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Morphine disinhibits glutamatergic input to VTA dopamine neurons and promotes dopamine neuron excitation.

Chen M, Zhao Y, Yang H, Luan W, Song J, Cui D, Dong Y, Lai B, Ma L, Zheng P - Elife (2015)

Bottom Line: However, it is not known whether morphine has an additional strengthening effect on excitatory input.We also studied the contribution of the morphine-induced disinhibitory effect on the presynaptic glutamate release to the overall excitatory effect of morphine on VTA-DA neurons and related behavior.Our results suggest that the disinhibitory action of morphine on presynaptic glutamate release might be the main mechanism for morphine-induced increase in VTA-DA neuron firing and related behaviors.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, School of Basic Medical Sciences and Institutes of Brain Science, Fudan Univeristy, Shanghai, China.

ABSTRACT
One reported mechanism for morphine activation of dopamine (DA) neurons of the ventral tegmental area (VTA) is the disinhibition model of VTA-DA neurons. Morphine inhibits GABA inhibitory neurons, which shifts the balance between inhibitory and excitatory input to VTA-DA neurons in favor of excitation and then leads to VTA-DA neuron excitation. However, it is not known whether morphine has an additional strengthening effect on excitatory input. Our results suggest that glutamatergic input to VTA-DA neurons is inhibited by GABAergic interneurons via GABAB receptors and that morphine promotes presynaptic glutamate release by removing this inhibition. We also studied the contribution of the morphine-induced disinhibitory effect on the presynaptic glutamate release to the overall excitatory effect of morphine on VTA-DA neurons and related behavior. Our results suggest that the disinhibitory action of morphine on presynaptic glutamate release might be the main mechanism for morphine-induced increase in VTA-DA neuron firing and related behaviors.

No MeSH data available.


Related in: MedlinePlus

Effect of exogenous application of GABA and 470 nm light stimulation on the frequency of sEPSCs in the presence of intracellularly applied PTX in VTA-DA neurons.(A) Effect of exogenous application of GABA on the frequency of sEPSCs of VTA-DA neurons in rats. Left panel: typical current traces of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX. Middle panel: typical time course of the frequency of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX. Right panel: average frequency of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX (n = 6 cells from four rats, p < 0.05, compared to control before GABA). (B) Effect of 470 nm light stimulation on the frequency of sEPSCs of VTA-DA neurons in mice. Panel 1: coronal image showing the expression of ChR2-mCherry (red) following injection of the viral construct bilaterally into the ventral tegmental area (VTA) of GADcre+ mice. Scale bar: 500 µm. Panel 2: 470 nm light (20 Hz)-induced firing of VTA GABA neurons in current-clamp mode. Panel 3: typical current traces of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX. Panel 4: typical time course of the frequency of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX. Panel 5: average frequency of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX (n = 6 cells from five mice, p < 0.05, compared to control before light stimulation). Data are shown as the mean ±s.e.m. *p < 0.05.DOI:http://dx.doi.org/10.7554/eLife.09275.007
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fig5: Effect of exogenous application of GABA and 470 nm light stimulation on the frequency of sEPSCs in the presence of intracellularly applied PTX in VTA-DA neurons.(A) Effect of exogenous application of GABA on the frequency of sEPSCs of VTA-DA neurons in rats. Left panel: typical current traces of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX. Middle panel: typical time course of the frequency of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX. Right panel: average frequency of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX (n = 6 cells from four rats, p < 0.05, compared to control before GABA). (B) Effect of 470 nm light stimulation on the frequency of sEPSCs of VTA-DA neurons in mice. Panel 1: coronal image showing the expression of ChR2-mCherry (red) following injection of the viral construct bilaterally into the ventral tegmental area (VTA) of GADcre+ mice. Scale bar: 500 µm. Panel 2: 470 nm light (20 Hz)-induced firing of VTA GABA neurons in current-clamp mode. Panel 3: typical current traces of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX. Panel 4: typical time course of the frequency of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX. Panel 5: average frequency of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX (n = 6 cells from five mice, p < 0.05, compared to control before light stimulation). Data are shown as the mean ±s.e.m. *p < 0.05.DOI:http://dx.doi.org/10.7554/eLife.09275.007

