Generation and genotyping of TrkA conditional knockout mice
targeting vector was constructed with a loxP
site within the promoter region and another in the first intron of TrkA
to remove 0.25 kb of promoter sequence immediately 5' to the transcriptional start site of TrkA
and exon 1 which includes the translation initiation site upon Cre recombination (). Embryonic stem cells derived from 129/SvJ mice were transfected with the targeting construct and recombinant clones were obtained by neomycin-resistance (neoR
) positive selection, and then injected into C57BL/6 blastocysts to generate TrkAflox
mice. Upon successful homologous recombination, correctly targeted TrkA
) had a 43-bp oligonucleotide containing a loxP site
inserted into the promoter region and a PGKneoR
fragment flanked by FRT
sequences, respectively, which were inserted into the first intron. Mice heterozygous for the TrkAneo
allele were mated with FlpE transgenic mice that express Flp recombinase to bring about Flp-mediated deletion of the neoR
cassette and generate animals carrying the TrkAflox
allele (Rodriguez et al., 2000
). The dlx5/6i-cre
mice used in this study were generated in the laboratory of M. Ekker (University of Ottawa). These transgenic mice express Cre recombinase driven by the enhancer fragment derived from the zebrafish intergenic region between the dlx4
genes, the orthologs of mammalian dlx5
. This enhancer has previously been shown to direct Cre expression specifically to the subcortical telencephalon which later gives rise to cells in the ventral telencephalon, including the BFCNs and the striatum (Zerucha et al., 2000
; Kohwi et al., 2007
). These transgenic mice were crossed with TrkAflox
mice to generate TrkAcKO
mice. The TrkA
mutant mice were routinely genotyped by PCR using a mix of oligos: TrkA-wt-5': 5'-TGTACGGCCATAGATAAGCAT-3'; TrkA-wt-3': 5'-TGCATAACTGTGTATTTCAC-3'; 3202: 5'-CGCCTTCTTGACGAGTTCTTCTG-3'. The PCR conditions are as follows; preheat 94°C for 2 min, [94°C for 30 sec, 55°C for 45 sec, and 72°C for 30 sec] × 35 cycles. All mouse protocols were approved by the UCSF Institutional Animal Care and Use Committee (IACUC) and the UTSW IACUC. Both male and female mice were used in all experiments, except for behavioral tests in which male mice were used.
Figure 1 Generation of TrkAcKO mice. A, Targeting strategy for conditional deletion of TrkA in the forebrain. Diagram shows the TrkA locus and targeted alleles. B, Dlx5/6i-cre -mediated activation of R26R, as identified by the expression of β-galactosidase (more ...)
Mice were intra-cardially perfused with ice-cold phosphate buffer saline (PBS) followed by 4% (W/V) ice-cold paraformaldehyde (PFA) in PBS. For H&E staining and immunohistochemistry, the dissected brains were then post-fixed overnight with 4% PFA at 4 °C.
DiI crystal (total diameter of approximately 0.5 mm, Molecular Probes; Eugene, OR) was inserted into the dissected brain at the depth of the medial septum (stereotaxic coordinates:1.2 mm anterior to bregma, 0.0 mm medial lateral, 4.5 mm ventral from dura) with forceps such that the ventral edges of basal forebrain covered the crystal. The brain was then placed in the dark in 2% PFA in PBS at 37°C for 5 weeks. The vibratome sagittal sections (50 μm) were examined for DiI labeling using a fluorescence microscope (Olympus BX50,Tokyo, Japan) equipped with a Coolsnap CCD camera (Roper Scientific, Trenton, NJ) and the images were analyzed using ImageJ software (National Institutes of Health, USA).
Golgi staining was performed as previously described (Glaser and Van der Loos, 1981
; Luikart et al., 2005
). In brief, mice were perfused with PBS and the dissected brains were incubated with Golgi-Cox solution for 12 days. Vibratome coronal sections (100 μm) were processed and examined under an Olympus optical microscope. Images were analyzed with ImageJ software.
