Protocol: epileptogenic in amygdala stimulation model; Protocol: unpaired in fear conditioning learning.
Biomaterial provider
A.I. Virtanen Institute for Molecular Sciences, Kuopio, Finland
Treatment protocol
Laser-capture microdissection of the lateral and basal nuclei of the amygdala for molecular profiling: Three hours after termination of the fear conditioning procedure animals were decapitated and the ipsilateral temporal lobe was dissected and embedded in Tissue- TekO.C.T. Compund (Sakura Finetek, McGaw Park, IL, USA), frozen on dry ice, and stored at -80oC. Then cryomolds containing the temporal lobe were cut into 7 μm thick serial sections and collected on Slides for Membrane-based Laser Microdissection (#11505158, Leica Microsystems, Germany). The slides were then stained with 1% cresyl violet (for protocol see www.ambion.com) and the lateral and basal nuclei of the amygdala (basolateral amygdala), delineated according to the description of Pitkänen and coworkers (1997), were dissected using Leica AS LMD Laser Microdissection System (Leica Microsystems). For each animal, around 60-70 sections containing basolateral amygdala were collected and used for laser-microdissection.
Growth protocol
Amygdala stimulation model (Nissinen et al., 2000): epileptogenic (E) animals = stimulated, responded with "good" status epilepticus (at least 40HAFDs within first 3h of SE), not expressing spontaneous seizures through the whole study. Fear conditioning: unpaired group; Animals received 5 habituation session to the fear conditioning apparatus lasting 10 min, starting on day 5 (2 sessions on day 5 and 6 and 1 session on day 7) after stimulation (induction of SE). On 8th day after stimulation: unpaired (U) = twice received: 2min in apparatus → footshock (1.5mA, 1s)¬→ tone (75dB for 20 sec).
Extracted molecule
total RNA
Extraction protocol
RNA isolation and amplification: Total cellular RNA was isolated using the PicoPure RNA isolation kit (#KIT0204, Molecular Devices, Sunnyvale, CA, USA) according to the protocol provided by the manufacturer. The quantity of isolated RNA was measured using NanoDrop-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Thereafter, 30 ng of total cellular RNA from each rat underwent 2 rounds of amplification using the MessageAmp II aRNA kit (#AM1751, Ambion, Austin, TX, USA), according to protocol provided by the manufacturer (in vitro transcription time was 14 h in both rounds). The quality of amplified aRNA was assessed on 1.5% agarose gel after each round of amplification.
Label
biotin
Label protocol
aRNAs from two (of four) rats belonging to this experimental group were pooled (3 μg aRNA from each animal). The array experiments were conducted in the Finnish DNA-Microarray Centre, Centre for Biotechnology, University of Turku, Finland. For microarray analysis, 5 μg of the pooled aRNA was first converted to cDNA using the one-cycle protocol (Affymetrix, Santa Clara,CA, USA), then to biotinylated cRNA.
Hybridization protocol
Biotynylated cRNA probe was hybridized to Rat Genome 230 2.0 Gene Chip (Affymetrix) according to protocols recommended by the manufacturer and used in the Finnish DNA-Microarray Centre (http://microarrays.btk.fi/index.php?id=874).