Mentions: To explore how morphine promoted presynaptic glutamate release in VTA-DA neurons, we hypothesized that glutamatergic input to VTA-DA neurons was inhibited by GABAergic interneurons and morphine disinhibited glutamatergic input by removing this inhibition, thus promoting glutamate release. To test this hypothesis, we first studied whether GABA could inhibit presynaptic glutamate release in VTA-DA neurons by examining the effect of exogenous applied of GABA on the frequency of sEPSCs of VTA-DA neurons in rats. From raw current traces (left panel of Figure 5A) and the time course of sEPSCs (middle panel of Figure 5A), we could see that GABA (10 μM) apparently decreased the frequency of sEPSCs. The average frequency of sEPSCs decreased from 6.7 ± 0.7 Hz before to 4.7 ± 0.6 Hz for 10–15 min after GABA application (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before GABA, right panel of Figure 5A). Then, we observed whether the activation of intrinsic GABAergic neurons could inhibit presynaptic glutamate release of VTA-DA neurons in mice. To do this, AAV virus expressing a double floxed-stopped channelrhodopsin-2 (ChR2)-mCherry was stereotaxically injected into the VTA of mice expressing Cre recombinase in GABA neurons. 2 weeks after infection, expression of ChR2–mCherry was observed in the VTA (panel 1 of Figure 5B). We then performed whole-cell patch-clamp recording in GABA neurons of the VTA and observed the light-induced action potentials in GABA neurons in order to confirm that ChR2 was indeed expressed in the GABA neurons of the VTA in mice. The results showed that 470 nm light stimulation (20 Hz) elicited action potentials in GABA neurons (panel 2 of Figure 5B). On this basis, whole-cell patch-clamp recording was performed in VTA-DA neurons to observe the effect of 470 nm light stimulation on the frequency of sEPSCs in the presence of intracellularly applied PTX (100 µM). Following 5–10 min of baseline recording of sEPSCs, twenty 470 nm light pulses of 5 ms at 20 Hz were delivered every 4 s for 15 min. Raw current traces (panel 3 of Figure 5B) and the time course of sEPSCs (panel 4 of Figure 5B) before and after light stimulation showed that the light stimulation apparently decreased the frequency of sEPSCs. The average frequency of sEPSCs decreased from 4.1 ± 0.7 Hz before to 2.7 ± 0.3 Hz for 10–15 min after light stimulation (n = 6 cells from five mice, paired t test, p < 0.05, compared to control before light stimulation, panel 5 of Figure 5B).10.7554/eLife.09275.007Figure 5.Effect of exogenous application of GABA and 470 nm light stimulation on the frequency of sEPSCs in the presence of intracellularly applied PTX in VTA-DA neurons.


Morphine disinhibits glutamatergic input to VTA dopamine neurons and promotes dopamine neuron excitation.

Chen M, Zhao Y, Yang H, Luan W, Song J, Cui D, Dong Y, Lai B, Ma L, Zheng P - Elife (2015)

