Cytosolic phospholipase A2 alpha/arachidonic acid signaling mediates depolarization-induced suppression of excitation in the cerebellum

Abstract

Background: Depolarization-induced suppression of excitation (DSE) at parallel fiber-Purkinje cell synapse is an endocannabinoid-mediated short-term retrograde plasticity. Intracellular Ca(2+) elevation is critical for the endocannabinoid production and DSE. Nevertheless, how elevated Ca(2+) leads to DSE is unclear.

Methodology/principal findings: We utilized cytosolic phospholipase A(2) alpha (cPLA(2)α) knock-out mice and whole-cell patch clamp in cerebellar slices to observed the action of cPLA(2)α/arachidonic acid signaling on DSE at parallel fiber-Purkinje cell synapse. Our data showed that DSE was significantly inhibited in cPLA(2)α knock-out mice, which was rescued by arachidonic acid. The degradation enzyme of 2-arachidonoylglycerol (2-AG), monoacylglycerol lipase (MAGL), blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine (AEA), fatty acid amide hydrolase (FAAH), did not affect DSE. These results suggested that 2-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K(+), indicating that large conductance Ca(2+)-activated potassium channel (BK) is sufficient to inhibit cPLA(2)α/arachidonic acid-mediated DSE. In addition, we showed that the release of 2-AG was independent of soluble NSF attachment protein receptor (SNARE), protein kinase C and protein kinase A.

Conclusions/significance: Our data first showed that cPLA(2)α/arachidonic acid/2-AG signaling pathway mediates DSE at parallel fiber-Purkinje cell synapse.

Figure 1. cPLA₂α deficiency inhibits DSE at parallel fiber-Purkinje cell synapse.

(A) The stimulus protocol with the holding potential (hp) of Purkinje cells and the stimulation timing (stim). The duration of depolarization to 0 mV was 50 ms. Δt between the depolarization and the test stimulus was 5 s. The numbers 1, 2, 3 index control parallel fiber stimuli and 4 labels the test stimulation. The intervals between indexed 1, 2, 3 were 20 s. The intervals between 3 and depolarization was 10 s. (B) Amplitudes of parallel fiber EPSCs derived from one WT Purkinje cell plotted over time for control responses with no preceding prepulse to 0 mV (open circles) and test responses following depolarization (closed circles). Numbered circles (1, 2, 3, 4) correspond to the control and test stimuli in (A), respectively. Representative EPSCs are shown at the right. Stimulus artifacts are blanked for clarity. (C) EPSCs of one KO Purkinje cell plotted over time for control responses with no preceding prepulse to 0 mV (open circles) and test responses following depolarization (closed circles). Representative EPSCs are shown at the right. (D) EPSCs derived from one WT Purkinje cell plotted over time. AACOCF₃ was perfused throughout the experiment, as shown by the bar at top. Control and test responses are shown by open and closed circles, respectively. Representative EPSCs are shown at the right. (E) EPSCs derived from one KO Purkinje cell plotted over time. AACOCF₃ was perfused throughout the experiment, as shown by the bar at top. Control and test responses are shown by open and closed circles, respectively. Representative EPSCs are shown at the right. (F) Bar graphs show the percentage inhibitions of test EPSCs in WT, KO, WT+AACOCF₃ and KO+AACOCF₃. ctrl: control responses (n = 88). WT: 28.3±5.4%; n = 26. KO: 79.4±5.8%; n = 20. WT+AACOCF₃: 75.7±8.3%; n = 22. KO+AACOCF₃: 80.7±6.7%; n = 20. *, P<0.05.

Figure 2. Arachidonic acid rescues DSE in cPLA₂α knock-out mice.

(A) Time courses of percentage changes of parallel fiber EPSC amplitudes derived from WT (open circles) or KO (filled circles) mice. Arachidonic acid was applied in the bath as indicated by the bar. Arachidonic acid depressed EPSCs in both KO and WT cells. (B) Representative parallel fiber EPSCs from WT and KO cells at the time points indicated in (A). Stimulus artifacts are blanked for clarity. (C) Arachidonic acid restored DSE in KO cells. DSE was induced by the protocol indicated in Figure 1A with Δt 5 s. Each data point represents the percentage inhibition of test EPSC every 2 min. Arachidonic acid was applied in the bath as indicated by the bar.

