2.9.4
Mini Recording
Miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presence of TTX (1 µM, Biotrend, Cologne, Germany). All voltage clamp recordings were performed at a holding potential of -70 mV. mEPSC events were detected using the software MiniAnalysis (Synaptosoft) with a 5 pA amplitude threshold and all mEPSCs were proof-verified manually. Access resistance (Ra) and membrane resistance were regularly monitored during voltage clamp recording to be in the range of 10 - 28 and 150 - 650 MΩ, respectively. If changes larger than 30% occurred, the data was rejected from analysis. Cumulative probability curve was generated from more than 300 mEPSCs. Mean values for mEPSC parameters between groups were analyzed using ANOVA and a post-hoc Turkey’s test. All results presented were as mean ± s.e.m.
2.10
Cellular Compartment Analysis of Temporal Activity by
Fluorescence In Situ Hybridization (catFISH)
Expression of immediate early genes (IEG) is induced in brain neurons by synaptic activity and is frequently used to monitor neuronal activation. Cellular compartment analysis of temporal antivity by fluorescence in situ hybridization (catFISH) uses fluorescence in situ hybridization (FISH) of IEG RNAs to fixed cultured neurons, and confocal microscope to identify the activity requirement for IEG transcription initiation. The temporal resolution properties of catFISH derive from the time course of IEG transcriptional activation (and subsequent termination) and the translocation of the nascent mRNA to the cytoplasm. Cells active shortly before fixation ( 2 to 15 min) are distinguished by the presence of one or two areas of intense intranuclear fluorescence; these foci typically disappear within 20 min and the mRNA becomes prominent within the cytoplasm. This cytoplasmic RNA signal is used to identify neurons active 30 to 45 min earlier. Thus, the sub- cellular location of an IEG RNA signal can be used to discriminate cells activated by two distinct experiences separated by 30 min.
Activation of mouse IEG Arc was examined in this study. The plasmid pBSII(SK)-mArc was from Paul F. Worley lab (Johns Hopkins University, School of Medicine, Baltimore, Maryland). The protocol was based on the "catFISH, Neuroanatomical Methods, Current Protocols in Neuro- science" [54], with minor modification.
2.10.1
Plasmid Linearization and Purification
The template plasmid, pBluescript II SK (+)-Arc (pBSII(SK)-Arc)had cDNA for mouse or rat Arc gene between EcRI and XhoI restriction sites. This plasmid had T3 and T7 RNA polymerase promoter sequences at the two sides of Arc gene (Figure 2.3). Riboprobes were generated from linearized plasmids by in vitro transcription using fluorescein-labeled dUTPs. So the combination of EcoRI digestion and T7 polymerase transcription would produce the antisense probe. Similarly,
XhoI digestion and T3 polymerase transcription would produce the sense probe.
pBSII SK (+)-
Arc
EcoRI
Arc cDNA
XhoI
~ 3kb
T3
T7
Figure 2.3: Arc cDNA in pBSII SK(+) vector. Coding sequence of Arc gene was cloned in pBluescript SII SK(+) vector flanked with EcoRI and XhoI restriction sites and T3 and T7 RNA polymerase promoters at each end (from Paul F. Worley, Johns Hopkins University,Baltimore). Restriction enzyme digestion and RNA polymerase are used for in vitro synthesis of RNA: EcoRI digested plasmid followed with T7 RNA polymerase in vitro transcription produce the anti-sense RNA probe against Arc mRNA; XhoI digestion and T3 RNA polymerase transcription make the sense RNA probe.
4.5 µg of the template DNA was digested with EcoRI and XhoI, respectively. An example digestion setup was shown in Table 2.26.
