Animals
All experimental procedures were performed under a protocol approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham. Mice were housed 5–7 per cage on a 12 h light/dark cycle and had ad libitum access to food and water. Both male and female mice were used. ROCK2fl/fl mice were generated as follows: C57BL/6N-Rock2tm1a(KOMP)Wtsi mice were made from ES cells purchased from the International Mouse Phenotyping Consortium at the University of California, Davis. ES cell injections were performed by the UAB Transgenic & Genetically Engineered Models Core. The C57BL/6N-Rock2tm1a(KOMP)Wtsi ES cells used for this research project were generated by the trans-NIH Knock-Out Mouse Project (KOMP) and obtained from the KOMP Repository (www.komp.org). NIH grants to Velocigene at Regeneron Inc (U01HG004085) and the CSD Consortium (U01HG004080) funded the generation of gene-targeted ES cells for 8500 genes in the KOMP Program and archived and distributed by the KOMP Repository at UC Davis and CHORI (U42RR024244).
To generate ROCK2fl/fl mice, C57BL/6N-Rock2tm1a heterozygous mice were crossed to B6N.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/J (JAX Stock No: 016226) to remove the KOMP selection cassette and generate C57BL/6N-Rock2tm1c mice (Fig. 1a). To remove the FLP1 transgene, C57BL/6N-Rock2tm1c heterozygous mice (ROCK2fl/−) were crossed with C57BL/6NJ (JAX Stock No: 005304) for two generations and absence of the FLP1 transgene was confirmed by PCR genotyping. Then, ROCK2fl/− mice were interbred to generate ROCK2fl/fl mice. ROCK2fl/fl mice were crossed with B6.Cg-Tg(Camk2a-cre)T29-1Stl/J (JAX Stock No: 005359) (CaMKII-Cre) for one generation to generate Cre:ROCK2fl/− mice, followed by interbreeding of offspring to yield Cre:ROCK2fl/fl mice or ROCK2fl/fl littermate controls (Fig. 1a). Henceforth, all experimental mice were on a mixed B6J; B6N genetic background. Mice were genotyped using DNA extracted from tail biopsies and PCR with gene-specific primers. All mice were 8–9 months old (unless otherwise indicated in the figure legend), and both males and females were used in each experiment. No statistically significant differences in sex were observed; therefore, all data is a combination of male and female mice (details are given in the figure legends). All experimenters were blind to genotypes until the conclusion of the study.
For experiments to evaluate the range of CaMKII-Cre expression in our colony, CaMKII-Cre mice, that were being used to mate with ROCK2fl/fl mice, were crossed with B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (JAX Stock No: 007909). Offspring were used for experiments to observe the red fluorescent protein variant (tdTomato) and assess spread of Cre expression in the forebrain (Fig. 1b).
Behavioral assessment
Mice were allowed to acclimate to the testing room for approximately 1 h prior to testing. All testing was performed at the same time each day within each cohort of animals. The apparatus was disinfected with 2% chlorhexidine prior to testing and cleaned with 70% ethanol between animals.
Elevated plus maze
The elevated plus maze apparatus (EPM; Med Associates) was 1 m high with 2 in wide arms. Two opposite arms had 8 in high black walls, while the other two opposing arms were open. Each mouse was placed one at a time in the center of the maze and the mouse was allowed to freely explore for 5 min. Exploration into arms was recorded and traced by the manufacturer’s software (CleverSys). Percent time in open arms was calculated by dividing the time in open arms by total time.
Light dark box test
The light dark box test was performed in a rectangular plexiglass box consisting of two compartments (one light, one dark). A removable dark partition was used to divide the box into a light and dark side. Mice were placed in the light compartment facing away from the dark partition and allowed to explore both compartments for 5 min. Activity was recorded and tracked with video tracking software (Cleversys).
Open field test
Mice were placed one at a time into a 16 in × 16 in plexiglass box (Med Associates) with opaque walls. Mice explored for 10 min. Ambulatory distance and ambulatory counts were determined by the manufacturer’s software (CleverSys).
