Cocaine enhances NMDA receptor-mediated currents in ventral tegmental area cells via dopamine D5 receptor-dependent redistribution of NMDA receptors - PubMed (original) (raw)

Cocaine enhances NMDA receptor-mediated currents in ventral tegmental area cells via dopamine D5 receptor-dependent redistribution of NMDA receptors

Björn Schilström et al. J Neurosci. 2006.

Erratum in

J Neurosci. 2006 Sep 13;26(37):9604

Abstract

Cocaine-induced plasticity of glutamatergic synaptic transmission in the ventral tegmental area (VTA) plays an important role in brain adaptations that promote addictive behaviors. However, the mechanisms responsible for triggering these synaptic changes are unknown. Here, we examined the effects of acute cocaine application on glutamatergic synaptic transmission in rat midbrain slices. Cocaine caused a delayed increase in NMDA receptor (NMDAR)-mediated synaptic currents in putative VTA dopamine (DA) cells. This effect was mimicked by a specific DA reuptake inhibitor and by a DA D1/D5 receptor agonist. The effect of cocaine was blocked by a DA D1/D5 receptor antagonist as well as by inhibitors of the cAMP/cAMP-dependent protein kinase A (PKA) pathway. Furthermore, biochemical analysis showed an increase in the immunoreactivity of the NMDAR subunits NR1 and NR2B and their redistribution to the synaptic membranes in VTA neurons. Accordingly, NMDAR-mediated EPSC decay time kinetics were significantly slower after cocaine, suggesting an increased number of NR2B-containing NMDARs. Finally, pharmacological analysis indicates that NR2B subunits might be incorporated in triheteromeric NR1/NR2A/NR2B complexes rather than in "pure" NR1/NR2B NMDA receptors. Together, our data suggest that acute cocaine increases NMDAR function in the VTA via activation of the cAMP/PKA pathway mediated by a DA D5-like receptor, leading to the insertion of NR2B-containing NMDARs in the membrane. These results provide a potential mechanism by which acute cocaine promotes synaptic plasticity of VTA neurons, which could ultimately lead to the development of addictive behaviors.

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Figures

Figure 1.

Cocaine causes a delayed enhancement of NMDAR EPSCs. A, Example traces (top) and time-course graph (bottom; n = 6) showing the effects of cocaine superfusion (1–3 μ

; 10 min) on mean NMDAR EPSCs in cells voltage clamped at +40 mV. B, Example traces (top) and time-course graph (bottom; n = 6) showing the effects of cocaine on AMPAR-mediated EPSCs (AMPAR EPSCs) recorded in cells voltage clamped at −70 mV. Averaged sample EPSCs were taken 5 min before (1) and 25 min after (2) cocaine application. Horizontal bars represent the time that cocaine was included in the superfusion medium. Error bars represent SEM.

Figure 2.

Cocaine enhances NMDAR EPSCs via activation of DA D1/D5 receptors. A, Time course of mean NMDAR EPSCs shows that application of the specific DA reuptake inhibitor GBR12909 (10 n

; 10 min; n = 6) mimics the effect of cocaine on NMDAR EPSCs. B, The D1/D5 agonist SKF81297 (10 μ

; 10 min; n = 8) increases NMDAR EPSCs without the delay seen with cocaine and GBR12909. C, The DA D1/D5 receptor antagonist SCH23390 (10 μ

; n = 4) inhibited cocaine enhancement of NMDAR EPSCs. D, The DA D2-like receptor antagonist eticlopride (3 μ

; n = 6) accelerates the effect of cocaine on NMDAR EPSCs but does not affect the magnitude. E, No enhancement of NMDAR EPSCs develops during continuous application of cocaine (1–3 μ

; 50 min; n = 4). Horizontal bars represent the time that drugs were included in the superfusion medium. Error bars represent SEM.

Figure 3.

DA D1/D5 receptor activation transiently decreases mEPSC frequency. A, SKF81297 superfusion (10 μ

; 10 min) had no significant effect on paired-pulse ratio (right; n = 6). Sample traces show EPSCs evoked by paired pulses delivered 50 ms apart in cells voltage clamped at −70 mV, before (top left) and during (bottom left) drug superfusion. B, SKF81297 superfusion (10 μ

; 10 min) had no effect on mEPSC amplitude but transiently decreased mEPSC frequency. Example mEPSC traces recorded in lidocaine before (top left) and during (bottom left) drug superfusion. The bar histograms represent mean mEPSC frequency (top right) and amplitude (bottom right) ± SEM. The asterisk indicates significant differences between treatment and control samples (*p < 0.05; Student’s t test; n = 5).

Figure 4.

The enhancement of NMDAR EPSCs by cocaine involves the cAMP/PKA signaling pathway. A, The effect of cocaine on NMDAR EPSCs was inhibited when the PKA inhibitor Rp-cAMPs (100 μ

; n = 4) was included in the recording pipette. B, The effect of cocaine was also inhibited when the membrane-impermeable PKA inhibitor PKI(6-22)-amide (20 μ

; n = 4) was included in the recording pipette. Horizontal bars represent the time that cocaine was included in the superfusion medium. Error bars represent SEM.

