Mechanisms of endocannabinoid mobilization in hippocampus

Endocannabinoids (eCBs) act as retrograde messengers at inhibitory synapses of the hippocampal CA1 region. Current models place eCB synthesis in the postsynaptic pyramidal cell and the site of eCB action at cannabinoid (CB1) receptors located on presynaptic interneuron terminals. Four responses at the CA1-interneuron synapse are attributed to eCBs; depolarization-induced suppression of inhibition (DSI), G-protein-coupled receptor mediated enhancement of DSI (∆DSI), persistent suppression of eIPSCs, and finally, mGluR-dependent long-term depression (iLTD). It has been proposed that all are mediated by the eCB, 2-arachidonoyl glycerol (2-AG), yet there is evidence that DSI does not arise from the same underlying biochemical processes as the other responses. In view of the increasing importance of eCB effects in the brain, it will be essential to understand the mechanisms by which eCB effects are produced. Our results reveal new differences in the biochemical pathways by which the eCB-dependent responses are initiated. Both U73122, a phospholipase C (PLC) antagonist and RHC-80267, a diacylglycerol (DAG) lipase antagonist, prevented eCB-dependent iLTD induction by DHPG. However, mAChR activation does not cause eCB-dependent iLTD. Neither enzyme inhibitor affects DSI, and persistent eCB-dependent eIPSC suppression induced by either mGluRs or mAChRs is unaffected by U73122. On the other hand, inhibition of DAG lipase prevents persistent eCB-dependent eIPSC suppression triggered by mAChRs. The results show that the biochemical pathways for the various eCB-dependent responses differ and might therefore be independently manipulated. Introduction The active component of marijuana, ∆-tetrahydrocannbinol (THC), has many effects on the brain: it alters cognition, induces euphoria and relaxation, produces hypothermia, and prevents nausea (Ameri, 1999). These actions are the result of THC binding to a cannabinoid receptor (CB1) in brain regions that subserve these functions. In the CNS, there exist natural ligands to CB1, the 3 endocannabinoids (eCBs). Anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are the two predominant eCBs discovered so far (Devane et al., 1992; Mechoulam et al., 1996; Sugiura et al., 1995). In the hippocampus 2-AG is presently thought to be the predominant eCB (Stella et al., 1997; Kim and Alger, 2004; Makara et al. 2005), however this has not yet been definitively established, and a role for AEA or other messengers cannot be entirely ruled out. The CB1 receptor is found in high concentrations in the hippocampus, an area of the brain involved in learning and memory. In the hippocampal CA1 region, CB1 is found in highest concentration on the axon terminals of a subset of inhibitory interneurons, the CCK-containing cells that synapse onto the pyramidal cells (Freund et al., 2003). Regulation of inhibitory synaptic inputs onto pyramidal cells can significantly affect CA1 excitability as well as plasticity at excitatory synapses. ECBs are thought to be produced and released from the postsynaptic CA1 pyramidal cells, and then to suppress GABA release from CB1-expressing presynaptic interneuron terminals (Wilson and Nicoll, 2001a, 2001b; Ohno-Shosaku et al., 2001). By binding to CB1, eCBs can suppress inhibition, and therefore play an important role in regulating synaptic plasticity of both inhibitory (Chevaleyre and Castillo, 2003) and excitatory (Carlson et al., 2002; Chevaleyre and Castillo, 2004) synapses onto CA1 pyramidal cells. A great deal is known about the molecular basis of the eCB system (Piomelli, 2003, for review). ECB–mediated responses are produced following two distinct kinds of cellular stimulation: a strong increase in [Ca ]i and activation of certain G-protein coupled receptors, including group I mGluRs (Maejima et al., 2001; Varma et al., 2001), mAChRs (Kim et al., 2002; Ohno-Shosaku et al., 2003) and D2 receptors (Giuffrida et al., 1999). At the interneuron synapses in CA1, four different eCB-mediated effects can be observed following different stimuli. First, a post-synaptic rise in [Ca]i causes a transient, eCB-dependent reduction of GABA release. This is called depolarizationinduced suppression of inhibition (DSI). Second, low-to-moderate activation of post-synaptic mAChRs or group I mGluRs enhances DSI (∆DSI). Third, activation of mAChRs or mGluRs with a 4 higher concentration of agonist causes a persistent, relatively Cainsensitive, eCB-dependent suppression of eIPSCs. Fourth, prolonged activation of group I mGluRs produces a long-term depression of GABA release (iLTD) that is dependent on CB1 activation for induction, but not maintenance. These four eCB-mediated responses enable the CA1 pyramidal cell to regulate inhibitory synaptic inputs under a variety of stimulus conditions. The responses are distinguished by differences in their induction mechanisms and their durations. They are all thought to reflect eCB actions, and all may be mediated by 2-AG, and yet there is evidence that they may not all be mediated by the same underlying processes. Differences in these responses could reflect differences in the eCB induction pathways or