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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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Proportion of DARPP-32-positive cells in the striatum.Low-magnification confocal section of a mouse striatal slice doubly stained with NeuN antibodies (red) and TO-PRO-3 (blue) (A), or with DARPP-32 antibodies (green) and TO-PRO-3 (blue) (B). Scale bars: 25 µm. (C) Bar graphs representing the proportion of cells identified as neurons by NeuN immunoreactivity (NeuN+), of DARPP-32-immunoreactive neurons (DARPP-32+) and of nuclei classified into A or B–E categories. The total number of cells (neuronal and non-neuronal) was determined by nuclear staining with TO-PRO-3 (TP3) in mouse dorsal striatum (DStr) and nucleus accumbens core (Core) and shell (Shell). The small percentage of cells classified in B–E categories, corresponds to striatal interneurons. Data are means±SEM; n = 3 mice; 2908 (NeuN count), 2091 (DARPP-32 count) and 1517 (nuclei categories count) cells counted.
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pone-0004770-g004: Proportion of DARPP-32-positive cells in the striatum.Low-magnification confocal section of a mouse striatal slice doubly stained with NeuN antibodies (red) and TO-PRO-3 (blue) (A), or with DARPP-32 antibodies (green) and TO-PRO-3 (blue) (B). Scale bars: 25 µm. (C) Bar graphs representing the proportion of cells identified as neurons by NeuN immunoreactivity (NeuN+), of DARPP-32-immunoreactive neurons (DARPP-32+) and of nuclei classified into A or B–E categories. The total number of cells (neuronal and non-neuronal) was determined by nuclear staining with TO-PRO-3 (TP3) in mouse dorsal striatum (DStr) and nucleus accumbens core (Core) and shell (Shell). The small percentage of cells classified in B–E categories, corresponds to striatal interneurons. Data are means±SEM; n = 3 mice; 2908 (NeuN count), 2091 (DARPP-32 count) and 1517 (nuclei categories count) cells counted.

Mentions: We used the tools validated above to accurately estimate the proportion of MSNs in the striatum, taking into account both DARPP-32 immunoreactivity and nuclear morphology. We determined the total number of cells in striatal sections by double staining with the pan-neuronal marker NeuN and TO-PRO-3 (Fig. 4A), or with DARPP-32 and TO-PRO-3 (Fig. 4B). We then counted for each double staining the number of cells identified by their TO-PRO-3-stained nuclei that were NeuN-positive or DARPP-32-positive (Fig. 4C). In parallel we counted the number of neurons in categories A–E defined in Table 1. The percentage of NeuN positive cells was 70±0.4% in the dorsal striatum, 80±1.4% in the core of the nucleus accumbens (NAc), and 75±3.1% in the shell (means±SEM, 3 mice) (Fig. 4C). DARPP-32-positive neurons accounted for 68±2.3% of the cells in dorsal striatum, 78±1.4% in NAc core and 73±2% in NAc shell (Fig. 4C) (means±SEM, 3 mice). Thus, as expected, DARPP-32-positive neurons formed the largest population of striatal neurons (i.e. about 97% of the neurons in the 3 striatal regions). Similar results were obtained when classification of all nuclei was done following the five categories described previously (Fig. 4C). Note that the percentages of cells in category A was consistently slightly lower than those obtained with DARPP-32 immunolabeling because identification on the basis of nuclear morphology required a section in which the nucleus was well visible and its features identifiable, which was not the case in all DARPP-32-positive cells. However, it should be pointed out that when the nucleus of DARPP-32-positive cells was clearly visible it always had the typical morphology described in Fig. 3A. All nuclei classified in category A were DARPP-32-immunolabeled (100±0%) and virtually no DARPP-32-positive neuron was misclassified into the other categories (1±1%). Nuclei classified in categories B–E constituted approximately 5% of the total. These results combined with our previous observation that DARPP-32-positive neurons are not labeled with markers for striatal interneurons [13] demonstrate that DARPP-32 antibodies label all MSNs and only MSNs in the striatum.


