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
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-CrecKO 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).