DGL-α , or rabbit anti-MGL , or rabbit anti-MGL primary antibodies for 48 h. Afterward, the sections were washed again in TBS three times for 15 min each, then incubated with secondary antibodies Alexa 594- conjugated goat anti-mouse IgG and DyLight 488- conjugated donkey antirabbit IgG for 2 h.
Excess secondary antibody was washed out three times in TBS, and three times in 0.1 M PB for 15 min each. Finally, the sections were mounted in Vectashield onto glass slides, and the coverslips were sealed with nail polish. Image acquisition was performed with an inverted Nikon Eclipse Ti-E microscope equipped with an A1R confocal system . Images of double labeling were obtained of a single focal plane by a 4× objective in sequential scanning mode using a four channel PMT detector. For the adjustment of digital light and electron micrographs, Adobe Photoshop CS2 was used. In all imaging processes, adjustments were done in the whole frame and no part of an image was modified separately in any way. 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 wild type 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-immunore activity, 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,vertical urban farming we incubated hippocampal sections derived from human subjects together with hippocampal 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 hippocampal formation . This pattern followed the laminar organization of the hippocampus and was found to be largely similar in mice . At higher magnification, 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 hippocampal formation, but its distribution varied with regard to given subcellular profiles. For example, in the stratum radiatum of the CA1 subfield, DGL-α-positive granules were distributed along the main trunk of the apical dendrites of pyramidal cells, whereas the trunk itself was devoid of immuno staining . 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 high resolution electron microscopic analysis in samples taken from the stratum radiatum of the CA1 subfield 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 immuno peroxidase reaction end product in sections taken from the DGL-α knockout mouse , indicating the low level of background in this immuno staining 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.