All animal procedures were approved by the local Animal Ethics Committee and the Ministry of Health. Animals were maintained according to institutional animal welfare guidelines and legislation, under veterinarian surveillance. The Arhgap15 ^LacZ/LacZ mouse strain has been previously described (Costa et al., 2011; Zamboni et al., 2016). Heterozygous and homozygous mutant mice are born at normal Mendelian frequency, appear overall normal, are viable and fertile, mate at regular rates, and do not show evident neurological or motor impairments. Animals were maintained in a mixed c57/bl6 genetic background. The GAD67-eGFP reporter mouse strain was generated by homologous recombination, introducing the enhanced-GFP (eGFP) cDNA cassette into the murine GAD1 locus, coding for GAD67 (glutamic acid-decarboxylase-67), which is expressed by GABAergic neurons starting from early developmental stages (DeDiego et al., 1994; Sakai and Miyazaki, 1997; Tamamaki et al., 2003). Heterozygous progeny was obtained from chimeric males and backcrossed to the C57BL/6 background. The loxP-flanked PGK-neo cassette, used as a selection marker for screening the recombinant embryonic stem cells, was successfully excised by mating GAD67-eGFP mice with CAG-cre transgenic mice (Tamamaki et al., 2003).

For the collection of postnatal brains, mice were anesthetized with Avertin (30 μl of pure Avertin in 400 μl of PBS) and transcardially perfused with 10 ml of PBS (pH 7.4) and 10 ml of 4% (w/v) PFA in PBS (pH 7.4, adjusted with NaOH). Brains were removed, post-fixed overnight at 4°C in 4% PFA, placed overnight at 4°C in 30% (w/v) sucrose in PBS for cryoprotection, embedded in OCT blocks, and stored at −80°C until analysis. OCT blocks were cut into 30 μm-thick coronal sections using a cryotome (Leica CM 1950). Free-floating sections were collected in PBS in multiwell plates and stored at −20°C in a cryoprotectant solution (30% (v/v) glycerol and 30% (v/v) ethylene glycol in 0.2 M phosphate buffer, pH 7.4) until processed. The range of sections used for analysis corresponds to coronal sections 44–54 of the reference Allen Brain Atlas (Allen Reference Atlas–Mouse Brain [brain atlas]. Available from atlas.brain-map.org). For the analysis, we selected neurons in the somatosensory cortical region. For the collection of embryonic brains, embryos were obtained through cesarean section at E11.5, 12.5, E14.5, or E17.5 (considering the day of the vaginal plug as E0.5) from anesthetized pregnant dams and transferred in PBS. Embryonic brains used for immunohistochemistry were dissected and fixed overnight at 4°C in 4% PFA, then placed overnight at 4°C in 30% (w/v) sucrose in PBS for cryoprotection, embedded in OCT blocks, and stored at -80°C until analysis. OCT blocks were cut into 15 μm-thick coronal sections and collected on super-adhesive glass slides.

Round glass slides were incubated with 80% nitric acid overnight, washed with deionized water several times, sterilized by autoclaving, coated with 1 mg/ml poly-l-lysine (Sigma) in borate buffer (pH 8.5) in a 12-well plate, and washed again with deionized water. One day before establishing the culture, glass slides were rinsed in a MEM (Gibco) solution with 1% (v/v) pyruvate 100X (Gibco), 7% (w/v) glucose, 1% (v/v) Penicillin-Streptomycin, and 10% (v/v) horse serum (Gibco). GAD67-eGFP and Arhgap15 ^LacZ/LacZ ;GAD67-eGFP E15.5 embryos were used to establish primary cultures of cortical neurons. Embryonic heads were dissected in sterile conditions in a cold solution of 1% (v/v) HEPES in HBSS with calcium and magnesium (Gibco). Cortices were dissected free of the rest of the brain, deprived of the meninges, washed with a cold solution containing 1% Penicillin-Streptomycin and 1% HEPES in HBSS with calcium and magnesium (Gibco), and incubated in 500 μl of Trypsine 0.05% (Gibco). Cortices were washed 5 times in HBSS at room temperature and disaggregated in a solution containing DNAase (used 1:1,000; Promega) by pipetting. Cells were counted and 80.000 cells were plated on each glass slide in a 12-well plate containing Neurobasal medium (Gibco) additioned with 1% Penicillin-Streptomycin, 2% (v/v) B27 (Gibco), and 0.25% (v/v) GlutaMAX (Gibco). Neurons were incubated at 37°C in a 5% CO₂ saturation atmosphere.

