Gephyrin immunoreactivity was evaluated in three groups: pilocarpine-treated controls (n = 6), rats 5 days after status epilepticus (n = 6), and epileptic rats (n = 6) 83 ± 3 days after status epilepticus (108 ± 4 days old at perfusion). Animals were evaluated 5 days after status epilepticus to allow time for synapses to degenerate after status epilepticus-induced neuron loss but to precede the onset of extensive, reactive synaptogenesis. Rats were killed with urethane (2 g/kg i.p.) and lightly fixed by perfusion through the ascending aorta at 30 mL/min for 2 minutes with 0.9% sodium chloride and for 10 minutes with 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at 4°C. Brains were postfixed for 1 hour at 4°C, then equilibrated in 30% sucrose in PB at 4°C. Right hippocampi were isolated and stored at −80°C. Hippocampi were thawed 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 30% ethylene glycol and 25% glycerol in 50 mM PB. Beginning at a random starting point near the septal pole and extending through the entire septotemporal length, a 1-in-24 series of sections was rinsed in PB and mounted on gelatin-coated microscope slides. Tissue was processed in batches that included balanced numbers of animals from each experimental group. Sections were rinsed in 0.1 M Tris-buffered saline (TBS, pH 7.4) and placed for 2 hours in blocking solution consisting of 3% goat serum, 2% bovine serum albumin (BSA), and 0.3% Triton X-100 in 0.05 M TBS. After rinsing in TBS, sections incubated overnight at 4°C in 1% goat serum, 0.2% BSA, 0.3% Triton X-100, 0.05 M TBS, and gephyrin antiserum (). Sections were rinsed in TBS and incubated for 3 hours in 2% BSA, 0.3% Triton X-100, 0.05 M TBS, and 10 μg/mL Alexa Fluor 488 goat antimouse serum (Invitrogen, Carlsbad, CA). Sections were rinsed in TBS and coverslipped with Vectashield (Vector Laboratories, Burlingame, CA).
Electron microscopy, Nissl staining, and in situ hybridization were evaluated in another set of rats consisting of three groups: controls (n = 6), rats 5 days after status epilepticus (n = 3), and epileptic rats (n = 5) 101 ± 60 days after status epilepticus (172 ± 31 days old at perfusion). Rats were killed with urethane (2 g/kg i.p.) and perfused through ascending aorta at 30 mL/min for 1 minute with 0.9% sodium chloride and 30 minutes with 2.5% paraformaldehyde and 1% glutaraldehyde in 0.1 M PB (pH 7.4) at 4°C. Brains were postfixed overnight at 4°C. Left hippocampi were isolated, equilibrated in 30% sucrose in PB at 4°C, gently straightened, frozen, and sectioned perpendicular to septotemporal axis with a sliding microtome set at 40 μm. Beginning at random starting points near the septal pole and extending through the entire septotemporal length, 1-in-12 series of sections were Nissl-stained with 0.25% thionin. Other 1-in-12 series of sections were stored at −20°C in 30% ethylene glycol and 25% glycerol in 50 mM PB and later processed for in situ hybridization for glutamic acid decarboxylase-65 (GAD). GAD65-cDNA (kindly provided by Drs. A. Tobin and N. Tillakaratne, University of California at Los Angeles) was ≈2.3 kb, isolated from a λ ZapII library from adult rat hippocampus (Erlander et al., 1991
), and included the entire coding region (≈1,755 bp), ≈74 bp of the 5′ untranslated region, and ≈467 bp of the 3′ untranslated region (Dr. N. Tillakaratne, University of California at Los Angeles, pers. commun.). RNA probes were produced by transcription of GAD65-cDNA using a nonradioactive RNA labeling kit (Roche, Indianapolis, IN). Sections were washed in 10 mM phosphate-buffered saline (PBS) and incubated sequentially in 0.02 N HCl, 0.01% Triton X-100 in PBS, 0.2 μg/mL proteinase K (Roche) in 50 mM Tris (pH 7.4), 5 mM EDTA, and 2 mg/mL glycine (Roche) in PBS. Sections were prehybridized for 1 hour in a solution containing 50% formamide, 750 mM NaCl, 25 mM EDTA, 25 mM piperazine-N,N
’-bis 2-ethanesulfonic acid (Roche), 0.2% sodium do-decyl sulfate, 250 μg/mL poly A (Roche), and 250 μg/mL salmon sperm DNA (Roche). Sections were hybridized for 2 days in a humid chamber at 50°C in a solution consisting of the prehybridization solution with digoxygenin-labeled RNA probe at a concentration of 2–4 μL/mL, 100 mM dithiothreitol, 4% dextran sulfate, and 250 μg/mL tRNA (Roche). After hybridization, sections were subjected to RNase treatment and stringency washes. Sections were processed for immunodetection of digoxygenin label with reagents of a nonradioactive nucleic acid detection kit (Roche), mounted on gelatin-coated slides, and cover-slipped with Crystalmount (Biomedia, Foster City, CA) and Permount.
