Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer's disease methods
Aim. Evidence-backed execution summary for Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer's disease methods from Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer's disease.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Statistical analysis
reagent used in the protocol.
- Use
- LTP: data for sub-chronic L -DOPA and selegiline treatment were analysed by two-way ANOVAs for genotype (Tg2576 versus WT) and treatment (sham versus drug). LTP data for the effect of bath administration of L -DOPA and DA receptor agonists on Tg2576 mice were analysed with one-way ANOVA. In both cases post hoc compa...
L -DOPA and selegiline restore memory and reward deficits
reagent used in the protocol.
- Use
- Previous studies have shown that pharmacological manipulations aimed at increasing the DAergic transmission in the hippocampus and cortex could improve synaptic functions, cognitive impairments and memory deficits in AD patients and AD-like experimental models. Prompted by the finding that DAergic cell death progre...
L -DOPA and selegiline restore memory and reward deficits
reagent used in the protocol.
- Use
- To confirm these data and examine the long-term effects of the increased availability of endogenous DA, we treated 6-month-old animals with selegiline, an irreversible selective inhibitor of monoamine oxidase-B (ref.; ). Microdialysis experiments on freely moving animals confirmed that sub-chronic treatment with se...
Immunohistochemistry and immunofluorescence
reagent used in the protocol.
- Use
- Mice were anaesthetized with Rompun (20 mg ml -1, 0.5 ml kg -1, i.p., Bayer) and Zoletil (100 mg ml -1, 0.5 ml kg -1, Virbac) and perfused transcardially with 50 ml saline followed by 50 ml of 4% paraformaldehyde in phosphate buf...
Immunohistochemistry and immunofluorescence
reagent used in the protocol.
- Use
- The sections selected for immunohistochemistry (every second slice was processed for a total of 9 sections) were processed with a rabbit anti-TH antibody. The endogenous peroxidise was neutralized with a 0.3% H 2 O 2 solution in PB. The sections were incubated overnight at 4 °C with the primary antibody d...
Immunohistochemistry and immunofluorescence
reagent used in the protocol.
- Use
- 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...
In situ end labelling of DNA fragmentation (TUNEL)
reagent used in the protocol.
- Use
- For TUNEL staining, brains were removed as before and cut in coronal slices (10 µm thickness). Every tenth slice was processed for TUNEL. After removal of paraffin with xylene and rehydration in ethanol solutions of decreasing concentration, sections were digested with proteinase K (20 µg...
Total protein extraction
reagent used in the protocol.
- Use
- The striatum was dissected from acute coronal brain slices; the hippocampus was isolated from the entire brain. Tissues were homogenized in lysis buffer containing (in mM) 320 sucrose, 50 NaCl, 50 Tris-HCl pH 7.5, 1% Triton X-100, 1 sodium orthovanadate, 5 β-glycerophosphate, 5 NaF and protease inhibitor cockta...
Reduced DA in the NAc shell and food reward deficits
Due to the importance of the mesolimbic system for reward and motivation processing, we examined whether the reduced outflow of DA from the VTA to the NAc shell could be associated with dysfunctional mesolimbic reward-associated processing in Tg2576 mice. To this end, we measured the responses of 6-month-old mice i...
- Use
- Due to the importance of the mesolimbic system for reward and motivation processing, we examined whether the reduced outflow of DA from the VTA to the NAc shell could be associated with dysfunctional mesolimbic reward-associated processing in Tg2576 mice. To this end, we measured the responses of 6-month-old mice i...
L -DOPA and selegiline restore memory and reward deficits
In view of the fact that treatments with L -DOPA or selegiline improve hippocampal synaptic plasticity and PSD composition, we then asked whether the impaired memory function in Tg2576 mice could be ameliorated with treatment. Indeed, both selegiline ( ) and L -DOPA ( ) treatments increased the total freezing time e...
