Brains were collected, dissected in slices of 200µm and mounted to Superfrost microscope slides (Menzel, Braunschweig, Germany) in a cryostat (Microm MH50, Microm, Walldorf, Germany) from rostral to caudal. From these frozen slices the brain areas of interest were acquired by micropuncture as described before (Palkovits, 1973) utilizing punchers with a diameter of 0.5 or 1mm (Fine Science Tools, Heidelberg, Germany). The brain regions collected included the anterior part of the cingulate cortex (Cg), the nucleus accumbens core and shell (NAc), the PVN, the supraoptic nucleus (SON) and the basolateral/lateral (BLA/LA), central (CeA) and medial (MeA) amygdala. Punches of 1mm diameter were collected from Bregma +1.3mm to +0.9mm, twice sampling the tissue medially about 0.5mm from the dorsal tissue border to receive the Cg and bilaterally sampling the NAc core and unpreventably a minor part of its shell around the anterior commissure. Further tissue was collected medially 0.8mm above the ventral tissue limit (Ø=1mm) and bilaterally from the optic tract (Ø=0.5mm) to acquire tissue from the PVN and SON, from Bregma -0.56mm to -0.96mm. Amygdala tissue samples were collected bilaterally from two slides for each region acquiring punches of 1mm diameter. CeA was collected from Bregma -0.96mm to -1.36mm dorsomedially from the ventral end of the external capsule, MeA from -1.16mm to -1.56mm dorsolaterally from the optic tract and BLA/LA was collected from Bregma -1.36mm to -1.76mm from in between the bifurcation of the external capsule. Coordinates are according to the Mouse brain Atlas (Paxinos and Franklin, 2001).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted in presterilized 1.5ml Safelock tubes (Eppendorf, Hamburg, Germany) using a TRIzol (Invitrogen, Karlsruhe, Germany) chloroform standard protocol. After tissue homogenization in 300µl of TRIzol by pipetting, 1µl linear acrylamide (5mg/ml, Ambion, Austin, TX) and 60µl of chloroform (Carl Roth, Karlsruhe, Germany) were added and the samples vortexed. Centrifugation for 5min at 18°C and 13,000rpm followed, then RNA was precipitated with 180µl isopropanol (Carl Roth) overnight at -20°C, centrifuged at 4°C and 13,000rpm for 30min and washed twice in 500µl 70% ethanol (Carl Roth) with centrifugation steps at 4°C and 13,000rpm of 10min in between. Following the last centrifugation step, all remaining liquid was removed with a pipet, pellets were dried in an incubator for 15min at 45°C and resolved in 13µl of autoclaved bidistilled water.
Label
Cy5
Label protocol
Extracted total RNA was amplified and dye coupled in two rounds with Ambion’s Amino Allyl MessageAmp aRNA kit (Ambion) according to the manufacturer’s protocol using T7 oligo(dT) primers for the first round to select specifically for mRNA for reverse transcription and random hexamer primers for the second round of reverse transcription. The procedure included the synthesis of a first then a second strand of cDNA and an in vitro transcription step overnight. cDNA synthesis was carried out in PCR tubes (Abgene, Hamburg, Germany) using a thermal cycler (GeneAmp PCR System 9700, PE Applied Biosystems, Foster City, CA). For in vitro transcription, the tubes provided by the kit’s manufacturer were used, after pipetting all reagents, tubes were sealed with parafilm (American National Can, Neenah, WI) and incubated at 37°C in a warm room constantly heated. In the second round of in vitro transcription, an amino allyl extension to every second uracyl base was incorporated to receive the amino allyl RNA (aRNA) for the following indirect dye coupling reaction. Before aRNA purification the filters were equilibrated with 100µl aRNA binding buffer and incubated for 5min. All samples were pooled to one sample per line and brain region. The dye swapped experimental design required coupling of one half of each pooled sample to cyanine 3 (Cy3, Amersham Biosciences, Buckinghamshire, UK) the other half to cyanine 5 (Cy5, Amersham Biosciences). Correct quantification of aRNA was ensured by photometric assessment of the optic density in a photometer (Ultrospec II, Pharmacia LKB Biochrom, Cambridge, United Kingdom) and additional analysis on agarose gel.
Brains were collected, dissected in slices of 200µm and mounted to Superfrost microscope slides (Menzel, Braunschweig, Germany) in a cryostat (Microm MH50, Microm, Walldorf, Germany) from rostral to caudal. From these frozen slices the brain areas of interest were acquired by micropuncture as described before (Palkovits, 1973) utilizing punchers with a diameter of 0.5 or 1mm (Fine Science Tools, Heidelberg, Germany). The brain regions collected included the anterior part of the cingulate cortex (Cg), the nucleus accumbens core and shell (NAc), the PVN, the supraoptic nucleus (SON) and the basolateral/lateral (BLA/LA), central (CeA) and medial (MeA) amygdala. Punches of 1mm diameter were collected from Bregma +1.3mm to +0.9mm, twice sampling the tissue medially about 0.5mm from the dorsal tissue border to receive the Cg and bilaterally sampling the NAc core and unpreventably a minor part of its shell around the anterior commissure. Further tissue was collected medially 0.8mm above the ventral tissue limit (Ø=1mm) and bilaterally from the optic tract (Ø=0.5mm) to acquire tissue from the PVN and SON, from Bregma -0.56mm to -0.96mm. Amygdala tissue samples were collected bilaterally from two slides for each region acquiring punches of 1mm diameter. CeA was collected from Bregma -0.96mm to -1.36mm dorsomedially from the ventral end of the external capsule, MeA from -1.16mm to -1.56mm dorsolaterally from the optic tract and BLA/LA was collected from Bregma -1.36mm to -1.76mm from in between the bifurcation of the external capsule. Coordinates are according to the Mouse brain Atlas (Paxinos and Franklin, 2001).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted in presterilized 1.5ml Safelock tubes (Eppendorf, Hamburg, Germany) using a TRIzol (Invitrogen, Karlsruhe, Germany) chloroform standard protocol. After tissue homogenization in 300µl of TRIzol by pipetting, 1µl linear acrylamide (5mg/ml, Ambion, Austin, TX) and 60µl of chloroform (Carl Roth, Karlsruhe, Germany) were added and the samples vortexed. Centrifugation for 5min at 18°C and 13,000rpm followed, then RNA was precipitated with 180µl isopropanol (Carl Roth) overnight at -20°C, centrifuged at 4°C and 13,000rpm for 30min and washed twice in 500µl 70% ethanol (Carl Roth) with centrifugation steps at 4°C and 13,000rpm of 10min in between. Following the last centrifugation step, all remaining liquid was removed with a pipet, pellets were dried in an incubator for 15min at 45°C and resolved in 13µl of autoclaved bidistilled water.