Brain sections were washed three times in PBS, incubated for 1 h at room temperature with a blocking solution (10% goat or donkey serum and 0.2% Triton-X 100 in PBS), and incubated overnight at 4°C with the primary antibodies diluted in a solution composed of 0.5% Triton-X100 and 5% goat or donkey serum. Then, sections were incubated for 2 h at room temperature with fluorophore-conjugated secondary antibodies diluted in a solution composed of 0.2% Triton-X100 and 3% goat or donkey serum and washed three times in PBS. Finally, sections were counterstained with DAPI and mounted with Mowiol onto super-adhesive glass slides. Double-immunostainings performed using two antibodies from the same host species were performed using the Tyramide SuperBoost™ kit with AlexaFluor™ Tyramides (Invitrogen). Primary cortical cultures were fixed at 3, 10, and 18 days in vitro (DIV) with 4% PFA in PBS for 20 min at room temperature. Neurons were incubated for 1 h at room temperature with a blocking solution containing 5% goat serum and 0.1% Triton-X in PBS. The primary antibody was diluted in a solution containing 3% goat serum and 0.1% Triton-X in PBS and incubated overnight at 4°C. Secondary antibodies were incubated for 1 h at room temperature. Coverslips were mounted with Mowiol onto glass slides. Primary antibodies: rabbit anti-CALB2 (Calretinin; used 1:500; Swant, 7,697), rabbit anti-PVALB (Parvalbumin; used 1:1,000; Swant, PV27), goat anti-SST (Somatostatin; used 1:500; SantaCruz, sc-7819s), rabbit anti-β-GAL (β-Galactosidase; used 1:1,000; MP Biomedicals, SKU:085597-CF), and rabbit anti-VIP (Vasoactive intestinal peptide; used 1:500; Invitrogen, PA5-78224). Secondary antibodies: Alexa Fluor 488 donkey anti-rabbit IgG and AlexaFluor 568 goat anti-mouse IgG (used 1:500; Invitrogen). Slides were examined with a Leica SP8 confocal microscope. Raw images were digitally processed to normalize the background and optimize the contrast, rotated, and sized with ImageJ (NIH, Bethesda, Maryland; http://imagej.nih.gov/ij/). For expression and layering analysis, cortices were divided into 10 horizontal bins of equal thickness (bin one is the outermost and closest to the pia, bin 10 is the innermost and closest to the ventricle). For tangential migration analysis, polar plots were generated by using matplotlib Python library (https://matplotlib.org/stable/#). Morphological analysis on primary cultures was performed using ImageJ (NIH, Bethesda, Maryland; http://imagej.nih.gov/ij/); arborization of each neuron was quantified by performing Sholl analysis (Sholl, 1953) by ImageJ plugin Sholl Analysis Plugin (v1.0), Ghosh Lab Software (http://ghoshlab.org/software/index.html).

300 μm-thick brain slices were prepared by vibratome sectioning from E17.5 GAD67-eGFP and Arhgap15 ^LacZ/LacZ ;GAD67-eGFP embryos. Slices were kept in cold PBS for 20 min, then transferred in Neurobasal medium (Gibco) additioned with 1% Penicillin-Streptomycin, 2% B27 (Gibco), and 0.25% GlutaMAX (Gibco) and maintained in culture for 6 h at 37°C and 5% CO₂. The organotypic slice cultures were imaged, while kept at 37°C and 5% CO₂, by time-lapse video imaging for about 5 h with a frame interval of 5 min using a ×20 objective. The acquired movies were used to determine the migration trajectory of individual CINs. Videos were analyzed using the Manual Tracking plugin of ImageJ (NIH, Bethesda, Maryland; http://imagej.nih.gov/ij/) and Photoshop (Adobe). A virtual grid with lines perpendicular to the SVZ and the pial surfaces was superimposed in each frame. Neurons were considered to be in radial orientation if their leading process (the longest and widest forward branch) formed an angle between 0° and 25° with the perpendicular lines of the grid, as previously done (Martini et al., 2009). The paths were measured by tracking the movements of the leading process of each neuron throughout the entire time-lapse.

