Morphine-induced synaptic plasticity in the VTA is reversed by HDAC inhibition

Abstract

Dopamine (DA) dysfunction originating from the ventral tegmental area (VTA) occurs as a result of synaptic abnormalities following consumption of drugs of abuse and underlies behavioral plasticity associated with drug abuse. Drugs of abuse can cause changes in gene expression through epigenetic mechanisms in the brain that underlie some of the lasting neuroplasticity and behavior associated with addiction. Here we investigated the function of histone acetylation and histone deacetylase (HDAC)2 in the VTA in recovery of morphine-induced synaptic modifications following a single in vivo exposure to morphine. Using a combination of immunohistochemistry, Western blot, and whole cell patch-clamp recording in rat midbrain slices, we show that morphine increased HDAC2 activity in VTA DA neurons and reduced histone H3 acetylation at lysine 9 (Ac-H3K9) in the VTA 24 h after the injection. Morphine-induced synaptic changes at glutamatergic synapses involved endocannabinoid signaling to reduce GABAergic synaptic strength onto VTA DA neurons. Both plasticities were recovered by in vitro incubation of midbrain slices with a class I-specific HDAC inhibitor (HDACi), CI-994, through an increase in acetylation of histone H3K9. Interestingly, HDACi incubation also increased levels of Ac-H3K9 and triggered GABAergic and glutamatergic plasticities in DA neurons of saline-treated rats. Our results suggest that acute morphine-induced changes in VTA DA activity and synaptic transmission engage HDAC2 activity locally in the VTA to maintain synaptic modifications through histone hypoacetylation.

Keywords: electrophysiology; histone deacetylase; synaptic plasticity; synaptic transmission; ventral tegmental area.

Fig. 1.

Morphine increases HDAC2 expression in VTA DA neurons. A: examples of brain sections stained with antibodies to TH (green), HDAC2 (red), and DAPI (blue) with merged panels, which show the expression of HDAC2 in TH neurons in the VTA of saline (top)- and morphine (bottom)-treated rats. Scale bar, 20 μm. B: averaged levels of nuclear and somatic HDAC2 expression at 3 AP levels from saline- and morphine-treated rats. In this and all subsequent figures, numbers in parentheses indicate the number of animals examined. *Statistical significance.

Fig. 2.

Histone acetylation at H3K9 is decreased after a single exposure to morphine and is recovered by in vitro HDAC inhibition with CI-994. A: representative Western blots (top) and quantitative data (bottom) of Ac-H3K9 and β-actin (control) in VTA homogenates from saline- and morphine-treated rats. B and C: representative Western blots and quantitative data of Ac-H3K9 and β-actin in VTA homogenates from saline (B)- and morphine (C)-treated rats incubated either in DMSO or CI-994. *Statistical significance.

Fig. 3.

Morphine-induced glutamatergic plasticity is reversed with HDAC inhibition. A: sample AMPAR-mediated mEPSC traces and average bar graphs of mEPSC amplitude and frequency from saline-treated and morphine-treated rats. Calibration bars, 10 pA, 5 s. B: cumulative probability plots of amplitude and frequency (interevent interval) for all mEPSCs in saline-treated and morphine-treated rats. Morphine induced an increase in amplitude and frequency. C: baseline PPRs from paired evoked EPSCs in saline- and morphine-treated rats. There was no significant difference in PPRs between the 2 groups. D: sample traces of AMPA and NMDA EPSCs recorded at +40 mV from VTA DA neurons of saline-treated and morphine-treated rats. Calibration bars, 50 pA, 25 ms. E: summary of AMPAR-to-NMDAR ratios obtained from saline-treated and morphine-treated rats with DMSO (vehicle) or CI-994 (20 μM). The morphine-induced increase in AMPAR-to-NMDAR ratio was reversed by incubation with CI-994. F: summary of AMPAR EPSC rectification indexes obtained from saline-treated and morphine-treated rats with DMSO (vehicle) and CI-994. CI-994 did not reverse the morphine-induced increase in rectification index but increased this index in slices from saline-treated rats. All recordings (D–F) were performed with intrapipette inclusion of spermine. *Statistical significance.

Fig. 4.

Morphine-induced changes of GABAergic synaptic transmission onto VTA DA neurons are reversed by HDAC inhibition. A: sample GABA_AR-mediated mIPSC traces from saline-treated and morphine-treated rats. Calibration bars, 30 pA, 5 s. B: average mIPSC amplitude and frequency from saline-treated and morphine-treated rats with DMSO and CI-994. C, insets: representative traces from saline with bath application of WIN 55212-2 (top) or DHPG (bottom): averaged IPSCs before or the peak response 35 min after WIN 55212-2 or DHPG. Calibration bars, 100 pA, 25 ms. Averaged experiments with bath-applied WIN 55212-2 (2 μM) or DHPG (50 μM) in saline or morphine. WIN 55212-2 induced a significant rundown of IPSCs in saline- but not morphine-treated rats. DHPG induced a significant LTD in both saline- and morphine-treated rats. D–F: cumulative probability plots of amplitude and frequency (interevent interval) for all mIPSCs in saline-treated and morphine-treated rats. CI-994 (20 μM) reversed morphine-induced pre- and postsynaptic LTD. *Statistical significance.