Generation of NPN2 knockout mice
The animal experiments were performed under protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. We received NPN2 tau-green fluorescent protein male heterozygotes (+/−; one of the two NPN2 alleles knocked out) from Drs. Andreas Walz and Peter Mombaerts (Rockefeller University, New York, NY, U.S.A.) on a C57BL/6J X B129 mixed genetic background (Walz et al., 2002
). These were backcrossed five generations; each time a heterozygous male was mated with a pure FVB/NJ wild-type female (Jackson Laboratory colony; The Jackson Laboratory, Bar Harbor, ME, U.S.A.). The progeny were genotyped according to the previously published polymerase chain reaction (PCR) protocol (Walz et al., 2002
). The resulting 1,500 pups are approximately 40% wild-type (−/−), 50% heterozygous (+/−), and 10% homozygous (−/−). From the N1 to N5 generation, handling induced seizures, and SE was noticed only in mice either heterozygous or homozygous for NPN2 knockout (KO) allele.
Kainic acid-induced SE
Male, 25–30 g, >2 month old +/+ and +/− littermates (n = 9/group) were injected subcutaneously with vehicle or kainic acid (KA) (Yang et al. 2005
). As noted in Results, −/− littermates were also injected with KA, but none survived beyond 1 week. In all experiments, the mice were observed for at least 6 h for evidence of SE, 4 h of which either continuous or intermittent class IV or V seizures were displayed (Racine, 1972
; Pitkanen et al., 2002
). Class IV or higher seizures were achieved after 2–3 doses (cumulative dose of 20–25 mg/kg with 15 mg/kg more common). The latency to class III or higher seizure onset, numbers of seizures, and duration of KA-induced SE (KA-SE) by genotype were recorded. Mice (n = 3) that did not develop KA-SE as noted by behavioral seizures were excluded from the analyses. SE was terminated after 6 h by subcutaneous injection (SC) injection of 40 mg/kg pentobarbital. Animals were anesthetized by injection of 100 mg/kg pentobarbital and decapitated. Brains were removed and frozen by slow immersion in −50°C dry ice/methanol/2-methylbutane bath to preserve morphology.
Quantification of KA-induced epileptogenesis
We investigated the long-term effects of KA-SE on cell death and epileptogenesis in the +/+ and +/− mice (n = 9/group). Systematic, treatment-blinded observations were made over 8 months via videotaping (10 h/month from 3 days to 240 days after KA-SE) to determine whether mice developed spontaneous recurrent behavioral seizures (>class II) indicating the presence of epilepsy (defined as >2 class IV or higher seizures in 80 h video period). The number of seizures/80 h of videotape/mouse was noted.
Pentylenetretrazol (PTZ) dissolved in buffered saline was administered SC at a dose of 30 mg/kg. Animals were monitored for 30 min after injection. Behavioral responses were scored using the following scale: class 1, hypoactive; class 2, partial clonus (clonic seizure activity affecting face, head, and/or forelimb or forelimbs); class 3, generalized clonus (sudden loss of upright posture, whole body clonus involving all four limbs and tail, rearing, and autonomic signs); and class 4, tonic–clonic seizure (generalized seizure characterized by tonic hindlimb extension) (Ferraro et al., 1999
). Latency to first seizure as well as the class of that seizure were analyzed [one-way analysis of variance (ANOVA) with Student-Newman-Keuls post hoc analysis; mice that did not develop seizures during the observation period were excluded from analysis].
Electrophysiology of NPN2 KO mice
Male +/+ and +/− mice (n = 3/group) were anesthetized in a CO2
chamber prior to rapid decapitation with a small animal guillotine. Brains were rapidly removed and transverse hippocampal slices (350 μ
m) were cut with a Vibratome 3000 (TPI, Saint Louis, MO, U.S.A.) in cold oxygenated artificial cerebrospinal fluid (ACSF) of the following composition: 128 mM NaCl, 1.25 mM KH2
, 10 mM glucose, 26 mM NaHCO3
, 3 mM KCl, 0.1 mM CaCl2
, and 2 mM MgCl2
(Thibault et al., 2001
). Intact slices were placed in an interface-type chamber containing ACSF with 2 mM CaCl2
(Ca-ACSF) at 32°C and gassed with 95% O2
for at least 1 h. Individual slices were then transferred to an RC-22C perfusion chamber (Warner Instruments, Hamden, CT, U.S.A.) equipped with a bottom net for ACSF perfusion beneath the slice. The oxygenated Ca-ACSF was delivered at 1.5–2 ml/min and warmed to 33° ± 1°C using an TC2
Bip inline heater (Cell Micro Controls, Norfolk, VA, U.S.A.).
