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A glutamatergic reward input from the dorsal raphe to ventral tegmental area dopamine neurons.

Qi J, Zhang S, Wang HL, Wang H, de Jesus Aceves Buendia J, Hoffman AF, Lupica CR, Seal RP, Morales M - Nat Commun (2014)

Bottom Line: Here we report rewarding effects following activation of a DR-originating pathway consisting of vesicular glutamate transporter 3 (VGluT3) containing neurons that form asymmetric synapses onto VTA dopamine neurons that project to nucleus accumbens.Activation also reinforces instrumental behaviour and establishes conditioned place preferences.These findings indicate that the DR-VGluT3 pathway to VTA utilizes glutamate as a neurotransmitter and is a substrate linking the DR-one of the most sensitive reward sites in the brain--to VTA dopaminergic neurons.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Drug Abuse, Neuronal Networks Section, National Institutes of Health, Baltimore, Maryland, USA.

ABSTRACT
Electrical stimulation of the dorsal raphe (DR) and ventral tegmental area (VTA) activates the fibres of the same reward pathway but the phenotype of this pathway and the direction of the reward-relevant fibres have not been determined. Here we report rewarding effects following activation of a DR-originating pathway consisting of vesicular glutamate transporter 3 (VGluT3) containing neurons that form asymmetric synapses onto VTA dopamine neurons that project to nucleus accumbens. Optogenetic VTA activation of this projection elicits AMPA-mediated synaptic excitatory currents in VTA mesoaccumbens dopaminergic neurons and causes dopamine release in nucleus accumbens. Activation also reinforces instrumental behaviour and establishes conditioned place preferences. These findings indicate that the DR-VGluT3 pathway to VTA utilizes glutamate as a neurotransmitter and is a substrate linking the DR-one of the most sensitive reward sites in the brain--to VTA dopaminergic neurons.

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Glutamate release onto mesoaccumbens neurons is driven by light-evoked activation of fibers from DR VGluT3 neurons(a) DR VGluT3 neurons were infected by AAV5-DIO-ChR2-eYFP injected into the DR of VGluT3∷Cre mice. The retrograde tracer DiI was injected into the nAcc to label individual mesoaccumbens neurons for intracellular recordings. (b) Brain horizontal section, containing biocytin filled cells (white cells) in VTA (TH immunoreactivity, blue). (c) At higher magnification note a biocytin cell with both DiI (red dots) and TH (blue), and DR VGluT3 terminals containing eYFP (green). (d) Responses of two TH-positive, mesoaccumbens neurons to hyperpolarizing voltage steps (-70 mV, -90 mV, and -110 mV), and (e)light-evoked EPSCs from the same neurons (blue boxes; onset of the laser pulses). Larger light-evoked EPSCs were observed in cells lacking large hyperpolarization-activated currents (Pearson correlation coefficient r = -0.5145, P= 0.07). (f) Traces from an individual neuron obtained prior to (control) and during 10 μM DNQX application. DNQX eliminated currents in all tested cells (P< 0.001, t3= 53.47, n = 4, paired t-test). Individual (gray) and averaged (black) current clamp traces from a single neuron (-55 mV) during light stimulation at 5 Hz (g, upper panel) and 20 Hz (g, lower panel). DNQX eliminated firing elicited by light. (h) Summary of the average number of action potentials observed during light stimulation in a group of TH-positive mesoaccumbens neurons (treatment × frequency:F1,28= 15.76, P= 0.005, n = 5, 5 trials per cell; two-way ANOVA) before and after DNQX application. Data are presented as mean +s.e.m. Bars: (b) 100 μm; (c) 10 μm.
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Figure 4: Glutamate release onto mesoaccumbens neurons is driven by light-evoked activation of fibers from DR VGluT3 neurons(a) DR VGluT3 neurons were infected by AAV5-DIO-ChR2-eYFP injected into the DR of VGluT3∷Cre mice. The retrograde tracer DiI was injected into the nAcc to label individual mesoaccumbens neurons for intracellular recordings. (b) Brain horizontal section, containing biocytin filled cells (white cells) in VTA (TH immunoreactivity, blue). (c) At higher magnification note a biocytin cell with both DiI (red dots) and TH (blue), and DR VGluT3 terminals containing eYFP (green). (d) Responses of two TH-positive, mesoaccumbens neurons to hyperpolarizing voltage steps (-70 mV, -90 mV, and -110 mV), and (e)light-evoked EPSCs from the same neurons (blue boxes; onset of the laser pulses). Larger light-evoked EPSCs were observed in cells lacking large hyperpolarization-activated currents (Pearson correlation coefficient r = -0.5145, P= 0.07). (f) Traces from an individual neuron obtained prior to (control) and during 10 μM DNQX application. DNQX eliminated currents in all tested cells (P< 0.001, t3= 53.47, n = 4, paired t-test). Individual (gray) and averaged (black) current clamp traces from a single neuron (-55 mV) during light stimulation at 5 Hz (g, upper panel) and 20 Hz (g, lower panel). DNQX eliminated firing elicited by light. (h) Summary of the average number of action potentials observed during light stimulation in a group of TH-positive mesoaccumbens neurons (treatment × frequency:F1,28= 15.76, P= 0.005, n = 5, 5 trials per cell; two-way ANOVA) before and after DNQX application. Data are presented as mean +s.e.m. Bars: (b) 100 μm; (c) 10 μm.

