Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer's disease methods
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Immunohistochemistry and immunofluorescence
For TH/GFAP, TH/Iba1, TH/NeuroTrace staining and for TH/APPswe, NeuroTrace/APPswe deposition, slices were incubated overnight with primary antibodies in PB containing 0.3% Triton X-100 and then incubated for 2 h at room temperature with secondary antibodies.
Immunohistochemistry and immunofluorescence
For DAT quantification, acute coronal brain slices (see below) were fixed in 4% paraformaldehyde in PB and transferred to 30% sucrose in PB at 4 °C. Slices were incubated for 2 days with primary antibodies in PB containing 1% Triton X-100 and then incubated for 2 h at room temperature with secondary antibodies. DAT levels (F/A) and TH levels (F/A) were quantified with ImageJ ( http://imagej.nih.gov/ij/ ) as mean signal fluorescence intensity (F) on a defined area (A). Quantification was done on eight samples per mouse.
Immunohistochemistry and immunofluorescence
The sections were counterstained with NeuroTrace 640/660 deep-red Fluorescent Nissl Stain (1:200, Invitrogen), mounted using an anti-fade medium (Fluoromount, Sigma-Aldrich) and examined under a confocal laser-scanning microscope (LSM700, Zeiss). The specificity of the immunohistochemical labelling was confirmed by the omission of primary antibodies and the use of normal serum instead (negative controls).
Microdialysis
Mice, anaesthetized with Zoletil and Rompun, were mounted on a stereotaxic frame (David Kopf Instruments) and implanted unilaterally with microdialysis probes 24-36 h before experiments. The concentric dialysis probes (AN69 fibres, Hospal Dasco) were implanted vertically at the level of the hippocampus (AP -3.0, ML±2.7 from bregma), NAc shell (AP +1.6, ML±0.2) and striatum (AP +1.0, ML±1.8). The probe lengths were 5 mm for hippocampus (3 mm membrane), 5.5 mm for NAc shell (1 mm) and 4.5 mm for striatum (2 mm). Each probe was fixed and the skin was sutured. Mice were returned to their home cages and the outlet and inlet probe tubing were protected by locally applied parafilm. Membranes were tested for in vitro recovery before surgery. On the day of the experiment each animal was placed in a circular cage containing m...
Acute brain slice preparation for electrophysiology
Acute brain slices (250-300 µm) were obtained following halothane anaesthesia and decapitation. The brain was rapidly removed and coronal slices containing the striatum and NAc core/shell or parasagittal slices containing the dorsal hippocampus were cut with a vibratome (VT1200S, Leica) in chilled bubbled (95% O 2, 5% CO 2 ) aCSF containing (in mM): NaCl 124, KCl 3, NaH 2 PO 4 1.25, NaHCO 3 26, MgCl 2 1, CaCl 2 2, glucose 10 (∼290 mOsm, pH 7.4). Slices were incubated for 1 h in aCSF at 32 °C and then transferred at room temperature for at least 30 min before recordings. A single brain slice was transferred to a recording chamber and completely submerged in aCSF (3-4 ml min -1; 32 °C).
Constant potential amperometry
Amperometric detection of DA in acute brain slices containing the striatum and the NAc was performed using carbon fibre electrodes (diameter 30 µm, length 100 µm, World Precision Instruments) positioned near a bipolar Ni/Cr stimulating electrode, to a depth of 50-150 µm into the coronal slice. The imposed voltage (MicroC potentiostat, World Precision Instruments) between the carbon fibre electrode and the Ag/AgCl pellet was 0.55 V. For stimulation, a single rectangular electrical pulse was applied using a DS3 Stimulator (Digitimer) every 5 min along a range of stimulation intensities (20-1,000 µA, 20-40 µs duration). In response to a protocol of increasing stimulation, a plateau of DA release was reached at the maximal stimulation intensity (1,000 µA, 40 µs). Signals were digitiz...
