Orexin Signaling in the VTA Gates Morphine-Induced Synaptic Plasticity

Abstract

Dopamine neurons in the ventral tegmental area (VTA) are a key target of addictive drugs, and neuroplasticity in this region may underlie some of the core features of addiction. From the very first exposure, all drugs of abuse induce synaptic plasticity in the VTA. However, it is not well understood how this diverse group of drugs brings about common synaptic change. Orexin (also known as hypocretin) is a lateral hypothalamic neuropeptide released into the VTA that promotes drug-seeking behaviors and potentiates excitatory synaptic transmission onto VTA dopamine neurons. Here we show that signaling at orexin receptor type 1 (OxR1) in the VTA is required for morphine-induced plasticity of dopamine neurons. Systemic or intra-VTA administration of the OxR1 antagonist SB 334867 in rats blocked a morphine-induced increase in the AMPAR/NMDAR ratio, an increase in presynaptic glutamate release, and a postsynaptic change in AMPAR number or function, including a switch in subunit composition. Furthermore, SB 334867 blocked a morphine-induced decrease in presynaptic GABA release, and a morphine-induced shift in the balance of excitatory and inhibitory synaptic inputs to dopamine neurons. These findings identify a novel role for orexin in morphine-induced plasticity in the VTA and provide a mechanism by which orexin can gate the output of dopamine neurons.

Keywords: AMPA; NMDA; dopamine; morphine; orexin; ventral tegmental area.

Figure 1.

Systemic administration of the OxR1 antagonist SB 334867 blocks morphine-induced potentiation of excitatory transmission onto VTA dopamine neurons. A, Example recordings of evoked NMDAR (light) and AMPAR (dark) EPSCs at +40 mV, from VTA dopamine neurons of rats 24 h after exposure to morphine or saline in naive (top), vehicle-treated (middle), and SB 334867-treated (10 mg/kg; bottom) rats. Calibration: 50 pA, 20 ms. B, Treatment with morphine (10 mg/kg, filled bars), but not saline (open bars), increased the AMPAR/NMDAR ratio in naive and vehicle-treated, but not in SB 334867-treated rats (p < 0.05, two-way ANOVA). C, Example traces of AMPAR mEPSCs recorded at −70 mV 24 h after morphine or saline treatment in naive (left), vehicle-treated (center), and SB 334867-treated (right) rats. Calibration: 50 pA, 100 ms. D, Left, AMPAR mEPSC frequency was increased in morphine-treated rats compared with saline-treated rats in naive and vehicle-treated, but not in SB 334867-treated rats (p < 0.05, two-way ANOVA). Right, Cumulative probability plots comparing morphine or saline exposure on mEPSCs for naive, vehicle-treated, and SB 334867-treated animals. E, Left, Morphine increased AMPAR mEPSC amplitude compared with saline in naive and vehicle-treated, but not SB 334867-treated rats (p < 0.05, two-way ANOVA). Right, Cumulative probability plots comparing morphine or saline exposure on mEPSC amplitude for naive, vehicle-treated, and SB 334867-treated rats. F, Morphine (filled bars) induced a paired-pulse depression of evoked AMPAR EPSCs in naive and vehicle-treated rats, but not in SB 334867-treated rats (p < 0.05, two-way ANOVA). Inset, Sample traces of evoked AMPAR EPSC paired pulses recorded at −70 mV. Calibration: 50 pA, 20 ms. G, Pretreatment with SB 334867 blocked a morphine-induced increase in the rectification index (p < 0.05, two-way ANOVA). Inset, Sample traces of AMPAR EPSCs recorded at −70, 0, and +40 mV with spermine in the pipette solution. Current–voltage relationship of AMPAR EPSCs for morphine and saline in vehicle-treated and SB 334867-treated rats. Calibration: 50 pA, 10 ms. n/N = cells/rats. Bars represent the mean ± SEM. *p < 0.05, **p < 0.01.

Figure 2.

