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Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice.

Matamales M, Bertran-Gonzalez J, Salomon L, Degos B, Deniau JM, Valjent E, Hervé D, Girault JA - PLoS ONE (2009)

Bottom Line: Retrograde labeling showed that all MSNs projecting to the SNr expressed D1R and very few D2R (<1%).In contrast, our results were compatible with the existence of some D1R-EGFP-expressing fibers giving off terminals in the LGP.Thus, our study shows that nuclear staining is a simple method for identifying MSNs and other striatal neurons.

View Article: PubMed Central - PubMed

Affiliation: Inserm UMR-S 839, Paris, France.

ABSTRACT
Precise identification of neuronal populations is a major challenge in neuroscience. In the striatum, more than 95% of neurons are GABAergic medium-sized spiny neurons (MSNs), which form two intermingled populations distinguished by their projections and protein content. Those expressing dopamine D(1)-receptors (D1Rs) project preferentially to the substantia nigra pars reticulata (SNr), whereas those expressing dopamine D(2)- receptors (D2Rs) project preferentially to the lateral part of the globus pallidus (LGP). The degree of segregation of these populations has been a continuous subject of debate, and the recent introduction of bacterial artificial chromosome (BAC) transgenic mice expressing fluorescent proteins driven by specific promoters was a major progress to facilitate striatal neuron identification. However, the fraction of MSNs labeled in these mice has been recently called into question, casting doubt on the generality of results obtained with such approaches. Here, we performed an in-depth quantitative analysis of striatal neurons in drd1a-EGFP and drd2-EGFP mice. We first quantified neuronal and non-neuronal populations in the striatum, based on nuclear staining with TO-PRO-3, and immunolabeling for NeuN, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein Mr approximately 32,000), and various markers for interneurons. TO-PRO-3 staining was sufficient to identify MSNs by their typical nuclear morphology and, with a good probability, interneuron populations. In drd1a-EGFP/drd2-EGFP double transgenic mice all MSNs expressed EGFP, which was driven in about half of them by drd1a promoter. Retrograde labeling showed that all MSNs projecting to the SNr expressed D1R and very few D2R (<1%). In contrast, our results were compatible with the existence of some D1R-EGFP-expressing fibers giving off terminals in the LGP. Thus, our study shows that nuclear staining is a simple method for identifying MSNs and other striatal neurons. It also unambiguously confirms the degree of segregation of MSNs in the mouse striatum and allows the full exploitation of results obtained with BAC-transgenic mice.

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All striatonigral projection neurons express EGFP under the control of drd1a promoter.(A) Retrograde labeling of striatonigral neurons in drd1a-EGFP mice after unilateral FluoroGold injection in the substantia nigra pars reticulata (SNr). FluoroGold immunoreactivity (red) in the SNr (scale bar: 200 µm; insets: 4× magnification) and the dorsal striatum (DStr) of drd1a-EGFP mice in the hemisphere ipsilateral (injected side) or controlateral (non-injected side) to the injection site. In the injected side FluoroGold-immunoreactive neurons always contained EGFP fluorescence (i.e. expressed D1R). No FluoroGold-positive neurons were observed in the non-injected side. Scale bars: 40 µm. (B) The same experiment was carried out in drd2-EGFP mice. EGFP fluorescence (1) was detected together with FluoroGold immunoreactivity (2) and DARPP-32 immunoreactivity (3), in the DStr and NAc core (Core) and shell (Shell). FluoroGold-immunoreactive neurons did not contain EGFP in all striatal regions (Merge 1+2). All EGFP and FluoroGold-positive neurons were DARPP-32-immunoreactive MSNs (Merge 1+2+3). Images are single confocal sections. Scale bar: 40 µm.
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pone-0004770-g006: All striatonigral projection neurons express EGFP under the control of drd1a promoter.(A) Retrograde labeling of striatonigral neurons in drd1a-EGFP mice after unilateral FluoroGold injection in the substantia nigra pars reticulata (SNr). FluoroGold immunoreactivity (red) in the SNr (scale bar: 200 µm; insets: 4× magnification) and the dorsal striatum (DStr) of drd1a-EGFP mice in the hemisphere ipsilateral (injected side) or controlateral (non-injected side) to the injection site. In the injected side FluoroGold-immunoreactive neurons always contained EGFP fluorescence (i.e. expressed D1R). No FluoroGold-positive neurons were observed in the non-injected side. Scale bars: 40 µm. (B) The same experiment was carried out in drd2-EGFP mice. EGFP fluorescence (1) was detected together with FluoroGold immunoreactivity (2) and DARPP-32 immunoreactivity (3), in the DStr and NAc core (Core) and shell (Shell). FluoroGold-immunoreactive neurons did not contain EGFP in all striatal regions (Merge 1+2). All EGFP and FluoroGold-positive neurons were DARPP-32-immunoreactive MSNs (Merge 1+2+3). Images are single confocal sections. Scale bar: 40 µm.

