Generation of progranulin-deficient mice
Mice with a floxed Grn
mice) were generated by homologous recombination in RF8 ES cells (129Sv/Jae) (Martens et al., 2012
mice were produced by breeding Grn+/F
mice with mice expressing Cre recombinase in the germline (Tg(ACTB-cre)2Mrt/J, JAX #003376). Deletion of progranulin was observed at the mRNA and protein levels in multiple tissues (Martens et al., 2012
); progranulin mRNA levels in brain and protein levels in brain and plasma were approximately 50% of normal in Grn+/−
mice (). Mice for this study were generated by breeding Grn+/−
mice, except , for which the mice were a product of a cross between Grn+/+
mice that had not been crossed to Cre and, thus, had a floxed Grn
allele but normal progranulin levels were used as controls in . An independent line of progranulin-deficient mice (Kayasuga et al., 2007
) was used to confirm our hippocampal spine density results in .
Fig. 1 Progranulin levels in progranulin-deficient mice. A–D, Progranulin mRNA levels in cortex (A), hippocampus (B), thalamus (C), and amygdala (D). Relative expression levels quantified by qPCR were calculated using the ΔΔCt method (more ...)
Fig. 2 Progranulin haploinsufficiency causes social deficits. A,C,D, Three-chamber sociability test, with sociability ratio (time spent investigating another mouse divided by time spent investigating an inanimate object) expressed as percent of control. A, On (more ...)
Fig. 4 Progranulin-deficient mice have normal hippocampal function and spine density. A, B, Box-and-whisker plots representing the age at which early (A) and late (B) milestones were attained for each genotype. Boxes indicate the 75th and 25th percentiles, with (more ...)
Males and females were used for all experiments except electrophysiology and Golgi staining, which used only males. Males and females were analyzed separately and then presented together as there were no significant main effects of sex on any of the outcome measures. Two cohorts of mice were tested on a mixed background that included 129Sv (on which the floxed line was originally created), FVB/N (the background strain of the Cre transgenic mice), and C57BL/6 (to which the original Grn+/−
mice were crossed to expand the line for experiments). Subsequently, after 10 generations of backcrosses, a third cohort was tested on a congenic C57BL/6 background. All mice used were Cre-negative and verified Disc-1 wild-type (Disc-1 deletion is endogenous to 129 substrains (Clapcote and Roder, 2006
)). Animals were housed in a pathogen-free barrier facility with a 12-h light/12-h dark cycle and ad libitum
access to food (NIH-31 Open Formula Diet, Harlan #7917) and water. All behavior experiments were conducted during daylight hours under normal room lighting conditions unless otherwise specified. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
Progranulin protein levels from mouse cortex and plasma were quantified using an ELISA kit (Adipogen) according to manufacturer's protocol. To isolate protein, mouse cortex was dissected and homogenized in lysis buffer [10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM EGTA and 0.05% (v/v) NP-40] supplemented with protease inhibitors (HALT; ThermoFisher). Samples were then spun at 16,000 × g for 10 minutes at 4°C. The supernatant was collected and the concentration of soluble protein in each sample was determined using Coomassie Plus Protein Assay reagent (ThermoFisher). 100μg of protein was added to 1× diluent and samples were run in duplicate. To isolate plasma, blood was collected from the right ventricle of the heart using a 1 mL syringe and a 23G, ¾″ needle. 250mM EDTA was included in the collection syringe to avoid clotting. To separate plasma from the cellular phase, samples were spun at 1000 × g for 10 minutes at 4°C in a swinging bucket centrifuge. Mouse plasma was then diluted 1:200 in 1× diluent and run in duplicate. Recombinant mouse progranulin supplied with the kit was used for generating a standard curve.
Mice were tested for social interaction time in a three-chamber sociability test adapted from Moy et al. (2004)
with minor modifications. Testing was conducted under red light. The test boxes were fabricated from white plexiglass at the UAB machine shop and did not contain bedding. After 30–90 minutes of habituation to the testing room, a mouse was placed in the center chamber with the two outside chambers containing empty wire cages (Spectrum Diversified Designs). Mice were allowed to explore all three chambers for 10 minutes. After 10 minutes, a novel mouse (8-month-old, male, C57BL/6 mouse that had been habituated to being in the wire cage) and an inanimate object (matchbox car or block) were placed under the wire cages. The test mouse was allowed to explore for 10 minutes. The time spent interacting with the novel mouse and the inanimate object was scored either by a blinded observer with a stopwatch or a video tracking system (CleverSys, Inc.). A sociability ratio was calculated for each mouse by dividing the time spent interacting with the other mouse by the time spent interacting with the inanimate object. To show data from multiple cohorts, the sociability ratio was normalized with 100% as the average sociability ratio of wild-type mice in each cohort.