- Use
- In view of the fact that treatments with L -DOPA or selegiline improve hippocampal synaptic plasticity and PSD composition, we then asked whether the impaired memory function in Tg2576 mice could be ameliorated with treatment. Indeed, both selegiline ( ) and L -DOPA ( ) treatments increased the total freezing time e...
L -DOPA and selegiline restore memory and reward deficits
Finally, we investigated whether the selegiline treatment could restore the defects in CPP and food consumption observed in Tg2576 mice during the CPP test ( ). As expected, although saline-treated Tg2576 animals were unable to show increased preference for the chamber associated with the rewarding food and consumed...
- Use
- Finally, we investigated whether the selegiline treatment could restore the defects in CPP and food consumption observed in Tg2576 mice during the CPP test ( ). As expected, although saline-treated Tg2576 animals were unable to show increased preference for the chamber associated with the rewarding food and consumed...
Immunohistochemistry and immunofluorescence
For the analysis of GFAP, Iba1 and DAT markers, images were taken as Z-stacks and these Z-stack images were then processed by maximum intensity projection. All samples were acquired with the same Z-stack thickness and the same laser settings. Data collection for densitometry was done by a researcher blind to the gen...
- Use
- For the analysis of GFAP, Iba1 and DAT markers, images were taken as Z-stacks and these Z-stack images were then processed by maximum intensity projection. All samples were acquired with the same Z-stack thickness and the same laser settings. Data collection for densitometry was done by a researcher blind to the gen...
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...
- Use
- 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...
Stereological analysis
Sections processed for immunohistochemistry were used for obtaining unbiased estimates of total number of TH + and TH - neurons in the SNpc and VTA and TH + neurons in the LC. The boundaries of these areas in the mouse brain were defined by TH staining, and area distinction was performed according to published...
- Use
- Sections processed for immunohistochemistry were used for obtaining unbiased estimates of total number of TH + and TH - neurons in the SNpc and VTA and TH + neurons in the LC. The boundaries of these areas in the mouse brain were defined by TH staining, and area distinction was performed according to published...
In situ end labelling of DNA fragmentation (TUNEL)
For TUNEL staining, brains were removed as before and cut in coronal slices (10 µm thickness). Every tenth slice was processed for TUNEL. After removal of paraffin with xylene and rehydration in ethanol solutions of decreasing concentration, sections were digested with proteinase K (20 µg...
- Use
- For TUNEL staining, brains were removed as before and cut in coronal slices (10 µm thickness). Every tenth slice was processed for TUNEL. After removal of paraffin with xylene and rehydration in ethanol solutions of decreasing concentration, sections were digested with proteinase K (20 µg...
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 (...
- Use
- 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 (...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All other data were analysed with a two-tailed paired or unpaired Student's t -test, as described in Figure legends. Data are presented as mean±s.e.m. Except for CPP, all statistical analysis was performed using GraphPad Prism (v5.00). Values of P ≤0.05 were considered to be statistically significant (sho...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- CPP: data were analysed by two-way analysis of variance (ANOVAs) with the genotype (Tg2576 versus WT) and chambers (paired versus unpaired) as independent factors. CPP data for selegiline treatment were analysed by two-way ANOVAs for treatment (sham versus sel) and chambers (paired versus unpaired). Chocolate consum...
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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...
Measurement outputs
What raw and processed outputs should exist?
LTP: data for sub-chronic L -DOPA and selegiline treatment were analysed by two-way ANOVAs for genotype (Tg2576 versus WT) and treatment (sham versus drug). LTP data for the eff...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
All other data were analysed with a two-tailed paired or unpaired Student's t -test, as described in Figure legends. Data are presented as mean±s.e.m. Except for CPP, all s...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
As PSD modifications precede functional alterations of synapses, we analysed protein expression in the hippocampal PSD fraction in 6-month-old Tg2576 mice. Consistent with our...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
As expected, selegiline failed to rescue TH levels in 6-month-old Tg2576 mice (, ). However, the selegiline-induced increase in DA availability restored GluA1 phosphorylation (...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
LTP: data for sub-chronic L -DOPA and selegiline treatment were analysed by two-way ANOVAs for genotype (Tg2576 versus WT) and treatment (sham versus drug).