Mentions: Previous reports with BAC transgenic mice have shown that striatonigral neurons are essentially D1R-expressing neurons [12], [13], [19]. Because the present study lifted the uncertainty about the existence of non-EGFP-expressing MSNs in the BAC transgenic mice, we used these mice to reassess the nature of striatonigral neurons by retrograde labeling. FluoroGold, a retrograde tracer, was stereotactically injected in the SNr of drd1a-EGFP and drd2-EGFP mice. FluoroGold-immunoreactive neurons were detected in the striatum ipsilaterally to the injection side, but not on the contralateral side (Fig. 6A). In drd1a-EGFP mice, 100±0% of FluoroGold-positive neurons were labeled with EGFP, indicating that all striatonigral neurons express the D1R (298 neurons examined from two different mice, Fig. 6A). In drd2-EGFP mice, 99.3±0.6% of FluoroGold-immunoreactive neurons did not contain EGFP (566 neurons examined from 2 different mice, Fig. 6B). Thus only 4 out of 566 retrograde-labeled neurons (0.7%) expressed EGFP in drd2-EGFP neurons. Since in drd1a-EGFP mice FluoroGold was completely colocalized with EGFP, these results suggest that the few neurons which contained both EGFP and FluoroGold in drd2-EGFP mice expressed both D1 and D2 receptors. As expected, immunolabeling with DARPP-32 showed that both EGFP-neurons and FluoroGold-neurons were MSNs (Fig. 6B). Interestingly, detailed analysis of the LGP of drd1a-EGFP mice revealed a loose weave of intermingled fibers that could account for sparse terminals of striatonigral neurons targeting the pallidum (Fig. 7), in agreement with the basal fluorescent signal previously observed in the LGP of drd1a-EGFP mice [12], [13]. Such fibers might account for the few branched neurons projecting to both SNr and GP reported in rats [31], [32].


Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice.

Matamales M, Bertran-Gonzalez J, Salomon L, Degos B, Deniau JM, Valjent E, Hervé D, Girault JA - PLoS ONE (2009)

All striatonigral projection neurons express EGFP under the control of drd1a promoter.(A) Retrograde labeling of striatonigral neurons in drd1a-EGFP mice after unilateral FluoroGold injection in the substantia nigra pars reticulata (SNr). FluoroGold immunoreactivity (red) in the SNr (scale bar: 200 µm; insets: 4× magnification) and the dorsal striatum (DStr) of drd1a-EGFP mice in the hemisphere ipsilateral (injected side) or controlateral (non-injected side) to the injection site. In the injected side FluoroGold-immunoreactive neurons always contained EGFP fluorescence (i.e. expressed D1R). No FluoroGold-positive neurons were observed in the non-injected side. Scale bars: 40 µm. (B) The same experiment was carried out in drd2-EGFP mice. EGFP fluorescence (1) was detected together with FluoroGold immunoreactivity (2) and DARPP-32 immunoreactivity (3), in the DStr and NAc core (Core) and shell (Shell). FluoroGold-immunoreactive neurons did not contain EGFP in all striatal regions (Merge 1+2). All EGFP and FluoroGold-positive neurons were DARPP-32-immunoreactive MSNs (Merge 1+2+3). Images are single confocal sections. Scale bar: 40 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651623&req=5

