Supplemental Data Figure S1. Protein expression for markers of GABA interneuron populations (PV – parvalbumin, CB – calbindin, CR – calretinin) was assessed by western blot. Figure S2. Effects of the subunit-selective GABAA-receptor agonist L-838,417 and the antipsychotic risperidone on behavioral phenotypes relevant to schizophrenia, autism, and intellectual disability. (A) Social preference was assessed in a three-chambered social approach/avoidance paradigm. NR1neo-/- mice showed significantly reduced social preference (group: F1,34=31.5, P<0.0001), but this deficit was not affected by risperidone (drug: F2,34=0.49, P=0.62) or L-838,417 (drug: F2,26=2.49, P>0.1). (B) Impairments in T- maze spatial memory performance in NR1neo-/- mice (group: F1,31=50.0, P<0.0001) were not improved by risperidone (drug: F1,31=0.67, P=0.42) or L-838,417 (F1,30=2.1, P=0.16). (C) NR1neo-/- mice had a significant deficit in PPI (F1,14=10.75, P<0.006), as observed in schizophrenia and autism. PPI deficits in NR1neo-/- mice were reversed by L-838,418 (group x drug: F2,24=8.3, P<0.002) and risperidone (group x drug: F2,26=4.1, P<0.03). (D) NR1neo-/- mice had elevated acoustic startle responses (F1,14=22.4, P<0.0003). Neither L- 838,417 (F2,24=2.7, P=0.09) nor risperidone (F2,26=0.14, P=0.9) had any effect on this phenotype. (E) NR1neo-/- mice displayed locomotor hyperactivity in a novel environment at baseline (F1,13=30.8, P<0.0001; n=7-8/group). Risperidone significantly reduced LMA (F2,36=31.1, P<0.0001, n=10/group). L-838,417 had no effect (F2,20=1.6, P>0.2; n=6/group) consistent with its pharmacologic profile as a non-sedating anxiolytic drug. Figures show mean +/- S.E.M., *P<0.05, **P<0.01, ***P<0.001. Supplemental Experimental Procedures Parvalbumin Immunohistochemistry: NR1neo-/- and wildtype littermates were used for stereological population estimates of PV+ cells. Frozen coronal sections (45m thick) were cut from fixed tissue. Immunolabeling was conducted on every fourth section using a parvalbumin monoclonal antibody (MAB1572, Chemicon, 1:1000) and an HRP- conjugated secondary antibody (EnVision + Dual Link HRP, DakoCytomation, Carpinteria, CA). DAB substrate was added to sections to visualize parvalbumin immunolabeling. Quantification of stereological population estimates was performed in two regions: (1) prefrontal cortex (PFC; e.g., prelimbic and infralimbic regions 1.34 to 1.78 mm relative to bregma AP) and (2) a broad cortical region (cortical layers I-VI from -0.82 to 1.54mm relative to bregma AP), encompassing motor and somatosensory cortex as our region of interest (ROI). Stereological counting was performed by a blinded experimenter using a computer-assisted microscope (Nikon E800, Stereoinvestigator, v8.0, MBF Biosciences, Inc.). The optical fractionator method was employed to generate population estimates of PV+ cells in this ROI 1, 2. Systematic random sampling was performed on every fourth section. For all sections, the optical disector height was 12m, with a guard zone height of 2m. The disector counting frame size was 30m² and the grid size was 150x200 m. Significance was assessed by a two-tailed unpaired t-test. Parvalbumin In Situ Hybridization: For riboprobe synthesis, total RNA was extracted from frozen WT murine brain tissues using Trizol (Invitrogen), treated with DNase I (Promega) then reverse transcribed using Superscript II reverse transcriptase according to the manufacturer’s instructions (Invitrogen). cDNA fragment corresponding to parvalbumin was cloned by PCR amplification using the following primers: F- CCATGGAGGCGATAGGAGCCTTTGCTGC and R- GTCGACGTCTCCAGCGGCCAGAAGCG. The amplified fragment was cloned into the pGEMT easy vector (Promega). Digoxigenin-labeled sense and antisense riboprobes were obtained by in vitro transcription using T7 and SP6 transcriptase (Promega) and digoxigenin- UTP (Roche). They were stored in hybridization buffer at the concentration of 10 ug/ml. NR1neo-/- and WT littermate mice were transcardially perfused with iced cold saline solution followed by 4% PFA. Brains were removed, post-fixed in 4% PFA for 1 h at 4°C, cryoprotected in 10 to 30% sucrose/PBS, frozen and cryosectioned at 50 μm. The floating sections were stored at -20°C in a cryopreservation solution until being processed for in situ hybridization. The sections were washed in PBS, then RIPA (150 mM of NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM of