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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: 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").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

Illustration of the analysis of subpopulations in the ventral midbrain. a Schematic drawing of an E12 mouse embryo (seen in sagittal view) in which the ventral midbrain (VM) area, where the dopamine (DA) neurons develop, is highlighted in red. In our analysis (Fig. 1), we looked at the colocalization of Vglut2 mRNA with TH immunoreactivity and found cells expressing both and either of these genes, i.e. TH only, Vglut2 only and TH–Vglut2 together. At this stage, DAT is not expressed, and was therefore not analysed. b Representation of the two known phases of TH promoter activity, a transient phase which is not specific to cells that will develop into catecholaminergic neurons, and a stable phase which gives rise to the stable TH expression in catecholaminergic neurons. c Schematic drawing of the single cell selection we performed at P1 (Fig. 3) to analyse gene expression in TH-Cre cells by RT-PCR. Coronal midbrain slices of Ctrl-Cre-GFP and cKO-Cre-GFP mice, in which TH-Cre activity has led to expression of the Cre-double reporter TaumGFP thus enabling localization of these cells by GFP in fluorescent microscope, were prepared on vibratome and the entire GFP-positive ventral area [encompassing the A10 area (the rostral linear nucleus of the raphé nuclei (RLi); paranigral nucleus (PN); interfascicular nucleus (IF); medial parabrachial pigmented nucleus (PbP)] together with the lateral VTA and substantia nigra pars compacta (SNc), but not substantia nigra pars compacta (SNr) was dissected and triturated into single cell solution from which single GFP-positive cells were picked. d The subsequent RT-PCR analysis using Vglut2 primers (designed to recognize mRNA both from the wildtype and knockout in the same reaction), DAT primers and TH primers led us to conclude that TH-Cre is active in several populations of cells in the GFP-positive dissected area of the ventral midbrain. Based on ours (Fig. 2) and previous studies that have shown that, in the adult, Vglut2 is most highly expressed in the A10 nuclei and not in the lateral VTA and SNc, and our observation of TH-Cre activity (detected as β-galacosidase in Fig. 3) suggest that the Vglut2 mRNA we detect is derived from the A10 nuclei. We cannot rule out that the Vglut2 we observe is derived from the lateral structures (shadowed in grey) that were also dissected, although this seems less likely. In summary, we found single GFP-positive cells expressing the combination of TH, Vglut2 and DAT as illustrated by the green dots in the figure: TH only; TH and DAT; TH and DAT and Vglut2; TH and Vglut2 but no DAT; Vglut2 only. Cells showing TH and DAT and Vglut2 have previously been termed “TH–Vglut2 Class 1” and cells showing TH and Vglut2 but no DAT have been termed “TH–Vglut2 Class 2”, a terminology we adapted and refer to in the current study. All literature referred to is listed in text
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Fig8: Illustration of the analysis of subpopulations in the ventral midbrain. a Schematic drawing of an E12 mouse embryo (seen in sagittal view) in which the ventral midbrain (VM) area, where the dopamine (DA) neurons develop, is highlighted in red. In our analysis (Fig. 1), we looked at the colocalization of Vglut2 mRNA with TH immunoreactivity and found cells expressing both and either of these genes, i.e. TH only, Vglut2 only and TH–Vglut2 together. At this stage, DAT is not expressed, and was therefore not analysed. b Representation of the two known phases of TH promoter activity, a transient phase which is not specific to cells that will develop into catecholaminergic neurons, and a stable phase which gives rise to the stable TH expression in catecholaminergic neurons. c Schematic drawing of the single cell selection we performed at P1 (Fig. 3) to analyse gene expression in TH-Cre cells by RT-PCR. Coronal midbrain slices of Ctrl-Cre-GFP and cKO-Cre-GFP mice, in which TH-Cre activity has led to expression of the Cre-double reporter TaumGFP thus enabling localization of these cells by GFP in fluorescent microscope, were prepared on vibratome and the entire GFP-positive ventral area [encompassing the A10 area (the rostral linear nucleus of the raphé nuclei (RLi); paranigral nucleus (PN); interfascicular nucleus (IF); medial parabrachial pigmented nucleus (PbP)] together with the lateral VTA and substantia nigra pars compacta (SNc), but not substantia nigra pars compacta (SNr) was dissected and triturated into single cell solution from which single