All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals
and approved by the Stanford University Institutional Animal Care and Use Committee. GIN mice (FVB-Tg(GadGFP)4570Swn/J, The Jackson Laboratory) (Oliva et al., 2000
) were treated with pilocarpine (300 mg/kg, i.p.) 20 min after atropine methylbromide (5 mg/kg, i.p.) at 27–69 d old. Diazepam (10 mg/kg, i.p.) was administered 2 h after the onset of stage 3 or greater seizures (Racine, 1972
) and repeated as needed to suppress convulsions. During recovery, mice received lactated ringers with dextrose subcutaneously. Control mice included animals that were treated identically but did not develop status epilepticus, as well as naïve mice. There were no significant differences in results of GFP-positive hilar neurons (GPHNs) in naïve and pilocarpine-treated control mice, so data were combined. Video monitoring, beginning at least 10 d after pilocarpine treatment, verified spontaneous motor convulsions in all epileptic mice. No controls were observed to have spontaneous seizures.
Green fluorescent protein (GFP)-immunocytochemistry
For GFP-immunocytochemistry experiments, the control group consisted of 8 mice: half female, half male, half naïve, and half pilocarpine-treated controls. The epileptic group consisted of 8 mice (3 female, 5 male). Mice were killed with urethane (2 gm/kg i.p.) at an average of 88 d old (range 55–219 d old), which was an average of 50 d (range 24–154 d) after pilocarpine-induced status epilepticus. They were perfused through the ascending aorta at 15 ml/min for 2 min with 0.9% sodium chloride and 30 min with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at 4°C. Brains were postfixed overnight at 4°C. Left hippocampi were isolated, equilibrated in 30% sucrose in PB, gently straightened, frozen, and sectioned perpendicular to septotemporal axis with a sliding microtome set at 40 µm. Sections were stored at −20°C in preservation solution consisting of 30% ethylene glycol and 25% glycerol in 50 mM PB. Balanced numbers of control and epileptic mice were processed together in each batch. Beginning at random points near the septal pole of the hippocampus and extending through the entire septotemporal length, 1-in-6 series of sections were rinsed in PB and treated with 1% H2O2 for 2 h. After rinses in PB and 0.1 M tris-buffered saline (TBS, pH 7.4), sections were treated with blocking solution consisting of 3% goat serum (Vector Laboratories), 2% bovine serum albumin (BSA), and 0.3% Triton X-100 in 0.05 M TBS for 1 h. Sections were rinsed in TBS and incubated for 40 h at 4°C in rabbit anti-GFP serum (1:2000, Invitrogen) diluted in 1% goat serum, 0.2% BSA, and 0.3% Triton X-100 in 0.05 M TBS. After rinses in TBS, sections incubated for 2 h in biotinylated goat anti-rabbit serum (1:500, Vector Laboratories) in secondary diluent consisting of 2% BSA, and 0.3% Triton X-100 in 0.05 M TBS. After rinses in TBS, sections incubated for 2 h in avidin-biotin-horseradish peroxidase complex (1:500, Vector Laboratories) in secondary diluent. After rinses in TBS and 0.1 M tris buffer (TB, pH 7.6), sections were placed for 5 min in chromogen solution consisting of 0.02% diaminobenzidine, 0.04% NH4Cl, and 0.015% glucose oxidase in TB and then transferred to fresh chromogen solution with 0.1% β-d-glucose for 13 min. The reaction was stopped in rinses of TB, and sections were mounted and dried on gelatin-coated slides. Sections dehydrated in a series of ethanols and xylenes and were coverslipped with DPX. Somatostatin-immunocytochemical processing was identical, except rabbit anti-somatostatin serum was used (1:4000, Peninsula Laboratories).
