|Viral Clostridial Light Chain Gene-based Control of Penicillin-induced Neocortical Seizures
Supplemental methods online
Animal surgery: Animals were anesthetized with ketamine (80 mg/kg, i.p.) and zylazine (5 mg/kg) and placed in the Kopf stereotaxic frame (David Kopf Instruments, Tujunga, CA). A midline sagittal incision was made and the underlying skull was exposed. Four epidural stainless steel electrodes (MX-080-2FL-1M, Small Part Inc., Miami Lakes, FL) were implanted for the purpose of EEG recording. Screws were placed on the dura mater bilaterally over the frontal and parietal cortex (LF, left frontal cortex; RF, right frontal cortex; AP: +1.0mm, L: 2.5mm from bregma, LP, left parietal cortex; RP, right parietal cortex; AP: -2.0mm, L 2.5mm from bregma)[1, 2]. An additional epidural screw electrode was placed at the tip of left frontal region to serve as reference electrode (REF). A burr hole was performed over the area of the right frontal motor cortex and the dura mater was gently opened under surgical microscope (Stereozoom 6; Leica, Buffalo, NY). A stainless-steel cannula (0.6 mm in diameter, C313G, Plastic One, Roanoke, VA) connected to an electrode was stereotaxically implanted in the right motor cortex at coordinate of AP: + 2mm, L: +2mm from bregma; D: +1.5mm from dura using the photographic atlas of rat brain. This cannula was used for both local injection and EEG recording. All of the electrodes and the cannulae were connected to a plastic connector (SMP-06V-BC, Japan Solderless Terminal MFG, Tokyo, Japan) that was fixed to the skull using dental cement (Hygienic Ortho Resin, H02100, Coltene-Whaldent, Cuyahoga Falls, OH).
Induction of seizures: Under a light dose of ketamine anesthesia (40 mg/kg, i.p), the rat was placed in the EEG monitoring cage. An inner stainless-steel injection cannula (0.3 mm in diameter, C313I, Plastic One, Roanoke, VA) connected to a tubing system was introduced into the cannula of the rat. A total dose of 1125 UI (3.75 l of 3000,000 UI/ml) of penicillin solution (NDC10515-500-10, Hanford Mfg. Co., Syracuse, NY) was injected over 5 min with a mechanical pump (NP 55-2222, Harvard Apparatus, Holliston, MA) using a Hamilton syringe to induce seizures. The cannula was removed 5 minutes after the end of injection and the rat was free to move in the recording cage.
EEG and video monitoring: After injection of penicillin, continuous EEG monitoring and simultaneous video recording were performed to record EEG activity. EEG monitoring using the implanted epidural electrodes was performed in three custom-made electrically shielded boxes[1, 2]. Implanted recording electrodes were connected to a pendulous electro-slip ring (MRS 35-06P, MT GIKEN, Tokyo, Japan), which permits the acquisition of real-time noise artifact-free EEGs in freely moving rats or during the seizures. Digital EEG recordings (6 channels per rat) were performed using a Vangard system (Vangard, Cleveland, Ohio), which consists of a recording Hewlett-Packard workstation and a similar review system connected to a 21-inch monitors. The acquisition sampling rate was set at 100 Hz. The high and low frequency filter settings were 1 Hz and 70 Hz, respectively. The EEG data were reformatted to a referential montage. The EEG recording was time locked with an analog digital video recording, which permitted the analysis of the behavioral aspects of all the seizures during each experiment.
Evaluation of the impact of cortical LC expression on motor performance: To assess possible side effects of LC gene expression on motor performance, the Basso–Beattie–Bresnahan (BBB) locomotor score, rotarod assay, and grip strength meter were used for behavioral and neuromuscular function testing before and after viral vector administration[4-6]. The criteria of BBB were rated on the 21-point scale that awards points for observable spontaneous movement at various limb joints (21=normal motor function, 0=no observable limb movement). The second motor function assay tested the maximal rotational velocity (Vmax) at which rat was able to remain on the rotarod apparatus (Columbus Instruments, Columbus, OH). Performance on the rotarod tests forelimb and hindlimb function as well as balance. Following establishment of the baseline motor performance at 5 r.p.m. (rotations per minute), the rotarod was accelerated by 0.1 r.p.m./second increment and the time required for the animal to fall off the rotarod was recorded. The Vmax at which the rat was able to remain on the rotarod was calculated with the following formula: Vmax= (ta) + s, where t=the time required for the animal to fall from the rotarod, a=the acceleration of the rotarod apparatus and, s=the start up speed. In addition, muscle strength of forelimb was measured using an automated Grip Strength Meter (GSM, Columbus Instruments, Columbus, OH). The Grip Strength Meter provides a validated measurement of motor function. The instrument uses an electronic digital force gauge that measures the peak force exerted upon it by the action of the animal.
Immunohistochemistry (IHC): Tissue sections identified as having GFP expression were immunohistochemically stained for LC and synaptobrevin/VAMP-1 (vesicle-associated membrane protein) with Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) according to the protocol of the manufacturer. After rinsed with PBS, sections were incubated in 0.002% proteinase K solution for 10 minutes at 370C, followed by rinsing again and incubation in 1% H2O2 in methanol for 10 minutes. Tissue sections were then blocked with blocking serum for 20 minutes. Rabbit anti-synaptobrevin/VAMP-1 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in 1:300 blocking solution or mouse anti-LC primary antibody diluted in 1:300 blocking solution was applied to sections in a humid chamber on a shaker for one hour at room temperature. Negative controls were performed by applying blocking solution alone to different sections in the same slide.
Sections were washed with PBS before diluted biotinylated secondary goat anti-rabbit antibody (for synaptobrevin/VAMP-1) or horse anti-mice antibody (for LC) was applied for 30 minutes at room temperature, followed by PBS rinses and incubation with avidin-HRP (ABC mix of VECTASTAIN Elite ABC Reagent) for 30 minutes at room temperature. Following washing with PBS, immunohistochemical staining was visualized with 3’3-diaminobenzidine (DAB) mixed solution (DAB Substrate Kit, Vector Laboratories, Burlingame, CA) for 5-10 minutes. After washing with water, slides were counterstained with Vector Methyl Green Solution (Vector Laboratories, Burlingame, CA) and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA).
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