OxR1 signaling in the VTA is required for morphine-induced plasticity at glutamatergic synapses. A, Sample traces of evoked AMPAR (dark) and NMDAR (light) EPSCs recorded at +40 mV, 24 h after exposure to morphine or saline in rats microinfused with intra-VTA vehicle or SB 334867 rats. Calibration: 50 pA, 20 ms. B, Morphine (filled bars) potentiated the AMPAR/NMDAR ratio compared with saline (open bars) in intra-VTA vehicle-treated animals, but not intra-VTA SB 334867-treated animals (p < 0.05, two-way ANOVA). SB 334867 was ineffective when infused outside the VTA or when the dose was lowered to 0.03 nmol/0.3 μl. C, Sample traces of AMPAR mEPSCs recorded at −70 mV, 24 h after morphine or saline exposure in intra-VTA vehicle-treated and intra-VTA SB 334867-treated rats. Calibration: 50 pA, 100 ms. D, Left, Morphine increased the frequency of AMPAR mEPSC relative to saline in intra-VTA vehicle-treated rats, but not intra-VTA SB-334867-treated rats (p < 0.05, two-way ANOVA). Right, Cumulative probability plot comparing morphine and saline exposure on mEPSCs for intra-VTA vehicle- or SB 334867-treated rats. E, Left, Intra-VTA SB 334867 inhibited a morphine-induced increase in the amplitude of AMPAR mEPSCs in VTA dopamine neurons (p < 0.05, two-way ANOVA). Right, Cumulative probability plot comparing morphine and saline exposure on mEPSC amplitude for intra-VTA vehicle- or SB 334867-treated rats. n/N = cells/rats. Bars represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Morphine decreases the probability of presynaptic GABA release in an OxR1-dependent manner. A, Example recordings of GABA_A mIPSCs recorded at −70 mV, 24 h after exposure to morphine or saline in naive, vehicle-treated, or SB 334867-treated rats. Calibration: 50 pA, 200 ms. B, Left, Morphine (filled bars) decreased the frequency of GABA_A mIPSCs relative to saline (open bars) in naive and vehicle-treated rats, but not in SB 334867-treated rats (p < 0.05, two-way ANOVA). Right, Cumulative probability plots comparing morphine or saline exposure on mIPSC for naive, vehicle-treated, and SB 334867-treated animals. C, Left, Morphine exposure did not alter GABA_A mIPSC amplitude compared with saline exposure in naive, vehicle-treated, or SB 334867-treated animals (p > 0.05, two-way ANOVA). Right, Cumulative probability plots comparing morphine or saline exposure on mIPSC amplitude for naive, vehicle-treated, and SB 334867-treated animals. n/N = cells/rats. Bars represent mean ± SEM. *p < 0.05.

Figure 4.

OxR1 signaling in the VTA is necessary for a morphine-induced suppression of presynaptic GABA release. A, Sample recordings of GABA_A mIPSCs at −70 mV, from rats that were exposed to morphine or saline after pretreatment with intra-VTA vehicle or intra-VTA SB 334867. Calibration: 50 pA, 200 ms. B, Left, Morphine (filled bars) decreased GABA_A mIPSC frequency relative to saline (open bars) in intra-VTA vehicle-treated rats, but not intra-VTA SB 334867-treated rats (p < 0.05, two-way ANOVA). Right, Cumulative probability plot comparing morphine and saline exposure on mIPSC frequency for intra-VTA vehicle-treated rats or SB 334867-treated rats. C, Left, There was no effect of morphine on the amplitude of GABA_A mIPSCs (p > 0.05, two-way ANOVA). Right, Cumulative probability plots comparing morphine and saline exposure on mIPSC amplitude for intra-VTA vehicle-treated rats or SB 334867-treated rats. n/N = cells/rats. Bars represent the mean ± SEM. **p < 0.01.

Figure 5.

OxR1 signaling is required for a morphine-induced shift in the balance of excitatory and inhibitory synaptic transmission onto dopamine neurons. A, Example recordings of EPSCs (dark) at −67 mV, and IPSCs (light) at +8 mV from VTA dopamine neurons in naive, vehicle-treated, and SB 334867-treated animals. Calibration: 200 pA, 20 ms. B, Morphine (filled), compared with saline (open), induced a switch in the ratio of excitatory and inhibitory conductances onto dopamine neurons from naive or vehicle-treated animals. This shift was inhibited by systemic SB 334867 (p < 0.05, two-way ANOVA). C, SB 334867 inhibited the morphine-induced increased G_e/G_i ratio in seven of nine identified tyrosine hydroxylase (TH)-positive neurons. TH was labeled with anti-TH antibodies and FITC. Biocytin was labeled with streptavidin-conjugated Texas Red. n/N = cells/rats. Bars represent the mean ± SEM. *p < 0.05, ***p < 0.001.