other factors. For example, both M1/M3 mAChRs and group I mGluRs are G-protein coupled receptors that are linked to the phosphatidyl inositol/phospholipase C (PI/PLC) pathway, and the production of inositol trisphosphate (IP3) and diacyglycerol (DAG). The synthesis of 2-AG can proceed from the conversion of phosphatidyl inositol to DAG by PLC and from DAG to 2-AG by DAG lipase (Di Marzo et al, 1998). Both mGluRand mAChR-activated, persistent eCB responses are absent in mutant mice genetically engineered to lack PLCβ1 (Hashimotodani et al., 2005). Nevertheless, the mAChR and the mGluR pathways for inducing eCB responses might not be exactly the same (e.g. Kim et al., 2002). In addition, DSI is unaffected by pharmacological inhibition of PLC or DAG lipase (Chevaleyre and Castillo, 2003), and is intact in the PLCβ1 -/mouse. Prolonged activation of mGluR-induced eCB release can lead to iLTD (Chevaleyre and Castillo, 2003), however, it is not clear if induction of iLTD is exclusively due to CB1 activation. Because of the numerous roles they serve, it will be important to understand the various pathways involved in eCB-dependent responses. We have therefore investigated these and other questions involved in eCB actions in rat hippocampal CA1 pyramidal cells. Our findings suggest that multiple pathways may be involved in producing eCB-dependent responses. In addition to the difference between the DSI pathway and the GPCR-mediated responses 5 we find that the mGluR and mAChR-dependent eCB pathways are biochemically distinct. The data also suggest that, like some forms of LTD (Sjostrom et al., 2003; Ronesi et al., 2004), hippocampal mGluR-dependent iLTD may require the participation of additional factors that work in conjunction with eCBs, as eCB-dependent activation of CB1 appears necessary but not sufficient for iLTD induction. Materials and Methods Preparation of slices: After deeply sedating and decapitating 4to 6-week-old male SpragueDawley rats, we removed the hippocampi and cut them into 400-μm-thick slices in ice-cold bath solution [see below, excluding NBQX (Tocris, Ballwin, MO) and AP5 (Tocris)] using a vibratome (Technical Products International, St. Louis, MO). The protocols were approved by the Institutional Animal Care and Use Committee of the University Of Maryland School Of Medicine. The slices were maintained at room temperature for > 1hr in an interface holding chamber filled with humid 95% O2/5% CO2. Electrophysiology: Whole-cell voltage-clamp recordings of CA1 pyramidal cells were carried out at 30 – 31 C. Electrode resistances in the bath were 3 – 6 MΩ, and series resistances of <30 MΩ were accepted. If the series resistance, when checked by –1 or –2mV steps, changed significantly (~15%), we discarded the data. At the holding potential of –70 mV, monosynaptic evoked IPSCs (eIPSCs) were elicited by 100-μsec extracellular stimuli delivered with concentric bipolar stimulating electrodes placed in s. pyramidale between CA3 and CA1. In Fig. 1EF, s. radiatum was also stimulated alternately, 1.5 sec apart from s. pyramidale stimulation, every 6 sec. Data were collected using an Axopatch 1C amplifier (Axon Instruments, Union City, CA), filtered at 2 kHz and digitized at 5 kHz using a Digidata 1200 and Clampex 8 software (Axon Instruments). The pipette solution contained (in mM): 88 CsCH3SO3, 50 CsCl2, 1 MgCl2, 2 Mg-ATP, 0.3 tris-GTP, 0.2 Cs4-BAPTA, 10 HEPES, and 5 QX-314 (pH = 7.20 with CsOH, and 295 mOsm with 6 sucrose). The bath solution contained (in mM): 120 NaCl, 3 KCl, 25 NaHCO3, 1 NaH2PO4, 2.5 CaCl2, 2 MgSO4, 20 glucose, 0.01 NBQX and 0.05 DL-AP5 (or 0.02 D-AP5) (300 mOsm). The bath solution was oxygenated with 95% O2/5% CO2, and flowed through the recording chamber at a rate of ~1 ml/min. In Fig. 1F, 2B and 5BC, the slices were preincubated with AM251 (Tocris), RHC80267 (Biomol, Plymouth Meeting, PA) or U73122 (Tocris) for >1 hr, and the corresponding drug was present in the bath solution throughout recording. The final concentration of DMSO, a solvent, was 0.1% (v/v) for RHC-80267, U73122, THL, and 0.01% for AM251. All other chemicals were purchased from Sigma (St. Louis, MO). Data Analysis: To measure DSI, we evoked eIPSCs at 4 sec intervals and depolarized the postsynaptic cell to 0 mV for 1 sec at 89or 120-sec intervals. When DSI was not measured, eIPSCs were evoked every 6 sec. DSI was calculated as follows: DSI (%) = 100 x [mean amplitude of 4 eIPSCs after depolarization / mean amplitude of 5 eIPSCs before depolarization]. Values of 2 – 3 DSI trials were averaged to obtain a mean DSI in a given condition. Changes in DSI (∆DSI) were calculated as ∆DSI = test DSI – control DSI. Evoked IPSC suppression was calculated as follows: 100 x [mean amplitude of 15 eIPSCs following drug treatment/mean amplitude of 15 eIPSCs before]. Inhibitory long-term depression (iLTD) was determined to be present if eIPSCs were significantly suppressed after a 20-min washout of the agonist. Statistical tests were done in Excel XP (Microsoft Corp., Redmund, WA) and the p value for significance was <0.05. Paired t tests were used, unless otherwise stated. For multiple comparisons, we used ANOVA followed by multiple t tests or HolmSidak tests.