Abstract
Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. Their medical significance may be best explained by the emerging concept that endocannabinoids are essential modulators of synaptic transmission throughout the central nervous system. However, the precise molecular architecture of the endocannabinoid signaling machinery in the human brain remains elusive. To address this issue, we investigated the synaptic distribution of metabolic enzymes for the most abundant endocannabinoid molecule, 2-arachidonoylglycerol (2-AG), in the postmortem human hippocampus. Immunostaining for diacylglycerol lipase-α (DGL-α), the main synthesizing enzyme of 2-AG, resulted in a laminar pattern corresponding to the termination zones of glutamatergic pathways. The highest density of DGL-α-immunostaining was observed in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum moleculare of the dentate gyrus. At higher magnification, DGL-α-immunopositive puncta were distributed throughout the neuropil outlining the immunonegative main dendrites of pyramidal and granule cells. Electron microscopic analysis revealed that this pattern was due to the accumulation of DGL-α in dendritic spine heads. Similar DGL-α-immunostaining pattern was also found in hippocampi of wild-type, but not of DGL-α knockout mice. Using two independent antibodies developed against monoacylglycerol lipase (MGL), the predominant enzyme inactivating 2-AG, immunostaining also revealed a laminar and punctate staining pattern. However, as observed previously in rodent hippocampus, MGL was enriched in axon terminals instead of postsynaptic structures at the ultrastructural level. Taken together, these findings demonstrate the post- and presynaptic segregation of primary enzymes responsible for synthesis and elimination of 2-AG, respectively, in the human hippocampus. Thus, molecular architecture of the endocannabinoid signaling machinery supports retrograde regulation of synaptic activity, and its similar blueprint in rodents and humans further indicates that 2-AG's physiological role as a negative feed-back signal is an evolutionarily conserved feature of excitatory synapses.
Highlights
Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders
To reveal the site of synthesis of the endocannabinoid 2-AG in the human hippocampus by determining the localization of its predominant synthesizing enzyme diacylglycerol lipase-α (DGL-α) (Bisogno et al, 2003; Gao et al, 2010; Tanimura et al, 2010), we first sought to identify an antibody with unequivocal specificity for this transmembrane serine hydrolase
Using an affinity-purified antibody raised against a large intracellular loop on the C-terminus of DGL-α (“DGL-α INT”; Katona et al, 2006), 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 (Fig. 1A)
Summary
Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. In contrast to conventional neurotransmitters, endocannabinoids are primarily synthesized and released by postsynaptic neurons in an on-demand manner (Kreitzer and Regehr, 2001; Ohno-Shosaku et al, 2001; Wilson and Nicoll, 2001), and subsequently activate presynaptically located CB1 cannabinoid receptors, thereby regulating neurotransmitter release from several types of axon terminals (Freund et al, 2003; Kano et al, 2009) This retrograde manner of synaptic endocannabinoid signaling is indispensable for various forms of homo- and heterosynaptic plasticity throughout the central nervous system (Chevaleyre et al, 2006; Kano et al, 2009), and probably accounts for the extensive involvement of the endocannabinoid system in brain disorders (Katona and Freund, 2008). The aim of our study was 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
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