To test for pheromone preference, time spent investigating stimuli containing urine and water was measured. Urine was collected and combined from 10 male C57BL/6 mice and subsequently frozen until testing. Urine or water (50 μL) was spotted onto small pieces of filter paper, which were then dried. The filter papers were inserted into histology cassettes to prevent direct contact, and cassettes containing urine and water were placed into opposite corners of a standard mouse cage with bedding. The test mouse was placed into the cage for 5 minutes and time spent within 5 cm of each stimulus was recorded with video tracking software (Clever Sys., Inc.).
Tube test of social dominance
Mice were tested for social dominance in a tube test adapted from Lindzey et al. (1961)
. Mice were placed, head first, into opposing sides of a clear plastic tube (3.81 cm I.D. × 30.5 cm in length) and released simultaneously. The trial ended when two paws of one mouse left the tube. The mouse remaining in the tube at the end of the trial was deemed the winner. A trial was aborted if the mice crossed each other or no mouse left the tube in two minutes. The tube was cleaned with 70% ethanol and dried between trials. Data were analyzed using a chi-squared test.
Experiments were conducted in Quick Change Test chambers (Med Associates). For the acquisition of fear conditioning, mice were placed in the chambers and allowed to acclimate for 180 seconds. After acclimation, a conditioned stimulus (white noise at 75 Db) was given for 20 seconds co-terminating with a 0.5-mA foot shock (2 seconds). This stimulus was given a total of three times with a 40-second inter-stimulus interval. Videos were recorded and the time the mice spent immobile was measured by a blinded observer. The mice were then returned to their home cage. To test for cued-based conditioning, mice were tested 24 hours after training. For this test, the chambers' context was made novel with a triangular, opaque white plexiglass insert and a spearmint scent. Mice were placed in the chamber for 6 minutes with the white noise auditory cue given for the last 3 minutes. Freezing was measured before and during the cue. The chamber was cleaned with 70% ethanol between mice during the training and with isopropanol during the cued testing.
The assessment of developmental milestones began on postnatal day two. Pups were monitored daily for weight and attainment of physical milestones, such as incisor eruption, pinnae detachment, and opening of eyes. Further, pups were evaluated for reflexes and coordinated movements. For grasp reflex, forepaws were gently stroked with the wooden end of a swab; achievement of the milestone was registered if a grasping motion was observed. For surface righting, pups were placed in bedding with their ventral side up and timed until they successfully righted themselves. To assess cliff avoidance, pups were placed at the edge of a 6-cm high Styrofoam platform, with head and paws hanging over the edge. The time it took to back away from the edge was measured and achievement of the milestone was recorded if the time was less than 10 seconds. Negative geotaxis was measured by placing a pup facing downward on a coarse surface at an angle of 30°. The time it took for a pup to right itself was scored and achievement of the milestone was recorded when the time was less than 10 seconds. To examine visual placing, pups were lowered downward by their tails toward a surface. When the pup raised their head and extended their forelimbs towards the surface, achievement of the milestone was recorded. To evaluate air righting, pups were held 30cm above a well bedded surface and dropped ventral side up. Achievement of the milestone was recorded on the day the pups were able to land on their feet. The bar hanging milestone was tested by allowing the mice to grasp a small bar and measuring the amount of time they could remain suspended without falling. Achievement of the milestone was scored if they could remain suspended.
Total ambulatory distance and anxiety were measured on the open field (Med Associates). Mice were placed in the open field for 15-minute trials. Activity was measured via infrared beam breaks by automated tracking software. The apparatus was cleaned with 70% ethanol between trials.
