Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity
- 1.7k Downloads
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.
KeywordsReward Oscillations Development Midbrain Mouse genetics
Caudal linear nucleus
Dorsal root ganglia
Elevated plus maze
Forced swim test
Green fluorescent protein
Local field potential
Later ventral tegmental area
Medial forebrain bundle
Rostral linear nucleus
Reference memory error
Substantia nigra pars compacta
Vesicular glutamate transporter 2
Vesicular inhibitory amino acid transporter
Ventral tegmental area
Working memory error
The A10 area of the ventral midbrain is the home of the classical dopamine (DA) neurons that are important for motivation, reward, learning and memory via substantial projections into limbic and cognitive regions. A10 consists of the ventral tegmental area (VTA) and three midline nuclei, known as the interfascicular (IF), rostral linear (RLi) and caudal linear (CLi) nuclei, respectively (Fields et al. 2007; Ikemoto 2007). Recent studies have shown that the medial VTA together with IF and RLi, which collectively often are referred to simply as the VTA, are more heterogeneous in terms of cell populations than previously thought (Li et al. 2013). For example, three distinct neuronal populations expressing the vesicular glutamate transporter 2 (Vglut2, aka Slc17A6) gene, which confers a glutamatergic phenotype (Fremeau et al. 2004), have been identified within the VTA of the adult rat and mouse. Of these three VTA Vglut2-expressing (VTA-VGLUT2) populations, one appears to consist of purely glutamatergic neurons (“Glu-only” or “Vglut2-only”) that show similar electrophysiological properties and projections as the neighbouring DA neurons (Hnasko et al. 2012; Kawano et al. 2006; Yamaguchi et al. 2007). The other two populations both express tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis, along with Vglut2, but differ with regards to their expression of the dopamine transporter (DAT) (Li et al. 2013). Thus, the neurons in one VTA-VGLUT2 population coexpress TH and DAT and have been named “TH–Vglut2 Class 1” neurons, while the other population contains neurons that do not express DAT and are referred to as “TH–Vglut2 Class 2” neurons (Li et al. 2013). Both of these TH–Vglut2 populations presumably represent DA neurons that corelease glutamate [reviewed in (El Mestikawy et al. 2011; Hnasko and Edwards 2012)]. In addition to the unique property of expression in DA neurons, Vglut2 is distinct from its sister molecules Vglut1 and Vglut3 in that Vglut2 is the only isoform which can be broadly detected already at midgestation, including within the developing VTA (Birgner et al. 2010). The functional role of Vglut2 expression in DA neurons has been investigated by different laboratories using the Cre-LoxP-based conditional knockout technique (Wallén-Mackenzie et al. 2010) targeting DA neurons via DAT promoter-driven expression of Cre recombinase. Briefly summarized, these studies demonstrated that DAT-Cre-mediated deletion of Vglut2 expression left motivation and memory parameters intact, while causing altered responses to sweet food and psychostimulants, thereby revealing a role for the glutamate-DA cophenotype in certain aspects of reward processing (Alsiö et al. 2011; Birgner et al. 2010; Fortin et al. 2012; Hnasko et al. 2010). Spatially, DAT-Cre is to date the most appropriate Cre-driver for targeting gene expression in DA neurons, but temporally, Vglut2 expression, detected in the VTA at embryonic day (E) 11, is more tuned with TH expression (E11; Zetterström et al. (1997)) which precedes that of DAT (E14; Fauchey et al. (2000)) by several days. To explore the possibility of targeting Vglut2 in DA neurons already from midgestation, we therefore turned to a previously validated TH-ires-Cre knock-in mouse (Lindeberg et al. 2004) known to recapitulate endogenous TH expression, and we could indeed verify this expected Cre activity. Somewhat surprisingly, ectopic Cre activity was also detected in the “Vglut2-only” neurons of the VTA. Thus, based on the promiscuity of the TH promoter, we hypothesized that we had a genetic tool at hand which we could use for targeting neurons within all three VTA-VGLUT2 populations, i.e. “Vglut2-only”, “TH–Vglut2 Class 1” and “TH–Vglut2 Class 2” neurons. Upon verification, we used behavioural, electrochemical and electrophysiological techniques to analyse this new conditional Vglut2 knockout mouse.
Materials and methods
All mice used in the study were housed in the animal facility at the BMC, Uppsala University, in accordance with the Swedish regulation guidelines (Animal Welfare Act SFS 1998:56) and European Union legislation (Convention ETS123 and Directive 2010/63/EU). Ethical approval was obtained from the Uppsala Animal Ethical Committee. The animals were housed by sex in standard Makrolon cages (59 × 38 × 20 cm) with aspen wood bedding (Scanbur AB Sollentuna, Sweden) and a wooden house. The temperature was kept at 21–22 °C with a humidity of 45–55 %. A 12 h light/12 h dark cycle was used, with lights on at 07.00 h. The animals had ad libitum access to food (R36, Labfor, Lactamin, Vadstena, Sweden) and water.