Electrophysiological data were acquired and analyzed using pCLAMP 8, a sharp-electrode amplifier (2A), and a DigiData 1320 digitization board (Molecular Devices, Union City, CA, U.S.A.). Extracellular recording electrodes were pulled from microhematocrit glass capillaries (Fisher Scientific, Pittsburgh, PA, U.S.A.) on a P80 pipet puller (Sutter Instruments, Novato, CA, U.S.A.). Electrodes had a 5–15 MΩ, tip resistance when filled with 2 M KCH3SO4 and 10 mM HEPES, pH 7.4. All experiments were conducted in current clamp mode. Voltage records were digitized at 2–20 kHz and low-pass-filtered at 1 kHz.
Synaptic stimulation was accomplished using a twisted bipolar stainless steel stimulation electrode (0.0045″ coated; A-M Systems, Everett, WA, U.S.A.) positioned in the Schaffer-collaterals/commissural fibers of stratum radiatum, approximately 500 μm from the recording electrode. Somatic field CA1 potentials were recorded (20 kHz sampling rate) from 4–6 slices per animal. Input-output (I/O) relationships were determined in every slice during baseline periods (0.2 Hz) prior to frequency facilitation (FF) runs. Data from these I/O analyses were used to determine the ratio of population spike (PS) over fiber potential at multiple stimulation intensities and were plotted and fit by linear regression. The slope and x-axis intercept of the linear fit from each slice were used for further analysis. First, the x-axis intercepts were used to determine the excitability threshold by estimating the fiber potential amplitude at which population spikes begin to appear. Second, the slope was used to measure the excitability across a range of stimulus intensities. Stimulus intensity during the FF paradigm was set to deliver pulses at 50% of the maximum PS amplitude measured during I/O and was delivered at 7 Hz for 18 s. Synaptic activation and stimulation protocols were accomplished using a SD9K stimulator (Astro Med, Grass Instrument, Warwick, RI, U.S.A.). Amplitude of the fiber volley was determined by comparison to prestimulus baseline. PS amplitudes were calculated as the difference between the maximum positive deflection of the field excitatory postsynaptic potential (EPSP) before the PS and the maximum negative deflection of the PS. The degree of FF was determined by calculating the ratio of difference to the first primary PS elicited in the FF stimulation period to all other PSs elicited throughout the stimulus train [(subsequent PS-1st PS)/1st PS]. Each second of ratios included seven measures that were then binned per second and reported as an average difference ratio per second. As FF stimulation progressed, a secondary PS gradually appeared. Again, the degree of facilitation of the secondary PS was determined by calculating the ratio increase compared to the first observed secondary PS.
Data was analyzed using Clampfit (v 8.0; Axon Instruments, Union City, CA, U.S.A.) and routines written in Igor Pro (v 5.0; Wavemetrics, Lake Oswego, OR, U.S.A.). Group comparisons were analyzed for main effects using ANOVA. Statistical analysis of the FF measures was performed using a ranked two-way repeated measures ANOVA. Means ± SEM were reported.
KA-SE-treated animals were killed 7 or 240 days after treatment, and their hippocampi was sectioned. For excitatory cells, the soma were counted in the cell layers of three major regions: Dentate gyrus/hilar (DG/H) region, CA3, and CA1 regions (see Supplementary Fig. 1
for demarcation) (Yang et al., 2005
). Within these same demarcated regions, nissl cell counts (presumed interneurons) were quantified in molecular layer, stratum radiatum, and stratum oriens of each subregion. Five cresyl violet-stained hippocampal sections (at 200-μ
m intervals) per animal were counted using the Image ProPlus program (Media Cybernetics, Bethesda, MD, U.S.A.). Both hippocampi were counted. Experimenters were blinded to genotype and treatment group, and average measures per animal were used for statistical analysis (n = at least 5/group). The cell density was calculated for 200 μ
of a given hippocampal region in each of five sections that were averaged together to establish a single value of cell density per hippocampal region for each mouse. For GABA, GAD-67, parvalbumin, calretinin, somatostatin, and neuropeptide Y cell counts, the total number of cells counted in each hippocampal subregion is noted instead of cell density (see Supplementary Fig. 1
for demarcation). The averages per region in and Supplementary Table 3
are calculated from at least five mice. The statistical analysis was performed using ANOVA followed by a Student-Newman-Keuls post hoc analysis. Similar analyses were done for both pyramidal and interneuron cell counts of drug naive (+/+), (+/−), and (−/−) mice.
