Lipid signaling in cytosolic phospholipase A2α– cyclooxygenase-2 cascade mediates cerebellar long- term depression and motor learning Tung Dinh Lea,1, Yoshinori Shiraia, Takehito Okamotob, Tetsuya Tatsukawab, Soichi Nagaob, Takao Shimizuc, and Masao Itoa,2 aIto Laboratory and bNagao Laboratory, RIKEN Brain Science Institute, Saitama 351-0198, Japan; and cDepartment of Biochemistry, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan Contributed by Masao Ito, December 31, 2009 (sent for review June 30, 2009) In this study, we show the crucial roles of lipid signaling in long- surge, self-regeneration may develop by positive feedback through term depression (LTD), that is, synaptic plasticity prevailing in this closed loop, leading to a rapid, intense activation of PKCα that cerebellar Purkinje cells. In mouse brain slices, we found that switches the status of PF-AMPARs from a basal state to the per- cPLA2α knockout blocked LTD induction, which was rescued by sistently depressed state for LTD (8, 20). replenishing arachidonic acid (AA) or prostaglandin (PG) D2 or E2. In this study, we demonstrate the critical requirement of – Moreover, cyclooxygenase (COX) 2 inhibitors block LTD, which is cPLA2α-COX-2 cascade for LTD induction. Incorporating the rescued by supplementing PGD2/E2. The blockade or rescue occurs cascade and its downstream pathways, we modify the LTD- when these reagents are applied within a time window of 5–15 inducing signal transduction model (Fig. 1). Furthermore, to test min following the onset of LTD-inducing stimulation. Furthermore, the current hypothesis that LTD underlies motor learning, we PGD2/E2 facilitates the chemical induction of LTD by a PKC activa- examine whether the deficiency of cPLA2α or the i.p.admin- tor but is unable to rescue the LTD blocked by a PKC inhibitor. We istration of a COX-2 inhibitor affects the adaptation of opto- conclude that PGD2/E2 mediates LTD jointly with PKC, and suggest kinetic eye movement response (OKR) in awake mice (21). Part possible pathways for their interaction. Finally, we demonstrate in fi of the present ndings have been reported preliminarily.* NEUROSCIENCE awake mice that cPLA2α deficiency or COX-2 inhibition attenuates short-term adaptation of optokinetic eye movements, supporting Results the view that LTD underlies motor learning. For slice experiments, we used six mouse types: (i) wild-type C3H/HeN strain (abbreviated as C3H WT), (ii) wild-type PLA2 | prostaglandin | Purkinje | arachidonic | OKR −/− C57BL/6J strain innately being sPLA2IIA (C57BL WT) (22), −/− −/− (iii) cPLA2α C3H/HeN strain (C3H KO), (iv) cPLA2α −/− lthough lipid signaling is implicated in diverse biological sPLA2IIA C57BL/6J strain (C57BL KO), (v) littermate of Afunctions, its importance in synaptic mechanisms has become C3H KO (C3H littermate), and (vi) littermate of C57BL KO increasingly evident (1–3). In the first step of lipid signaling, (C57BL littermate). C57BL/6Cr WT mice were also used for phospholipase A2 (PLA2) acts on membrane phospholipids to testing OKR. In acute slices obtained at PW5-12, we recorded release arachidonic acid (AA) and certain lysophospholipid from PCs with patch pipettes in a current clamp configuration. mediators. We previously reported that, among the more than 20 We recorded PF stimulation–evoked excitatory postsynaptic fi fi PLA2 subtypes so far identi ed (2, 4), ve subtypes are located in potentials (PF-EPSPs) (Fig. 2A, Inset). Various reagents (Table α the cerebellum, three of which (cPLA2 ,sPLA2IIA, and iPLA2) S1) were applied to PCs by intracellular infusion through a patch are expressed with different patterns of localization in Purkinje pipette or bath applied to reach PCs under observation by – cells (PCs) (5). It has also been reported (6 8) that PLA2 is involved extracellular perfusion. LTD was induced by conjunction (Cj) of in cerebellar postsynaptic long-term depression (hereafter referred double-shock PF stimulation and current pulse–induced mem- to as LTD) that is a persistent attenuation of L-α-amino-3-hydroxy- brane depolarization (200 ms, 0.8–1.2 nA), repeated at 1 Hz for – 5-methylisoxazole-4-propionate receptor (AMPAR) mediated 5 min (23). Cj induced LTD similar to that induced by PF-CF fi transmission in parallel ber (PF) synapses on PCs (9). In the conjunction (Table S2). PFs were stimulated with double pulses second step of lipid signaling, AA is converted by cyclooxygenases (each 0.1 ms in duration) paired at 50-msec intervals timed in (COXs) to prostaglandins (PGs) (10) or by lipoxygenases (LOs) to such a way that the first pulse fell 30 ms later than the onset of hydroxy fatty acids and leukotrienes (11). In the present study on each depolarizing pulse. Separate application of double-shock mouse cerebellar slices, using genetic and pharmacological PF stimulation or membrane depolarization induced no appre- manipulations, we show the involvement of these enzymes and ciable LTD (Fig. 2A). LTD developed during the initial 10 min their products in LTD induction. and was followed by a slow phase continuing for another 40 min. In LTD induction, the conjunction of PF and climbing fiber (CF) impulses evokes a strong Ca2+ surge in PC dendrites. This is caused by a supralinear addition of CF impulse–induced Ca2+ t 2+ – Author contributions: T.D.L., Y.S., S.N., T.S., and M.I. designed research; T.D.L., T.O., and entry hrough voltage-gated Ca channels and PF impulse T.T. performed research; T.D.L., Y.S., S.N., and M.I. analyzed data; and T.D.L. and M.I. 2+ induced Ca release from the intracellular store via metabotropic wrote the paper. 