Physical Exercise Prevents Stress-Induced Activation of Granule Neurons and Enhances Local Inhibitory Mechanisms in the Dentate Gyrus

Immediate early gene expression in GABAergic interneurons of the dentate gyrus.

To test whether GABAergic interneurons are differentially affected by cold water swim stress in runners compared with sedentary mice, IEG expression was examined in mice exposed to a running wheel or sedentary living for 6 weeks. After a 24 h break from running, mice were subjected to cold water swim stress or no stress for 5 min as described above. A 2 h poststress survival period was used for this experiment to maximize IEG expression in inhibitory neurons (Peng and Houser, 2005). After this time, mice were perfused as described above and their brains were processed for markers of inhibitory interneurons—GAD67, a global marker of GABAergic cells, or parvalbumin (PV), a marker of basket cells and chandelier cells, interneurons known to exert a profound inhibitory influence over granule cells, combined with c-fos. Previous studies (Vazdarjanova et al., 2006; Mainardi et al., 2009) as well as our preliminary data have shown that inhibitory interneurons do not express arc, so only c-fos was used for this experiment.

Extracellular GABA release in the ventral hippocampus.

To test whether runners and sedentary mice differ in stress-induced release of GABA in the ventral hippocampus, chronic guide shafts were implanted in the ventral hippocampus of mice as described below before 6 weeks of running or sedentary living. Extracellular microdialysis samples were obtained (Rada et al., 2010) before, during, and after cold water swim stress at 30 s intervals. These samples were analyzed for GABA content using micellar electrokinetic chromatography (MEKC) (Skirzewski et al., 2011) as described below.

Anxiety-like behavior.

To determine whether blockade of GABAA receptors, an abundant GABA receptor in the hippocampus (Atack, 2011), can eliminate the anxiolytic action of running, runner and sedentary mice were subjected to implantation of cannulae in the ventral hippocampus after 3 weeks of running. After 1 week of recovery from surgery, the GABAA receptor antagonist bicuculline or vehicle was infused directly into the ventral hippocampus. Ten minutes following injection, mice were tested on the elevated plus maze as described below (Cornélio and Nunes-de-Souza, 2007; Rezvanfard et al., 2009). A shorter period of running was used for this study because 3 weeks of running was sufficient to produce an anxiolytic response comparable in magnitude to that of 6 weeks of running. For comparison purposes, a similar experiment was conducted involving infusions of bicuculline or vehicle into the dorsal hippocampus after 3 weeks of running. It should be noted that, unlike the experiments included in this report, our pilot studies included bicuculline infusions into the hippocampus of sedentary mice. Many of the sedentary mice in our pilot studies had obvious seizures after bicuculline infusion while none of the runner mice showed this effect. Because we could not assess anxiety-like behavior in mice with seizures, we did not include bicuculline infused sedentary groups in our subsequent studies. Bicuculline has been shown to induce seizures (Freund et al., 1987) in control (sedentary) mice and our data raise the possibility that running increases the seizure threshold in the hippocampus.

Behavioral manipulations and measures.

For cold water swim stress, mice were placed in rectangular containers (20 × 20 × 20 cm) filled with ice water (∼5°C) for 5 min. After 5 min, mice were taken out of the cold water, dried with paper towel, and returned to their home cage until perfusion.

For anxiety testing at 3 and 6 weeks after running, the elevated plus-maze, a common test of anxiety-like behavior in rodents, was used (Lister, 1987; Walf and Frye, 2007). Mice were placed on an elevated (50 cm) plus-shaped track. Two of the arms had high walls (30 cm) while the other two arms and central intersection were open. All arms were 50 cm in length, dimly lit by reflected light. Exploratory behavior was measured for 5 min and analyzed. Number of entries into the open and closed arms, and time spent in the open arms were measured for each mouse. Because the open arms are more exposed to the light and open air, more time spent in the open arms is considered a measure of reduced anxiety (Heldt et al., 2012; O'Leary et al., 2013; Pritchard et al., 2013; Zheng and Rinaman, 2013). Total entries into both closed and open arms are considered a measure of locomotion (File and Aranko, 1988; Clénet et al., 2006; Hayase, 2013).

Craniotomy.

Mice were anesthetized with ketamine/xylazine, shaved and placed in a stereotaxic instrument (David Kopf Instruments). Cannulae were implanted using the following coordinates: for ventral hippocampus, 3.16 mm posterior from bregma, ±3.0 mm lateral to midline, and 4.2 ventral from the dura; for dorsal hippocampus, 2.0 mm posterior from bregma, ±2.0 mm lateral to midline, and 1.5 ventral from the dura (Adhikari et al., 2010). Bilateral cannulae were inserted at depths of 0.5 mm ventral to dura in an effort to prevent excessive damage and injector needles were lowered the remaining depth during microinfusion. Surgical screws were inserted in the skull around the guide cannulae, and the cannulae (26 ga, 5 mm pedestal, 3 mm tubing length; Plastics One) were lowered into place. Dental acrylic was applied to stabilize and seal the wound and dummy caps (Plastics One) were used to block cannulae from clogging.