Generation of transgenic mice
The Vglut2 f/f;TH-Cre mouse line was produced using the breeding procedure established for conditional knockout mice to ensure identical genetic background (Crusio 2004) by breeding the Th-IRES-Cre (here abbreviated as TH-Cre) knock-in mouse line (on a C57BL/6 J genetic background) (Lindeberg et al. 2004) to the Vglut2 f/f mouse line (on a C57BL/6 J-SV129 genetic background) (Wallén-Mackenzie et al. 2009) thereby generating cKO (Vglut2 f/f;TH-Cre+) and control (Vglut2 f/f;TH-Cre−) mice as littermates which allows for behavioural phenotyping and comparison between genotype groups (Wolfer et al. 2002). The generation of Vglut2 f/f;TH-Cre mouse line was described previously and it was demonstrated that a subset of Vglut2 f/f;TH-Cre cKO mice have an itch phenotype at a late adult stage (Lagerstrom et al. 2010). None of the analyses in the current study included adult mice showing an itch phenotype. The Vglut2 f/f mouse line was bred with the Tau mGFP reporter mouse line (Hippenmeyer et al. 2005) to allow histological and single-cell RT-PCR analyses with selectivity for the TH-Cre-expressing cells, thus producing the Vglut2 f/f;Tau-mGFP mouse, which was subsequently crossed with the TH-Cre mice to produce Vglut2 f/f;TH-Cre;Tau-mGFP (cKO-Cre-GFP) and Vglut2 f/+;TH-Cre;Tau-mGFP littermate controls (Ctrl-Cre-GFP). The Tau mGFP Cre-reporter mouse allows visualization of Cre-expressing cell nuclei by detection of β-galactosidase (β-gal) protein and projections of the corresponding cells by green fluorescent protein (GFP). Littermate control mice were used in all experiments to ensure that any aberrant phenotypes were specifically dependent on the deletion of Vglut2 expression in TH-Cre-expressing neurons. Further, the observer was blind to the genotype of the mice until the final analysis stage.
In situ hybridization and immunohistochemistry
Animals were mated overnight for production of cKO-Cre-GFP and Ctrl-Cre-GFP offspring, and females were checked for a vaginal plug the following morning. Embryos were collected at embryonic (E) day 12 and 14. In the morning of E19, pups were born and staged as P0. For dissection of embryos, pregnant females were sacrificed by cervical dislocation and embryos were removed. For collection of adult brains, mice were sacrificed by cervical dislocation and brains removed. Amniotic sac (from embryos) and tails (from pups) were collected for genotyping according to protocols previously described (Wallén-Mackenzie et al. 2006). The tissue was fixed in zinc formalin (Richard-Allan Scientific, Kalamazoo, MI) for 18–24 h at room temperature before dehydration and paraffin infusion (Tissue Tek vacuum infiltration processor; Miles Scientific, Elkhart, IN). Sections (7 μm thick) were cut on a Microm microtome, attached to Superfrost slides (Menzel-Gläser, Braunschweig, Germany) and stored at 4 °C until usage. Slides were then deparaffinized in X-tra solve (MediteHistotechnic, Burgdorf, Germany) and rehydrated in ethanol/water before subsequent treatments.
In situ hybridization histochemistry
For paraffin in situ hybridization histochemistry, rehydrated paraffin sections were fixed for 10 min in 4 % formaldehyde, washed in phosphate-buffered saline (PBS), and treated with proteinase K (Sigma; 27 μg/ml diluted in 10 mM Tris–HCl/1 mM EDTA, pH 8.0) for 5 min. After refixation and washes in PBS, the slides were acetylated for 10 min in a mixture of 1.3 % triethanolamine (Sigma), 0.2 % acetic anhydride (Fluka, Neu-Ulm, Germany), and 0.06 % HCl diluted in water. Slides were then incubated for 30 min in PBS containing 1 % Triton X-100 (Sigma). After subsequent washes in PBS, slides were prehybridized for 2–5 h in hybridization solution without probe [50 % formamide (Fluka), 5× saline-sodium citrate (SSC), 5× Denhardt’s, 250 μg/ml yeast transfer RNA (Sigma) and 500 μg/ml sheared salmon sperm DNA (Ambion, Austin, TX, USA) diluted in water]. The probe for Vglut2 (covering nucleotides 1,616–2,203) was diluted to 0.1–1 μg/ml in hybridization solution and heated to 80 °C. Sections were then hybridized with 100 μl of hybridization solution for 16 h at 70 °C. The next day, slides were dipped in prewarmed 5× SSC, transferred to 0.2× SSC, and incubated for 2 h at 70 °C. After one wash in 0.2× SSC at room temperature and one wash in B1 solution (0.1 M Tris–HCl, pH 7.5, and 0.15 M NaCl), sections were immuno-blocked with 10 % foetal calf serum in B1, and then incubated overnight at 4 °C with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Roche, Mannheim, Germany) diluted 1:5,000 in B1 containing 1 % foetal calf serum. The following day, slides were washed in B1, equilibrated in B3 (0.1 M Tris–HCl, pH 9.5, 0.1 M NaCl, 50 mM MgCl2), and colour developed in a 10 % polyvinyl alcohol (Sigma) solution also containing 100 mM Tris–HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2, 0.17 % nitroblue tetrazolium (Roche), 0.17 % 5-bromo-4-chloro-3-indolyl phosphate (Roche), and 1 mM levamisole (Sigma). Staining was sufficient after 6–24 h, whereupon slides were washed in PBS and incubated overnight with primary mouse TH (Chemicon) antibody and processed as described below (omitting the boiling procedure).