Mentions: VGluT3 belongs to a family of three vesicular glutamate transporters (VGluT1, VGluT2 and VGluT3). While VGluT1 and VGluT2 are restricted to known glutamatergic neurons, VGluT3 is found in hippocampal GABAergic neurons, striatal cholinergic interneurons, monoamine neurons and glia14, 15, 16. To determine the capacity of DR VGluT3 efferents to release glutamate on mesoaccumbens neurons, intracellular recordings were performed in VTA slices from VGluT3-ChR2-eYFP mice injected in the nAcc with the retrograde tract tracer Dil (n = 6; Fig. 4a and Supplementary Fig. 7a,b). A total of 37 DiI-labeled VTA neurons were recorded, and were classified into two subpopulations based on action potential width during cell attached recordings, and peak firing frequency in response to depolarizing current (Supplementary Fig. 7). Light-activated excitatory postsynaptic currents (EPSCs) were observed in nearly one-half of the Dil-labeled neurons (18/37; Supplementary Fig. 7); consistent with anatomical data demonstrating VGluT3 inputs on mesoaccumbens neurons from the DR nucleus. The majority of the neurons (Type 1; n = 13) exhibited long duration action potentials (2.36 ± 0.28 ms) and low to moderate firing frequencies during depolarization (6.92 ± 0.71 Hz). Type 1 neurons exhibited large inward “sag” currents during strong membrane hyperpolarization (117.67 ± 30.28 pA at -110 mV), consistent with the activation of hyperpolarization-activated cation (Ih) currents. The remaining neurons (Type 2; n = 5) exhibited short duration action potentials (1.20 ± 0.08 ms), fast firing frequencies during depolarization (95.40 ± 6.94 Hz), and small inward currents during hyperpolarization (46.09 ± 21.24 pA). By immunohistochemistry, we found that Type 1 neurons were TH-positive, and Type 2 neurons were TH-negative (Fig. 4b,d and Supplementary Fig. 7). TH-positive neurons showed larger mean light-evoked EPSCs (-37.53 ± 11.71 pA) using maximal stimulation parameters (10 mW, 5ms) than the TH-negative neurons (-9.85 ± 4.45 pA; Supplementary Fig. 7i). However, there was still considerable heterogeneity within individual TH-positive neurons (range -5 to -133 pA). Input-output curves (Supplementary Fig. 7) and pharmacological manipulations were only performed in TH-positive neurons with reliably detectable, larger responses (≥ 20 pA). Larger light-activated currents in TH-positive neurons were negatively correlated with the amplitude of the Ih currents, although this did not achieve statistical significance (Fig. 4d,e; Pearson correlation coefficient r = -0.5145, P = 0.07). Consistent with the presence of GluR1 at postsynaptic sites, light evoked currents were blocked by the AMPA/kainate receptor antagonist DNQX (Fig. 4g). In current clamp recordings, light-evoked EPSPs elicited in TH-positive neurons were capable of driving action potentials across a range of frequencies (n = 5), and firing was prevented by DNQX application (Fig. 4g,h). To further determine the monosynaptic nature of the DR VGluT3-VTA projections, VTA EPSCs evoked by light stimulation of DR inputs were recorded in the presence of TTX (500 nM). We found that TTX eliminated the optical-evoked EPSCs, which were restored by subsequent application of 4-AP (200 μM) in the presence of TTX (Supplementary Fig. 8a, b, and c). We also observed that the light-evoked EPSCs exhibited short latency (2.08 ± 0.13 ms, n = 7) and low synaptic jitter (standard deviation of latency) (0.22 ± 0.05 ms, n = 7) (Supplementary Fig. 8d). From these electrophysiological findings, we inferred that some mesoaccumbens DA neurons receive monosynaptic glutamatergic signaling from DR-VGluT3 fibers, which are capable of driving mesoaccumbens neuronal firing. In vivo this would be expected to evoke DA release within the nAcc.