from paperScoring or quantification
Quantify the primary readouts for this experiment: LTP: data for sub-chronic L -DOPA and selegiline treatment were analysed by two-way ANOVAs for genotype (Tg2576 versus WT) and treatment (sham versus drug). LTP data for the eff...; All other data were analysed with a two-tailed paired or unpaired Student's t -test, as described in Figure legends. Data are presented as mean±s.e.m. Except for CPP, all s...; As PSD modifications precede functional alterations of synapses, we analysed protein expression in the hippocampal PSD fraction in 6-month-old Tg2576 mice. Consistent with our...; As expected, selegiline failed to rescue TH levels in 6-month-old Tg2576 mice (, ). However, the selegiline-induced increase in DA availability restored GluA1 phosphorylation (....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
LTP: data for sub-chronic L -DOPA and selegiline treatment were analysed by two-way ANOVAs for genotype (Tg2576 versus WT) and treatment (sham versus drug). LTP data for the eff...; All other data were analysed with a two-tailed paired or unpaired Student's t -test, as described in Figure legends. Data are presented as mean±s.e.m. Except for CPP, all s...; Due to the importance of the mesolimbic system for reward and motivation processing, we examined whether the reduced outflow of DA from the VTA to the NAc shell could be associ...; Previous studies have shown that pharmacological manipulations aimed at increasing the DAergic transmission in the hippocampus and cortex could improve synaptic functions, cogni...
from paperReporting output
Report representative outputs alongside summary comparisons for LTP: data for sub-chronic L -DOPA and selegiline treatment were analysed by two-way ANOVAs for genotype (Tg2576 versus WT) and treatment (sham versus drug). LTP data for the eff..., All other data were analysed with a two-tailed paired or unpaired Student's t -test, as described in Figure legends. Data are presented as mean±s.e.m. Except for CPP, all s..., As PSD modifications precede functional alterations of synapses, we analysed protein expression in the hippocampal PSD fraction in 6-month-old Tg2576 mice. Consistent with our..., As expected, selegiline failed to rescue TH levels in 6-month-old Tg2576 mice (, ). However, the selegiline-induced increase in DA availability restored GluA1 phosphorylation (....
inferred from protocolStructured statistical methods
LTP: data for sub-chronic L -DOPA and selegiline treatment were analysed by two-way ANOVAs for genotype (Tg2576 versus WT) and treatment (sham versus drug). LTP data for the eff...; All other data were analysed with a two-tailed paired or unpaired Student's t -test, as described in Figure legends. Data are presented as mean±s.e.m. Except for CPP, all s...; Due to the importance of the mesolimbic system for reward and motivation processing, we examined whether the reduced outflow of DA from the VTA to the NAc shell could be associ...; Previous studies have shown that pharmacological manipulations aimed at increasing the DAergic transmission in the hippocampus and cortex could improve synaptic functions, cogni...
source structuredSource and audit
What supports the facts on this page?
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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"@type": "HowTo",
"name": "Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer's disease methods",
"description": "Evidence-backed execution summary for Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer's disease methods from Dopamine neuronal loss contributes to memory and reward dysfunction in a model of Alzheimer's disease.",
"totalTime": "PT3840M",
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{
"@type": "HowToStep",
"position": 1,
"name": "Immunohistochemistry and immunofluorescence",
"text": "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."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Immunohistochemistry and immunofluorescence",
"text": "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."
},
{
"@type": "HowToStep",
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"name": "Immunohistochemistry and immunofluorescence",
"text": "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)."
},
{
"@type": "HowToStep",
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"name": "Microdialysis",
"text": "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..."
},
{
"@type": "HowToStep",
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"name": "Acute brain slice preparation for electrophysiology",
"text": "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)."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Constant potential amperometry",
"text": "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..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Multielectrode array recordings",
"text": "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..."
},
{
"@type": "HowToStep",
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