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)

Proportion of DARPP-32-positive cells in the striatum.Low-magnification confocal section of a mouse striatal slice doubly stained with NeuN antibodies (red) and TO-PRO-3 (blue) (A), or with DARPP-32 antibodies (green) and TO-PRO-3 (blue) (B). Scale bars: 25 µm. (C) Bar graphs representing the proportion of cells identified as neurons by NeuN immunoreactivity (NeuN+), of DARPP-32-immunoreactive neurons (DARPP-32+) and of nuclei classified into A or B–E categories. The total number of cells (neuronal and non-neuronal) was determined by nuclear staining with TO-PRO-3 (TP3) in mouse dorsal striatum (DStr) and nucleus accumbens core (Core) and shell (Shell). The small percentage of cells classified in B–E categories, corresponds to striatal interneurons. Data are means±SEM; n = 3 mice; 2908 (NeuN count), 2091 (DARPP-32 count) and 1517 (nuclei categories count) cells counted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651623&req=5

pone-0004770-g004: Proportion of DARPP-32-positive cells in the striatum.Low-magnification confocal section of a mouse striatal slice doubly stained with NeuN antibodies (red) and TO-PRO-3 (blue) (A), or with DARPP-32 antibodies (green) and TO-PRO-3 (blue) (B). Scale bars: 25 µm. (C) Bar graphs representing the proportion of cells identified as neurons by NeuN immunoreactivity (NeuN+), of DARPP-32-immunoreactive neurons (DARPP-32+) and of nuclei classified into A or B–E categories. The total number of cells (neuronal and non-neuronal) was determined by nuclear staining with TO-PRO-3 (TP3) in mouse dorsal striatum (DStr) and nucleus accumbens core (Core) and shell (Shell). The small percentage of cells classified in B–E categories, corresponds to striatal interneurons. Data are means±SEM; n = 3 mice; 2908 (NeuN count), 2091 (DARPP-32 count) and 1517 (nuclei categories count) cells counted.
Mentions: We used the tools validated above to accurately estimate the proportion of MSNs in the striatum, taking into account both DARPP-32 immunoreactivity and nuclear morphology. We determined the total number of cells in striatal sections by double staining with the pan-neuronal marker NeuN and TO-PRO-3 (Fig. 4A), or with DARPP-32 and TO-PRO-3 (Fig. 4B). We then counted for each double staining the number of cells identified by their TO-PRO-3-stained nuclei that were NeuN-positive or DARPP-32-positive (Fig. 4C). In parallel we counted the number of neurons in categories A–E defined in Table 1. The percentage of NeuN positive cells was 70±0.4% in the dorsal striatum, 80±1.4% in the core of the nucleus accumbens (NAc), and 75±3.1% in the shell (means±SEM, 3 mice) (Fig. 4C). DARPP-32-positive neurons accounted for 68±2.3% of the cells in dorsal striatum, 78±1.4% in NAc core and 73±2% in NAc shell (Fig. 4C) (means±SEM, 3 mice). Thus, as expected, DARPP-32-positive neurons formed the largest population of striatal neurons (i.e. about 97% of the neurons in the 3 striatal regions). Similar results were obtained when classification of all nuclei was done following the five categories described previously (Fig. 4C). Note that the percentages of cells in category A was consistently slightly lower than those obtained with DARPP-32 immunolabeling because identification on the basis of nuclear morphology required a section in which the nucleus was well visible and its features identifiable, which was not the case in all DARPP-32-positive cells. However, it should be pointed out that when the nucleus of DARPP-32-positive cells was clearly visible it always had the typical morphology described in Fig. 3A. All nuclei classified in category A were DARPP-32-immunolabeled (100±0%) and virtually no DARPP-32-positive neuron was misclassified into the other categories (1±1%). Nuclei classified in categories B–E constituted approximately 5% of the total. These results combined with our previous observation that DARPP-32-positive neurons are not labeled with markers for striatal interneurons [13] demonstrate that DARPP-32 antibodies label all MSNs and only MSNs in the striatum.

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