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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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Most DR neurons projecting to the VTA express VGluT3 mRNA(a) Retrograde tracer FG was delivered into the VTA. (b) FG injection site in the VTA. (c) DR diagram. (d-f) Low magnification of a DR coronal section showing brown cells with FG-immunoreactivity (FG-IR, d), expression of VGluT3 mRNA (white aggregated grains; e) or TPH-immunoreactivity (TPH-IR, green; f). Four individual cells are seen at higher magnification. A FG neuron expressing VGluT3 mRNA without TPH-IR (arrow), a FG neuron co-expressing VGluT3 mRNA and TPH-IR (double arrow), a FG neuron expressing TPH-IR only (double arrowheads) and a FG neuron lacking both VGluT3 mRNA and TPH-IR (arrowhead). (g) Frequency of FG phenotypes (mean + s.e.m.). Among all DR neurons projecting to the VTA, 46% expressed only VGluT3 mRNA, 14% co-expressed VGluT3 mRNA and TPH-IR, 13% expressed TPH-IR alone, and 28% lacked both VGluT3 mRNA and TPH-IR. FG cell counting was made between bregma -6.84 and -8.40 mm (n = 4, 10-15 sections per rat). (h-l) Phenotype map of DR neurons projecting to the VTA. Each dot represents the average of DR neurons projecting to the VTA. Many VGluT3 expressing neurons without TPH-IR were concentrated in the dorsal part (DRD) and ventral part (DRV) of the DR (green; i) intermixed with neurons that either co-expressed TPH-IR with VGluT3 (yellow; j) or expressed only TPH-IR (pink; k). DR neurons projecting to the VTA lacking both VGluT3 and TPH-IR were frequently observed in the DRD, DRV, lateral (DRL) and posterodorsal (PDR) aspects of the DR (grey; l). Aq, aqueduct; fr, fasciculus retroflexus; mlf, medial longitudinal fasciculs; mp, mammillary peduncle; SNR, substantia nigra reticulata. Diagrams were adapted from rat brain atlas57. Bars: (b) 400 μm; (f) 110 μm; 10 μm for high magnification.
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Figure 1: Most DR neurons projecting to the VTA express VGluT3 mRNA(a) Retrograde tracer FG was delivered into the VTA. (b) FG injection site in the VTA. (c) DR diagram. (d-f) Low magnification of a DR coronal section showing brown cells with FG-immunoreactivity (FG-IR, d), expression of VGluT3 mRNA (white aggregated grains; e) or TPH-immunoreactivity (TPH-IR, green; f). Four individual cells are seen at higher magnification. A FG neuron expressing VGluT3 mRNA without TPH-IR (arrow), a FG neuron co-expressing VGluT3 mRNA and TPH-IR (double arrow), a FG neuron expressing TPH-IR only (double arrowheads) and a FG neuron lacking both VGluT3 mRNA and TPH-IR (arrowhead). (g) Frequency of FG phenotypes (mean + s.e.m.). Among all DR neurons projecting to the VTA, 46% expressed only VGluT3 mRNA, 14% co-expressed VGluT3 mRNA and TPH-IR, 13% expressed TPH-IR alone, and 28% lacked both VGluT3 mRNA and TPH-IR. FG cell counting was made between bregma -6.84 and -8.40 mm (n = 4, 10-15 sections per rat). (h-l) Phenotype map of DR neurons projecting to the VTA. Each dot represents the average of DR neurons projecting to the VTA. Many VGluT3 expressing neurons without TPH-IR were concentrated in the dorsal part (DRD) and ventral part (DRV) of the DR (green; i) intermixed with neurons that either co-expressed TPH-IR with VGluT3 (yellow; j) or expressed only TPH-IR (pink; k). DR neurons projecting to the VTA lacking both VGluT3 and TPH-IR were frequently observed in the DRD, DRV, lateral (DRL) and posterodorsal (PDR) aspects of the DR (grey; l). Aq, aqueduct; fr, fasciculus retroflexus; mlf, medial longitudinal fasciculs; mp, mammillary peduncle; SNR, substantia nigra reticulata. Diagrams were adapted from rat brain atlas57. Bars: (b) 400 μm; (f) 110 μm; 10 μm for high magnification.

