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Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity.

Nordenankar K, Smith-Anttila CJ, Schweizer N, Viereckel T, Birgner C, Mejia-Toiber J, Morales M, Leao RN, Wallén-Mackenzie Å - Brain Struct Funct (2014)

Bottom Line: The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout.Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus.In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area.

View Article: PubMed Central - PubMed

Affiliation: Unit of Functional Neurobiology and Unit of Developmental Genetics, Biomedical Center, Department of Neuroscience, Uppsala University, Box 593, S-751 24, Uppsala, Sweden.

ABSTRACT
Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations ("TH-Vglut2 Class1") also expressed the dopamine transporter (DAT) gene while one did not ("TH-Vglut2 Class2"), and the remaining population did not express TH at all ("Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area.

No MeSH data available.


Related in: MedlinePlus

TH-Cre transgene analysis in the adult mouse brain via the tau-mGFP double Cre-reporter. Cell nuclei marked with DAPI (blue) ( a). Projections from the ventral tegmental area and substantia nigra pars compacta (VTA/SNc) to the entire striatal area (left) via the median forebrain bundle (MFB) visualized by immunofluorescence for the green fluorescent protein (GFP), close-up of GFP fibres in the hippocampus (right) (a). Nuclear β-galactosidase (β-gal; red) shows the position of cells in which TH-Cre activity has enabled β-gal expression; endogenous TH protein expression is indicated by TH immunoreactivity (green); co-localization (β-gal (red) nuclei in centre of TH (green) cytoplasm) is mainly seen in medial (m) and lateral (l) VTA, less so in the medially located rostral linear nucleus (RLi) and Interfascicular nucleus (IF) where many cells show β-gal (red) only (b, c). Triple β-gal (red)/TH (blue)/GFP (green) immunofluorescence in RLi shows some β-gal/GFP-positive TH-Cre-reporting cells that immunopositive for TH (hash) and some that do not have TH (single asterisk) (d left and close-up to the right)
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Fig2: TH-Cre transgene analysis in the adult mouse brain via the tau-mGFP double Cre-reporter. Cell nuclei marked with DAPI (blue) ( a). Projections from the ventral tegmental area and substantia nigra pars compacta (VTA/SNc) to the entire striatal area (left) via the median forebrain bundle (MFB) visualized by immunofluorescence for the green fluorescent protein (GFP), close-up of GFP fibres in the hippocampus (right) (a). Nuclear β-galactosidase (β-gal; red) shows the position of cells in which TH-Cre activity has enabled β-gal expression; endogenous TH protein expression is indicated by TH immunoreactivity (green); co-localization (β-gal (red) nuclei in centre of TH (green) cytoplasm) is mainly seen in medial (m) and lateral (l) VTA, less so in the medially located rostral linear nucleus (RLi) and Interfascicular nucleus (IF) where many cells show β-gal (red) only (b, c). Triple β-gal (red)/TH (blue)/GFP (green) immunofluorescence in RLi shows some β-gal/GFP-positive TH-Cre-reporting cells that immunopositive for TH (hash) and some that do not have TH (single asterisk) (d left and close-up to the right)

