Activation of the Ghrelin Receptor Mediates NMDA Receptor Phosphorylation at the NR1 Subunit in the Rat Hippocampus

Abstract

Ghrelin is a 28 amino acid peptide hormone. Although the primary role of ghrelin is to initiate feeding behavior, ghrelin has many other functions in the brain. Ghrelin promotes learning and memory in the hippocampus, where the receptor of ghrelin (GHSR1a) is highly localized. A cellular mechanism underlying learning and memory is the strengthening of synapses by the n-methyl-d-aspartate (NMDA) receptor. Since phosphorylation of the NR1 subunit up-regulates the receptor activity, we hypothesized that ghrelin stimulated the phosphorylation of NR1 (pNR1). One to 1000nM ghrelin or non-peptide agonist (L-692,585) were bath-applied to acutely-prepared and cultured hippocampal slices. In some experiments, the GHSR1a antagonist, [D-Lys3]-GHRP-6, or inverse agonist, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P, was co-applied with ghrelin. Immediately after the experiments, slices were processed for pNR1 immunoreativity. pNR1 was visualized as fluorescent signals with the Olympus FV300 confocal system and quantified using the IPlab imaging software by selecting regions of interest. pNR1, detected as discrete puncta, increased in response to 1-1000nM ghrelin in a dose-dependent manner. Similar dose-responses were obtained with L-692,585. With 100nM ghrelin, pNR1 increased by 18.4% in acutely-prepared slices and by 16.3% in cultured slices. With 100nM L-692,585, pNR1 increased by 40.8% in cultured slices. These responses were blocked by the [D-Lys3]-GHRP-6 (10µM) and D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P (10µM). Our results suggest that the magnitude of NR1 phosphorylation is tightly regulated by GHSR1a. This work is supported by APS Undergraduate Summer Research Fellowship (BGM) and NIH2R15DA021683 (MI).