Spatial learning and memory were assessed with the Morris Water Maze as described (Scearce-Levie, 2011
). All mice were single-housed for at least 5 days before testing. Hidden platform training consisted of 4 days of training with six trials each day (three in the morning and three in the afternoon). The platform sat 1.5 cm below the surface of the opaque water (20°C) in a fixed location. Each mouse was placed in the maze, initially facing the outer wall, at a drop-off point that changed semi-randomly for each trial. A trial ended when the mouse located the platform and remained motionless on the platform for 2 seconds. If the mouse did not find the platform in 60 seconds, they were led to the platform then removed from the pool. Performance was measured with an EthoVision video-tracking system (Noldus Information Technology). A probe trial was conducted 1 day after conclusion of training. The platform was removed and each mouse was placed into the maze opposite of where the platform resided. Each trial lasted 60 seconds. The latency to find the platform was analyzed using a two-way ANOVA with repeated measures.
Electrophysiology was performed on acute hippocampal slices from 12-month-old mice. Mice were sacrificed by decapitation and brains immediately placed in ice-cold cutting solution (110 μM sucrose, 60 μM NaCl, 3 μM KCl, 1.25 μM NaH2PO4, 28 μM NaHCO3, 2 μM CaCl2, 7 μM MgCl2, 5 μM glucose, and 0.6 μM ascorbate). Transverse slices (400 μm thick) were prepared on a Vibratome (The Vibratome Company) and the hippocampus was dissected from the slice in a room temperature solution of 50% cutting solution and 50% ACSF (125 μM NaCl, 2.5 μM KCl, 1.25 μM NaH2PO4, 25 μM NaHCO3, 2 μM CaCl2, 1 μM MgCl2, 25 μM glucose). Slices were then equilibrated for 45 minutes in room temperature ACFS, followed by 1 hour in 32°C ACSF. All solutions were saturated with 95% O2
and 5% CO2
. Electrophysiology measurements were made in an interface chamber (Fine Science Tools) as described (Levenson et al., 2004
). Hippocampal slices were constantly perfused at a rate of 1 mL/min with 30°C ACSF. Extracellular stimuli were applied to the Schaffer-collaterals along the border of areas CA1 and CA3 (Model 2200 stimulus isolator, A–M Systems) with an enameled, bipolar platinum-tungsten (92%:8%) electrode. fEPSPs were recorded in stratum radiatum with a glass recording electrode (1–3 MΩ) filled with ACSF. Clampex was used for data acquisition and Clampfit used for data analysis (Molecular Devices). Input/output curves were generating by applying a range of stimuli from 0.5V to 30V in 1V increments. Intensities were averaged over three sweeps. The stimulus intensity that evoked 50% of the maximal fEPSP slope was used for subsequent stimulations. Paired-pulse facilitation was measured by applying consecutive stimuli at a range of interstimulus intervals (10, 20, 50, 100, 150, 200, 250, 300 ms). After 20 minutes of baseline recording, LTP was induced by applying two 100-Hz, 1-second stimulus trains, 20 seconds apart. Recordings were taken every 20 seconds, and data were averaged into 2-minute intervals. Data from paired pulse facilitation and LTP were analyzed using a two-way ANOVA with repeated measures.
Mouse brains were processed for morphological assessment with the FD Rapid GolgiStain Kit (FD NeuroTechnologies) according to the manufacturer's protocol. Mice were sacrificed with 100 mg/kg Fatal-Plus (Vortech Pharmaceuticals) and decapitated, and brains were removed. Hemibrains were submerged in impregnation solution for 14 days then transferred to a cryoprotectant for an additional 7 days. Next, brains were cut into 200-μm sections with a cryostat (Leica) and mounted on gelatin-coated slides. After mounting, the slides were rinsed in ddH2O and incubated in developing solution. After development, the sections were rehydrated with increasing concentrations of ethanol and cleared with xylene. Lastly, slides were coverslipped using EUKITT mounting medium (Electron Microscopy Sciences).
Microscopy was performed on a MicroBrightField system (MBF Bioscience). Image stacks were taken of 10–40 μm segments of second-order apical dendrites on CA1 pyramidal neurons at 100× (oil-immersion). All image stacks were manually traced using Neurolucida and analyzed using Neurolucida Explorer.