For immunofluorescence histochemistry, rehydrated paraffin sections were boiled (this step was omitted in experiments of combined in situ hybridization and immunofluorescence) for 10 min in 0.1 M citric acid (VWR International, Leicestershire, UK), pH 6.0, left to cool for 20–30 min, washed in PBS, and incubated with primary mouse TH (Chemicon), rabbit β-gal (ICN/Cappel), chicken GFP (Abcam), chicken and guinea pig VGLUT2 [own production based on peptide sequences described in (Hioki et al. 2003)] via Innovagen, Lund, Sweden), rat Nestin (Dev Studies Hybridoma Bank), rabbit vesicular inhibitory amino acid transporter (VIAAT; gift from Prof Bruno Gasnier (McIntire et al. 1997; Sagne et al. 1997), mouse alpha-Internexin (Chemicon), mouse β-III-Tubulin (TUJ1; Chemicon), rabbit Synapsin (Chemicon), respectively, in PBS with 0.3 % Triton X-100 at room temperature overnight. The following day, slides were washed in PBS and incubated with Alexa fluorescent secondary antibodies (Invitrogen, San Diego, CA) diluted 1:200 in PBS with 0.3 % Triton X-100 and 10 % goat serum for 2 h at room temperature. Slides were then washed in PBS, incubated with 1 μg/ml 4′,6′-diamidino-2-phenylindole (DAPI) (Sigma), washed again, and mounted. Images were captured on a Zeiss LSM 510 Meta confocal microscope and analysed using Volocity software (Improvision).
Combination of Vglut2 in situ hybridization and TH immunolabeling
Coronal free-floating sections (10, 12 or 16 µm in thickness) were processed as described previously (Wang and Morales 2008). Sections were incubated for 10 min in phosphate buffer (PB) containing 0.5 % Triton X-100, rinsed 2 × 5 min with PB, treated with 0.2 N HCl for 10 min, rinsed 2 × 5 min with PB and then acetylated in 0.25 % acetic anhydride in 0.1 M triethanolamine, pH 8.0 for 10 min. Sections were rinsed 2 × 5 min with PB, post-fixed with 4 % paraformaldehyde (PFA) for 10 min. Prior to hybridization and after a final rinse with PB, the free-floating sections were incubated in hybridization buffer (50 % formamide; 10 % dextran sulfate; 5× Denhardt’s solution; 0.62 M NaCl; 50 mM DTT; 10 mM EDTA; 20 mM PIPES, pH 6.8; 0.2 % SDS; 250 µg/ml salmon sperm DNA; 250 µg/ml tRNA) for 2 h at 55 °C. Sections collected on glass slides were dehydrated through a series of graded ethanol (50, 70 and 95 %, 5 min for each concentration). Sections were hybridized for 16 h at 55 °C in hybridization buffer containing [35S]- and [33P]-labelled single-stranded antisense or sense Vglut2 (nucleotides 317–2,357, Accession # NM_053427) probes at 107 cpm/ml. Sections were treated with 4 µg/ml RNase A at 37 °C for 1 h, washed with 1× SSC, 50 % formamide at 55 °C for 1 h, and with 0.1× SSC at 68 °C for 1 h. After the last SSC wash, sections were rinsed with PB and incubated for 1 h in PBS supplemented with 4 % bovine serum albumin and 0.3 % Triton X-100. This was followed by overnight incubation at 4 °C with an anti-TH mouse monoclonal antibody (1:500, MAB 318, Millipore, Billerica, MA) for which specificity has been documented (Tagliaferro and Morales 2008). After rinsing 3 × 10 min in PB, sections were processed with an ABC kit (Vector Laboratories, Burlingame, CA). The material was incubated for 1 h at RT in a 1:200 dilution of the biotinylated secondary antibody, rinsed with PB, and incubated with avidin-biotinylated horseradish peroxidase for 1 h. Sections were rinsed and the peroxidase reaction was then developed with 0.05 % 3, 3-diaminobenzidine-4 HCl (DAB) and 0.03 % hydrogen peroxide. Free-floating sections were mounted on coated slides. Slides were dipped in Ilford K.5 nuclear tract emulsion (Polysciences, Inc., Warrington; 1:1 dilution in double distilled water) and exposed in the dark at 4 °C for 4 weeks prior to development. Slides processed for fluorescent and bright-field histochemistry were analysed using an Olympus (Tokyo, Japan) microscope with an Optigrid system (Thales, Fairport, NY) and by confocal microscopy using the Zeiss (Oberkochen, Germany) LSM 510 META system. Images were captured using Volocity software (Improvision, Lexington, MA) and captured images were auto-levelled using Adobe Photoshop software. For cell counting within the IF and RLi areas, an observer blind to the genotype of the mice (1 cKO and 1 control) counted TH-expressing, Vglut2-expressing and TH/Vglut2-expressing cells in 11 cKO and 9 control sections.