Other 1-in-12 series of sections from the same hippocampi from which sections were obtained for Nisslstaining and GAD in situ hybridization were prepared for electron microscopy. Sections were postfixed with 1% OsO4 in sodium cacodylate buffer (pH 7.2) for 1 hour, dehydrated in a series of ethanols, placed in propylene oxide, gradually transferred to pure Araldite/Eponate-12 (Ted Pella, Redding, CA), and flat-embedded between sheets of ACLAR at 60°C for 24 hours. Sample areas on flatembedded tissue sections were isolated and mounted on blank epoxyresin capsules. Block faces were trimmed and ultrathin sections of silver interference color were cut from reembedded blocks with an ultramicrotome (Reichert Ul-tracut S, Leica, Vienna, Austria). Serial sections were collected on coated, nickel single-slot grids. Postembedding GABA-immunocytochemistry was performed on ultrathin sections by blocking nonspecific labeling with 0.8% ovalbumin and 5% fetal calf serum in 0.05 M TBS (pH 7.6) for 1 hour followed by incubation overnight in GABA antiserum () in blocking solution. Grids were gently rinsed and then incubated in antirabbit colloidal gold (10-nm-diameter, 1:80, Ted Pella) in 0.1% Triton X-100 and 0.05 M Tris buffer (pH 8.2) for 90 minutes. Sections were post-stained with 2% aqueous uranyl acetate for 6 minutes and Sato’s lead stain for 4 minutes.
Investigators were blind to experimental groups during analysis. The optical fractionator method (West et al., 1991
) was used as described previously to estimate numbers of neurons (Buckmaster and Jongen-Rêlo, 1999
) and immunoreactive punctae (Kumar and Buckmaster, 2006
). Sampling parameters are listed in . To estimate total numbers of gephyrin-immunoreactive punctae in the granule cell layer plus molecular layer of the dentate gyrus, borders were outlined in sections and sample sites determined randomly and systematically with Stereo Investigator software (MBF Bioscience, Williston, VT). Sample site locations in the granule cell layer, inner third of the molecular layer, or outer two-thirds of the molecular layer were recorded on low-magnification images of sections. Landmarks in the tissue that were visible in low-magnification images were used to relocate sample sites and collect high-magnification images with a confocal laser-scanning microscope (LSM 5 Pascal, Zeiss, Oberkochen, Germany) equipped with a 100× objective and set at 10× zoom. An average of 3,230 ± 373 gephyrin-immunoreactive punctae were counted per rat. A total of 58,140 gephyrin-positive punctae were counted at 1,469 sample sites in 162 sections from 18 rats. To estimate punctae numbers per dentate gyrus, fractions of area sampled were calculated by multiplying numbers of sample sites by counting frame area and dividing by total areas of analyzed regions per section. Gephyrin-positive punctae were counted within sample sites, which consisted of image stacks (6 × 0.4 μm separation). All gephyrin-positive punctae were counted unless they were visible in the most superficial section of a stack or touched left or bottom borders of counting frames. The superficial guard zone was the most superficial section of the stack. Beneath the analyzed stack, an additional optical section was included to provide a deep guard zone.
