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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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VGluT3 neurons make asymmetric synapses on TH neurons having GluR1 postsynaptic to VGluT3 terminals(a-c) VGluT3 terminals make synapses mostly on TH-positive dendrites (electron microscopy). (a) An axon terminal (AT) containing VGluT3 (scattered dark material) makes an asymmetric synapse (green arrow) with a TH-positive (gold particles, blue arrowhead) dendrite. (b) An axon terminal containing VGluT3 makes an asymmetric synapse (green arrow) with a TH-negative dendrite. (c) The bars indicate the frequency (mean + s.e.m.) of VGluT3 terminals making asymmetric synapses on TH-positive or TH-negative dendrites. From a total of 466 VGluT3 terminals, 62.51 ± 0.44% (n = 291) make synapses on TH-positive dendrites and 37.49 ± 0.44% (n = 175) on TH-negative dendrites (* P< 0.0001, t3= 28.65, two-tailed paired t-test, terminals collected from 4 rats, n = number of counted terminals). (d) A TH-positive dendrite containing GluR1 signal postsynaptic to a VGluT3 terminal (immunofluorescence microscopy). Note the presence of GluR1 (green, arrow) located within a TH-positive dendrite (blue) adjacent to a VGluT3-positive terminal (red). (e) The association between the postsynaptic GluR1 within a TH-dendrite and a VGluT3 terminal is better seen after 3-D reconstruction of confocal images. (f) At the electron microscopic level, GluR1 signal is seen as gold particles (arrowheads) along the postsynaptic membrane of two dendrites making asymmetric synapses (arrows) with a single AT (pink outline) containing VGluT3 (scattered dark material). Bars: (a, b, and f) 200 nm; (d) 2 μm.
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Figure 2: VGluT3 neurons make asymmetric synapses on TH neurons having GluR1 postsynaptic to VGluT3 terminals(a-c) VGluT3 terminals make synapses mostly on TH-positive dendrites (electron microscopy). (a) An axon terminal (AT) containing VGluT3 (scattered dark material) makes an asymmetric synapse (green arrow) with a TH-positive (gold particles, blue arrowhead) dendrite. (b) An axon terminal containing VGluT3 makes an asymmetric synapse (green arrow) with a TH-negative dendrite. (c) The bars indicate the frequency (mean + s.e.m.) of VGluT3 terminals making asymmetric synapses on TH-positive or TH-negative dendrites. From a total of 466 VGluT3 terminals, 62.51 ± 0.44% (n = 291) make synapses on TH-positive dendrites and 37.49 ± 0.44% (n = 175) on TH-negative dendrites (* P< 0.0001, t3= 28.65, two-tailed paired t-test, terminals collected from 4 rats, n = number of counted terminals). (d) A TH-positive dendrite containing GluR1 signal postsynaptic to a VGluT3 terminal (immunofluorescence microscopy). Note the presence of GluR1 (green, arrow) located within a TH-positive dendrite (blue) adjacent to a VGluT3-positive terminal (red). (e) The association between the postsynaptic GluR1 within a TH-dendrite and a VGluT3 terminal is better seen after 3-D reconstruction of confocal images. (f) At the electron microscopic level, GluR1 signal is seen as gold particles (arrowheads) along the postsynaptic membrane of two dendrites making asymmetric synapses (arrows) with a single AT (pink outline) containing VGluT3 (scattered dark material). Bars: (a, b, and f) 200 nm; (d) 2 μm.

Mentions: A prior study using pressure injections and non-radioactive detection of VGluT3 mRNA reported that all VGluT3 afferents to VTA were from the DR and the bed nucleus of the stria terminalis4. In the present study by using iontophorectic application of FG (to minimize labeling of fibers of passage) and radioactive detection of VGluT3 mRNA (to maximize detection of mRNA), we confirmed that DR VGluT3 neurons project to the VTA, found a very few VGluT3 neurons from the medial raphe projecting to the VTA, and did not find VGluT3 neurons from the bed nucleus of the stria terminalis projecting to the VTA (Supplementary Fig. 2). These findings together with prior studies showing lack of VGluT3 mRNA in the VTA12, 13 suggest that the major source of VGluT3 afferents, if not the only one, to the VTA is from the DR. To determine whether DR VGluT3 terminals form synapses on VTA tyrosine hydroxylase (TH) neurons, we performed double VGluT3 and TH immuno-electron microscopy with an anti-VGluT3 antibody that in the DR labeled only those neurons that co-expressed VGluT3 mRNA (Supplementary Fig. 3). By using this anti-VGluT3 antibody, we found that VGluT3-terminals established asymmetric synapses mostly on TH-positive dendrites (62.51 ± 0.44%; 291 terminals out of 466; Fig. 2a,c), and less frequently on TH-negative dendrites (37.49 ± 0.44%; 175 terminals out of 466; Fig. 2b,c). The presence of VGluT3-axon terminals making asymmetric synapses in the VTA suggests that these terminals provide an excitatory input to VTA neurons. Thus, we next examined whether glutamatergic receptors were found postsynaptic to VGluT3 terminals. By fluorescence microscopy, we detected in the VTA the AMPA receptor subunit GluR1 postsynaptic to VGluT3 punctuate structures that were distributed within TH-positive neurons (Fig. 2d,e). By immuno-electron microscopy, we confirmed the presence of GluR1 along the postsynaptic membrane of asymmetric synapses established by VGluT3 axon terminals (Fig. 2f). These anatomical findings indicate that the DR provides a major glutamatergic input to TH neurons in the VTA.