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´.
Spontaneous and amphetamine-induced locomotor activity
To assess spontaneous and amphetamine-induced activity, mice were placed in automated activity chambers, so-called Locoboxes, consisting of a plastic cage (55 × 55 × 22 cm) inside a ventilated and illuminated (10 lux) cabinet (Locobox, KungsbackaMät- ochReglerteknik AB) in which activity was recorded as the following parameters: corner time, horizontal locomotion, vertical locomotion (rearing) and peripheral activity. After 30 min of baseline recording, mice received an intraperitoneal injection of saline and their locomotive activity was recorded for a further 90 min. To assess their drug-induced behaviour, 2–3 days after the saline injection, the same procedure was followed, but the mice were instead given an intraperitoneal injection of 1.5 mg/kg amphetamine. Data analysis was performed using the GraphPad Prism software (GraphPad Software Inc., La Jolla, USA).
The radial arm maze
The radial arm maze is a hippocampus-dependent task used to record spatial working memory (WM), in which the ability of the mouse to remember the location of food-baited versus unbaited arms is measured (Meck et al. 1984). Spatial memory performance was examined in 24 cKO mice and littermate controls, all males using an eight-armed radial maze (see Fig. 5 for illustration of the radial maze and the parameters scored). The maze, elevated 45 cm above the floor, consisted of eight open arms (60 cm long and 12.5 cm wide, surrounded by inclining walls at a height of 13 cm at the centre and 3 cm at the end of the maze arms) radiating from a central compartment (30 cm in diameter). A podium (10 × 4 cm) with a recessed food plate (diameter 3 cm) was fixed at 1.5 cm from the end of each of the maze arms. Three days prior to the beginning of the experiment, the mice were schedule-fed for 6 h/day, which was reduced to 2 h/day at the start of the behavioural studies. Four of the eight arms were baited with the preferred food, R6-38, consisting of a high content of theobroma cacao. For the acquisition period, the animals were placed individually in the centre of the maze once each day for 5 days. The animals were allowed to perform for 10 min on the first trial day and thereafter the animals were allowed to remain in the apparatus until all reinforcements were obtained or until 10 min had elapsed, whichever occurred first. The same four arms were baited with a small piece of reinforcement food pellet each day. Nineteen days after the last acquisition session a retention test was performed. The same arms were baited and the mice again the same procedure was followed. The mice were manually scored for performance during the trial time. For scoring, an entry half way into an arm was defined as an arm entry. For each trial, a reference memory error (RME) was defined as a visit into an unbaited or incorrect arm. A working memory error (WME) was defined as a re-entry into an arm in which the reward was already obtained during the session. The total number of entries into each arm and the percentage of correct responses were also scored. Results were analysed using StatView 5.0 for Windows. A 2 × 2 × 6 (genotype × sex × trial days) two-way repeated-measures ANOVA was used to assess RME, WME, total number of arm entries and the percentage of correct responses obtained during trial period. Test day was analysed by Student’s t test.