Results and Parameters of Stereological Neuron and Punctae Counts in the Dentate Gyrus
To estimate numbers of GAD-positive neurons per dentate gyrus, Nissl-stained granule cells, and Nissl-stained hilar neurons, sample sites were randomly and systematically determined by Stereo Investigator software (MBF Bioscience) within borders drawn around corresponding regions: entire dentate gyrus, granule cell layer, and hilus, respectively. The hilus was defined by its border with the granule cell layer and straight lines drawn from the ends of the granule cell layer to the proximal end of the CA3 pyramidal cell layer. The dentate gyrus was defined by the hilar border with CA3 and by the edge of the molecular layer, which was the edge of the tissue in the inferior blade and the hippocampal fissure in the superior blade. Total section thickness was used for dissector height, and only labeled somata or nuclei that were not cut at the upper surfaces of sections were counted. This modification of the optical fractionator method facilitates analysis of tissue sectioned thinly to enhance staining; however, it might increase the probability of underestimating cell numbers. There would be no effect on relative values of control versus other groups, because all were analyzed identically. GAD-positive somata and large Nissl-stained nuclei (neuronal, not glial) in the granule cell layer and hilus were counted using a 100× objective. A total of 3,135 granule cells were counted at 1,029 sample sites in 156 sections from 14 rats (224 ± 10 counted granule cells/rat). In the same sections, 7,104 Nissl-stained neurons were counted at 3,456 sample sites in the hilus (507 ± 41 counted hilar neurons/rat). A total of 3,475 GAD-positive neurons were counted at 10,605 sample sites in 156 sections in the entire dentate gyrus (248 ± 20 counted GAD-positive neurons/rat).
To estimate numbers of synapses in the dentate gyrus, we modified a stereological method developed originally to estimate total numbers of synapses in stratum radiatum of the CA1 field in hippocampus (Geinisman et al., 1996, 2000). Parameters of the stereological electron microscopic synapse counts are listed in . To measure areas of strata within the dentate gyrus, embedded sections were analyzed with Neurolucida (MBF Bioscience) and a microscope (Nikon, Melville, NY) equipped with a 10× objective, and Lucivid (MBF Bioscience). Contours were drawn around the granule cell layer, inner third of the molecular layer, and outer two-thirds of the molecular layer. Borders between the inner third and outer molecular layer were determined by measuring total height of the molecular layer and placing a series of points one-third of the way from the granule cell layer toward the outer border of molecular layer. As in previous studies (Geinisman et al., 1996, 2000), six sites per hippocampus were sampled for electron microscopy. To ensure that synapses in all parts of the dentate gyrus had equal probability of being sampled, sample sites were distributed systematically and randomly. For each animal, cumulative length of the granule cell layer was measured and divided into six uniform intervals. Placements of first sample sites were randomly located within first intervals, and subsequent samples were at equal intervals. For example, shows the 12 embedded sections of a control rat. For each section, granule cell layer length was measured beginning at the tip of the superior blade to the apex and then to the tip of the inferior blade. In this example the cumulative length of the granule cell layer was 73.7 arbitrary units, which was divided by 6, yielding 12.3. A random number between 0 and 12.3 was generated (2.8, in this case), and the first sample site was 2.8 units from the starting point, which was the tip of superior blade of the granule cell layer in the first section. The next sample point was 12.3 units farther along the granule cell layer, which extended through the rest of the granule cell layer in the first section and the granule cell layer length of the second section and was located in the inferior blade of the granule cell layer in the third section. The next sample point was 12.3 units farther along the granule cell layer, and so on, until six sample sites were identified. At each sample site, tissue was trimmed to include the entire height of the molecular layer and granule cell layer. For each hippocampus, the fraction of area sampled was calculated by multiplying the number of sample sites per stratum (six) by counting frame area and dividing by area of that stratum.
Parameters of Stereological Electron Microscopic (EM) Synapse Counts in the Dentate Gyrus
Figure 1 Sampling scheme for stereological analysis of synapse numbers in the dentate gyrus. A: Starting from a random section near the septal pole, a 1-in-12 series of 40-μm-thick transverse sections was embedded. Cumulative length of the granule cell (more ...)
At each sample site, counting frames were located in the granule cell layer, inner molecular layer, and outer molecular layer. Counting frames were randomly determined by locations of score marks made by small imperfections in the cutting edges of diamond knives used to make ultrathin sections. Low-magnification electron micrographs were used to verify score-mark locations. Counting frames were the area of electron micrographs (3.69 × 5.11 μm) photographed using a transmission electron microscope (JEOL 100CX; JEOL, Peabody, MA) at 25,000× magnification and printed at a final magnification of 48,400×. Counting frames were aligned across 100 consecutive ultrathin sections for the inner and outer molecular layer and 150 consecutive sections in the granule cell layer, where synapse density is lower. Counting frames were aligned at one axis by the edge of the tissue block and at the other axis by a score-mark. These fiduciary marks were determined without bias and were independent of tissue-based landmarks. Between fiduciary marks and photographed areas there were borders of constant dimensions, so tissue within photographed areas was not disrupted. Tissue samples were sectioned completely, and total numbers of ultrathin sections determined, so sampled fractions of tissue thickness could be calculated.