EDTA, pH 8, 50 mM of Tris, pH 8), post-fixed in 4% PFA for 5 min and treated with 0.25 acetic anhydre in 0.1 M of triethanolamine. Hybridization was performed with digoxigenin-labeled sense or antisense riboprobes (1 μg/ml) overnight at 70°C in hybridization buffer (50% formamide, 5X SSC, 5X Denhardt's, 250 μg/ml yeast tRNA, 125 mg/l salmon sperm DNA). The sections were washed with 50% formamide/2X SSC/0.1%, Tween 20 for 2 h at 70°C then MABT (100 mM of maleic acid and 150 mM of NaCl at pH 7.5, 0.1% Tween 20) at room temperature. After blocking with 10% goat serum/MABT, hybridization was revealed by incubation with an alkaline phosphatase (AP) coupled antibody (Roche, dilution 1:1,000 in MABT, 10% heat-inactivated goat serum blocking buffer) overnight at 4°C. The slides were washed with MABT and with AP buffer (100 mM of Tris, pH 9, 100 mM of NaCl, 50 mM of MgCl2, 0.24 mg/ml levamisole/0.1%, Tween 20). The sections were then incubated in a 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma) solution. Using the same ROI as described above, PV cell density was determined at 100X using Image ProPlus software (Media Cybernetics Inc., Silver Springs, MD). Western Blotting: Brains from NR1neo-/- and WT littermate mice were surgically removed and flash frozen on dry ice. Left hemispheres were dissected and homogenized in homogenization buffer [25 mM Hepes pH 7.5, 300 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.1% Triton supplemented with 0.5 mM DTT, protease and phosphatase inhibitors]. Homogenates were centrifuged at 16,000g for 20 min at 4oC and protein concentrations of the supernatants were determined by Bradford assay. Protein extracts (50 µg) were loaded onto NuPage Novex 4-12% Bis-Tris precast gels (Invitrogen) and transferred onto PVDF membrane (Millipore). The membrane was blocked in 3% nonfat milk and probed with anti-NMDAR1 (1:500 goat polyclonal, sc1468, Santa Cruz Biotechnology), anti-Parvalbumin (1:1000 mouse monoclonal, MAB1572, Millipore), anti-Calbindin (1:2000 rabbit polyclonal, AB1778, Millipore), anti-Calretinin (1:10000 goat polyclonal, AB1550, Millipore), anti-GAD65 (1:500 rabbit monoclonal, ab75750, Abcam), anti-GAD67 (1:1000 rabbit polyclonal, 5305S, Cell Signaling Technology), and anti-β-Actin (1:50000 mouse monoclonal, AB6276, Abcam), followed by secondary antibodies goat anti-mouse (12-349, Millipore), donkey anti-rabbit (711-035-152, Jackson), bovine anti-mouse (sc-2375, Santa Cruz Biotechnology), and bovine anti-goat (sc-2352, Santa Cruz Biotechnology). Blots were developed with chemiluminescence reagent ECL (Thermo Scientific). Group differences were assessed with a group x marker ANOVA, followed by Bonferroni post-tests where appropriate. RNA Analysis: Given lower protein levels, expression of post-synaptic GABAA- and GABAB-receptor subunits was assessed using qPCR. RNAs were purified using Trizol from WT and NR1 transgenic mice. Quantitative reverse transcription-PCR (RT-PCR) was done using the following primers: (GABA) B receptor-1, PrimerBank ID:131888528b2, F-CTCCTGGACGGATATGGACAC and R- GTCGGGGTCACATCGGAAAT; (GABA) B receptor-2, PrimerBank ID:124487262b1 F-CTTCCTGGACCTGCGACTCTA and R-CGGCCCATACTTTATTGCATCAT; (GABA) A receptor, subunit alpha 1, PrimerBank ID:145966746b2 F- CGACTGCTGGACGGTTATGAC and R-CACTTCAGTTACACGCTCTCC; (GABA) A receptor, subunit alpha 2, PrimerBank:ID 142353792b2 F- TTACAGTCCAAGCCGAATGTC, R- AGTGGGCATGAATGAGCATCC and GAPDH F- GGTGGAGGTCGGTGTGAACG and R- CTCGCTCCTGGAAGATGGTG. For both receptors, group differences were assessed with a group x subunit ANOVA, followed by Bonferroni post-tests where appropriate. Ex Vivo Electrophysiology: Mice were decapitated following isoflurane anesthesia. The brain was removed and coronal slices (300 μm) containing the hippocampus were cut with a Vibratome (VT1000S, Leica Microsystems) in an ice-cold artificial cerebrospinal fluid solution (ACSF), in which NaCl was replaced by an equiosmolar concentration of sucrose. ACSF consisted of 130 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, 1 mM MgCl2 and 2 mM CaCl2 (pH 7.2–7.4 when saturated with 95% O2/5% CO2). Slices were incubated in ACSF at 32–34 °C for 45 min and kept at 22-25 °C thereafter, until transfer to the recording chamber. The osmolarity of all solutions was 305–315 mOsm. Slices were viewed using infrared differential interference contrast optics under an upright microscope (Eclipse FN1, Nikon