12-lipoxygenase metabolites of arachidonic acid mediate metabotropic glutamate receptor-dependent long-term depression at hippocampal CA3-CA1 synapses

Abstract

Arachidonic acid metabolites have been proposed as signaling molecules in hippocampal long-term potentiation (LTP) and long-term depression (LTD) for >15 years. However, the functional role of these molecules remains controversial. Here we used a multidisciplinary biochemical, electrophysiological, and genetic approach to examine the function of the 12-lipoxygenase metabolites of arachidonic acid in long-term synaptic plasticity at CA3-CA1 synapses. We found that the 12-lipoxygenase pathway is required for the induction of metabotropic glutamate receptor-dependent LTD (mGluR-LTD), but is not required for LTP: (1) Hippocampal homogenates were capable of synthesizing the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid (HETE). (2) Stimulation protocols that induce mGluR-LTD lead to a release of 12-(S)-HETE from acute hippocampal slices. (3) A mouse in which the leukocyte-type 12-lipoxygenase (the neuronal isoform) was deleted through homologous recombination was deficient in mGluR-LTD, but showed normal LTP. (4) Pharmacological inhibition of 12-lipoxygenase also blocked induction of mGluR-LTD. (5) Finally, direct application of 12(S)-HPETE, but not 15(S)-HPETE, to hippocampal slices induced a long-term depression of synaptic transmission that mimicked and occluded mGluR-LTD induced by synaptic stimulation. Thus, 12(S)-hydroperoxyeicosa-5Z, 8Z, 10E, 14Z-tetraenoic acid (12(S)-HPETE), a 12-lipoxygenase metabolite of arachidonic acid, satisfies all of the criteria of a messenger molecule that is actively recruited for the induction of mGluR-LTD.

Figure 1.

Activation of the mGluR₅ receptor is required for the induction of mGluR-LTD. a, In a typical experiment, application of MPEP (200 μm) during the LTD induction protocol (first arrow, 5 Hz stimulation of the Schaffer collateral pathway for 3 min) blocked the induction of LTD. In the same slice, the 5 Hz stimulation protocol induced LTD after MPEP was washed out (second arrow). Horizontal bar indicates period of drug application. b, Average responses from all trials with MPEP (200 μm; n = 4) showing blockade of induction of LTD induced by presynaptic stimulation at 5 Hz frequency for 3 min (arrow). Horizontal bar indicates period of drug application. c, Summary of LTD experiments. LTD induced with a 3 min period of 5 Hz stimulation (n = 4) or LTD induced with a 15 min period of 1 Hz stimulation in normal solution (n = 9), or in the presence of MPEP (n = 6; mean ± SEM).

Figure 2.

“Leukocyte-type” 12-LO enzyme activity is present in wild-type murine hippocampus. When hippocampal homogenates from wild-type (solid line) or 12-LO KO mice (broken line) were incubated with [³H]AA (25 μCi; 2.5 μCi/nmol), a single peak of radiolabeled material corresponding to 12-HETE was generated. The bulk of the enzyme activity in the homogenate is attributable to the leukocyte-type enzyme with the remainder arising from contamination of the preparation with platelets which express a unique 12-LO isoform. In this trial, typical of four, 7890 cpm of 12-HETE were generated per milligram of protein. The radioactivity eluting at the solvent front (∼6 min) is a mixture of polar lipids unrelated to LO metabolism.

Figure 3.

12-HETE production was enhanced during induction of mGluR-LTD. a, Normalized values of 12-HETE production are plotted as a function of time in 5 min bins during baseline conditions (1), during 1 Hz stimulation (2), and after LTD induction (3). LTD-inducing stimulation (1 Hz/15 min) significantly enhanced the amount of 12-HETE released from the stimulated slice into the bath solution (p < 0.05 versus baseline; n = 9). b, LTD of fEPSP recorded from the same slices that were used for the biochemical experiments shown in a (n = 9). c, mGluR₅ antagonist MPEP (200 μm) blocked the increase in 12-HETE production during induction of mGluR-LTD (n = 6). d, LTD of fEPSP in the same experiments as in c was blocked in the presence of MPEP (n = 6). Error bars indicate SEM.

Figure 4.