Mice were anesthetized with isoflurane (2% (v/v) in 1 L/min O₂). Four screw electrodes (Bilaney Consultants GMBH) were inserted bilaterally through the skull (anteroposterior +2.0–3.0 mm, mediolateral 2.0 mm from bregma). A grounding electrode was placed into the nasal bone. The five electrodes were connected to a pedestal and fixed with acrylic cement (Palavit), as previously described (Manfredi et al., 2009). EEGs were recorded from eighteen (ten wild-type and eight Arhgap15 ^LacZ/LacZ ) freely moving awake animals in a Faraday chamber, using a Power-Lab digital acquisition system (AD Instruments) with a sampling rate of 100 Hz and a resolution of 0.2 Hz. The basal cerebral activity was recorded continuously for 6 h. EEG tracings were analyzed and scored for the presence of rhythmic 4–6 Hz sharp waves of rhythmic spindle-like events (Erbayat-Altay et al., 2008), spikes and solitary spikes followed by slow waves activity, and trains of spikes. Spikes were recognized as having a duration <200 ms with a baseline amplitude set to 4.5 times the standard deviation of the EEG signal (determined during interspike activity periods). Repetitive spiking activity (trains of spikes) was defined by the presence of at least five consecutive spikes in less than 5 s (Berretta et al., 2022). The classification “trains of spikes” was attributed to mice showing at least four events. Spike activity was quantified using LabChart 8 (AD Instruments). Segments with movement artifacts or electrical noise were excluded from statistical analysis.

Eight (five females and three males) P90 mice for each genotype were transferred to individual cages in a quiet room. Atropine (1 mg/kg; Sigma) was administered by intraperitoneal injection to limit the peripheral side effects of pilocarpine. After 30 min, pilocarpine hydrochloride (350 mg/kg, Sigma) was administered by intraperitoneal injection. Animals were monitored every 10 min for 90 min after pilocarpine administration. We used a seizure staging system adapted from the established rodent seizure Racine’s scale (Racine, 1972): stage 0, no abnormality; stage 1, exploring, sniffing, and grooming ceased, becoming motionless; stage 2, forelimb and/or tail extension, appearance of rigid posture; stage 3, myoclonic jerks of the head and neck, with brief twitching or repetitive movements with head bobbing; stage 4, forelimb clonus and partial or occasional rearing and falling; stage 5, forelimb clonus, continuous rearing and falling; stage 6, tonic-clonic movements with loss of posture tone; stage 7, death.

For acute slices, 4 GAD67-eGFP and 4 Arhgap15 ^LacZ/LacZ ;GAD67-eGFP P150 mice were killed by cervical dislocation. Brains were removed and placed at 4°C in oxygenated (95%O₂–5%CO₂) adapted artificial cerebrospinal fluid (ACSF), containing 120 mM choline chloride, 3.5 mM KCl, 0.5 mM CaCl₂, 6 mM MgSO₄, 1.25 mM NaH₂PO₄, 25 mM d-glucose and 25 mM NaHCO₃. Somatosensory cortex coronal slices (300 μm) were cut in ice-cold ACSF using a vibratome (Microm HM 650 V, Thermo Scientific) and subsequently placed for 30 min in ACSF containing 120 mM NaCl, 3.5 mM KCl, 25 mM, NaHCO₃, 25 mM d-glucose, 2.5 mM CaCl₂, 1.3 mM MgSO₄, and 1.25 mM NaH₂PO₄, at 32 °C. Slices were kept at room temperature for at least 1 h before recording. Patch electrodes of borosilicate glasses (Hilgenberg, Mansfield, Germany) were pulled to a final resistance of 5–9 MΩ. For current-clamp recordings in both brain slice and primary cultured neurons, the internal solution contained: 135 mM gluconic acid (potassium salt: K-gluconate), 5 mM NaCl, 2 mM MgCl₂, 10 mM HEPES, 0.5 mM EGTA, 2 mM ATP-Tris, and 0.4 mM Tris-GTP.

