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. Author manuscript; available in PMC: 2011 Apr 6.
Published in final edited form as: J Neurosci. 2010 Oct 6;30(40):13419–13430. doi: 10.1523/JNEUROSCI.2966-10.2010

Figure 5. Morphine treatment increases 5-HT induced GIRK currents and Ba2+-sensitive basal but reduces GABAB receptor-activated GIRK currents.

Figure 5

Immunostaining of GIRK2 and PSD95 in untreated hippocampal neurons (10–14 DIV) or after ~20 h morphine treatment (100 µm). A–C) Colocalization between GIRK2 and PSD95 increased with morphine, demonstrating a response in 11–14 DIV that was similar to 21 DIV neurons. C) Zoom of dendrites from control and morphine-treated neurons. Scale bars: 10 and 2 µm. D–F) Whole-cell patch-clamp recordings show changes in GIRK currents with morphine treatment. The 5-HT induced GIRK currents and the Ba2+ -sensitive basal currents increased following 20 h morphine treatment. The GABAB-activated GIRK current decreased in morphine-treated neurons. Representative current-voltage plots show the macroscopic currents recorded in 20K, 20K + 1 mM Ba2+, 20K + 1 µm 5-HT, or 20K + 100 µm baclofen for control (D) and morphine (E) treated neurons. Arrow marks the equilibrium potential for potassium (EK). F) Bar graphs show mean current density (± SEM) for baclofen, 5-HT and Ba2+sensitive basal K+ currents. *Student’s t-test for significance (P < 0.05). N represents the number of recordings. G) Western staining shows increase in total protein for GIRK2 and decrease in GABAB2 from hippocampal neurons exposed to morphine for ~20 h. Representative immunoblots for GIRK2, GABAB2 and GAPDH (loading control) are shown. Bar graph shows quantification of optical density for indicated protein (see Methods). H) Surface biotinylation experiment shows increase in surface expression of GIRK2 in neurons exposed to morphine. Representative immunoblots for NMDA receptor, GABAB2, GIRK2, and GAPDH (lack of GAPDH confirms isolation of plasma membrane fraction). Bar graph shows quantification of surface expression and increase in GIRK2 (n=3 separate experiments). *Student’s t-test for significance (P < 0.05). I) Systemic morphine increases GIRK2 protein levels in vivo. Western blot shows increase in total protein for GIRK2 in rat hippocampi isolated 5 hr following a single morphine injection (10 mg/kg). Bar graph shows relative change in GIRK2 (mean optical density normalized to GAPDH) for three different morphine concentrations (n = 3) vs. saline injection (n = 4). **One-way ANOVA followed by Bonferroni's multiple comparison test for significance (P < 0.05).

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