The group I metabotropic glutamate receptor agonist (RS)-3, 5-dihydroxyphenylglycine (DHPG) (100 µM, 10 min) has been shown to induce a novel form of long-term depression (LTD) of synaptic transmission in the CA1 region of the rat hippocampus in vitro (Palmer et al. 1997). We have shown that neither activation of PKC nor release of Ca2+ from intracellular stores is required for DHPG-induced LTD (Schnabel et al. 1999). We have now investigated the role of tyrosine phosphorylation in the induction of DHPG-induced LTD.
Adult, female Wistar rats (160-190 g) were killed by cervical dislocation in accordance with Home Office regulations and transverse hippocampal slices obtained. Extracellular field EPSPs were evoked by stimulation of the Schaffer collateral pathway and recorded using a grease-gap technique (Blake et al. 1988). Results were analysed with Student’s unpaired t tests. n signifies the number of times a result was obtained, which is the same as the number of slices tested and all results are quoted as a percentage of baseline EPSP slope ± standard error of mean.
Initial experiments were performed in Mg2+-free medium as we have previously shown this to facilitate the magnitude of LTD (Palmer et al. 1997). DHPG (100 µM, 10 min) induced LTD, measured 30 min following washout of DHPG, of 65 ± 5 % (n = 5). Neither a tyrosine kinase inhibitor, lavendustin A (10 µM), nor a tyrosine phosphatase inhibitor, orthovanadate (1 mM), affected basal synaptic transmission. Lavendustin A also had no significant effect on DHPG-induced LTD (55 ± 11 %, n = 5). In contrast, orthovanadate significantly reduced the level of DHPG-induced LTD (109 ± 14 %, n = 5; P ▓le│ 0.05).
To exclude effects mediated via NMDA receptors, these experiments were repeated in 1 mM Mg2+-containing medium in the presence of the glycine site, NMDA receptor antagonist, L-689, 560 (5 µM), and in the presence of the GABAA receptor antagonist picrotoxin (50 µM). Under these conditions DHPG (100 µM, 10 min) induced LTD of 71 ± 5 % (n = 5). Neither lavendustin A (10 µM) nor orthovanadate (1 mM) affected basal synaptic transmission. Lavendustin A, again, had no significant effect on DHPG-induced LTD (65 ± 6 %, n = 6), whilst orthovanadate significantly reduced the level of DHPG-induced LTD (88 ± 3 %, n = 5; P ▓le│ 0.05).
These results suggest a role for tyrosine dephosphorylation in the induction of DHPG-induced LTD.This work was supported by the MRC.
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Palmer, M.J., Irving, A.J., Seabrook, G.R., Jane, D.E. & Collingridge, G.L. (1997). Neuropharmacology 36, 1517-1532.
Schnabel, R., Kilpatrick, I.C. & Collingridge, G.L. (1999). Neuropharmacology 38, 1585-1596.