Male and female mice (B6: 129-Grm5m I Rod
/J) weighing 20–32g were used for this study. Status epilepticus (SE) was induced according to our established procedures (28
). Briefly, mice were given a single subcutaneous injection of methyl-scopolamine nitrate (1mg/kg) 30 min before the injection of either saline in the control or pilocarpine in the experimental groups. In the latter groups, mice were given a single i.p. injection of pilocarpine (300 mg/kg) and experienced status epilepticus for about 4 hrs. Pilocarpine-induced behavioral changes, including hypoactivity, tremor, head bobbing, and myoclonic movements of the limbs, progressing to recurrent myoclonic convulsions with rearing, falling, and status epilepticus, were similar [similar to what in previous reports?] to our previous reports (28
All experiments were approved by the Tan Tock Seng Hospital – National Neuroscience Institutional Animal Care & Use Committee. The mice were kept in a specific pathogen-free room and maintained with free access to food and water on a 12 hr light-dark cycle. Efforts were made throughout the study to minimize animal suffering and to use the minimum number of animals.
NeuN, CB, CR and PV immunocytochemistry
For NeuN, parvalbumin (PV), calbindin (CB), and calretinin (CR) immunocytochemical studies, 7 experimental mice at 2 months after SE and 6 age-matched control mice were used. They were deeply anesthetized with chloral hydrate (0.40 g/kg), perfused transcardially with 10 ml of saline initially, and followed by 100 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 20 min. After perfusion, the brains were removed and kept overnight in 30% sucrose in 0.1 M PB. Coronal sections at 40 µm thicknesses were cut in a cryostat (HM505E, Microm, Zeiss, Oberkochen, Germany). A set of five serial sections was prepared from each brain and placed in different wells of a 24-well tissue culture dish for the control, NeuN, CB, CR and PV immunocytochemical reactions. For immunocytochemical studies, freely-floating sections were treated in 4% normal goat serum for 2 hrs at room temperature, washed in 0.1 M phosphate-buffered saline (PBS) containing 0.1% Triton-X 100, and placed overnight in primary rabbit antibodies for NeuN (1:1000) (Chemicon International, Inc., CA, USA), PV (1:3000), CB (1:3000) and CR (1:3000) (Swant Inc, Switzerland). After incubation, sections were washed in PBS and placed in biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) diluted 1:2000 in PBS/Triton X-100 for 1 hr. They were placed in avidin–biotin complex (ABC) reagent (Vector Laboratories) in PBS/Triton X-100 for 1 hr, washed in PBS, and reacted in a solution of 0.12% H2O2 and 0.05% 3,3-diaminobenzidine (DAB) (Sigma-Aldrich, Missouri, USA) in Tris buffer (TB) for 15 min. Sections were then mounted, dehydrated, covered, and photographed by using an image analysis system.
Mice (30 SE and 22 controls) were anesthetized with 20% urethane (0.75g/kg, i.p.) and fixed in a stereotactic apparatus. A concentric stainless steel stimulating electrode (World Precision Instruments Inc, USA) with tip diameter of 0.1mm was implanted into the lateral entorhinal cortex (LEnt) with coordinates (mean value ± standard deviation) posterior to the bregma: 2.7 ± 0.1 mm; lateral to the midline: 4.3 ± 0.05mm; and ventral to the dura: 2.8 ± 0.10mm (44
). Extracellular recordings of neurons from the dorsal subiculum were performed using single-barrel glass micropipettes (external diameter of the tip: 2–3µm, impedance from 5 to 15MΩ.) filled with biotinylated dextran amine (BDA, Molecular Probes Inc, USA) solution (5%) for juxtacellular labeling of neurons. The stereotaxic coordinates (mean value ± standard deviation) for all recording sites were: posterior to bregma: 3.4 ± 0.2 mm; lateral to midline: 2.0 ± 0.15mm; and ventral from the dura1.35 ± 0.20mm (44
). Signals were amplified (P16; Grass Instruments; Astro-Med, Inc, West Warwick, U.S.A.) and fed to storage oscilloscopes. Single-cell signals were filtered at a range of 30Hz-3k Hz. Local field activity was filtered at a range of 0.1Hz-1k Hz. Single-cell and field activities were stored simultaneously on a computer via a Cambridge Electronic Design (Cambridge, UK) interface using Spike 2 software. The spontaneous activity of a single neuron was recorded for 10–20 min after stable firing. The lateral entorhinal cortex was electrically stimulated with single pulses at 0.2mA, 0.2ms, 0.1–0.2Hz and delivered with a S88K stimulator (Grass Instrument, Astro-Med Inc, West Warwick, USA). To confirm the stimulation site, positive electric current was administered (1~2mA) for 30~60 sec through the stimulating electrode after the experiment to produce a local lesion of the LEnt.