Mentions: The tau-mGFP floxed Cre-reporter (Hippenmeyer et al. 2005) was used to enable validation of Cre activity in the previously described TH-Cre knock-in mouse (Lindeberg et al. 2004) by IHC for β-galactosidase (β-gal) and green fluorescent protein (GFP) in the same sections to detect TH-Cre-expressing cell nuclei (β-gal) and projections(GFP) throughout the adult brain. By analysis of Ctrl-Cre-GFP mice (see Materials and Methods for description), we could confirm the previous characterization of the TH-Cre mice (Lindeberg et al. 2004) which, using the loxP-STOP-loxP-LacZ (Tsien et al. 1996), showed TH-Cre-activity in all catecholaminergic cell groups, including the noradrenergic neurons of the A1, A2 and locus coeruleus groups, the adrenergic neurons of the C1, C2 and C3 groups in the medulla, in addition to the DA cell groups in the ventral midbrain (A8, A9, A10) and hypothalamus (A11-13) (data not shown). We could also confirm previous observations of TH-Cre activity in the dorsal root ganglia (Lagerström et al. 2010; Lindeberg et al. 2004), but in contrast to the previous characterization (Lindeberg et al. 2004), we did not detect TH-Cre activity, assessed by β-gal-IHC, in the cortex or hippocampus (data not shown). However, as described below, the hippocampus did contain GFP-immunoreactive fibres, confirming innervation from TH-Cre-expressing neurons located elsewhere (shown in Fig. 2b and also in Fig. 7a). We then focused our analyses on the ventral midbrain. By analysing GFP IHC in sagittal sections, we detected median forebrain bundle projections reaching from the VTA/SNc area to the dorsal and ventral striatum (Fig. 2a). Combined β-gal (“reporting” TH-Cre transgene activity) and TH (endogenous TH protein) IHC on coronal sections visualized the two markers in the A10 area (2C-D), with high-level magnification showing β-gal in the nuclei of TH-immunoreactive neurons (Fig. 2c–e). In addition, in the IF and RLi areas, where we (Fig. 3) and others (Hnasko et al. 2012; Li et al. 2013) detected Vglut2-expressing cells that did not express endogenous TH, several β-gal-positive cell nuclei were detected that lacked TH-immunoreactivity (Fig. 2b–d). Although somewhat surprising, these findings are in accordance with the original characterization of the TH-Cre transgene (Lindeberg et al. 2004)which found that due to early and transient expression from the TH promoter during development, the Cre transgene will be active early on in cells not expressing stable endogenous TH later in life (see Fig. 8 for illustration of the TH promoter activity). This seemingly ectopic Cre activity will give rise to early deletion of any floxed alleles present and which will remain gene targeted throughout their life, thereby providing a molecular mechanism for how adult A10 cells can be immunopositive for β-gal but not TH. Based on this finding, we hypothesized that mice expressing both TH-Cre and the floxed Vglut2 allele, i.e. Vglut2f/f;TH-Cre+ mice, should have Vglut2 gene targeted in both “TH–Vglut2” cells and in “Vglut2-only” cells.Fig. 2


Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity.

Nordenankar K, Smith-Anttila CJ, Schweizer N, Viereckel T, Birgner C, Mejia-Toiber J, Morales M, Leao RN, Wallén-Mackenzie Å - Brain Struct Funct (2014)