Drug preparation and microinfusion.

Bicuculline (National Cancer Institute; #32192/2), a GABAA receptor antagonist (Curtis et al., 1970), was dissolved in 0.1 m HCl:saline, and titrated with 0.1 m NaOH:saline until a pH of 5.6 was achieved (de Feo et al., 1985), then diluted with saline until the concentration was 1 mg/ml. Mice were manually restrained and vehicle or bicuculline was injected using a Hamilton syringe through guide cannulae with internal injectors (33 ga, Plastics One) at a rate of 0.5 μl/min for 1 min for a total volume of 1 μl per brain. After the experiment was over, mice were perfused and cannulae placement was verified.

Microdialysis.

Mice were anesthetized and a guide shaft was implanted aimed at the ventral hippocampus (coordinates: 3.16 mm posterior from bregma, 3.0 mm lateral to midline, and 4.2 mm ventral from the dura). Concentric microdialysis probes were constructed in the laboratory as previously described (Rada et al., 2010). Mice had probes lowered through the guide shaft into the ventral hippocampus and fixed in place 14 h before experimentation. Probes were perfused with a modified Ringer's solution (136.0 mm NaCL, 3.7 mm KCl, 1.2 mm CaCl₂, 1.0 mm MgCl₂, and 10.0 mm NaHCO₃ at pH 7.4) at a flow rate of 1 μl/min. Each sample was collected during 30 s, taking 3 samples before the stressor (baseline) followed by 3 samples throughout the stressor, and 5 samples after the end of stressor. Microdialysis samples were analyzed using MEKC (Rada et al., 1999; Skirzewski et al., 2011). Samples were prepared by mixing a 66 μm solution of fluorescein (Sigma) in 20 mm carbonate buffer at pH 9.4 with a 2.5 mm solution of fluorescein isothiocyanate isomer-I (FITC) (Sigma) in acetone in a 1:1 (v/v) ratio. The samples reacted overnight (16 h) in dark at room temperature and in a water saturated chamber to avoid evaporation. Ten microliters of derivatizing solution were mixed with 1 ml of a 9.7 mm GABA solution to react overnight. We used a Meridialysis capillary zone electrophoresis system (Model R2D2, Mérida) equipped with an argon laser (20 mW) (Omnichrom) tuned to 488 nm. A fiber optic conducted the laser beam onto a dichroic mirror centered at 510 nm and focused on the window of a capillary (Polymicro Technologies) by a 0.85-numeric aperture objective. The window was made by burning out the polyimide coating at 35 cm from the anodic end of a 45 cm long capillary, with a 26 μm internal diameter and 358 of outside diameter, fused-silica capillary. The fluorescence was captured through the objective and focused on a multialkali photomultiplier tube.

The running buffer was a 40 mm borate buffer with 20 mm SDS. The sample was loaded at the anodic end by a negative pressure (12 p.s.i. for 0.5 s) applied at the cathodic end of the capillary. Electrophoretic separation was obtained by applying 28 kV between the anode and cathode for 25 min. Before each run, the capillary was rinsed with 1 m NaOH for 5 min followed by water for 5 min and running buffer for 5 min. Data were normalized as the percentage of baseline. After the experiment was over, mice were perfused and the brains examined for probe placement.

Immunohistochemistry.

Coronal sections (1:8) were cut at 40 μm throughout the entire hippocampus with a Vibratome. For BrdU peroxidase staining, sections were mounted onto slides and allowed to dry overnight. Sections were pretreated by heating in 0.1 m citric acid, pH 6.0. Slides were rinsed with PBS, digested in trypsin for 10 min, rinsed, denatured in 2N HCl:PBS for 30 min, rinsed, incubated with mouse anti-BrdU antibody (1:250 with 10% Tween 20; BD Bioscience), and stored at 4°C. The following day, slides were rinsed with PBS, incubated in biotinylated horse anti-mouse (1:200; Vector) for 60 min, rinsed, incubated in avidin-biotin complex for 60 min, rinsed, and reacted with diaminobenzidine (DAB) for 6 min.

For c-fos and arc protein peroxidase immunostaining, free-floating sections were rinsed in PBS, incubated in 0.3% H₂O₂ in absolute methanol for 20 min to block endogenous peroxidase activity, rinsed, incubated in 3% normal goat serum (Vector) to prevent nonspecific binding, transferred directly into rabbit anti-c-fos (1:1000; Santa Cruz Biotechnology) or rabbit anti-arc (1:1000; Synaptic Systems) antibody and stored at 4°C. Two days later, tissue was rinsed, incubated in biotinylated goat anti-rabbit (1:200; Vector) for 60 min, rinsed, incubated in avidin-biotin complex for 60 min, rinsed, and reacted with DAB for 10 min. Tissue was mounted to suprafrost slides and allowed to dry for a minimum of 2 h. All peroxidase tissue was counterstained with 0.5% cresyl violet and coverslipped under Permount (Fisher Scientific).