After the digestion, linearization result was tested by running a 1% agrose gel together with the non-digested plasmid. The rest of the linearization product was purified with Phenol/Chloroform method. The cut constructs were filled up to 200 µl with DEPC-treated H2O and the same volume (200 µl) of Phenol/Chloroform buffer (Tris-buffered 50% phenol, 48% chloroform, 2% isoamyl alcohol solution, so called "25:24:1" solution) was added in. The mixture was mixed with vortex and centrifuged with speed 1300 g at room temperature for 10 min. The supernatant was transferred to a new RNase free tube and the 25:24:1 solution treatment was repeated once again. About 200 µl (as much as possible) supernatant was collected and mixed with same volume of Chloroform. The solution was mixed with vortex and centrifuged at 1300 g, room temperature for 10 min. The supernatant after Chloroform treatment was mixed with 1 µl of glycogen (20mg/ml), 0.1 volume of
2.10 catFISH 35
Table 2.26: DNA Linearization
Component 60 µl Reaction (µl) DNA 45a Red Buffer (10x) 6 Enzymeb 2 H2O up to 60 µl
aDNA concentration was adjusted to 100
ng/µl.
bEcoRI and XhoI digestion were made
separately and parallely.
3 M Na-acetate and 2.5 volumes of absolute ethanol. This mixture was put on dry ice for the DNA to precipitate for 10 to 15 min. After a few seconds of warm up, it was spun down in a 4◦C cooled centrifuge at 1400 g for 20 min. This time, the supernatant was discarded and the pellet was washed with 650 µl 70% ethanol twice, both with centrifugation at 4◦C , 1400 g for 10 min. The pellet was dried up at room temperature and resuspended with 25 µl DEPC-treated H2O. Concentration of the purified DNA probe was measured with a photometer (Pharmacia Biotech Ultraspec 2000 Spectrophotometer).
2.10.2
RNA Probes
For the in vitro transcription, SP6/T7 Transcription Kit (DIG RNA Labeling Kit (SP6/T7), Roche 11175025910) and T3 polymerase (Roche 11031163001) were used to get the antisense and sense RNA probe for Arc gene. The procedure was as follows.
Step 1 1 µg purified template DNA or 4 µl control DNA (vial 3 or 4) and DEPC-treated H2O was added into a RNase-free tube to the final volume of 13 µl.
Step 2 Following reaction mix (Table 2.27) was prepared on ice.
Table 2.27: In vitro Transcription
Reagent Vial Volume (µl)
NTP labeling mixture, 10x 7 2
Transcription buffer, 10x 8 2
RNase inhibitor 10 1
RNA Polymerase T7 or T3a 12 2
aT3 RNA polymerase was purchased separately.
The mixture was mixed gently and briefly spun down.
Step 3 After the transcription, 2 µl DNase I, RNase-free was added to remove template DNA.
This was incubated at 37◦C for 15 min.
Step 4 The whole reaction was stopped with adding 2 µl 0.2M EGTA (pH 8.0).
After in vitro transcription, the products were purified further with columns and the final RNA probe concentration was measured.
2.10.3
Fixation and Prehybridization
After different treatments, cells were fixed with ice-cold 4% PFA (pH 7.2 to pH 7.6) for 10 min. The fixation, following washing and prehybridization steps were summarized in Table 2.28.
Table 2.28: catFISH-Fixation & Prehybridization
Procedure Solution Time (min)
Incubation 4% PFA,ice-cold 10 1x PBS 2 1x PBS 2 70% EtOH 1 95% EtOHa 1 Rinse 2x SSCb 2
Incubation Acetic anhydride solution 10
Rinse DEPC-H2O several times
Incubation 1:1 acetone/methanol solution, ice-cold 5
Rinse 2x SSC 5
Incubation 1x Prehybridization solution 30
aPoint to stop. Coverslips could be stored in 95% EtOH for several days. bSSC: saline-sodium citrate buffer.
For the prehybridization, 1x prehybridization solution (Sigma P1415, 2x concentrate. Diluted 1:1 with deionized Formamide to prepare 1x pre-hybridization solution immediately before use.) was applied to each coverslip on parafilm in the chamber. Some Whatman 3 MM filter paper was saturated with 2x SSC / 50% deionized formamide and put at the bottom of the chamber. Cover- slips were incubated in this humid chamber for 30 min at room temperature.