High-pressure liquid chromatography (HPLC) with electrochemical detection
Brains were obtained from cKO and littermate control mice (7 Ctrl, 7cKO) following euthanasia by cervical dislocation. The brains were put in a 1 mm coronal mouse brain matrix (Ted Pella Inc, Redding, CA, USA) kept on ice and sliced. The olfactory bulb, a combination of the substantia nigra and the ventral tegmental area (VTA/SNc), hypothalamus, nucleus accumbens (NAcc), caudate putamen (CaPu), prefrontal cortex (PFC), amygdala and hippocampus were excised from the slices, weighed and stored at −80 °C. The tissue was subsequently homogenized by sonification in 0.1 M perchloric acid solution (50 mg (1.6 mM) glutathione; 1.5 g (149 mM) 70 % PCA; 100 ml H2O). The homogenates were centrifuged at 4 °C for 15 min at 12,000×g and the supernatant recovered for analysis of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) content by HPLC. 40–50 µl of the supernatant was injected onto the HPLC column with the current set to 50 nA for all samples. A mobile phase (containing 55 mM sodium acetate, 0.01 mM EDTA, 1.16 mM 1-octanesulfonic acid sodium salt, 10 % methanol, pH 4) was used to separate the analytes. Chromatograms were captured using the Azure program (Kromatek, Essex, England) and the pmol values of the peaks were calculated from a standard curve for each DA, DOPAC and HVA. All statistical analyses of the data were performed using GraphPad Prism 5.0 software. Outliers were identified using the Grubbs test and excluded from the analysis. The concentration of each metabolite was compared between the cKO and littermate control mice using the Mann–Whitney U test.
In vitro local field potential (LFP) recordings from hippocampus slices were performed as previously described (Leao et al. 2009). Following decapitation under isoflurane anaesthesia, brains of P18-P25 cKO and littermate control mice were removed from the skull and placed in ice-cold high-sucrose artificial cerebrospinal fluid (ACSF) (in mM: KCl, 2.49; NaH2PO4, 1.43; NaHCO3, 26; glucose, 10; sucrose, 252; CaCl2, 1; MgCl2, 4). A vibratome (VT1000, Leica Microsystems) was used to obtain horizontal hippocampal slices that were moved to a submerged recording chamber containing ACSF (in mM: NaCl, 124; KCl, 3.5; NaH2PO4, 1.25; MgCl2, 1.5; CaCl2, 1.5; NaHCO3, 30; glucose, 10), constantly bubbled with 95 % O2 and 5 % CO2 and kept at 35 °C for 1 h then maintained at room temperature. For LFP recordings, slices were transferred to an interface-type chamber and kept at 35 °C (Zelano et al. 2013). A recording glass pipette filled with ACSF was placed in the stratum radiatum of CA3. LFP signals were amplified 100× using custom-made amplifier (John Curtin School of Medical Research, Australian National University), low-pass filtered at 3 kHz and digitized at 10 kHz by a National Instruments DAQ card. Data were analysed using Matlab (Mathoworks).
Vglut2 expression in TH and non-TH-expressing cells in the embryonic VTA area
TH-Cre activity detected in “TH-only”, “TH–Vglut2” and “Vglut2-only” VTA neurons
Vglut2 mRNA in the adult VTA area verified in TH- and non-TH-expressing cells
Previous studies have identified highest ventral midbrain Vglut2 expression in DA neurons of the medial A10 area, including the IF and RLi (see Fig. 3a for illustration) of the adult mouse and rat, intermingled with Vglut2-only expressing cells (Hnasko et al. 2012; Li et al. 2013; Yamaguchi et al. 2007). Based on these studies, we addressed Vglut2 expression in the ventral midbrain of the adult mouse of C57/BL6-Sv129 mixed genetic background and could confirm these previous reports (Hnasko et al. 2012; Li et al. 2013; Yamaguchi et al. 2007). Vglut2 in situ hybridization combined with IHC for TH showed a mixture of cells expressing both Vglut2 and TH (“TH–Vglut2”), cells expressing Vglut2 but not TH (“Vglut2-only”), and cells expressing TH but not Vglut2 (“TH-only”, presumably classical DA neurons) in the adult A10 area (Fig. 3b–d). We next counted the number of TH-expressing (Mean/SEM IF: Ctrl 33.6/8.6; cKO 34.3/10.0, RLi: Ctrl 10.9/4.8; cKO 5.1;1.2), Vglut2-expressing (Mean/SEM IF: Ctrl 8.3/3.4; cKO 4.1/1.1, RLi: Ctrl 29.0/8.6; cKO 16.5/3.3) and TH/Vglut2-expressing (Mean/SEM IF: Ctrl 0.3/0.2; cKO 0.3/0.1, RLi: Ctrl 1.8/0.5; cKO 2/1.4) cells, respectively, in the IF and RLi areas of control (9 sections) and cKO (11 sections) mice in the Vglut2 f/f;TH-Cre mouse line, but due to loss of material failed to reach a statistically relevant evaluation.