Immunohistochemistry was performed on free-floating 30-μm thick coronal sections as described (Palop et al., 2011
). To obtain sections, mice were sacrificed as described above and transcardially perfused with normal saline. Brains were removed, drop-fixed in 4% paraformaldehyde in phosphate buffer (PB: 80 mM Na2
, 200 mM NaH2
, pH 7.4), and placed at 4°C for 48 hours. After two phosphate-buffered saline (PBS) washes, the brains were transferred to 30% sucrose in PBS for 48 hours or until they sank. 30-μm sections were then cut on a sliding microtome with freezing stage (Leica). The sections were placed in cryoprotectant (30% ethylene glycol, 30% glycerol, 40% PBS, v/v/v) and held at −20°C until use.
Sections were washed free of cryoprotectant with PBS. Endogenous peroxidase activity was quenched with 3% H2O2 and 10% methanol in PB, followed by blocking with 10% normal goat serum (Vector Laboratories), 1% dry milk, and 0.2% gelatin to prevent nonspecific binding. Primary antibodies were used at the following concentrations diluted in PBS: 3% normal goat serum and 0.2% gelatin: Iba1 (1:5000; Wako), GFAP (1:5000; Dako), c-fos (1:10,000; EMD Millipore). Species-specific biotinylated secondary IgG (1:5000; Vector Laboratories) was applied and the Vectastain Elite avidin-biotin horseradish peroxidase complex kit (Vector Laboratories) was used for amplification. Diaminobenzidine:tetrahydrochloride was added for detection and sections were mounted with cover glass using Cytoseal 60 (Electron Microscopy Sciences). For analysis of Iba1 and GFAP, photomicrographs of 3 sections per mouse were collected on an upright microscope (Zeiss) with a 10× objective. Thresholds were set to include only GFAP or Iba1 positive pixels. The total number of GFAP or Iba1 positive pixels per image were quantified then averaged for the 3 images. Autofluorescent images were taken on an upright microscope (Zeiss) equipped with a FITC (Ex 480/30 Em 535/40) filter cube. Pixel densities were calculated using Image J software.
Quantitative real-time PCR
TNF-α mRNA levels were quantified using qPCR. RNA was first prepared using Trizol reagent (Invitrogen), according to manufacturer's protocol. From the prepared RNA, cDNAs were generated with SuperScript III and random primers (Invitrogen). cDNAs were quantified using LightCycler 480 Probes master mix (Roche) and Taqman Gene Expression Assays for TNF-α(Mm00443258_m1), progranulin (Mm00433848_m1), and β-actin (Mm00607939_s1). Amplification was performed on a Roche LightCycler 480, and data were quantified using the ΔΔCt method (User Bulletin #2, Applied Biosystems).
Mice were exposed to a novel and social environment as previously described (Scearce-Levie et al., 2008
). Briefly, two mice of the same genotype and opposite sex were placed in a cage with a new type of bedding and novel olfactory, tactile, and visual stimuli for 2 hours prior to sacrificing. Control mice were left in their home cage, undisturbed for 3 days prior to sacrifice.
Sections were immunostained for c-fos or NeuN as described above, then scanned with a slide scanner (Electron Microscopy Sciences). Three serial sections, 300 microns apart, in which the caudate/putamen and amygdala were clearly defined (between 1.0 and 1.8 mm posterior to bregma) were chosen. For the caudate/putamen and the central amygdala (including amygdalostriatral transition area), the lateral boundary was the external capsule and the medial boundary was the globus pallidus and internal capsule (). A horizontal line extending medially from the bifurcation of the external capsule separated caudate/putamen from amygdala. The ventral border of the amygdala was defined by a line drawn between the tips of the external capsule and extending medially to either the internal capsule or optic tract, depending on the anterior/posterior location. Cells were manually counted by an observer blind to genotype. Any cell touching a line was excluded from the count. Data are represented as a sum of all cells counted through the 3 sections.
Fig. 7 Decreased neuronal activation in the amygdala of Grn+/− mice. A, Regions counted for c-Fos– and NeuN-positive neurons, including central amygdala (CeA), caudate/putamen (CPu), and dentate gyrus (DG). B–D, Active c-Fos–positive (more ...)
All experiments were conducted by observers blinded to genotype. All data represent means ± SEM. Data were analyzed using GraphPad Prism or SPSS, and statistical comparisons were made with ANOVA with Dunnet's post-hoc test for multiple comparisons unless otherwise stated. A p < 0.05 was considered significant. For the tube test of social dominance, statistical comparisons were made with a two-tailed binomial test against an expected outcome (50%). Outliers more than two standard deviations from the mean were excluded for all behavioral assays.