Mentions: VGluT3 neurons and serotonergic neurons from DR are known to innervate the rat VTA4, 7, 8, 9, 10, 11. To determine the relative proportions of rat DR neurons projecting to VTA expressing VGluT3, the rate-limiting enzyme for serotonin production (tryptophan hydroxylase; TPH), or both markers, we first injected the retrograde tracer Fluoro-Gold (FG) into the rat VTA (Fig. 1a,b and Supplementary Fig. 1). We then looked for co-localization of FG (Fig. 1c,d) with VGluT3 mRNA, using in situ hybridization (Fig. 1e), or TPH, using immunohistochemistry (Fig. 1f). We found that about half of all FG neurons expressed VGluT3 mRNA without TPH (45.54 ± 1.24%; 1,329 cells out of 2,968 FG neurons; Fig. 1g and Supplementary Table 1). Some FG neurons co-expressed VGluT3 mRNA and TPH (13.99 ± 0.81%; 415 cells out of 2,968 FG neurons), whereas others expressed TPH without VGluT3 mRNA (12.90 ± 0.91%; 389 cells out of 2,968 FG neurons). A fourth subpopulation of FG neurons lacked both VGluT3 mRNA and TPH (27.65 ± 1.36%; 835 cells out of 2,968 FG neurons). Most of the FG neurons expressing VGluT3 without TPH were concentrated in the dorsal and ventral aspects of the DR (Fig. 1h,i), and were intermingled with FG-TPH neurons co-expressing (Fig. 1j) or lacking VGluT3 (Fig. 1k). The FG neurons lacking both VGluT3 and TPH were observed in all aspects of the DR (Fig. 1l). Although it is well documented that the VTA receives a major serotonergic input from the DR7, 8, 9, 10, our findings indicate that the major projection from rat DR to VTA consists of VGluT3 neurons (58.76%), with a minor pathway arising from a population of TPH neurons without VGluT3 (12.90%). Although most of the DR VGluT3 neurons projecting to the VTA lacked TPH, nearly half of all TPH neurons co-expressed VGluT3 mRNA (51.61%). These data suggest that the majority of inputs from the DR to the VTA have the capacity to accumulate glutamate into synaptic vesicles via VGluT3.


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)

Most DR neurons projecting to the VTA express VGluT3 mRNA(a) Retrograde tracer FG was delivered into the VTA. (b) FG injection site in the VTA. (c) DR diagram. (d-f) Low magnification of a DR coronal section showing brown cells with FG-immunoreactivity (FG-IR, d), expression of VGluT3 mRNA (white aggregated grains; e) or TPH-immunoreactivity (TPH-IR, green; f). Four individual cells are seen at higher magnification. A FG neuron expressing VGluT3 mRNA without TPH-IR (arrow), a FG neuron co-expressing VGluT3 mRNA and TPH-IR (double arrow), a FG neuron expressing TPH-IR only (double arrowheads) and a FG neuron lacking both VGluT3 mRNA and TPH-IR (arrowhead). (g) Frequency of FG phenotypes (mean + s.e.m.). Among all DR neurons projecting to the VTA, 46% expressed only VGluT3 mRNA, 14% co-expressed VGluT3 mRNA and TPH-IR, 13% expressed TPH-IR alone, and 28% lacked both VGluT3 mRNA and TPH-IR. FG cell counting was made between bregma -6.84 and -8.40 mm (n = 4, 10-15 sections per rat). (h-l) Phenotype map of DR neurons projecting to the VTA. Each dot represents the average of DR neurons projecting to the VTA. Many VGluT3 expressing neurons without TPH-IR were concentrated in the dorsal part (DRD) and ventral part (DRV) of the DR (green; i) intermixed with neurons that either co-expressed TPH-IR with VGluT3 (yellow; j) or expressed only TPH-IR (pink; k). DR neurons projecting to the VTA lacking both VGluT3 and TPH-IR were frequently observed in the DRD, DRV, lateral (DRL) and posterodorsal (PDR) aspects of the DR (grey; l). Aq, aqueduct; fr, fasciculus retroflexus; mlf, medial longitudinal fasciculs; mp, mammillary peduncle; SNR, substantia nigra reticulata. Diagrams were adapted from rat brain atlas57. Bars: (b) 400 μm; (f) 110 μm; 10 μm for high magnification.
