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Research ArticleResearch Article: New Research, Sensory and Motor Systems

Whole-Brain Mapping of Direct Inputs to Dopamine D1 and D2 Receptor-Expressing Medium Spiny Neurons in the Posterior Dorsomedial Striatum

Jiayi Lu, Yifeng Cheng, Xueyi Xie, Kayla Woodson, Jordan Bonifacio, Emily Disney, Britton Barbee, Xuehua Wang, Mariam Zaidi and Jun Wang
eNeuro 30 December 2020, 8 (1) ENEURO.0348-20.2020; https://doi.org/10.1523/ENEURO.0348-20.2020
Jiayi Lu
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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Yifeng Cheng
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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Xueyi Xie
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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Kayla Woodson
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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Jordan Bonifacio
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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Emily Disney
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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Britton Barbee
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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Xuehua Wang
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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Mariam Zaidi
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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Jun Wang
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX 77807
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  • ORCID record for Jun Wang
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  • Figure 1.
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    Figure 1.

    Rabies virus-mediated retrograde monosynaptic whole-brain tracing of neurons projecting to D1-MSNs and D2-MSNs in the pDMS. A, B, The experimental design used to trace neurons with afferent inputs to D1-MSNs in the pDMS. The design shows how we employed D1-Cre;Snap25 transgenic mice, in which D1-MSNs selectively expressed Cre and GFP (green). Note that the same approach was repeated in D2-Cre;Snap25 and D1-tdTomato;D2Cre mice, where we traced afferent inputs to D2-MSNs. Selective rabies infection of D1-MSNs was achieved by injecting the pDMS of these mice with Cre-dependent helper viruses expressing an avian membrane EnvA receptor protein (TVA) and RG (AAV-Flex-TVA-mCherry and AAV-Flex-RG) three weeks before injection of a modified rabies virus (EnvA-SADΔG-mCherry) into the same area at a 10° angle. C, One week after rabies injection, the rabies virus had specifically infected TVA-expressing D1-MSNs and spread retrogradely from RG-expressing D1-MSNs to neurons with monosynaptic connections with them. Note that extrastriatal neurons with monosynaptic connections to RG-expressing D1-MSNs expressed rabies-derived mCherry (red). mCherry-expressing neurons that also contained D1Rs and thus expressed Cre-driven GFP (green) appeared yellow. Extrastriatal neurons that expressed D1Rs but did not make any connections with RG-expressing D1-MSNs were labeled green. D, Representative confocal images of rabies virus-labeled mCherry-expressing neurons (red) that project to D1-MSNs (i–vii) or D2-MSNs (viii–xiv) throughout the brain in the D1-Cre;Snap25 or D2-Cre;Snap25 mice, respectively. Rows 2 and 4 show enlarged images of the boxed areas in rows 1 and 3, respectively. Note that there were extensive mCherry-positive neurons in the cortex (i–xiv), BNST (iii, x), GPe (iv, xi), amygdala (v, xii), thalamus (vi, xiii), and midbrain (vii, xiv). M2, secondary motor cortex; Cg, cingulate cortex; PrL, prelimbic cortex; S1, primary sensory cortex; M1, primary motor cortex; BNST, bed nucleus of the stria terminalis; GPe, globus pallidus external; AD, anterior dorsal thalamus; AV, anterior ventral thalamus; BLA, basolateral amygdala; CeA, central amygdala; EP, entopeduncular nucleus; Pf, parafascicular thalamic nucleus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; inj. site, injection site. Scale bars: 500 μm (rows 1 and 3) and 200 μm (rows 2 and 4). E, There was no significant difference between the total number of extrastriatal neurons with projections to D1-MSNs or D2-MSNs; unpaired t test. F, Extrastriatal inputs onto D1-MSNs (blue) versus D2-MSNs (orange); *p < 0.05, **p < 0.01, ***p < 0.001; two-way RM ANOVA followed by Tukey’s test for the indicated comparisons; n = 5 mice for D1-MSNs (D1-Cre;Snap25 mice) and n = 8 mice for D2-MSNs (4 D2-Cre;Snap25 mice and 4 D1-tdTomato;D2-Cre mice; E, F).

  • Figure 2.
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    Figure 2.