Instruments Inc.) with a 40x water-immersion objective. The recording chamber was continuously perfused (1–2 ml/min) with oxygenated ACSF heated to 32±10 C using an automatic temperature controller (Warner Instruments). Picrotoxin (100 μM) was added to all solutions to block the GABAA receptor-mediated currents. The intracellular solution contained (in mM): 145 potassium gluconate, 2 MgCl2, 2.5 KCl, 2.5 NaCl, 0.1 BAPTA, 10 HEPES, 2 Mg-ATP, 0.5 GTP- Tris (pH 7.2–7.3 with KOH, osmolarity 280–290 mOsm). Recordings were performed in whole-cell voltage-clamp (Vh=-65 mV) or whole-cell current-clamp (at resting potential) modes as indicated. The data was acquired through a MultiClamp700B amplifier (Molecular Devices). Currents were low-pass filtered at 2 kHz and digitized at 20 kHz using a Digidata 1440A acquisition board and pClamp10 software (both from Molecular Devices). Access resistance was monitored throughout the recordings by injection of 10 mV hyperpolarizing pulses (in voltage-clamp) and data was discarded if the access resistance changed by >25% over the course of data acquisition. Evoked responses were triggered by constant-current pulses generated by an A310 Accupulser (World Precision Instruments) and delivered at 0.2 Hz via a bipolar tungsten stimulation electrode positioned within 100 μm of the recorded cell. The amplitude of the current pulses was controlled by a stimulus isolator (ISO-Flex, A.M.P.I.) and was adjusted in each cell to evoke a minimal response with a 25 μs current pulse. The current duration was then increased to 50, 75, 100, and 125 μs to construct an input-output curve for the evoked synaptic events. All analyses of intracellular recordings were carried out with Clampfit 10 (Molecular Devices). The time constant of decay was based on a monoexponential fit to the decay phase of an average spontaneous excitatory postsynaptic current (sEPSC) trace computed from a minimum of 50 individual sEPSCs. Mean sEPSC frequencies were analyzed from 10–20 s long trace segments. Evoked EPSC amplitudes were computed from an average of 5-10 responses at each stimulation intensity. Rheobase was measured as the minimal current (advanced in 10 pA steps) necessary to trigger an action potential. In Vivo Electrophysiology: Recording of auditory ERPs was performed at least 1 week after electrode implantation in a home-cage environment, as previously described 3-6. Cages were placed in a sound attenuated recording chamber inside a Faraday electrical isolation cage. Electrode pedestals were connected to a 30cm tripolar electrode cable that exited the chamber to connect to a high impedance differential AC amplifier (A-M Systems, Carlsborg, WA, USA). Stimuli were generated by Micro1401 hardware with Spike2 software (Cambridge Electronic Design, Cambridge, UK) and were delivered through speakers attached to the cage top. For assessment of gamma phase synchrony in NR1neo-/- mice and WT littermates, the sequencer file consisted of a series of 200 paired white-noise stimuli presented 500 ms apart at 85 dB (10 ms in duration) with a 8-s interstimulus (ISI) interval. Recording sessions were preceded by a 15 minute acclimation phase. Raw EEG was filtered between 1 and 500 Hz. Individual sweeps were rejected for movement artifact based on a criterion of 2 times the root mean squared amplitude per mouse. Signal Processing: Time-frequency decomposition of EEG signal was performed with the EEGLAB toolbox in Matlab 7. Single trial epochs between -0.3 and 0.8 sec relative were extracted from the continuous data sampled at 1667 Hz. Total power and evoked (i.e., phase-locked) power were calculated using Morlet wavelets in 100 linearly spaced frequency bins between 5-100 Hz, with wavelet cycles linearly increasing from 3 to 6, as published 8. For each subject, evoked power was averaged from 0-100 ms post-stimulus in the theta (4-12 Hz), beta (12-25 Hz), and gamma frequency ranges (30-80 Hz). Pre- stimulus total power was calculated in the same frequency ranges from -300 to -100 ms relative to the stimulus onset. Finally, gamma signal-to-noise ratio (SNR) was calculated as the maximal peak in the post-stimulus gamma-band response from 0-100 ms divided by the average pre-stimulus gamma power. T-Maze Alternation: Spatial working memory was assessed in a spontaneous alternation T-maze paradigm. The maze consisted of three identical arms (30 cm long, 10 cm wide, 12 cm tall). Wild-type