Patch-clamp recordings from CINs (somatosensory cortex, layer IV-VI) were performed in whole-cell configuration using an EPC-10 amplifier (HEKA Elektronic, Lambrecht, Germany). Traces were sampled at 10 kHz and filtered using a low-pass Bessel filter set at 2 kHz. All the experiments were performed at room temperature (22–24°C). Resting membrane potential (Vrest) and membrane capacitance (Cm) were routinely acquired when the whole-cell patch-clamp configuration was established. The membrane time constant (tm) was calculated by Clampfit software following a step current injection of −30 pA. The membrane capacitance (Cm) was calculated by applying the formula Cm = τm/Rin.

The action potential (AP) parameters were obtained by analyzing a series of spikes recorded during tonic firing of 1–2 min duration. Tonic firing was elicited by depolarizing membranes with a minimum amount of current corresponding to the rheobase value (Marcantoni et al., 2014). After reaching steady-state condition during tonic firing, at least five APs were selected and averaged for each cell, then the measures of AP peak amplitude, half-width, maximum rising slope, and maximum repolarizing slope were performed with Clampfit software (Axon Instruments). The peak amplitude of AP was measured from the threshold to the AP peak and the half-width was calculated at half-maximal AP height.

To analyze the relationship between firing frequency and injected current, the membrane potential was adjusted to −70 mV and then 20 pulses of increasing intensity (from -30–160 pA, 500 ms duration) were injected. The mean firing frequency of each current step was calculated as the number of spikes per second. The rheobase was determined as the minimum amount of current required to trigger one spike. Input resistance (Rin) was calculated in a linear region of the membrane voltage-injected current curve centered at the holding potential (−70 mV), through the injection of hyperpolarizing and depolarizing current steps (from −30 to 30 pA; 10 pA steps).

For primary neuronal cultures, the extracellular solution for current recordings (Tyrode’s solution) contained: 2 mM CaCl₂, 10 mM HEPES, 130 mM NaCl, 4 mM KCl, 2 mM MgCl₂, and 10 mM d-glucose (Tomagra et al., 2019).

Patch-clamp recordings were performed using an EPC-9 amplifier (HEKA Elektronic, Lambrecht, Germany) and pClamp software (Molecular Devices, Silicon Valley, CA, United States). Analysis of firing activity was performed with Clampfit software (Axon Instruments).

For the statistical comparisons, GraphPad Prism software (GraphPad Software Inc.) was used. For each experiment, the statistical test used is reported in the figure legends. Shapiro-Wilk and Kolmogorov-Smirnov tests were used to test for normality, F test was used to test for equality of variance, and results were evaluated to choose the appropriate statistical test. The results are shown as mean ± Standard Error of Mean (SEM). The threshold for statistical significance was set at p < 0.05.