The TH-Cre transgene mediates deletion in “TH–Vglut2” and “Vglut2-only” neurons
These results show that in cells expressing the TH-Cre transgene in the VTA/SNc area, as identified by green fluorescence, Vglut2 expression is found in “Vglut2-only” cells and in both populations of cells expressing TH, i.e. those expressing Vglut2 together with both TH and DAT, and those expressing Vglut2 together with TH only (See Fig. 8 for illustration). Vglut2 was identified as gene targeted (knocked out) within all three populations of VTA-VGLUT2 neurons. Furthermore, the results indicate that recombination has occurred in nearly 100 % of all picked GFP-positive neurons of the mesencephalon expressing Vglut2. This level of recombination is in accordance with our previous characterization showing gene targeting in 74 % of the Vglut2-expressing cells of the dorsal root ganglia (DRG) in the same mouse line (Lagerström et al. 2010). In comparison, 82 % of DAT-Cre-expressing cells which also expressed Vglut2 showed targeting of the Vglut2 allele in the previously characterized Vglut2 f/f;DAT-Cre mouse line (Birgner et al. 2010).
Behavioural analysis reveals a normal amphetamine-induced activational response but decreased spatial memory
The eight-armed radial maze (illustration Fig. 5c) was utilized to determine whether there was any impairment in hippocampus-dependent spatial memory (Meck et al. 1984) performance in the cKO mice. The overall performance of the mice in the maze task was determined by the percentage of correct responses. Both the cKO and the littermate control mice increased their efficiency throughout the trials [F(1,100) = 12.558, p < 0.0001]. However, cKO mice took significantly longer to acquire the task in comparison to the littermate controls [F(1,20) = 8.369, p = 0.009] (Fig. 5d). There was no difference in the retention (18 days post-acquisition) of the learned task between the two genotypes (Fig. 5d). Reference memory error (RME) and working memory error (WME) were also investigated in the task (Fig. 5e, f). There was a significant overall difference in spatial processing throughout the trial days for both RME [F(5,100) = 12,234, p < 0.0001] and WME [F(5,75) = 3.645, p = 0.0053]. cKO mice made significantly more RMEs in comparison to the littermate control mice across the trial days [F(1,20) = 5.605, p = 0.0281] (Fig. 5e), whereas WMEs were not significantly different in the two groups [F(1,15) = 3.504, p = 0.0809] (Fig. 5f). No differences were seen between the two genotypes in either the RME or WME on day 23 (Fig. 5e, f).
Taken together, this data show that Vglut2 f/f;TH-Cre cKO mice lack alterations in depression-like and anxiety-like behaviour as measured by the FST and the EPM paradigm, respectively. These behavioural phenotypes mimic those of the Vglut2 f/f;DAT-Cre cKO mice characterized previously (Birgner et al. 2010), as does the finding of normal levels of total activity, detected both in the activity chambers prior to injection of psychostimulant, and in the EPM. In contrast, Vglut2 f/f;TH-Cre cKO mice lack the altered response to the psychostimulant amphetamine, which is evident in the Vglut2 f/f;DAT-Cre cKO mice, but show an impairment in the acquisition of the radial maze task and performed more spatial RMEs than control mice. Spatial memory functions are mediated by the hippocampus and were previously found normal in the Vglut2 f/f;DAT-Cre cKO mice. Thus, the targeting of Vglut2 expression by TH-Cre does not produce the identical phenotype as corresponding targeting by DAT-Cre, a finding likely reflected by the herein identified fact that TH-Cre and DAT-Cre differ quite substantially in their targeting of floxed Vglut2 cells.
HPLC analysis reveals altered DA levels in the hippocampus
The observations of reduced DA tissue levels in the hippocampus support our behavioural observation of a possible alteration in the hippocampus due to the lack of Vglut2 expression in TH-Cre active cells.
Hippocampus slices from cKO mice show increased kainate-induced excitability
These profound differences in CA3 responses to kainate challenge indicate an impairment of cKO mice in the ability to generate stable network oscillations.
The present study focused on investigating the function of Vglut2 expression in neurons that either transiently or stably also express TH, the rate-limiting enzyme for catecholamine synthesis. We found that the targeted deletion of Vglut2 in TH-expressing neurons resulted in a memory formation-deficit observed as an increased time required to acquire hippocampus-dependent spatial memory and elevated amount of errors produced in the radial arm maze, a finding which could possibly be associated with the observations of decreased DA levels and altered firing pattern in CA3 neurons of the hippocampus.
