An excised patch membrane sensor for arachidonic acid released in mouse hippocampal slices under stimulation of L-glutamate

An excised patch membrane sensor for arachidonic acid (AA) is described, whose response stems from AA-induced channel-type transport of ions across the excised patch membrane. The patch membrane sensor was prepared in situ by excising mouse hippocampal cell membranes with patch pipets having a tip diameter of <0.5μm. The sensor responds to AA, giving rise to a channel-type current, and its magnitude (apparent conductance) increased with increasing AA concentration in the range from 10 to 30nM. The detection limit was 2.1nM (S/N=3). The induction of channel-type currents was selective to AA over fatty acids such as palmitic acid, stearic acid, oleic acid, γ-linolenic acid, and docosahexaenoic acid and AA metabolites such as 12-HETE, 5-HETE, and prostaglandin D₂. The sensor was applied to quantification of AA released from various neuronal regions (CA1, CA3, and DG) of mouse hippocampus under stimulation of 100μM L-glutamate. The release of AA from each region was observed 1min after the stimulation and the concentration of AA 5min after the stimulation varied among the neuronal sites, i.e., 8±1 nM (n=5) for CA1, 15±3 nM (n=3) for CA3, and 6±2 nM (n=9) for DG. The L-glutamate-evoked release of AA was partly inhibited by ionotropic glutamate receptor antagonists (APV and DNQX) and completely blocked by phospholipase A₂ (PLA₂) inhibitor (MAFP), suggesting that the release of AA occurred by glutamate receptor-mediated activation of PLA₂. The potential use of the present sensor for detecting local concentration of AA at various neuronal sites is discussed.