A glutamatergic reward input from the dorsal raphe to ventral tegmental area dopamine neurons.

Qi J, Zhang S, Wang HL, Wang H, de Jesus Aceves Buendia J, Hoffman AF, Lupica CR, Seal RP, Morales M - Nat Commun (2014)

Glutamate release onto mesoaccumbens neurons is driven by light-evoked activation of fibers from DR VGluT3 neurons(a) DR VGluT3 neurons were infected by AAV5-DIO-ChR2-eYFP injected into the DR of VGluT3∷Cre mice. The retrograde tracer DiI was injected into the nAcc to label individual mesoaccumbens neurons for intracellular recordings. (b) Brain horizontal section, containing biocytin filled cells (white cells) in VTA (TH immunoreactivity, blue). (c) At higher magnification note a biocytin cell with both DiI (red dots) and TH (blue), and DR VGluT3 terminals containing eYFP (green). (d) Responses of two TH-positive, mesoaccumbens neurons to hyperpolarizing voltage steps (-70 mV, -90 mV, and -110 mV), and (e)light-evoked EPSCs from the same neurons (blue boxes; onset of the laser pulses). Larger light-evoked EPSCs were observed in cells lacking large hyperpolarization-activated currents (Pearson correlation coefficient r = -0.5145, P= 0.07). (f) Traces from an individual neuron obtained prior to (control) and during 10 μM DNQX application. DNQX eliminated currents in all tested cells (P< 0.001, t3= 53.47, n = 4, paired t-test). Individual (gray) and averaged (black) current clamp traces from a single neuron (-55 mV) during light stimulation at 5 Hz (g, upper panel) and 20 Hz (g, lower panel). DNQX eliminated firing elicited by light. (h) Summary of the average number of action potentials observed during light stimulation in a group of TH-positive mesoaccumbens neurons (treatment × frequency:F1,28= 15.76, P= 0.005, n = 5, 5 trials per cell; two-way ANOVA) before and after DNQX application. Data are presented as mean +s.e.m. Bars: (b) 100 μm; (c) 10 μm.
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Figure 4: Glutamate release onto mesoaccumbens neurons is driven by light-evoked activation of fibers from DR VGluT3 neurons(a) DR VGluT3 neurons were infected by AAV5-DIO-ChR2-eYFP injected into the DR of VGluT3∷Cre mice. The retrograde tracer DiI was injected into the nAcc to label individual mesoaccumbens neurons for intracellular recordings. (b) Brain horizontal section, containing biocytin filled cells (white cells) in VTA (TH immunoreactivity, blue). (c) At higher magnification note a biocytin cell with both DiI (red dots) and TH (blue), and DR VGluT3 terminals containing eYFP (green). (d) Responses of two TH-positive, mesoaccumbens neurons to hyperpolarizing voltage steps (-70 mV, -90 mV, and -110 mV), and (e)light-evoked EPSCs from the same neurons (blue boxes; onset of the laser pulses). Larger light-evoked EPSCs were observed in cells lacking large hyperpolarization-activated currents (Pearson correlation coefficient r = -0.5145, P= 0.07). (f) Traces from an individual neuron obtained prior to (control) and during 10 μM DNQX application. DNQX eliminated currents in all tested cells (P< 0.001, t3= 53.47, n = 4, paired t-test). Individual (gray) and averaged (black) current clamp traces from a single neuron (-55 mV) during light stimulation at 5 Hz (g, upper panel) and 20 Hz (g, lower panel). DNQX eliminated firing elicited by light. (h) Summary of the average number of action potentials observed during light stimulation in a group of TH-positive mesoaccumbens neurons (treatment × frequency:F1,28= 15.76, P= 0.005, n = 5, 5 trials per cell; two-way ANOVA) before and after DNQX application. Data are presented as mean +s.e.m. Bars: (b) 100 μm; (c) 10 μm.
Mentions: VGluT3 belongs to a family of three vesicular glutamate transporters (VGluT1, VGluT2 and VGluT3). While VGluT1 and VGluT2 are restricted to known glutamatergic neurons, VGluT3 is found in hippocampal GABAergic neurons, striatal cholinergic interneurons, monoamine neurons and glia14, 15, 16. To determine the capacity of DR VGluT3 efferents to release glutamate on mesoaccumbens neurons, intracellular recordings were performed in VTA slices from VGluT3-ChR2-eYFP mice injected in the nAcc with the retrograde tract tracer Dil (n = 6; Fig. 4a and Supplementary Fig. 7a,b). A total of 37 DiI-labeled VTA neurons were recorded, and were classified into two subpopulations based on action potential width during cell attached recordings, and peak firing frequency in response to depolarizing current (Supplementary Fig. 7). Light-activated excitatory postsynaptic currents (EPSCs) were observed in nearly one-half of the Dil-labeled neurons (18/37; Supplementary Fig. 7); consistent with anatomical data demonstrating VGluT3 inputs on mesoaccumbens neurons from the DR nucleus. The majority of the neurons (Type 1; n = 13) exhibited long duration action potentials (2.36 ± 0.28 ms) and low to moderate firing frequencies during depolarization (6.92 ± 0.71 Hz). Type 1 neurons exhibited large inward “sag” currents during strong membrane hyperpolarization (117.67 ± 30.28 pA at -110 mV), consistent with the activation of hyperpolarization-activated cation (Ih) currents. The remaining neurons (Type 2; n = 5) exhibited short duration action potentials (1.20 ± 0.08 ms), fast firing frequencies during depolarization (95.40 ± 6.94 Hz), and small inward currents during hyperpolarization (46.09 ± 21.24 pA). By immunohistochemistry, we found that Type 1 neurons were TH-positive, and Type 2 neurons were TH-negative (Fig. 4b,d and Supplementary Fig. 7). TH-positive neurons showed larger mean light-evoked EPSCs (-37.53 ± 11.71 pA) using maximal stimulation parameters (10 mW, 5ms) than the TH-negative neurons (-9.85 ± 4.45 pA; Supplementary Fig. 7i). However, there was still considerable heterogeneity within individual TH-positive neurons (range -5 to -133 pA). Input-output curves (Supplementary Fig. 7) and pharmacological manipulations were only performed in TH-positive neurons with reliably detectable, larger responses (≥ 20 pA). Larger light-activated currents in TH-positive neurons were negatively correlated with the amplitude of the Ih currents, although this did not achieve statistical significance (Fig. 4d,e; Pearson correlation coefficient r = -0.5145, P = 0.07). Consistent with the presence of GluR1 at postsynaptic sites, light evoked currents were blocked by the AMPA/kainate receptor antagonist DNQX (Fig. 4g). In current clamp recordings, light-evoked EPSPs elicited in TH-positive neurons were capable of driving action potentials across a range of frequencies (n = 5), and firing was prevented by DNQX application (Fig. 4g,h). To further determine the monosynaptic nature of the DR VGluT3-VTA projections, VTA EPSCs evoked by light stimulation of DR inputs were recorded in the presence of TTX (500 nM). We found that TTX eliminated the optical-evoked EPSCs, which were restored by subsequent application of 4-AP (200 μM) in the presence of TTX (Supplementary Fig. 8a, b, and c). We also observed that the light-evoked EPSCs exhibited short latency (2.08 ± 0.13 ms, n = 7) and low synaptic jitter (standard deviation of latency) (0.22 ± 0.05 ms, n = 7) (Supplementary Fig. 8d). From these electrophysiological findings, we inferred that some mesoaccumbens DA neurons receive monosynaptic glutamatergic signaling from DR-VGluT3 fibers, which are capable of driving mesoaccumbens neuronal firing. In vivo this would be expected to evoke DA release within the nAcc.