mice have a natural tendency to alternate when placed in such a chamber, achieving spontaneous alternation rates up to 75% 9. This paradigm measures spatial working memory behavior, which is highly sensitive to dysfunction of the hippocampus and the prefrontal cortex, among other brain regions 10, 11. Two versions of the T-maze task were performed; a discrete-trial (5 second delay) spontaneous alternation paradigm was carried out as described 9, and a continuous alternation (8 min) version of the task, as described 12. NR1neo-/- showed significant deficits in both versions of this task, so we proceeded with the continuous alternation paradigm, as it is more amenable to acute pharmacologic testing. Mice were well handled and acclimated to the room prior to testing, which occurred in low-lux red light to minimize anxiety. In addition, mice had been well habituated to the T-maze apparatus prior to testing, to avoid neophobia. Mice were tracked with an overhead video camera and manually scored. A mouse was scored as having entered an arm when all four paws were located within the runway. An alternation was defined as sequential entries into each of the three different arms (e.g., ABC, BCA, etc.). As such, a non-alternation behavior occurred when a mouse re-entered an arm that it had visited in the previous trial (e.g., BAB, CBC, etc.). Spontaneous alternation percentage was calculated as the total number of alternations divided by (total arm entries – 2). Prior to drug administration, NR1neo-/- and WT mice were tested several times over the course of two weeks, demonstrating a stable baseline level of performance. Pharmacology: Acute effects of the pharmacologic compounds baclofen, risperidone, and L-838,417 were assessed on electrophysiological and behavioral measures. All drugs were administered via intraperitoneal injections, 15-30 minutes before testing sessions. The metabotropic GABA(B)-receptor agonist baclofen was dissolved in saline to achieve concentrations of 1-5 mg/kg. The D2-receptor antagonist risperidone was dissolved in saline at 0.05 and 0.1 mg/kg. L-838,417, the selective GABA(A)-alpha-2/3/5 partial agonist and GABA(A)-alpha-1 inverse agonist, was obtained from Tocris and dissolved at 10-30 mg/kg in a 10% DMSO solution in saline. Control experiments consisted of saline and 10% DMSO in saline vehicles. Statistics: Unless stated otherwise above, statistical significance between groups was assessed with group x drug repeated-measures ANOVA, followed by Bonferroni post- tests where appropriate, using GraphPad Prism software. In the case of a main drug effect, planned comparisons were used to assess if the drug significantly altered the NR1neo-/- group from vehicle control levels. Pearson’s r correlations were calculated to assess the relationship between electrophysiological and behavioral measures and were Bonferroni corrected for multiple comparisons. Before applying any T-test, normality of the distribution was assessed with a K-S test. In the absence of normality, a non- parametric Mann-Whitney U test was performed. 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Genes Brain Behav 2009; 8(7): 661-675. 6. Lazarewicz MT, Ehrlichman RS, Maxwell CR, Gandal MJ, Finkel LH, Siegel SJ. Ketamine modulates theta and gamma oscillations. J Cogn Neurosci 2010; 22(7): 1452-1464. 7. Delorme A, Makeig S. EEGLAB: an open source toolbox for analysis of single- trial EEG dynamics including independent component analysis. Journal of neuroscience methods 2004; 134(1): 9-21. 8. Gandal MJ, Edgar JC, Ehrlichman RS, Mehta M, Roberts TP, Siegel SJ. Validating gamma oscillations and delayed auditory responses as translational biomarkers of autism. Biol Psychiatry 2010; 68(12): 1100-1106. 9. Deacon RM, Rawlins JN. T-maze alternation in the rodent. Nat Protoc 2006; 1(1): 7-12. 10. Deacon RM, Rawlins JN. Hippocampal lesions, species-typical behaviours and anxiety in mice. Behav Brain Res 2005; 156(2): 241-249. 11. Wall PM, Blanchard RJ, Yang M, Blanchard DC. Infralimbic D2 receptor influences on anxiety-like behavior and active memory/attention in CD-1 mice. Prog Neuropsychopharmacol Biol Psychiatry 2003; 27(3): 395-410. 12. Belforte JE, Zsiros V, Sklar ER, Jiang Z, Yu G, Li Y et al. Postnatal NMDA receptor ablation in corticolimbic interneurons confers schizophrenia-like phenotypes. Nat Neurosci 2010; 13(1): 76-83.
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