Passive avoidance test
A 3-day passive avoidance protocol was employed. On the first day, mice were habituated to the apparatus, which consisted of two compartments (one light, one dark) separated by a guillotine door. Mice were placed in the illuminated compartment, with the guillotine door closed, and allowed to explore for 30 s. The guillotine door was opened, and latency to enter the dark compartment was recorded. An event was considered an entry only when all four paws were in the dark compartment, and at this point, the door was closed to confine the mouse to the dark compartment. On the second day, mice were again placed in the illuminated compartment for 30 s, after which the guillotine door was opened. Two seconds after entry into the dark compartment, mice received a 0.5 mA foot shock, which lasted for 2 s. After 30 s, mice were removed from the apparatus. Latency to enter the dark compartment was recorded. On the third day, mice were placed in the illuminated compartment. After 30 s, the guillotine door was opened. Latency to enter the dark compartment was recorded, with a maximum of 5 min. Any mice that did not enter the dark compartment were assigned a latency of 5 min.
Perfusions and brain tissue processing
For iontophoretic microinjections, mice were anesthetized with Fatal Plus (Vortech Pharmaceuticals, Catalog #0298-9373-68) and transcardially perfused with cold 1% paraformaldehyde (PFA; Sigma Aldrich, Catalog #P6148) for 1 min, followed by 4% PFA with 0.125% glutaraldehyde (Fisher Scientific, Catalog #BP2547) for 10 min. A peristaltic pump (Cole Parmer) was used for consistent administration of cold PFA. Immediately following perfusion, each brain was extracted and drop-fixed in 4% PFA with 0.125% glutaraldehyde for 8–12 h at 4 °C. After fixation, brains were coronally sliced in 250 µm thick sections using a Leica vibratome (VT1000S, speed 70, frequency 7). Brains were sliced in 0.1 M PB and stored at 4 °C, one slice per well in a 48-well plate, in 0.1 M PB with 0.1% sodium azide (Fisher, Catalog #BP922I).
For tdTomato experiments, mice were anesthetized with Fatal Plus and transcardially perfused with cold 1× phosphate-buffered saline (PBS) for 2 min. Immediately following perfusion, each brain was extracted and dissected into two hemispheres. Both hemispheres were placed in 0.1 M PB with 0.1% sodium azide and stored at 4 °C. Right hemispheres were sliced in 50 µm thick sagittal sections using a Leica vibratome (VT1000S, speed 70, frequency 7). Brains were sliced in 0.1 M PB and stored at − 20 °C in cryoprotectant solution with 0.01% sodium azide (300 g sucrose, 500 mL 0.1 M phosphate buffer, 300 mL ethylene glycol, 0.1 g sodium azide, bring to 1 L with phosphate buffer). Sagittal sections were washed in 5 µL of 4ʹ,6-diamidino-2-phenylindole (DAPI) per 1.5 mL of 1× PBS for 15 min. Finally, slices were mounted on glass slides using Vectashield mounting medium (Vector Laboratories, Catalog#H-1000), sealed with clear nail polish, and stored at 4 °C.
For biochemistry, mice were anesthetized with Fatal Plus and transcardially perfused with cold 1× phosphate-buffered saline (PBS) for 2 min. Immediately following perfusion, each brain was extracted and dissected into two hemispheres. Both hemispheres were flash frozen in 2-methylbutane (Sigma, Catalog #320404), placed on dry ice, and stored at − 80 °C.
Iontophoretic microinjections
Iontophoretic microinjections were performed based on methods described previously [27, 31]. A Nikon Eclipse FN1 upright microscope with a 10× objective and a 40× water objective was placed on an air table. The tissue chamber used consisted of a 50 × 75 mm plastic base with a 60 × 10 mm petri dish epoxied to the base. A platinum wire was attached so that the ground wire could be connected to the bath by an alligator clip. The negative terminal of the electric current source was connected to a glass micropipette filled with 2 µL of 8% Lucifer yellow dye (ThermoFisher, Catalog#L453). Micropipettes (A-M Systems, Catalog #603500) with highly tapered tips were pulled fresh at the time of use. A manual micromanipulator was secured on the air table with magnets that provided a 45° angle for injection. Brain slices were placed into a small petri dish containing 1× PBS and DAPI for 5 min at room temperature. After incubation in DAPI, slices were placed on dental wax, and then a piece of filter paper was used to adhere the tissue. The filter paper was then transferred to the tissue chamber filled with 1× PBS, and weighted down for stability. The 10× objective was used to visualize advancement of the tip of the micropipette in X, Y, and Z planes until the tip was just a few micrometers above the tissue. The 40× objective was then used while advancing the tip into layer 2/3 of the mPFC, dorsal and ventral CA1 regions of the hippocampus, or the basolateral amygdala. Once the microelectrode contacted a neuron, 2 nA of negative current were applied for 5 min to fill the neuron with Lucifer yellow. After 5 min, the current was turned off and the micropipette was removed from the neuron. Neuron impalement within the mPFC, CA1, and basolateral amygdala occurred randomly in a blind manner. If the entire neuron did not fill with dye after penetration, the electrode was removed and the neuron was not used for analysis. Multiple neurons were injected in each hemisphere of each animal. After injection, the filter paper containing the tissue was moved back into the chamber containing 1× PBS. The tissue was carefully lifted off the paper and placed on a glass slide with two 125 µm spacers (Electron Microscopy Sciences, Catalog #70327-20S). Excess PBS was carefully removed with a Kimwipe, and the tissue was air-dried for 1 min. One drop of Vectashield (Vector Labs, Catalog #H1000) was added directly to the slice, and the coverslip (Warner, Catalog #64-0716) was added and sealed with nail polish. Injected tissue was stored at 4 °C in the dark.