pone-0004770-g006: All striatonigral projection neurons express EGFP under the control of drd1a promoter.(A) Retrograde labeling of striatonigral neurons in drd1a-EGFP mice after unilateral FluoroGold injection in the substantia nigra pars reticulata (SNr). FluoroGold immunoreactivity (red) in the SNr (scale bar: 200 µm; insets: 4× magnification) and the dorsal striatum (DStr) of drd1a-EGFP mice in the hemisphere ipsilateral (injected side) or controlateral (non-injected side) to the injection site. In the injected side FluoroGold-immunoreactive neurons always contained EGFP fluorescence (i.e. expressed D1R). No FluoroGold-positive neurons were observed in the non-injected side. Scale bars: 40 µm. (B) The same experiment was carried out in drd2-EGFP mice. EGFP fluorescence (1) was detected together with FluoroGold immunoreactivity (2) and DARPP-32 immunoreactivity (3), in the DStr and NAc core (Core) and shell (Shell). FluoroGold-immunoreactive neurons did not contain EGFP in all striatal regions (Merge 1+2). All EGFP and FluoroGold-positive neurons were DARPP-32-immunoreactive MSNs (Merge 1+2+3). Images are single confocal sections. Scale bar: 40 µm.
Mentions: Previous reports with BAC transgenic mice have shown that striatonigral neurons are essentially D1R-expressing neurons [12], [13], [19]. Because the present study lifted the uncertainty about the existence of non-EGFP-expressing MSNs in the BAC transgenic mice, we used these mice to reassess the nature of striatonigral neurons by retrograde labeling. FluoroGold, a retrograde tracer, was stereotactically injected in the SNr of drd1a-EGFP and drd2-EGFP mice. FluoroGold-immunoreactive neurons were detected in the striatum ipsilaterally to the injection side, but not on the contralateral side (Fig. 6A). In drd1a-EGFP mice, 100±0% of FluoroGold-positive neurons were labeled with EGFP, indicating that all striatonigral neurons express the D1R (298 neurons examined from two different mice, Fig. 6A). In drd2-EGFP mice, 99.3±0.6% of FluoroGold-immunoreactive neurons did not contain EGFP (566 neurons examined from 2 different mice, Fig. 6B). Thus only 4 out of 566 retrograde-labeled neurons (0.7%) expressed EGFP in drd2-EGFP neurons. Since in drd1a-EGFP mice FluoroGold was completely colocalized with EGFP, these results suggest that the few neurons which contained both EGFP and FluoroGold in drd2-EGFP mice expressed both D1 and D2 receptors. As expected, immunolabeling with DARPP-32 showed that both EGFP-neurons and FluoroGold-neurons were MSNs (Fig. 6B). Interestingly, detailed analysis of the LGP of drd1a-EGFP mice revealed a loose weave of intermingled fibers that could account for sparse terminals of striatonigral neurons targeting the pallidum (Fig. 7), in agreement with the basal fluorescent signal previously observed in the LGP of drd1a-EGFP mice [12], [13]. Such fibers might account for the few branched neurons projecting to both SNr and GP reported in rats [31], [32].

Bottom Line: Retrograde labeling showed that all MSNs projecting to the SNr expressed D1R and very few D2R (<1%).In contrast, our results were compatible with the existence of some D1R-EGFP-expressing fibers giving off terminals in the LGP.Thus, our study shows that nuclear staining is a simple method for identifying MSNs and other striatal neurons.

View Article: PubMed Central - PubMed

Affiliation: Inserm UMR-S 839, Paris, France.

ABSTRACT
Precise identification of neuronal populations is a major challenge in neuroscience. In the striatum, more than 95% of neurons are GABAergic medium-sized spiny neurons (MSNs), which form two intermingled populations distinguished by their projections and protein content. Those expressing dopamine D(1)-receptors (D1Rs) project preferentially to the substantia nigra pars reticulata (SNr), whereas those expressing dopamine D(2)- receptors (D2Rs) project preferentially to the lateral part of the globus pallidus (LGP). The degree of segregation of these populations has been a continuous subject of debate, and the recent introduction of bacterial artificial chromosome (BAC) transgenic mice expressing fluorescent proteins driven by specific promoters was a major progress to facilitate striatal neuron identification. However, the fraction of MSNs labeled in these mice has been recently called into question, casting doubt on the generality of results obtained with such approaches. Here, we performed an in-depth quantitative analysis of striatal neurons in drd1a-EGFP and drd2-EGFP mice. We first quantified neuronal and non-neuronal populations in the striatum, based on nuclear staining with TO-PRO-3, and immunolabeling for NeuN, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein Mr approximately 32,000), and various markers for interneurons. TO-PRO-3 staining was sufficient to identify MSNs by their typical nuclear morphology and, with a good probability, interneuron populations. In drd1a-EGFP/drd2-EGFP double transgenic mice all MSNs expressed EGFP, which was driven in about half of them by drd1a promoter. Retrograde labeling showed that all MSNs projecting to the SNr expressed D1R and very few D2R (<1%). In contrast, our results were compatible with the existence of some D1R-EGFP-expressing fibers giving off terminals in the LGP. Thus, our study shows that nuclear staining is a simple method for identifying MSNs and other striatal neurons. It also unambiguously confirms the degree of segregation of MSNs in the mouse striatum and allows the full exploitation of results obtained with BAC-transgenic mice.

Show MeSH
Related in: MedlinePlus