We previously reported ARHGAP15 expression in the cortex, hippocampus, and olfactory bulbs of early postnatal and adult mice (Zamboni et al., 2016, 2018). To evaluate the expression of ARHGAP15 in embryonic and adult CINs, we examined the expression of the LacZ knock-in reporter in brain sections of Arhgap15 ^LacZ/+ ;GAD67-eGFP animals by immunostaining for β-GAL. LacZ expression was not detected in E11.5 and E12.5 brains (Figure 1A). In sections of E14.5 brains, LacZ expression was detected in 26 ± 0.9%, 23 ± 1.7%, and 18 ± 2% of CINs in the marginal zone (MZ), cortical plate (CP), and intermediate zone (IZ)/subventricular zone (SVZ), respectively (Figures 1B,E). No LacZ expression was detected in the ganglionic eminences, indicating that embryonic CINs start expressing ARHGAP15 only after they start migrating and enter the neocortex (NCX). In sections of early postanal (P2) and adult (P45) brains, LacZ expression was detected in 35 ± 1.3% and 56 ± 2.6% of CINs, respectively (Figures 1C,F,G). To determine the expression of ARHGAP15 in the major CIN subtypes, we carried out double immunostainings for β−GAL and either PVALB, CALB2, SST, or VIP on sections of P45 Arhgap15 ^LacZ/+ brains. The results show that ARHGAP15 is expressed by 57 ± 3%, 53 ± 4%, 53 ± 1%, and 52 ± 1% of PVALB-, CALB2-, SST-, and VIP-positive neurons, respectively (Figures 2A–E). These results indicate that ARHGAP15 starts to be expressed in migrating CINs between the embryonic stages E13.5 and E14.5, and it is expressed by most (about 60%) of adult CINs, not being specific for any of the major CIN subtypes.

RAC1 is critical for neuritogenesis during development (Sayyad et al., 2016). To determine whether loss of ARHGAP15, hence hyperactive RAC1, affects CINs morphology (i.e., neurite elongation and complexity), we examined cultures of dissociated neurons from cortices of E15.5 GAD67-eGFP and Arhgap15 ^LacZ/LacZ;GAD67-eGFP embryos at 3 and 10 DIV. We selected eGFP-positive neurons and examined their neurites length and complexity (Figures 3A,B). At 3 DIV, Arhgap15 ^LacZ/LacZ eGFP-positive neurons displayed a reduced number of branches (Figure 3C). At 3 DIV, but not at 10 DIV, Arhgap15 ^LacZ/LacZ eGFP-positive neurons also showed a reduced length of the longest neurite compared to the GAD67-eGFP (Figures 3D,E). At both 3 and 10 DIV, Arhgap15 ^LacZ/LacZ eGFP-positive neurons showed a reduced number of primary neurites (Figures 3F,G). Moreover, at 10 DIV, but not at 3 DIV, a reduction in soma diameter was also observed in Arhgap15 ^LacZ/LacZ eGFP-positive neurons (Figures 3H,I). Sholl analysis revealed that, at 3 DIV, Arhgap15 ^LacZ/LacZ eGFP-positive neurons showed a lower number of intersections at various distances from the cell body (Figure 3J), indicating a reduced efficiency in neuritogenesis and branching, whereas at 10 DIV they showed a lower number of intersections in the region more proximal to the soma, but a higher number of distal intersections (Figure 3K). These results suggest that Arhgap15 ^LacZ/LacZ CINs fail to undergo an efficient neuritogenesis and branching in the first maturation steps in vitro, but they later develop an increased number and/or branching of secondary neurites, possibly to compensate for their defective number of primary neurites.

To confirm that cultured eGFP-positive neurons express ARHGAP15, we performed immunostainings for β−GAL on primary cultures derived from Arhgap15 ^LacZ/+ ;GAD67-eGFP embryos at 3, 10, and 18 DIV. This analysis showed that about half of eGFP-positive neurons express ARHGAP15 also in culture (Supplementary Figure S1).