Juxtacellular injection of BDA, BDA immunostaining, and characterization of neurons with different patterns of spontaneous activity by immunofluorescence double labeling for BDA with PV, CB, or CR
After the electrophysiological recording, the neurons were labeled by juxtacellular injection of biotinylated dextran amine (BDA) with a modification of the protocol reported previously (24
). Briefly, positive current at 10–30nA, 500ms was applied at a frequency of 1 Hz through an iontophoresis pump (Kation Scientific, Minneeapolis, MN, USA) for 20 min. One day after BDA delivery, mice were deeply anaesthetized, and perfused with 10ml of saline, followed by 100ml of 4% paraformaldehyde and 0.2% picric acid in 0.1 M PB (pH 7.4) for 20 min. Coronal sections of the brain were cut at 40 µm thickness, serial sections were transferred to different wells of a 24-well tissue culture dish, and sections were placed overnight in avidin-horseradish peroxidase (HRP) conjugate (1:1000) and reacted in a solution of 0.12% H2
and 0.05 DAB in TB for 20 min for single-labeling of recorded cells according to the previous reports (40
To correlate electrophysiological properties to different subtypes of interneurons, immunofluorescence double-labeling for BDA with PV, CB, or CR was done. To efficiently label recorded neurons, we developed a new method to double-amplify BDA immunopositive product using avidin-horseradish peroxidase conjugate and primary mouse antibody for HRP. Sections were incubated overnight in avidin-horseradish peroxidase conjugate (1:1000), washed in 0.1% TBS-TX (TBS containing 0.05% Triton X-100), and blocked in 4% normal goat serum for 2 hrs, and then placed in a primary mouse antibody for HRP (1:300) (ABcam, Cambridge, UK) and rabbit antibodies for PV (1:150), CB (1:200) or CR (1:150) for 24 h. After washing in 0.1% TBS-TX, sections were incubated in Cy3-conjugated goat anti-mouse IgG (1:100) for 4 hrs and followed in FITC-conjugated goat anti-rabbit IgG (1:100) for 4 hrs. The sections were mounted, dried, and covered with FluorSave TM Reagent (Calbiochem-Novabiochem, CA, USA) to retard fading, and then examined with an Olympus Fluoview FV500 Confocal Laser Scanning Biological Microscope.
Iontophretic injection of phaseolus vulgaris leucoagglutinin (PHA-L), PHA-L single or PHA-L with CB, CR, or PV double immunostaining
Twenty mice (8 for control and 12 at 2 months after pilocarpine injection) were used. Mice were anesthetized with chloral hydrate (40mg/kg) and fixed in a Stoelting stereotaxic apparatus. A small hole was drilled into the skull above the intended injection sites, and a glass micropipette (with a diameter of 20–30 µm) containing a 2.5% solution of PHA-L (Vector Laboratories, Burlingame, CA, USA) in 0.1M phosphate buffered saline (pH 7.4) was lowered into the superficial layer (mainly in layers II and III) of the lateral entorhinal cortex at 2.7 mm posterior to the bregma, 4.3 mm lateral to the midline, and 2.8 mm ventral to the dura (44
). PHA-L was delivered iontophoretically with positive current (5µA: 7 seconds on, 7 seconds off) for 10 minutes. Seven days after PHA-L delivery, animals were deeply anaesthetized, and perfused transcardially with 50 ml of saline, followed by 100 ml of 4% paraformaldehyde and 0.15% picric acid in 0.1M PB (pH 7.4) for 30 minutes. Coronal sections at 40 µm thickness were cut in a cryostat, and serial sections were transferred to different wells of a 24-well tissue culture dish for control, PHA-L single staining, and PHA-L with PV, CB, or CR double-labeling.
For the PHA-L immunocytochemical study, freely-floating sections were placed overnight in primary goat antibody for PHA-L (1: 5,000) (Vector Laboratories, Burlingame, CA, USA), and for 2 hours in biotinylated horse anti-goat IgG for PHA-L diluted 1: 500 in TBS/Triton X-100. Sections were then incubated in an avidin-biotin complex (ABC) reagent in TBS/Triton X-100 for 2 hours, reacted in a solution of 0.12% H2O2 and 0.05% 3,3’-diaminobenzidine (DAB) (Sigma-Aldrich, Missouri, USA) in TB for 20 minutes, and mounted. Alternative PHA-L-stained sections were counterstained with cresyl fast violet (CFV), coverslipped, and photographed using image analysis system.
