Thy1 GFP-expressing mice
. Mice expressing GFP under control of the Thy1 promoter were bred from the M line, which has been described previously (Feng et al., 2000
). Thy1 belongs to the Ig superfamily and is expressed in many neuronal as well as some non-neuronal cells. Importantly, the dentate granule cells labeled by GFP in the M line in our study appear to represent typical granule cells in that their somatic, dendritic, and axonal architectures replicate patterns revealed by other techniques (Ramon y Cajal, 1911).
Status epilepticus induction. All procedures conformed to National Institutes of Health and institutional guidelines for the care and use of animals. Mice were maintained on a 12 hr light/dark cycle. Eleven male GFP-expressing mice 2–3 months old were injected with 1 mg/kg methyl scopalamine nitrate intraperitoneally (Sigma, St. Louis, MO). Fifteen minutes later, six mice were injected intraperitoneally with 340 mg/kg pilocarpine (Sigma), and five littermates were injected with saline. Pilocarpine was administered between 10 A.M. and noon. Mice were observed after the injections for the appearance of seizure activity and onset of status epilepticus. Status epilepticus was defined as occasional or frequent myoclonic jerks, partial- or whole-body clonus, shivering, loss of posture, and/or rearing and falling that was not interrupted by periods of normal behavior. Animals were allowed to remain in status epilepticus for 3 hr before the condition was terminated by injecting 10 mg/kg diazepam. Control animals also received diazepam injections.
Forty-eight hours after the termination of seizure activity, mice were anesthetized with 100 mg/kg pentobarbital intraperitoneally and perfused through the ascending aorta at 10 ml/min for 30 sec with ice-cold PBS with 1 U/ml heparin followed by room temperature 2.5% paraformaldehyde and 4% sucrose, pH 7.4, for 10 min. The brains were removed and postfixed in ice-cold 2.5% paraformaldehyde and 4% sucrose, pH 7.4, for 1 hr, after which they were cryoprotected in 10% sucrose in PBS overnight, for 24 hr in 20% sucrose in PBS, and finally for 48 hr in 30% sucrose in PBS. After cryoprotection, brains were snap-frozen in isopentane cooled to –25°C with dry ice and stored at –80°C until cryosectioning. Sections were cut at 40 μm, wet-mounted to Superfrost Plus slides (Port City Diagnostics, Wilmington, NC), and stored at –80°C until immunostaining.
BDNF and GFP immunohistochemistry
. The BDNF primary antibody (generously donated by Amgen, Thousand Oaks, CA) was used at a concentration of 1 μg/ml. This polyclonal rabbit antibody has been extensively characterized, and its specificity for BDNF has been established (Conner et al., 1997
; Yan et al., 1997
). Antibody specificity was further confirmed in our hands by preincubating the primary antibody with BDNF peptide (Danzer et al., 2004a
) and by using conditional BDNF knock-out animals (Danzer et al., 2004b
; He et al., 2004
). GFP expression was simultaneously enhanced by incubating the sections with mouse anti-GFP monoclonal antibodies (1:500; Chemicon, Temecula, CA). After a 1 hr incubation at room temperature in PBS, 5% NGS, and 0.5% Igepal (Sigma), both primary antibodies were applied simultaneously at 4°C for 24 hr in PBS and 5% normal goat serum. Alexa Fluor 594 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse secondary antibodies were used at concentrations of 1:750 (Molecular Probes, Eugene, OR). Adjacent sections not treated with the BDNF primary antibody were also run for each animal. In all cases, sections from pilocarpine-treated mice were processed simultaneously in the same incubation dishes with their littermate controls so that valid comparisons could be made.