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)

VGluT3 neurons make asymmetric synapses on TH neurons having GluR1 postsynaptic to VGluT3 terminals(a-c) VGluT3 terminals make synapses mostly on TH-positive dendrites (electron microscopy). (a) An axon terminal (AT) containing VGluT3 (scattered dark material) makes an asymmetric synapse (green arrow) with a TH-positive (gold particles, blue arrowhead) dendrite. (b) An axon terminal containing VGluT3 makes an asymmetric synapse (green arrow) with a TH-negative dendrite. (c) The bars indicate the frequency (mean + s.e.m.) of VGluT3 terminals making asymmetric synapses on TH-positive or TH-negative dendrites. From a total of 466 VGluT3 terminals, 62.51 ± 0.44% (n = 291) make synapses on TH-positive dendrites and 37.49 ± 0.44% (n = 175) on TH-negative dendrites (* P< 0.0001, t3= 28.65, two-tailed paired t-test, terminals collected from 4 rats, n = number of counted terminals). (d) A TH-positive dendrite containing GluR1 signal postsynaptic to a VGluT3 terminal (immunofluorescence microscopy). Note the presence of GluR1 (green, arrow) located within a TH-positive dendrite (blue) adjacent to a VGluT3-positive terminal (red). (e) The association between the postsynaptic GluR1 within a TH-dendrite and a VGluT3 terminal is better seen after 3-D reconstruction of confocal images. (f) At the electron microscopic level, GluR1 signal is seen as gold particles (arrowheads) along the postsynaptic membrane of two dendrites making asymmetric synapses (arrows) with a single AT (pink outline) containing VGluT3 (scattered dark material). Bars: (a, b, and f) 200 nm; (d) 2 μm.
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Figure 2: VGluT3 neurons make asymmetric synapses on TH neurons having GluR1 postsynaptic to VGluT3 terminals(a-c) VGluT3 terminals make synapses mostly on TH-positive dendrites (electron microscopy). (a) An axon terminal (AT) containing VGluT3 (scattered dark material) makes an asymmetric synapse (green arrow) with a TH-positive (gold particles, blue arrowhead) dendrite. (b) An axon terminal containing VGluT3 makes an asymmetric synapse (green arrow) with a TH-negative dendrite. (c) The bars indicate the frequency (mean + s.e.m.) of VGluT3 terminals making asymmetric synapses on TH-positive or TH-negative dendrites. From a total of 466 VGluT3 terminals, 62.51 ± 0.44% (n = 291) make synapses on TH-positive dendrites and 37.49 ± 0.44% (n = 175) on TH-negative dendrites (* P< 0.0001, t3= 28.65, two-tailed paired t-test, terminals collected from 4 rats, n = number of counted terminals). (d) A TH-positive dendrite containing GluR1 signal postsynaptic to a VGluT3 terminal (immunofluorescence microscopy). Note the presence of GluR1 (green, arrow) located within a TH-positive dendrite (blue) adjacent to a VGluT3-positive terminal (red). (e) The association between the postsynaptic GluR1 within a TH-dendrite and a VGluT3 terminal is better seen after 3-D reconstruction of confocal images. (f) At the electron microscopic level, GluR1 signal is seen as gold particles (arrowheads) along the postsynaptic membrane of two dendrites making asymmetric synapses (arrows) with a single AT (pink outline) containing VGluT3 (scattered dark material). Bars: (a, b, and f) 200 nm; (d) 2 μm.
Mentions: A prior study using pressure injections and non-radioactive detection of VGluT3 mRNA reported that all VGluT3 afferents to VTA were from the DR and the bed nucleus of the stria terminalis4. In the present study by using iontophorectic application of FG (to minimize labeling of fibers of passage) and radioactive detection of VGluT3 mRNA (to maximize detection of mRNA), we confirmed that DR VGluT3 neurons project to the VTA, found a very few VGluT3 neurons from the medial raphe projecting to the VTA, and did not find VGluT3 neurons from the bed nucleus of the stria terminalis projecting to the VTA (Supplementary Fig. 2). These findings together with prior studies showing lack of VGluT3 mRNA in the VTA12, 13 suggest that the major source of VGluT3 afferents, if not the only one, to the VTA is from the DR. To determine whether DR VGluT3 terminals form synapses on VTA tyrosine hydroxylase (TH) neurons, we performed double VGluT3 and TH immuno-electron microscopy with an anti-VGluT3 antibody that in the DR labeled only those neurons that co-expressed VGluT3 mRNA (Supplementary Fig. 3). By using this anti-VGluT3 antibody, we found that VGluT3-terminals established asymmetric synapses mostly on TH-positive dendrites (62.51 ± 0.44%; 291 terminals out of 466; Fig. 2a,c), and less frequently on TH-negative dendrites (37.49 ± 0.44%; 175 terminals out of 466; Fig. 2b,c). The presence of VGluT3-axon terminals making asymmetric synapses in the VTA suggests that these terminals provide an excitatory input to VTA neurons. Thus, we next examined whether glutamatergic receptors were found postsynaptic to VGluT3 terminals. By fluorescence microscopy, we detected in the VTA the AMPA receptor subunit GluR1 postsynaptic to VGluT3 punctuate structures that were distributed within TH-positive neurons (Fig. 2d,e). By immuno-electron microscopy, we confirmed the presence of GluR1 along the postsynaptic membrane of asymmetric synapses established by VGluT3 axon terminals (Fig. 2f). These anatomical findings indicate that the DR provides a major glutamatergic input to TH neurons in the VTA.

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