    Summary of brain-wide monosynaptic inputs to pDMS D1-MSNs and D2-MSNs. The majority of direct synaptic inputs to the pDMS arose in the cortex and thalamus. Analysis of the normalized distribution of rabies virus-labeled neurons showed that the orbital frontal cortex, secondary motor cortex, cingulate cortex, and secondary visual cortex preferentially projected to D1-MSNs versus D2-MSNs. The primary motor cortex, primary sensory cortex, central thalamic nucleus, ventromedial thalamic nucleus, and globus pallidus preferentially innervated D2-MSNs versus D1-MSNs. The extrastriatal inputs onto D1-MSNs (blue) or D2-MSNs (orange) are expressed as a percentage of the total inputs to these cell types; *p < 0.05, **p < 0.01, ***p < 0.001; two-way RM ANOVA followed by Tukey’s test; n = 5 mice for D1-MSNs (D1-Cre;Snap25 mice) and n = 8 mice for D2-MSNs (4 D2-Cre;Snap25 mice and 4 D1-tdTomato;D2-Cre mice).

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    Figure 3.

    The BNST and CeA preferentially express D2Rs versus D1Rs. A, Representative fluorescent image of a coronal section of the BNST from a D1-tdTomato;D2-Cre;Snap25 mouse. The atlas skeleton (left) shows the BNST location at +0.26 mm relative to bregma. B, Representative dual-channel higher magnification fluorescent images of the boxed region of panel A showing abundant GFP-expressing (D2R-positive) neurons (left), a few tdTomato-expressing (D1R-positive) neurons (middle), and some colocalization (right). The bottom panels show an enlarged image of the boxed region from the top panels. Str, striatum; aca, anterior commissure area; acp, posterior commissure area. C, Schematic representation of the BNST starting at +0.26 mm and ending at −0.34 mm relative to bregma. D, Bar graph summarizing the numbers of D1R-expressing and D2R-expressing neurons in the BNST; **p < 0.01, unpaired t test; n = 18 sections from three mice. E, Representative fluorescent image of a coronal section of the CeA from a D1-tdTomato;D2-Cre;Snap25 mouse. The atlas skeleton (left) shows the location of the CeA at −1.22 mm relative to bregma. F, Representative dual-channel higher magnification fluorescent images of the boxed region of panel E showing abundant GFP-expressing D2R-positive neurons (left), a few tdTomato-expressing D1R-positive neurons (middle), and some colocalization (right). The bottom panels show enlarged images of the boxed region of the top panels. GPe, globus pallidus external; tDS; tail of the striatum; BLA, basolateral amygdala. G, Schematic representation of the CeA starting at −0.82 mm and ending at −1.46 mm relative to bregma. H, Bar graph depicting the higher average number of D2R-expressing neurons than D1R-expressing neurons in the CeA; ***p < 0.001, unpaired t test; n = 18 sections from three mice. Scale bars: 1 mm (A, E), 250 μm (B, F, top panels), 20 μm (B, F, bottom panels).

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    Figure 4.