Label
Cy3
Label protocol
Extracted total RNA was amplified and dye coupled in two rounds with Ambion’s Amino Allyl MessageAmp aRNA kit (Ambion) according to the manufacturer’s protocol using T7 oligo(dT) primers for the first round to select specifically for mRNA for reverse transcription and random hexamer primers for the second round of reverse transcription. The procedure included the synthesis of a first then a second strand of cDNA and an in vitro transcription step overnight. cDNA synthesis was carried out in PCR tubes (Abgene, Hamburg, Germany) using a thermal cycler (GeneAmp PCR System 9700, PE Applied Biosystems, Foster City, CA). For in vitro transcription, the tubes provided by the kit’s manufacturer were used, after pipetting all reagents, tubes were sealed with parafilm (American National Can, Neenah, WI) and incubated at 37°C in a warm room constantly heated. In the second round of in vitro transcription, an amino allyl extension to every second uracyl base was incorporated to receive the amino allyl RNA (aRNA) for the following indirect dye coupling reaction. Before aRNA purification the filters were equilibrated with 100µl aRNA binding buffer and incubated for 5min. All samples were pooled to one sample per line and brain region. The dye swapped experimental design required coupling of one half of each pooled sample to cyanine 3 (Cy3, Amersham Biosciences, Buckinghamshire, UK) the other half to cyanine 5 (Cy5, Amersham Biosciences). Correct quantification of aRNA was ensured by photometric assessment of the optic density in a photometer (Ultrospec II, Pharmacia LKB Biochrom, Cambridge, United Kingdom) and additional analysis on agarose gel.
Hybridization protocol
Ten array slides per brain region (MPI24K Arrays, MPI of Psychiatry, Munich, Germany) – serving as technical replicates - were prehybridized prior to the experiment in the prehybridization buffer for 1h at 42°C, then washed in water and isopropanol and dried by centrifugation (Megafuge 1.0R, Heraeus, Hanau, Germany) at 1500g for 3min in 50ml tubes (Sarstedt). Dye coupled aRNA samples were mixed with the contrary dye coupled sample from the other mouse line of the specific regions and loaded with a hybridization buffer to five arrays each under m-Series LifterSlips (Menzel). All arrays were hybridized at 50°C over 16-17h in separate hybridization chambers. Then arrays were washed in 2xSSC and 0.1% SDS for 5min at 42°C, 10min room temperature in 0.1xSSC and 0.1% SDS, four times in 0.1xSSC for 1min and finally for 20s in 0.01xSSC solutions. Slides were dried by centrifugation at 1,500rpm for 3min.
Scan protocol
The arrays were scanned on a PerkinElmer ScanArray 4000 (PerkinElmer Life and Analytical Sciences, Shelton, CT) laser scanner using automatic focusing and adapting laser power between 60 and 80 for Cy3 and 40 and 70 for Cy5. This ensured that for both dyes - in average - the same fluorescence intensities were reached and that not more than 1-2% of spots showed fluorescence intensities over the point of saturation.
Description
technical replicate dye-swapped 1 of 5 from pooled samples
Data processing
Quantification of all arrays was performed with QuantArray-software (GSI Lumonics, Billerica, MA) applying a fixed-circle quantification protocol and manual positioning of all grids over the hybridized spots. To provide a negative control for the hybridization and evaluation procedure, excess aRNA was used for an additional hybridization in both Cy3 and Cy5 combinations. For statistical evaluation, analytic methods were applied as previously described (Dudoit et al., 2002; Yang et al., 2002). In brief, first an MA-plot was generated to display raw fluorescence intensities of Cy5 (R) and Cy3 (G) with M = log2 R/G and A = log2sqrt(RG). Data were normalized to exclude systematic and technical errors. For two normalization steps, a function c was subtracted from the logarithmized fluorescence intensities (log2 R and log2 G). First a global normalization was performed, based on the assumption that R and G correlate. In a second normalization step, an intensity-dependent normalization was added, which was performed applying a LOESS smooth, as it is implied in the R software package (http://www.r-project.org). From the MA-plot, 40% of values were used to calculate the smooth. In several other normalization steps, differences were minimized that resulted from unequal distribution of probes from the array production or from unbalanced fluorescence intensities within one array slide. All data were merged in a matrix, p-values for multiple testing were calculated by permutation and are therefore called adjusted p-values.