For double-labeling of PHA-L with CB, CR, and PV, sections were incubated in primary goat antibody for PHA-L (1:1000) and rabbit antibodies for CB (1:500), CR (1:500) and PV (1:500) (Swant, Switzerland) for 48 hours, washed in TBS/Triton X-100, and placed in biotinylated horse anti-goat IgG (1:200) and swine anti-rabbit IgG (1:100) for 4 hours. They were then incubated in ABC solution for 2 hours, and reacted in DAB-Nickel solution for 20 minutes. Sections were then incubated in rabbit peroxidase anti-peroxidase (PAP) (1:100) solution overnight, and reacted with DAB alone.
To further confirm possible synaptic contacts between PHA-L immunopositive en passant or terminal boutons and CB, CR, or PV immunopositive neurons observed from DAB immunostaining, immunofluorescence double-labeling was performed. Sections were incubated in a primary goat antibody for PHA-L (1:1000) and rabbit antibodies for CB (1:500), CR (1:500), and PV (1:500) (Swant, Switzerland) for 48 hours, washed in TBS/Triton X-100, and placed for 4 hrs in FITC-conjugated goat anti-rabbit IgG against CB, CR, PV and Cy3-conjugated donkey anti-goat IgG for PHA-L. The sections were then mounted, dried, and coverslipped by using FluorSaveTM Reagent (Calbiochem-Novabiochem, CA) to retard fading. The tissue preparations were digitized and reconstructed three-dimensionally using a Confocal Laser Scanning Biological Microscope.
Electrophysiological data were analyzed off-line using Spike2 and DataPac software. The original data were separated on three-frequency bands by digital filtering (FIR 555 Rolloff): 1) 2 Hz low pass band for slow-wave activity; 2) 20–60 Hz band pass for beta-gamma activity; and 3) 500 Hz high pass for unit activity. The sharp increase in the amplitude of beta-gamma activity was considered a beginning of the Up phase. The flattening of beta-gamma activity was considered the Down phase.
Discriminated unit discharges were acquired and further analyzed for interspike interval and firing rate. In previous studies (2
), burst analysis was performed using the bursts v1.25.s2s script
of Spike2. Burst events were identified by the following parameters: the maximum interval between the first two spikes (30 ms), and the minimum number of spikes within a burst (n=2). Burst rate, percentage of burst discharges /total discharges, and the duration of each burst were acquired. To analyze field activity, Fast Fourier transformation (FFT) of waveform data into power spectrums was performed. FFT size was set to 128 Hz, as determined by Nyquist criteria, with hamming windowing of root mean square data. Evoked responses of neurons (latent period and duration) were analyzed on the basis of peristimulus time histograms (PSTH). The latent period was defined as the time period from stimulation to the beginning of evoked discharges. The duration was indicated as the time period for evoked discharges.
For cell counting of NeuN, CB, CR, and PV immunopositive neuronal profiles, three sections from the dorsal subiculum of each animal were selected. Neuronal number in the dorsal subiclum was counted manually and the respective area for those counted neurons was measured using the KS 100 Imaging System (Carl Zeiss Vision, Germany). The cell number was then indicated as the number per square millimeter (no/mm2). Counting of neuronal profiles was done by an investigator blinded to the experimental conditions to which the mice were subjected. The density of cells was indicated as the mean value ± standard deviation (SD). In the present study, in the same manner as that employed in a comparative study between the control and SE mice, we measured the relative number of NeuN, CB, CR and PV immunopositive neurons, but not an absolute number of neurons as measured by stereological techniques. No calibration was made because the chances of neuronal counting errors might occur equally in both groups of the control and SE mice. Statistical significance for two groups of control and experimental mice was determined by a Student’s t-Test, whereas for those with more than two [more than two what?], One-way ANOVA followed by post-hoc test was used. A P-value of less than 0.05 was considered statistically significant.
Juxtacellularly-labeled neurons with BDA were examined under light microscope with high magnification of ×1000. The number of dendritic spines in the 2nd and 3rd order dendrites was counted and indicated as the number per millimeter (No/mm) and compared between normal and SE groups. In the present study, since a comparison study was made between the control and SE mice, no absolute number was needed. Therefore the calibration of the data was not done.