GFP-positive cells were picked. d The subsequent RT-PCR analysis using Vglut2 primers (designed to recognize mRNA both from the wildtype and knockout in the same reaction), DAT primers and TH primers led us to conclude that TH-Cre is active in several populations of cells in the GFP-positive dissected area of the ventral midbrain. Based on ours (Fig. 2) and previous studies that have shown that, in the adult, Vglut2 is most highly expressed in the A10 nuclei and not in the lateral VTA and SNc, and our observation of TH-Cre activity (detected as β-galacosidase in Fig. 3) suggest that the Vglut2 mRNA we detect is derived from the A10 nuclei. We cannot rule out that the Vglut2 we observe is derived from the lateral structures (shadowed in grey) that were also dissected, although this seems less likely. In summary, we found single GFP-positive cells expressing the combination of TH, Vglut2 and DAT as illustrated by the green dots in the figure: TH only; TH and DAT; TH and DAT and Vglut2; TH and Vglut2 but no DAT; Vglut2 only. Cells showing TH and DAT and Vglut2 have previously been termed “TH–Vglut2 Class 1” and cells showing TH and Vglut2 but no DAT have been termed “TH–Vglut2 Class 2”, a terminology we adapted and refer to in the current study. All literature referred to is listed in text

Mentions: Brains were obtained from P1 cKO-Cre-GFP and Ctrl-Cre-GFPmice following decapitation. A 1 mm thick coronal slice, which contained the mesencephalon was prepared under fluorescent microscope. Excess tissue was removed until only the substantia nigra pars compacta (SNc) and A10 areas, visualized by TH-Cre-driven GFP fluorescence, remained. The tissue was collected in ice-cold dissociation solution (90 mM Na2SO4, 30 mM K2SO4, 5.8 mM MgCl2, 0.25 mM CaCl2, 10 mM HEPES, 20 mM glucose, and 0.001 % phenol red, pH 7.4) then digested with papain for 20 min at 37 °C with agitation. The tissue was then triturated by several passages through glass pipettes of decreasing diameter to obtain a cell suspension (see inset in Fig. 8 for illustration of this procedure). The cells were then centrifuged through a differential gradient to eliminate dead cells and debris. Cells were plated on poly-l-lysine-coated coverslips and left to adhere for 30 min at 37 °C. The coverslips were then washed with Krebs–Ringer buffer (KRB) (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose, 6 mM sucrose, pH 7.35) to eliminate non-attached cells and in KRB during single cell collection. GFP-expressing cells were randomly collected to avoid a selection bias towards cells that express high levels of GFP. All cells were collected individually using autoclaved borosilicate patch pipettes under RNAse-free conditions; each cell was collected by applying light negative pressure to the pipette, no intracellular pipette solution was used. The content of each pipette was transferred into individual pre-chilled tubes containing a freshly prepared solution of 20 U of RNase inhibitor and 8.3 mM DTT, samples were frozen immediately on dry ice and stored at −80 °C until use. The samples were thawed on ice and the RNA converted to cDNA by reverse transcription for 1 h using 0.5 mM dNTPs mix, 1.25 μM random primers, 40 U of RNase inhibitor, 100 U of M-MLV RT (Invitrogen), 50 mM Tris-HCl, 75 mM KCl and 3 mM MgCl2, pH 8.3. The RT enzyme was denatured and the cDNAs stored at −80 °C until use. A first round of PCR was performed using 1.5 mM MgCl2, 10 pmol of each primer, 1.0 U of platinum Taq-DNA polymerase (Invitrogen), 20 mM Tris–HCl and 50 mM KCl pH 8.4. Thermal cycles consisted of an initial denaturation step of 94 °C for 2 min, followed by 35 cycles of 94 °C for 50 s, 55 °C for 45 s and 72 °C for 45 s. A second nested PCR was then performed as mentioned above using 10 % of the first PCR reaction as template. All PCR products were resolved on 2.5 % agarose gels. Primers were designed based upon sequences deposited in the GenBank database (www.ncbi.nlm.nih.gov/nucleotide). The Vglut2 primers were designed around exons 4, 5 and 6 to detect both the wildtype and the knockout allele. TH and DAT mRNA expressions were also investigated. The oligonucleotides used were Vglut2: first round sense 5´-gccgctacatcatagccatc-3´ and antisense 5´-gctctctccaatgctctcctc-3´, nested sense 5´-acatggtcaacaacagcactatc-3´ and antisense 5´-ataagacaccagaagccagaaca-3´; TH: first round sense 5´-gttctcaacctgctcttctcctt-3´ and antisense 5´-ggtagcaatttcctcctttgtgt-3´, nested sense 5´-gtacaaaaccctcctcactgtctc-3´ and antisense 5´-cttgtattggaaggcaatctctg-3´; DAT: first round sense 5´-ttcactgtcatcctcatctctttc-3´ and antisense 5´-gaagctcgtcagggagttaatg-3´, nested sense 5´-gtattttgagcgtggtgtgct-3´ and antisense 5´-gatccacacagatgcctcac-3´.