Multielectrode array recordings
A parasagittal acute slice containing the dorsal hippocampus was placed over an 8 × 8 array of planar electrodes, each 50 × 50 µm in size, with an interpolar distance of 150 µm (MED-P5155, Alpha MED Sciences), adjusted so that the entire CA1 pyramidal layer and stratum radiatum were covered with underlying electrodes. The slice was kept submerged using a platinum ring covered with nylon mesh. Voltage signals were recorded with the MED64 System (Alpha MED Sciences) and digitized at 20 kHz followed by filtering at 0.1-1 Hz with a 6071E Data Acquisition Card (National Instruments), using Mobius software (Alpha MED Sciences). Field excitatory post-synaptic potentials (fEPSPs) were evoked by Schaffer collateral stimulation (100 µs duration) through one of the 64 planar electrodes placed in the stratum radiatum. The recording chan...
Place conditioning task
To evaluate palatable food-induced CPP, we used a two-chamber apparatus made of two Plexiglas chambers (15 × 15 × 20 cm) that differ in the pattern and the colour of the grid, connected by a central compartment (15 × 5 × 20 cm) with two sliding doors (4 × 20 cm). In each chamber two triangular parallelepipeds (5 × 5 × 20 cm), made of black Plexiglas and arranged in various patterns, were used as conditioned stimuli. The lighting conditions (visual and tactile cues) were adjusted to prevent preference for a certain chamber. On the pre-conditioning day, each mouse was left in the central alley and allowed free access and exploration of the adjacent chambers of the apparatus, in the absence of food for 15 min. During the following 6 days (conditioning session), each mouse was confined daily for 30 min alternately in o...
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Evidence quotes (8)
For TH/GFAP, TH/Iba1, TH/NeuroTrace staining and for TH/APPswe, NeuroTrace/APPswe deposition, slices were incubated overnight with primary antibodies in PB containing 0.3% Triton X-100 and then incubated for 2 h at room temperature with secondary antibodies.
For DAT quantification, acute coronal brain slices (see below) were fixed in 4% paraformaldehyde in PB and transferred to 30% sucrose in PB at 4 °C. Slices were incubated for 2 days with primary antibodies in PB containing 1% Triton X-100 and then incubated for 2 h at room temperature with secondary antibodies. DAT levels (F/A) and TH levels (F/A) were quantified with ImageJ ( http://imagej.nih.gov/ij/ ) as mean signal fluorescence intensity (F) on a defined area (A). Quantification was done on eight samples per mouse.
The sections were counterstained with NeuroTrace 640/660 deep-red Fluorescent Nissl Stain (1:200, Invitrogen), mounted using an anti-fade medium (Fluoromount, Sigma-Aldrich) and examined under a confocal laser-scanning microscope (LSM700, Zeiss). The specificity of the immunohistochemical labelling was confirmed by the omission of primary antibodies and the use of normal serum instead (negative controls).
Mice, anaesthetized with Zoletil and Rompun, were mounted on a stereotaxic frame (David Kopf Instruments) and implanted unilaterally with microdialysis probes 24-36 h before experiments. The concentric dialysis probes (AN69 fibres, Hospal Dasco) were implanted vertically at the level of the hippocampus (AP -3.0, ML±2.7 from bregma), NAc shell (AP +1.6, ML±0.2) and striatum (AP +1.0, ML±1.8). The probe lengths were 5 mm for hippocampus (3 mm membrane), 5.5 mm for NAc shell (1 mm) and 4.5 mm for striatum (2 mm). Each probe was fixed and the skin was sutured. Mice were returned to their home cages and the outlet and inlet probe tubing were protected by locally applied parafilm. Membranes were tested for in vitro recovery before surgery. On the day of the experiment each animal was placed in a circular cage containing microdialysis equipment: the microdialysis probe was connected to a CMA/100 pump (Carnegie Medicine) through PE-20 tubing and an ultra low torque multichannel power-assisted swivel (Model MCS5, Instech Laboratories) to allow free movement. Artificial cerebrospinal fluid (aCSF; in mM: NaCl 140; KCl 4;...