Bottom Line: Here we report rewarding effects following activation of a DR-originating pathway consisting of vesicular glutamate transporter 3 (VGluT3) containing neurons that form asymmetric synapses onto VTA dopamine neurons that project to nucleus accumbens.Activation also reinforces instrumental behaviour and establishes conditioned place preferences.These findings indicate that the DR-VGluT3 pathway to VTA utilizes glutamate as a neurotransmitter and is a substrate linking the DR-one of the most sensitive reward sites in the brain--to VTA dopaminergic neurons.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Drug Abuse, Neuronal Networks Section, National Institutes of Health, Baltimore, Maryland, USA.

ABSTRACT
Electrical stimulation of the dorsal raphe (DR) and ventral tegmental area (VTA) activates the fibres of the same reward pathway but the phenotype of this pathway and the direction of the reward-relevant fibres have not been determined. Here we report rewarding effects following activation of a DR-originating pathway consisting of vesicular glutamate transporter 3 (VGluT3) containing neurons that form asymmetric synapses onto VTA dopamine neurons that project to nucleus accumbens. Optogenetic VTA activation of this projection elicits AMPA-mediated synaptic excitatory currents in VTA mesoaccumbens dopaminergic neurons and causes dopamine release in nucleus accumbens. Activation also reinforces instrumental behaviour and establishes conditioned place preferences. These findings indicate that the DR-VGluT3 pathway to VTA utilizes glutamate as a neurotransmitter and is a substrate linking the DR-one of the most sensitive reward sites in the brain--to VTA dopaminergic neurons.

Show MeSH