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)

Illustration of the analysis of subpopulations in the ventral midbrain. a Schematic drawing of an E12 mouse embryo (seen in sagittal view) in which the ventral midbrain (VM) area, where the dopamine (DA) neurons develop, is highlighted in red. In our analysis (Fig. 1), we looked at the colocalization of Vglut2 mRNA with TH immunoreactivity and found cells expressing both and either of these genes, i.e. TH only, Vglut2 only and TH–Vglut2 together. At this stage, DAT is not expressed, and was therefore not analysed. b Representation of the two known phases of TH promoter activity, a transient phase which is not specific to cells that will develop into catecholaminergic neurons, and a stable phase which gives rise to the stable TH expression in catecholaminergic neurons. c Schematic drawing of the single cell selection we performed at P1 (Fig. 3) to analyse gene expression in TH-Cre cells by RT-PCR. Coronal midbrain slices of Ctrl-Cre-GFP and cKO-Cre-GFP mice, in which TH-Cre activity has led to expression of the Cre-double reporter TaumGFP thus enabling localization of these cells by GFP in fluorescent microscope, were prepared on vibratome and the entire GFP-positive ventral area [encompassing the A10 area (the rostral linear nucleus of the raphé nuclei (RLi); paranigral nucleus (PN); interfascicular nucleus (IF); medial parabrachial pigmented nucleus (PbP)] together with the lateral VTA and substantia nigra pars compacta (SNc), but not substantia nigra pars compacta (SNr) was dissected and triturated into single cell solution from which single GFP-positive cells were picked. d The subsequent RT-PCR analysis using Vglut2 primers (designed to recognize mRNA both from the wildtype and knockout in the same reaction), DAT primers and TH primers led us to conclude that TH-Cre is active in several populations of cells in the GFP-positive dissected area of the ventral midbrain. Based on ours (Fig. 2) and previous studies that have shown that, in the adult, Vglut2 is most highly expressed in the A10 nuclei and not in the lateral VTA and SNc, and our observation of TH-Cre activity (detected as β-galacosidase in Fig. 3) suggest that the Vglut2 mRNA we detect is derived from the A10 nuclei. We cannot rule out that the Vglut2 we observe is derived from the lateral structures (shadowed in grey) that were also dissected, although this seems less likely. In summary, we found single GFP-positive cells expressing the combination of TH, Vglut2 and DAT as illustrated by the green dots in the figure: TH only; TH and DAT; TH and DAT and Vglut2; TH and Vglut2 but no DAT; Vglut2 only. Cells showing TH and DAT and Vglut2 have previously been termed “TH–Vglut2 Class 1” and cells showing TH and Vglut2 but no DAT have been termed “TH–Vglut2 Class 2”, a terminology we adapted and refer to in the current study. All literature referred to is listed in text
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Related In: Results  -  Collection

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Fig8: Illustration of the analysis of subpopulations in the ventral midbrain. a Schematic drawing of an E12 mouse embryo (seen in sagittal view) in which the ventral midbrain (VM) area, where the dopamine (DA) neurons develop, is highlighted in red. In our analysis (Fig. 1), we looked at the colocalization of Vglut2 mRNA with TH immunoreactivity and found cells expressing both and either of these genes, i.e. TH only, Vglut2 only and TH–Vglut2 together. At this stage, DAT is not expressed, and was therefore not analysed. b Representation of the two known phases of TH promoter activity, a transient phase which is not specific to cells that will develop into catecholaminergic neurons, and a stable phase which gives rise to the stable TH expression in catecholaminergic neurons. c Schematic drawing of the single cell selection we performed at P1 (Fig. 3) to analyse gene expression in TH-Cre cells by RT-PCR. Coronal midbrain slices of Ctrl-Cre-GFP and cKO-Cre-GFP mice, in which TH-Cre activity has led to expression of the Cre-double reporter TaumGFP thus enabling localization of these cells by GFP in fluorescent microscope, were prepared on vibratome and the entire GFP-positive ventral area [encompassing the A10 area (the rostral linear nucleus of the raphé nuclei (RLi); paranigral nucleus (PN); interfascicular nucleus (IF); medial parabrachial pigmented nucleus (PbP)] together with the lateral VTA and substantia nigra pars compacta (SNc), but not substantia nigra pars compacta (SNr) was dissected and triturated into single cell solution from which single GFP-positive cells were picked. d The subsequent RT-PCR analysis using Vglut2 primers (designed to