Acute brain slices (250-300 µm) were obtained following halothane anaesthesia and decapitation. The brain was rapidly removed and coronal slices containing the striatum and NAc core/shell or parasagittal slices containing the dorsal hippocampus were cut with a vibratome (VT1200S, Leica) in chilled bubbled (95% O 2, 5% CO 2 ) aCSF containing (in mM): NaCl 124, KCl 3, NaH 2 PO 4 1.25, NaHCO 3 26, MgCl 2 1, CaCl 2 2, glucose 10 (∼290 mOsm, pH 7.4). Slices were incubated for 1 h in aCSF at 32 °C and then transferred at room temperature for at least 30 min before recordings. A single brain slice was transferred to a recording chamber and completely submerged in aCSF (3-4 ml min -1; 32 °C).
Amperometric detection of DA in acute brain slices containing the striatum and the NAc was performed using carbon fibre electrodes (diameter 30 µm, length 100 µm, World Precision Instruments) positioned near a bipolar Ni/Cr stimulating electrode, to a depth of 50-150 µm into the coronal slice. The imposed voltage (MicroC potentiostat, World Precision Instruments) between the carbon fibre electrode and the Ag/AgCl pellet was 0.55 V. For stimulation, a single rectangular electrical pulse was applied using a DS3 Stimulator (Digitimer) every 5 min along a range of stimulation intensities (20-1,000 µA, 20-40 µs duration). In response to a protocol of increasing stimulation, a plateau of DA release was reached at the maximal stimulation intensity (1,000 µA, 40 µs). Signals were digitized with Digidata 1440A coupled to a computer running pClamp 10 (both from Molecular Devices). Electrode calibration was performed at the end of each experiment by bath-perfused DA (0.3-10 µM).
A parasagittal acute slice containing the dorsal hippocampus was placed over an 8 × 8 array of planar electrodes, each 50 × 50 µm in size, with an interpolar distance of 150 µm (MED-P5155, Alpha MED Sciences), adjusted so that the entire CA1 pyramidal layer and stratum radiatum were covered with underlying electrodes. The slice was kept submerged using a platinum ring covered with nylon mesh. Voltage signals were recorded with the MED64 System (Alpha MED Sciences) and digitized at 20 kHz followed by filtering at 0.1-1 Hz with a 6071E Data Acquisition Card (National Instruments), using Mobius software (Alpha MED Sciences). Field excitatory post-synaptic potentials (fEPSPs) were evoked by Schaffer collateral stimulation (100 µs duration) through one of the 64 planar electrodes placed in the stratum radiatum. The recording channel with the highest fEPSP amplitude, at a distance of at least 300 µm from the stimulation site, was chosen as recording site. Input-output curves were obtained by measuring the fEPSP initial slope at increasing 5 µA steps of afferent stimulation, delivered every 30̴...
To evaluate palatable food-induced CPP, we used a two-chamber apparatus made of two Plexiglas chambers (15 × 15 × 20 cm) that differ in the pattern and the colour of the grid, connected by a central compartment (15 × 5 × 20 cm) with two sliding doors (4 × 20 cm). In each chamber two triangular parallelepipeds (5 × 5 × 20 cm), made of black Plexiglas and arranged in various patterns, were used as conditioned stimuli. The lighting conditions (visual and tactile cues) were adjusted to prevent preference for a certain chamber. On the pre-conditioning day, each mouse was left in the central alley and allowed free access and exploration of the adjacent chambers of the apparatus, in the absence of food for 15 min. During the following 6 days (conditioning session), each mouse was confined daily for 30 min alternately in one of the two chambers. One of the patterns was consistently paired with palatable food (0.5 g milk chocolate, Milka Alpine Milk Chocolate providing 5.31 kcal g -1 of energy; paired chamber) and the other with regular chow (RC, Mucedola 4RF21 diet; unpaired chamber). The RC p...
Machine-readable layer
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