Microscopy and data collection. All data collection and analysis were conducted with the experimenter blinded to treatment group. GFP-expressing dentate granule cell terminals in the stratum lucidum of CA3b were imaged using a Leica (Nussloch, Germany) TCS SL confocal system set up on a Leica DMIRE2 inverted microscope equipped with epifluorescent illumination and a 63× oil immersion objective (numerical aperture, 1.4). CA3a and CA3b pyramidal cell thorny excrescences were also imaged. Images were captured using 6× optical zoom. GFP (Alexa Fluor 488) and BDNF (Alexa Fluor 594) immunoreactivity signals were captured using sequential line scanning. Z-series “stacks” of neuronal processes were collected at 0.4 μm increments with the pinhole set to 0.7 Airy units. Four times line averaging was used to improve image quality. Neuronal structures were identified from the z-series stacks and scored as either BDNF-negative or -positive. Colocalization was confirmed in x, y, and z dimensions. The thickness of the focal plane of the microscope becomes significant for colocalization in the z dimension. For the objective used in the present study, z resolution is stated to be 235 nm (Leica). (This assumes ideal conditions and 488 nm light, and, in practice, z resolution will be somewhat larger.) Maximum resolution in the x–y plane is 180 nm.
Mossy fiber boutons, en passant terminals, and thorny excrescence spine heads were scored as positive for BDNF immunoreactivity if at least 50% of the structure was immunoreactive and if the immunoreactive signal was at least twice as intense as the area immediately surrounding the terminal. Filipodia were scored as positive if they either met the above criteria or if three regions of a filipodium were twice as intense as background. These criteria for filipodia were used because BDNF-immunoreactive particles <1 μm in diameter were often seen to “track” the path of the filipodia (see , right column), indicating that the filipodium was almost certainly BDNF-immunoreactive but not always meeting the 50% filled criterion. The experimenter's ability to consistently distinguish structures as twice as intense as background was confirmed with the Leica software channel dye separator, which can quantify BDNF and GFP signals from captured images. To confirm that the settings used to capture the signal for BDNF immunoreactivity did not also capture the GFP immunoreactivity, sections immunostained as above but with the BDNF antibody excluded from the immunostaining procedure were also examined. GFP immunoreactivity did not interfere with the BDNF signal. Finally, we note that the criteria used for determining whether a structure is immunopositive are deliberately conservative. Because this is the first study to directly examine BDNF immunoreactivity in many of these structures, we did not want to falsely identify a structure as containing BDNF. A consequence of this approach, however, is that our values likely underestimate the number of immunoreactive structures. We also recommend caution when comparing the different percentages of BDNF-immunopositive structures (e.g., boutons vs filipodia). The different sizes and shapes of these terminal structures may differentially affect our ability to detect BDNF immunoreactivity in them.
Figure 3. BDNF-immunoreactive giant mossy fiber boutons and mossy fiber bouton filipodia. Top, GFP in granule cell giant mossy fiber boutons (arrows) and mossy fiber bouton filipodia (arrowheads). Middle, BDNF-immunoreactive clusters that colocalize with giant (more ...)
Identification of dentate granule cell giant mossy fibers boutons was based on their location in the stratum lucidum and their unique structure: their unusually large size (8–17 μm 2
) and their continuity with the mossy fiber axon (Amaral and Dent, 1981
). En passant terminals were distinguished from giant mossy fiber boutons by their smaller size (0.5–2 μm) (Acsády et al., 1998
) and smoother appearance (see ). Expansions had to be at least twice the diameter of the parent axon to be counted as an en passant terminal (Claiborne et al., 1986
). CA3 pyramidal cell thorny excrescences, which are located on the proximal dendrite, were identified based on their unique structure: single or clusters of large postsynaptic spines attached to a single stalk (see ).
Figure 2. Composite confocal image showing a dentate granule cell giant mossy fiber bouton (white arrow), a dentate granule cell MFB filipodia (green arrowhead), dentate granule cell en passant terminals (yellow arrowheads, EPT), a CA3b pyramidal cell apical dendrite (more ...)
Statistics. To avoid pseudoreplication, each animal was given a single percentage score for each structure assessed for BDNF immunoreactivity. Comparisons used Mann–Whitney rank–sum tests run on SigmaStat software (Jandel Scientific, San Rafael, CA).