LTD and LTP in wild-type versus 12-LO knock-out mice. a, Examples of EPSCs recorded in slices from wild-type mice (left) and mutant mice (right). b, LTD of EPSCs from whole-cell recordings in CA1 neurons after 5 Hz stimulation for 3 min. LTD was absent in slices from 4- to 10-d-old 12-LO knock-out mice (filled symbols; n = 12 cells, 5 mice). The same stimulation protocol induced robust LTD in slices from wild-type mice of the same age (open symbols; n = 6 cells, 4 mice). c, LTP summary graphs for wild-type (n = 10 slices, 4 mice) and 12-LO knock-out adult mice (n = 13 slices, 4 mice). LTP of field EPSPs was induced by two trains of 100 Hz tetanic stimulation, each lasting 1 sec, spaced 20 sec apart. d, Low-frequency stimulation (1 Hz for 15 min) induced LTD of the field EPSPs in slices from wild-type mice (n = 9 slices, 6 mice), but not in slices from 12-LO knock-out mice (n = 4 slices, 4 mice). e, Low-frequency stimulation (1 Hz for 15 min) induced LTD of the field EPSPs in slices from wild-type mice under control conditions (n = 5 slices, open symbols), but not in the presence of the 12-LO inhibitor CDC (10 μm) (n = 5 slices, filled symbols). Error bars indicate SEM.

Figure 5.

12(S)-HPETE mimicked and occluded LTD. a, Effect of 12(S)-HPETE on EPSC in slices from wild-type mice (n = 6). b, 12(S)-HPETE-induced depression of EPSC in slices from 12-LO knock-out mice (n = 6). c, Application of 15(S)-HPETE did not affect the EPSC amplitude in slices from wild-type mice (n = 6). d, Induction of LTD with 5 Hz stimulation (arrow) occluded the subsequent effect of 12(S)-HPETE (100 nm) on the EPSC amplitude. Representative experiment showing EPSC traces during baseline recording (1), during LTD (2), and during 12(S)-HPETE application (3). e, 12(S)-HPETE (100 nm) application occluded electrically induced LTD (arrow). f, Summary of LTD data and effects of 12(S)-HPETE in wild-type and 12-LO knock-out mice. g, 12(S)-HPETE does not block adenosine-induced synaptic depression. After LTD was induced by application of 12(S)-HPETE (100 nm), subsequent addition of adenosine (50 μm) induced an additional depression (n = 4).

Figure 6.

12(S)-HPETE-induced synaptic depression produces a significant change in coefficient of variation, consistent with a presynaptic effect. a, Superimposed EPSCs were recorded under baseline conditions (left) and after application of 12(S)-HPETE (100 nm) (right). b, Scatterplot showing effects of lipoxygenase metabolite application on values of 1/CV² (I²/σ², where I is mean EPSC and σ² is EPSC variance) versus effects on I. Data shown for 12(S)-HPETE application in slices from wild-type mice (open triangles; n = 6) or 12-LO knock-out mice (filled triangles; n = 6), and 15(S)-HPETE application in slices from wild-type mice (open squares; n = 6). Each symbol is a measurement from a separate slice. All values were normalized by their baseline values before lipid application. There was a significant correlation between changes in I and I²/σ² (solid line; correlation coefficient; r = 0.86), consistent with presynaptic changes. Horizontal dotted line demonstrates the predicted relation between changes in I and I²/σ² if the effect of the metabolite was purely postsynaptic.

Figure 7.

12(S)-HPETE had no effect on the mEPSC amplitude. a, Representative mEPSCs recorded from a CA1 cell at a holding potential of -70 mV under baseline conditions (left) and after 12(S)-HPETE (100 nm) was added (right). b, Cumulative amplitude histograms of mEPSCs recorded from the same cell as in a under baseline conditions (solid line) and after 12(S)-HPETE was applied (dashed line). c, Averaged amplitude histograms of mEPSCs recorded before (open symbols) and after (filled symbols) 12(S)-HPETE application (n = 6 cells). For the cumulative graphs, responses were normalized by the median amplitude of the mEPSCs, collected under baseline conditions, in each individual experiment. d, Summary plot of mEPSC data. Averaged values of mEPSC parameters, mean peak amplitude (left), and CV (right). Error bars indicate SEM.