All procedures conformed to NIH and institutional guidelines for the care and use of animals.
Thy1-GFP Expressing Mice
Mice expressing green fluorescent protein (GFP) under control of the Thy1 promoter were obtained as a generous gift from Dr. Guoping Feng (Duke University). The mice used in the present study were bred from the M line on a C57BL/6 background, and have been described previously (Feng et al., 2000
; Danzer and McNamara, 2004
; Walter et al., 2007
; Danzer et al., 2008
). Importantly, the subset of granule cells (~11%) that express GFP in this mouse line is morphologically and physiologically indistinguishable from unlabeled granule cells (Vuksic et al., 2008
), and therefore is likely representative of the entire population.
Two to three-month-old GFP-expressing male and female mice were injected with 1 mg/kg methyl scopolamine nitrate intraperitoneally (i.p.). Fifteen minutes later, mice were injected i.p. with either 340 mg/kg pilocarpine or saline (control mice). All treatments occurred between 10 a.m. and noon. Mice were observed following the injections for the onset of status epilepticus, which typically occurred within 1 h. Status epilepticus was defined behaviorally by continuous tonic/clonic convulsions. Mice received 10 mg/kg diazepam 3 h after the onset of status epilepticus. Control mice received diazepam 4 h after saline injections. Pilocarpine-treated animals that failed to develop or did not survive status epilepticus were excluded from the study. Following status epilepticus, and for the next 2 days (once per day), animals were weighed and given Ringers solution as needed to maintain pre-seizure body weight. Each pilocarpine-treated animal was paired to a control animal, and the control animal received 0.5 ml Ringers if the pilocarpine-treated animal received an injection. Mice were perfused with paraformaldehyde 2 days (control, N = 4; status epilepticus, N = 6) or 1 month (control, N = 7; status epilepticus, N = 12) after treatment.
Two to three-month-old GFP-expressing mice underwent stereotaxic implantation of a bipolar stimulation-recording electrode in the right amygdala under pentobarbital anesthesia (60 mg/kg, i.p.). The following coordinates were used, with bregma as reference: 1.0 mm posterior, 2.9 mm lateral, and 4.6 mm below dura. A wire secured to the skull overlying the left frontal cortex was used as a ground electrode. Electrographic seizure threshold was determined 1 week after surgery by application of a 1 s train of 1 ms biphasic rectangular pulses at 60 Hz beginning at 60 μA. Additional stimulations increasing by 20 μA were delivered at 1 min intervals until an electrographic seizure lasting at least 5 s was detected. Subsequently, experimental animals were stimulated twice a day at stimulus intensities 100 μA above the electrographic seizure threshold. Interstimulus interval was at least 4 h, and animals were stimulated each day until five consecutive seizures involving at least 12 s of limb clonus and/or tonus were evoked. Sham kindled animals were connected twice daily to the stimulation apparatus, but did not receive stimulations. Mice were perfused with paraformaldehyde 1 day (control, N = 7; kindled, N = 7) or 1 month (control, N = 8; kindled, N = 7) after the last kindled seizure or sham stimulation.
Animals were overdosed with 100 mg/kg pentobarbital administered i.p. and perfused through the ascending aorta at 10 ml/min for 30 s with ice-cold phosphate-buffered saline [phosphate buffered saline (PBS)] with 1 mM sodium orthovanadate (NaOV) and 1 U/ml heparin. Mice were then perfused for 10 min with 25°C, 2.5% paraformaldehyde, 4% sucrose, and 1 mM NaOV, pH 7.4. The brains were removed and post-fixed in the same fixative for 1 h at 4°C. Brains were cryoprotected in an ascending sucrose series (10%, 20%, 30%) in 1 mM NaOV in PBS, snap-frozen in isopentane cooled to −25°C with dry ice and stored at −80°C until cryosectioning. Forty micron coronal sections corresponding roughly to figure 46–48 of Paxinos and Franklin’s mouse brain atlas (2001)
were used for GFP, Nissl, and Fluoro-Jade B staining.
Microscopy and Data Collection
The following general guidelines were used for all image collection and data analysis. All images and analysis were collected with the investigator blinded to treatment group. Only bright GFP-labeled granule cells, in which processes could be followed to their natural terminations, were selected for analysis. GFP-expressing dentate granule cells were imaged using a Leica TCS SL confocal system set up on a Leica DMIRE2 inverted microscope equipped with epifluorescent illumination and a 63X oil immersion objective (NA 1.4). Using this system, three-dimensional Z-series stacks were captured at 0.2 μm increments with two to six times optical zoom. No corrections were made for shrinkage of the tissue. To avoid pseudoreplication, multiple measurements from individual animals were averaged and used as a single data point for statistical analysis. Significance was determined by Student’s t-test. Nonparametric versions of tests and medians and ranges are reported, as noted, for data that was not normally distributed. Finally, all comparisons were made between an experimental group and an age and condition-matched littermate control group. Control groups for the kindling and pilocarpine models differ by a number of variables (surgery, handling, etc.), so comparisons between controls should be avoided.