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Figure 1: Most DR neurons projecting to the VTA express VGluT3 mRNA(a) Retrograde tracer FG was delivered into the VTA. (b) FG injection site in the VTA. (c) DR diagram. (d-f) Low magnification of a DR coronal section showing brown cells with FG-immunoreactivity (FG-IR, d), expression of VGluT3 mRNA (white aggregated grains; e) or TPH-immunoreactivity (TPH-IR, green; f). Four individual cells are seen at higher magnification. A FG neuron expressing VGluT3 mRNA without TPH-IR (arrow), a FG neuron co-expressing VGluT3 mRNA and TPH-IR (double arrow), a FG neuron expressing TPH-IR only (double arrowheads) and a FG neuron lacking both VGluT3 mRNA and TPH-IR (arrowhead). (g) Frequency of FG phenotypes (mean + s.e.m.). Among all DR neurons projecting to the VTA, 46% expressed only VGluT3 mRNA, 14% co-expressed VGluT3 mRNA and TPH-IR, 13% expressed TPH-IR alone, and 28% lacked both VGluT3 mRNA and TPH-IR. FG cell counting was made between bregma -6.84 and -8.40 mm (n = 4, 10-15 sections per rat). (h-l) Phenotype map of DR neurons projecting to the VTA. Each dot represents the average of DR neurons projecting to the VTA. Many VGluT3 expressing neurons without TPH-IR were concentrated in the dorsal part (DRD) and ventral part (DRV) of the DR (green; i) intermixed with neurons that either co-expressed TPH-IR with VGluT3 (yellow; j) or expressed only TPH-IR (pink; k). DR neurons projecting to the VTA lacking both VGluT3 and TPH-IR were frequently observed in the DRD, DRV, lateral (DRL) and posterodorsal (PDR) aspects of the DR (grey; l). Aq, aqueduct; fr, fasciculus retroflexus; mlf, medial longitudinal fasciculs; mp, mammillary peduncle; SNR, substantia nigra reticulata. Diagrams were adapted from rat brain atlas57. Bars: (b) 400 μm; (f) 110 μm; 10 μm for high magnification.
Mentions: VGluT3 neurons and serotonergic neurons from DR are known to innervate the rat VTA4, 7, 8, 9, 10, 11. To determine the relative proportions of rat DR neurons projecting to VTA expressing VGluT3, the rate-limiting enzyme for serotonin production (tryptophan hydroxylase; TPH), or both markers, we first injected the retrograde tracer Fluoro-Gold (FG) into the rat VTA (Fig. 1a,b and Supplementary Fig. 1). We then looked for co-localization of FG (Fig. 1c,d) with VGluT3 mRNA, using in situ hybridization (Fig. 1e), or TPH, using immunohistochemistry (Fig. 1f). We found that about half of all FG neurons expressed VGluT3 mRNA without TPH (45.54 ± 1.24%; 1,329 cells out of 2,968 FG neurons; Fig. 1g and Supplementary Table 1). Some FG neurons co-expressed VGluT3 mRNA and TPH (13.99 ± 0.81%; 415 cells out of 2,968 FG neurons), whereas others expressed TPH without VGluT3 mRNA (12.90 ± 0.91%; 389 cells out of 2,968 FG neurons). A fourth subpopulation of FG neurons lacked both VGluT3 mRNA and TPH (27.65 ± 1.36%; 835 cells out of 2,968 FG neurons). Most of the FG neurons expressing VGluT3 without TPH were concentrated in the dorsal and ventral aspects of the DR (Fig. 1h,i), and were intermingled with FG-TPH neurons co-expressing (Fig. 1j) or lacking VGluT3 (Fig. 1k). The FG neurons lacking both VGluT3 and TPH were observed in all aspects of the DR (Fig. 1l). Although it is well documented that the VTA receives a major serotonergic input from the DR7, 8, 9, 10, our findings indicate that the major projection from rat DR to VTA consists of VGluT3 neurons (58.76%), with a minor pathway arising from a population of TPH neurons without VGluT3 (12.90%). Although most of the DR VGluT3 neurons projecting to the VTA lacked TPH, nearly half of all TPH neurons co-expressed VGluT3 mRNA (51.61%). These data suggest that the majority of inputs from the DR to the VTA have the capacity to accumulate glutamate into synaptic vesicles via VGluT3.

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
Related in: MedlinePlus