Collecting large series of consecutive ultrathin sections and processing them for postembedding immunocytochemistry is technically demanding. Occasional sections were damaged or lost. If five or more consecutive sections were unavailable for analysis in a series, an equal number was added to the end of the series to compensate. This occurred an average of 0.48 times per series, and there were no significant differences between experimental groups (P = 1, analysis of variance [ANOVA]). Gaps consisting of four or fewer consecutive sections also occurred: 78% one section, 14% two sections, 5% three sections, and 3% four sections. An average of eight sections per series was missing, and there were no significant differences between experimental groups (P = 0.48, ANOVA). These missing sections were not compensated, because gaps were small relative to synapse size, making it unlikely that synapses were missed.
Synapses were identified by parallel membranes, concentration of presynaptic vesicles, and postsynaptic densities (Gray, 1959
). All synapses that appeared at least partially within fields of view in series of consecutive sections were counted, unless they were evident in the first section of a series or touched the upper or left-hand borders of prints. The superficial guard zone was the most superficial ultrathin section of the series. Beneath the analyzed series, additional ultrathin sections were included to provide a deep guard zone.
GABA-immunoreactive structures were identified by comparing densities of gold particles to background levels. Pilot tests verified the specificity of GABA labeling. The density of colloidal gold particles was measured over a series of representative sections containing putative GABA-negative and GABA-positive axon terminals and dendrites in the molecular layer. Putative GABA-positive axon terminals were identified by the presence of a symmetric synapse. Putative GABA-negative axon terminals were identified by the presence of an asymmetric synaptic contact with a dendritic spine. Putative GABA-positive dendrites were identified by a high density of asymmetric synaptic contacts with an aspiny dendritic shaft. Putative GABA-negative dendrites were identified by the presence of spines. Using a Neurolucida system (MBF Bioscience) equipped with a data tablet, areas of these structures and the numbers of gold particles overlying them were measured. Gold particle density was highest in putative GABA-positive axon terminals and lowest in putative GABA-negative axon terminals. Relative gold particle densities were 1.0 in GABA-negative axon terminals, 1.4 in GABA-negative dendrites; 10.5 in GABA-positive dendrites, and 28.8 in GABA-positive axon terminals. Therefore, the postembedding GABA immunocytochemistry protocol labeled GABA-positive axons and dendrites with intensities over 10-fold higher than background levels. This level of labeling specificity obviated the need for measuring gold particle density in all of the electron micrographs.
Postsynaptic structures were identified by neurochemical (GABA-negative or GABA-positive) and ultrastructural characteristics. Ultrastructural characteristics included microtubules and mitochondria for dendritic shafts; a spine apparatus, and lack of other organelles for dendritic spines; a nucleus and organelles, including Golgi apparatus, for somata; and microtubule fascicles and membrane undercoating for axon initial segments (Peters et al., 1991
Sizes of GABA-negative asymmetric synapses were measured for a randomly sampled subset of counted synapses whose postsynaptic densities were within or touchinga1 × 1 μm area centered in counting frames of entire series except the last 25 prints. Lengths of postsynaptic densities were measured in each section, summed, and multiplied by ultrathin section thickness to yield synapse area.
Totals of 16,335 GABA-positive and 43,023 GABA-negative synapses (1,167 ± 103 and 3,073 ± 184 per rat, respectively) were counted at 252 sample points consisting of over 29,000 electron micrographs from 14 rats. GABA-negative synapse size was measured at 3,157 synapses (226 ± 12 per rat).
Images were prepared using Adobe Photoshop (San Jose, CA). Only brightness and contrast were adjusted. All chemicals and drugs were obtained from Sigma (St. Louis, MO) unless specified otherwise. Results are reported as mean ± SEM. Statistics were performed using Sigma Stat (Systat, San Jose, CA) with P < 0.05 considered significant.