BDNF is a negative modulator of morphine action

Abstract

Brain-derived neurotrophic factor (BDNF) is a key positive regulator of neural plasticity, promoting, for example, the actions of stimulant drugs of abuse such as cocaine. We discovered a surprising opposite role for BDNF in countering responses to chronic morphine exposure. The suppression of BDNF in the ventral tegmental area (VTA) enhanced the ability of morphine to increase dopamine (DA) neuron excitability and promote reward. In contrast, optical stimulation of VTA DA terminals in nucleus accumbens (NAc) completely reversed the suppressive effect of BDNF on morphine reward. Furthermore, we identified numerous genes in the NAc, a major target region of VTA DA neurons, whose regulation by BDNF in the context of chronic morphine exposure mediated this counteractive function. These findings provide insight into the molecular basis of morphine-induced neuroadaptations in the brain's reward circuitry.

Fig. 1. Effects of BDNF-TrkB signaling within the VTA-NAc on morphine reward

Localized knockout (KO) of BDNF (A) or TrkB (B) from VTA neurons enhances morphine conditioned place conditioning (CPP, 15 mg/kg, sc). Student’s t-test, *p < 0.05, n = 8–12. (C) DAT-cre/flTrkB (TrkB^lx/lx;DAT^cre/wt) mice also displayed enhanced morphine CPP (10 mg/kg, ip). One-way ANOVA, Fisher’s PLSD post-hoc test, *p < 0.05 compared to controls; ^#p < 0.05 compared with TrkB^lx/lx;DAT^cre/wt mice, n = 6–11. (D) A single infusion of BDNF into VTA (0.25 μg/side) suppressed morphine CPP (15 mg/kg, sc). t-test, *p < 0.05, n = 7–8. (E) Localized TrkB KO in NAc and (F) intra-NAc BDNF infusion (1.0 μg/side) had no effect on morphine CPP (15 mg/kg, sc), n = 8–9.

Fig. 2. Regulation of VTA DA neuron excitability by morphine and BDNF

(A) Sample traces of in vivo recordings from VTA DA neurons from control (top), morphine-treated (middle), and BDNF+morphine-treated mice (bottom). (B to E) Morphine (25 mg pellet, sc; animals analyzed 48 hr later) increases (B) basal firing rate, (C) burst firing rate, and (D) burst duration in VTA DA neurons, which were normalized by intra-VTA infusion of BDNF (0.25 μg/side). One-way ANOVA, Fisher’s PLSD test, *p < 0.05, ***p < 0.001, compared with control; ^##p < 0.01, ^###p < 0.001, compared with morphine group. n = 4–6. (E) Sample traces of K⁺ conductance recorded from VTA DA neurons in brain slices from control, morphine-treated, and BDNF+morphine-treated mice. (F) Morphine treatment as in (A) significantly decreased both peak and sustained phases of K⁺ currents in VTA DA neurons, an effect that was reversed by BDNF. Two-way ANOVA, Fisher’s PLSD test, *p < 0.05, **p < 0.01, ***p < 0.001, compared with control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with morphine group. n = 5–9. Localized BDNF KO from VTA increases (G) basal firing rate, (H) burst firing rate, and (I) burst duration in VTA DA neurons. t-test, *p < 0.05, **p < 0.01 compared with AAV-GFP controls, n = 7.

Fig. 3. Modulation of morphine reward by optogenetic activation of DA terminals in NAc

(A) Experimental paradigm for optogenetic stimulation during morphine CPP. Mice were conditioned to a morphine/light chamber and a saline/no-light chamber for 30 min. 470 nm phasic light pulses (20 Hz, 5 pulses, 40 ms duration) were delivered during the 30-min conditioning session. (B to J) Immunostaining for ChR2-EYFP (B, E, and H, green), DAT (C, red), GAD67 (F, red), and VGLUT2 (I, red) in NAc. (D, G, and J) Confocal microscopy shows that ChR2-EYFP puncta in NAc co-label for DAT, but not GAD67 or VGLUT2. Scale bar, 10 μm. (K) In vivo optogenetic stimulation of VTA DA nerve terminals in NAc enhances morphine reward (10 mg/kg, ip) and prevents VTA BDNF-induced impairment of morphine reward, (L) while D1 receptor antagonism (SCH 23390, 1 μg) blocks light potentiation, and VTA BDNF-induced impairment, of morphine reward. t-test, **p < 0.01, **p < 0.001, n = 7–11.

Fig. 4. Morphine-regulated NAc gene expression after VTA BDNF deletion: Identification of novel NAc mediators of BDNF-morphine interactions

Microarray analysis was performed on NAc of sham- and morphine-pelleted mice under control or VTA BDNF knockdown conditions. (A) Heatmap of up (red)- or down (green)-regulated NAc genes upon knockdown of VTA BDNF. (B) Heatmap of up- or down-regulated NAc genes by morphine regardless of knockdown of VTA BDNF. (C) Venn diagrams of genes that were uniquely regulated by morphine (red) or by knockdown of VTA BDNF (blue), and of genes that were regulated by morphine and knockdown of VTA BDNF in an interactive manner (green). (D to G) Alterations of sox11 (D and E) and gadd45g (F and G) expression in NAc from heatmap of microarray analysis (D and F) and qRT-PCR validation (E and G). One-way ANOVA for qRT-PCR validation, Fisher’s PLSD test, ^τp < 0.1, *p < 0.05, ***p < 0.001 compared to sham+AAV-GFP controls; ^$p < 0.1, ^#p < 0.05, ^###p < 0.001 compared to sham+AAV-CreGFP mice, n = 9–12. (H) Reduction of sox11 expression using AAV-shRNA-Sox11 increases morphine CPP (10 mg/kg, sc). Fisher’s PLSD test, *p < 0.05 compared with AAV-GFP controls; ^#p < 0.05 compared with AAV-scrambled controls, n = 11–12. (I) Sox11 overexpression in NAc using HSV-Sox11 decreases morphine CPP (15 mg/kg, sc). t-test, *p < 0.05, n = 8. (J) Enhancement of morphine reward (15 mg/kg, sc) induced by knockdown of VTA TrkB is counteracted by sox11 overexpression in NAc. Fisher’s PLSD test, *p < 0.05 compared with HSV-tomato (TMT) (NAc)+AAV-GFP (VTA) controls; ^#p < 0.05 compared with HSV-TMT (NAc)+AAV-CreGFP (VTA) mice. (K) Enhancement of morphine reward (12.5 mg/kg, sc) induced by knockdown of VTA TrkB is further enhanced by gadd45g overexpression in NAc. Fisher’s PLSD test, *p < 0.05 compared to HSV-GFP+AAV-GFP controls; ^#p < 0.05, ^###p < 0.001 compared to HSV-Gadd45g+AAV-CreGFP.