Effect of exogenous application of GABA and 470 nm light stimulation on the frequency of sEPSCs in the presence of intracellularly applied PTX in VTA-DA neurons.(A) Effect of exogenous application of GABA on the frequency of sEPSCs of VTA-DA neurons in rats. Left panel: typical current traces of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX. Middle panel: typical time course of the frequency of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX. Right panel: average frequency of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX (n = 6 cells from four rats, p < 0.05, compared to control before GABA). (B) Effect of 470 nm light stimulation on the frequency of sEPSCs of VTA-DA neurons in mice. Panel 1: coronal image showing the expression of ChR2-mCherry (red) following injection of the viral construct bilaterally into the ventral tegmental area (VTA) of GADcre+ mice. Scale bar: 500 µm. Panel 2: 470 nm light (20 Hz)-induced firing of VTA GABA neurons in current-clamp mode. Panel 3: typical current traces of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX. Panel 4: typical time course of the frequency of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX. Panel 5: average frequency of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX (n = 6 cells from five mice, p < 0.05, compared to control before light stimulation). Data are shown as the mean ±s.e.m. *p < 0.05.DOI:http://dx.doi.org/10.7554/eLife.09275.007
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fig5: Effect of exogenous application of GABA and 470 nm light stimulation on the frequency of sEPSCs in the presence of intracellularly applied PTX in VTA-DA neurons.(A) Effect of exogenous application of GABA on the frequency of sEPSCs of VTA-DA neurons in rats. Left panel: typical current traces of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX. Middle panel: typical time course of the frequency of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX. Right panel: average frequency of sEPSCs before and after GABA (10 μM) in the presence of intracellularly applied PTX (n = 6 cells from four rats, p < 0.05, compared to control before GABA). (B) Effect of 470 nm light stimulation on the frequency of sEPSCs of VTA-DA neurons in mice. Panel 1: coronal image showing the expression of ChR2-mCherry (red) following injection of the viral construct bilaterally into the ventral tegmental area (VTA) of GADcre+ mice. Scale bar: 500 µm. Panel 2: 470 nm light (20 Hz)-induced firing of VTA GABA neurons in current-clamp mode. Panel 3: typical current traces of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX. Panel 4: typical time course of the frequency of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX. Panel 5: average frequency of sEPSCs before and after blue light (470 nm) stimulation in the presence of intracellularly applied PTX (n = 6 cells from five mice, p < 0.05, compared to control before light stimulation). Data are shown as the mean ±s.e.m. *p < 0.05.DOI:http://dx.doi.org/10.7554/eLife.09275.007
Mentions: To explore how morphine promoted presynaptic glutamate release in VTA-DA neurons, we hypothesized that glutamatergic input to VTA-DA neurons was inhibited by GABAergic interneurons and morphine disinhibited glutamatergic input by removing this inhibition, thus promoting glutamate release. To test this hypothesis, we first studied whether GABA could inhibit presynaptic glutamate release in VTA-DA neurons by examining the effect of exogenous applied of GABA on the frequency of sEPSCs of VTA-DA neurons in rats. From raw current traces (left panel of Figure 5A) and the time course of sEPSCs (middle panel of Figure 5A), we could see that GABA (10 μM) apparently decreased the frequency of sEPSCs. The average frequency of sEPSCs decreased from 6.7 ± 0.7 Hz before to 4.7 ± 0.6 Hz for 10–15 min after GABA application (n = 6 cells from four rats, paired t test, p < 0.05, compared to control before GABA, right panel of Figure 5A). Then, we observed whether the activation of intrinsic GABAergic neurons could inhibit presynaptic glutamate release of VTA-DA neurons in mice. To do this, AAV virus expressing a double floxed-stopped channelrhodopsin-2 (ChR2)-mCherry was stereotaxically injected into the VTA of mice expressing Cre recombinase in GABA neurons. 2 weeks after infection, expression of ChR2–mCherry was observed in the VTA (panel 1 of Figure 5B). We then performed whole-cell patch-clamp recording in GABA neurons of the VTA and observed the light-induced action potentials in GABA neurons in order to confirm that ChR2 was indeed expressed in the GABA neurons of the VTA in mice. The results showed that 470 nm light stimulation (20 Hz) elicited action potentials in GABA neurons (panel 2 of Figure 5B). On this basis, whole-cell patch-clamp recording was performed in VTA-DA neurons to observe the effect of 470 nm light stimulation on the frequency of sEPSCs in the presence of intracellularly applied PTX (100 µM). Following 5–10 min of baseline recording of sEPSCs, twenty 470 nm light pulses of 5 ms at 20 Hz were delivered every 4 s for 15 min. Raw current traces (panel 3 of Figure 5B) and the time course of sEPSCs (panel 4 of Figure 5B) before and after light stimulation showed that the light stimulation apparently decreased the frequency of sEPSCs. The average frequency of sEPSCs decreased from 4.1 ± 0.7 Hz before to 2.7 ± 0.3 Hz for 10–15 min after light stimulation (n = 6 cells from five mice, paired t test, p < 0.05, compared to control before light stimulation, panel 5 of Figure 5B).10.7554/eLife.09275.007Figure 5.Effect of exogenous application of GABA and 470 nm light stimulation on the frequency of sEPSCs in the presence of intracellularly applied PTX in VTA-DA neurons.

Bottom Line: However, it is not known whether morphine has an additional strengthening effect on excitatory input.We also studied the contribution of the morphine-induced disinhibitory effect on the presynaptic glutamate release to the overall excitatory effect of morphine on VTA-DA neurons and related behavior.Our results suggest that the disinhibitory action of morphine on presynaptic glutamate release might be the main mechanism for morphine-induced increase in VTA-DA neuron firing and related behaviors.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medical Neurobiology, Collaborative Innovation Center for Brain Science, School of Basic Medical Sciences and Institutes of Brain Science, Fudan Univeristy, Shanghai, China.

ABSTRACT
One reported mechanism for morphine activation of dopamine (DA) neurons of the ventral tegmental area (VTA) is the disinhibition model of VTA-DA neurons. Morphine inhibits GABA inhibitory neurons, which shifts the balance between inhibitory and excitatory input to VTA-DA neurons in favor of excitation and then leads to VTA-DA neuron excitation. However, it is not known whether morphine has an additional strengthening effect on excitatory input. Our results suggest that glutamatergic input to VTA-DA neurons is inhibited by GABAergic interneurons via GABAB receptors and that morphine promotes presynaptic glutamate release by removing this inhibition. We also studied the contribution of the morphine-induced disinhibitory effect on the presynaptic glutamate release to the overall excitatory effect of morphine on VTA-DA neurons and related behavior. Our results suggest that the disinhibitory action of morphine on presynaptic glutamate release might be the main mechanism for morphine-induced increase in VTA-DA neuron firing and related behaviors.

No MeSH data available.


Related in: MedlinePlus