Mean cell counts of interneurons in hippocampal subregions
For counting of GABAergic synapses, hippocampal sections (at 200-μm intervals) per animal were counted using the Image ProPlus program. Confocal microscopy [Zeiss Laser Confocal Scanning Microscope (LSM); Carl Zeiss, Oberkochen, Germany] 0.5-μm sections (Z-series stacks) were taken along the z-axis in the CA3b region using the 100× oil immersion objective. The images taken from different sections were compared under constant conditions by adjusting the LSM parameters (pinhole size, laser power, detector voltage). Punctata below 0.1 μm2 were eliminated. Cells were counted only if the clearly defined largest diameter of the cell and clearly defined puntata could be visualized. The CA3b region of both hippocampi was counted. The numbers of Parv+ of GAD-65+ punctata around 50 pyramidal cells were counted, as well as the percent double labeling with both markers. Experimenters were blinded to genotype and treatment group, and average measures per animal were used for statistical analysis (n = 5/group). The Statistical analysis was performed using ANOVA followed by a Student-Newman-Keuls post hoc analysis.
Immunocytochemistry of mouse hippocampus
Drug naive adult mice (>P60) were perfused transcardially with 4% buffered paraformaldehyde. Free floating coronal sections (30 μm) were cut throughout the rostral to caudal extent of the hippocampus. Sections were washed with 1× phosphate-buffered saline (PBS), blocked with blocking buffer [1× PBS, 0.25% Triton X-100, 8% bovine serum albumin (BSA)] for 1 h, and incubated for 1–4 days at 4°C with 1:5000 GABA and 1:1000 parvalbumin (Sigma, St. Louis, MO, U.S.A.), 1:1000 calretinin, neuropeptide Y 1:2000, GAD-65 1:1000, GAD-67 1:500, 1:500 somatostatin (all from Chemicon, Millipore, Billerica, MA, U.S.A.), and 1:100 GABAA receptor α1 (Millipore, Billerica, MA, U.S.A.). Following incubation with primary antibody, sections were rinsed 6× with blocking buffer, then with biotinylated goat secondary antibody (1:1000; Jackson ImmunoResearch, West Grove, PA, U.S.A.) for 2 h at room temperature, and finally 1 h in 1% ABC reagent (Vector Laboratory, Burlingame, CA, U.S.A.). Antibodies were visualized with 3,3 diaminobenzidine (DAB; Sigma, St. Louis, MO, U.S.A.) as a peroxidase substrate with 0.01% H2O2 added as a catalyst. Immunocytochemical controls were performed using the same reaction procedure with nonspecific immunoglobulin G (IgG) or without the primary antibody. For neuropeptide Y experiments, a rhodamine-labeled donkey anti-sheep antibody (1:1000: Jackson ImmunoResearch) was used. For GAD-65/parvalbumin and GABAA receptor α1 double labeling experiments, a Texas Red goat anti-mouse or Cy5 goat anti-rabbit (1:1000; Jackson ImmunoResearch) was used. Cell counts were performed as described above.
To assess the distribution of various nerve fiber layers, four stained sections per mouse were photographed under bright field illumination with an Olympus BX-51 microscope (Olympus, Tubingen, Germany). Optical density (O.D.) measurements (four per subregion) were assessed based on average per subregion using the Image ProPlus analysis program. The O.D. of the fimbrae served as an internal constant control in each slide, and average measurement per subregion per animal was used for statistical analysis. Results were expressed as a mean ± SEM. Statistical analysis was performed using ANOVA followed by a Student-Newman-Keuls post hoc analysis. Sample sizes are noted in .