Immunohistochemistry and antibodies
For DAB staining, the fixed brains were paraffin-embedded, coronally sectioned (10 μm) and analyzed using an Olympus BX50 microscope equipped with a color CCD camera (DXM1200; Nikon, Tokyo, Japan). For the immunofluorescent staining, 40 μm vibratome sections were analyzed by confocal microscopy (LSM-510; Zeiss, Oberkochen, Germany). X-gal, DAB and immunofluorescent staining were performed as previously described (Lei et al., 2005
; Luikart et al., 2005
). Antibodies used for immunostaining were anti-TrkA (1:1000; Advanced Targeting Systems, San Diego, CA); anti-p75 (1:500; Promega, Madison, WI); anti-ChAT (1:400; Chemicon, Temecula, CA); anti-phospho-ERK (1:200; Cell Signaling, Danvers, MA); anti-phospho AKT (1:200, Cell Signaling); anti-β-galactosidase (1:1000; Chemicon); and anti-DARPP-32 (1:200; BD Biosciences, San Diego, CA). Primary antibodies were visualized by secondary antibodies conjugated with horseradish peroxidase (HRP, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), Cy2 and Cy3 (1:400; Jackson Laboratory, Bar Harbor, ME).
Quantification of cholinergic cell number and size
To quantify cholinergic cell numbers in BF and striatum, 6 coronal vibratome sections per animal (240 μm apart, bregma: 0.38 mm – 1.34 mm) were immunostained for ChAT, TrkA, or p75. Cell counts in BF were restricted to the MS/VDB region. The MS/VDB area encompasses the triangular region containing ≥95% of cholinergic neurons, is devoid of DARPP-32 immunostaining, and starts at the midline, 200 μm below the corpus callosum. It ends at a line drawn 310 μm below and parallel to the anterior commissures. The area of sections that contained MS, but not VDB, were defined as the triangular region beginning at midline, 200 μm below the corpus callosum and ending in a line drawn across the tops of the anterior commissures. The striatum was identified and defined by DARPP-32 immunostaining and by morphological criteria (superior boundary: corpus callosum; lateral boundary: external capsule; medial boundary: lateral ventricle and corpus callosum). Images were analyzed with ImageJ software using the cell counter macro to quantify the ChAT-immunoreactive (IR) cells and quantification was expressed as cells/area. A minimum of 200 cells were counted per section. To measure cell size of cholinergic neurons, pictures were taken of ChAT-IR neurons at 100× magnification. Two measurements were recorded for each neuronal soma (length and width) and averaged using ImageJ software.
Quantification of MS/VDB, striatum, and cortex volume
Serial Golgi-stained sections were used to measure the areas of the MS/VDB, striatum, and cortex regions with Image J software from which the volume was calculated. The aforementioned defined morphological criteria were used to evaluate the area of the MS/VDB and striatum. The cortex was evaluated within the same sections and was defined as follows: medial boundary, corpus callosum; lateral boundary, corpus callosum and external capsule; inferior boundary, anterior commissures. The volume of the striatum and cortex was analyzed in both hemispheres.
Retrograde tracing of BFCNs
The BFCNs were retrogradely traced in vivo by stereotactic injection of 50.6 nL of 20% (W/V) Texas Red-conjugated dextran amine (3,000 MW; Molecular Probe) in distilled water into the frontal cortex (Bregma 0.26, 1.0 mm lateral from midline, 1.0 mm below dura) using a Nanoliter 2000 nano-injector (World Precision Instruments, Sarasota, FL) at postnatal day (P) 21. The mice were intra-cardially perfused at 2 weeks after the injection. Dissected brains were coronally sectioned (40 μm) and analyzed by confocal microscopy.
The basal forebrain including the medial septum, nucleus basalis and diagonal band, as well as the striatum were dissected under a stereoscopic microscope (Leica M420; Leica, Bensheim, Germany), immediately frozen in liquid nitrogen, and homogenized in lysis buffer containing 1% Triton X-100, 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail (Roche, Indianapolis, IN), and 50 mM sodium fluoride. Tissues were then sonicated using Vibra Cell (Sonics & Materials, Newtown, CT) and insoluble material was removed from the protein extracts by centrifugation at 10,000 rpm for 20 min at 4°C. Protein content in the supernatant was quantified with the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA). Equivalent amounts of total protein (10 – 25 μg) were separated in either 10% or 4 – 20% gradient SDS-PAGE gels (BioRad, Richmond, CA) and transferred to nitrocellulose membranes. Membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% nonfat milk for 1 hr, then incubated with primary antibody to TrkA (1:4000), ChAT (1:1000), p75 (1:1000, rabbit polyclonal, Millipore, Billerica, MA), DARPP-32 (1:1000; Cell Signaling, Danvers, MA) phospho-Thr34-DARPP-32 (1:500; Cell Signaling), phospho-ERK1/2 (1:2000, Cell Signaling), ERK1/2 (1:400, Santa Cruz Biotechnology, Santa Cruz, CA), phospho-AKT (1:1000, Cell Signaling), AKT (1:1000, Cell Signaling), or β-actin (1:5,000; Sigma, Saint Louis, MO) and were then visualized with appropriate HRP-conjugated secondary antibodies (1:10,000; Santa Cruz) followed by Chemiglow West reagents (Cell Biosciences, Santa Clara, CA). Blots were stripped using Re-Blot Plus stripping solution (Millipore) and reprobed with the indicated primary antibodies as indicated in figure legends. Band densitometry in blots was performed using ImageJ software. Protein levels were determined by band densitometry and normalized to the band intensity values of β-Actin for TrkA, ChAT, and DARPP-32; phospho-proteins were normalized to intensity values of the corresponding total protein bands.