recognize mRNA both from the wildtype and knockout in the same reaction), DAT primers and TH primers led us to conclude that TH-Cre is active in several populations of cells in the GFP-positive dissected area of the ventral midbrain. Based on ours (Fig. 2) and previous studies that have shown that, in the adult, Vglut2 is most highly expressed in the A10 nuclei and not in the lateral VTA and SNc, and our observation of TH-Cre activity (detected as β-galacosidase in Fig. 3) suggest that the Vglut2 mRNA we detect is derived from the A10 nuclei. We cannot rule out that the Vglut2 we observe is derived from the lateral structures (shadowed in grey) that were also dissected, although this seems less likely. In summary, we found single GFP-positive cells expressing the combination of TH, Vglut2 and DAT as illustrated by the green dots in the figure: TH only; TH and DAT; TH and DAT and Vglut2; TH and Vglut2 but no DAT; Vglut2 only. Cells showing TH and DAT and Vglut2 have previously been termed “TH–Vglut2 Class 1” and cells showing TH and Vglut2 but no DAT have been termed “TH–Vglut2 Class 2”, a terminology we adapted and refer to in the current study. All literature referred to is listed in text
Mentions: Brains were obtained from P1 cKO-Cre-GFP and Ctrl-Cre-GFPmice following decapitation. A 1 mm thick coronal slice, which contained the mesencephalon was prepared under fluorescent microscope. Excess tissue was removed until only the substantia nigra pars compacta (SNc) and A10 areas, visualized by TH-Cre-driven GFP fluorescence, remained. The tissue was collected in ice-cold dissociation solution (90 mM Na2SO4, 30 mM K2SO4, 5.8 mM MgCl2, 0.25 mM CaCl2, 10 mM HEPES, 20 mM glucose, and 0.001 % phenol red, pH 7.4) then digested with papain for 20 min at 37 °C with agitation. The tissue was then triturated by several passages through glass pipettes of decreasing diameter to obtain a cell suspension (see inset in Fig. 8 for illustration of this procedure). The cells were then centrifuged through a differential gradient to eliminate dead cells and debris. Cells were plated on poly-l-lysine-coated coverslips and left to adhere for 30 min at 37 °C. The coverslips were then washed with Krebs–Ringer buffer (KRB) (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose, 6 mM sucrose, pH 7.35) to eliminate non-attached cells and in KRB during single cell collection. GFP-expressing cells were randomly collected to avoid a selection bias towards cells that express high levels of GFP. All cells were collected individually using autoclaved borosilicate patch pipettes under RNAse-free conditions; each cell was collected by applying light negative pressure to the pipette, no intracellular pipette solution was used. The content of each pipette was transferred into individual pre-chilled tubes containing a freshly prepared solution of 20 U of RNase inhibitor and 8.3 mM DTT, samples were frozen immediately on dry ice and stored at −80 °C until use. The samples were thawed on ice and the RNA converted to cDNA by reverse transcription for 1 h using 0.5 mM dNTPs mix, 1.25 μM random primers, 40 U of RNase inhibitor, 100 U of M-MLV RT (Invitrogen), 50 mM Tris-HCl, 75 mM KCl and 3 mM MgCl2, pH 8.3. The RT enzyme was denatured and the cDNAs stored at −80 °C until use. A first round of PCR was performed using 1.5 mM MgCl2, 10 pmol of each primer, 1.0 U of platinum Taq-DNA polymerase (Invitrogen), 20 mM Tris–HCl and 50 mM KCl pH 8.4. Thermal cycles consisted of an initial denaturation step of 94 °C for 2 min, followed by 35 cycles of 94 °C for 50 s, 55 °C for 45 s and 72 °C for 45 s. A second nested PCR was then performed as mentioned above using 10 % of the first PCR reaction as template. All PCR products were resolved on 2.5 % agarose gels. Primers were designed based upon sequences deposited in the GenBank database (www.ncbi.nlm.nih.gov/nucleotide). The Vglut2 primers were designed around exons 4, 5 and 6 to detect both the wildtype and the knockout allele. TH and DAT mRNA expressions were also investigated. The oligonucleotides used were Vglut2: first round sense 5´-gccgctacatcatagccatc-3´ and antisense 5´-gctctctccaatgctctcctc-3´, nested sense 5´-acatggtcaacaacagcactatc-3´ and antisense 5´-ataagacaccagaagccagaaca-3´; TH: first round sense 5´-gttctcaacctgctcttctcctt-3´ and antisense 5´-ggtagcaatttcctcctttgtgt-3´, nested sense 5´-gtacaaaaccctcctcactgtctc-3´ and antisense 5´-cttgtattggaaggcaatctctg-3´; DAT: first round sense 5´-ttcactgtcatcctcatctctttc-3´ and antisense 5´-gaagctcgtcagggagttaatg-3´, nested sense 5´-gtattttgagcgtggtgtgct-3´ and antisense 5´-gatccacacagatgcctcac-3´.

Bottom Line: 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").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