Confocal microscopy
Confocal microscopy was used to capture images of Lucifer yellow-filled dendrites on layer 2/3 pyramidal neurons in the mPFC, pyramidal neurons in CA1 of the hippocampus, and amygdala neurons. Our methods are based on previously described methods [27, 31], and are detailed as follows. Imaging was performed by a blinded experimenter on a Nikon (Tokyo, Japan) Ti2 C2 confocal microscope, using a Plan Apo 60×/1.40 NA oil-immersion objective. Three-dimensional z-stacks were obtained of secondary dendrites from dye-impregnated neurons that met the following criteria: (1) within 80 µm working distance of microscope, (2) relatively parallel with the surface of the coronal section, (3) no overlap with other branches, (4) located between 40 and 120 µm from the soma. Nikon Elements 4.20.02 image capture software was used to acquire z-stacks with a step size of 0.1 µm, image size of 1024 × 512 px, zoom of 4.8×, line averaging of 4×, and acquisition rate of 1 frame/s.
Confocal microscopy was also used to capture images of slices from Cre/tdTomato and Cre-negative/tdTomato littermates. Imaging was performed on a Nikon Ti2 C2 confocal microscope, using a Plan Fluor 10×/0.75 NA air objective. Nikon Elements 4.20.02 image capture software was used to acquire stitched images (20 × 10 field) by automatically stitching multiple adjacent 10× images using 25% blending overlap. Each frame had an image size of 1024 × 1024 px, and acquisition rate of 1 frame/s. Subsequently, 10× stitched images were acquired, with all other imaging parameters remaining the same. Following image acquisition, post-image processing was performed using Fiji ImageJ. Each image file was a two channel image. The Split Channels function was used to separate the two channels. Brightness was adjusted for each channel separately, and then the Merge Channels function was used to overlay the DAPI and TRITC images.
Dendritic spine morphometry analysis
Confocal z-stacks of dendrites were deconvolved using Huygens Deconvolution System (16.05, Scientific Volume Imaging, the Netherlands). The following settings were used: deconvolution algorithm: GMLE; maximum iterations: 10; signal to noise ratio: 15; quality: 0.003. Deconvolved images were saved in .tif format. Deconvolved image stacks were imported into Neurolucida 360 (2.70.1, MBF Biosciences, Williston, Vermont) for dendritic spine analysis. For each image, the semi-automatic directional kernel algorithm was used to trace the dendrite. The outer 5 µm of each dendrite were excluded from the trace. All assigned points were examined to ensure they matched the dendrite diameter and position in X, Y, and Z planes, and were adjusted if necessary. Dendritic spine reconstruction was performed automatically using a voxel-clustering algorithm, with the following parameters: outer range = 5 µm, minimum height = 0.3 µm, detector sensitivity = 80%, minimum count = 8 voxels. The semi-automatically identified spines were examined to ensure that all identified spines were real, and that all existing spines had been identified. If necessary, spines were added by increasing the detector sensitivity. Additionally, merge and slice tools were used to correct errors made in the morphology and backbone points of each spine. Each dendritic spine was automatically classified as a dendritic filopodium, thin spine, stubby spine, or mushroom spine based on constant parameters [27, 31]. Three-dimensional dendrite reconstructions were exported to Neurolucida Explorer (2.70.1, MBF Biosciences, Williston, Vermont) for branched structure analysis, which provides measurements of dendrite length; number of spines; spine length; number of thin, stubby, and mushroom spines, and filopodia; spine head diameter; and spine neck diameter, among other measurements. The output was exported to Microsoft Excel (Redmond, WA). Spine density was calculated as the number of spines per 10 µm of dendrite length. Values per mouse were calculated by averaging the values for all dendrites corresponding to that mouse. To assess apical and basal dendrites separately, values for either apical or basal dendrites were averaged per mouse. For a mouse to be included in statistical analyses for dendritic spine density and morphology a minimum of 100 µm of dendrite length had to be reconstructed and analyzed. The number of neurons, dendrites, dendritic length, and spines analyzed per animal are provided in Additional file 1: Table S1.
