Disruption of ArhGAP15 results in hyperactive Rac1, affects the architecture and function of hippocampal inhibitory neurons and causes cognitive deficits

During brain development, the small GTPases Rac1/Rac3 play key roles in neuronal migration, neuritogenesis, synaptic formation and plasticity, via control of actin cytoskeleton dynamic. Their activity is positively and negatively regulated by GEFs and GAPs molecules, respectively. However their in vivo roles are poorly known. The ArhGAP15 gene, coding for a Rac-specific GAP protein, is expressed in both excitatory and inhibitory neurons of the adult hippocampus, and its loss results in the hyperactivation of Rac1/Rac3. In the CA3 and dentate gyrus (DG) regions of the ArhGAP15 mutant hippocampus the CR+, PV+ and SST+ inhibitory neurons are reduced in number, due to reduced efficiency and directionality of their migration, while pyramidal neurons are unaffected. Loss of ArhGAP15 alters neuritogenesis and the balance between excitatory and inhibitory synapses, with a net functional result consisting in increased spike frequency and bursts, accompanied by poor synchronization. Thus, the loss of ArhGAP15 mainly impacts on interneuron-dependent inhibition. Adult ArhGAP15−/− mice showed defective hippocampus-dependent functions such as working and associative memories. These findings indicate that a normal architecture and function of hippocampal inhibitory neurons is essential for higher hippocampal functions, and is exquisitely sensitive to ArhGAP15-dependent modulation of Rac1/Rac3.

The reaction was continued for 3-10 hrs, depending on signal intensity, at 32°C in dark. Subsequent to Xgal staining, sections were mounted on slides.

Pull-down assay for Rac1/Rac3 activity
Rac1/Rac3 activity was measured by pull-down assay. A glutathione-S-transferase-PAK-CD (PAK-CRIB domain) fusion protein, containing the Rac binding region from human PAK1, was used to determine Rac activity. Lysates of the embryonic cortices were centrifuged at 4°C for 10 min at 13,000 RPM, and the supernatant was incubated with glutathione S-transferase PAK glutathione-coupled Sepharose 4B beads (GE Healthcare) for 30 min. at 4°C. Proteins bound to the beads were washed 3 times in lysis buffer, and then quantified by polyacrylamide gel electrophoresis and Western Blot analysis using a mouse anti-Rac1 antibody (clone 23A8, Upstate Biotech., used 1:2000) which, however, also recognizes Rac3 in Western blot analyses (data not shown). Images were quantified by densitometric analysis using Quantity One software (BioRad, CA, USA).

Analysis of neuritogenesis and neuronal morphology
Neuritogenesis was assayed by measuring the following parameters: the length of the longest neurite, the number and distribution of branches (indicating the arborization rate) and the number of secondary neuritis departing from the main neurite of at least 150 GFP+ neurons per genotype. To perform this analysis, we used basic functions of ImageJ software. In addition, the global arborization and complexity of each neuron was assessed by "Sholl Analysis", done on a 100 GFP+ neurons per genotype: this computer-assisted method uses a series of concentric circles around the soma of each neuron, and determines how many times its neurites intersect each of these circles (plugin ImageJ), expressed as a mean value.
For neuronal morphology, a minimum of 150 GFP+ neurons were photographed and classified as unipolar, bipolar and multipolar, according to the number of visible primary neuritis departing from the soma. The analysis was done using the CellCounter plugin of ImageJ. Finaly, for the multipolar neurons the number of primary neurites was also counted.
All statistical analyses were done with the Student's T-test.

Electrical recording from primary cultures
WT and ArhGAP15 -/hippocampi (E18) were enzymatically dissociated and plated at density 1200 cells/mm 2 on poly-L-lysin/laminin coated MEA devices, and maintained for up to 18 DIV in neurobasal medium supplemented with 1% Penn/Strep, 1% Glutamine, 2.5% FBD, 2% B-27 neurobasal, in a humidified 5% CO 2 atmosphere at 37°C. One third of the culture medium was changed once a week. To examine synchronicity, cross-correlation probability vs. time diagrams were constructed by means of the Neuroexplorer software, using ± 0.5 s and ± 3.5 s and 5 ms bin size. Data are expressed as means ± S.E.M. and statistical significance was calculated with the Student's unpaired T-test.