A deeper understanding of the observed complex hippocampal phenotype will require additional approaches, but will be discussed briefly here. Taken together, three independent results point towards a hippocampus-related phenotype in the TH-Cre-mediated cKO mice; the slower acquisition and the accentuated error rate in the radial arm maze, a spatial memory test designed to assay hippocampus function (Meck et al. 1984); the significantly lower DA tissue levels observed by electrochemical detection in homogenates prepared from the hippocampus (while no other region containing either DA cell bodies or projection terminals showed similar alterations); and the increased sensitivity to the ionotropic glutamate receptor agonist kainate, leading to seizure-like activity in slice preparations obtained from cKO brains. In recent studies, it was shown in rats that severing of the signalling from the VTA to the hippocampus via the local administration of lidocaine (Mahmoodi et al. 2011) or baclofen (Martig and Mizumori 2011), resulted in a decreased performance in memory tests. Neither study showed motivational or movement disruptions, but an increase in errors and an elevated number of trials required to reach the acquisition criterion in memory tasks (Mahmoodi et al. 2011; Martig and Mizumori 2011). It could therefore be proposed that the memory deficits seen in our cKO mice may be caused by the disruption of the DA signalling between the VTA and hippocampus. Further, in a previous study, 6-hydroxydopamine lesioning of the VTA in rats resulted in the loss of approximately 50 % of DA neurons leading to a 74 % reduction in DA levels in the hippocampus as well as an 83 % loss in the frontal cortex (Wisman et al. 2008). The loss of DA was associated with a poorer reference memory performance, with repeated trials required for the rats to acquire the task (Wisman et al. 2008). These findings highlight the important role of the VTA DA system in learning and consolidation of memory. Importantly, our observation of a decreased capability of the cKO mice during the acquisition phase in the radial arm maze, and the observations we make in the seizure-prone slice preparation could also be due to a loss of glutamatergic signalling, by the targeting of “Vglut2-only” cells and the glutamatergic, instead of the dopaminergic, component of the glutamate/DA cophenotypic cells. Electrical stimulation of the VTA has previously been shown to elicit theta oscillations, important for long-range synchronization of neuronal activity, in the hippocampus (Orzeł-Gryglewska et al. 2012). Therefore, disruption of glutamatergic signalling originating from the VTA can lead to reduced activity in the hippocampal area. Known effects of activity deprivation in the hippocampus are downregulation of GABAA receptors (Kilman et al. 2002) and reduction of GABA immunoreactivity, as shown in neocortical and hippocampal culture neurons (Marty et al. 1997). Maintenance of homeostasis is likely driven by a similar mechanism in the hippocampus of cKO mice. This would, as a consequence, lead to less activation of the hippocampal circuits through the VTA and an adaption of the circuitry that might already start during embryonal development. Since projections from the VTA are severed in hippocampal slice preparations, the remaining circuit is solely dependent on inhibitory and excitatory connections within the hippocampus. As a putative result of a compensatory downregulation of inhibitory components in the cKO mice upon the deprivation of VTA-derived activity, isolated hippocampal could exhibit a general overexcitation. In the presence of 100 nM kainate, a possible imbalance in the excitation/inhibition ratio could therefore lead to the generation of epileptiform discharges observed in the cKO mice, while control mice show normal gamma oscillations. Notably, given the promiscuous nature of the TH promoter as discussed above, it is conceivable that the hippocampus-related effects are independent of any alterations occurring in Vglut2 expression in the VTA area, but are derived from alterations in Vglut2 expression elsewhere. A broader range of genetic tools for ascertaining more selective gene targeting events, both spatially and temporally, should prove helpful in resolving the functional correlation between glutamate/dopamine transmission from the VTA and the herein described hippocampus-related phenotypes.
In summary, using a TH-Cre knock-in strategy which gives rise to Cre activity both in stable TH-expressing neurons and in cells that only transiently express TH during early development, we found a way to target Vglut2 gene expression within all three VTA-VGLUT2 populations identified in the medial aspects of the A10 area. Both the unexpected absence of altered psychostimulant-induced behavioural activation and the identification of the strong deficiency in hippocampus function require further investigation, not least due to the promiscuity of the TH-Cre transgene which is expressed in several non-neuronal and neuronal populations in addition to the VTA. Today, most studies, even those using optogenetics to control neural activity, rely on DAT-Cre or TH-Cre for targeting of DA cells. With the accelerating gain of knowledge of various subpopulations within the A10 area, it is becoming increasingly evident that the field would benefit from a broader selection of genetic tools to enable further characterization of the physiological roles of these different neuronal groups.
We thank Prof. Ted Ebendal (Univ. of Uppsala, Sweden) and Prof. Sylvia Arber (Univ. of Basel, Switzerland) for generously sharing the TH-ires-Cre and tau-mGFP mouse lines, respectively; Prof. Bruno Gasnier for the kind gift of VIAAT antibody; Dr. Madeleine le Grevés for experimental advice; and Li Li and Emily Sällström for technical assistance. This work was supported by grants from the Swedish Medical Research Council (2007-5742, 2011-4747), Uppsala University, the Swedish Brain Foundation, Parkinsonfonden, and the foundations of Major Gösta Lind, Åke Wiberg, Åhlén, and Bertil Hållsten. MM and JMT were supported by the Intramural Research Program of the National Institute on Drug Abuse.
Conflict of interest
The authors declare no conflict of interest.
