Soluble epoxide hydrolase inhibitor enhances synaptic neurotransmission and plasticity in mouse prefrontal cortex

Abstract

Background: The soluble epoxide hydrolase (sEH) is an important enzyme chiefly involved in the metabolism of fatty acid signaling molecules termed epoxyeicosatrienoic acids (EETs). sEH inhibition (sEHI) has proven to be protective against experimental cerebral ischemia, and it is emerging as a therapeutic target for prevention and treatment of ischemic stroke. However, the role of sEH on synaptic function in the central nervous system is still largely unknown. This study aimed to test whether sEH C-terminal epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) affects basal synaptic transmission and synaptic plasticity in the prefrontal cortex area (PFC). Whole cell and extracellular recording examined the miniature excitatory postsynaptic currents (mEPSCs) and field excitatory postsynaptic potentials (fEPSPs); Western Blotting determined the protein levels of glutamate receptors and ERK phosphorylation in acute medial PFC slices.

Results: Application of the sEH C-terminal epoxide hydrolase inhibitor, AUDA significantly increased the amplitude of mEPSCs and fEPSPs in prefrontal cortex neurons, while additionally enhancing long term potentiation (LTP). Western Blotting demonstrated that AUDA treatment increased the expression of the N-methyl-D-aspartate receptor (NMDA) subunits NR1, NR2A, NR2B; the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR1, GluR2, and ERK phosphorylation.

Conclusions: Inhibition of sEH induced an enhancement of PFC neuronal synaptic neurotransmission. This enhancement of synaptic neurotransmission is associated with an enhanced postsynaptic glutamatergic receptor and postsynaptic glutamatergic receptor mediated synaptic LTP. LTP is enhanced via ERK phosphorylation resulting from the delivery of glutamate receptors into the PFC by post-synapse by treatment with AUDA. These findings provide a possible link between synaptic function and memory processes.

Fig. 1

The sEH inhibitor, AUDA, alters synaptic response in PFC pyramidal neurons. a Schematic illustration of prefrontal cortex slices and placement of electrodes. The stimulating electrode was placed on layer II and the recording electrode was placed on layer V. Evoked EPSC was recorded of AMPA EPSC which was isolated in the presence of NMDA antagonist (APV 50 μM) and GABA_A receptor (bicuculline 10 μM) antagonists. (scale 40 ms, 50 pA). Time course showed that treatment of 10 μM AUDA increased the EPSC amplitude. b Traces showed that the number of spikes evoked by a current pulse during control/ACSF and perfusion with 10 μM AUDA (scale 100 ms ,20 pA). c The percentage of fEPSP response with 1 μM, 5 μM or 10 μM AUDA treatment. ***p < 0.001 vs. vehicle control

Fig. 2

Enhancement of synaptic response by EETs at prefrontal cortex synapse. a PFC slice was perfused with 14,15-EET (10 μM, n = 5), and the percentage of fEPSPs response was measured. b The percentage of fEPSPs response was measured in the presence of vehicle combination with AUDA or 14,15-EEZE in combination with AUDA. *p < 0.05 vs. control, **p < 0.01 vs. vehicle/AUDA group

Fig. 3

The sEH inhibitor, AUDA, increases postsynaptic efficiency of I-O glutamatergic transmission but not presynaptic glutamate release in PFC slice. a The I-O relationship was measured by the stimulation intensity and amplitude of fEPSP in the different concentration of 1 μM, 5 μM or 10 μM AUDA. b Mean value of I-O curves of glutamatergic transmission in PFC slices. c Sample traces were an average of 5-10 successive responses. The paired-pulse fEPSPs were evoked with intervals of 30 ms and 90 ms. Calibration; 50 pA, 30 ms. d Paired-pulse ratio was evoked at 30 ms, 60 ms, 90 ms, 120 ms, 150 ms intervals in the different concentration of AUDA. **p < 0.01, ***p < 0.001 vs. vehicle control

Fig. 4

Enhancement of miniature excitatory postsynaptic current (mEPSC) amplitude in the PFC neurons by AUDA treatment. a Sample traces of mEPSCs were taken from brainslices of vehicle control, AUDA treatment in PFC neurons. mEPSCs were recorded in the PFC neurons at a holding potential of -70 mV in the presence of TTX (1 μM). Calibration: 50 pA, 100 ms. (b and c) The summary frequency and amplitude were measured for vehicle control and AUDA groups. (d) The AUDA-induced changes were measured in percentage of frequency-/amplitude- of mEPSCs response at 10 μM of AUDA *p < 0.05 vs. vehicle control

Fig. 5

sEH inhibitor, AUDA, facilitates LTP in PFC slice. a The graph represents the mean ± SEM slope of fEPSPs plotted against time. Applied with 3× high-frequency stimulation of 100 Hz for 1 s induced LTP. b In the presence of 1 μM AUDA, the enhancement of LTP was observed. c Comparison of fEPSPs slope potentiation 10 min after tetanus in the absence or presence AUDA is shown. d Comparison of fEPSPs slope potentiation 60 min after tetanus in the absence or presence AUDA is shown. **p < 0.01 vs. vehicle control

Fig. 6

Synaptic receptors expression in PFC slices could be increased by the sEH inhibitor, AUDA. a The PFC slices were incubated with AUDA (10 μM) for 10 min and then washed to remove AUDA. One hour later, homogenate from the PFC was prepared and blotted with antibodies AMPA receptors subunit GluR1, GluR2, NMDA receptor subunits NR1, NR2A, NR2B and dopamine receptor D2 in the PFC slices. b The bar graph showed the normalized band intensity of synaptic receptors in PFC slice. *p < 0.05, **p < 0.01 vs. vehicle control

Fig. 7

The sEH inhibitor AUDA does not affect the total amount of NR1, NR2A, NR2B, GluR1, GluR2 mRNA levels. a, b Total RNA was isolated from the PFC slices by treating with AUDA and mRNA expression for NR1, NR2A, NR2B, GluR1, GluR2 were analyzed with RT-PCR and (c) Real-Time qPCR. The mRNA expression for β-actin was used as an internal control

Fig. 8

The sEH inhibitor AUDA increases the phosphorylation of protein levels by AUDA. a The tissue lysate of PFC area was prepared and blotted with antibodies against the phosphorylated  Ser1303 on NR2B, phosphorylated  Ser831 on GluR1 and phosphorylated  Tyr876 on GluR2. b The bar graph showed the normalized band intensity of phosphorylated synaptic receptors in PFC slice. *p <0.05, **p < 0.01 vs. vehicle control

Fig. 9

The sEH inhibitor AUDA induced the phosphorylation of ERK in PFC slices. a The PFC slices were incubated with AUDA (10 μM) for 10 min and then washed to remove AUDA. One hour later, homogenate from the PFC was prepared and blotted with antibodies directly against the active form of ERKs. AUDA-induced increase of ERKs phosphorylation in the PFC slices is shown. AUDA treatment increased the phosphorylated levels of ERK42 and ERK44 relative to the vehicle controls (ACSF). No change was observed when the cellular extract was blotted with an antibody that recognizes total ERKs, suggesting that the observed pERKs increments were not due to an increase in the total amount of ERKs. b Here shows the phosphorylated levels of ERK42 and ERK44 among the vehicle control, vehicle/HFS and AUDA/HFS groups. c The homogenate from the PFC following AUDA treatment was prepared and blotted with antibodies directly against the COX-2. GAPDH was the internal control *p < 0.05, **p < 0.01 vs. vehicle control; #p <0.05 vs. vehicle HFS group