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Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-␣ (DAGL␣) and -␤ (DAGL␤) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show ϳ80% reductions in 2-AG levels in the brain and spinal cord in DAGL␣ Ϫ/Ϫ mice and a 50% reduction in the brain in DAGL␤ Ϫ/Ϫ mice. In contrast, DAGL␤ plays a more important role than DAGL␣ in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGL␤ Ϫ/Ϫ mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABAmediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGL␣ Ϫ/Ϫ animals and relatively unaffected in DAGL␤ Ϫ/Ϫ animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGL␣ Ϫ/Ϫ and/or DAGL␤ Ϫ/Ϫ mice. These findings provide the first evidence that DAGL␣ is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.

Fusion of spleen cells from a mouse immunized with chicken embryo retina cells with clonal mouse myeloma cells yielded a lymphocyte hybrid cell line that produced antibody that bound to neural tissue such as retina, brain, spinal cord, and dorsal root ganglia but not to other tissues tested. The antigen was shown by indirect immunofluorescence to be associated with plasma membranes of most, or all, neuron cell bodies in chicken retina, but little or no antigen was detected on axons or dendrites, Muller cells, or retina pigment cells. The activity of antigen A2B5 is relatively stable at 100°C, is insensitive to trypsin, exhibits the solubility properties of a ganglioside, and is destroyed by neuraminidase. Antibody A2B5 cytotoxicity against retina cells is inhibited by a GQ ganglioside fraction from bovine brain (estimated half-maximal inhibition at 0.2 AM) or by N-acetylneuraminic acid (half-maximal inhibition at 5000 MM) but not by other purified gangliosides tested. These results suggest that the antigen is a complex ganglioside in plasma membranes of retina neuron cell bodies but not axons or dendrites. Chicken embryo retina cells have been used to study biological processes that require interactions between neurons, such as cell aggregation (1, 2), adhesiveness (3), and synapse formation (4-7). To define surface molecules of retina neurons, we have used the technique, introduced by Milstein and coworkers (8-10), of antibody production by hybrid cells formed by fusion of mouse myeloma cells with spleen cells from mice immunized with chicken embryo retina cells. This approach is useful because lymphocyte hybridomas can synthesize large quantities of monoclonal antibody with specificity for a single antigen determinant. Hybridoma cell lines have been reported which synthesize antibodies specific for cell surface antigens as diverse as the Forsmann antigen (11), HLA determinants (12), tumor-specific antigens (13), and an antigen detected on human neuroblastoma cells and fetal brain (14) (also see ref. 15).Other techniques also have been used to produce antisera against nervous system antigens. These include xenogenic immunization followed by extensive absorption with non-neuronal tissues (16,17) and immunization with neural cell lines (18-20), partially purified synaptosomes, plasma membranes, and protein fractions (21,22).In this report we describe the characterization of a monospecific antibody synthesized by lymphocyte hybrid A2B5 cells that recognizes a surface antigen restricted to cell bodies of most, or all, retina neurons. Flow General, Hamden, CT). The cell suspension was incubated for 30 min at 37°C; cells then were sedimented at 1000 X g; the supernatant solution was harvested by using Titertek harvesting filters (Flow General, and the radioactivity in the supernatant solutions that was absorbed by the filters was determined. Saturating concentrations of antibody A2B5 in the presence of complement released 50-60% of radioactive material from cells compared to that released by 0.3% Triton X-...

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