However, we cannot exclude the possibility that neurons lacking expression of SST and CCK represent another subtype of LTS cells. recordings SAPK3 from LTS interneurons in acute neocortical slices showed that inclusion of DAGL inhibitors in the whole-cell pipette prevented the long-lasting hyperpolarization triggered by LTS cell Angiotensin III (human, mouse) repetitive firing. Similarly, extracellular applications of a PLC inhibitor prevented SSI in LTS interneurons. Moreover, metabotropic glutamate receptor-dependent activation of PLC produced a long-lasting hyperpolarization which was prevented by the CB1 antagonist AM251, as well as by PLC and DAGL inhibitors. The loss of SSI in the presence of intracellular DAGL blockers confirms that endocannabinoid production occurs in the same interneuron undergoing the persistent hyperpolarization. Since DAGLs produce no endocannabinoid other than 2-AG, these results identify this compound as the autocrine mediator responsible for the postsynaptic slow self-inhibition of neocortical LTS interneurons. Keywords:neocortex, endocannabinoids, interneurons, 2-AG, inhibition, SSI == Introduction == Endocannabinoids are the endogenous ligands of the cannabinoid receptor CB1 and CB2. These receptors are the target of 9-tetrahydrocannabinol, the active principle of the recreational drug marijuana, well known for its psychoactive and analgesic Angiotensin III (human, mouse) effects (Freund et al., 2003;Iversen, 2003). Activation of the cannabinoid system typically leads to disruption of psychomotor behavior, short-term memory impairment, intoxication, and stimulation of appetite (Iversen, 2003;Di Marzo and Matias, 2005). At the cellular level, activation of CB1 receptors activates potassium channels (Deadwyler et al., 1995;Mackie Angiotensin III (human, mouse) et al., 1995) and inhibits calcium channel function (Mackie and Hille, 1992;Kreitzer and Regehr, 2001). Since CB1 receptors are highly expressed in presynaptic terminals, CB1-mediated effects on calcium channels can result in a reduced release of either GABA or glutamate (Hjos et al., 2000;Kreitzer and Angiotensin III (human, mouse) Regehr, 2001;Ohno-Shosaku et al., 2001;Wilson et al., 2001;Freund et al., 2003). We have recently found that, in layer V of rat neocortex, a subtype of GABAergic interneurons, classified as low-threshold spiking (LTS), undergoes a prominent and long-lasting hyperpolarization following their own repetitive firing. This slow self-inhibition (SSI) is due to the postsynaptic activation of a G-protein-coupled inward rectifier potassium (GIRK) conductance triggered by an activity-dependent autocrine action of endocannabinoids (Bacci et al., 2004). Endogenous cannabinoids are identified mainly in two endogenous lipids: the ethanolamide of arachidonic acid, anandamide (Devane et al., 1992;Mechoulam et al., 1994), and 2-arachidonoylglycerol (2-AG) (Mechoulam et al., 1995;Sugiura et al., 1995). The on demand biosynthesis of anandamide and 2-AG is strongly dependent on rises of intracellular Ca2+concentration (Di Marzo et al., 1994;Cadas et al., 1996;Bisogno et al., Angiotensin III (human, mouse) 1997;Cadas et al., 1997;Sasaki and Chang, 1997;Stella et al., 1997;Stella and Piomelli, 2001), such as occur during sustained neuronal activity. Anandamide and 2-AG are synthesized through different biochemical pathways, both using phospholipids as precursors. Anandamide is synthesized via hydrolysis of the phospholipid precursorN-arachidonoyl phosphatidylethanolamine via potentially alternative pathways (Di Marzo et al., 1994;Sugiura et al., 1996a,b;Liu et al., 2008;Simon and Cravatt, 2008). Two possible pathways involving phospholipase C (PLC), or phosphatidic acid hydrolase are believed to lead to the synthesis of 2-AG (Freund et al., 2003;Piomelli, 2003;Di Marzo et al., 2005). Diacylglycerols (DAGs), a major source of 2-AG for these pathways, are always converted into the endocannabinoid by diacylglycerol lipases (DAGLs) (Bisogno et al., 1997,1999;Stella et al., 1997), and are in turn produced via either PLC-dependent or -independent pathways. The last step of 2-AG biosynthesis is catalyzed by a specific enzyme,sn-1-DAG lipase, which is present in two isoforms in neurons (DAGL and DAGL). The expression ofsn-1-DAG lipase- in postsynaptic cells of adult rodents is correlated with 2-AG biosynthesis (Bisogno et al., 2003;Yoshida et al., 2006). DAGL substrates can be produced by PLC, which cleaves membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3and DAG (Freund et al., 2003;Piomelli, 2003;Bisogno et al., 2005). In neurons, PLC can be activated by group I metabotropic glutamate receptors (mGluRs). Indeed, endocannabinoid-dependent modulation of both short- and long-term synaptic transmission relies on mGluR activation in several brain areas (Varma et al., 2001;Brown et al., 2003;Freund et al., 2003;Piomelli, 2003;Jung et al., 2005;Maejima et al., 2005;Chevaleyre et al., 2006;Jung et al., 2007). Although the active players in the synthetic pathways of both anandamide and 2-AG are known, specific pharmacological agents that can be used to block the biosynthesis of endocannabinoids are lacking. Recently, specific inhibitors for the DAGL have been synthesized (Bisogno et al., 2006). Here we show that SSI can be robustly induced in LTS interneurons that express the neuropeptide somatostatin (SST), extending our previous findings demonstrating SSI expression in CCK-containing LTS cells. Blocking the last step in 2-AG production prevented SSI. Moreover, PLC blockade prevented SSI, whereas PLC activation by a mGluR agonist mimicked SSI in LTS interneurons. Together these results identify 2-AG as the endogenous cannabinoid responsible for the.