2+ glutamate receptor type 1 (8, 12, 13). A Ca -sensitive protein The authors declare no conflict of interest. α kinase C subtype (PKC ) (14) is thus activated and in turn phos- Freely available online through the PNAS open access option. phorylates AMPARs at serine 880 of the GluR2 C terminus (15, 1T.D.L. is on leave from the Department of Physiology, Hanoi Medical University, Hanoi, 16). The phosphorylated AMPARs are subsequently dissociated Vietnam. from anchoring proteins and eliminated from the synaptic mem- 2To whom correspondence should be addressed. E-mail: [email protected]. brane via endocytosis, thus causing LTD. In neurochemistry, AA This article contains supporting information online at www.pnas.org/cgi/content/full/ activates PKC, which in turn activates a kinase cascade sequen- 0915020107/DCSupplemental. fi tially involving Raf, MEK, ERK1/2, and nally PLA2, which pro- *T.D. Le, Y. Shirai, T. Shimizu, and M. Ito, seventh IBRO Congress in Melbourne 2007 Pos- 2+ duces AA (Fig. 1, blue arrows) (17–19). Hence, after the Ca Mon-344; Society for Neuroscience Annual Meeting in Chicago 2009 Poster 320. www.pnas.org/cgi/doi/10.1073/pnas.0915020107 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 P3 cPLA2 㻍 ERK1/2 AA㻍 PGD2/E2 conjunction COX-2 Ca2+ M 㻍 surge P1 P2 Raf㻍 PKC PF-AMPAR LTD Fig. 1. Signal transduction pathways activated by intracellular Ca2+ surge and leading to LTD. Green lines, major pathways. Blue arrows, loop connections. Red arrows, cPLA2α–COX-2 cascade. Brown lines (P1, P2, P3), possible pathways downstream of PGD2/E2. As a convention in this study, “LTD magnitude” represents the LTD (25.7% and 26.3%, respectively). The failure in LTD average decrease in PF-EPSP slopes at 41–50 min relative to induction and lack of cPLA2α in the two mutants strongly indi- their average during the 5 min preconjunction period, and is cate that cPLA2α is required for LTD induction. indicated as mean ± SEM % (number of cells tested) in Table S2 Pyrrolidine-1, a cPLA2α-specific inhibitor (24, 25), effectively (values given in the text are mean values only). LTD magnitudes blocked LTD induction. Under infusion of 0.4 μM pyrrolidine-1 in slices obtained from C3H WT and C57BL WT mice at PW 5–6 dissolved in 0.01% DMSO, the LTD magnitude decreased to are 26.3 and 29.2%, respectively (Fig. 2A and Table S2), showing 4.7% as compared with 27.8% attained under infusion of 0.01% no significant difference (Student t test, P = 0.234). Hence, we DMSO alone (Fig. S1 A and B). Mapping with 5- or 10-min conclude that sPLA2IIA, innately lacking in the genotype of the perfusions of pyrrolidine-1 revealed its time-limited effect on C57BL strain but present in that of the C3H strain (22), does not LTD (Fig. 2C). As summarized in Fig. 2D, LTD suppression is significantly contribute to LTD. maximal when the perfusion of pyrrolidine-1 overlaps or imme- diately succeeds the conjunction, and diminishes when the LTD Blockade by cPLA2α KO or Inhibitors. In C3H KO and C57BL inhibitor perfusion is more delayed. A modest effect of the KO mice, the basic electrophysiological properties of PCs at inhibitor perfusion preceding conjunction could be due to a postnatal weeks (PWs) 5–12 were comparable to those in lit- delay in washing out the inhibitor. Statistically significant termate mice. Nevertheless, we regularly failed to induce LTD in decreases in the LTD magnitude (open squares in Fig. 2D) are either of these two strains of mutant mice (Fig. 2B). In contrast, associated with the inhibitor perfusions covering the 10-min in C3H and C57BL littermate mice, PCs showed substantial period after the onset of conjunction (indicated by double- % % A 140 C57BL WT 2PF (10) x B 140 120 y 2mV 120 C57BL littermate (14) 10 ms 100 100 y 80 x 80 60 60 α C57BL WT md (11) C57BL cPLA2 KO (31) 40 C57BL WT 2PF + md (9) 40 C3H littermate (10) PF-EPSP slope PF-EPSP slope 20 20 α C3H WT 2PF + md (5) C3H cPLA2 KO (13) 0 0 -10 0 10 20 30 40 50 min -10 0 10 20 30 40 50 min % % C 140 10 min perfusion of D 40 120 0.4 μM pyrrolidine-1 30 100 80 20 (6) (5) (6) (6) 60 th 10 40 Perfusion from 0 min (6) (5) (5) th LTD magnitude PF-EPSP slope Perfusion from 5 min (5) 0 (7) 20 Perfusion from 10th min (5) (6) 0 -10 -10 0 10 20 30 40 50 60 70 80 min -10 0 10 20 30 40 min % % E 140 α F C57BL cPLA2 KO 40 120 Perfusion of 10 μMAA 30 100 (7) 80 20 60 10 40 Perfusion from 0 th min (7) (5) (6) LTD magnitude PF-EPSP slope th 20 Perfusion from 5 min (6) 0 (6) 0 -10 -10 0 10 20 30 40 50 60 min -10 0 10 20 30 40 min Fig.