    Rabies virus-mediated retrograde monosynaptic whole-brain labeling of D1R-expressing and D2R-expressing neurons projecting to D1-MSNs and D2-MSNs in the pDMS. A–C, Schematic showing the experimental approach used to label extrastriatal D1R-expressing or D2R-expressing neurons with projections to pDMS D1-MSNs or D2-MSNs. D1-Cre;Snap25 mice were employed to identify D1→D1 connections (A), D2-Cre;Snap25 mice for D2→D2 connections (B), and D1-tdTomato;D2-Cre mice for D1→D2 connections (C). D1-Cre;Snap25 and D2-Cre;Snap25 mice expressed GFP in D1R-expressing and D2R-expressing neurons, respectively (A, B). In D1-tdTomato;D2-Cre mice, D1R-expressing neurons were labeled red (C). Injection of Cre-dependent helper viruses (AAV-Flex-TVA-mCherry and AAV-Flex-RG) into the pDMS induced selective expression of TVA and RG in Cre-expressing D1-MSNs (A), D2-MSNs (B), and D2-MSNs (C). Three weeks after helper virus infusion, injection of EnvA-SADΔG-mCherry into the same site of the D1-Cre;Snap25 (A) and D2-Cre;Snap25 (B) mice, and of EnvA-SADΔG-GFP into the same site of D1-tdTomato;D2-Cre mice (C) caused selective rabies infection and expression of mCherry by D1-MSNs (A) or D2-MSNs (B), and expression of rabies-GFP by D2-MSNs (C). Retrograde spread of rabies virus then occurred from D1-MSNs (A) or D2-MSNs (B, C) to extrastriatal presynaptic neurons. This facilitated identification of D1→D1 (A), D2→D2 (B), and D1→D2 (C) connections, as indicated. D, Representative confocal images of the cortex in the indicated mice following injection of either EnvA-SADΔG-mCherry or EnvA-SADΔG-GFP. White arrows indicate colocalization of D1→D1 (left), D2→D2 (middle), and D1→D2 (right). Scale bars: 10 μm. E, The relative levels of the indicated connections, where D1→D1 is expressed as a percentage of the total number of D1-MSN inputs, and D2→D2 or D1→D2 is expressed as a percentage of the total number of D2-MSN inputs; *p < 0.05, **p < 0.01 for the indicated comparisons, one-way ANOVA followed by Tukey’s test. F, Bar graph comparing D1R-expressing and D2R-expressing inputs from the cortex, amygdala, and thalamus onto MSNs. D1R-expressing or D2R-expressing inputs from the indicated brain regions were normalized to the total extrastriatal D1R-expressing or D2R-expressing inputs, as appropriate. Note that the cortex exhibited high percentages of D1→D1 and D1→D2 connections, whereas the thalamus showed a high percentage of D2→D2 connections; ***p < 0.001, *p < 0.05, two-way RM ANOVA followed by Tukey’s test. G, Bar graph comparing D1R-expressing and D2R-expressing inputs from the SNc, lateral habenula (LHb), and BNST onto MSNs. D1R-expressing or D2R-expressing inputs from these brain regions were normalized to the total extrastriatal D1R-expressing or D2R-expressing inputs; **p < 0.01, *p < 0.05, two-way RM ANOVA followed by Tukey’s test; n = 5 (D1→D1), n = 4 (D2→D2), n = 4 (D1→D2) mice (E–G).

  • Figure 5.
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    Figure 5.

    Distribution of extrastriatal D1R-expressing or D2R-expressing inputs onto MSNs from the cortex, amygdala, and thalamus. D1R-expressing or D2R-expressing inputs from the indicated brain regions were normalized to the total extrastriatal D1R-expressing or D2R-expressing inputs, as appropriate. A, Bar graph of cortical inputs, segregated into ten major subregions, indicating that the frontal associate cortex had a high proportion of D1→D2 connections; *p < 0.05, two-way RM ANOVA followed by Tukey’s test. B, No significant differences were found in the proportions of connection types between the BLA or CeA and the pDMS. C, No significant differences were found in the proportions of connection types between the paraventricular thalamic nucleus (PVT), anterior thalamic nucleus (ATN), anteroventral thalamic nucleus (AV), intermediodorsal thalamic nucleus (IMD), or posterior thalamic nucleus (Po) and the pDMS. However, the mediodorsal thalamic nucleus (MD), central thalamic nucleus (CM), and ventromedial thalamic nucleus (VM) were found to have high percentages of D2→D2 connections, while the parafascicular thalamic nucleus (Pf) had a high percentage of D1→D1 connections; ***p < 0.001, **p < 0.01, *p < 0.05, two-way RM ANOVA followed by Tukey’s test; n = 5 (D1→D1), n = 4 (D2→D2), n = 4 (D1→D2) mice.

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Whole-Brain Mapping of Direct Inputs to Dopamine D1 and D2 Receptor-Expressing Medium Spiny Neurons in the Posterior Dorsomedial Striatum
Jiayi Lu, Yifeng Cheng, Xueyi Xie, Kayla Woodson, Jordan Bonifacio, Emily Disney, Britton Barbee, Xuehua Wang, Mariam Zaidi, Jun Wang
eNeuro 30 December 2020, 8 (1) ENEURO.0348-20.2020; DOI: 10.1523/ENEURO.0348-20.2020

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Whole-Brain Mapping of Direct Inputs to Dopamine D1 and D2 Receptor-Expressing Medium Spiny Neurons in the Posterior Dorsomedial Striatum
Jiayi Lu, Yifeng Cheng, Xueyi Xie, Kayla Woodson, Jordan Bonifacio, Emily Disney, Britton Barbee, Xuehua Wang, Mariam Zaidi, Jun Wang
eNeuro 30 December 2020, 8 (1) ENEURO.0348-20.2020; DOI: 10.1523/ENEURO.0348-20.2020
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Keywords

  • BNST
  • CeA
  • dopamine D1 and D2 receptors
  • posterior dorsomedial striatum
  • rabies virus-mediated retrograde monosynaptic tracing
  • whole-brain mapping

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