Mossy Fiber Axons
Confocal microscopy was used to image mossy fiber axons of dentate granule cells in stratum lucidum of CA3b (as defined by Lorente de Nó, 1934
). Mossy fiber axons are decorated with three morphologically distinct types of presynaptic terminals; giant mossy fiber boutons, filopodial extensions arising from these giant boutons, and en passant
terminals. Mossy fiber terminal structure was quantified by importing confocal image stacks into Neurolucida (Microbrightfield, Williston, VT) or Metamorph (Universal Imaging Corporation, West Chester, PA, version 4.5r6) imaging software. For this analysis, giant mossy fiber boutons were defined as expansions of the mossy fiber axon with a cross-sectional area that exceeded 4 μm2
(Claiborne et al., 1986
; Acsády et al., 1998
). Electron microscopy studies indicate that these larger expansions are giant mossy fiber boutons, whereas expansions with a cross-sectional area of less than 4 μm2
are en passant
terminals, accumulations of mitochondria, or tissue artifacts (Acsády et al., 1998
). Given the ambiguity about the identity of these smaller structures, only the larger giant mossy fiber boutons were examined here. Filopodia were defined as extensions arising from a giant mossy fiber bouton, but distinct from the main mossy fiber axon, that were less than 1 μm in diameter and greater than 1 μm in length (Amaral, 1979
; Amaral and Dent, 1981
). Filopodia were not included as part of the giant mossy fiber boutons cross sectional area. Recent data also demonstrate that giant mossy fiber boutons can be arranged into local terminal arborization complexes, consisting of a core mossy fiber giant bouton connected to one or more satellite boutons by axonal fibers originating from the core bouton (Galimberti et al., 2006
). For the present study, satellites were defined as mossy fiber expansions connected to a core giant bouton by thin axonal processes. A profile area greater than 4 μm2
was required for an expansion to qualify as a satellite, thereby distinguishing satellites from filopodia. Furthermore, only giant boutons in which all the emanating processes could be identified as either the primary axon, or followed to a natural termination as either a filopodium or satellite, were scored for determining the frequency of complexes. Because larger complexes, with more distant satellites, are more likely to be truncated in brain tissue sections, this conservative approach likely underestimates the frequency of complexes with satellites. Conservative criteria are necessary, however, since giant boutons are frequently penetrated by other axons (Rollenhagen et al., 2007
), which could lead to overestimates of complex frequency.
Approximately 15–30 randomly-selected giant mossy fiber boutons from each animal were examined to determine mean cross sectional area, the percentage of giant mossy fiber boutons with satellites, filopodia number, and length. Two to four adjacent brain sections from each animal were used. To be selected for analysis, giant mossy fiber boutons had to be contained within the tissue section examined. The density of giant mossy fiber boutons per length of axon was determined from five to six axonal segments per animal, with a combined length of ~500–1,000 μm.
GFP Positive Cell Counts
The number and location of GFP-expressing granule cells in kindled and control mice was determined using Neurolucida software to analyze confocal image stacks. Images were collected with a Leica SP5 confocal microscope set up on a DMI6000 stand equipped with 10X objective (NA 0.3). Images were collected at 2 μm increments through the Z-depth of the DG. Neurolucida software was used to generate 3-dimensional reconstructions encoding the number of GFP-expressing granule cells in the upper blade of the dentate in each section, the distance of each cell from the granule cell layer-hilar border and the distance of each cell from the crest of the DG. Importantly, measurements of GFP cell number and position were conducted in the same sections used for morphological studies. Finally, measurements presented here should not be construed as being representative of regions of the dentate not examined.
Cell Loss Scoring
Sections adjacent to those used for morphological studies were stained for Nissl substance with cresyl violet and cell loss was scored under 10× magnification in four regions: the hilus, dentate granule cell layer, CA3 pyramidal cell layer, and CA1 pyramidal cell layer. Regions were scored using a semiquantitative system as follows: 0: no obvious cell loss. 1: less than 25% cell loss. 2: ~50% cell loss. 3: greater than 90% cell loss. Two adjacent hippocampal sections per animal were scored, the scores for each region averaged and a cumulative cell loss score given to the animal (cumulative cell loss = hilus + dentate + CA3 + CA1; minimum possible score = 0, maximum = 12).
Fluoro-Jade B Staining
Fluoro-Jade B is a sensitive marker for dead and dying neurons (Schmued and Hopkins, 2000
). To stain sections with Fluoro-Jade B, slides were immersed in 100% EtOH for 3 min, 70% EtOH for 1 min, dH2O for 1 min, 0.06% potassium permanganate for 15 min, dH2O for 1 min, 0.001% Fluoro-Jade B + 0.1% acetic acid for 30 min, rinsed in dH2O, air dried, and coverslipped with Crystalon. For each time point and epilepsy model, sections from control and experimental animals were stained simultaneously in the same containers. Sections from animals previously demonstrated to exhibit extensive cell loss, and sections from untreated animals were included with each reaction as positive and negative controls, respectively.
Confocal image stacks representing the z-depth of a neuronal structure were captured using a Leica TCS SL confocal microscope. Image stacks were used to generate maximum projections in Leica’s LCS Lite Confocal software (version 2.61). In some cases, images are montages generated from the confocal z-series of a structure. This processing was done to remove adjacent structures located above or below the observed structure, which would obscure the two-dimensional representation. Montages, contrast, and brightness adjustments and figure preparation were conducted using Adobe Photoshop (version 7.0). Contrast and brightness were adjusted identically for images meant for comparison.