Biochemical fractionation and western blotting
Flash-frozen hemispheres were sub-dissected to obtain the prefrontal cortex and hippocampus. Samples were thawed in 75 µL of ice cold TEVP buffer + 320 mM sucrose and homogenized. The homogenate was centrifuged for 10 min at 800×g at 4 °C. The supernatant was saved as a whole homogenate and stored at − 80 °C. Protein concentrations were determined by BCA protein assay (Pierce). GAPDH-enriched and PSD95-enriched fractionations were performed, based on previously described methods [34]. Samples were homogenized in 75 µL of sucrose buffer and centrifuged at 3000×g at 4 °C. The resulting supernatant was collected and centrifuged at 10,000×g at 4 °C. Following centrifugation, the supernatant was saved as the GAPDH-enriched fraction. The remaining pellet was washed in 50 µL of sucrose buffer, resuspended in 60 µL of HBS with 2% Triton X-100 and incubated on ice for 30 min. Followed by, centrifugation at 10,000×g for 20 min at 4 °C. The remaining supernatant was saved as the synaptophysin-enriched fraction and the pellet was resuspended in 30 µL of 1× PBS and saved as the PSD95-enriched fraction. Twenty-five micrograms of protein per sample were loaded into each well for SDS-PAGE. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature. Images were captured using an Odyssey Image Station (Li-Cor), and band intensities were quantified using Odyssey Application Software (3.0, Li-Cor). Primary antibodies: ROCK2 (D1B1) Rabbit mAb (Cell Signaling, Catalog #9029S), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (6C5) Mouse mAb (MilliporeSigma, Catalog #MAB374), Synaptophysin (D35E4) Rabbit mAB (Cell Signaling, Catalog #5461), PSD-95 (D27E11) Rabbit mAb (Cell Signaling, Catalog #3450), Phospho-mTOR (Ser2448) Rabbit mAb (Cell Signaling, Catalog #2971S), mTOR Rabbit mAb (Cell Signaling, Catalog #2972S), AMPA Receptor 1 Rabbit mAb (Cell Signaling, Catalog #13185), Phospho-LIMK Rabbit mAb (Cell Signaling, Catalog #3841), SQSTM1/p62 Rabbit mAb (Cell Signaling, Catalog #5114S), NMDA Receptor 1 Rabbit mAb (Cell Signaling, Catalog #5704), and α-tubulin Mouse mAb clone #12G010 (Developmental Studies Hybridoma Bank, University of Iowa). Secondary antibody: AlexaFluor 680 goat anti-rabbit (Life Technologies, Catalog #A21109) and AlexaFluor 488 donkey anti-mouse IgG (H + L) (Life Technologies, Catalog #A21202).
Statistical analysis
Statistical analyses were conducted using GraphPad Prism 8.4.0 (GraphPad Software, La Jolla, CA). Data are presented as mean ± SEM, and all graph error bars represent SEM. Assuming Gaussian distribution, two-tailed unpaired t-tests with a 95% confidence interval were used for comparison of biochemistry, behavior, and dendritic spine morphometry between ROCK2fl/fl and Cre/ROCK2fl/fl mice. Significance was defined as p < 0.05. ROUT’s method with a false discovery rate specified to 5% was used to identify outliers in biochemistry analysis. To compare spine densities or morphologies among experimental conditions, the mean spine density or morphologic measurement was calculated per experimental replicate (or N). These experiment means were then averaged per experimental condition and reported as a condition mean. See figure legends for details on N per experiment.