2.10.4
Hybridization
After the prehybridization, coverslips were rinsed in 2x SSC. A final concentration of 1 ng/µl RNA probe (both antisense and sense, separately) were prepared in 1x hybridization solution (Sigma H7140, 2x concentrate. Diluted 1:1 with deionized Formamide to prepare 1x hybridization solution
2.10 catFISH 37
immediately before use.). The probe was denatured by being heated for 5 min at 90 ◦C and followed immediately by rapid cooling in an ice bath. Hybridization was performed in the humid chamber in a 56◦C oven overnight for at least 16 hours.
Following hybridization, coverslips were washed (Table 2.29) to remove unbound probe or probe which had loosely bound to imperfectly matched sequences. During all the subsequent washing steps, coverslips were gently agitated.
Table 2.29: Wash after Hybridization
Procedure Solution Time (min)
Wash 2x SSCa 5
2x SSC 5
2x SSC 5
Treatment 10 µg/ml RNasaeA in 2x SSC, 37◦C 15
Wash 2x SSC 10
Treatment 10 U/ml DNase Ib, 37◦C 15
Wash 2x SSC 10 0.5x SSC 10 0.5x SSC, 56◦C 30 0.5x SSC 10 Treatment 2% H2O2in 1x SSC 15 Wash 1x SSC 5 1x SSC 5 1x SSC 5 TBS 5
aAt room temperature if not stated.
bDNaseI treatment is needed for transfected or infected conditions only.
2.10.5
Signal Detection
FISH signals were detected with horseradish peroxidase (HRP) conjugated antibody to digoxi- genin and amplified using a Cyanine-3 Tyramide Signal Amplification kit ( TSA kit, PerkinElmer, NEL704A). Briefly, coverslips were blocked with TNB blocking buffer (0.1 M Tris-HCl, pH 7.5; 0.15 M NaCl; 0.5% Blocking reagent supplied by the TSA kit) at room temperature for 30 min. HRP conjugated DIG antibody (Anti-Digoxigenin-POD, Fab fragments from sheep, Roche 11207733910) was applied to coverslips at 1:100 dilution, 4 ◦C overnight. On the next day, cov- erslips were washed three times with TNT wash buffer (0.1 M Tris-HCl, pH 7.5; 0.15 M NaCl; 0.05% Tween 20) at room temperature with 5 min interval. Coverslips were then incubated with Fluorophore Tyramide for 30 min at room temperature. Fluorophore Tyramide was 1:87.5 diluted in Amplification Diluent. After three times wash with TNT wash buffer, coverslips were coun- terstained with nuclear fluorescent Hoechst 33258 (Serva, 2 µg/ml in PBS) for 5 min at room
temperature. Cells were then washed with PBS for three times with 5 min interval and mounted with Mowiol.
2.10.6
Acquisition of Confocal Image Series
catFISH results were examined with Leica SP2 confocal microscope. The setting up for the con- focal microscope was: 63x objective/Numerical aperture 1.32 oil, Zoom 2, 1024 x 1024 format, 8 bit resolution, xyz scan mode, 400 Hz speed, 1.0 airy pinhole, line average 2, frame average 1. Z dimension depth was defined with the Cy-3 signal and normally it spanned 7 to 10 µm. Intensity settings of photomultiplier tubes (PTM) for Hoechst and Cy-3 were set to get the minimal signal but not saturated for the highest. Signals from catFISH and Hoechst staining were scanned sequen- tially between frames with a ∆Z of 1 µm. For the quantitative analysis, confocal settings was adjust from above with the changes in: 40x objective, Zoom 1, 512 x 512 format, line average 1, and ∆Z 3 µm. These images were merged to get the maximal signal with ImageJ software and analyzed for positive Arc signal or signal location.