Neuronal migration requires extensive cytoskeletal reorganization at the growth cone, a process in which small Rho GTPases play a critical role (Liaci et al., 2021). Since interneurons cover a longer and more complex migratory path as compared to other neurons (Cooper, 2013) and were shown to express ARHGAP15 during their migration from the GE to the NCX (Figure 1B), we looked at their cortical migration. We determined the number and position of tangentially migrating CINs in the cortices of GAD67-eGFP and Arhgap15 ^LacZ/LacZ ;GAD67-eGFP embryos by analyzing coronal sections of E14.5 brains (Figure 4A). No significant changes in the number of tangentially migrating CINs were detected between the two genotypes (Figure 4B). We examined the orientation of eGFP-positive neurons by determining the angle between their leading process and the canonical direction of their tangential migration (i.e., parallel to the pial and ventricular surfaces). In the Arhgap15 ^LacZ/LacZ ;GAD67-eGFP embryonic cortex, the leading process of migrating CINs displayed a mean angle relative to the tangential trajectory significantly wider than that of GAD67-eGFP control neurons (Figure 4C), indicating that Arhgap15 ^LacZ/LacZ CINs tend to deviate from the canonical tangential direction of migration. No differences were observed comparing the length of the leading processes of GAD67-eGFP and Arhgap15 ^LacZ/LacZ ;GAD67-eGFP tangentially migrating CINs (Figure 4D). Thus, the absence of ARHGAP15 alters the control of leading process directionality during tangential migration.

After E15.5, eGFP-positive tangentially migrating neurons present in the MZ and the IZ/SVZ routes have colonized all the cortical areas (Martini et al., 2009). Subsequently, they activate a different migration mode, namely the radial migration, perpendicular to the cortical and ventricular surfaces. Specifically, neurons in the MZ and IZ/SVZ migrate deeply to occupy the CP. Only a small fraction of CINs migrate through the subplate (Tanaka and Nakajima, 2012; Marín, 2013). We looked at CINs trajectory during their radial migration in coronal sections of E17.5 GAD67-eGFP and Arhgap15 ^LacZ/LacZ ;GAD67-eGFP brains. The magnifications of CPs suggested that radially migrating CINs may have altered trajectories with respect to the canonical radial direction (Figure 5A). To precisely determine CINs trajectory in the cortical primordium, we monitored single eGFP-positive CINs by time-lapse video imaging in 300 μm-thick slices of E17.5 GAD67-eGFP and Arhgap15 ^LacZ/LacZ ;GAD67-eGFP cortices maintained in organotypic cultures. We examined the trajectory of GAD67-eGFP and Arhgap15 ^LacZ/LacZ ;GAD67-eGFP CINs after their tangential-to-radial switch and tracked their leading process movements for at least 100 min. Most of them were stationary after 65 min. The criteria used for identifying radially migrating neurons are graphically illustrated in Figure 5B (Martini et al., 2009). We calculated the ratio between the path length and linear distance between the initial and final positions of the leading process of each neuron (Figure 5C, Supplementary Videos S1, 2). We observed that Arhgap15 ^LacZ/LacZ ;GAD67-eGFP neurons deviate from the physiological radial direction of migration significantly more often than GAD67-eGFP (Figure 5D). This result suggests a loss of directional control (or a gain of directional flexibility) in Arhgap15-KO CINs, in accordance with data shown in Figure 4.

Given the alterations in CIN migration described above, we examined the laminar distribution of CINs in WT and Arhgap15 ^LacZ/LacZ adult (P45) cortices. We examined the laminar organization of the most common CIN subtypes in the somatosensory cortical area of WT and Arhgap15 ^LacZ/LacZ cortices, by immunostaining coronal sections for PVALB, CALB2, SST, and VIP. We observed a significant increase in the number of CALB2-positive neurons in bins 5, 6, and 7 of Arhgap15 ^LacZ/LacZ mice (Figures 6A,C). Subtle changes were observed in the distribution of SST- and VIP-positive neurons in bins 10 and 6, respectively (Figures 6A,D,E). No significant alterations were observed in the distribution of PVALB-positive neurons in the absence of ARHGAP15 (Figures 6A,B). We found a slight increase in the total number of CALB2-positive CIN in Arhgap15 ^LacZ/LacZ mice, which, however, does not reach statistical significance. The total number of PV-positive, SST-positive, and VIP-positive CINs is unaltered in the Arhgap15 ^LacZ/LacZ mice. These results show that the stratification of adult CALB2-positive CINs in the forebrain cortex is altered in the absence of ARHGAP15, along with milder alteration in the layering of SST- and VIP-positive cells, probably due to the migratory defects observed.