TH-Cre transgene analysis in the adult mouse brain via the tau-mGFP double Cre-reporter. Cell nuclei marked with DAPI (blue) ( a). Projections from the ventral tegmental area and substantia nigra pars compacta (VTA/SNc) to the entire striatal area (left) via the median forebrain bundle (MFB) visualized by immunofluorescence for the green fluorescent protein (GFP), close-up of GFP fibres in the hippocampus (right) (a). Nuclear β-galactosidase (β-gal; red) shows the position of cells in which TH-Cre activity has enabled β-gal expression; endogenous TH protein expression is indicated by TH immunoreactivity (green); co-localization (β-gal (red) nuclei in centre of TH (green) cytoplasm) is mainly seen in medial (m) and lateral (l) VTA, less so in the medially located rostral linear nucleus (RLi) and Interfascicular nucleus (IF) where many cells show β-gal (red) only (b, c). Triple β-gal (red)/TH (blue)/GFP (green) immunofluorescence in RLi shows some β-gal/GFP-positive TH-Cre-reporting cells that immunopositive for TH (hash) and some that do not have TH (single asterisk) (d left and close-up to the right)
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Fig2: TH-Cre transgene analysis in the adult mouse brain via the tau-mGFP double Cre-reporter. Cell nuclei marked with DAPI (blue) ( a). Projections from the ventral tegmental area and substantia nigra pars compacta (VTA/SNc) to the entire striatal area (left) via the median forebrain bundle (MFB) visualized by immunofluorescence for the green fluorescent protein (GFP), close-up of GFP fibres in the hippocampus (right) (a). Nuclear β-galactosidase (β-gal; red) shows the position of cells in which TH-Cre activity has enabled β-gal expression; endogenous TH protein expression is indicated by TH immunoreactivity (green); co-localization (β-gal (red) nuclei in centre of TH (green) cytoplasm) is mainly seen in medial (m) and lateral (l) VTA, less so in the medially located rostral linear nucleus (RLi) and Interfascicular nucleus (IF) where many cells show β-gal (red) only (b, c). Triple β-gal (red)/TH (blue)/GFP (green) immunofluorescence in RLi shows some β-gal/GFP-positive TH-Cre-reporting cells that immunopositive for TH (hash) and some that do not have TH (single asterisk) (d left and close-up to the right)
Mentions: The tau-mGFP floxed Cre-reporter (Hippenmeyer et al. 2005) was used to enable validation of Cre activity in the previously described TH-Cre knock-in mouse (Lindeberg et al. 2004) by IHC for β-galactosidase (β-gal) and green fluorescent protein (GFP) in the same sections to detect TH-Cre-expressing cell nuclei (β-gal) and projections(GFP) throughout the adult brain. By analysis of Ctrl-Cre-GFP mice (see Materials and Methods for description), we could confirm the previous characterization of the TH-Cre mice (Lindeberg et al. 2004) which, using the loxP-STOP-loxP-LacZ (Tsien et al. 1996), showed TH-Cre-activity in all catecholaminergic cell groups, including the noradrenergic neurons of the A1, A2 and locus coeruleus groups, the adrenergic neurons of the C1, C2 and C3 groups in the medulla, in addition to the DA cell groups in the ventral midbrain (A8, A9, A10) and hypothalamus (A11-13) (data not shown). We could also confirm previous observations of TH-Cre activity in the dorsal root ganglia (Lagerström et al. 2010; Lindeberg et al. 2004), but in contrast to the previous characterization (Lindeberg et al. 2004), we did not detect TH-Cre activity, assessed by β-gal-IHC, in the cortex or hippocampus (data not shown). However, as described below, the hippocampus did contain GFP-immunoreactive fibres, confirming innervation from TH-Cre-expressing neurons located elsewhere (shown in Fig. 2b and also in Fig. 7a). We then focused our analyses on the ventral midbrain. By analysing GFP IHC in sagittal sections, we detected median forebrain bundle projections reaching from the VTA/SNc area to the dorsal and ventral striatum (Fig. 2a). Combined β-gal (“reporting” TH-Cre transgene activity) and TH (endogenous TH protein) IHC on coronal sections visualized the two markers in the A10 area (2C-D), with high-level magnification showing β-gal in the nuclei of TH-immunoreactive neurons (Fig. 2c–e). In addition, in the IF and RLi areas, where we (Fig. 3) and others (Hnasko et al. 2012; Li et al. 2013) detected Vglut2-expressing cells that did not express endogenous TH, several β-gal-positive cell nuclei were detected that lacked TH-immunoreactivity (Fig. 2b–d). Although somewhat surprising, these findings are in accordance with the original characterization of the TH-Cre transgene (Lindeberg et al. 2004)which found that due to early and transient expression from the TH promoter during development, the Cre transgene will be active early on in cells not expressing stable endogenous TH later in life (see Fig. 8 for illustration of the TH promoter activity). This seemingly ectopic Cre activity will give rise to early deletion of any floxed alleles present and which will remain gene targeted throughout their life, thereby providing a molecular mechanism for how adult A10 cells can be immunopositive for β-gal but not TH. Based on this finding, we hypothesized that mice expressing both TH-Cre and the floxed Vglut2 allele, i.e. Vglut2f/f;TH-Cre+ mice, should have Vglut2 gene targeted in both “TH–Vglut2” cells and in “Vglut2-only” cells.Fig. 2

Bottom Line: The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout.Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus.In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area.

View Article: PubMed Central - PubMed

Affiliation: Unit of Functional Neurobiology and Unit of Developmental Genetics, Biomedical Center, Department of Neuroscience, Uppsala University, Box 593, S-751 24, Uppsala, Sweden.

ABSTRACT
Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations ("TH-Vglut2 Class1") also expressed the dopamine transporter (DAT) gene while one did not ("TH-Vglut2 Class2"), and the remaining population did not express TH at all ("Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area.

No MeSH data available.


Related in: MedlinePlus