Figure 3. MAGL blocks the action of arachidonic acid in DSE.

(A) EPSCs from one WT Purkinje cell plotted over time for control responses (open circles) and test responses (closed circles). Cells were filled with MAGL as indicated by the bar. Representative EPSCs are shown at the right. The percentage inhibition of test EPSCs (89.7±9.1%; n = 21) is shown in (A1). (B) EPSCs from one WT Purkinje cell plotted over time for control (open circles) and test responses (closed circles). Cells were filled with FAAH as indicated by the bar. Representative EPSCs are shown at the right. The percentage inhibition of test EPSCs (28.7±7.1%; n = 23) is shown in (B1). (C) Time courses of percentage changes of parallel fiber EPSC amplitudes derived from KO cells filled with either MAGL (filled circles) or FAAH (open circles). Arachidonic acid (AA) was applied in the bath as indicated by the bar. Arachidonic acid depressed EPSCs in FAAH-filled cells but not MAGL-filled cells. (D) MAGL blocked the rescue of DSE by arachidonic acid in KO cells. KO cells filled with either MAGL (filled circles) or FAAH (open circles) Arachidonic acid restored DSE in KO cells. DSE was induced by the protocol indicated in Figure 1A with Δt 5 s. Each data point represents the average percentage inhibition of test EPSC every 2 min. Arachidonic acid was applied in the bath as indicated by the bar. *, P<0.05.

Figure 4. Paxilline reverses the blockade of DSE by internal K⁺.

(A) EPSCs from one WT Purkinje cell plotted over time for control responses (open circles) and test responses (closed circles). Representative EPSCs are shown at the right. Internal K⁺ was applied as indicated by the bar. The percentage inhibition of test EPSCs (89.3±10.4%; n = 26) is shown in (A1). (B) EPSCs from one WT Purkinje cell plotted over time for control (open circles) and test responses (closed circles). Representative EPSCs are shown at the right. Internal K⁺ plus external apamin was applied as indicated by the bar. The percentage inhibition of test EPSCs (89.2±9.9%; n = 20) is shown in (B1). (C) EPSCs from one WT Purkinje cell plotted over time for control (open circles) and test responses (closed circles). Representative EPSCs are shown at the right. Internal K⁺ plus external paxilline was applied as indicated by the bar. The percentage inhibition of test EPSCs (36.6±8.4%; n = 22) is shown in (C1). *, P<0.05.

Figure 5. BoTx, PPADS, chelerythrine and KT5720 do not influence DSE.

(A) EPSCs from one WT Purkinje cell plotted over time for control (open circles) and test responses (closed circles). Representative EPSCs are shown at the right. Internal BoTx was applied as indicated by the bar. The percentage inhibition of test EPSCs (27.8±9.5%; n = 17) is shown in (A1). (B) EPSCs from one WT Purkinje cells plotted over time for control (open circles) and test responses (closed circles). Representative EPSCs are shown at right. The percentage inhibition of test EPSCs (29.2±9.1%; n = 25) is shown in (B1). (C) and (D), Control (open circles) and test (closed circles) EPSCs from two WT Purkinje cells are plotted over time. Representative EPSCs are shown at the right. Representative EPSCs are shown at the right. (C1) and (D1) show DSE amplitudes in chelerythrine (28.7±10.3%; n = 18) and KT5720 (29.2±10.1%; n = 18), respectively. DSE in WT cells (Figure 1E; gray bar) is replotted in (C1) and (D1) for comparison. Applications of BoTx, PPADS, chelerythrine and KT5720 are indicated by bars. Stimulus artifacts of EPSCs are blanked for clarity. *, P<0.05.

Figure 6. A proposed model for DSE.

A proposed model for 2-AG release and DSE at parallel fiber-Purkinje cell synapse. Strikethrough texts show the molecules unrelated to DSE, as demonstrated in the present work. See Discussion for explanation.