Quantitative analysis of synaptosomal proteins on immunoblots
Synaptosomes were prepared from +/+, +/−, and −/− adult mice (>P60, n = 3 per group) as previously described (Barnes et al., 1995
). Briefly, 5 μ
g hippocampal synaptosomal protein were used on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels followed by transfer to Immobilon P membranes (Millipore). After blocking the membranes with 1× PBS, 0.1% Tween-20, and 5% milk, proteins were visualized by primary antibody [1:4000 NPN2 antiserum (gift of Alex Kolodkin, Johns Hopkins University), 1:1000 GABAA
2/3 (Upstate, Danvers, MA, U.S.A.), 1:5000 GAD-67, GAT-1, GluR1, NMDA2A/2B, PSD-95 (all from Chemicon), 1:1000 Fyn (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), 1:5000 synapsin I and β
tubulin (National Hybridoma Core Bank, Iowa City, IA, U.S.A.)] followed by secondary antibody detection with horseradish peroxidase (HRP)-linked goat anti-rabbit or anti-mouse IgG 1:5000 (Jackson ImmunoResearch) using the Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, U.S.A.). Light emission was detected by BioMax film (Kodak, Rochester, NY, U.S.A.). O.D. of detected bands were expressed as a mean of the ratios of −/− versus +/+ mouse values ± SEM. Statistical analysis was performed using ANOVA and Newman-Keuls post hoc test.
Rapid Golgi staining and quantitative analysis
Brains isolated from +/+ and +/− mice (n = 7/group) were fixed with 10% formalin in PBS and processed using a modified rapid Golgi impregnation technique (Valverde, 1976
). Briefly, coronal tissue blocks of cerebrum (2–3 mm thick) were impregnated using solutions containing osmium tetroxide plus potassium dichromate (for 5–7 days) followed by immersion in silver nitrate (for 36–42 h). After dehydration, blocks were embedded in nitrocellulose, and coronal sections were cut at 120 μ
m, cleared using α
-terpineol and xylenes, and coverslipped under Permount (Fisher Scientific, Fair Lawn, NJ, U.S.A.). All slides were coded, and randomly selected neurons (6–8 per brain from each cell population; see below) meeting staining criteria were evaluated. Camera lucida drawings of individual neurons were used to quantitate the number and length of dendritic branches and the complexity of the dendritic tree. Only neurons that fulfilled all of the following criteria were analyzed: (1) Cell type must be identifiable; (2) neurons must be completely impregnated with the silver-chromate Golgi precipitate throughout all dendrites and spines; (3) soma of the selected neurons had to be located in the middle third of the thickness of the 120-μ
m section; and (4) all dendrites must be relatively free of neurites from nearby cells, from blood vessels, and from nondescript precipitate.
For dendritic branching analysis, using Zeiss bright-field microscopes equipped with drawing tubes, we first prepared camera lucida drawings of the basilar trees of randomly selected CA1 neurons at a magnification of 400×. A total of 5–6 CA1 neurons that met the above criteria were selected from each hippocampus. The camera lucida drawings were then quantified using Sholl analysis (Sholl, 1953
). Using a template comprised of a series of enlarging concentric circles that originated at the soma, by quantifying the number of dendritic intersections with each circle, the Sholl analysis provides a profile of the amount and distribution of dendritic material measured at 10-μ
m intervals. We compared the branching and total length of the CAl dendrites as a function of genotype. An estimate of total dendritic length was obtained from the total number of dendritic intersections in the Sholl analysis to microns using a previously generated conversion factor. Dendritic complexity was assessed using a branch point analysis that ascertained the number, location, and branch order of dendritic branch bifurcations. Statistical significance was determined using the Wilcoxon rank-sign test. An adjusted α
for significance for multiple comparisons was used.
Dendritic spines were quantified on the terminal tip segments of the CA1 basilar trees. At a magnification of 1,200×, using special long-working distance oil immersion lenses, dendritic spines were counted directly from the microscope along four segments of the 30-μm long terminal tip. These spine counts provide an accurate comparison of data between groups, but underestimate the actual total numbers of spines since only flanking spines were counted. (Spines directed either up toward the observer or away from the observer—on the underside of the dendritic branch segment—would be partially obscured by the Golgi stain within the branch).
In addition to total spine density, we also evaluated spine configurations. Spines may be designated by shape into three basic morphological categories: (1) L-spines, which look like a lollipop and have a well-defined spine head and a narrower definitive spine neck. These may be further subdivided into Ls spines (with a small spine head) or LL spines (with a large spine head). (The “L” type spine has also been designated by other investigators as an “M” spine for its “mushroom”-like appearance.) (2) “N” (nubby) spines, which lack a definitive spine head and typically have a thickened spine neck. (3) “D” (dimple) type spines, which are small and have neither a well-defined head nor neck. Spine data was evaluated for statistical significance using the unpaired Student's t-test.