- Alsiö J, Nordenankar K, Arvidsson E, Birgner C, Mahmoudi S, Halbout B, Smith C, Fortin GM, Olson L, Descarries L, Trudeau LE, Kullander K, Levesque D, Wallén-Mackenzie Å (2011) Enhanced sucrose and cocaine self-administration and cue-induced drug seeking after loss of VGLUT2 in midbrain dopamine neurons in mice. J Neurosci 31(35):12593–12603. doi: 10.1523/JNEUROSCI.2397-11.2011 PubMedCrossRefGoogle Scholar
- Birgner C, Nordenankar K, Lundblad M, Mendez JA, Smith C, le Greves M, Galter D, Olson L, Fredriksson A, Trudeau LE, Kullander K, Wallén-Mackenzie Å (2010) VGLUT2 in dopamine neurons is required for psychostimulant-induced behavioral activation. Proc Natl Acad Sci U S A 107(1):389–394. doi: 10.1073/pnas.0910986107 PubMedCentralPubMedCrossRefGoogle Scholar
- Fortin GM, Bourque MJ, Mendez JA, Leo D, Nordenankar K, Birgner C, Arvidsson E, Rymar VV, Berube-Carriere N, Claveau AM, Descarries L, Sadikot AF, Wallén-Mackenzie Å, Trudeau LE (2012) Glutamate corelease promotes growth and survival of midbrain dopamine neurons. J Neurosci 32(48):17477–17491. doi: 10.1523/JNEUROSCI.1939-12.2012 PubMedCrossRefGoogle Scholar
- Gras C, Amilhon B, Lepicard ÈM, Poirel O, Vinatier J, Herbin M, Dumas S, Tzavara ET, Wade MR, Nomikos GG, Hanoun N, Saurini F, Kemel M-L, Gasnier B, Giros B, Mestikawy SE (2008) The vesicular glutamate transporter VGLUT3 synergizes striatal acetylcholine tone. Nat Neurosci 11(3):292–300. doi: 10.1038/nn2052 PubMedCrossRefGoogle Scholar
- Lagerström MC, Rogoz K, Abrahamsen B, Persson E, Reinius B, Nordenankar K, Olund C, Smith C, Mendez JA, Chen ZF, Wood JN, Wallén-Mackenzie Å, Kullander K (2010) VGLUT2-dependent sensory neurons in the TRPV1 population regulate pain and itch. Neuron 68(3):529–542. doi: 10.1016/j.neuron.2010.09.016 PubMedCentralPubMedCrossRefGoogle Scholar
- Stornetta RL, Sevigny CP, Schreihofer AM, Rosin DL, Guyenet PG (2002b) Vesicular glutamate transporter DNPI/VGLUT2 is expressed by both C1 adrenergic and nonaminergic presympathetic vasomotor neurons of the rat medulla. J Comp Neurol 444(3):207–220. doi: 10.1002/cne.10142 PubMedCrossRefGoogle Scholar
- Wallén-Mackenzie Å, Gezelius H, Thoby-Brisson M, Nygard A, Enjin A, Fujiyama F, Fortin G, Kullander K (2006) Vesicular glutamate transporter 2 is required for central respiratory rhythm generation but not for locomotor central pattern generation. J Neurosci 26(47):12294–12307. doi: 10.1523/JNEUROSCI.3855-06.2006 PubMedCrossRefGoogle Scholar
- Wallén-Mackenzie Å, Nordenankar K, Fejgin K, Lagerstrom MC, Emilsson L, Fredriksson R, Wass C, Andersson D, Egecioglu E, Andersson M, Strandberg J, Lindhe O, Schioth HB, Chergui K, Hanse E, Langstrom B, Fredriksson A, Svensson L, Roman E, Kullander K (2009) Restricted cortical and amygdaloid removal of vesicular glutamate transporter 2 in preadolescent mice impacts dopaminergic activity and neuronal circuitry of higher brain function. J Neurosci 29(7):2238–2251. doi: 10.1523/JNEUROSCI.5851-08.2009 PubMedCrossRefGoogle Scholar
- Wallén-Mackenzie Å, Wootz H, Englund H (2010) Genetic inactivation of the vesicular glutamate transporter 2 (VGLUT2) in the mouse: what have we learnt about functional glutamatergic neurotransmission? Ups J Med Sci 115(1):11–20. doi: 10.3109/03009730903572073 PubMedCentralPubMedCrossRefGoogle Scholar
- Zelano J, Mikulovic S, Patra K, Kuhnemund M, Larhammar M, Emilsson L, Leao R, Kullander K (2013) The synaptic protein encoded by the gene Slc10A4 suppresses epileptiform activity and regulates sensitivity to cholinergic chemoconvulsants. Exp Neurol 239:73–81. doi: 10.1016/j.expneurol.2012.09.006 PubMedCrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.