The total number of GPHNs per dentate gyrus was estimated using the optical fractionator method (West et al., 1991
). Investigators were blind to experimental groups during analysis. Starting at random points near the septal pole of the hippocampus, 1-in-6 series of GFP-immunostained sections were sampled. Using Stereo Investigator (MBF Biosciences) and a microscope equipped with a 10× objective, contours were drawn around the hilus, which was defined by its border with granule cell layer and straight lines drawn from the ends of granule cell layer to the proximal end of the CA3 pyramidal cell layer. The entire hilar area was sampled. Total section thickness was used for dissector height, and only labeled somata not cut at upper surfaces of sections were counted. This modification of the optical fractionator method facilitates analysis of tissue sectioned thinly to enhance staining; however, it increases the probability of underestimating cell numbers. Using a 100× objective, 1741 GFP-positive somata were counted in 246 sections from 16 mice. The total number of GPHNs per dentate gyrus was estimated by multiplying counted neurons by a factor for section sampling (6). To analyze GPHN soma size, one or two sections from the mid-septotemporal level of the hippocampus from each mouse were evaluated. Using Neurolucida (MBF Biosciences) and a microscope equipped with a 100× objective, an investigator blind to experimental groups drew contours around 123 somata in 16 mice and recorded areas and maximum diameters. Adjacent thionin-stained sections were analyzed similarly to measure areas of 657 Nissl-stained hilar somata.
The total length of GFP-positive axon in the granule cell layer plus molecular layer was estimated by investigators blind to experimental groups. Starting at random points near the septal pole of the hippocampus, 1-in-12 series of GFP-immunostained sections were sampled. Contours were drawn around the granule cell layer plus molecular layer in each section, and areas were recorded. Using Stereo Investigator (MBF Biosciences), sample points were determined randomly and systematically. Counting grids were 350 × 350 µm, and counting frames were 25 × 25 µm. All GFP-positive axons within counting frames and throughout the entire depth of sections were reconstructed using a 100× objective, and cumulative length at each sample site was recorded. A total of 164,767 µm of axon was measured at 341 sample sites in 124 sections from 16 mice. Total axon length per dentate gyrus (granule cell layer plus molecular layer) was estimated by multiplying measured axon length by factors for section sampling (12) and area sampling, which was the total area of the granule cell layer plus molecular layer divided by the analyzed area (number of counting frames per section times counting frame area).
Numbers of GFP-positive axon crossings of the granule cell layer were measured in a section from the mid-septotemporal level of the hippocampus from each mouse. Using Neurolucida (MBF Bioscience) and a microscope equipped with a 100× objective, a line was drawn along the middle of the granule cell layer starting at the tip of the superior blade, to the apex, and then to the tip of the inferior blade. All GFP-positive axon crossings were counted. Number of crossings per length of the granule cell layer was calculated. A total of 3201 crossings were counted in 16 sections from 16 mice.
Male and female mice were used for hippocampal slice experiments. There were no significant gender differences in any of the parameters analyzed in the present study. The control group consisted of 13 naïve and 13 pilocarpine-treated controls. The epileptic group consisted of 31 mice, which were used for slice experiments when they were 92 ± 4 d old, an average of 48 d (range 12–82 d) after pilocarpine-induced status epilepticus. Mice were deeply anesthetized with urethane (1.5 g/kg, i.p.) and decapitated. Tissue blocks including the dentate gyrus were removed rapidly and stored for 3 min in ice-cold modified artificial cerebrospinal fluid (mACSF) containing (in mM): 230 sucrose, 2.5 KCl, 10 MgSO4
, 1.25 NaH2
, 26 NaHCO3
, 2.5 CaCl2
, and 10 d
-glucose. Horizontal slices (300 µm) were prepared with a microslicer (Leica VT1000S). All slices were from the same septotemporal level of the hippocampus, which corresponded to 3.6–4.4 mm below the dorsal surface of the brain (4.08 ± 0.04 mm and 4.10 ± 0.04 mm in control and epileptic mice, respectively, p=0.74, t test) in the atlas of Franklin and Paxinos (2007)
. Slices were incubated at 32°C for 30 min in a submersion-type holding chamber that contained 50% mACSF and 50% normal ACSF, which consisted of (in mM): 126 NaCl, 3 KCl, 2 MgSO4
, 1.25 NaH2
, 26 NaHCO3
, 2 CaCl2
, and 10 d
-glucose. After that, slices were transferred to normal ACSF at 32°C for 1 h. ACSF was aerated continuously with a mixture of 95% O2
and 5% CO2
. Slices were maintained at room temperature until used for recording.
