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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.


Targeting construct and single-cell RT-PCR analysis. a Illustration of the genomic DNA sequence showing the Vglut2 locus in a wildtype mouse (uppermost panel), followed by the mRNA generated from the wildtype (WT) floxed allele that has not seen TH-Cre activity (middle), and the mRNA of the conditional knockout allele generated after TH-Cre-activity has excised the DNA sequence between the LoxP (P) sites (lower panel). The Vglut2 primers for RT-PCR analysis on single cell material were designed around exons 4 and 6 to detect both the wildtype and knockout (KO) allele, the primer annealing sites for nested RT-PCR are indicated below the transcript as follows: first round in middle panel, second round in lower panel. b Representative example of a gel picture showing final RT-PCR products in single cell material (derived from GFP fluorescent, dissected VTA/SNc P1 conditional knockout (cKO) and control brains, resp.);Vglut2 (WT allele 506 bp, uppermost band; KO allele 372 bp, lowermost band), TH and DAT, all as indicated to the right of the picture
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Fig4: Targeting construct and single-cell RT-PCR analysis. a Illustration of the genomic DNA sequence showing the Vglut2 locus in a wildtype mouse (uppermost panel), followed by the mRNA generated from the wildtype (WT) floxed allele that has not seen TH-Cre activity (middle), and the mRNA of the conditional knockout allele generated after TH-Cre-activity has excised the DNA sequence between the LoxP (P) sites (lower panel). The Vglut2 primers for RT-PCR analysis on single cell material were designed around exons 4 and 6 to detect both the wildtype and knockout (KO) allele, the primer annealing sites for nested RT-PCR are indicated below the transcript as follows: first round in middle panel, second round in lower panel. b Representative example of a gel picture showing final RT-PCR products in single cell material (derived from GFP fluorescent, dissected VTA/SNc P1 conditional knockout (cKO) and control brains, resp.);Vglut2 (WT allele 506 bp, uppermost band; KO allele 372 bp, lowermost band), TH and DAT, all as indicated to the right of the picture

Mentions: To further analyse gene expression in TH-Cre-active cells, we turned to single-cell RT-PCR experiments in freshly dissociated GFP-positive cells derived from the dissected VTA/SNc area using Vglut2 primers designed to distinguish between the wildtype and knockout alleles (Fig. 4a). Sixty GFP-expressing cells were collected and prepared from P1 Ctrl-Cre-GFP and cKO-Cre-GFP mice, respectively (Fig. 8 for illustration). As expected, none of the green cells picked from the Ctrl-Cre-GFP mice expressed the Vglut2 knockout allele, instead all 12 cells expressing Vglut2 showed the wildtype allele (representative examples shown in Fig. 4b). In contrast, Vglut2 expression in the cKO-Cre-GFP was represented almost exclusively by the knockout allele; in 28 out of 29 (97 %) Vglut2-expressing cells recombination had occurred leading to a deletion of Vglut2, thus verifying the conditional targeting of Vglut2 (representative examples shown in Fig. 4b). In addition, primers for endogenous TH and DAT expression were used to further characterize the GFP-expressing cells. Out of 60 cells, 48 cells (80 %) were found to be expressing endogenous TH. Of these 48 cells, 13 cells (22 % of all cells) were coexpressing TH and DAT but did not contain Vglut2 (presumably classical DA neurons), 5 cells (8 % of all cells) were coexpressing TH, Vglut2 and DAT (possibly “TH–Vglut2 Class 1” neurons) and 18 cells (30 % of all cells) were found to be coexpressing TH and Vglut2 (possibly “TH–Vglut2 Class 2” neurons). Further, six GFP-expressing cells (10 %) were also identified which expressed Vglut2 but not TH or DAT (possibly “Vglut2-only” neurons). Representative examples of RT-PCR gene products are shown in Fig. 4b.Fig. 4


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)

Targeting construct and single-cell RT-PCR analysis. a Illustration of the genomic DNA sequence showing the Vglut2 locus in a wildtype mouse (uppermost panel), followed by the mRNA generated from the wildtype (WT) floxed allele that has not seen TH-Cre activity (middle), and the mRNA of the conditional knockout allele generated after TH-Cre-activity has excised the DNA sequence between the LoxP (P) sites (lower panel). The Vglut2 primers for RT-PCR analysis on single cell material were designed around exons 4 and 6 to detect both the wildtype and knockout (KO) allele, the primer annealing sites for nested RT-PCR are indicated below the transcript as follows: first round in middle panel, second round in lower panel. b Representative example of a gel picture showing final RT-PCR products in single cell material (derived from GFP fluorescent, dissected VTA/SNc P1 conditional knockout (cKO) and control brains, resp.);Vglut2 (WT allele 506 bp, uppermost band; KO allele 372 bp, lowermost band), TH and DAT, all as indicated to the right of the picture
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: Targeting construct and single-cell RT-PCR analysis. a Illustration of the genomic DNA sequence showing the Vglut2 locus in a wildtype mouse (uppermost panel), followed by the mRNA generated from the wildtype (WT) floxed allele that has not seen TH-Cre activity (middle), and the mRNA of the conditional knockout allele generated after TH-Cre-activity has excised the DNA sequence between the LoxP (P) sites (lower panel). The Vglut2 primers for RT-PCR analysis on single cell material were designed around exons 4 and 6 to detect both the wildtype and knockout (KO) allele, the primer annealing sites for nested RT-PCR are indicated below the transcript as follows: first round in middle panel, second round in lower panel. b Representative example of a gel picture showing final RT-PCR products in single cell material (derived from GFP fluorescent, dissected VTA/SNc P1 conditional knockout (cKO) and control brains, resp.);Vglut2 (WT allele 506 bp, uppermost band; KO allele 372 bp, lowermost band), TH and DAT, all as indicated to the right of the picture
Mentions: To further analyse gene expression in TH-Cre-active cells, we turned to single-cell RT-PCR experiments in freshly dissociated GFP-positive cells derived from the dissected VTA/SNc area using Vglut2 primers designed to distinguish between the wildtype and knockout alleles (Fig. 4a). Sixty GFP-expressing cells were collected and prepared from P1 Ctrl-Cre-GFP and cKO-Cre-GFP mice, respectively (Fig. 8 for illustration). As expected, none of the green cells picked from the Ctrl-Cre-GFP mice expressed the Vglut2 knockout allele, instead all 12 cells expressing Vglut2 showed the wildtype allele (representative examples shown in Fig. 4b). In contrast, Vglut2 expression in the cKO-Cre-GFP was represented almost exclusively by the knockout allele; in 28 out of 29 (97 %) Vglut2-expressing cells recombination had occurred leading to a deletion of Vglut2, thus verifying the conditional targeting of Vglut2 (representative examples shown in Fig. 4b). In addition, primers for endogenous TH and DAT expression were used to further characterize the GFP-expressing cells. Out of 60 cells, 48 cells (80 %) were found to be expressing endogenous TH. Of these 48 cells, 13 cells (22 % of all cells) were coexpressing TH and DAT but did not contain Vglut2 (presumably classical DA neurons), 5 cells (8 % of all cells) were coexpressing TH, Vglut2 and DAT (possibly “TH–Vglut2 Class 1” neurons) and 18 cells (30 % of all cells) were found to be coexpressing TH and Vglut2 (possibly “TH–Vglut2 Class 2” neurons). Further, six GFP-expressing cells (10 %) were also identified which expressed Vglut2 but not TH or DAT (possibly “Vglut2-only” neurons). Representative examples of RT-PCR gene products are shown in Fig. 4b.Fig. 4

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.