Morris Water Maze
A circular pool (142 cm diameter) was filled with room temperature water to a depth of approximately 30 cm. A platform (10 cm diameter) was placed in one quadrant of the pool with the top of the platform about 2 cm below the water level. White non-toxic paint was added to enhance the contrast with the animal and to hide the location of the platform. Each day the mice were placed in the pool and allowed to swim for 1 min to find the platform. The swim path and time until locating the platform was recorded via video camera and computer using EthoVision (Noldus, Leesburg, VA). If the mouse did not find the platform within 1 min, it was gently guided or placed on the platform for 10 sec, then removed from the pool and returned to its home cage. Each animal was placed in the pool for a total of 4 times each day for 10 days. Immediately following the training days, a probe test was conducted in which the platform was removed from the pool and each mouse was allowed to swim for 1 min to determine whether the animal had learned the location of the platform. To control for visual problems, the mice were given 4 trials a day for 3 days using the same pool and platform, however a large black block was placed on top of the platform to clearly mark the location. This control test found no visual problems in any of the mice examined.
The mice were placed in a chamber (31 × 25 × 25 cm; Med Associates, St. Albans, VT) possessing aluminum side walls and Plexiglass rear and front walls with a dim light and constant white noise. The presentation of tone and shock stimuli in all training and testing sessions was controlled by FreezeFrame software (Coulbourn Instruments, Allentown, PA). Training day: mice were allowed to explore for 120 sec, and then a tone (conditioned stimulus, CS) of 25 kHz and 75 dB was presented for 30 sec and co-terminated in the last 2 sec with a mild foot-shock (0.5 mA, constant current) (unconditioned stimulus, US). Another CS-US pairing was presented twice with 30 sec intervals each time, total 3 CS-US pairing for 5 min. Contextual and cued testing: to test for contextual memory, mice were placed in the same chamber 24 hr after training and allowed to explore for 4 min without CS or US. Freezing behavior, defined as complete absence of voluntary movements except for respiratory movement, was observed. 24 hr after the contextual test, the mice were tested for cue memory by returning them to the chamber, which was modified with a plastic floor and a vanilla scent without white noise. Freezing was scored for 2 min without the tone and another 2 min with the tone.
Novel object recognition
The mice were allowed to explore an empty chamber (70 × 70 × 30 cm) for 10 min per day for 3 days to minimize the disruption, stress, and novelty of handling, and also to become familiarized with the testing environment. In the sample phase, the mice were allowed to interact with the sample objects in the back left and right corner in the chamber. Exploration of an object was defined as directing the nose to the object at a distance of <2 cm and/or touching it with the nose. Turning around or sitting on the object was not considered exploratory behavior. The sample phase ended when the mouse had explored the identical objects for a total of 30 sec. Immediately after a 1 hr interval, mice were allowed to explore in the chamber with the previously familiarized object in one back corner and the novel object in the other back corner for 3 min. The time spent exploring each object was recorded via video camera to calculate the discrimination ratio = b/(a+b): a = exploration time of familiar object. b = exploration time of the novel object.
The same cohort of mice was used for all behavioral tests, with littermates used as controls.
All data values are presented as the mean ± SEM. At least 3 littermate mice per genotype were used per experiments, as indicated in figure legends. Student's t-tests were applied to data with two groups of samples. One-way ANOVA analyses were used for comparisons of data with greater than two groups and were followed by Tukey's-Kramer post hoc test for significance. A value of p<0.05 was considered statistically significant. Data were analyzed using GraphPad Prism, version 5.04 for Windows (GraphPad Software, San Diego, CA, USA).