Figure 5.

Acute exposure to cocaine induces an increase in NR1 and NR2B subunit immunoreactivity in the VTA. A, VTA and hippocampal slices (Hipp.) were incubated without [saline (S)] or with 1 μ

cocaine (C) for 10 min. After treatment, slices were homogenized, and samples (25 μg) were resolved by SDS-PAGE. The levels of NR2B (top), NR1 (middle), and NR2A and NR2C (bottom) were detected using specific antibodies and normalized to actin. The bar histograms represent normalized levels of NMDAR subunits plotted as percentage of control ± SD of six (NR2B), four (NR1), and three (NR2A and NR2C) independent experiments. Asterisks indicate significant differences between treatment and control samples (*p < 0.05; Student’s t test). B, VTA slices were preincubated for 60 min with the protein synthesis inhibitor anisomycin (20 μ

) followed by 10 min of 1 μ

cocaine (C+A) or with anisomycin alone (A). Slices were also incubated without or with 1 μ

cocaine for 10 min. After treatment, slices were homogenized, and samples (25 μg) were resolved by SDS-PAGE. The levels of NR1 and NR2B were detected using specific antibodies and normalized to actin. The bar histograms represent normalized levels of NR2B plotted as percentage of control ± SD of three independent experiments. Asterisks indicate significant differences between cocaine-treated slices and control slices or cocaine and anisomycin plus cocaine slices (*p < 0.05; Student’s t test). C, VTA slices were treated without or with 1 μ

cocaine for 10 min or with 1 μ

cocaine for 10 min followed by 30 min washout of cocaine (C+W). After treatments, the level of membranal NR2B in the samples (50 μg) was determined as in A. The bar histogram of normalized NR2B levels is plotted as percentage of control ± SD of three independent experiments (*p < 0.05; Student’s t test). D, VTA slices were treated as in A. Samples (25 μg) were resolved by SDS-PAGE, and the level of NR2B phosphorylation was detected using site-specific phospho-NR2B antibodies (P-1472) normalized to total NR2B. The bar histogram of normalized P-1472 NR2B levels is plotted as percentage of control ± SD of six independent experiments.

Figure 6.

Systemic administration of cocaine induces a redistribution of NR1 and NR2B subunits in the VTA in vivo. A, Levels of NMDAR subunits NR1, NR2B, NR2A, and NR2C in the VTA in the crude synaptosomal (P2), light membrane (P3), synaptosomal membrane (LP1), and synaptic vesicle-enriched (LP2) fractions from saline (S) or 30 min after intraperitoneal injection of cocaine (C; 15 mg/kg), resolved by SDS-PAGE (50 μg per lane). The histograms depict the relative levels of NR1, NR2B, and NR2A proteins, given as percentage of the control samples. Data are means ± SEM of three independent experiments. Asterisks indicate significant differences between treatment and control samples (*p < 0.05; ANOVA). B, Animals were treated as in A, and levels of GluR1, PSD-95, and synaptophysin (Syn.) in the different subcellular membranal fractions (50 μg per lane) were detected using specific antibodies as indicated. No change in protein levels was found after normalization to anti-actin antibodies (data not shown).

Figure 7.

Acute cocaine increases expression of NR2B subunits likely in triheteromeric NMDAR complexes. A, NMDAR EPSC enhancement by cocaine requires activation of NR2B receptors. Time course of mean NMDAR EPSCs shows that in the continuous presence of 3 μ

ifenprodil, cocaine did not cause an enhancement of NMDAR EPSCs (n = 4). The horizontal bar represents the time that cocaine was included in the superfusion medium. B, NR2B-specific antagonists ifenprodil (0.003–10 μ

; left) and Ro25-6981 (0.005–5 μ

; right) inhibit NMDAR EPSCs to the same extent in control slices (white bars; ifenprodil, n = 4; Ro25-6981, n = 5) and in slices pretreated with cocaine (black bars; ifenprodil, n = 11; Ro25-6981, n = 7) (p > 0.05; two-way ANOVA). C, Analysis of NMDAR EPSC decay time suggests a cocaine-induced switch toward a higher synaptic NR2B/NR2A ratio. Left, Sample traces of NMDAR EPSCs before and after cocaine recorded at +40 mV and normalized to 100 pA for comparison. Each trace is the average of 15 consecutive sweeps. Middle, Summary scatter plots of τw (weighted decay time constant) of NMDAR EPSCs from individual VTA cells before (open circle) and 25 min after (filled circle) cocaine superfusion. Right, Mean decay time constant value of NMDAR EPSCs increases significantly after cocaine application (*p < 0.005; Student’s t test; n = 22). Error bars represent SEM.

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Cocaine enhances NMDA receptor-mediated currents in ventral tegmental area cells via dopamine D5 receptor-dependent redistribution of NMDA receptors - PubMed (original) (raw)