For combined BrdU-IEG immunofluorescence, free-floating sections were rinsed in 0.1 m TBS, pH 7.5, denatured in 2N HCl:TBS for 30 min, rinsed, and incubated with rat anti-BrdU (1:200 with 0.5% Tween 20; Accurate Chemical) plus rabbit anti-c-fos (1:200; Santa Cruz Biotechnology) or rabbit anti-arc (1:1000; Synaptic Systems) and stored at 4°C. Three days later, sections were rinsed, incubated in biotinylated goat anti-rat (1:250; Millipore Bioscience Research Reagents) for 90 min, rinsed, and incubated in streptavidin-conjugated Alexa 568 (1:1000; Invitrogen) to visualize BrdU and goat anti-rabbit Alexa 488 (1:500; Invitrogen) to visualize c-fos and arc, in the dark for 60 min. Sections were rinsed and mounted onto suprafrost slides, and coverslipped using glycerol in TBS (3:1). Slides were kept in dark at 4°C until microscopic analyses.

For interneuron-IEG immunofluorescence, free-floating sections were rinsed in 0.1 m TBS, pH 7.5, and incubated with mouse anti-PV (1:1000, Sigma) or mouse anti-GAD67 (1:2000; Millipore) plus rabbit anti-c-fos (1:200 with 0.5% Tween 20;Santa Cruz Biotechnology), and stored at 4°C. Arc was not used because inhibitory interneurons in the hippocampus do not express this immediate early gene (Vazdarjanova et al., 2006; Mainardi et al., 2009). Two days later, sections were rinsed, incubated in goat anti-mouse Alexa 568 (1:500; Invitrogen) to visualize interneurons and goat anti-rabbit Alexa 488 (1:500; Invitrogen) to visualize c-fos, in the dark for 60 min. Sections were rinsed and mounted onto suprafrost slides, and coverslipped using glycerol in TBS (3:1). Slides were kept in dark at 4°C until microscopic analyses.

For vGAT or vGLUT immunofluorescence, free-floating sections were rinsed in 0.1 m TBS, pH 7.5, incubated in 3% normal goat serum (Vector) to prevent nonspecific binding, and incubated with guinea pig anti-vGLUT1 (1:5000; Millipore) or rabbit anti-vGAT (1:500, Synaptic Systems) with 0.5% Tween 20 and stored at 4°C. Three (vGLUT1) or one day(s) (vGAT) later, sections were rinsed, incubated in goat anti-guinea pig Alexa 488 (1:500; Invitrogen) to visualize vGLUT1 or goat anti-rabbit Alexa 488 (1:500; Invitrogen) to visualize vGAT, in the dark for 120 min. Sections were rinsed and mounted onto suprafrost slides, and coverslipped using glycerol in TBS (3:1). Slides were kept in dark at 4°C until microscopic analyses.

Microscopic data analysis.

Slides were coded until completion of the data analysis. Sections through the hippocampus were designated dorsal or ventral before analyzing (Banasr et al., 2006). Other hippocampal regions (CA1 and CA3) were designated using the Allen Brain Atlas (http://mouse.brain-map.org/). For the dentate gyrus, estimates of the total number of BrdU, c-fos, and arc-labeled cells in the granule cell layer were determined using peroxidase stained tissue. For CA1 and CA3 regions, estimates of the total number of c-fos and arc-labeled cells were determined throughout the pyramidal cell layer. Labeled cells were counted in one hemisphere and counts were multiplied by 16 to estimate the number of labeled cells in the entire area.

All BrdU+, PV+, or GAD67+ cells on every eighth section through the entire dentate gyrus, including the granule cell layer, hilus, and molecular layer, were examined for colabeling with c-fos or arc with a Zeiss Axiovert confocal laser-scanning microscope (510 LSM; lasers, argon 458/488, and HeNe 543). Each possible double-labeled cell was scanned by obtaining optical stacks of 1-μm-thick sections and analyzed. To verify double-labeling, cells were examined in orthogonal planes. For optical intensity measurements of vGAT and vGLUT, confocal analysis was performed as previously described (Singer et al., 2011). Ten representative 1 μm optical slices from the granule cell layer and molecular layer of the dorsal and ventral dentate gyrus were scanned and measured for optical intensity from each brain. Raw intensity values were divided by average intensity measurements from the fornix of each brain to control for variability in staining intensity between brains.