Learning and memory tests
The dark-light box test for anxiety-like behaviour was carried out using a 20 x 30 x 20 cm chamber with transparent Perspex walls and open top was connected to a 20 x 15 x 20 cm plastic dark box that was completely enclosed except for a 7.5 x 7.5-cm connection door. The transparent chamber was illuminated by direct light (500 lux). Each mouse was released in the middle of the lit transparent chamber and tracked for 5 min.
For the emergence test, frames of non-reflective black Perspex walls (37-cm high) were used to partition a round open field arena (diameter of 150 cm and 35-cm high walls) into four square 50×50 cm arenas, allowing for concurrent tracking of four animals at one time. Illumination in the room was provided by indirect diffuse light (4x40-W bulbs, 12 lx). The day before the test a plastic box (12x8x4 cm with an aperture of 8x4 cm) was inserted in each mouse's home cage. The same box was then placed in a corner of the arena the testing day. Each box was placed 5 cm from the central walls' corner, with the opening facing the centre of the arena. The mice were released in the centre and tracked for 30 min.
For the water maze test, mice were trained in a circular pool (150 cm diameter and 50 cm height) according to standardized protocols (Wolfer DP and HP Lipp, 1992). The wire-mesh platform was 14x14 cm. In the hidden-platform version of the water maze, mice had to locate a hidden platform in a fixed position. The test included an acquisition phase (18 trials, 6/day, intertrial time 30-40 min) followed by a reversal phase during which the platform was moved to the opposite position (12 trials, 6/day). For the analysis the trials were averaged in blocks of two trials.
The following measures were calculated to assess acquisition: escape latency, swim speed, time floating, wall hugging and the percentage of time in the current quadrant goal (excluding episodes of floating). Spatial selectivity during the probe trial was quantified using the following parameters: For the eight-arms radial maze test, the apparatus consisted of eight arms (38 cm long, 7 cm wide) extending from an octagonal centre platform (diameter 18.5 cm) with 5-cm transparent plastic walls. The distance from the platform centre to the end of each arm was 47 cm. At the end of each arm is present a cup with a food pellet. Food-deprived mice (maintained at 85% of their freefeeding weight) were were placed in the centre platform and allowed to collect pellets placed at the end of each arm for 10 min. The animals were adapted to the maze for 1 day and then tested for 10 days. For each trial, the total number of arm choices, number of correct choices before the first error, total numbers of errors were recorded.
The trace fear conditioning tests were performed by placing the mice in an opaque conditioning chamber (LxWxH: 25x17x23 cm) with a grid floor through which scrambled foot shocks could be delivered as unconditioned stimuli (US; 0.26 mA average intensity). The chamber was placed into a dimly lit (<5 lux) sound attenuating box (background noise level 55 dB), and a speaker on top of the chamber allowed to deliver sounds as conditioning stimuli (CS; 2000 Hz). All mice were pre-exposed to the test chamber for 10 min on the day preceding conditioning. On day two, the training session, 5 trials were presented: the first trial was preceded by a 60-sec baseline period (no tone), followed by the presentation of a 15-sec tone (CS, 85 dB 2000 Hz) and 15-seconds later the CS offset (which was the trace) by a 2-seconds foot-shock (US, 0.2 mA). After US offset there was a 60-sec interval here regarded as inter trial interval (ITI) before the next trial begun.
Twenty-four hrs after fear conditioning, mice were placed again in the conditioning box and scored for freezing behaviour in both the context test (2 min without CS "context induced freezing") and subsequently in the cue test (new environment, 2 min divided in: first min as baseline and second min with CS "Cue induced freezing"). During the test, animals were continuously video-tracked using the ANY-maze system (Anymaze, Stoelting Co, Wood Dale, IL, USA, www.anymaze.com).
The frequency of freezing (absence of movements except respiration) was continuously recorded.
During the tests, animals were video-tracked using the EthoVision 2.3 system (Noldus Information Technology, Wageningen, the Netherlands, http://www.noldus.com) using an image frequency of 4.2/s. Raw data were transferred to Wintrack 2.4 (http://www.dpwolfer.ch/wintrack) (Wolfer and Lipp 1992)for off-line analysis. Statistical computations were done using Statview 5.0 (SAS Institute, Cary, NC, USA, www.statview.com). ANOVA was used to compare genotype effect. Repeated ANOVA was used to compare genotype effect across different sessions and to check session dependence of mutation effect, "session" was included as an additional betweensubject ANOVA factor.