Cells were visualized with Nomarski optics (40×, Nikon) and an infrared-sensitive video camera (Hamamatsu Photonics). Fluorescence was used to identify GFP-positive neurons in the hilus. Whole-cell patch-clamp recordings were obtained at 32 ± 1°C. Interneurons were recorded in current-clamp mode (Axopatch 1D, Molecular Devices). Interneuron pipette solution contained (in mM): 100 potassium gluconate, 40 HEPES, 20 biocytin, 10 EGTA, 5 MgCl2, 2 disodium ATP, and 0.3 sodium GTP. Granule cells were recorded in voltage-clamp mode (Axopatch 200B, Molecular Devices). Granule cell pipette solution contained (in mM): 120 cesium methanesulfonate, 20 biocytin, 10 HEPES, 5 NaCl, 5 QX-314, 2 magnesium ATP, 0.3 sodium GTP, and 0.1 BAPTA. The measured liquid junction potential was 7 mV, and all membrane potentials were corrected accordingly. Patch electrodes were pulled from borosilicate glass (1.5 mm outer diameter, 0.75 mm inner diameter, 3–4 MΩ). Seal resistance was >5 GΩ, and only data obtained from electrodes with access resistance <20 MΩ and <20% change during recordings were included. Series resistance was 80% compensated, and compensation was readjusted during experiments, when necessary. Data were acquired (pCLAMP, Molecular Devices) and stored on computer for off-line analysis. Membrane currents and potentials were low-pass filtered at 2 kHz and digitized at 10 kHz.
Input resistance was determined in interneurons by measuring peak voltage responses to current steps ≤ ±10 pA and calculating slopes of regression lines in current-voltage plots. Unitary inhibitory postsynaptic currents (uIPSCs) were recorded in granule cells. IPSCs were enhanced and EPSCs minimized by holding granule cells at 0 mV. Action potentials were evoked in GFP-positive hilar neurons by brief (1.2 ms) depolarizing (1.5 nA) current injection, while uIPSCs were recorded in granule cells. Trains of 20 action potentials at 50 Hz were evoked at 0.1 Hz in interneurons. IPSCs were analyzed using Mini Analysis (Synaptosoft, Fort Lee, NJ). The threshold for event detection was 3 times root mean square noise level. Latencies of uIPSCs were measured from the peak of presynaptic action potentials to 5% of uIPSC amplitude. Rise time was measured as the interval between points corresponding to 10 and 90% of peak amplitude during the rising phase. Amplitude was measured as the difference between peak amplitude and baseline. Decay time (τ) was measured as the interval between points corresponding to 100% and 37% of peak amplitude during the falling phase. Charge transfer was measured as the area under individual uIPSCs. For uIPSC analysis, parameters were measured from traces averaged after uIPSC failures were excluded. For paired-pulse and multi-pulse analysis, averaged traces included failures.
After recording, slices were placed in 4°C 4% paraformaldehyde in PB at least overnight and then stored at −20°C in preservation solution. Slices were rinsed in 0.5% Triton X-100 and 0.1 M glycine in PB and then placed in blocking solution containing 0.5% Triton X-100, 2% goat serum (Vector Laboratories), and 2% BSA in PB for 4 h. Slices incubated with mouse antibody to NeuN (1:1000, MAB377; Chemicon) in blocking solution overnight. After rinsing, slices incubated with Alexa Fluor 594 streptavidin (5 µg/ml) and Alexa Fluor 488 goat anti-mouse serum (10 µg/ml; Invitrogen) in blocking solution overnight. Slices were rinsed, mounted on slides, and coverslipped with Vectashield (Vector Laboratories).
For dendritic reconstruction, biocytin-labeled neurons were scanned with a confocal microscope (LSM 5 Pascal; Zeiss) at a magnification just large enough to include the entire dendritic arbor. Stack height was adjusted to include all dendritic processes. Optical section interval was 3 µm. For high-magnification reconstruction of axon-dendrite appositions, a 100× objective and 0.5 µm optical section intervals were used. For axon reconstruction, a 20× objective was used to collect image stacks at 1 µm optical section intervals through the entire slice depth containing axon. Montages were collected to sample the entire area of the dentate gyrus molecular layer containing axon. Somata, dendrites, and axons were reconstructed and measured using Neurolucida (MBF Biosciences).
All chemicals and drugs were obtained from Sigma unless specified otherwise. Results are reported as mean ± s.e.m. Statistics were performed using Excel (Microsoft) and Sigma Stat (Systat) with p<0.05 considered significant.