We previously observed an altered density of excitatory and inhibitory synapses on the soma of Arhgap15 ^LacZ/LacZ cortical pyramidal neurons, and also noticed significant differences between the electroencephalography (EEG) of sleeping WT and Arhgap15 ^LacZ/LacZ animals (Zamboni et al., 2018).

We noted that, occasionally, following moderate stress, Arhgap15 ^LacZ/LacZ mice (5 out of 150) manifested stiffening, leg extension, and absence of movements lasting less than 5 s, which were never seen in WT mice (n > 200), probably manifestations of tonic-clonic convulsions (i.e., spontaneous epileptic seizures). For this reason, we monitored and quantified the cortex-derived electrical activity of free-moving WT and Arhgap15 ^LacZ/LacZ adult (P120) mice. The majority of mutant mice showed an abnormal EEG with epileptiform discharges, usually associated with tonic-clonic convulsions, and/or a series of single spikes, associated with less severe myoclonus (Figure 7A) (Erbayat-Altay et al., 2008). The mean number of spikes per hour was significantly greater in Arhgap15 ^LacZ/LacZ , as compared to WT mice (Figure 7B) (Purtell et al., 2018). A significantly higher fraction of Arhgap15 ^LacZ/LacZ mice exhibited spikes and slow waves activity and/or trains of spikes, as compared to WT mice (spikes and slow waves: 1 out of 10 WT mice, 5 out of 8 KO mice; trains of spikes: 1 out of 10 WT mice, 3 out of 8 KO mice) (Figure 7C). These results indicate that Arhgap15 ^LacZ/LacZ mice show a subclinical epileptic-like phenotype characterized by spike activity.

We tested the susceptibility of Arhgap15 ^LacZ/LacZ mice to acute treatment with pilocarpine, a widely used inducer of epilepsy (Curia et al., 2008). Mice were administered with a single dose of atropine to prevent the peripheral side effects and, after 30 min, with a single dose of pilocarpine (Figure 7D). The epileptogenic activity of the drug was assessed by scoring 7 progressive epilepsy stages, according to the modified Racine scale (Racine, 1972) (Figure 7D). Arhgap15 ^LacZ/LacZ mice exhibited a higher likelihood to enter stages 2, 4, 5, and 6, as compared to control animals (Figure 7E). Moreover, they showed a significantly higher mortality rate at 90 min (stage 7; Figure 7E). These data confirm that the loss of ARHGAP15 increases susceptibility to drug-induced epilepsy.

To determine the impact of ARHGAP15 depletion on CINs electrophysiological properties, we performed whole-cell patch-clamp recordings in acute slices obtained from adult GAD67-eGFP and Arhgap15 ^LacZ/LacZ ;GAD67-eGFP mice. Interneurons showed similar input resistance (R_in) and resting membrane voltage (V_rest) (Figures 8A–C), whereas the membrane capacitance of Arhgap15 ^LacZ/LacZ ;GAD67-eGFP was significantly higher (Figure 8D). We observed a reduced firing rate of Arhgap15-KO CINs in response to the injection of current pulses of increasing amplitude (from 0 to 200 pA) till 130 pA of amplitude (Figures 8E,F). We also found that the rheobase was significantly higher in Arhgap15-KO interneurons, thus highlighting their low intrinsic excitability (Figure 8G). No differences were found in the single action potential (AP) properties (i.e., AP peak amplitude, AP half-width, AP max rising slope, and AP max repolarizing slope) (Figures 8H−L).

To confirm the altered phenotype in vitro, we examined the firing properties of CINs in dissociated primary cultures derived from E15.5 GAD67-eGFP and Arhgap15 ^LacZ/LacZ ;GAD67-eGFP embryos at 17 DIV. Consistent with previous results, we did not observe any differences in R_in and V_rest (Supplementary Figures S2A–C). We found a decrease in the response of Arhgap15-KO interneurons to current injections from 20 to 180 pA (Supplementary Figures S2D,E), therefore confirming the functional defect also in vitro.